Age-Related Loss of Innate Immune Antimicrobial Function of Dermal Fat Is Mediated by Transforming Growth Factor Beta.

Dermal fibroblasts (dFBs) resist infection by locally differentiating into adipocytes and producing cathelicidin antimicrobial peptide in response to Staphylococcus aureus (S. aureus). Here, we show that neonatal skin was enriched with adipogenic dFBs and immature dermal fat that highly expressed cathelicidin. The pool of adipogenic and antimicrobial dFBs declined after birth, leading to an age-dependent loss of dermal fat and a decrease in adipogenesis and cathelidicin production in response to infection. Transforming growth factor beta (TGF-ß), which acted on uncommitted embryonic and adult dFBs and inhibited their adipogenic and antimicrobial function, was identified as a key upstream regulator of this process. Furthermore, inhibition of the TGF-ß receptor restored the adipogenic and antimicrobial function of dFBs in culture and increased resistance of adult mice to S. aureus infection. These results provide insight into changes that occur in the skin innate immune system between the perinatal and adult periods of life.

Retinoids Enhance the Expression of Cathelicidin Antimicrobial Peptide during Reactive Dermal Adipogenesis.

A subset of dermal fibroblasts undergo rapid differentiation into adipocytes in response to infection and acutely produce the cathelicidin antimicrobial peptide gene Camp Vitamin A and other retinoids inhibit adipogenesis yet can show benefit to skin disorders, such as cystic acne, that are exacerbated by bacteria. We observed that retinoids potently increase and sustain the expression of Camp in preadipocytes undergoing adipogenesis despite inhibition of markers of adipogenesis, such as Adipoq, Fabp4, and Rstn Retinoids increase cathelicidin in both mouse and human preadipocytes, but this enhancement of antimicrobial peptide expression did not occur in keratinocytes or a sebocyte cell line. Preadipocytes undergoing adipogenesis more effectively inhibited growth of Staphylococcus aureus when exposed to retinoic acid. Whole transcriptome analysis identified hypoxia-inducible factor 1-α (HIF-1α) as a mechanism through which retinoids mediate this response. These observations uncouple the lipid accumulation element of adipogenesis from the innate immune response and uncover a mechanism, to our knowledge previously unsuspected, that may explain therapeutic benefits of retinoids in some skin disorders.

PIKfyve regulates melanosome biogenesis.

PIKfyve, VAC14, and FIG4 form a complex that catalyzes the production of PI(3,5)P2, a signaling lipid implicated in process ranging from lysosome maturation to neurodegeneration. While previous studies have identified VAC14 and FIG4 mutations that lead to both neurodegeneration and coat color defects, how PIKfyve regulates melanogenesis is unknown. In this study, we sought to better understand the role of PIKfyve in melanosome biogenesis. Melanocyte-specific PIKfyve knockout mice exhibit greying of the mouse coat and the accumulation of single membrane vesicle structures in melanocytes resembling multivesicular endosomes. PIKfyve inhibition blocks melanosome maturation, the processing of the melanosome protein PMEL, and the trafficking of the melanosome protein TYRP1. Taken together, these studies identify a novel role for PIKfyve in controlling the delivery of proteins from the endosomal compartment to the melanosome, a role that is distinct from the role of PIKfyve in the reformation of lysosomes from endolysosomes.

Comparison of sporulation and germination conditions for Clostridium perfringens type A and G strains.

Clostridium perfringens is a spore forming, anaerobic, Gram-positive bacterium that causes a range of diseases in humans and animals. C. perfringens forms spores, structures that are derived from the vegetative cell under conditions of nutrient deprivation and that allows survival under harsh environmental conditions. To return to vegetative growth, C. perfringens spores must germinate when conditions are favorable. Previous work in analyzing C. perfringens spore germination has produced strain-specific results. Hence, we analyzed the requirements for spore formation and germination in seven different C. perfringens strains. Our data showed that C. perfringens sporulation conditions are strain-specific, but germination responses are homogenous in all strains tested. C. perfringens spores can germinate using two distinct pathways. The first germination pathway (the amino acid-only pathway or AA) requires L-alanine, L-phenylalanine, and sodium ions (Na+) as co-germinants. L-arginine is not a required germinant but potentiates germination. The AA pathway is inhibited by aromatic amino acids and potassium ions (K+). Bicarbonate (HCO3-), on the other hand, bypasses potassium-mediated inhibition of C. perfringens spore germination through the AA pathway. The second germination pathway (the bile salt / amino acid pathway or BA) is more promiscuous and is activated by several bile salts and amino acids. In contrast to the AA pathway, the BA pathway is insensitive to Na+, although it can be activated by either K+ or HCO3-. We hypothesize that some C. perfringens strains may have evolved these two distinct germination pathways to ensure spore response to different host environments.

Antimicrobial production by perifollicular dermal preadipocytes is essential to the pathophysiology of acne.

Innate immune defense against deep tissue infection by Staphylococcus aureus is orchestrated by fibroblasts that become antimicrobial when triggered to differentiate into adipocytes. However, the role of this process in noninfectious human diseases is unknown. To investigate the potential role of adipogenesis by dermal fibroblasts in acne, a disorder triggered by Cutibacterium acnes, single-cell RNA sequencing was performed on human acne lesions and mouse skin challenged by C. acnes. A transcriptome consistent with adipogenesis was observed within specific fibroblast subsets from human acne and mouse skin lesions infected with C. acnes. Perifollicular dermal preadipocytes in human acne and mouse skin lesions showed colocalization of PREF1, an early marker of adipogenesis, and cathelicidin (Camp), an antimicrobial peptide. This capacity of C. acnes to specifically trigger production of cathelicidin in preadipocytes was dependent on TLR2. Treatment of wild-type mice with retinoic acid (RA) suppressed the capacity of C. acnes to form acne-like lesions, inhibited adipogenesis, and enhanced cathelicidin expression in preadipocytes, but lesions were unresponsive in Camp-/- mice, despite the anti-adipogenic action of RA. Analysis of inflamed skin of acne patients after retinoid treatment also showed enhanced induction of cathelicidin, a previously unknown beneficial effect of retinoids in difficult-to-treat acne. Overall, these data provide evidence that adipogenic fibroblasts are a critical component of the pathogenesis of acne and represent a potential target for therapy.

Progesterone analogs influence germination of Clostridium sordellii and Clostridium difficile spores in vitro.

Clostridium sordellii and Clostridium difficile are closely related anaerobic Gram-positive, spore-forming human pathogens. C. sordellii and C. difficile form spores that are believed to be the infectious form of these bacteria. These spores return to toxin-producing vegetative cells upon binding to small molecule germinants. The endogenous compounds that regulate clostridial spore germination are not fully understood. While C. sordellii spores require three structurally distinct amino acids to germinate, the occurrence of postpregnancy C. sordellii infections suggests that steroidal sex hormones might regulate its capacity to germinate. On the other hand, C. difficile spores require taurocholate (a bile salt) and glycine (an amino acid) to germinate. Bile salts and steroid hormones are biosynthesized from cholesterol, suggesting that the common sterane structure can affect the germination of both C. sordellii and C. difficile spores. Therefore, we tested the effect of sterane compounds on C. sordellii and C. difficile spore germination. Our results show that both steroid hormones and bile salts are able to increase C. sordellii spore germination rates. In contrast, a subset of steroid hormones acted as competitive inhibitors of C. difficile spore germination. Thus, even though C. sordellii and C. difficile are phylogenetically related, the two species' spores respond differently to steroidal compounds.

Cutaneous innate immune tolerance is mediated by epigenetic control of MAP2K3 by HDAC8/9.

The skin typically tolerates exposure to various microbes and chemicals in the environment. Here, we investigated how the epidermis maintains this innate immune tolerance to stimuli that are recognized by Toll-like receptors (TLRs). Loss of tolerance to TLR ligands occurred after silencing of the histone deacetylases (HDACs) HDAC8 and HDAC9 in keratinocytes. Transcriptional analysis identified MAP2K3 as suppressed by HDAC8/9 activity and a potential key intermediary for establishing this tolerance. HDAC8/9 influenced acetylation at H3K9 and H3K27 marks in the MAP2K3 promoter. Proteomic analysis further identified SSRP1 and SUPT16H as associated with HDAC8/9 and responsible for transcriptional elongation of MAP2K3. Silencing of MAP2K3 blocked the capacity of HDAC8/9 to influence cytokine responses. Relevance in vivo was supported by observations of increased MAP2K3 in human inflammatory skin conditions and the capacity of keratinocyte HDAC8/9 to influence dendritic cell maturation and T cell proliferation. Keratinocyte-specific deletion of HDAC8/9 also increased inflammation in mice after exposure to ultraviolet radiation, imiquimod, or Staphylococcus aureus These findings define a mechanism for the epidermis to regulate inflammation in the presence of ubiquitous TLR ligands.

Skin inflammation activates intestinal stromal fibroblasts and promotes colitis.

Inflammatory disorders of the skin are frequently associated with inflammatory bowel diseases (IBDs). To explore mechanisms by which these organs communicate, we performed single-cell RNA-Seq analysis on fibroblasts from humans and mice with IBD. This analysis revealed that intestinal inflammation promoted differentiation of a subset of intestinal stromal fibroblasts into preadipocytes with innate antimicrobial host defense activity. Furthermore, this process of reactive adipogenesis was exacerbated if mouse skin was inflamed as a result of skin wounding or infection. Since hyaluronan (HA) catabolism is activated during skin injury and fibroblast-to-adipocyte differentiation is dependent on HA, we tested the hypothesis that HA fragments could alter colon fibroblast function by targeted expression of human hyaluronidase-1 in basal keratinocytes from mouse skin. Hyaluronidase expression in the skin activated intestinal stromal fibroblasts, altered the fecal microbiome, and promoted excessive reactive adipogenesis and increased inflammation in the colon after challenge with dextran sodium sulfate. The response to digested HA was dependent on expression of TLR4 by preadipocytes. Collectively, these results suggest that the association between skin inflammation and IBD may be due to recognition by mesenchymal fibroblasts in the colon of HA released during inflammation of the skin.

Kinetic evidence for the presence of putative germination receptors in Clostridium difficile spores.

Clostridium difficile is a spore-forming bacterium that causes Clostridium difficile-associated disease (CDAD). Intestinal microflora keeps C. difficile in the spore state and prevents colonization. Following antimicrobial treatment, the microflora is disrupted, and C. difficile spores germinate in the intestines. The resulting vegetative cells are believed to fill empty niches left by the depleted microbial community and establish infection. Thus, germination of C. difficile spores is the first required step in CDAD. Interestingly, C. difficile genes encode most known spore-specific protein necessary for germination, except for germination (Ger) receptors. Even though C. difficile Ger receptors have not been identified, taurocholate (a bile salt) and glycine (an amino acid) have been shown to be required for spore germination. Furthermore, chenodeoxycholate, another bile salt, can inhibit taurocholate-induced C. difficile spore germination. In the present study, we examined C. difficile spore germination kinetics to determine whether taurocholate acts as a specific germinant that activates unknown germination receptors or acts nonspecifically by disrupting spores' membranes. Kinetic analysis of C. difficile spore germination suggested the presence of distinct receptors for taurocholate and glycine. Furthermore, taurocholate, glycine, and chenodeoxycholate seem to bind to C. difficile spores through a complex mechanism, where both receptor homo- and heterocomplexes are formed. The kinetic data also point to an ordered sequential progression of binding where taurocholate must be recognized first before detection of glycine can take place. Finally, comparing calculated kinetic parameters with intestinal concentrations of the two germinants suggests a mechanism for the preferential germination of C. difficile spores in antibiotic-treated individuals.

Quorum sensing between bacterial species on the skin protects against epidermal injury in atopic dermatitis.

Colonization of the skin by Staphylococcus aureus is associated with exacerbation of atopic dermatitis (AD), but any direct mechanism through which dysbiosis of the skin microbiome may influence the development of AD is unknown. Here, we show that proteases and phenol-soluble modulin α (PSMα) secreted by S. aureus lead to endogenous epidermal proteolysis and skin barrier damage that promoted inflammation in mice. We further show that clinical isolates of different coagulase-negative staphylococci (CoNS) species residing on normal skin produced autoinducing peptides that inhibited the S. aureus agr system, in turn decreasing PSMα expression. These autoinducing peptides from skin microbiome CoNS species potently suppressed PSMα expression in S. aureus isolates from subjects with AD without inhibiting S. aureus growth. Metagenomic analysis of the AD skin microbiome revealed that the increase in the relative abundance of S. aureus in patients with active AD correlated with a lower CoNS autoinducing peptides to S. aureus ratio, thus overcoming the peptides' capacity to inhibit the S. aureus agr system. Characterization of a S. hominis clinical isolate identified an autoinducing peptide (SYNVCGGYF) as a highly potent inhibitor of S. aureus agr activity, capable of preventing S. aureus-mediated epithelial damage and inflammation on murine skin. Together, these findings show how members of the normal human skin microbiome can contribute to epithelial barrier homeostasis by using quorum sensing to inhibit S. aureus toxin production.