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Mammalian injury responses are predominantly characterized by fibrosis and scarring rather than functional regeneration. This limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we screened transcripts in human subjects following laser rejuvenation treatment and compared them to mice with enhanced Wound Induced Hair Neogenesis (WIHN), a rare example of mammalian organogenesis. We found that Rnasel-/- mice exhibit an increased regenerative capacity, with elevated WIHN through enhanced IL-36α. Consistent with RNase L's known role to stimulate caspase-1, we found that pharmacologic inhibition of caspases promoted regeneration in an IL-36 dependent manner in multiple epithelial tissues. We identified a negative feedback loop, where RNase L activated caspase-1 restrains the pro-regenerative dsRNA-TLR3 signaling cascade through the cleavage of toll-like adaptor protein TRIF. Through integrated single-cell RNA sequencing and spatial transcriptomic profiling, we confirmed Oas & Il36 genes to be highly expressed at the site of wounding and are elevated in Rnasel-/- mice wounds. This work suggests that RNase L functions as a regeneration repressor gene, in a functional tradeoff that tempers immune hyper-activation during viral infection at the cost of inhibiting regeneration.

Introduction: The U.S. Military members experiencing combat-related injuries have a higher chance of developing infections by multidrug-resistant (MDR) bacteria at admission to military hospitals. MDR wound infections result in higher amputation rates and greater risks for subsequent or chronic infections that require readmission or extended stay in the hospital. Currently, there is no FDA-clear, deployable early diagnostic system for suitable field use.We are reporting our efforts to improve a previously developed Rapid Label-free Pathogen Identification (RAPID) system to detect viable MDR bacteria in wound infections and perform antibiotic susceptibility testing (AST). Specifically, we added multiplex and automation capability and significantly simplified the sample preparation process. A functional prototype of the improved system was built, and its performance was validated using a variety of lab-prepared spiked samples and real-world samples., Materials and Methods: To access the baseline performance of the improved RAPID system in detecting bacteria presence, we selected 17 isolates, most of them from blood or wound infections, and prepared mono-strain spiked samples at 104 to 106 cfu/mL concentration. These samples were processed and analyzed by the RAPID system. To demonstrate the AST capability of the system, we selected 6 strains against 6 different antibiotics and compared the results from the system with the ones from the gold standard method.To validate the system's performance with real-world samples, we first investigated its performance on 3 swab samples from epicutaneous methicillin-resistant Staphylococcus aureus-exposed mouse model. The AST results from our system were compared with the ones from the gold standard method. All animal experiments were approved by the Johns Hopkins University Animal Care and Use Committee (Protocol No. MO21M378). Then, we obtained swab samples from 7 atopic dermatitis (AD) patients and compared our AST results with the ones from the gold standard method. The human subject protocol was approved by the Johns Hopkins Medicines Institutional Review Boards (Study No. CR00043438/IRB00307926) and by USAMRDC (Proposal Log Number/Study Number 20000251)., Results: High-quality data were obtained from the spiked samples of all 17 strains. A quantitative analysis model built using these data achieved 94% accuracy in predicting the species ID in 8 unknown samples. The AST results on the spiked samples had shown 100% matching with the gold standard method. Our system successfully detects the presence/absence of viable bacteria in all 3 mouse and 7 AD patient swab samples. Our system shows 100% and 85.7% (6 out of 7) accuracy when compared to the oxacillin susceptibility testing results for the mouse and the AD patient swabs, respectively., Conclusions: Our system has achieved excellent performance in detecting viable bacteria presence and in performing AST in a multiplex, automated, and easy-to-operate manner, on both lab-prepared and real samples. Our results have shown a path forward to a rapid (sample-to-answer time ≤3 hours), accurate, sensitive, species-specific, and portable system to detect the presence of MDR combat-related wound infections in the field environment. Our future efforts involve ruggedizing the RAPID system and evaluating performance under relevant environmental conditions., (© The Association of Military Surgeons of the United States 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Skin homeostasis relies on a delicate balance between host proteases and protease inhibitors along with those secreted from microbial communities, as disruption to this harmony contributes to the pathogenesis of inflammatory skin disorders, including atopic dermatitis and Netherton's syndrome. In addition to being a prominent cause of skin and soft tissue infections, the gram-positive bacterium Staphylococcus aureus is a key player in inflammatory skin conditions due to its array of 10 secreted proteases. Herein we review how S. aureus proteases augment the development of inflammation in skin disorders. These mechanisms include degradation of skin barrier integrity, immune dysregulation and pruritis, and impairment of host defenses. Delineating the diverse roles of S. aureus proteases has the potential to reveal novel therapeutic strategies, such as inhibitors of proteases or their cognate target, as well as neutralizing vaccines to alleviate the burden of inflammatory skin disorders in patients.

Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs). NETs counteract microbes but are also linked to inflammation in atherosclerosis, arthritis, or psoriasis by unknown mechanisms. Here, we report that NET-associated RNA (naRNA) stimulates further NET formation in naive PMNs via a unique TLR8-NLRP3 inflammasome-dependent pathway. Keratinocytes respond to naRNA with expression of psoriasis-related genes (e.g., IL17, IL36) via atypical NOD2-RIPK signaling. In vivo, naRNA drives temporary skin inflammation, which is drastically ameliorated by genetic ablation of RNA sensing. Unexpectedly, the naRNA-LL37 'composite damage-associated molecular pattern (DAMP)' is pre-stored in resting neutrophil granules, defining sterile NETs as inflammatory webs that amplify neutrophil activation. However, the activity of the naRNA-LL37 DAMP is transient and hence supposedly self-limiting under physiological conditions. Collectively, upon dysregulated NET release like in psoriasis, naRNA sensing may represent both a potential cause of disease and a new intervention target., (© 2024. The Author(s).)

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) in the U.S. as well as more serious invasive diseases, including bacteremia, sepsis, endocarditis, surgical site infections, osteomyelitis, and pneumonia. These infections are exacerbated by the emergence of antibiotic-resistant clinical isolates such as methicillin-resistant S. aureus (MRSA), highlighting the need for alternatives to antibiotics to treat bacterial infections. We have previously developed a multi-component toxoid vaccine (IBT-V02) in a liquid formulation with efficacy against multiple strains of Staphylococcus aureus prevalent in the industrialized world. However, liquid vaccine formulations are not compatible with the paucity of cold chain storage infrastructure in many low-to-middle income countries (LMICs). Furthermore, whether our IBT-V02 vaccine formulations are protective against S. aureus isolates from LMICs is unknown. To overcome these limitations, we developed lyophilized and spray freeze-dried formulations of IBT-V02 vaccine and demonstrated that both formulations had comparable biophysical attributes as the liquid formulation, including similar levels of toxin neutralizing antibodies and protective efficacy against MRSA infections in murine and rabbit models. To enhance the relevancy of our findings, we then performed a multi-dimensional screen of 83 S. aureus clinical isolates from LMICs (e.g., Democratic Republic of Congo, Palestine, and Cambodia) to rationally down-select strains to test in our in vivo models based on broad expression of IBT-V02 targets (i.e., pore-forming toxins and superantigens). IBT-V02 polyclonal antisera effectively neutralized toxins produced by the S. aureus clinical isolates from LMICs. Notably, the lyophilized IBT-V02 formulation exhibited significant in vivo efficacy in various preclinical infection models against the S. aureus clinical isolates from LMICs, which was comparable to our liquid formulation. Collectively, our findings suggested that lyophilization is an effective alternative to liquid vaccine formulations of our IBT-V02 vaccine against S. aureus infections, which has important implications for protection from S. aureus isolates from LMICs., Competing Interests: Authors IM, AV, RO, MM, SS, TK, RA, HK and MJ were employed by company Integrated Biotherapeutics Inc. Author MK was employed by company Engineered Biopharmaceuticals. Author NA has received previous grant support from Pfizer and Boehringer Ingelheim and has been a paid consultant for Janssen Pharmaceuticals. MJ and RA have stocks in Razi Holdings, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Wang, Mukherjee, Venkatasubramaniam, Dikeman, Orlando, Zhang, Ortines, Mednikov, Sherchand, Kanipakala, Le, Shukla, Ketner, Adhikari, Karauzum, Aman and Archer.)

Musculoskeletal infections (MSKI), which are a major problem in orthopedics, occur when the pathogen eludes or overwhelms the host immune system. While effective vaccines and immunotherapies to prevent and treat MSKI should be possible, fundamental knowledge gaps in our understanding of protective, nonprotective, and pathogenic host immunity are prohibitive. We also lack critical knowledge of how host immunity is affected by the microbiome, implants, prior infection, nutrition, antibiotics, and concomitant therapies, autoimmunity, and other comorbidities. To define our current knowledge of these critical topics, a Host Immunity Section of the 2023 Orthopaedic Research Society MSKI International Consensus Meeting (ICM) proposed 78 questions. Systematic reviews were performed on 15 of these questions, upon which recommendations with level of evidence were voted on by the 72 ICM delegates, and another 12 questions were voted on with a recommendation of "Unknown" without systematic reviews. Two questions were transferred to another ICM Section, and the other 45 were tabled for future consideration due to limitations of available human resources. Here we report the results of the voting with internet access to the questions, recommendations, and rationale from the systematic reviews. Eighteen questions received a consensus vote of ≥90%, while nine recommendations failed to achieve this threshold. Commentary on why consensus was not achieved on these questions and potential ways forward are provided to stimulate specific funding mechanisms and research on these critical MSKI host defense questions., (© 2023 Orthopaedic Research Society.)

Biofilm-associated bacterial infections are the major reason for treatment failure in many diseases including burn trauma infections. Uncontrolled inflammation induced by bacteria leads to materiality, tissue damage, and chronic diseases. Specialized proresolving mediators (SPMs), including maresin-like lipid mediators (MarLs), are enzymatically biosynthesized from omega-3 essential long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), by macrophages and other leukocytes. SPMs exhibit strong inflammation-resolving activities, especially inflammation provoked by bacterial infection. In this study, we explored the potential direct inhibitory activities of three MarLs on Gram-positive (Staphylococcus aureus ) and Gram-negative ( Pseudomonas aeruginosa and Escherichia coli ) bacteria in their biofilms that are leading bacteria in burn trauma-related infections. We also examined the effects of MarLs on the bactericidal activities of a typical broad-spectrum antibiotic, carbenicillin (carb), on these bacteria in their preformed biofilms. The results revealed that MarLs combined with carbenicillin can inhibit the survival of Gram-positive and Gram-negative bacteria in their biofilms although MarLs alone did not exhibit bactericidal activity. Thus, our findings suggest that the combination of MarLs and carbenicillin can lower the antibiotic requirements to kill the bacteria in preformed biofilms.

Staphylococcus aureus skin colonization and eosinophil infiltration are associated with many inflammatory skin disorders, including atopic dermatitis, bullous pemphigoid, Netherton's syndrome, and prurigo nodularis. However, whether there is a relationship between S. aureus and eosinophils and how this interaction influences skin inflammation is largely undefined. We show in a preclinical mouse model that S. aureus epicutaneous exposure induced eosinophil-recruiting chemokines and eosinophil infiltration into the skin. Remarkably, we found that eosinophils had a comparable contribution to the skin inflammation as T cells, in a manner dependent on eosinophil-derived IL-17A and IL-17F production. Importantly, IL-36R signaling induced CCL7-mediated eosinophil recruitment to the inflamed skin. Last, S. aureus proteases induced IL-36α expression in keratinocytes, which promoted infiltration of IL-17-producing eosinophils. Collectively, we uncovered a mechanism for S. aureus proteases to trigger eosinophil-mediated skin inflammation, which has implications in the pathogenesis of inflammatory skin diseases., Competing Interests: Competing interests statement:N.K.A. has received previous grant support from Pfizer and Boehringer Ingelheim and was a paid consultant for Janssen Pharmaceuticals. The remaining authors state no conflict of interest.

Background Context: Bacterial infection of spinal instrumentation is a significant challenge in spinal fusion surgery. Although the intraoperative local application of powdered vancomycin is common practice for mitigating infection, the antimicrobial effects of this route of administration are short-lived. Therefore, novel antibiotic-loaded bone grafts as well as a reliable animal model to permit the testing of such therapies are needed to improve the efficacy of infection reduction practices in spinal fusion surgery., Purpose: This study aims to establish a clinically relevant rat model of spinal implant-associated infection to permit the evaluation of antimicrobial bone graft materials used in spinal fusion., Study Design: Rodent study of chronic spinal implant-associated infection., Methods: Instrumentation anchored in and spanning the vertebral bodies of L4 and L5 was inoculated with bioluminescent methicillin-resistant Staphylococcus aureus bacteria (MRSA). Infection was monitored using an in vivo imaging system (IVIS) for 8 weeks. Spines were harvested and evaluated histologically, and colony-forming units (CFUs) were quantified in harvested implants and spinal tissue., Results: Postsurgical analysis of bacterial infection in vivo demonstrated stratification between MRSA and phosphate-buffered saline (PBS) control groups during the first 4 weeks of the 8-week infection period, indicating the successful establishment of acute infection. Over the 8-week chronic infection period, groups inoculated with 1 × 10 5 MRSA CFU and 1 × 10 6 MRSA CFU demonstrated significantly higher bioluminescence than groups inoculated with PBS control (p = 0.009 and p = 0.041 respectively). Histological examination at 8 weeks postimplantation revealed the presence of abscesses localized to implant placement in all MRSA inoculation groups, with the most pervasive abscess formation in samples inoculated with 1 × 10 5 MRSA CFU and 1 × 10 6 MRSA CFU. Quantification of CFU plated from harvested spinal tissue at 8 weeks post-implantation revealed the 1 × 10 5 MRSA CFU inoculation group as the only group with a significantly greater average CFU count compared to PBS control (p = 0.017). Further, CFU quantification from harvested spinal tissue was greater than CFU quantification from harvested implants across all inoculation groups., Conclusion: Our model demonstrated that the inoculation dosage of 1 × 10 5 MRSA CFU exhibited the most robust chronic infection within instrumented vertebral bodies. This dosage had the greatest difference in bioluminescence signal from control (p < 0.01), the lowest mortality (0% compared to 50% for samples inoculated with 1 × 10 6 MRSA CFU), and a significantly higher amount of CFUs from harvested spine samples than CFUs from control harvested spine samples. Further, histological analysis confirmed the reliability of this novel rodent model of implanted-associated infection to establish infection and biofilm formation of MRSA for all inoculation groups., Clinical Significance: This model is intended to simulate the infection of instrumentation used in spinal fusion surgeries concerning implant locality and material. This model may evaluate potential antimicrobial and osteogenic biomaterials and investigate the relationship between implant-associated infection and failed fusion., Competing Interests: Declarations of competing interests Dr. Witham is a medical advisory board member and investor in Augmedics, Inc. He has received grant support from the Gordon and Marilyn Macklin Foundation. Dr. Archer has received previous grant support from Pfizer and Boehringer Ingelheim and is a paid consultant for Janssen Pharmaceuticals. One or more of the authors declare financial or professional relationships on ICMJE-TSJ disclosure forms., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Staphylococcus aureus is a leading cause of bacteremia, further complicated by the emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA). A better understanding of host defense mechanisms is needed for the development of host-directed therapies as an alternative approach to antibiotics. The levels of IL-1, IL-17, and TNF-α cytokines in circulation have been associated with predictive outcomes in patients with S. aureus bacteremia. However, their causative role in survival and the cell types involved in these responses during bacteremia is not entirely clear. Using a mouse model of S. aureus bacteremia, we demonstrated that IL-17A/F and TNF-α had no significant impact on survival, whereas IL-1R signaling was critical for survival during S. aureus bacteremia. Furthermore, we identified that T cells, but not neutrophils, monocytes/macrophages, or endothelial cells were the crucial cell type for IL-1R-mediated survival against S. aureus bacteremia. Finally, we determined that the expression of IL-1R on γδ T cell, but not CD4 + or CD8 + T cells was responsible for survival against the S. aureus bacteremia. Taken together, we uncovered a role for IL-1R, but not IL-17A/F and TNF-α in protection against S. aureus bacteremia. Importantly, γδ T cell-intrinsic expression of IL-1R was crucial for survival, but not on other immune cells or endothelial cells. These findings reveal potential cellular and immunological targets for host-directed therapies for improved outcomes against S. aureus bacteremia., Competing Interests: NA has received previous grant support from Pfizer and Boehringer Ingelheim and has been a paid consultant for Janssen Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 Wang, Ahmadi, Dikeman, Youn and Archer.)

Staphylococcus aureus is the leading cause of skin and soft tissue infections and is a major health burden due to the emergence of antibiotic-resistant strains. To address the unmet need of alternative treatments to antibiotics, a better understanding of the protective immune mechanisms against S. aureus skin infection is warranted. Here, we report that tumor necrosis factor (TNF) promoted protection against S. aureus in the skin, which was mediated by bone marrow-derived immune cells. Furthermore, neutrophil-intrinsic TNF receptor (TNFR) signaling directed immunity against S. aureus skin infections. Mechanistically, TNFR1 promoted neutrophil recruitment to the skin, whereas TNFR2 prevented systemic bacterial dissemination and directed neutrophil antimicrobial functions. Treatment with a TNFR2 agonist showed therapeutic efficacy against S. aureus and Pseudomonas aeruginosa skin infections, which involved increased neutrophil extracellular trap formation. Our findings revealed nonredundant roles for TNFR1 and TNFR2 in neutrophils for immunity against S. aureus and can be therapeutically targeted for protection against bacterial skin infections.

In this issue of Cell Host & Microbe, Kashaf et al. and Key et al. examine isolates of Staphylococcus aureus among individuals with atopic dermatitis, revealing insights into evolution, antibiotic resistance, transmission mechanisms, skin colonization, and virulence factors. These findings further our understanding of disease pathogenesis and potential treatments., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Phosphodiesterase 4 (PDE4) is highly expressed in keratinocytes and immune cells and promotes pro-inflammatory responses upon activation. The activity of PDE4 has been attributed to various inflammatory conditions, leading to the development and approval of PDE4 inhibitors as host-directed therapeutics in humans. For example, the topical PDE4 inhibitor, crisaborole, is approved for the treatment of mild-to-moderate atopic dermatitis and has shown efficacy in patients with psoriasis. However, the role of crisaborole in regulating the immunopathogenesis of inflammatory skin diseases and infection is not entirely known. Therefore, we evaluated the effects of crisaborole in multiple mouse models, including psoriasis-like dermatitis, AD-like skin inflammation with and without filaggrin mutations, and Staphylococcus aureus skin infection. We discovered that crisaborole dampens myeloid cells and itch in the skin during psoriasis-like dermatitis. Furthermore, crisaborole was effective in reducing skin inflammation in the context of filaggrin deficiency. Importantly, crisaborole reduced S. aureus skin colonization during AD-like skin inflammation. However, crisaborole was not efficacious in treating S. aureus skin infections, even as adjunctive therapy to antibiotics. Taken together, we found that crisaborole reduced itch during psoriasis-like dermatitis and decreased S. aureus skin colonization upon AD-like skin inflammation, which act as additional mechanisms by which crisaborole dampens the immunopathogenesis in mouse models of inflammatory skin diseases. Further examination is warranted to translate these preclinical findings to human disease., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Tissue injury induces metabolic changes in stem cells, which likely modulate regeneration. Using a model of organ regeneration called wound-induced hair follicle neogenesis (WIHN), we identified skin-resident bacteria as key modulators of keratinocyte metabolism, demonstrating a positive correlation between bacterial load, glutamine metabolism, and regeneration. Specifically, through comprehensive multiomic analysis and single-cell RNA sequencing in murine skin, we show that bacterially induced hypoxia drives increased glutamine metabolism in keratinocytes with attendant enhancement of skin and hair follicle regeneration. In human skin wounds, topical broad-spectrum antibiotics inhibit glutamine production and are partially responsible for reduced healing. These findings reveal a conserved and coherent physiologic context in which bacterially induced metabolic changes improve the tolerance of stem cells to damage and enhance regenerative capacity. This unexpected proregenerative modulation of metabolism by the skin microbiome in both mice and humans suggests important methods for enhancing regeneration after injury.

Despite meritorious attempts, a S. aureus vaccine that prevents infection or mitigates severity has not yet achieved efficacy endpoints in prospective, randomized clinical trials. This experience underscores the complexity of host- S. aureus interactions, which appear to be greater than many other bacterial pathogens against which successful vaccines have been developed. It is increasingly evident that S. aureus employs strategic countermeasures to evade or exploit human immune responses. From entering host cells to persist in stealthy intracellular reservoirs, to sensing the environmental milieu and leveraging bacterial or host metabolic products to reprogram host immune responses, S. aureus poses considerable challenges for the development of effective vaccines. The fact that this pathogen causes distinct types of infections and can undergo transient genetic, transcriptional or metabolic adaptations in vivo that do not occur in vitro compounds challenges in vaccine development. Notably, the metabolic versatility of both bacterial and host immune cells as they compete for available substrates within specific tissues inevitably impacts the variable repertoire of gene products that may or may not be vaccine antigens. In this respect, S. aureus has chameleon phenotypes that have alluded vaccine strategies thus far. Nonetheless, a number of recent studies have also revealed important new insights into pathogenesis vulnerabilities of S. aureus . A more detailed understanding of host protective immune defenses versus S. aureus adaptive immune evasion mechanisms may offer breakthroughs in the development of effective vaccines, but at present this goal remains a very high bar. Coupled with the recent advances in human genetics and epigenetics, newer vaccine technologies may enable such a goal. If so, future vaccines that protect against or mitigate the severity of S. aureus infections are likely to emerge at the intersection of precision and personalized medicine. For now, the development of S. aureus vaccines or alternative therapies that reduce mortality and morbidity must continue to be pursued., Competing Interests: RP is a consultant for IBT which is producing a multi-valent antitoxin vaccine and is a member of the review board for the University of Rochester which is involved in an anti-Gmd vaccine effort. MY is a consultant for Genentech-Roche, Alexion/AstraZeneca and Horizon Pharmaceuticals, which are involved in discovery and development of immunotherapeutic agents and strategies. NA is a consultant for Janssen Pharmaceuticals and has received previous grant support from Pfizer and Boehringer Ingelheim, which are involved in discovery and development of immunotherapeutic agents and strategies. MA has stocks in Integrated Biotherapeutics, a company engaged in development of vaccines and antibody therapies for bacterial infections. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wong Fok Lung, Chan, Prince, Yeaman, Archer, Aman and Proctor.)

Staphylococcus aureus is an important cause of various infections in humans, including bacteremia, skin and soft tissue infections, and infections associated with implanted medical devices. The emergence of hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA) underscores the urgent and unmet need to develop novel, safe, and effective antibiotics against these multidrug-resistant clinical isolates. Oxazolidinone antibiotics such as linezolid have excellent oral bioavailability and provide coverage against MRSA infections. However, their widespread and long-term use is often limited by adverse effects, especially myelosuppression. TBI-223 is a novel oxazolidinone with potentially reduced myelosuppression, compared to linezolid, but its efficacy against MRSA infections is unknown. Therefore, the preclinical efficacy of TBI-223 (80 and 160 mg/kg twice daily) was compared with that of linezolid (40 and 80 mg/kg twice daily) and sham treatment in mouse models of MRSA bacteremia, skin wound infection, and orthopedic-implant-associated infection. The dosage was selected based on mouse pharmacokinetic analysis of both linezolid and TBI-223, as well as measurement of the MICs. In all three models, TBI-223 and linezolid had comparable dose-dependent efficacies in reducing bacterial burden and disease severity, compared with sham-treated control mice. Taken together, these findings indicate that TBI-223 represents a novel oxazolidinone antibiotic that may provide an additional option against MRSA infections. Future studies in larger animal models and clinical trials are warranted to translate these findings to humans. IMPORTANCE Staphylococcus aureus is the predominant cause of bloodstream, skin, and bone infections in humans. Resistance to commonly used antibiotics is a growing concern, making it more difficult to treat staphylococcal infections. Use of the oxazolidinone antibiotic linezolid against resistant strains is hindered by high rates of adverse reactions during prolonged therapy. Here, a new oxazolidinone named TBI-223 was tested against S. aureus in three mouse models of infection, i.e., bloodstream infection, skin infection, and bone infection. We found that TBI-223 was as effective as linezolid in these three models. Previous data suggest that TBI-223 has a better safety profile than linezolid. Taken together, these findings indicate that this new agent may provide an additional option against MRSA infections. Future studies in larger animal models and clinical trials are warranted to translate these findings to humans.

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1β and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense., Competing Interests: Declaration of interests Xinzhong Dong is a co-founder and a scientific advisory board member of Escient Pharmaceuticals, a company focused on developing small molecules targeting MRGPRs. N.K.A. has received previous grant support from Pfizer and Boehringer Ingelheim and was a paid consultant for Janssen Pharmaceuticals. L.S.M. is currently a full-time paid employee of Janssen Research & Development, the pharmaceutical companies of Johnson & Johnson, and owns Johnson & Johnson stock. L.S.M. performed all work at his prior affiliation at Johns Hopkins University School of Medicine and he has received prior grant support from AstraZeneca, Pfizer, Boehringer Ingelheim, Regeneron Pharmaceuticals, and Moderna Therapeutics; was a paid consultant for Armirall and Janssen Research and Development; was on the scientific advisory board of Integrated Biotherapeutics; and is a shareholder of Noveome Biotherapeutics, which are all developing therapeutics against infections (including S. aureus and other pathogens) and/or inflammatory conditions. B.S.K. has served as a consultant for AbbVie, ABRAX Japan, Almirall, Cara Therapeutics, Maruho, Menlo Therapeutics, Pfizer, and Third Rock Ventures. He has also participated on the advisory board for Almirall, Boehringer Ingelheim, Cara Therapeutics, Kiniksa Pharmaceuticals, Menlo Therapeutics, Regeneron Pharmaceuticals, Sanofi Genzyme, and Trevi Therapeutics. He is also Founder, Chief Scientific Officer, and stockholder of Nuogen Pharma, Inc. He is stockholder of Locus Biosciences. C.V. is a paid consultant and stockholder of AstraZeneca., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Competing Interests: Conflict of Interest SGK is an advisory board member/consultant for AbbVie, Celldex Therapeutics, Galderma, Incyte Corporation, Pfizer, Regeneron Pharmaceuticals, and Kiniksa Pharmaceuticals and has served as an investigator for Galderma, Kiniksa Pharmaceuticals, Pfizer, and Sanofi. NKA has received financial support from Pfizer. The remaining authors state no conflict of interest.

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Allergens have been identified as potential triggers in patients with atopic dermatitis (AD). Patients with AD are highly sensitive to cockroach allergen. The underlying mechanism, however, remains undetermined. Here, we established a cockroach allergen-induced AD-like mouse model, and we demonstrate that repeated exposure to cockroach allergen led to aggravated mouse skin inflammation, characterized by increased type 2 immunity, type 2 innate lymphoid cells (ILC2s), and mast cells. Increased mast cells were also observed in patients with AD. Mast cell-deficient mice (KitW-sh/W-sh) showed diminished skin inflammation, suggesting that mast cells are required in allergen-induced skin inflammation. Furthermore, DC immunoreceptor (DCIR) is upregulated in skin mast cells of patients with AD and mediates allergen binding and uptake. DCIR-/- mice or reconstituted KitW-sh/W-sh mice with DCIR-/- mast cells showed a significant reduction in AD-like inflammation. Both in vitro and in vivo analyses demonstrate that DCIR-/- mast cells had reduced IgE-mediated mast cell activation and passive cutaneous anaphylaxis. Mechanistically, DCIR regulates allergen-induced IgE-mediated mast cell ROS generation and oxidation of calmodulin kinase II (ox-CaMKII). ROS-resistant CaMKII (MM-VVδ) prevents allergen-induced mast cell activation and inflammatory mediator release. Our study reveals a DCIR/ROS/CaMKII axis that controls allergen-induced mast cell activation and AD-like inflammation.

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., (© 2021 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Staphylococcus aureus is a major human pathogen causing serious implant–associated infections. Combination treatment with rifampin (10 to 15 mg/kg per day), which has dose-dependent activity, is recommended to treat S. aureus orthopedic implant–associated infections. Rifampin, however, has limited bone penetration. Here, dynamic 11 C-rifampin positron emission tomography (PET) performed in prospectively enrolled patients with confirmed S. aureus bone infection ( n = 3) or without orthopedic infection ( n = 12) demonstrated bone/plasma area under the concentration-time curve ratio of 0.14 (interquartile range, 0.09 to 0.19), exposures lower than previously thought. PET-based pharmacokinetic modeling predicted rifampin concentration-time profiles in bone and facilitated studies in a mouse model of S. aureus orthopedic implant infection. Administration of high-dose rifampin (human equipotent to 35 mg/kg per day) substantially increased bone concentrations (2 mg/liter versus <0.2 mg/liter with standard dosing) in mice and achieved higher bacterial killing and biofilm disruption. Treatment for 4 weeks with high-dose rifampin and vancomycin was noninferior to the recommended 6-week treatment of standard-dose rifampin with vancomycin in mice (risk difference, −6.7% favoring high-dose rifampin regimen). High-dose rifampin treatment ameliorated antimicrobial resistance (0% versus 38%; P = 0.04) and mitigated adverse bone remodeling ( P < 0.01). Last, whole-genome sequencing demonstrated that administration of high-dose rifampin in mice reduced selection of bacterial mutations conferring rifampin resistance ( rpoB ) and mutations in genes potentially linked to persistence. These data suggest that administration of high-dose rifampin is necessary to achieve optimal bone concentrations, which could shorten and improve treatments for S. aureus orthopedic implant infections.

Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound-induced hair follicle neogenesis (WIHN) as a model of non-fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We previously demonstrated that TLR3 promotes WIHN through binding wound-associated dsRNA, the source of which is still unclear. Here, we find that multiple distinct contexts of high WIHN all show a strong neutrophil signature. Given the correlation between neutrophil infiltration and endogenous dsRNA release, we hypothesized that neutrophil extracellular traps (NETs) likely release nuclear spliceosomal U1 dsRNA and modulate WIHN. However, rather than enhance regeneration, we find mature neutrophils inhibit WIHN such that mice with mature neutrophil depletion exhibit higher WIHN. Similarly, Pad4 null mice, which are defective in NET production, show augmented WIHN. Finally, using single-cell RNA sequencing, we identify a dramatic increase in mature and activated neutrophils in the wound beds of low regenerating Tlr3-/- mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro-regenerative cue, their presence and NETs hinder regeneration., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Staphylococcus aureus causes most skin infections in humans, and the emergence of methicillin-resistant S. aureus (MRSA) strains is a serious public health threat. There is an urgent clinical need for nonantibiotic immunotherapies to treat MRSA infections and prevent the spread of antibiotic resistance. Here, we investigated the pan-caspase inhibitor quinoline-valine-aspartic acid-difluorophenoxymethyl ketone (Q-VD-OPH) for efficacy against MRSA skin infection in mice. A single systemic dose of Q-VD-OPH decreased skin lesion sizes and reduced bacterial burden compared with vehicle-treated or untreated mice. Although Q-VD-OPH inhibited inflammasome-dependent apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) speck formation and caspase-1-mediated interleukin-1β (IL-1β) production, Q-VD-OPH maintained efficacy in mice deficient in IL-1β, ASC, caspase-1, caspase-11, or gasdermin D. Thus, Q-VD-OPH efficacy was independent of inflammasome-mediated pyroptosis. Rather, Q-VD-OPH reduced apoptosis of monocytes and neutrophils. Moreover, Q-VD-OPH enhanced necroptosis of macrophages with concomitant increases in serum TNF and TNF-producing neutrophils, monocytes/macrophages, and neutrophils in the infected skin. Consistent with this, Q-VD-OPH lacked efficacy in mice deficient in TNF (with associated reduced neutrophil influx and necroptosis), in mice deficient in TNF/IL-1R and anti-TNF antibody-treated WT mice. In vitro studies revealed that combined caspase-3, caspase-8, and caspase-9 inhibition reduced apoptosis, and combined caspase-1, caspase-8, and caspase-11 inhibition increased TNF, suggesting a mechanism for Q-VD-OPH efficacy in vivo. Last, Q-VD-OPH also had a therapeutic effect against Streptococcus pyogenes and Pseudomonas aeruginosa skin infections in mice. Collectively, pan-caspase inhibition represents a potential host-directed immunotherapy against MRSA and other bacterial skin infections., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases.

Malaria infection starts with the injection of Plasmodium sporozoites into the host's skin. Sporozoites are motile and move in the skin to find and enter blood vessels to be carried to the liver. Here, we present the first characterization of P. falciparum sporozoites in vivo, analyzing their motility in mouse skin and human skin xenografts and comparing their motility to two rodent malaria species. These data suggest that in contrast to the liver and blood stages, the skin is not a species-specific barrier for Plasmodium. Indeed, P. falciparum sporozoites enter blood vessels in mouse skin at similar rates to the rodent malaria parasites. Furthermore, we demonstrate that antibodies targeting sporozoites significantly impact the motility of P. falciparum sporozoites in mouse skin. Though the sporozoite stage is a validated vaccine target, vaccine trials have been hampered by the lack of good animal models for human malaria parasites. Pre-clinical screening of next-generation vaccines would be significantly aided by the in vivo platform we describe here, expediting down-selection of candidates prior to human vaccine trials., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

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.

Atopic dermatitis (AD) is a common, chronic, inflammatory skin condition characterized by recurrent and pruritic skin eruptions. Multiple factors contribute to the pathogenesis of AD, including skin barrier dysfunction, microbial dysbiosis, and immune dysregulation. Interactions among these factors form a complex, multidirectional network that can reinforce atopic skin disease but can also be ameliorated by targeted therapies. This review summarizes the complex interactions among contributing factors in AD and the implications on disease development and therapeutic interventions., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Bacterial skin infections are a major societal health burden and are increasingly difficult to treat owing to the emergence of antibiotic-resistant strains such as community-acquired methicillin-resistant Staphylococcus aureus. Understanding the immunologic mechanisms that provide durable protection against skin infections has the potential to guide the development of immunotherapies and vaccines to engage the host immune response to combat these antibiotic-resistant strains. To this end, mouse skin infection models allow researchers to examine host immunity by investigating the timing, inoculum, route of infection and the causative bacterial species in different wild-type mouse backgrounds as well as in knockout, transgenic, and other types of genetically engineered mouse strains. To recapitulate the various types of human skin infections, many different mouse models have been developed. For example, four models frequently used in dermatological research are based on the route of infection, including (i) subcutaneous infection models, (ii) intradermal infection models, (iii) wound infection models, and (iv) epicutaneous infection models. In this article, we will describe these skin infection models in detail along with their advantages and limitations. In addition, we will discuss how humanized mouse models such as the human skin xenograft on immunocompromised mice might be used in bacterial skin infection research., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Psoriasis is an inflammatory skin disease with strong neutrophil (PMN) infiltration and high levels of the antimicrobial peptide, LL37. LL37 in complex with DNA and RNA is thought to initiate disease exacerbation via plasmacytoid dendritic cells. However, the source of nucleic acids supposed to start this initial inflammatory event remains unknown. We show here that primary murine and human PMNs mount a fulminant and self-propagating neutrophil extracellular trap (NET) and cytokine response, but independently of the canonical NET component, DNA. Unexpectedly, RNA, which is abundant in NETs and psoriatic but not healthy skin, in complex with LL37 triggered TLR8/TLR13-mediated cytokine and NET release by PMNs in vitro and in vivo. Transfer of NETs to naive human PMNs prompts additional NET release, promoting further inflammation. Our study thus uncovers a self-propagating vicious cycle contributing to chronic inflammation in psoriasis, and NET-associated RNA (naRNA) as a physiologically relevant NET component.

In vivo whole-animal optical (bioluminescence and fluorescence) imaging of Staphylococcus aureus infections has provided the opportunity to noninvasively and longitudinally monitor the dynamics of the bacterial burden and ensuing host immune responses in live anesthetized animals. Herein, we describe several different mouse models of S. aureus skin infection, skin inflammation, incisional/excisional wound infections, as well as mouse and rabbit models of orthopedic implant infection, which utilized this imaging technology. These animal models and imaging methodologies provide insights into the pathogenesis of these infections and innate and adaptive immune responses, as well as the preclinical evaluation of diagnostic and treatment modalities. Noninvasive approaches to investigate host-pathogen interactions are extremely important as virulent community-acquired methicillin-resistant S. aureus strains (CA-MRSA) are spreading through the normal human population, becoming more antibiotic resistant and creating a serious threat to public health.

In vivo bioluminescence imaging has been used to monitor Staphylococcus aureus infections in preclinical models by employing bacterial reporter strains possessing a modified lux operon from Photorhabdus luminescens. However, the relatively short emission wavelength of lux (peak 490 nm) has limited tissue penetration. To overcome this limitation, the gene for the click beetle (Pyrophorus plagiophtalamus) red luciferase (luc) (with a longer >600 emission wavelength), was introduced singly and in combination with the lux operon into a methicillin-resistant S. aureus strain. After administration of the substrate D-luciferin, the luc bioluminescent signal was substantially greater than the lux signal in vitro. The luc signal had enhanced tissue penetration and improved anatomical co-registration with infected internal organs compared with the lux signal in a mouse model of S. aureus bacteremia with a sensitivity of approximately 3 × 10 4 CFU from the kidneys. Finally, in an in vivo mixed bacterial wound infection mouse model, S. aureus luc signals could be spectrally unmixed from Pseudomonas aeruginosa lux signals to noninvasively monitor the bacterial burden of both strains. Therefore, the S. aureus luc reporter may provide a technological advance for monitoring invasive organ dissemination during S. aureus bacteremia and for studying bacterial dynamics during mixed infections.

Psoriasis is a frequent systemic inflammatory autoimmune disease characterized primarily by skin lesions with massive infiltration of leukocytes, but frequently also presents with cardiovascular comorbidities. Especially polymorphonuclear neutrophils (PMNs) abundantly infiltrate psoriatic skin but the cues that prompt PMNs to home to the skin are not well-defined. To identify PMN surface receptors that may explain PMN skin homing in psoriasis patients, we screened 332 surface antigens on primary human blood PMNs from healthy donors and psoriasis patients. We identified platelet surface antigens as a defining feature of psoriasis PMNs, due to a significantly increased aggregation of neutrophils and platelets in the blood of psoriasis patients. Similarly, in the imiquimod-induced experimental in vivo mouse model of psoriasis, disease induction promoted PMN-platelet aggregate formation. In psoriasis patients, disease incidence directly correlated with blood platelet counts and platelets were detected in direct contact with PMNs in psoriatic but not healthy skin. Importantly, depletion of circulating platelets in mice in vivo ameliorated disease severity significantly, indicating that both PMNs and platelets may be relevant for psoriasis pathology and disease severity.

How developmental programs reactivate in regeneration is a fundamental question in biology. We addressed this question through the study of Wound Induced Hair follicle Neogenesis (WIHN), an adult organogenesis model where stem cells regenerate de novo hair follicles following deep wounding. The exact mechanism is uncertain. Here we show that self-noncoding dsRNA activates the anti-viral receptor toll like receptor 3 (TLR3) to induce intrinsic retinoic acid (RA) synthesis in a pattern that predicts new hair follicle formation after wounding in mice. Additionally, in humans, rejuvenation lasers induce gene expression signatures for dsRNA and RA, with measurable increases in intrinsic RA synthesis. These results demonstrate a potent stimulus for RA synthesis by non-coding dsRNA, relevant to their broad functions in development and immunity.

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., Competing Interests: Conflict of interest statement: M.R.Y. is a cofounder of NovaDigm Therapeutics, which is developing novel vaccines and immunotherapeutics for infectious diseases, including S. aureus. L.S.M. has received grant support for work unrelated to the work reported in this manuscript from AstraZeneca, Pfizer, Regeneron Pharmaceuticals, Moderna Therapeutics, and Boehringer Ingelheim, is on the scientific advisory board for Integrated Biotherapeutics, and is a shareholder of Noveome Biotherapeutics, which are each developing vaccines and therapeutics against S. aureus and other pathogens.

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., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Background: In vivo bioluminescence imaging (BLI) provides noninvasive monitoring of bacterial burden in animal models of orthopaedic implant-associated infection (OIAI). However, technical limitations have limited its use to mouse and rat models of OIAI. The goal of this study was to develop a larger, rabbit model of OIAI using in vivo BLI to evaluate the efficacy of an antibiotic-releasing implant coating., Methods: A nanofiber coating loaded with or without linezolid-rifampin was electrospun onto a surgical-grade locking peg. To model OIAI in rabbits, a medial parapatellar arthrotomy was performed to ream the femoral canal, and a bright bioluminescent methicillin-resistant Staphylococcus aureus (MRSA) strain was inoculated into the canal, followed by retrograde insertion of the coated implant flush with the articular surface. In vivo BLI signals were confirmed by ex vivo colony-forming units (CFUs) from tissue, bone, and implant specimens., Results: In this rabbit model of OIAI (n = 6 rabbits per group), implants coated without antibiotics were associated with significantly increased knee width and in vivo BLI signals compared with implants coated with linezolid-rifampin (p < 0.001 and p < 0.05, respectively). On day 7, the implants without antibiotics were associated with significantly increased CFUs from tissue (mean [and standard error of the mean], 1.4 × 10 ± 2.1 × 10 CFUs; p < 0.001), bone (6.9 × 10 ± 3.1 × 10 CFUs; p < 0.05), and implant (5.1 × 10 ± 2.2 × 10 CFUs; p < 0.05) specimens compared with implants with linezolid-rifampin, which demonstrated no detectable CFUs from any source., Conclusions: By combining a bright bioluminescent MRSA strain with modified techniques, in vivo BLI in a rabbit model of OIAI demonstrated the efficacy of an antibiotic-releasing coating., Clinical Relevance: The new capability of in vivo BLI for noninvasive monitoring of bacterial burden in larger-animal models of OIAI may have important preclinical relevance.

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., (Copyright © 2018 by The American Association of Immunologists, Inc.)

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.

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., (Copyright © 2018 American Society for Microbiology.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Deletion of late cornified envelope (LCE) genes LCE3B and LCE3C (LCE3B/C-del) is a psoriasis risk factor linked to the major psoriasis risk gene HLA-C*06. Niehues et al. demonstrate that LCE3B/C-del leads to increased keratinocyte LCE3A expression. They also show that LCE3A/B/C possess antimicrobial activity but do not obviously regulate epidermal barrier integrity. These findings implicate LCE proteins in psoriasis pathogenesis via a new functional role., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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 S aureus hematogenous implant-related infections., Competing Interests: Conflict of interest statement: L.S.M. has received grant support from MedImmune, LLC for the work reported in this paper; grant support from Pfizer, Regeneron, and the Chan Soon-Shiong Nanthealth Foundation; and consulting fees from Noveome Biotherapeutics and the Chan Soon-Shiong Nanthealth Foundation that are unrelated to the work reported in this paper. L.I.C., T.S.C., C.T., C.K.S., and B.R.S. are associated with MedImmune, LLC, a subsidiary of AstraZeneca, and may hold AstraZeneca stock.

Approximately 20% of the population is persistently colonized by Staphylococcus aureus in the nares. Th17-like immune responses mediated by the interleukin-17 (IL-17) family of cytokines and neutrophils are becoming recognized as relevant host defense mechanisms for resolution of S. aureus mucocutaneous infections. Since antimicrobial peptides are regulated by the IL-17 cytokines, we sought to determine the role of IL-17 cytokines in production of antimicrobial peptides in a murine model of S. aureus nasal carriage. We discovered that nasal tissue supernatants have antistaphylococcal activity, and mice deficient in both IL-17A and IL-17F lost the ability to clear S. aureus nasal colonization. IL-17A was found to be sufficient for nasal mBD-3 production ex vivo and was required for CRAMP, mBD-3, and mBD-14 expression in response to S. aureus colonization in vivo These data were confirmed in a clinical study of nasal secretions in which elevated levels of the human forms of these antimicrobial peptides were found in nasal secretions from healthy human subjects when they were colonized with S. aureus but not in secretions from noncolonized subjects. Together, these data provide evidence for the importance of IL-17A regulation of antimicrobial peptides and IL-17F in the clearance of S. aureus nasal carriage., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Infections with Staphylococcus aureus are a continuing and growing problem in community and hospital settings. Preclinical animal modeling of S. aureus relies on experimental infection, which carries some limitations. We describe here a novel, spontaneous model of oral staphylococcal infection in double knockout mice, deficient in the receptors for IL-17 (IL-17RA) and interferon (IFN)-γ (IFNγRI), beginning at 6 to 8 weeks of age. IFNγRI(-/-)IL17RA(-/-) (GRAKO) mice developed progressive oral abscesses. Cytometric methods revealed extensive neutrophilic infiltration of oral tissues in GRAKO mice; further investigation evidenced that IL-17 predominated neutrophil defects in these mice. To investigate the contribution of IFN-γ signaling to this native host defense to S. aureus, we observed perturbations of monocyte recruitment and macrophage differentiation in the oral tissues of GRAKO mice, and CXCL9/chemokine ligand receptor (CXCR)3-driven recruitment of T-cell oral tissues and draining lymph nodes. To address the former finding, we depleted macrophages and monocytes in vivo from IL17RA(-/-) mice using liposomes loaded with clodronate. This treatment elicited oral abscesses, recapitulating the phenotype of GRAKO mice. From these findings, we propose novel collaborative functions of IL-17 and IFN-γ, acting through neutrophils and macrophages, respectively, in native mucocutaneous host defenses to S. aureus., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The anterior nares of humans are the major reservoir for Staphylococcus aureus colonization. Approximately 20% of the healthy human population is persistently and 80% is intermittently colonized with S. aureus in the nasal cavity. Previous studies have shown a strong causal connection between S. aureus nasal carriage and increased risk of nosocomial infection, as well as increased carriage due to immune dysfunction. However, the immune responses that permit persistence or mediate clearance of S. aureus on the nasal mucosa are fundamentally undefined. In this study, we developed a carriage model in C57BL/6J mice and showed that clearance begins 14 days postinoculation. In contrast, SCID mice that have a deficient adaptive immune response are unable to eliminate S. aureus even after 28 days postinoculation. Furthermore, decolonization was found to be T cell mediated but B cell independent by evaluating carriage clearance in T-cell receptor β/δ (TCR-β/δ) knockout (KO) and IgH-μ KO mice, respectively. Upregulation of the cytokines interleukin 1β (IL-1β), KC (also termed CXC ligand 1 [CXCL1]), and IL-17A occurred following inoculation with intranasal S. aureus. IL-17A production was crucial for clearance, since IL-17A-deficient mice were unable to effectively eliminate S. aureus carriage. Subsequently, cell differential counts were evaluated from nasal lavage fluid obtained from wild-type and IL-17A-deficient colonized mice. These counts displayed IL-17A-dependent neutrophil migration. Antibody-mediated depletion of neutrophils in colonized mice caused reduced clearance compared to that in isotype-treated controls. Our data suggest that the Th17-associated immune response is required for nasal decolonization. This response is T cell dependent and mediated via IL-17A production and neutrophil influx. Th17-associated immune responses may be targeted for strategies to mitigate distal infections originating from persistent S. aureus carriage in humans.