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Orthopedic implant-associated infection (OIAI) is a major complication that leads to implant failure. In preclinical models of Staphylococcus aureus OIAI, osteomyelitis and septic arthritis, interleukin-1α (IL-1α), IL-1β, and tumor necrosis factor (TNF) are induced, but whether they have interactive or distinctive roles in host defense are unclear. Herein, a S. aureus OIAI model was performed in mice deficient in IL-1α, IL-1β, or TNF. Mice deficient in IL-1β or TNF (to a lesser extent) but not IL-1α had increased bacterial burden at the site of the OIAI throughout the 28-day experiment. IL-1β and TNF had a combined and critical role in host defense as mice deficient in both IL-1R and TNF (IL-1R/TNF-deficient mice) had a 40% mortality rate, which was associated with markedly increased bacterial burden at the site of the OIAI infection. Finally, IL-1α- and IL-1β-deficient mice had impaired neutrophil recruitment whereas IL-1β-, TNF-, and IL-1R/TNF-deficient mice all had impaired recruitment of both neutrophils and monocytes. Therefore, IL-1β and TNF contributed to host defense against S. aureus OIAI and neutrophil recruitment was primarily mediated by IL-1β and monocyte recruitment was mediated by both IL-1β and TNF.

Patients with prurigo nodularis (PN) suffer from intractable itch and dramatic reduction in QOL. Although there is significant clinical heterogeneity in the presentation of PN, disease endotypes remain unknown. We assayed circulating plasma cytokine concentrations in patients with PN (n = 20) along with matched healthy controls and utilized an unsupervised machine learning algorithm to identify disease endotypes. We found two distinct clusters of patients with PN with noninflammatory (cluster 1) and inflammatory (cluster 2) plasma profiles. Cluster 2 had more African Americans (82%, n = 9 vs. 33%, n = 3; P = 0.028), higher Worst Itch Numeric Rating Scale scores (9.5 ± 0.9 vs. 8.3 ± 1.2; P = 0.036), and lower QOL as reflected by higher Dermatology Life Quality Index scores (21.9 ± 6.4 vs. 13.0 ± 4.1; P = 0.015). In addition, cluster 1 had a higher rate of myelopathy (67%, n = 6 vs. 18%, n = 2; P = 0.028). Compared with cluster 1, cluster 2 had higher levels of IL-1α, IL-4, IL-5, IL-6, IL-10, IL-17A, IL-22, IL-25, and IFN-α. With population-level analysis, African American patients with PN had higher erythrocyte sedimentation rate, C-reactive protein, ferritin, and eosinophils and lower transferrin than Caucasian patients with PN. These findings indicate discrete clusters of patients with PN with plasma biomarker profiles corresponding to distinct demographic and clinical characteristics, potentially allowing for precision medicine approaches to treat PN.

Atopic dermatitis (AD) often presents more severely in African Americans (AAs) and with greater involvement of extensor areas. To investigate immune signatures of AD in AAs with moderate to severe pruritus, lesional and non-lesional punch biopsies were taken from AA patients along with age-, race-, and sex-matched controls. Histology of lesional skin showed psoriasiform dermatitis and spongiotic dermatitis, suggesting both Th2 and Th17 activity. Gene Set Variation Analysis showed upregulation of Th2 and Th17 pathways in both lesional versus non-lesional and lesional versus control (p

IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL‑36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL‑36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL‑36 cytokines in human atopic dermatitis skin and in IL‑36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL‑36 responses represent a key mechanism and potential therapeutic target against allergic diseases.

C-C motif chemokine receptor 2 (CCR2) is an important mediator of myeloid cell chemotaxis during inflammation and infection. Myeloid cells such as monocytes, macrophages, and neutrophils contribute to host defense during orthopedic implant-associated infections (OIAI), but whether CCR2-mediated chemotaxis is involved remains unclear. Therefore, a Staphylococcus aureus OIAI model was performed by surgically placing an orthopedic-grade titanium implant and inoculating a bioluminescent S. aureus strain in knee joints of wildtype (wt) and CCR2-deficient mice. In vivo bioluminescent signals significantly increased in CCR2-deficient mice compared with wt mice at later time points (Days 14-28), which was confirmed with ex vivo colony-forming unit enumeration. S. aureus γ-hemolysin utilizes CCR2 to induce host cell lysis. However, there were no differences in bacterial burden when the OIAI model was performed with a parental versus a mutant γ-hemolysin-deficient S. aureus strain, indicating that the protection was mediated by the host cell function of CCR2 rather than γ-hemolysin virulence. Although CCR2-deficient and wt mice had similar cellular infiltrates in the infected joint tissue, CCR2-deficient mice had reduced myeloid cells and γδ T cells in the draining lymph nodes. Taken together, CCR2 contributed to host defense at later time points during an OIAI by increasing immune cell infiltrates in the draining lymph nodes, which likely contained the infection and prevented invasive spread.

Signal transducer and activator of transcription 3 (STAT3) is important for psoriasis pathogenesis because STAT3 signaling downstream of IL-6, IL-21, IL-22, and IL-23 contributes to T helper type 17 cell development and because transgenic mice with keratinocyte (KC) STAT3 expression (K14-Stat3C mice) develop psoriasis-like dermatitis. In this study, the relative contribution of STAT3 signaling in KCs versus in T cells was evaluated in the imiquimod model of psoriasis-like dermatitis. Mice with STAT3-inducible deletion in KCs (K5-Stat3-/- mice) had decreased psoriasis-like dermatitis and epidermal STAT3 phosphorylation compared with wild-type mice, whereas mice with constitutive deletion of STAT3 in all T cells were similar to wild-type mice. Interestingly, mice with KC-inducible deletion of IL-6Rα had similar findings to those of K5-Stat3-/- mice, identifying IL-6/IL-6R as a predominant upstream signal for KC STAT3-induced psoriasis-like dermatitis. Moreover, psoriasis-like dermatitis inversely associated with type 1 immune gene products, especially CXCL10, whereas CXCL10 limited psoriasis-like dermatitis, suggesting that KC STAT3 signaling promoted psoriasis-like dermatitis by restricting downstream CXCL10 expression. Finally, treatment of mice with the pan-Jak inhibitor, tofacitinib, reduced psoriasis-like dermatitis and epidermal STAT3 phosphorylation. Taken together, STAT3 signaling in KCs rather than in T cells was a more important determinant for psoriasis-like dermatitis in a mechanism that involved upstream KC IL-6R signaling and downstream inhibition of type 1 immunity‒associated CXCL10 responses.

Mottled skin pigmentation and solar lentigines from chronic photodamage with aging involve complex interactions between keratinocytes and melanocytes. However, the precise signaling mechanisms that could serve as therapeutic targets are unclear. Herein, we report that expression of nuclear factor erythroid 2–related factor 2 (NRF2), which regulates reduction-oxidation reactions, is altered in solar lentigines and photodamaged skin. Moreover, mottled skin pigmentation in humans could be treated with topical application of the NRF2 inducer sulforaphane (SF). Similarly, UV light–induced pigmentation of WT mouse ear skin could be treated or prevented with SF treatment. Conversely, SF treatment was unable to reduce UV-induced ear skin pigmentation in mice deficient in NRF2 or in mice with keratinocyte-specific conditional deletion of IL-6Rα. Taken together, NRF2 and IL-6Rα signaling are involved in the pathogenesis of UV-induced skin pigmentation, and specific enhancement of NRF2 signaling could represent a potential therapeutic target., Pharmacological activation of NRF2-signaling resulted in improved skin hyperpigmentation in human and mouse skin.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

Staphylococcus aureus infections are a major threat in healthcare, requiring adequate early-stage diagnosis and treatment. This calls for novel diagnostic tools that allow noninvasive in vivo detection of staphylococci. Here we performed a preclinical study to investigate a novel fully-human monoclonal antibody 1D9 that specifically targets the immunodominant staphylococcal antigen A (IsaA). We show that 1D9 binds invariantly to S. aureus cells and may further target other staphylococcal species. Importantly, using a human post-mortem implant model and an in vivo murine skin infection model, preclinical feasibility was demonstrated for 1D9 labeled with the near-infrared fluorophore IRDye800CW to be applied for direct optical imaging of in vivo S. aureus infections. Additionally, 89Zirconium-labeled 1D9 could be used for positron emission tomography imaging of an in vivo S. aureus thigh infection model. Our findings pave the way towards clinical implementation of targeted imaging of staphylococcal infections using the human monoclonal antibody 1D9.

The mechanisms that mediate durable protection against Staphylococcus aureus skin reinfections are unclear, as recurrences are common despite high antibody titers and memory T cells. Here, we developed a mouse model of S. aureus skin reinfection to investigate protective memory responses. In contrast with WT mice, IL-1β-deficient mice exhibited poor neutrophil recruitment and bacterial clearance during primary infection that was rescued during secondary S. aureus challenge. The γδ T cells from skin-draining LNs utilized compensatory T cell-intrinsic TLR2/MyD88 signaling to mediate rescue by trafficking and producing TNF and IFN-γ, which restored neutrophil recruitment and promoted bacterial clearance. RNA-sequencing (RNA-seq) of the LNs revealed a clonotypic S. aureus-induced γδ T cell expansion with a complementarity-determining region 3 (CDR3) aa sequence identical to that of invariant Vγ5+ dendritic epidermal T cells. However, this T cell receptor γ (TRG) aa sequence of the dominant CDR3 sequence was generated from multiple gene rearrangements of TRGV5 and TRGV6, indicating clonotypic expansion. TNF- and IFN-γ-producing γδ T cells were also expanded in peripheral blood of IRAK4-deficient humans no longer predisposed to S. aureus skin infections. Thus, clonally expanded γδ T cells represent a mechanism for long-lasting immunity against recurrent S. aureus skin infections.

Prurigo nodularis is an understudied, chronic inflammatory skin disease that disproportionately affects African-Americans and presents with intensely pruritic nodules of unknown etiology. To better characterize immune dysregulation in prurigo nodularis, peripheral blood mononuclear cells and skin biopsies were obtained from majority African American patients with prurigo nodularis and healthy subjects matched by age, race, and sex. Flow cytometric analysis of functional T-cell response comparing prurigo nodularis to healthy subjects identified increased γδT-cells (CD3(+)CD4(−)CD8(−)γδTCR(+)) with Vδ2(+) γδT-enrichment. Activated T-cells demonstrated uniquely increased IL-22 cytokine expression in prurigo nodularis patients when compared to healthy controls. CD4+ and CD8+ T-cells were identified as the source of increased circulating IL-22. Consistent with these findings, RNA-sequencing of lesional prurigo nodularis skin compared to non-lesional prurigo nodularis skin and biopsy site matched control skin demonstrated robust up-regulation of Th22-related genes and signaling networks implicated in impaired epidermal differentiation. Th22-related cytokine upregulation remained significant with stratifications by race and biopsy site. Importantly, the expression of the IL-22 receptors IL22RA1/A2 was significantly elevated in lesional prurigo nodularis skin. These results indicate both systemic and cutaneous immune responses in prurigo nodularis patients are skewed towards a Th22/IL-22 profile. Prurigo nodularis may benefit from immunomodulatory therapies directed at Th22-mediated inflammation.

Staphylococcus aureus is a leading cause of hospital-acquired infections in the United States and is a major health concern as methicillin-resistant S. aureus (MRSA) and other antibiotic resistant strains are common. Compounds that inhibit the S. aureus sortase (SrtA) cysteine transpeptidase may function as potent anti-infective agents as this enzyme attaches virulence factors to the bacterial cell wall. While a variety of SrtA inhibitors have been discovered, the vast majority of these small molecules have not been optimized using structure-based approaches. Here we have used NMR spectroscopy to determine the molecular basis through which pyridazinone-based small molecules inhibit SrtA. These inhibitors covalently modify the active cysteine thiol and partially mimic the natural substrate of SrtA by inducing the closure of an active site loop. Computational and synthetic chemistry methods led to second generation analogs that are ~70-fold more potent than the lead molecule. These optimized molecules exhibit broad-spectrum activity against other types of class A sortases, have reduced cytotoxicity and impair SrtA-mediated protein display on S. aureus cell surface. Our work shows that pyridazinone analogs are attractive candidates for further development into anti-infective agents, and highlights the utility of employing NMR spectroscopy and solubility-optimized small molecules in structure-based drug discovery.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hidradenitis suppurativa (HS) is a chronic inflammatory disorder characterized by painful nodules, sinus tracts, and scars occurring predominantly in intertriginous regions. The prevalence of HS is currently 0.053–4%, with a predominance in African-American women and has been linked to low socioeconomic status. The majority of the reported literature is retrospective, population based, epidemiologic studies. In this regard, there is a need to establish a repository of biospecimens, which represent appropriate gender and racial demographics amongst HS patients. These efforts will diminish knowledge gaps in understanding the disease pathophysiology. Hence, we sought to outline a step-by-step protocol detailing how we established our HS biobank to facilitate the formation of other HS tissue banks. Equipping researchers with carefully detailed processes for collection of HS specimens would accelerate the accumulation of well-organized human biological material. Over time, the scientific community will have access to a broad range of HS tissue biospecimens, ultimately leading to more rigorous basic and translational research. Moreover, an improved understanding of the pathophysiology is necessary for the discovery of novel therapies for this debilitating disease. We aim to provide high impact translational research methodology for cutaneous biology research and foster multidisciplinary collaboration and advancement of our understanding of cutaneous diseases.

T cell cytokines contribute to immunity against Staphylococcus aureus , but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL-17 response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1β, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4 . However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6 + T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6 + Vδ4 + T cells in immunity against S. aureus skin infections.

Industrial hog operation (IHO) workers are at increased risk of carrying Staphylococcus aureus in their nares, particularly strains that are livestock-associated (LA) and multidrug-resistant. The pathogenicity of LA-S. aureus strains remains unclear, with some prior studies suggesting reduced transmission and virulence in humans compared to community-associated methicillin-resistant (CA-MRSA) S. aureus. The objective of this study was to determine the degree to which LA-S. aureus strains contracted by IHO workers cause disease relative to a representative CA-MRSA strain in a mouse model of skin and soft tissue infection (SSTI). Mice infected with CC398 LA-S. aureus strains (IHW398-1 and IHW398-2) developed larger lesion sizes with higher bacterial burden than mice infected with CA-MRSA (SF8300) (p S. aureus infected mice had decreased IL-1β protein levels compared with CA-MRSA-infected mice (p S. aureus SSTIs. WGSA revealed heterogeneity in virulence factor and antimicrobial resistance genes carried by LA-S. aureus and CA-MRSA strains. The observed pathogenicity suggest that more attention should be placed on preventing the spread of LA-S. aureus into human populations.

Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N‐acetyl‐glucosamine) oligomers, we here identify six‐subunit‐long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll‐like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size‐dependent system of immuno‐modulation that appears conserved in plants and humans. Since blocking of the chitin‐TLR2 interaction effectively prevents chitin‐mediated inflammation in vitro and in vivo, our study highlights the chitin‐TLR2 interaction as a potential target for developing novel therapies in chitin‐related pathologies and fungal disease.

Bacterial biofilm infections of implantable medical devices decrease the effectiveness of antibiotics, creating difficult-to-treat chronic infections. Prosthetic joint infections (PJI) are particularly problematic because they require prolonged antibiotic courses and reoperations to remove and replace the infected prostheses. Current models to study PJI focus on Gram-positive bacteria, but Gram-negative PJI (GN-PJI) are increasingly common and are often more difficult to treat, with worse clinical outcomes. Herein, we sought to develop a mouse model of GN-PJI to investigate the pathogenesis of these infections and identify potential therapeutic targets. An orthopedic-grade titanium implant was surgically placed in the femurs of mice, followed by infection of the knee joint with Pseudomonas aeruginosa or Escherichia coli. We found that in vitro biofilm-producing activity was associated with the development of an in vivo orthopedic implant infection characterized by bacterial infection of the bone/joint tissue, biofilm formation on the implants, reactive bone changes, and inflammatory immune cell infiltrates. In addition, a bispecific antibody targeting P. aeruginosa virulence factors (PcrV and Psl exopolysaccharide) reduced the bacterial burden in vivo. Taken together, our findings provide a preclinical model of GN-PJI and suggest the therapeutic potential of targeting biofilm-associated antigens.

Chitin is a highly abundant polysaccharide and linked to fungal infection and asthma. Unfortunately, its polymeric structure has hampered the identification of immune receptors directly binding chitin and signaling immune activation and inflammation, because purity, molecular structure and molarity are not well definable for a polymer typically extracted from biomass. Therefore, by using defined chitin (N-acetyl-glucosamine) oligomers, we identified six subunit long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR) 2 as the primary fungal chitin receptor on human and murine immune cells. Chitin oligomers directly bound TLR2 with nanomolar affinity and showed both overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Conversely, chitin oligomers shorter than 6 subunits were inactive or showed antagonistic effects on chitin/TLR2-mediated signaling, hinting to a size-dependent sensing/activation system unexpectedly conserved in plants and humans. Since blocking the chitin-TLR2 interaction effectively prevented chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease.

IL-17 is a potent proinflammatory cytokine that drives pathogenesis of multiple autoimmune diseases, including psoriasis. A major source of pathogenic IL-17 is a subset of γδ T cells (Tγδ17) that acquires the ability to produce IL-17 while developing in the thymus. The mechanisms that regulate homeostasis of Tγδ17 cells and their roles in psoriasis, however, are not fully understood. In this paper, we show that the heparan sulfate proteoglycan syndecan-1 (sdc1) plays a critical role in regulating homeostasis of Tγδ17 cells and modulating psoriasis-like skin inflammation in mice. sdc1 was predominantly expressed by Tγδ17 cells (but not IL-17− Tγδ cells) in the thymus, lymph nodes, and dermis. sdc1 deficiency significantly and selectively increased the frequency and absolute numbers of Tγδ17 cells by mechanisms that included increased proliferation and decreased apoptosis. Adoptive transfer experiments ruled out a significant role of sdc1 expressed on nonhematopoietic cells in halting expansion and proliferation of sdc1-deficient Tγδ17 cells. When subjected to imiquimod-induced psoriasiform dermatitis, Tγδ17 cells in sdc1KO mice displayed heightened responses accompanied by significantly increased skin inflammation than their wild-type counterparts. Furthermore, transferred sdc1-deficient γδ T cells caused more severe psoriasiform dermatitis than their sdc1-sufficient counterparts in TCR–βδ KO hosts. The results uncover a novel role for sdc1 in controlling homeostasis of Tγδ17 cells and moderating host responses to psoriasis-like inflammation.

Staphylococcus aureus wound infections delay healing and result in invasive complications such as osteomyelitis, especially in the setting of diabetic foot ulcers. In preclinical animal models of S. aureus skin infection, antibody neutralization of alpha-toxin (AT), an S. aureus -secreted pore-forming cytolytic toxin, reduces disease severity by inhibiting skin necrosis and restoring effective host immune responses. However, whether therapeutic neutralization of alpha-toxin is effective against S. aureus -infected wounds is unclear. Herein, the efficacy of prophylactic treatment with a human neutralizing anti-AT monoclonal antibody (MAb) was evaluated in an S. aureus skin wound infection model in nondiabetic and diabetic mice. In both nondiabetic and diabetic mice, anti-AT MAb treatment decreased wound size and bacterial burden and enhanced reepithelialization and wound resolution compared to control MAb treatment. Anti-AT MAb had distinctive effects on the host immune response, including decreased neutrophil and increased monocyte and macrophage infiltrates in nondiabetic mice and decreased neutrophil extracellular traps (NETs) in diabetic mice. Similar therapeutic efficacy was achieved with an active vaccine targeting AT. Taken together, neutralization of AT had a therapeutic effect against S. aureus -infected wounds in both nondiabetic and diabetic mice that was associated with differential effects on the host immune response.

The immune response to Staphylococcus aureus infection in skin involves the recruitment of polymorphonuclear neutrophils (PMNs) from the bone marrow via the circulation and local granulopoiesis from hematopoietic stem and progenitor cells (HSPCs) that also traffic to infected skin wounds. We focus on regulation of PMN number and function and the role of pore-forming α-toxin (AT), a virulence factor that causes host cell lysis and elicits inflammasome-mediated IL-1β secretion in wounds. Infection with wild-type S. aureus enriched in AT reduced PMN recruitment and resulted in sustained bacterial burden and delayed wound healing. In contrast, PMN recruitment to wounds infected with an isogenic AT-deficient S. aureus strain was unimpeded, exhibiting efficient bacterial clearance and hastened wound resolution. HSPCs recruited to infected wounds were unaffected by AT production and were activated to expand PMN numbers in proportion to S. aureus abundance in a manner regulated by TLR2 and IL-1R signaling. Immunodeficient MyD88-knockout mice infected with S. aureus experienced lethal sepsis that was reversed by PMN expansion mediated by injection of wild-type HSPCs directly into wounds. We conclude that AT-induced IL-1β promotes local granulopoiesis and effective resolution of S. aureus–infected wounds, revealing a potential antibiotic-free strategy for tuning the innate immune response to treat methicillin-resistant S. aureus infection in immunodeficient patients.

Hematogenous implant-related infections are an important clinical problem because bacteria spread from the bloodstream to a previously well-functioning implant and result in infectious complications and failure of a medical device or prosthesis. To study these infections, we developed a preclinical animal model of a Staphylococcus aureus hematogenous implant infection with the capability to monitor noninvasively and longitudinally the dissemination of the bacteria from the blood to a surgically placed orthopedic implant. Using this model, α-toxin and clumping factor A were identified as key factors that contributed to the pathogenesis of these infections by promoting biofilm formation. Finally, neutralizing antibodies against these factors provided a targeted, nonantibiotic alternative approach to help prevent these difficult-to-treat and costly infections.

Infection is a major complication of implantable medical devices, which provide a scaffold for biofilm formation, thereby reducing susceptibility to antibiotics and complicating treatment. Hematogenous implant-related infections following bacteremia are particularly problematic because they can occur at any time in a previously stable implant. Herein, we developed a model of hematogenous infection in which an orthopedic titanium implant was surgically placed in the legs of mice followed 3 wk later by an i.v. exposure to Staphylococcus aureus This procedure resulted in a marked propensity for a hematogenous implant-related infection comprised of septic arthritis, osteomyelitis, and biofilm formation on the implants in the surgical legs compared with sham-operated surgical legs without implant placement and with contralateral nonoperated normal legs. Neutralizing human monoclonal antibodies against α-toxin (AT) and clumping factor A (ClfA), especially in combination, inhibited biofilm formation in vitro and the hematogenous implant-related infection in vivo. Our findings suggest that AT and ClfA are pathogenic factors that could be therapeutically targeted against Saureus hematogenous implant-related infections.

The immune response to Staphylococcus aureus infection in skin involves the recruitment of neutrophils (PMN) from the bone marrow via the circulation and local granulopoiesis from hematopoietic stem and progenitor cells (HSPC) that also traffic to infected skin wounds. We focus on regulation of PMN number and function and the role of pore-forming alpha-toxin (AT), a virulence factor that causes host cell lysis and elicits inflammasome-mediated IL-1β secretion in wounds. Infection with wild type S. aureus enriched in AT reduced PMN recruitment and resulted in sustained bacterial burden and delayed wound healing. In contrast, PMN recruitment to wounds infected with an isogenic AT mutant strain (ΔAT) was unimpeded, exhibiting efficient bacterial clearance and hastened wound resolution. HSPC recruited to infected wounds was unaffected by AT production and were activated to expand PMN numbers in proportion to S. aureus abundance in a manner regulated by TLR2 and IL-1 receptor signaling. Immunodeficient MyD88 knockout mice infected with S. aureus experienced lethal sepsis that was reversed by PMN expansion mediated by injection of wild type HSPC directly into wounds. We conclude that AT induced IL-1β promotes local granulopoiesis and effective resolution of S. aureus-infected wounds, revealing a potential antibiotic free strategy for tuning the innate immune response to treat MRSA infection in immunodeficient patients.

Background Atopic dermatitis (AD) is associated with epidermal barrier defects, dysbiosis, and skin injury caused by scratching. In particular, the barrier-defective epidermis in patients with AD with loss-of-function filaggrin mutations has increased IL-1α and IL-1β levels, but the mechanisms by which IL-1α, IL-1β, or both are induced and whether they contribute to the aberrant skin inflammation in patients with AD is unknown. Objective We sought to determine the mechanisms through which skin injury, dysbiosis, and increased epidermal IL-1α and IL-1β levels contribute to development of skin inflammation in a mouse model of injury-induced skin inflammation in filaggrin-deficient mice without the matted mutation (ft/ft mice). Methods Skin injury of wild-type, ft/ft, and myeloid differentiation primary response gene–88–deficient ft/ft mice was performed, and ensuing skin inflammation was evaluated by using digital photography, histologic analysis, and flow cytometry. IL-1α and IL-1β protein expression was measured by means of ELISA and visualized by using immunofluorescence and immunoelectron microscopy. Composition of the skin microbiome was determined by using 16S rDNA sequencing. Results Skin injury of ft/ft mice induced chronic skin inflammation involving dysbiosis-driven intracellular IL-1α release from keratinocytes. IL-1α was necessary and sufficient for skin inflammation in vivo and secreted from keratinocytes by various stimuli in vitro. Topical antibiotics or cohousing of ft/ft mice with unaffected wild-type mice to alter or intermix skin microbiota, respectively, resolved the skin inflammation and restored keratinocyte intracellular IL-1α localization. Conclusions Taken together, skin injury, dysbiosis, and filaggrin deficiency triggered keratinocyte intracellular IL-1α release that was sufficient to drive chronic skin inflammation, which has implications for AD pathogenesis and potential therapeutic targets.

Bacterial biofilm formation is a major complication of implantable medical devices that results in therapeutically challenging chronic infections, especially in cases involving antibiotic-resistant bacteria. As an approach to prevent these infections, an electrospun composite coating comprised of poly(lactic-coglycolic acid) (PLGA) nanofibers embedded in a poly(ε-caprolactone) (PCL) film was developed to locally codeliver combinatorial antibiotics from the implant surface. The release of each antibiotic could be adjusted by loading each drug into the different polymers or by varying PLGA:PCL polymer ratios. In a mouse model of biofilm-associated orthopedic-implant infection, three different combinations of antibiotic-loaded coatings were highly effective in preventing infection of the bone/joint tissue and implant biofilm formation and were biocompatible with enhanced osseointegration. This nanofiber composite-coating technology could be used to tailor the delivery of combinatorial antimicrobial agents from various metallic implantable devices or prostheses to effectively decrease biofilm-associated infections in patients.

Background The Nod-like receptor NACHT, LRR, and PYD domain–containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. Objective We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. Methods After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. Results Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration–approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1β processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1β release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1β processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. Conclusion Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome–linked inflammation could potentially be targeted pharmacologically through BTK.

Summary Staphylococcus aureus colonization contributes to skin inflammation in diseases such as atopic dermatitis, but the signaling pathways involved are unclear. Herein, epicutaneous S. aureus exposure to mouse skin promoted MyD88-dependent skin inflammation initiated by IL-36, but not IL-1α/β, IL-18, or IL-33. By contrast, an intradermal S. aureus challenge promoted MyD88-dependent host defense initiated by IL-1β rather than IL-36, suggesting that different IL-1 cytokines trigger MyD88 signaling depending on the anatomical depth of S. aureus cutaneous exposure. The bacterial virulence factor PSMα, but not α-toxin or δ-toxin, contributed to the skin inflammation, which was driven by IL-17-producing γδ and CD4 + T cells via direct IL-36R signaling in the T cells. Finally, adoptive transfer of IL-36R-expressing T cells to IL-36R-deficient mice was sufficient for mediating S. aureus -induced skin inflammation. Together, this study defines a previously unknown pathway by which S. aureus epicutaneous exposure promotes skin inflammation involving IL-36R/MyD88-dependent IL-17 T cell responses.

Autosomal dominant hyper-IgE syndrome is caused by STAT3 loss-of-function mutations, which results in a constellation of clinical features, including highly elevated serum IgE levels, a severe eczema-like skin eruption, and an increased susceptibility to Candida albicans and Staphylococcus aureus skin infections. However, the mechanism by which these individuals have elevated IgE levels is not well understood. To evaluate the role of defective STAT3 signaling in contributing to IgE production, we used a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous exposure to S. aureus in cre/lox mice with cell specific deletion of STAT3. Unexpectedly, STAT3 deletion in keratinocytes (K5-creERT2 × STAT3fl/fl mice) but not in T cells (CD4-creERT2 × STAT3fl/fl mice) resulted in a substantial elevation of serum IgE levels. The increase in serum IgE involved concomitant increase in IgG and IgA levels that were also significantly higher in the keratinocyte-specific STAT3-deficient mice compared with wildtype mice. CD4+ T cells were required for the IgE production, since CD4+ T cell depletion resulted in significantly reduced level of serum IgE in the K5-creERT2 × STAT3fl/fl mice. Taken together, these results indicate that keratinocytes can modulate serum antibody levels, especially in the absence of STAT3 signaling. These results provide an explanation of the increased serum IgE levels in autosomal dominant hyper-IgE syndrome and more broadly into the mechanisms that promote antibody production in response to epicutaneous exposure to microbes and inflammation.

Staphylococcus aureus infection is a major complication of acute and chronic skin wounds that delays wound closure and healing. These infections are particularly problematic in diabetic foot ulcers as the infection can become invasive, leading to osteomyelitis that often necessitates limb amputation. Prior reports in S. aureus skin infection models have indicated that neutralizing S. aureus alpha-toxin decreases disease severity by reducing skin lesion size and restoring an appropriate host immune response, however whether alpha-toxin neutralization impacts S. aureus-infected skin wounds is not clear, especially in the setting of diabetes. Herein, we evaluated the efficacy of MEDI4893* a neutralizing human anti-alpha-toxin monoclonal antibody (mAb) in a S. aureus skin wound infection model. Three full-thickness cuts were made on the back skin of wildtype (wt) C57BL/6 and diabetic (TallyHo) mice and the cuts inoculated with a bioluminescent methicillin-resistant S. aureus (MRSA) strain. In both WT and diabetic mice, prophylaxis with MEDI4893* resulted in a significant reduction in skin lesion sizes, decreased bacterial burden, improved re-epithelialization, and a decreased percentage of neutrophils and an increased percentage of macrophages in the skin compared with control mAb treatment. Thus, in S. aureus-infected skin wounds, anti-alpha-toxin mAb treatment reduced disease severity by not only inhibiting dermonecrosis but also by resolving neutrophilic inflammation.

The immune mechanisms for durable immunity against S. aureus skin infections are unclear as reinfections are common despite specific antibodies and Th1/Th17 cells. Using a CA-MRSA skin reinfection model in mice involving a primary infection (1°) in the lower back (cleared by 14 days) followed by a secondary infection (2°) in the upper back on day 28, we found that 1° and 2° wt mice had similar responses. In contrast, 1° IL-1β−/− mice had increased lesions and bacterial burden with impaired neutrophil recruitment whereas 2° IL-1β−/− mice were protected and resembled wt mice. Transfer of serum containing CA-MRSA specific antibodies did not confer protection to 1° IL-1β−/− mice and depletion of CD4+ T cells did not alter the protection of 2° IL-1β−/− mice, indicating a lack of a role of antibodies or CD4+ T cells. However, 2° IL-1β−/− mice were no longer protected if the egress of lymphocytes from lymph nodes was blocked in 2° IL-1β−/− mice (FTY720 treatment) and the immune impairment in naïve IL-1β−/− mice could be rescued by adoptive transfer of total lymph nodes cells from d28 IL-1β−/− mice. Moreover, transfer of only 50,000 γδ T cells from lymph nodes of day 28 IL-1β−/− mice conferred protection to 1° IL-1β−/− mice. These γδ T cells produced IFNγ/TNF-α, but not IL-17/IL-22, and neutralizing IFNγ/TNF-α activity resulted in loss of protection in 2° IL-1β−/− mice. In humans, similar IFNγ/TNF-α-producing γδ T cells were found in individuals with IRAK4 deficiency whose susceptibility to S. aureus skin infections improves with age but not from individuals with chronic granulomatous disease whose susceptibility is lifelong. These findings define an alternative mechanism to therapeutically target for long-term protection against CA-MRSA skin infections.

Psoriasis is an autoimmune skin disease affecting 2–3% of the population that is driven by IL-17 responses. Although STAT3 promotes the development of IL-17-producing T cells, STAT3 overexpression in keratinocytes is sufficient to induce psoriasiform dermatitis in mice. To study the role of STAT3 in keratinocytes versus T cells in psoriasis, the imiquimod mouse model of psoriasiform dermatitis was performed on mice with specific deletion of STAT3 in either keratinocytes (K5-creERT2×STAT3fl/fl [K5-STAT3]) or T-cells (Lck-cre×STAT3fl/fl [Lck-STAT3]). Unexpectedly, K5-STAT3 mice but not Lck-STAT3 mice had reduced skin inflammation and inflammatory cell influx compared to wt mice, suggesting that STAT3 signaling in keratinocytes rather than T cells was more important determinant for mediating the skin inflammation. K5-STAT3 mice also had increased IFN-γ+ T cells but less IL-17+ T-cells compared to wt mice, indicating that loss of STAT3 signaling in keratinocytes dampened inflammation by inhibiting IL-17 responses while promoting IFN-γ responses. Since interferon/STAT1 signaling responses can be inhibited by STAT3, we hypothesized that loss of STAT3 signaling in keratinocytes would lead to enhanced interferon/STAT1-signaling and responses. Consistent with this possibility, histologic sections from the inflamed skin of K5-STAT3 mice had increased epidermal STAT1 phosphorylation and increased mRNA expression of interferon-regulated chemokines CXCL10 and CXCL11 compared with wt mice. Taken together, these findings indicate that keratinocytes can regulate T cell responses in the skin and STAT3 signaling in keratinocytes could represent a therapeutic target in psoriasis.