Highlights d Germ-free mice have abnormal skin barrier structure and function d Keratinocyte aryl hydrocarbon receptor (AHR) mediates barrier function and repair d Human skin commensal microbes activate keratinocyte AHR d Targeting the microbiota-AHR axis promotes skin barrier repair in disease models

Following mycobacterial entry into macrophages the ESX-1 type VII secretion system promotes phagosomal permeabilization and type I IFN production, key features of tuberculosis pathogenesis. The current model states that the secreted substrate ESAT-6 is required for membrane permeabilization and that a subsequent passive leakage of extracellular bacterial DNA into the host cell cytosol is sensed by the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) pathway to induce type I IFN production. We employed a collection of Mycobacterium marinum ESX-1 transposon mutants in a macrophage infection model and show that permeabilization of the phagosomal membrane does not require ESAT-6 secretion. Moreover, loss of membrane integrity is insufficient to induce type I IFN production. Instead, type I IFN production requires intact ESX-1 function and correlates with release of mitochondrial and nuclear host DNA into the cytosol, indicating that ESX-1 affects host membrane integrity and DNA release via genetically separable mechanisms. These results suggest a revised model for major aspects of ESX-1–mediated host interactions and put focus on elucidating the mechanisms by which ESX-1 permeabilizes host membranes and induces the type I IFN response, questions of importance for our basic understanding of mycobacterial pathogenesis and innate immune sensing.

Leishmania braziliensisinfection results in inflammation and skin injury, with highly variable and unpredictable clinical outcomes. Here, we investigated the potential impact of microbiota on infection-induced inflammatory responses and disease resolution by conducting an integrated analysis of the skin microbiome and host transcriptome on a cohort of 62L. braziliensis-infected patients. We found that overall bacterial burden and microbiome configurations dominated withStaphylococcus spp.were associated with delayed healing and enhanced inflammatory responses, especially by IL-1 family members. Dual RNA-seq of human lesions revealed that high lesionalS. aureustranscript abundance was associated with delayed healing and increased expression of IL-1β. This cytokine was critical for modulating disease outcome inL. braziliensis-infected mice colonized withS. aureus, as its neutralization reduced pathology and inflammation. These results implicate the microbiome in cutaneous leishmaniasis disease outcomes in humans and suggest host-directed therapies to mitigate the inflammatory consequences.

SUMMARYThe epidermis forms a barrier that defends the body from desiccation and entry of harmful substances, while sensing and integrating environmental signals. The tightly orchestrated cellular changes required for the proper formation and maintenance of this epidermal barrier occur in the context of the skin microbiome. Using germ free mice, we demonstrate the microbiota is necessary for proper differentiation and repair of the epidermal barrier. These effects were mediated by the aryl hydrocarbon receptor (AHR) in keratinocytes, a xenobiotic receptor also implicated in epidermal differentiation. Murine skin lacking keratinocyte AHR was more susceptible to barrier damage and infection, during steady state and epicutaneous sensitization. Colonization with a defined consortium of human skin isolates restored barrier competence in an AHR-dependent manner. We reveal a fundamental mechanism whereby the microbiota regulates skin barrier formation and repair, with far-reaching implications for the numerous skin disorders characterized by epidermal barrier dysfunction.

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Cutaneous leishmaniasis (CL) is a protozoal infection in which the immune response plays a major role in lesion development. The activation of local responses such as cytolysis by CD8+ T cells and release of IL-1β by myeloid cells is associated with poor clinical outcome in patients. We found that lesions from cutaneous leishmaniasis patients also exhibit a bacterial dysbiosis, but whether this dysbiosis affects immune responses and consequently impacts chemotherapy to heal the patients is unknown. We carried out an integrative multi-omics analysis of 64 patients, including RNA-seq and 16S-seq from lesion biopsies and clinical metadata. Staphylococcus was the dominant overrepresented genus in lesions from most patients, so we generated an in-house S. aureus pangenome to quantify transcript abundances through dual RNA-seq mapping analysis. Increased overall bacterial burden assessed by qPCR and the presence of S. aureus assessed by dual RNA-seq indicated enrichment for cytokines (e.g., IL1B), inflammatory responses (e.g., PTGS2), and chemotaxis (e.g., CXCL8) by Gene Ontology analysis. Of particular importance, we found that lesions with increased S. aureus transcript abundances exhibited high expression of inflammatory-related genes that have been associated with treatment failure, such as PRF1, GNLY, and GZMB. Correspondingly, we found that patients with a Staphylococcus spp. dysbiosis exhibited a delay in resolving their lesions following treatment. These results indicate that the microbiome influences the local immune responses in CL, affecting how patients respond to therapy by delaying the healing time. These results suggest that the use of antibiotics combined with chemotherapy may improve treatment outcome for CL. Supported by grant from NIH (R01 AI149456)

Diabetic foot ulcers (DFU) are a serious complication of diabetes mellitus that burden patients and health care systems. Staphylococcus aureus is prevalent and abundant in the DFU microbiome, and strain-level differences in S. aureus may drive clinical outcomes. To identify mechanisms underlying strain-specific outcomes in DFU with S. aureus, we performed high-throughput phenotyping screens on a collection of 221 S. aureus cultured isolates from clinically uninfected DFU. Of the 4 phenotypes examined (in vitro biofilm formation and production of staphylokinase, staphyloxanthin, and siderophores), we discovered that isolates from non-healing wounds produced more staphyloxanthin, a carotenoid cell membrane pigment. In a murine diabetic wound healing model, staphyloxanthin-producing isolates delayed wound closure significantly compared to staphyloxanthin-deficient isolates. Staphyloxanthin promoted resistance to oxidative stress in vitro and enhanced bacterial survival in human neutrophils. Comparative genomic and transcriptomic analysis of genetically similar clinical isolates with disparate staphyloxanthin phenotypes revealed a mutation in the Sigma B regulatory pathway that resulted in marked differences in stress response gene expression. Our findings suggest that staphyloxanthin production delays wound healing by protecting S. aureus from neutrophil-mediated oxidative stress, and may provide a target for therapeutic intervention in S. aureus-positive wounds.

Chronic wounds pose a major challenge on the health care system, with rising costs and associated morbidities. Better understanding of chronic wound biology and the advancement of technology have led to the use of an array of wound care therapies, including the off-label application of topical timolol for the healing of chronic wounds. Timolol is a non-selective, beta1/beta2 adrenergic receptor antagonist well established for the treatment of glaucoma. Clinical experience has broadened its use for a number of dermatologic indications such as infantile hemangiomas and recently chronic wounds. Although safety systemic absorption studies have been performed for the ocular administration, to date no studies have documented absorption of timolol post application to chronic wounds. Thus, a single center, open label, prospective, observational, cross-sectional comparative study was conducted to determine the blood plasma levels of timolol in patients after topical administration to a chronic wound, and compare to those after ocular topical administration of the same drug formulation for the indication of glaucoma. A total of 40 patients enrolled in the study that met inclusion criteria, 20 in each group diagnosed with either a chronic wound or glaucoma. The findings showed that there was no statistical significance in the average plasma level of timolol between the 2 groups. There were no cardiac or respiratory adverse events reported. Therefore, topical timolol for the use in chronic wounds may be safely applied in patients who have no contraindications, as an adjunctive therapy for chronic wounds with little potential for cardiac effects of systemic beta adrenergic receptor blockade.