Clarifying the Critical Factors for Th1 and Th17 Pathogenicity in an Animal Model of CNS-Targeted Autoimmune Disease.

Experimental autoimmune encephalitomyelitis (EAE) is a CD4+ T cell-mediated CNS-targeted autoimmune disease and a model of multiple sclerosis (MS). IL-12-polarized IFN-γ-producing Th1 cells and IL-23-polarized IL-17-producing Th17 cells have been implicated in EAE and MS pathogenesis. However, current dogma states that IL-23, and not IL-12, is absolutely critical for T cell encephalitogenicity. Furthermore, few reports measuring Th1 or Th17 cells in EAE have considered Th17 cell plasticity. IL-23-polarized Th17 cells can downregulate IL-17 and upregulate IFN-γ, which makes them indistinguishable from Th1 cells. This conversion to an “exTh17” is T-bet-dependent and promoted by IL-23. Though we have previously demonstrated that IL-12- or IL-23-polarized T cells can each induce EAE via distinct mechanisms, the contribution of IL-23 to IL-12-polarized disease, and vice versa, is unexamined. Therefore, we questioned whether IFN-γ-producing CD4+ T cells found during MS and EAE are actually IL-23-driven exTh17 cells and whether bona fide Th1 or stable Th17 cells are encephalitogenic independently of exTh17 cells. We also questioned whether distinctions seen between IL-12- and IL-23-mediated EAE could be found in MS patients. Here, we used adoptive transfer models of EAE to demonstrate that IL-12-polarized Th1 cell encephalitogenicity can be IL-23-independent. IL-23-independent Th1-mediated disease and IL-12-independent Th17-mediated disease had distinct cellular infiltration patterns and cytokine and chemokine expression profiles. Furthermore, we saw distinct cytokine and chemokine profiles in MS patients grouped by relative IL-12 and IL-23 expression. We also investigated the contribution of plasticity to Th17 pathogenicity. We demonstrated that IL-23-polarized T-bet-/- cells were stable Th17 cells. They induced EAE following adoptive transfer into wild-type and RAG2-/- hosts, though disease was milder and delayed relative to wild type Th17 cells. We also determined that the reduced potency of stable Th17 cells is not a result of poor proliferation or survival, rather due to altered trafficking molecules on stable Th17 cells. These data contribute to the understanding to the critical factors for CD4+ T cell encephalitogenicity, and suggest that Th1, Th17, and exTh17 cells are distinct effector lineages in EAE. These data have translational implications, which could result in the discovery of biomarkers in MS patient populations and targeted therapies.PHDImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108991/1/hgrifka_1.pd

IL‐12‐polarized Th1 cells produce GM‐CSF and induce EAE independent of IL‐23

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/115966/1/eji3410.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/115966/2/eji3410-sup-0002-PRC.pd

USRN Discovery Pilot: Increasing the Discoverability of Open Access Content Through a National Network

This paper presents the results of the USRN Discovery Pilot Project, a collaboration of SPARC, the Confederation of Open Access Repositories (COAR), CORE and Antleaf, to enhance the discoverability of research papers in US repositories leveraging CORE as an indexing service for USRN repositories. The project conducted actions in three strategic areas; - Assessing and quantitatively measuring discoverability and barriers to it at the beginning and end of the pilot project, - conducting interventions to increase discoverability, and - supporting interventions by technology and guidelines (provided by CORE services), to minimise effort and maximise effect. The key results of the project include: - Around three-quarters of a million research outputs held in the selected US repositories have been made discoverable (a 50% increase) compared to the year before; - The project has made available the CORE Data Provider’s Guide as well as a selection of new and improved tools to support repositories in increasing their discoverability. These include the CORE Reindexing Button and Index Notification modules, Fresh Finds and the USRN Desirable Characteristics for Digital Publication Repositories checking tool. The project team is now exploring ways to scale out this work to include more repositories

458 Influence of a Gastrointestinal Infection on Lung Immunity

OBJECTIVES/GOALS: We aim to characterize how Heligmosomoides polygyrus bakeri (H. poly) alleviates murine allergic asthma which shares many characteristics of human asthma. This approach of has already identified helminth-produced human immune cell ligand “mimics” that hold great potential for next-generation clinical biologics METHODS/STUDY POPULATION: We examined the lung tissue of C57BL/6 mice infected with H. poly for changes in the pulmonary microenvironment. At ten days post infection, four infected mice and two co-housed uninfected mice were sacrificed, and their lung tissue harvested for examination of RNA via RT-qPCR. This design allows for the comparison between the lung microenvironments of infected and naīve mice. In future experiments, we intend to characterize what small molecules produced by the helminth drive changes in the lung using germ-free models of H. poly infection. RESULTS/ANTICIPATED RESULTS: We found key differences in lung chemokines between mice infected with H. poly and naīve mice. Using a student t-test with naīve correction for variance, we were able to show significant differences in the expression of E cadherin (p = 0.0355), CXCL10 (p = 0.0025), CX3CL1 (p = 0.0029), CCR2 (p = 0.017), and IDO1 (0.0078). We also found that differences in the expression of CCL5 bordered on significant with a p-value of 0.066. The expression of most of these markers (CXCL10, CCR2, CCL5, and IDO1) was elevated in the lungs of infected mice compared to naīve controls. In contrast, E cadherin and CX3CL1 showed the opposite trend with naīve mice showing greater expression. These clear differences in lung tissue gene expression underscore the connection between the gastrointestinal and pulmonary mucosal immune compartments. DISCUSSION/SIGNIFICANCE: The changes are unexpected for an infection that has been shown to attenuate allergic inflammation in the lung with increases in the IFN-Y responsive genes IDO1 and CXCL10 and inflammatory lung markers, CCL5 and CCR2. In contrast, there were decreases in inflammatory lung marker CX3CL1 and the tight junction protein E cadherin in infected mice

Amino Acid Trp: The Far Out Impacts of Host and Commensal Tryptophan Metabolism

Tryptophan (Trp) is an essential amino acid primarily derived from the diet for use by the host for protein synthesis. The intestinal tract is lined with cells, both host and microbial, that uptake and metabolize Trp to also generate important signaling molecules. Serotonin (5-HT), kynurenine and its downstream metabolites, and to a lesser extent other neurotransmitters are generated by the host to signal onto host receptors and elicit physiological effects. 5-HT production by neurons in the CNS regulates sleep, mood, and appetite; 5-HT production in the intestinal tract by enterochromaffin cells regulates gastric motility and inflammation in the periphery. Kynurenine can signal onto the aryl hydrocarbon receptor (AHR) to elicit pleiotropic responses from several cell types including epithelial and immune cells, or can be further metabolized into bioactive molecules to influence neurodegenerative disease. There is a remarkable amount of cross-talk with the microbiome with regard to tryptophan metabolites as well. The gut microbiome can regulate the production of host tryptophan metabolites and can use dietary or recycled trp to generate bioactive metabolites themselves. Trp derivatives like indole are able to signal onto xenobiotic receptors, including AHR, to elicit tolerogenic effects. Here, we review studies that demonstrate that tryptophan represents a key intra-kingdom signaling molecule.</jats:p

A simplified protocol for deriving sterile, infectious murine Heligmosomoides polygyrus bakeri larvae

Summary: Gastrointestinal helminth infection occurs within a diverse microbiome, complicating the interpretation of whether effects are caused by the parasite versus the microbial community. Here, we present a protocol for deriving sterile larvae of the murine helminth, Heligmosomoides polygyrus bakeri (H. polygyrus), providing experimental control of the microbiome. We describe steps for sterilizing with a bleach solution and developing into infectious larvae using E. coli. We then detail procedures for removing bacterial contaminants before harvesting to ensure the generation of germ-free larvae. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Discovery of a protective, transmissible, and type 2 inflammation-skewing intestinal microbiome

Abstract We recently discovered a transmissible, dominant, and remarkably protective intestinal microbiome (a “Magical Microbiome,” MM) in the setting of a murine model of colitis. The objective of this project is to fill current knowledge gaps regarding the molecular mechanisms underlying protection in this novel model of microbiome-mediated protection. Protection can be transmitted to unmanipulated conventional or germ-free C57Bl/6 mice via cohousing or oral gavage of MM+ stool. During DSS-induced colitis, MM+ mice develop less weight loss, intestinal pathology, and production of proinflammatory molecules relative to genetically identical C57Bl/6 MM-controls. Preliminary results suggest that the eukaryotic and bacterial microbiome under investigation re-programs the host inflammation to prevent disease or promote repair. MM-mediated protection is IL-13-dependent and associated with enhanced type 2 inflammation, as transcript levels of type 2-associated genes including IL-4, IL-13, and Fizz1 were significantly higher in colons of MM+ mice. 16s rRNA gene sequencing and eukaryote screening demonstrated that MM+ mice have a bacterial microbiome distinct from controls and host a novel Tritrichomonas species. Transfer of Tritrichomonas to wild-type mice correlated with protection, but vancomycin treatment ameliorates the protection while maintaining Tritrichomonas, suggesting a possible interaction between bacteria and protist. Stool metabolites are also distinct between MM+ and MM− hosts, further supporting a distinct microbiome function. Overall, we possess a unique microbiome that can be used to understand protective host-microbe interactions and identify novel therapeutic targets for inflammatory diseases.</jats:p

Slug and Snail have differential effects in directing colonic epithelial wound healing and partially mediate the restitutive effects of butyrate

Restitution of wounds in colonic epithelium is essential in the maintenance of health. Microbial products, such as the short-chain fatty acid butyrate, can have positive effects on wound healing. We used an in vitro model of T84 colonic epithelial cells to determine if the Snail genes Slug ( SNAI2) and Snail ( SNAI1), implemented in keratinocyte monolayer healing, are involved in butyrate-enhanced colonic epithelial wound healing. Using shRNA-mediated Slug/Snail knockdown, we found that knockdown of Slug (Slug-KD), but not Snail (Snail-KD), impairs wound healing in scratch assays with and without butyrate. Slug and Snail had differential effects on T84 monolayer barrier integrity, measured by transepithelial resistance, as Snail-KD impaired the barrier (with or without butyrate), whereas Slug-KD enhanced the barrier, again with or without butyrate. Targeted transcriptional analysis demonstrated differential expression of several tight junction genes, as well as focal adhesion genes. This included altered regulation of Annexin A2 and ITGB1 in Slug-KD, which was reflected in confocal microscopy, showing increased accumulation of B1-integrin protein in Slug-KD cells, which was previously shown to impair wound healing. Transcriptional analysis also indicated altered expression of genes associated with epithelial terminal differentiation, such that Slug-KD cells skewed toward overexpression of secretory cell pathway-associated genes. This included trefoil factors TFF1 and TFF3, which were expressed at lower than control levels in Snail-KD cells. Since TFFs can enhance the barrier in epithelial cells, this points to a potential mechanism of differential modulation by Snail genes. Although Snail genes are crucial in epithelial wound restitution, butyrate responses are mediated by other pathways as well.NEW &amp; NOTEWORTHY Although butyrate can promote colonic mucosal healing, not all of its downstream pathways are understood. We show that the Snail genes Snail and Slug are mediators of butyrate responses. Furthermore, these genes, and Slug in particular, are necessary for efficient restitution of wounds and barriers in T84 epithelial cells even in the absence of butyrate. These effects are achieved in part through effects on regulation of β1 integrin and cellular differentiation state.</jats:p