Project description:Objectives: To define the inflammatory signature of healthy keratinocytes induced by gram-negative anaerobe bacteria commonly found in HS and investigate pathways of activation. Methods: Type strains of P. nigrescens, P. melanogenica, P. intermedia, P. asaccharolytica, F. nucleatum, as well as S. aureus and the normal skin commensal S. epidermidis were heat-killed and co-incubated with normal human keratinocytes. RNA was analyzed using RNAseq and RT-qPCR. TLR4 and JAK inhibitors were used to investigate mRNA and protein inhibition of inflammatory cytokines. Results: All GNAs tested induced significantly higher levels of CXCL8 than S. epidermidis or negative control. P. nigrescens, P. melanogenica, and F. nucleatum were the most stimulatory and produced CXCL8 levels much higher than S. aureus. RNASeq revealed a broad inflammatory profile most strongly induced by F. nucleatum. All three bacteria strongly activated the IL-17 pathway and correlated with HS skin transcriptomes. Both TLR4 and JAK inhibition significantly reduced keratinocyte inflammation. Results: All GNAs tested induced significantly higher levels of CXCL8 than S. epidermidis or negative control. P. nigrescens, P. melanogenica, and F. nucleatum were the most stimulatory and produced CXCL8 levels much higher than S. aureus. RNASeq revealed a broad inflammatory profile most strongly induced by F. nucleatum. All three bacteria strongly activated the IL-17 pathway and correlated with HS skin transcriptomes.
Project description:Type 1 diabetes (T1D) is a chronic autoimmune disease that results from destruction of pancreatic β-cells. T1D subjects were recently shown to harbor distinct intestinal microbiome profiles. Based on these findings, the role of gut bacteria in T1D is being intensively investigated. The mechanism connecting intestinal microbial homeostasis with the development of T1D is unknown. Specific gut bacteria such as Bacteroides dorei (BD) and Ruminococcus gnavus (RG) show markedly increased abundance prior to the development of autoimmunity. One hypothesis is that these bacteria might traverse the damaged gut barrier, and their constituents elicit a response from human islets that causes metabolic abnormalities and inflammation. We have tested this hypothesis by exposing human islets to BD and RG in vitro, after which RNA-Seq analysis was performed. The bacteria altered expression of many islet genes. The commonly upregulated genes by these bacteria were cytokines, chemokines and enzymes, suggesting a significant effect of gut bacteria on islet antimicrobial and biosynthetic pathways. Additionally, each bacteria displayed a unique set of differentially expressed genes (DEGs). Ingenuity pathway analysis of DEGs revealed that top activated pathways and diseases included TREM1 Signaling and Inflammatory Response, illustrating the ability of bacteria to induce islet inflammation. The increased levels of selected factors were confirmed using immunoblotting and ELISA methods. Our data demonstrate that islets produce a complex anti-bacterial response. The response includes both symbiotic and pathogenic aspects. Both oxidative damage and leukocyte recruitment factors were prominent, which could induce beta cell damage and subsequent autoimmunity.