WO2023018692A1

WO2023018692A1 - Compositions and methods for treating infections involving biofilm

Info

Publication number: WO2023018692A1
Application number: PCT/US2022/039797
Authority: WO
Inventors: Manu CAPOOR; Ondrej SLABY
Original assignee: ECM Diagnostics Inc
Current assignee: ECM Diagnostics Inc
Priority date: 2021-08-09
Filing date: 2022-08-09
Publication date: 2023-02-16
Anticipated expiration: 2024-02-09

Abstract

In the various aspects and embodiments, the invention provides compositions and methods for treating biofilm-associated bacterial infections, including but not limited to infections involving commensal pathogen. The invention provides compositions and methods for treating an infection involving bacterial biofilm in a patient, where the method comprises administering to the patient an antibiotic and a cytokine inhibitor.

Description

COMPOSITIONS AND METHODS FOR TREATING INFECTIONS INVOLVING BIOFILM

BACKGROUND

Biofilm-associated infections represent a serious health burden. They are a consequence of microorganisms that colonize biological interfaces or surfaces of indwelling medical devices upon which they produce an extracellular matrix. Embedded within this matrix, such microorganisms become resistant to antibiotic treatments and adapt to host immune system effector mechanisms. Antibiotic treatments alone are often ineffective in treating these infections.

For example, a substantial number of patients undergoing microdiscectomy surgery actually have an underlying occult biofilm-based infection by the opportunistic pathogen Propionibacterium acnes (P. acnes), recently renamed as Cutibacterium acnes (C acnes). C. acnes is an anaerobic bacterium that is involved in the inflammatory skin condition acne vulgaris. C. acnes can also be involved in postoperative device-related infections, infected tissues and joints, surgical site infections, and infections of wounds. Antibiotic treatment alone is often not effective for C. acnes- related infections, and/or requires a prolonged course of treatment, such as 3 to 6 months with oral therapy, sometimes with intravenous treatment with beta-lactam. Achermann et al., Propionibacterium acnes: from commensal to opportunistic biofilm-associated implant pathogen. Clin. Microbiol. Rev. Vol. 27, No. 3 (2014).

There is a need for improved therapies for biofilm-associated infections, including for treatment of degenerative disc disease (DDD) and chronic lower back pain, as well as infections associated with indwelling medical devices and opportunistic or chronic infections of various tissues and joints. In the various aspects and embodiments, the present invention meets these objectives.

SUMMARY OF THE INVENTION

In the various aspects and embodiments, the invention provides compositions and methods for treating biofilm-associated bacterial infections, including infections involving commensal pathogens, including but not limited to Cutibacterium acnes. The invention leverages synergy between antibiotic treatment and treatment with certain cytokine inhibitors. For example, in various embodiments treatment with a cytokine inhibitor (such as but not limited to an IL-ip inhibitor) reduces an antibiotic-resistant phenotype of biofilm-associated bacteria, resulting in a more antibiotic-sensitive phenotype.

Thus, in one aspect, the invention provides a method for treating an infection involving bacterial biofilm in a patient. The method comprises administering to the patient one or more antibiotics and an inhibitor of a cytokine, including but not limited to an inhibitor of a pro-inflammatory cytokine. In various embodiments, the infection is an infection of a commensal pathogen. In some embodiments, the infection is a low virulence infection. Exemplary conditions in which infection of a commensal pathogen or low virulence infection may be involved include infections of one or more organs or tissues selected from skin, prostate, eye, ear, nose, throat, breast, heart, vasculature, lung, intestinal tract, urogenital tract, oral cavity, central nervous system, bone, and joints. In some embodiments, the patient has an infection involving a commensal pathogen (which may be a low virulence infection), which can be manifest as or be associated with degenerative disc disease (DDD), chronic lower back pain (CLBP), orthopedic infections, implant infections, musculoskeletal conditions (osteitis, osteomyelitis, synovitis-acne-pustulosis-hyperostosis-osteitis (SA-PHO) syndrome, sarcoidosis, chronic prostatitis, and prostate cancer, among others. In some embodiments, the commensal pathogen is C. acnes. In other embodiments, the infection is a chronic infection involving a biofilm forming bacteria, including but not limited to Mycobacterium or Borrelia.

In some embodiments, the one or more antibiotics can be administered locally to the affected tissue. For example, antibiotic therapies can be administered by local injection or application to infected tissues or areas surrounding infected tissues, such as to the intervertebral disc region, or to periprosthetic tissues, or by intra-articular or percutaneous injection. Alternatively, the antibiotic may be administered systemically (e.g., orally or i.v.). In some embodiments, antibiotics (alone or with other therapies) are applied locally during a biopsy or surgical procedure (e.g., endoscopic surgery). In still other embodiments, the one or more antibiotics can be administered topically, for example to the infected portions of the skin or wound.

Exemplary antibiotics in accordance with various embodiments include betalactams, carbapenems, quinolones, macrolides, and cephalosporins, among others. In some embodiments, the patient receives chronic antibiotic therapy, for example, for about 100 days or more. In various embodiments, the antibiotic (or combination thereof) is administered orally one or more times daily. Alternatively or in addition, one or more antibiotics are administered by i.v. In some embodiments, intravenous antibiotic therapy (e.g., with beta lactam antibiotic) can be about once daily to about once weekly for 2 to about 6 weeks.

In accordance with the present disclosure, the patient will further receive a therapy inhibiting one or more cytokines. In various embodiments, the cytokine is a proinflammatory cytokine that is expressed at sites of infection, and/or one that induces bacterial virulence, antibiotic resistance, and/or bacterial growth. In some embodiments, the cytokine induces or sustains antibiotic resistance. In still other embodiments, the cytokine induces nerve growth. In various embodiments, the cytokine is one or more selected from IL-ip, IL-la, IL-2, IL-6, IL-8, IL-10, IL-12, IL-17, IL-23, INF-y, TNF-a, TGF-P, CCL-3, CCL-4, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF).

In some embodiments, the method involves administering an IL-ip inhibitor. For example, in various embodiments, the IL-ip inhibitor is a monoclonal anti-IL-ip antibody (or antigen-binding domain or antibody fragment thereof), a recombinant protein with IL-ip binding activity, or a small molecule inhibitor. In some embodiments, the IL-ip inhibitor is a monoclonal antibody or other recombinant protein (e.g., IL-1 trap) that binds to and blocks and/or neutralizes IL-ip. For example, in some embodiments, the anti-IL-ip antibody is administered by injection directly to intervertebral disc tissue, periprosthetic tissue, or by intra-articular injection to affected joints. In some embodiments, an IL- ip inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In certain embodiments, the IL-ip inhibitor is administered topically, for example, to infected areas of the skin or wound. In some embodiments, the IL-ip inhibitor is administered as a co-formulation with one or more antibiotics.

The synergistic action of an IL-ip inhibitor, together with an antibiotic, for example in a fixed dose combination, delivered at time of microdiscectomy can be a surprisingly effective therapy for an underlying disc infection. The underlying anti- inflammatory mechanisms of the IL-ip inhibitor not only targets the host but also targets the bacteria growing as an adherent biofilm. By treating the infection, the associated inflammation, and the biofilm, the therapy can significantly reduce the rate of follow-on discectomies, fusions, and resulting disability.

In some aspects, the invention provides a pharmaceutical composition comprising an effective amount of an antibiotic and an inhibitor or a cytokine, such as an inhibitor of a pro-inflammatory cytokine. Exemplary inhibitors of cytokines may be antibodies or recombinant proteins, or small molecule inhibitors, targeting one or more of IL-ip, IL- la, IL-2, IL-6, IL-8, IL-10, IL-12, IL-17, IL-23, INF-y, TNF-a, TGF-P, CCL-3, or CCL- 4, nerve growth factor (NGF), or brain-derived neurotrophic factor (BDNF). In some embodiments, the pharmaceutical composition comprises an IL-ip inhibitor, such as canakinumab, gevokizumab, LY2189102, CDP-484, and IL-1 trap. The pharmaceutical composition further comprises one or a combination of antibiotics, including but not limited to beta-lactams, carbapenems, quinolones, macrolides, and cephalosporins. In various embodiments, one or more antibiotics are selected from penicillin, benzylpenicillin, amoxicillin, ampicillin, dicloxacillin, methicillin, nafcillin, oxacillin, penicillin G, piperacillin-tazobactam, cephalexin, cefoxitin, cephalothin, ceftriaxone, ciprofloxacin, levofloxacin, chloramphenicol, erythromycin, tetracycline, tigecycline, minocycline, vancomycin, clindamycin, azithromycin, fusidic acid, doxycycline, moxifloxacin, linezolid, rifampicin, rifampin, telavancin, doripenem, ertapenem, imipenem, meropenem, taurolidine, daptomycin, metronidazole, trimethoprimsulfamethoxazole, or a combination thereof. In some embodiments, the composition comprises a beta-lactam antibiotic (such as but not limited to amoxicillin) and a betalactamase inhibitor (e.g., clavulanate). In some embodiments, the composition comprises anti-IL-ip antibody or IL-1 trap, and vancomycin.

The pharmaceutical composition may be formulated as a solution, suspension, dispersion, emulsion, or the like. In some embodiments, the composition is in the form of a sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. In some embodiments, the composition is formulated as a cream, gel, or ointment for topical application.

Embodiments of the invention will be further illustrated with the following nonlimiting examples. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 illustrates aP. acnes-d ven inflammatory state. P. acnes PAMPs bind to TLRs, which activate NF-KB, leading to the synthesis of pro-IL-i ; P. acnes PAMPs also activate inflammasomes (NLRP3) and caspase-1, which convert pro-IL-1 to IL-1P; P. acnes proteases directly convert pro-IL-1 P to IL-ip.

Figure 2 illustrates an IL-ip driven degenerative state. IL-ip stimulates production of CC-chemokines that recruit myeloid cells, neurotrophins NGF and BDNF that induce nerve ingrowth, and proteolytic enzymes (MMPs, ADAMTS4/5 aggrecanases) that degrade the ECM. Positive feedback loops arise when IL-ip binds to IL-1R and when extracellular matrix degradation products (DAMPs; e.g. fragmented collagen, aggrecan, and hyaluronic acid) activate TLRs. Both binding events further stimulate IL-ip production.

Figure 3 illustrates a P. acnes driven, pain generating state. P. acnes directly activates nociceptors through several distinct mechanisms: N-formylated peptides activate FPR on a subset of nociceptor neurons that mediate mechanical hyperalgesia; CAMPs and pore-forming hemolytic factors allow the entry of ions that lead to depolarization that induces mechanical and thermal hyperalgesia.

Figure 4 depicts relative lipase activity after 3h and 24h of 1 : 100 and 1 : 1000 NP cells/ . acnes co-cultures, confirming viability of P. acnes.

Figure 5A-F depicts average expression levels of pro-inflammatory cytokines:

(A) IL-ip, (B) IL-la, (C) IL-6, (D) IL-8, (E) CCL3, (F) CCL4, at four time-points (3h, 24h, 48h and 1 week). MOI100 and MOIIOOO were normalized to their levels in noninfected NP cells. LPS-treated cells were used as positive controls (* P<0.05, ** P<0.01).

Figure 6 illustrate an increase in the NGF and BDNF expression levels in P. ocwc.s'-infected NP cells after one week of infection (* P<0.05).

Figure 7 shows an increase in IL-1 P protein in NP cells after 24 hours of P. acnes infection, as detected by ELISA (* P<0.05).

Figure 8A-C depicts clindamycin suppression of the production of (A) IL-ip,

(B) IL-6 and (C) IL-8 in P. acnes -infected NP cells. Averages from the experiments with the three NP cell cultures are presented (* P<0.05, ** P<0.01). Abbreviations'. ADAMTS-4/5, a disintegrin and metalloproteinase with thrombospondin motifs 4/5; AF, anullus fibrosus; BDNF, brain-derived neurotrophic factor; CAMP, Christie, Atkins and Munch-Petersen factors; CLBP, chronic low back pain; DAMPs, damage-associated molecular patterns; DDD, degenerative disc disease; ECM, extracellular matrix; PAMPs, pathogen-associated molecular patterns; FPR, N- formylated peptide receptor; IL-1R, interleukin-1 receptor; MMPs, matrix metalloproteinases; NF-KB, nuclear factor KB; NGF, nerve growth factor; NP, nucleus pulposus; TLRs, Toll-like receptors, MOI, multiplicity of infection.

DETAILED DESCRIPTION

In the various aspects and embodiments, the invention provides compositions and methods for treating biofilm-associated bacterial infections, including infections involving commensal pathogens and other chronic biofilm-forming pathogens. As disclosed herein, the invention leverages synergy between antibiotic treatment and treatment with cytokine inhibitors. For example, in various embodiments treatment with a cytokine inhibitor (such as but not limited to IL-ip inhibitor) reduces the antibioticresistant phenotype of biofilm-associated bacteria, resulting in a more antibiotic-sensitive phenotype.

Thus, in one aspect, the invention provides a method for treating an infection involving bacterial biofilm in a patient. The method comprises administering to the patient an effective amount of one or more antibiotics and a cytokine inhibitor. The cytokine inhibitor may be an inhibitor of a pro-inflammatory cytokine.

In various embodiments, the infection is an infection of a commensal pathogen. Exemplary commensal pathogens include Propionibacterium sp., Staphylococcus sp., Corynebacterium sp., Lactobacillus sp., Pseudomonas sp., Enterococcus sp., Streptococcus sp., Bacillus sp., Citrobacter sp., E. coli, Moraxella sp., Haemophilus sp., Neisseria sp., Clostridium sp., Enterobacter sp., Helicobacter sp., and Klebsiella sp. In some embodiments, the commensal pathogen is Propionibacterium acnes (recently renamed Cutibacterium acnes or C. acnes; the designations of P. acnes and C. acnes are used interchangeably herein). Thus, the infection can be an infection in which such commensal pathogens (such as but not limited to C. acnes) are frequently implicated or associated, or one in which microbiological profiling has determined the commensal pathogen (such as but not limited to C. acnes) to be present in an infectious capacity. Such infections are sometimes referred to herein as a low virulence infection. In accordance with this disclosure, the anti-inflammatory properties of the cytokine inhibitor can impact both the host and the pathogen responsible for the low-virulence infections, facilitating more effective antibiotic treatment (i. e. , as compared to antibiotic treatment in the absence of the cytokine inhibitor).

A low- virulence infection is a chronic, low-grade, infection that is associated with a commensal microorganism. The clinical presentation is often missing characteristic signs of infection and can lead to delayed treatment and increased associated costs. In various embodiments, the low-virulence microorganism has a biofilm forming phenotype. In accordance with embodiments of the present invention, the low-virulence infection involves C. acnes. C. acnes is a gram-positive aerotolerant anaerobe that forms part of the normal resident microbiota of the skin, oral cavity and the gastrointestinal and genito-urinary tracts. It is an opportunistic pathogen that has been linked to a wide range of infections and conditions, including implant infections, discitis, musculoskeletal conditions (e.g., osteitis, osteomyelitis, synovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome), sarcoidosis, and chronic prostatitis. In accordance with embodiments of the invention, the low-virulence infection may be associated with any of a variety of organs or tissues, including but not limited to skin, prostate, eye, ears, nose, throat, breast, heart, vasculature, lung, intestinal tract, urogenital tract, oral cavity, central nervous system, bone, and joints. In some embodiments, the infection is associated with a surgical site or wound in the surgical environment (“surgical wound”). In various embodiments, the infection is a post-operative infection (such as a postoperative device-related infection, including but not limited to prosthetic shoulder joint, knee or hip joint, cerebrovascular device, cardiovascular device, breast implant, and spinal prosthesis). Exemplary low- virulence infections (i.e., involving a commensal pathogen) may be associated with an orthopedic device, cerebrospinal fluid shunt, external ventricular drainage, postoperative craniotomy, breast implant, pacemaker, Implantable Cardioverter Defibrillator (ICD), heart valve replacement or surgery, vascular graft, endophthalmitis (e.g., after lens implantation), peritoneal catheter, osteomyelitis (e.g., spinal osteomyelitis), sarcoidosis (including associated with lungs, heart, lymph nodes, or eyes), prostatitis, arteriosclerosis, atherosclerosis, rheumatoid arthritis, osteoarthritis, fibromyalgia, cystic fibrosis, and systemic lupus erythematosus. In some embodiments, the infection is an infection involving the skin, such as acne vulgaris, psoriasis, eczema, or atopic dermatitis. In still other embodiments, the infection by a commensal pathogen is an infection of the oral cavity such as periodontitis or endodontitis. In some embodiments, the infection is associated with esophagitis or gastric ulcer. In some embodiments, the infection by a commensal pathogen is a urinary tract infection (UTI), sinusitis, rhinosinusitis, or otitis media. In some embodiments, the infection involving biofilm is inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis), diverticulitis, or celiac disease. In still other embodiments, the subject is suspected of having a low- virulence infection of the central nervous system, and which may be an aggravating or causative element of a movement, neurodegenerative, or mental health condition (e.g., mood or behavioral disorder). Exemplary conditions include multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, chronic fatigue syndrome, chronic depression or anxiety, and schizophrenia, among others.

Thus, exemplary conditions in which infection of a commensal pathogen or low virulence infection may be involved include degenerative disc disease (DDD), chronic lower back pain (CLBP), orthopedic infection, implant infection, musculoskeletal condition (osteitis, osteomyelitis, SA-PHO syndrome, sarcoidosis, and chronic prostatitis). For example, the commensal pathogen or low-virulence infection can be associated with a prosthetic, such as a shoulder prosthetic (where C. acnes is frequently isolated) or other prosthetic joint such as knee or hip. In other embodiments, the commensal pathogen or low-virulence infection is associated with a cardiovascular device (e.g., cardiovascular implantable electronic device, CIED, such as a defibrillator or pacemaker).

In some embodiments, the patient has DDD or CLBP, and the subject is suspected of having an associated low-virulence infection. For example, the patient may have DDD or CLBP, and the patient may be suspected of having a low-virulence infection (such as an infection by a commensal pathogen). In accordance with this disclosure, the patient is administered one or more antibiotics and a cytokine inhibitor. In various embodiments, the method can be employed with or without surgical interventions to resolve radicular pain and/or nociceptor pain that may be associated with the condition. In some embodiments, the method can be employed in connection with a surgical intervention, by local injection of a pharmaceutical composition comprising the active agents.

The present disclosure is further applicable to other biofilm forming bacteria that are responsible for chronic and hard to treat infections, such as but not limited to Mycobacterium sp. (including Mycobacterium tuberculosis, Mycobacterium leprae, and non-tuberculous mycobacterium) as well as Borrelia sp. (e.g., Borrelia burgdorferi), Yersinia sp., and Chlamydia sp. (e.g., Chlamydia trachomatis).

The present disclosure demonstrates the upregulation of IL-ip, and other known IL-ip-induced inflammatory markers and neurotrophic factors, from nucleus-pulposus- derived disc cells infected in vitro with the commensal pathogen C. acnes, which can be responsible for low-virulence infection. Upon infection, significant upregulation of IL- ip, alongside IL-6, IL-8, chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-C motif) ligand 4 (CCL4), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), was observed with cells isolated from the degenerative discs of eight patients versus non-infected controls. Pre-treatment of cells with clindamycin prior to infection significantly reduced the production of pro-inflammatory mediators. These studies support the finding that C. acnes stimulates the expression of IL-ip and other host molecules previously associated with pathological changes in disc tissue, including neo-innervation.

In some embodiments, the patient has CLBP, and which is consistent with structural intervertebral disc damage and/or a low-virulence infection. In some embodiments, the patient may be a candidate for, and may be scheduled for, intervertebral disc surgery. Patients that have a low-virulence infection may be at increased risk of developing CLBP and/or may become “failed back surgery” patients, unless diagnosed correctly and treated appropriately. For example, some patients that undergo disc surgery will also suffer from CLBP prior to the acute condition necessitating their surgery. Also, a certain proportion (around 5 to 10%) of patients undergoing disc surgery will develop CLBP in the follow-up, which are sometimes referred to as “failed back surgery” patients or “post-discectomy syndrome”. These conditions are statistically associated with a low-virulence infection.

Stirling et al. (2001) published the first evidence of bacterial infection of the degenerated disc in 2001. Stirling et al. found that 19 of 36 (53%) sciatica patients who had undergone microdiscectomy tested positive for bacterial infection; 84% of these were P. acnes infections. Subsequent research yielded conflicting results, but two independent meta analyses (Urquhart 2015; Ganko 2015) reported the pooled prevalence of bacterial infection was 34% or 36.2% and P. acnes was identified as the major infecting species. They found moderate evidence that low virulent bacteria play a role in disc degeneration and moderate evidence of causation, but indicated that these observations could result from bacterial contamination. A 368-patient study has now confirmed the previously observed prevalence and found that P. acnes was the only significant species isolated from degenerated disc tissue. Importantly, this study documents the presence of P. acnes biofilm in the infected disc tissue; a result consistent with infection rather than perioperative contamination (See, Capoor MN, et al., Propionibacierium acnes biofilm is present in intervertebral discs of patients undergoing microdiscectomy. PLoS One 2017;12(4):e0174518).

While antibiotic therapy has seen some moderate results in the treatment of CLBP, in accordance with this disclosure, it is believed that antibiotic therapy alone will often not be effective to treat a low- virulence infection (e.g., involving C. acnes), in view of the pathology of these infections in which the pro-inflammatory state and bacterial virulence factors (including biofilm formation) work in concert to sustain the infection and maintain an antibiotic-resistant phenotype.

P. acnes plays a key role in the development of acne vulgaris, as well as roles in other chronic and recurrent infections, including implant infections, which are facilitated by the organism’s ability to form biofilms. In pilosebaceous units, P. acnes pathogen- associated molecular patterns (PAMPs) bind to toll-like receptor (TLR)2 on sebocytes. This activates NF-KB signaling and results in the production of pro-IL-ip. P. acnes PAMPs also trigger NLRP3 inflammasome activation, which facilitates pro-IL-ip cleavage and the excessive release of mature IL-ip. IL-ip promotes dermal matrix destruction through induction of matrix metalloproteases (MMPs). Progression of the inflammation results in follicular rupture, allowing P. acnes to leak out of the pilosebaceous unit and activate perifollicular myeloid cells. This results in further release of IL-ip and neutrophil-rich perifollicular inflammation. Thus, IL-ip is the driver of inflammatory responses to P. acnes in acne vulgaris.

Disc degeneration is mediated by the abnormal secretion of cytokines by the inner nucleus pulposus (NP) and outer annulus fibrosus (AF) cells of the intervertebral disc, as well as by immune cells attracted to the site of disc degeneration. The initiating insult is thought to trigger NF-KB signaling through TLRs and stimulate the production of pro- IL-ip. Increased levels ofNLRP3 and caspase-1 have also been described in degenerated disc tissue, indicating inflammasome activation and facilitated maturation of pro-IL-ip. In addition, P. acnes proteases can convert pro-IL-ip to mature IL-ip. Inflammatory actions are further amplified because IL-ip is not only an NF-KB target gene, but also an NF-KB activator, forming a positive feedback loop. This IL-ip-IL-Rl signaling promotes extracellular matrix degradation through induction of proteolytic enzymes, including MMPs 1, 2, 3, 9, and 13 and aggrecanases of ‘disintegrin and metalloproteinase with thrombospondin motifs’ (ADAMTS) families 4/5. Specific damage-associated molecular patterns (DAMPs), which include fragmented collagen, aggrecan, or hyaluronic acid, bind to TLRs on NP and AF cells and activate NF-KB, forming another positive feedback loop. IL-ip also promotes the production of chemotactic CC- chemokines, mainly CCL3 and CCL4, leading to the recruitment and activation of infiltrating immune cells that further amplify the inflammatory cascade. These pathways form the core of a model in which P. acnes infection causes IL-1 P release by NP, AF and immune cells, which leads to extracellular matrix degradation within the intervertebral disc. See FIGS. 1 and 2.

The ingrowth of nociceptive nerve fibers into the degenerated disc, usually accompanied by the presence of annular fissures can be a main source of nociception related to CLBP. IL-ip induces the expression of neurotrophin-like nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in both disc and immune cells, supporting nerve ingrowth into the degenerated disc. IL-ip-stimulated NGF and BDNF production further induces expression of pain-associated cation channels in the dorsal root ganglion, the depolarization of which is likely to promote low back and radicular pain. Finally, NGF has a direct activating or sensitizing effect on nociceptors, and its upregulation in CLBP has been demonstrated. FIG. 2.

Disc cells (NP and AF) make up a small portion of the disc tissue mass and primarily remove waste and receive nutrition through diffusion. In degenerative disc disease, there can be nerve ingrowth and likely some level of angiogenesis to support the nerve ingrowth. In accordance with this disclosure, P. acnes produces alpha-hemolysins and other virulence factors that interact with nociceptors in nerve in-growths, resulting in CLBP. Further, in accordance with this disclosure, P. acnes is believed to significantly amplify etiological factors that contribute to DDD through the promotion of IL-ip, which facilitates pathogenesis in synergy with other etiological factors of DDD. Since activation of IL-ip system promotes low back pain, P. acnes as extensive inducer of IL-ip indirectly participates in the pathogenesis of CLBP and DDD. Based on the ability of Staphylococcus aureus to activate sensory neurons by releasing formyl peptides and the pore-forming/hemolytic virulence factor a-haemolysin, it is believed that there is also a direct, IL-ip independent, mechanism of P. acnes involvement in CLBP realized through the release of pore-forming hemolysins that induce calcium flux and action potentials in nociceptive neurons. This is supported by the fact, that P. acnes genome contains several putative pore-forming/hemolytic virulence factors (e.g. genes PPA687, PPA1198, PPA1231, PPA1340, and PPA2108 encoding homologs of Christie, Atkins and Munch-Petersen (CAMP) factors, potentially hemolytic gene products including PPA565, which shares similarity with hemolysin III of B. cereus, and PA938 (COG1253) and PPA1396 (COG1189), which share similarity with the hemolysins TlyC and TlyA of Brachyspira hyodysenteriae, respectively). These hemolytic factors are clinically relevant, as hemolysis can be used as a clinical marker for the presence of P. acnes in orthopedic infections. Therefore, P. acnes can play a role in the pathogenesis of CLBP and other conditions described herein (including but not limited to orthopedic infections), and P. acnes bacterial load could be directly linked to clinical course. FIG. 3.

In some embodiments, the patient is suspected of having an associated infection based on the detection of a commensal pathogen (e.g., P. acnes) in a tissue of interest, such as intervertebral disc tissue, or other tissue biopsy including but not limited to synovial fluid or periprosthetic tissue. In some embodiments, the presence of the commensal pathogen is detected in biopsied tissue by one or more techniques selected from: the presence of microbial nucleic acid, host cell RNA profile, culturing of the commensal pathogen from the tissue, immunohistochemistry, microbial-specific staining of tissue, and presence of a metabolite signature in the tissue. In some embodiments, the infection is identified by the presence of one or more virulence factors in the patient’s biopsied tissue, where the virulence factors can be specific to a particular commensal microorganism, such as but not limited to P acnes. In some embodiments, the methods comprise detection or quantification (e.g., detection, identification, and quantification) of commensal microorganisms, such as but not limited to P. acnes. In some embodiments, the presence of biofilm in the biopsied tissue is confirmed. In some embodiments, the patient has intervertebral disc disease or CLBP and a fine needle biopsy is isolated for testing. In these embodiments, the invention involves analysis of disc tissue for the presence of infection of one or more commensal pathogens (e.g., P. acnes), and/or for the presence of an RNA signature indicative of a low-virulence infection. In some embodiments, the presence of infection is determined using disc tissue following discectomy. In some embodiments, the invention involves detecting or quantifying commensal pathogen(s) (including but not limited to P. acnes) in the disc tissue sample by microbiological cultivation or by genetic, microbial-specific stain, immunochemical, or spectroscopic analysis. In some embodiments, the invention further comprises evaluating the RNA from a sample (e.g., mRNA or miRNA) to classify the profile as being indicative of low virulence infection, or not being indicative of a low virulence infection. In some embodiments, an RNA profile is evaluated independently to determine the presence of a low virulence infection, with or without the use of other techniques such as PCR, culture, or microscopy.

In some embodiments, an RNA profile (of host tissue) is evaluated for the presence of an RNA signature (e.g., mRNA or miRNA signature) that is indicative of a low-grade or low-virulence infection. Exemplary RNA signatures are disclosed in WO 2017/019440, which is hereby incorporated by reference. An exemplary miRNA score for P. acnes infection can be determined by scoring the relative expression levels of miR- 29a-3p and miR-574-3p. For example, a diagnostic miRNA score (DMS) based on the following formula results in high specificity using a cut-off of -0.3: DMS = 18.71 - 11.24 * loglO (miR-29a-3p) + 10.4 * loglO (miR-574-3p). Other RNA signatures can be trained from RNA profiles of samples that are positive or abundant for P. acnes (or other commensal microorganism) and samples that test negative (or non-abundant) for P. acnes (or other commensal microorganisms). For example, samples can be binned based on detection (or detection level) of P. acnes by quantitative PCR and at least one other technique, such microbial culture or spectroscopy (including with microbial stains, FISH, or immunohistochemistry).

In some embodiments, the presence of a low-virulent infection is evaluated by culture, including aerobic and/or anaerobic cultivation and subsequent biochemical and spectroscopic (e.g., mass spec., MALDI-TOF MS, or NMR) identification of species. For genetic analysis, nucleic acids (e.g., DNA and/or RNA) are isolated from the sample for analysis. In still other embodiments, immunochemistry (e.g., immunohistochemistry or ELISA) can be used to detect protein or other epitopes of commensal organisms. For instance, without limitation, monoclonal antibodies against P. acnes antigens including virulence factors can be used, such as antibodies specific for epitopes of cell-membrane- bound lipoteichoic acid (PAB antibody) and ribosome-bound trigger factor protein (TIG antibody). In some embodiments, antibodies specific for P. acnes alpha-hemolysin or formylated peptide is used to identify these virulence factors in biopsy or disc material, which indicates that the patient is suffering from a low virulence infection. Other techniques such as microscopy on tissue samples can be used to identify positive samples, and may employ any appropriate staining reagent (e.g., gram stain, P. acnes- specific stain, or fluorescent in situ hybridization (FISH)) or other immunoreagents specific for the commensal microorganism of interest. Mass spectroscopy techniques can be used to identify P. oc s -speci fic molecules that are indicative of infection, including biofilm components.

In some embodiments, the presence or level of commensal pathogens is determined (alternatively or in addition to culturing) by hybridization or amplification of microbial nucleic acids. For example, detection assays include real-time or endpoint polymerase chain reaction (PCR), nucleic acid hybridization to microarrays, or nucleic acid sequencing. In some embodiments, the molecular detection assay detects microbial ribosomal RNA (rRNA) genes, such as 16S and/or 23S rRNA genes, and particularly the variable regions of 16S. In some embodiments, the presence of a commensal pathogen is determined by a microarray hybridization-based assay or DNA sequencing. Exemplary methods for diagnosing a low-virulence infection of an intervertebral disc are described in WO 2017/019440, which is incorporated herein by reference.

In some embodiments, the presence of the low-virulence infection is determined in a non-invasive manner. For example, in some embodiments an MR-spectroscopy signature is obtained to determine the chemical composition of the intervertebral disc. The chemical composition may comprise biofilm components specific for P. acnes biofilm, as well as other molecules indicative of P. acnes infection. Exemplary methods for performing MRS on intervertebral discs are described in US 2011/0087087, which is hereby incorporated by reference in its entirety. In some embodiments of the present invention, where the diagnostic test confirms a low-virulence infection, the patient may be treated for infection before any invasive surgery (such as but not limited to discectomy), and in some embodiments, invasive procedures may be entirely avoided. In still other embodiments, the patient is treated according to this disclosure in connection with a surgical procedure.

According to embodiments of the disclosure, therapeutic approaches combining appropriate antibiotic treatment with a therapy or therapies targeting pro-inflammatory cytokines (concomitantly or sequentially) are undertaken. A range of therapies targeting these factors are known, including several monoclonal antibody therapies.

Antibiotics can be administered locally to the affected tissue. For example, antibiotic therapies can be administered by local injection or application to infected tissues or areas surrounding infected tissues, such as to the intervertebral disc region, or to periprosthetic tissues, or by intra-articular injection. Alternatively, the antibiotic may be administered systemically (e.g., orally or i.v.). In some embodiments, one or more antibiotics (alone or with other therapies) are applied locally during a biopsy or surgical procedure. In some embodiments, one or more antibiotics are applied topically, for example, to infected portions of the skin or infected wound. Exemplary antibiotics (including for P. acnes infection) include beta-lactams, carbapenems, quinolones, macrolides, and cephalosporins, among others. Exemplary antibiotics for treatment of commensal pathogens (including but not limited to P. acnes) include clindamycin, erythromycin, vancomycin, and daptomycin. In some embodiments, the antibiotic is a tetracycline antibiotic, such as tetracycline, minocycline, doxycycline, oxy tetracycline and lymecycline. In various embodiments, depending on the etiology of the infection, the antibiotic can include one or more of a beta-lactam, a macrolide, and a tetracycline. In some embodiments, one or more antibiotics are selected from penicillin, benzylpenicillin, amoxicillin, ampicillin, dicloxacillin, methicillin, nafcillin, oxacillin, penicillin G, piperacillin-tazobactam, cephalexin, cefoxitin, cephalothin, ceftriaxone, ciprofloxacin, levofloxacin, chloramphenicol, erythromycin, tetracycline, tigecycline, minocycline, vancomycin, clindamycin, azithromycin, fusidic acid, doxycycline, moxifloxacin, linezolid, rifampicin, rifampin, telavancin, doripenem, ertapenem, imipenem, meropenem, taurolidine, daptomycin, metronidazole, trimethoprimsulfamethoxazole, or a combination thereof. In some embodiments, a beta-lactam antibiotic (such as but not limited to amoxicillin) is administered with a beta-lactamase inhibitor (e.g., clavulanate). The antibiotic to be given orally or by i.v. for DDD or CLBP are generally selected from those that can penetrate the intervertebral disc. Antibiotics given by local injection to infected tissue include those that have limitations in their oral bioavailability.

In some embodiments, the patient receives chronic antibiotic therapy, for example, for at least about 1 month, or at least about 2 months, or at least about 3 months (e.g., about 100 days or more). In various embodiments, the antibiotic (or combination thereol) is administered orally one or more times daily (e.g., 1-3 times daily). Alternatively or in addition, one or more antibiotics are administered by i.v. In some embodiments, intravenous antibiotic therapy (e.g., with beta lactam antibiotic) can be about once daily to about once weekly for 2 to about 6 weeks.

In accordance with the present disclosure, the patient will further receive a therapy inhibiting one or more cytokines. In various embodiments, the cytokine is a pro- inflammatory cytokine that is expressed at sites of infection, and/or one that induces bacterial virulence, antibiotic resistance, and/or bacterial growth. In some embodiments, the cytokine induces or sustains antibiotic resistance. In still other embodiments, the cytokine induces nerve growth. In various embodiments, the cytokine is selected from IL-ip, IL-la, IL-2, IL-6, IL-8, IL-10, IL-12, IL-17, IL-23, INF-y, TNF-a, TGF- , CCL- 3, CCL-4, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF).

In some embodiments, the method involves administering an IL-ip inhibitor. For example, in various embodiments, the IL-ip inhibitor is a monoclonal anti-IL-ip antibody (or antigen-binding domain or antibody fragment thereol), a recombinant protein with IL-ip binding activity, or a small molecule inhibitor. In some embodiments, the IL-ip inhibitor is an antisense oligonucleotide (e.g., targeting IL-ip mRNA), an siRNA (e.g., targeting IL- ip mRNA), or amiRNA (e.g., miR-9 ormiR-155) or antagomir that downregulates inflammasome expression or activation.

In some embodiments, the IL-ip inhibitor is a monoclonal antibody that binds to and blocks and/or neutralizes IL-ip, or blocks interaction between IL-ip and one or more microbial receptors. In some embodiments, the antibody is a single chain antibody, such as a scFv. In some embodiments, the antibody is an antibody fragment selected from F(ab')2, Fab, Fab' and Fv. Exemplary IL-ip inhibitors are selected from canakinumab, gevokizumab, LY2189102, and CDP-484 (pegylated F(ab') antibody fragment against IL-ip). These agents in various embodiments are administered systemically (e.g., intravenously or subcutaneously), or alternatively by local injection or topical application to the tissues having or suspected of having a low-virulence infection, or the surrounding area. For example, in some embodiments, the anti-IL-ip antibody is administered by injection directly to intervertebral disc tissue, periprosthetic tissue, or by intra-articular injection to affected joints.

In some embodiments, the IL-ip inhibitor is an IL-1 trap (e.g., rilonacept). IL-1 trap is a hybrid molecule containing the extracellular domain of IL-1 receptor accessory protein and IL-1 receptor type 1 arranged inline and fused to the Fc-portion of IgGl. IL- 1 trap is a dimeric inhibitor that neutralizes IL-ip. These agents in various embodiments are administered systemically (e.g., intravenously or subcutaneously), or alternatively by local injection to the tissues having or suspected of having a low- virulence infection, or the surrounding area. For example, in some embodiments, the IL-1 trap is administered by injection directly to intervertebral disc tissue, periprosthetic tissue, or by intraarticular injection to affected joints.

In still other embodiments, the IL-ip inhibitor comprises a soluble receptor for IL-ip, such as sIL-lRI or sIL-lRII. These agents in various embodiments are administered systemically (e.g., intravenously or subcutaneously), or alternatively by local injection or topical application to the tissues having or suspected of having a low- virulence infection, or the surrounding area. For example, in some embodiments, the soluble receptor is administered by injection directly to intervertebral disc tissue, periprosthetic tissue, or by intra-articular injection to affected joints.

In still other embodiments, the IL-ip inhibitor is a caspase inhibitor (or other inhibitor that prevents processing of proIL-ip), and which is optionally administered orally. Such inhibitors include caspase inhibitors (e.g., pralnacasan) or VX-765, as well as CP424174 and CP412245. Other cytokine production inhibitors that may be used include CJ14877 and CJ14897. These small molecule inhibitors can be administered systemically, including orally or parenterally, or in some embodiments, are administered locally to affected tissues. In some embodiments, inhibitors that target the IL-1 receptor may also be used, such as EBI-005, Anakinra (IL-IRa), MED-8968, or LL-Z1271a. However, in various embodiments it is preferred to target IL-ip directly.

Generally, the IL-ip antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous or percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure. In some embodiments, the IL-ip inhibitor is a small molecule (such as caspase inhibitor) and may be administered orally or transdermally in some embodiments.

In some embodiments, the therapy with the IL-ip inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the IL-ip inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an IL-ip inhibitor (e.g., anti-IL-ip antibody or IL-1 trap). In some embodiments, an IL-ip inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, or endoscopic surgery. In some embodiments, the IL-ip inhibitor is administered as a co-formulation with one or more antibiotics. In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of Nerve Growth Factor (NGF), which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the NGF inhibitor is selected from a monoclonal anti-NGF antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv), small-peptide mimetics of NGF, small-molecule TrkA antagonist, TrkA immunoadhesion molecule, soluble binding domain of NGF receptor, or monoclonal antibody or monoclonal antibody fragment against TrkA. Tropomyosin receptor kinase A (TrkA) is also known as high affinity nerve growth factor receptor. In some embodiments, the inhibitor of NGF is tanezumab, fulranumab, TrkAd5, AMG 403, Appha-Dl l, MNAC13, ALE0540, PD90780, or PPC-1807. In still other embodiments, the NGF inhibitor is an antisense oligonucleotide targeting the NGF mRNA, or an siRNA or miRNA targeting NGF mRNA expression.

Generally, the NGF antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous or percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure. In some embodiments, the NGF inhibitor is a small molecule, and may be administered orally or transdermally in some embodiments.

In some embodiments, the therapy with the NGF inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the NGF inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an NGF inhibitor. In some embodiments, an NGF inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery and endoscopic surgery. In some embodiments, the NGF inhibitor is administered as a co-formulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of BDNF, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the BDNF inhibitor is selected from a monoclonal anti-BDNF antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv), small-peptide mimetics of BDNF, small-molecule TrkB inhibitor, soluble binding domain of BDNF receptor, or monoclonal antibody or monoclonal antibody fragment against TrkB. In still other embodiments, the BDNF inhibitor is an antisense oligonucleotide targeting the BDNF mRNA, or an siRNA or miRNA targeting BDNF mRNA expression.

Generally, the BDNF antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous or percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure. In some embodiments, the BDNF inhibitor is a small molecule, and may be administered orally or transdermally in some embodiments.

In some embodiments, the therapy with the BDNF inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the BDNF inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of a BDNF inhibitor. In some embodiments, an BDNF inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery and endoscopic surgery. In some embodiments, the BDNF inhibitor is administered as a co-formulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of IL-8, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the IL-8 inhibitor is a monoclonal anti-IL-8 antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv). In some embodiments, the IL-8 inhibitor is HuMaxIL8 (BMS-986253). In still other embodiments, the IL8 inhibitor is an antisense oligonucleotide targeting the IL-8 mRNA, or an siRNA or miRNA targeting IL-8 mRNA expression.

Generally, the IL-8 antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure. In some embodiments, the therapy is administered during endoscopic surgery.

In some embodiments, the therapy with the IL-8 inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the IL-8 inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an IL-8 inhibitor. In some embodiments, an IL-8 inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the IL-8 inhibitor is administered as a co-formulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of IL-6, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the IL-6 inhibitor is a monoclonal anti-IL-6 antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv). In some embodiments, the IL-6 inhibitor is siltuximab, olokizumab, clazakizumab, or sirukumab. In some embodiments, the IL-6 inhibitor is a monoclonal antibody targeting IL-6R (e.g., tocilizumab or sarilumab). In still other embodiments, the IL6 inhibitor is an antisense oligonucleotide targeting the IL-6 mRNA, or an siRNA or miRNA targeting IL-6 mRNA expression. Generally, the IL-6 inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure.

In some embodiments, the therapy with the IL-6 inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the IL-6 inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an IL-6 inhibitor. In some embodiments, an IL-6 inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the IL-6 inhibitor is administered as a co-formulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of IL-2, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the IL-2 inhibitor is a monoclonal anti-IL-2 antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv). In still other embodiments, the IL-2 inhibitor is an antisense oligonucleotide targeting the IL-2 mRNA, or an siRNA or miRNA targeting IL-2 mRNA expression.

Generally, the IL-2 antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure. In some embodiments, the therapy with the IL-2 inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the IL-2 inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an IL-2 inhibitor. In some embodiments, an IL-2 inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the IL-2 inhibitor is administered as a co-formulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of IL-10, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the IL-10 inhibitor is a monoclonal anti-IL-10 antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv). In still other embodiments, the IL- 10 inhibitor is an antisense oligonucleotide targeting the IL-10 mRNA, or an siRNA or miRNA targeting IL-10 mRNA expression.

Generally, the IL-10 antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure.

In some embodiments, the therapy with the IL- 10 inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the IL- 10 inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an IL- 10 inhibitor. In some embodiments, an IL- 10 inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the IL-10 inhibitor is administered as a coformulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of IL- 12, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the IL-12 inhibitor is a monoclonal anti-IL-12 antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv). In some embodiments, the IL-12 inhibitor is ustekinumab. In still other embodiments, the IL-12 inhibitor is an antisense oligonucleotide targeting the IL-12 mRNA, or an siRNA or miRNA targeting IL- 12 mRNA expression.

Generally, the IL-12 antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure.

In some embodiments, the therapy with the IL- 12 inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the IL-12 inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an IL-12 inhibitor. In some embodiments, an IL-12 inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the IL-12 inhibitor is administered as a coformulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of IL-17, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the IL-17 inhibitor is a monoclonal anti-IL-17 antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv). In some embodiments, the IL-17 inhibitor is secukinumab or ixekixumab. In still other embodiments, the IL-17 inhibitor is an antisense oligonucleotide targeting the IL- 17 mRNA, or an siRNA or miRNA targeting IL- 17 mRNA expression.

Generally, the IL-17 antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure.

In some embodiments, the therapy with the IL- 17 inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the IL- 17 inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an IL- 17 inhibitor. In some embodiments, an IL- 17 inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the IL-17 inhibitor is administered as a coformulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure. In some embodiments, the patient receives therapy with an inhibitor of IL-23, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the IL-23 inhibitor is a monoclonal anti-IL-23 antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv). In some embodiments, the IL-23 inhibitor is risankizumab or ustekinumab. In still other embodiments, the IL-23 inhibitor is an antisense oligonucleotide targeting the IL-23 mRNA, or an siRNA or miRNA targeting IL-23 mRNA expression.

Generally, the IL-23 inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure.

In some embodiments, the therapy with the IL-23 inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the IL-23 inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an IL-23 inhibitor. In some embodiments, an IL-23 inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the IL-23 inhibitor is administered as a coformulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of interferongamma (INF-y), which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the INF-y inhibitor is a monoclonal anti-INF-y antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv). In still other embodiments, the INF-y inhibitor is an antisense oligonucleotide targeting the INF-y mRNA, or an siRNA or miRNA targeting INF-y mRNA expression.

Generally, the INF-y antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure.

In some embodiments, the therapy with the INF-y inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the INF-y inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an INF-y inhibitor. In some embodiments, an INF-y inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the INF-y inhibitor is administered as a coformulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of TGF-P, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the TGF-P inhibitor is a monoclonal anti-TGF-P antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv). In still other embodiments, the TGF-P inhibitor is an antisense oligonucleotide targeting the TGF-P mRNA, or an siRNA or miRNA targeting TGF-P mRNA expression.

Generally, the TGF-P antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure.

In some embodiments, the therapy with the TGF-P inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the TGF-P inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an TGF-P inhibitor. In some embodiments, an TGF-P inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the TGF-P inhibitor is administered as a co-formulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient receives therapy with an inhibitor of TNF-a, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy. In some embodiments, the TNF-a inhibitor is a monoclonal anti-TNF-a antibody or fragment thereof (including scFv, F(ab')2, Fab, Fab' and Fv). In some embodiments, the TNF-a inhibitor is infliximab, adalimumab, golimumab, certolizumab, or etanercept. In still other embodiments, the TNF-a inhibitor is an antisense oligonucleotide targeting the TNF-a mRNA, or an siRNA or miRNA targeting TNF-a mRNA expression.

Generally, the TNF-a antagonist or inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, percutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy or surgical procedure.

In some embodiments, the therapy with the TNF-a inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the TNF-a inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of a TNF-a inhibitor. In some embodiments, a TNF-a inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the TNF-a inhibitor is administered as a co-formulation with one or more antibiotics and/or TNF-a inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the patient further receives an anti-angiogenic therapy, which can be provided with antibiotic treatment, or in combination with antibiotic treatment and IL-ip inhibitor therapy (or therapy targeting other pro-inflammatory cytokine) and/or anti-NGF therapy. In some embodiments, the angiogenesis inhibitor is a VEGF pathway inhibitor, which can be a monoclonal anti-VEGF antibody or fragment thereof (including a scvl), small-peptide mimetics, small-molecule inhibitor (e.g., tyrosine kinase inhibitor), or soluble binding domain of VEGF receptor. Exemplary angiogenesis inhibitors include antibodies directed against VEGF or VEGFR, soluble VEGFR/VEGFR hybrids, and tyrosine kinase inhibitors. An exemplary VEGF pathway inhibitor is Bevacizumab. Bevacizumab binds to VEGF and inhibits it from binding to VEGF receptors.

Generally, the angiogenesis inhibitor can be administered by a route selected from parenteral, oral, topical, and transdermal. Generally, where the inhibitor is a biologic, such as an antibody or portion thereof or other recombinant protein, the inhibitor may be administered parenterally, including subcutaneous injection, intramuscular injection, intravenous injection, or local injection to affected tissue. In some embodiments, the therapy is provided during a biopsy procedure.

In some embodiments, the therapy with the angiogenesis inhibitor is concurrent with antibiotic therapy (e.g., for at least about 1 month, 2 months, or 3 months) or is administered before or after antibiotic therapy. In some embodiments, the course of therapy with the angiogenesis inhibitor is shorter or longer than antibiotic therapy, such as about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6 months or more. Administrations may be given daily, weekly, or every other week, or monthly. In some embodiments, the patient receives from 1 to 12, or from 1 to 8, or from 2 to 8 doses of an angiogenesis inhibitor. In some embodiments, an angiogenesis inhibitor is administered once locally to the affected tissue during a surgical procedure. The procedure may be selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. In some embodiments, the angiogenesis inhibitor is administered as a co-formulation with one or more antibiotics and/or IL-ip inhibitor (as described). In some embodiments, the patient may further receive oral antibiotic therapy after the surgical procedure.

In some embodiments, the cytokine inhibitor is an IL-ip inhibitor (e.g., an IL-1 trap or an antibody neutralizing IL-ip), and the antibiotic includes vancomycin or clindamycin. These agents can be administered by injection or infusion of the affected tissue optionally simultaneously. For example, the composition may be administered by injection or infusion directly to intervertebral disc tissue, periprosthetic tissue, or by intra-articular injection to affected joints. The composition may be administered during a surgical procedure selected from discectomy, debridement, arthroplasty, orthopedic surgery, and endoscopic surgery. For example, treatment can be provided post-surgery to facilitate recovery, prevent infection, or prevent infection progression or recurrence.

Dosage forms suitable for parenteral administration (e.g. intravenous, intramuscular, intraperitoneal, subcutaneous, percutaneous, and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

Compositions for topical application (e.g., including a cytokine inhibitor and an antibiotic) may be formulated as a cream, gel, solution, or ointment.

In some embodiments, the present disclosure provides an intradiscal injection system with a depot carrier to administer an antibiotic and cytokine inhibitor, such as an IL-1B inhibitor (as described). For example, upon concluding a discectomy, but prior to closure of the deep space, the surgeon would employ the injection system, formulated to minimize leakage, to administer the antibiotic with cytokine inhibitor. Accordingly, in some aspects, the invention provides a pharmaceutical composition comprising an effective amount of an antibiotic and an inhibitor or a pro-inflammatory cytokine. Exemplary inhibitors of cytokines may be antibodies or recombinant proteins, or a small molecule inhibitor, targeting one or more of IL-ip, IL-la, IL-2, IL-6, IL-8, IL-10, IL-12, IL-17, IL-23, INF-y, TNF-a, TGF- , CCL-3, CCL-4, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) (each as described above). In some embodiments, the pharmaceutical composition comprises an IL-ip inhibitor, such as canakinumab, gevokizumab, LY2189102, CDP-484, and IL-1 trap. The pharmaceutical composition further comprises one or a combination of antibiotics, including but not limited to beta-lactams, carbapenems, quinolones, macrolides, and cephalosporins. Exemplary antibiotics include one or more of clindamycin, erythromycin, vancomycin, and daptomycin. In some embodiments, the antibiotic is a tetracycline antibiotic, such as tetracycline, minocycline, doxycycline, oxytetracycline and lymecycline. In various embodiments, one or more antibiotics are selected from penicillin, benzylpenicillin, amoxicillin, ampicillin, dicloxacillin, methicillin, nafcillin, oxacillin, penicillin G, piperacillin-tazobactam, cephalexin, cefoxitin, cephalothin, ceftriaxone, ciprofloxacin, levofloxacin, chloramphenicol, erythromycin, tetracycline, tigecycline, minocycline, vancomycin, clindamycin, azithromycin, fusidic acid, doxycycline, moxifloxacin, linezolid, rifampicin, rifampin, telavancin, doripenem, ertapenem, imipenem, meropenem, taurolidine, daptomycin, metronidazole, trimethoprim-sulfamethoxazole, or a combination thereof. In some embodiments, the composition comprises a beta-lactam antibiotic (such as but not limited to amoxicillin) and a beta-lactamase inhibitor (e.g., clavulanate). In some embodiments, the composition comprises vancomycin.

The pharmaceutical composition may be formulated as a solution, suspension, dispersion, emulsion, or the like. In some embodiments, the composition is in the form of a sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Other suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

Embodiments of the invention will be further illustrated with the following nonlimiting examples.

EXAMPLES

Example 1. Ability of P. acnes to Induce IL-lB-based Pathogenesis

The ability of P. acnes PAMPs to induce IL-ip and trigger NLRP3 inflammasome assembly are demonstrated by monitoring of IL-1 P, NLRP3 and caspase 1 levels in ex vivo models based on NP and AF cell cultures derived from clinical specimens of degenerated disc tissue. In vivo models in which animals are experimentally infected with P. acnes strains isolated from human degenerated discs could also be employed. Further, short fragments of hyaluronic acid, P. ocwc.s-induced DAMPs, shown to interact with TLR2 of the NP cells in vitro, could also be confirmed to induce IL-ip in an animal experiment.

To further establish that P. acnes is a strong contributor to IL-ip-based pathogenesis of DDD, in vitro and in vivo experiments are performed to quantitatively compare the ability of P. acnes to induce IL-ip with other known activators of IL-ip used in the experimental studies of DDD. Ability of the P. acnes related formyl peptides and the pore-forming/hemolytic virulence factor to induce calcium flux and action potentials in nociceptive neurons could be evaluated in vitro using analogical designs as used in study of Staphylococcus aureus.¹⁹

Expression levels of pro-inflammatory cytokines and nerve growth factors were assessed in P. acnes -infected cell cultures derived from human tissue samples of degenerated nucleus pulposus (NP).

Eight NP cell primary cultures were established from human tissue samples of degenerated nucleus pulposus (NP) that were obtained from eight patients who underwent microdiscectomy for lumbar disc herniation. Further, a P. acnes strain (type II) derived from P. acnes strongly positive herniated disc tissue was used for in vitro infection of NP cells disc cultures.

Experiments were performed in triplicates with two multiplicities of infection (MOI), e.g., 100,1000, and expression levels of pro-inflammatory cytokines (e.g., IL-ip, IL- la, IL-6, IL-8, CCL3, CCL4) and nerve growth factors (e.g., NGF, BDNF) were quantified by quantitative real-time PCR at four time-points (3h, 24h, 48h and 1 week). Non-infected cells were used as negative controls and LPS-treated cells were used as positive controls. Figure 4 depicts the presence of viable P. acnes strain in the NP culture, as confirmed by P. acnes lipase assay. Specifically, relative lipase activity of 1: 100 and 1 : 1000 NP cells/ . acnes co-cultures was assayed after 3 hours and 24 hours, confirming the viability of P. acnes. Figure 5 shows the average expression levels of the tested genes: IL-ip, IL-la, IL-6, IL-8, CCL3, and CCL4. The data shows significant increase in expression of all tested cytokines with the most significant changes observed after 24 hours and MOI 1000. On the other hand, when the expression changes of the cytokines were evaluated at the level of the individual NP cell cultures, it was obvious that the dynamics of the inflammatory response differed between the cell cultures. For example, fold changes related to IL-ip presented across various cell lines and under different conditions are summarized in Table 1 below:

In some cases, an observed cytokine response did not present until after 24 hours.

The data further showed that no significant changes were found in NGF and BDNF expression levels after 3h, 24h and 48 hours of lasting infection. Figure 6 depicts an MOI-dependent significant increase in NGF and BDNF expression levels in P. acnes- infected NP cells observed after one week of infection. In the case of the disc degeneration driving the cytokine IL-ip, Figure 7 shows that its protein expression levels were also increased 24 hours post-infection, as detected by ELISA.

Since P. acnes is known to be sensitive to the antibiotic clindamycin in the therapy of acne vulgaris, this study further sought to assess the effect of clindamycin treatment in suppressing production and expression of pro-inflammatory cytokines in human NP cells induced by P. acnes.

Accordingly, NP cell cultures derived from three specimens of clinical disc tissue were infected with one of the P. acnes strains (type II) derived from P. acnes strongly positive herniated disc tissues (MOI 1000). Half of the plates were treated with 0.25pg/mL clindamycin, and expression levels of IL-ip, IL-6 and IL-8 were measured after 24 hours and 48 hours. Significantly lower expression levels of all cytokines at both time points were observed, as shown in Figure 8, indicating the ability of clindamycin to suppresses expression of pro-inflammatory cytokines in human NP cells induced by P. acnes.

Discussion

Biofilm-associated infections represent a serious health burden. They are a consequence of microorganisms that colonize biological interfaces or surfaces of indwelling medical devices upon which they produce an extracellular matrix. Embedded within this matrix, such microorganisms become resistant to antibiotic treatments and adapt to host immune system effector mechanisms. Antibiotic treatments alone are ineffective in treating these infections.

A substantial number of patients undergoing microdiscectomy surgery actually have an underlying occult biofilm-based infection by the opportunistic pathogen P. acnes, recently renamed Cutibacterium acnes. This anaerobic bacterium plays a key role in the inflammatory skin condition acne vulgaris, postoperative device-related infections, infected tissues (i.e. , prostate, eye, breast, heart) and joints (i.e., shoulder, hip, knee), surgical site infections, and wounds.

Certain opportunistic human pathogens are able to bind the cytokine IL-ip released by host cells, potentially facilitating proliferation and modulation of virulence properties (including biofilm formation). This bacterial growth enhancement in response to host IL-ip can be dependent upon gene products selectively expressed during growth as biofilms rather than as planktonic cells. Thus, this disclosure proposes that IL-ip can promote P. acnes growth and biofilm formation in vivo in degenerate tissues or on surfaces of indwelling medical devices, and resulting in an antibiotic resistant phenotype.

Using high resolution microscopy, the inventors have demonstrated the existence of P. acnes biofilm deep within degenerated intervertebral disc tissue. They have further demonstrated that P. acnes stimulates the expression of various inflammatory mediators from intervertebral disc cells, particularly IL-ip. As a seemingly master cytokine, IL-ip can play a central role in amplifying an inflammatory cascade within the intervertebral disc resulting in degenerative disc disease. Furthermore, the data are consistent with growth enhancing effects of IL-ip on P. acnes in multiple tissues.

The presence of viable, biofilm-embedded bacteria within intervertebral discs (traditionally viewed as sterile) represents an abnormal physiological situation. The ability to treat a chronically infected disc therefore provides a new opportunity to significantly reduce the disability associated with DDD and potentially represents a paradigm shift in the clinical management of patients with this condition, as well as other infected tissues. For patients with DDD who undergo microdiscectomy surgery, a significant proportion experience recurrent disc herniation and follow-on discectomy. They are at a heightened risk of further disc deterioration, increased loss of disc space height and potential disc fusion.

Accordingly, this disclosure proposes to employ an anti-IL-ip therapy in association with an antibiotic for the synergistic treatment of human biofilm-based infections, including those that might involve P. acnes or other commensal pathogens. The anti -biofilm properties of the anti-IL-ip agent has the potential to transform the resident bacteria to a more antibiotic-sensitive phenotype. Meanwhile, as demonstrated, antibiotic treatment can help suppress the production of pro-inflammatory cytokines, and in turn prevent the bacterial growth-and virulence-enhancing effects that these cytokines can induce. Thus, these agents can provide for a unique and synergistic treatment of bacterial infections involving biofilms, including but not limited to those infections that may involve P acnes.

Materials and methods

Microbiological culture

The disc fragment for culture was weighed, placed into a Micro Bag (Seward) containing 4 ml of Viande-Levure medium, and homogenized with a Stomacher 80 (Seward) under aseptic conditions. 100 pl of the resultant homogenate was inoculated onto Wilkins Chalgren Anaerobic Agar with 7% sheep's blood and vitamin K (Hi Media Laboratories). An Anaerobic Work Station Concept 400 (Ruskinn Technology) was utilized for culture; inoculated plates were incubated for 14 days at 37°C with an atmosphere of 80% N2, 10% CO2, and 10% H2. The same amount of the homogenate was also cultured aerobically on Columbia Blood Agar (Oxoid) for 7 days at 37°C in order to detect aerobic bacteria. Following incubation, the bacterial colonies were counted and the quantity of each colonial morphotype was expressed as colony forming units (CFU) per gram of tissue. In the case of P. acnes positivity, a single P. acnes colony was taken and inoculated on a new anaerobic plate and incubated under anaerobic condition at 35-37°C until colonies appeared. From inoculated plate, full sterile loop was taken and placed in glycerol serum broth media in 2mL sterile cryo tube and placed in -80°C freezer.

Study participants

Human tissue samples of degenerated Nucleus pulposus (NP) were obtained from 8 patients who underwent microdiscectomy at the University Hospital Brno, Czech Republic.

Isolation and culture of human nucleus pulposus (NP) cells

Fresh nucleus pulposus (NP) tissue samples were cut into small pieces using a sterile, individually packaged, gamma-irradiated scalpel and, a sterile, gamma-irradiated petri dish and then digested overnight with collagenase A (Roche) at 37°C. After the digestion, the cell suspensions with undigested tissues were filtered through a cell strainer with pores of 40 pm (Millipore) and centrifuged. The cell pellets were resuspended in Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12 (DMEM/F12 (1: 1) lx, Gibco) supplemented with 10% fetal bovine serum and antibiotics penicillin (200 U/ml) and streptomycin (100 U/mL). Cells were cultured at 37°C in a humified atmosphere with 5% CO2 and were maintained in monolayer culture. In the experiment with antibiotic, 0.25 pg/mL clindamycin treatment was used.

After a few passages, cells were seeded in 6-well plates without antibiotics, allowed to attach overnight and assigned to Propionibacterium acnes treatment (ratio of 1:100 and 1:1000). Supplementation with LPS (200 ng/pl, Sigma-Aldrich) served as a positive control, and cells cultured without P. acnes served as a negative control. Cells were harvested into Qiazol lysis reagent (Qiagen) and RIPA buffer (Sigma) at four time points: after 3 h, 24 h, 48 h and 1 week.

RNA isolation

RNA was extracted by use of the Direct-zol RNA kit (Zymo Research) as described in the manufacturer’s instructions. The concentration and purity of RNA were determined at 260 and 280 nm using a NanoDrop 2000 (Thermo Scientific).

Quantitative reverse transcription real-time PCR (qRT-PCR)

The total amount of RNA was subjected to reverse transcription (RT) with the use of High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT conditions were as follows: 10 min at 25 °C, 2 h at 37 °C, 5 min at 85 °C. Real-time PCR was performed using TaqMan Gene Expression Master Mix (Applied Biosystems) and primer/probe systems: (Thermo Fisher): interleukin- ip (IL-ip, Hs01555410_ml), interleukin- la (IL-la, Hs00174092_ml), interleukin 6 (IL-6, Hs00174131_ml), interleukin 8 (CXCL-8, Hs00174103_ml), chemokine (C-C motif) ligand 2 (CCL2, Hs00234140_ml), chemokine (C-C motif) ligand 3 (CCL3, HS00234142_ml), chemokine (C-C motif) ligand 4 (CCL4, Hs99999148_ml), nerve growth factor (NGF, Hs00171458_ml), Brain-derived neurotrophic factor (BDNF, Hs02718934_sl).

All experiments were performed with glyceraldehyde phosphate dehydrogenase (GAPDH) as internal control using the real-time QuantStudio 12K Flex system (Life Technologies). The fold-change of cDNA expression levels was determined from the obtained AACt values compared to AACt values of control samples. The thermal cycling amplification program was as follows: one cycle of 94°C for 10 min and 40 cycles of 95°C for 15 s, 60°C for 60 s. Each sample was tested in triplicate and all real-time PCR reactions were run in duplicates.

Lipase assay

Lipase activity was measured in cell-free culture supernatants collected at 3 h, 24 h and 48 h time points. The procedure was performed using Lipase Activity Assay Kit II (MAK047, Sigma-Aldrich) according to manufacturer protocol.

Enzyme-Linked ImmunoSorbent Assay (ELISA)

Cell lysates (RIP A, Sigma) were added in duplicates to ELISA plates, each with specific antibody against Human IL-beta (RAB0273A), Human IL-6 (RAB0306), Human IL-8/CXCL8 (RABIL8A), Human MCP-1/CCL2 (RAB0054), Human MIP-1 alpha/CCL3 (RAB0073), and Human MIP-1 beta/CCL-4 (RAB0075). Plates were kept at 4 °C overnight and further proceeded according to manufacturer protocol.

Data normalization and statistical analyses

Student t-test was used for statistical evaluation of qRT-PCR experiments (the values are given as the mean ± SD). In all tests the value of p < 0.05 (*), the value of p < 0.01 (**) or the value of p < 0.001 (***) was considered significant.

Example 2. Mechanism of nociceptor activation by P. acnes Live bacteria actively release formylated peptides and secrete a host of virulence factors including pore-forming toxins (PFTs) to facilitate tissue dissemination (See Chiu et al, Nature 2013; 501:52—7). Alpha-hemolysin (Hla) is a PFT secreted by nearly all 5. aureus strains, playing a role in tissue damage, bacterial spread, and inflammation and it was shown to be one of the mechanisms employed by 5. aureus to elicit sustained bursts of calcium flux, selectively in capsaicin-responsive neurons (See Chiu et al, Nature 2013; 501:52-7).

Type 1 secretion systems (T1SS) are wide-spread among Gram-negative bacteria. An important example is the secretion of the hemolytic toxin HlyA from uropathogenic strains. Secretion is achieved in a single step directly from the cytosol to the extracellular space. The translocation machinery is composed of three indispensable membrane proteins, two in the inner membrane, and the third in the outer membrane. The inner membrane proteins belong to the ABC transporter and membrane fusion protein families (MFPs), respectively, while the outer membrane component is a porin- like protein. Assembly of the three proteins is triggered by accumulation of the transport substrate (HlyA) in the cytoplasm, to form a continuous channel from the inner membrane, bridging the periplasm and finally to the exterior. Interestingly, the majority of substrates ofTlSS contain all the information necessary for targeting the polypeptide to the translocation channel — a specific sequence at the extreme C-terminus (See Thomas et al, Mol. Cell Res. 2014; 1843:1629-41). From . acnes, PPA1396 seems to resemble this operon structure more. It has also been annotated as hemolysin type A.

In bacteria, protein expression initiates with a formyl-methionine group, the formyl group is then removed post-translationally by peptide deformylase. Formyl peptides can be released from the bacteria either actively or passively as a result of cell death. Neutrophils execute a variety of antimicrobial functions, including the generation of reactive oxygen species (ROS), phagocytosis of pathogens and dead and dying tissue, degranulation with the release of a variety of toxic products, expulsion of neutrophil extracellular traps, and paracrine signaling to recruit other cell types. Neutrophils sense inflammatory stimuli principally within the G protein-coupled receptor (GPCR) family. The first GPCR to be described on the human neutrophil was formyl peptide receptor 1 (FPR1) which, when activated, triggers a wide variety of functions, including chemotaxis, degranulation, ROS production, and phagocytosis. The principal ligands for FPR1 are bacterial and mitochondrial formylated peptides, actively secreted by invading pathogens or passively released from dead and dying host cells after tissue injury.

Although it was initially thought that FPR1 only bound N-formylated peptides, it is now widely recognized that the formyl group is not a prerequisite for receptor binding. The N-formylated version of any peptide containing a methionine residue at the 5' terminus is at least 100-fold more potent than the identical nonformylated peptide.

Several short formylated peptide sequences have been identified to elicit a response e.g. fMet-Leu-Phe (fMLF) by activating the formyl peptide receptors, and used routinely as models for the study of these systems. Various other peptides have been identified by studying organisms such as 5. aureus (See Chiu et al, Nature 2013; 501:52-7) and L.monycogenes (See Rabiet et al, Eur. J. Immunol. 2005; 35:2486-95).

In this example, we show that a similar mechanism can exist in P. acnes, and which may contribute to chronic pain/inflammation.

Identification of P. acnes formyl peptides

P. acnes strains have a number of proteins that have the fMLF and fMLP pattern which has been identified in E.coli, but not fMIFL a pattern derived from S. aureus. Nevertheless, as in other bacterial species, P. acnes may release formylated peptides to its environment, which can trigger an inflammatory response.

Among these proteins that have the peptides of interest there are two membrane proteins and one secreted. Although these proteins are likely to serve a different function in the cell, it is possible that they can be released to the environment and elicit the neutrophil response. Similarly, a number of hypothetical proteins are detected in the genome, which may play a similar role, however they are not predicted to contain any release mechanism.

Table 2: Predicted proteins with formyl peptide motif

Locus Tag Gene Product Name Genome Name Formylated Gene Symbol peptide

Identification of P. acnes hemolysins

There are at least three proteins annotated as hemolysins in P. acnes, which are conserved in all sequenced genomes. Of the three, PPA1396 (and its orthologs) seem to better align to the operon structure of known hemolysins. The gene is predicted to encode an alpha hemolysin, which has been shown to activate nociceptors (See Chiu et al, Nature 2013; 501:52-7).

Notably, there is evidence, that the additional genes also exhibit hemolytic activity. Table 3: Predicted hemolysins

The three genes are present in all species/strains of Propionibacterium acnes (aka Cutibacterium acnes).

Table 4: Hemolysis genes across P. acnes strains

Publications by Nodzo et al (Am. J. Orthop. Belle Mead NJ. 2014; 43:E93-97) show that more than 50% of examined P. acnes strains exhibited hemolytic activity, a population enriched in samples with definite infection. Since hemolysin is ubiquitous in P. acnes this result is somewhat unexpected - one would expect that all strains are hemolytic. However, it has been shown that non-hemolytic strains may produce hemolysis when treated with antibiotics or other stress (See Wright et al, Infect. Dis. 2016; 9:39-44).

Conclusions

Based on bioinformatics analysis and correlation with 5. aureus findings we predict that P. acnes hemolysins play a role in causing chronic pain, and this prediction is supported by the presence of such genes in the P. acnes genome, as well as the evidence of their expression. This seems to be a ubiquitous phenotype.

The potential of having formylated peptides playing this role is also present. Formylated peptides are ubiquitous among bacteria, and P. acnes contains at least a few proteins with sequence similar to peptides that have been shown to elicit response. However, it is possible that other sequences can also play a similar role as it has been shown in other studies (See Rabiet et al, Eur. J. Immunol. 2005; 35:2486-95), either actively secreted by the bacterium or as byproducts of protein cleavage.

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23. Albert HB, Sorensen JS, Christensen BS, Manniche C. Antibiotic treatment in patients with chronic low back pain and vertebral bone edema (Modic type 1 changes): a double-blind randomized clinical trial. Eur Spine J 2013; 22: 697-707.

24. Palazzo C, Ferrari M, Lefevre-Colau M-M, Nguyen C, Rannou F, Poiraudeau S. Lack of effectiveness of antibiotics in chronic low back pain with Modic 1 changes. Joint Bone Spine 2017; 84: 507-508.

25. Zeller V, Ghorbani A, Strady C, Leonard P, Mamoudy P, Desplaces D. Propionibacterium acnes: An agent of prosthetic joint infection and colonization. J Infect 2007; 55:119-124. 26. Dinarello CA, Simon A, van der Meer JW. Treating inflammation by blocking interleukin-1 in abroad spectrum of diseases. Nat Rev Drug Discov 2012; 11: 633- 52.

27. Eibl JK, Strasser BC, Ross GM. Structural, biological, and pharmacological strategies for the inhibition of nerve growth factor. Neurochem Int 2012; 61 : 1266- 1275.

28. Katz N, Borenstein DG, Birbara C, Bramson C, Nemeth MA, Smith MD, Brown MT. Efficacy and safety of tanezumab in the treatment of chronic low back pain. Pain 2011; 152:2248-2258.

29. Thomas S, Holland IB, Schmitt L. The Type 1 secretion pathway — The hemolysin system and beyond. Biochim. Biophys. Acta BBA - Mol. Cell Res. 2014; 1843:1629-41.

30. Rabiet M-J, Huet E, Boulay F. Human mitochondria-derived N-formylated peptides are novel agonists equally active on FPR and FPRL1, while Listeria monocytogenes-derived peptides preferentially activate FPR. Eur. J. Immunol. 2005; 35:2486-95.

31. Wright TE, Boyle KK, Duquin TR, Crane JK. Propionibacterium acnes Susceptibility and Correlation with Hemolytic Phenotype. Infect. Dis. 2016; 9:39- 44.

Example 3: IL-1B Receptor identification in C. acnes - Exploring similarities with known receptors

It is proposed that the pro-inflammatory cytokine IL-1 P has a significant effect in the development of the bacterial biofilm, promoting their formation. IL-1 receptors have been identified in some bacterial species. It is believed that binding this or other human cytokine can result in increased growth of some bacterial pathogens, resulting in the formation of biofilms and changed virulence features. There isn’t a single protein family that can be attributed to this function, instead the function is attributed to proteins families including: channel-forming usher protein (Cafl A from Yersinia pestis). a gram-negative secretin (PilQ from Neisseria meningitidis), an outer membrane pore protein (porin OprF of Pseudomonas aeruginosa), a pilus subunit (PilE from Neiseria meningitidis), an intrinsically disordered outer membrane lipoprotein (BilRI from A. actinomycetemcomitans), and a secreted protein displaying structural similarity to human cytokine receptors (IrmA from Escherichia coli). See Hogbuom M. and Ihalin R. Functional and structural characteristics of bacterial proteins that bind host cytokines. Virulence 2017; 8(8): 1592-1601. These proteins don’t have any sequence or structural similarity to each other, suggesting scenarios of convergent evolution among various organisms.

Cutibacterium acnes does not have any proteins annotated as an IL-ip-binding protein. In this example we sought to identify putative genes that would encode such a receptor.

Using sequence similarity searches we tried to identity protein sequences in C. acnes with similarity to any of the IL-ip binding proteins from other organisms. In addition to sequence analysis, we looked for genes belonging to certain protein families (i.e., Pfam, COG etc.) since this is a more sensitive search. Finally, we looked for regions in the genomes sharing common gene organization. In this case we assumed that similar function genes are grouped together to functional clusters and the order of genes can help identity protein functions.

We identified OprF protein from P. aeruginosa, which is present in all C. acnes strains. There weren’t any high-quality homologs/orthologs of the identified IL-ip receptors in C. acnes, other than the OprF from P. aeruginosa which is similar to proteins annotated as OmpA family. These genes are shared among all C. acnes genomes with almost identical sequence.

Low similarities are not unexpected given the evolutionary distance between any of these organisms and the inherent variability observed in membrane proteins. While these cytokine receptors are generally observed in pathogenic organisms that have evolved towards living and exploiting an environment with interleukins and having such explicit function could be important for their survival, C. acnes is not generally considered as such an organism. It is of course possible that other proteins in C. acnes have cytokine-binding functions with entirely new sequence and structure.

EQUIVALENTS

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

Claims

1. A method for treating an infection involving bacterial biofilm in a patient, comprising administering to the patient one or more antibiotics and a cytokine inhibitor.

2. The method of claim 1, wherein the infection is an infection of a commensal pathogen.

3. The method of claim 2, wherein the commensal pathogen is one or more of Propionibacterium sp., Staphylococcus sp., Corynebacterium sp., Lactobacillus sp., Pseudomonas sp., Enterococcus sp., Streptococcus sp., Bacillus sp., Citrobacter sp., E. coli, Moraxella sp., Haemophilus sp., Neisseria sp., Clostridium sp., Enterobacter sp., Helicobacter sp., and Klebsiella sp.

4. The method of claim 3, wherein the commensal pathogen is Propionibacterium acnes.

5. The method of any one of claims 1 to 3, wherein the patient has degenerative disc disease or chronic lower back pain, and the subject is suspected of having an associated infection.

6. The method of any one of claims 1 to 3, wherein the patient has an orthopaedic infection.

7. The method of any one of claims 1 to 3, wherein the patient has an infection associated with an implanted device.

8. The method of any one of claims 1 to 3, wherein the patient has a musculoskeletal condition.

9. The method of any one of claims 1 to 3, wherein the patient has sarcoidosis.

10. The method of any one of claims 1 to 3, wherein the patient has chronic prostatitis.

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11. The method of any one of claims 1 to 3, wherein the infection is associated with an organ or tissue selected from skin, prostate, eye, ears, nose, throat, breast, heart, vasculature, lung, intestinal tract, urogenital tract, oral cavity, central nervous system, bone, and joints.

12. The method of claim 11, wherein the infection is associated with a surgical site or wound in the surgical environment.

13. The method of claim 11, wherein the infection is a post-operative infection, and optionally a post-operative device-related infection.

14. The method of claim 13, wherein the infection is associated with prosthetic shoulder joint, knee or hip joint, cerebrovascular device, cardiovascular device, breast implant, or spinal prosthesis.

15. The method of any one of claims 11 to 14, wherein the infection is associated with an orthopaedic device, cerebrospinal fluid shunt, external ventricular drainage, postoperative craniotomy, breast implant, pacemaker, Implantable Cardioverter Defibrillator (ICD), heart valve replacement or surgery, vascular graft, endophthalmitis, peritoneal catheter, osteomyelitis, sarcoidosis, prostatitis, arteriosclerosis, atherosclerosis, rheumatoid arthritis, osteoarthritis, fibromyalgia, cystic fibrosis, or systemic lupus erythematosus.

16. The method of claim 11, wherein the infection is associated with acne vulgaris, psoriasis, eczema, or atopic dermatitis.

17. The method of claim 11, wherein the infection is an infection of the oral cavity, which is optionally periodontitis or endodontitis.

18. The method of claim 11, wherein the infection is a urinary tract infection.

19. The method of claim 11, wherein the infection is associated with sinusitis, rhinosinusitis, otitis media, esophagitis, or gastric ulcer.

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20. The method of claim 11 , wherein the infection is associated with an inflammatory bowel disease, diverticulitis, or celiac disease.

21. The method of claim 11, wherein the infection is associated with a movement disorder, a neurodegenerative condition, mood disorder, or a mental health condition.

22. The method of any one of claims 1 to 21, wherein the patient is suspected of having an infection based on the detection of a commensal pathogen in tissue.

23. The method of claim 22, wherein the presence of the commensal pathogen is detected in tissue by one or more of: the presence of microbial nucleic acid, host cell RNA profile, culturing of the commensal pathogen from tissue, immunohistochemistry of tissue, microbial-specific staining of tissue, and presence of a metabolite signature in tissue.

24. The method of claim 22 or 23, wherein the infection is identified by the presence of one or more virulence factors in the patient’s tissue.

25. The method of any one of claims 1 to 3, wherein the infection involves a pathogen selected from Mycobacterium sp., Borrelia sp., Yersinia sp., and Chlamydia sp.

26. The method of any one of claims 1 to 25, wherein the one or more antibiotics includes a beta-lactam, carbapenem, quinolone, macrolide, or a cephalosporin.

27. The method of claim 26, wherein the one or more antibiotics include one or more of clindamycin, erythromycin, vancomycin, and daptomycin.

28. The method of any one of claims 1 to 25, wherein one or more antibiotics include a tetracycline antibiotic.

29. The method of any one of claims 1 to 25, wherein one or more antibiotics include one or more of penicillin, benzylpenicillin, amoxicillin, ampicillin, dicloxacillin, methicillin, nafcillin, oxacillin, penicillin G, piperacillin-tazobactam, cephalexin, cefoxitin, cephalothin, ceftriaxone, ciprofloxacin, levofloxacin, chloramphenicol,

50 erythromycin, tetracycline, tigecycline, minocycline, vancomycin, clindamycin, azithromycin, fusidic acid, doxycycline, moxifloxacin, linezolid, rifampicin, rifampin, telavancin, doripenem, ertapenem, imipenem, meropenem, taurolidine, daptomycin, metronidazole, trimethoprim-sulfamethoxazole, or a combination thereof.

30. The method of any one of claims 26 to 29, wherein one or more antibiotics are administered orally.

31. The method of any one of claims 26 to 29, wherein one or more antibiotics are administered by i.v.

32. The method of any one of claims 26 to 29, wherein one or more antibiotics are administered by local injection.

33. The method of any one of claims 26 to 29, wherein the one or more antibiotics are administered topically.

34. The method of any one of claims 1 to 33, wherein the cytokine inhibitor inhibits a cytokine selected from IL-10, IL-la, IL-2, IL-6, IL-8, IL-10, IL-12, IL-17, IL-23, INF- y, TNF-a, TGF-0, CCL-3, CCL-4, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF).

35. The method of claim 34, comprising administering an IL- 10 inhibitor.

36. The method of claim 35, wherein the IL-10 inhibitor is a monoclonal anti-IL-10 antibody or antigen-binding domain or antibody fragment thereof, a recombinant protein with IL- 10 binding activity, or a small molecule inhibitor.

37. The method of claim 35, wherein the IL-10 inhibitor is an antisense oligonucleotide targeting IL- 10 mRNA, an siRNA targeting IL- 10 mRNA, or a miRNA.

38. The method of claim 36, wherein the IL-10 inhibitor is a monoclonal antibody that binds to and blocks and/or neutralizes IL-10.

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39. The method of claim 36, wherein the IL-ip inhibitor is selected from canakinumab, gevokizumab, LY2189102, and CDP-484.

40. The method of any one of claims 35 to 39, wherein the IL-ip inhibitor is administered by parenterally, and optionally by injection directly to intervertebral disc tissue, periprosthetic tissue, or by intra-articular injection to affected joints.

41. The method of claim 36, wherein the IL-ip inhibitor is an IL-1 trap.

42. The method of claim 41, wherein the IL-ip inhibitor is administered by parenterally, and optionally by injection directly to intervertebral disc tissue, periprosthetic tissue, or by intra-articular injection to affected joints.

43. The method of claim 36, wherein the IL-ip inhibitor comprises a soluble receptor for IL-ip.

44. The method of claim 43, wherein the IL-ip inhibitor is administered by parenterally, and optionally by injection directly to intervertebral disc tissue, periprosthetic tissue, or by intra-articular injection to affected joints.

45. The method of claim 36, wherein the IL-ip inhibitor is a caspase inhibitor, or inhibitor that prevents processing of proIL-ip.

46. The method of claim 45, wherein the IL-ip inhibitor is administered orally or parenterally.

47. The method of any one of claims 35 to 46, wherein the IL-ip inhibitor is administered locally or topically to affected tissues.

48. The method of claim 34, comprising administering an inhibitor of Nerve Growth Factor (NGF).

49. The method of claim 48, wherein the NGF inhibitor is administered by a route selected from parenteral, oral, topically, and transdermal.

50. The method of claim 48, wherein the NGF inhibitor is administered locally to affected tissues.

51. The method of claim 34, comprising administering an inhibitor of BDNF.

52. The method of claim 51, wherein the BDNF inhibitor is administered by a route selected from parenteral, oral, topical, and transdermal.

53. The method of claim 51, wherein the BDNF inhibitor is administered locally to affected tissues.

54. The method of claim 34, comprising administering an inhibitor of IL-8.

55. The method of claim 54, wherein the IL-8 inhibitor is administered by a route selected from parenteral, oral, topical, and transdermal.

56. The method of claim 54, wherein the IL-8 inhibitor is administered locally to affected tissues.

57. The method of claim 34, comprising administering an inhibitor of IL-6.

58. The method of claim 57, wherein the IL-6 inhibitor is administered by a route selected from parenteral, oral, topical, and transdermal.

59. The method of claim 57, wherein the IL-6 inhibitor is administered locally to affected tissues.

60. The method of claim 34, comprising administering an inhibitor of TNF-a.

61. The method of claim 60, wherein the TNF-a inhibitor is administered by a route selected from parenteral, oral, topical, and transdermal.

62. The method of claim 60, wherein the TNF-a inhibitor is administered locally to affected tissues.

63. The method of any one of claims 1 to 62, wherein the at least one antibiotic and the cytokine inhibitor are administered following surgery by local injection of a pharmaceutical composition comprising the at least one antibiotic and the inhibitor.

64. A pharmaceutical composition comprising an effective amount of an antibiotic and an inhibitor or a pro-inflammatory cytokine, wherein the composition is optionally formulated for parenteral or topical administration.

65. The pharmaceutical composition of claim 64, wherein the inhibitor of a cytokine is an antibody or recombinant protein targeting one or more of IL-ip, IL- la, IL-2, IL-6, IL-8, IL-10, IL-12, IL-17, IL-23, INF-y, TNF-a, TGF- , CCL-3, CCL-4, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF).

66. The pharmaceutical composition of claim 65, wherein the inhibitor is a small molecule inhibitor of one or more of IL-ip, IL-la, IL-6, IL-8, TNF-a, CCL-3, CCL-4, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF).

67. The pharmaceutical composition of claim 65 or 66, comprising an IL-ip inhibitor.

68. The pharmaceutical composition of claim 65, wherein the IL-ip inhibitor is a monoclonal antibody, or antigen binding portion thereof, that binds to and neutralized IL-ip.

69. The pharmaceutical composition of claim 68, wherein the IL-ip inhibitor is selected from canakinumab, gevokizumab, LY2189102, CDP-484, and IL-1 trap.

70. The pharmaceutical composition of any one of claims 64 to 69, wherein one or more antibiotics are selected from a beta-lactam, a carbapenem, a quinolone, a macrolide, and a cephalosporin.

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71. The pharmaceutical composition of claim 70, wherein the one or more antibiotics comprises one or more of clindamycin, erythromycin, vancomycin, and daptomycin.

72. The pharmaceutical composition of any one of claims 64 to 69, wherein the one or more antibiotics is a tetracycline antibiotic.

73. The pharmaceutical composition of any one of claims 64 to 69, wherein the one or more antibiotics are selected from penicillin, benzylpenicillin, amoxicillin, ampicillin, dicloxacillin, methicillin, nafcillin, oxacillin, penicillin G, piperacillin-tazobactam, cephalexin, cefoxitin, cephalothin, ceftriaxone, ciprofloxacin, levofloxacin, chloramphenicol, erythromycin, tetracycline, tigecycline, minocycline, vancomycin, clindamycin, azithromycin, fusidic acid, doxycycline, moxifloxacin, linezolid, rifampicin, rifampin, telavancin, doripenem, ertapenem, imipenem, meropenem, taurolidine, daptomycin, metronidazole, trimethoprim-sulfamethoxazole, or a combination thereof.

74. The pharmaceutical composition of any one of claims 64 to 69, wherein the composition comprises a beta-lactam antibiotic and a beta-lactamase inhibitor.

75. The pharmaceutical composition of any one of claims 64 to 69, comprising vancomycin.

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