The role of intestinal mononuclear phagocytes in control of mucosal T cell homeostasis

The intestine is constantly exposed to a wide variety of dietary antigens, commensal bacteria and pathogens, toward which it has evolved complex immune responses to protect the host. The intestinal immune system relies on innate immune cells, such as mononuclear phagocytes (MNPs), that include dendritic cells (DCs), monocytes (Mo) and macrophages (Mfs), to sense and respond to luminal and mucosal challenges. MNPs are essential players as they instruct adaptive immune cells, in particular T cells, to discriminate between innocuous and harmful antigens. Generation of different CD4 T cell responses to commensal and pathogenic bacteria is crucial for maintaining a healthy gut environment, but the associated cellular mechanisms are poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by epithelium-associated commensal segmented filamentous bacteria (SFB). Several reports suggest that the cytokine environment induced by gut bacteria is sufficient to drive LP Th17 cell differentiation. In this context, intestinal DCs are proposed to facilitate the conversion of naïve CD4 T cells to Th17 cells within gut-draining lymph nodes. Whether such mechanisms control commensal-mediated Th17 cell differentiation has not been examined. In this work, I explore the mechanisms of induction of Th17 cells by SFB, with a particular focus on the role of antigen-presenting cells in this process. Initiation of CD4 T cell responses requires both major histocompatibility II (MHCII)-mediated antigen presentation and cytokine stimulation, which can be provided by the same or different subsets of intestinal MNPs. To test the requirement for either function in the induction of Th17 cells by SFB, we analyzed the role of SFB-induced cytokine environment in driving Th17 cell differentiation of non-SFB transgenic CD4 T cells. We find that although the cytokine environment is important, it is not sufficient to promote Th17 cell differentiation of activated CD4 T cells. In fact, we show that MHCII-dependent antigen presentation of SFB antigens by intestinal MNPs is crucial for Th17 cell induction. Expression of MHCII on CD11c+ cells was necessary and sufficient for SFB-induced Th17 cell differentiation. We also show that most SFB-induced Th17 cells respond to SFB antigens, which stressed that they carry T cell receptors that recognize SFB moieties. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. Our results outline the complex role of MNPs in the regulation of intestinal Th17 cell homeostasis, and we investigated the contribution of individual subsets to SFB-specific Th17 cell differentiation. Although the role of DCs in initiating T cell responses is well appreciated, how Mfs contribute to the generation of CD4 T cell responses to intestinal microbes is unclear. To this end, I examined the role of mucosal DCs and Mfs in Th17 induction by SFB in vivo. Employing DC and Mf subset-specific depletion and gain-of-function mouse models, I show that Mfs, and not conventional CD103+ DCs, are essential for generation of SFB-specific Th17 responses. Thus, Mfs drive mucosal T cell responses to certain commensal bacteria

Learning ‘‘graph-mer’’ Motifs that Predict Gene Expression Trajectories in Development

A key problem in understanding transcriptional regulatory networks is deciphering what cis regulatory logic is encoded in gene promoter sequences and how this sequence information maps to expression. A typical computational approach to this problem involves clustering genes by their expression profiles and then searching for overrepresented motifs in the promoter sequences of genes in a cluster. However, genes with similar expression profiles may be controlled by distinct regulatory programs. Moreover, if many gene expression profiles in a data set are highly correlated, as in the case of whole organism developmental time series, it may be difficult to resolve fine-grained clusters in the first place. We present a predictive framework for modeling the natural flow of information, from promoter sequence to expression, to learn cis regulatory motifs and characterize gene expression patterns in developmental time courses. We introduce a cluster-free algorithm based on a graph-regularized version of partial least squares (PLS) regression to learn sequence patterns—represented by graphs of k-mers, or “graph-mers”—that predict gene expression trajectories. Applying the approach to wildtype germline development in Caenorhabditis elegans, we found that the first and second latent PLS factors mapped to expression profiles for oocyte and sperm genes, respectively. We extracted both known and novel motifs from the graph-mers associated to these germline-specific patterns, including novel CG-rich motifs specific to oocyte genes. We found evidence supporting the functional relevance of these putative regulatory elements through analysis of positional bias, motif conservation and in situ gene expression. This study demonstrates that our regression model can learn biologically meaningful latent structure and identify potentially functional motifs from subtle developmental time course expression data

Nkx2.2 and Arx genetically interact to regulate pancreatic endocrine cell development and endocrine hormone expression

AbstractNkx2.2 and Arx are essential pancreatic transcription factors. Nkx2.2 is necessary for the appropriate specification of the islet alpha, beta, PP and epsilon cell lineages, whereas Arx is required to form the correct ratio of alpha, beta, delta and PP cells. To begin to understand the cooperative functions of Nkx2.2 and Arx in the development of endocrine cell lineages, we generated progenitor cell-specific deletions of Arx on the Nkx2.2 null background. The analysis of these mutants demonstrates that expansion of the ghrelin cell population in the Nkx2.2 null pancreas is not dependent on Arx; however, Arx is necessary for the upregulation of ghrelin mRNA levels in Nkx2.2 mutant epsilon cells. Alternatively, in the absence of Arx, delta cell numbers are increased and Nkx2.2 becomes essential for the repression of somatostatin gene expression. Interestingly, the dysregulation of ghrelin and somatostatin expression in the Nkx2.2/Arx compound mutant (Nkx2.2null;ArxΔpanc) results in the appearance of ghrelin+/somatostatin+ co-expressing cells. These compound mutants also revealed a genetic interaction between Nkx2.2 and Arx in the regulation of the PP cell lineage; the PP cell population is reduced when Nkx2.2 is deleted but is restored back to wildtype numbers in the Nkx2.2null;ArxΔpanc mutant. Moreover, conditional deletion of Arx in specific pancreatic cell populations established that the functions of Arx are necessary in the Neurog3+ endocrine progenitors. Together, these experiments identify novel genetic interactions between Nkx2.2 and Arx within the endocrine progenitor cells that ensure the correct specification and regulation of endocrine hormone-producing cells

Digestion of Chromatin in Apoptotic Cell Microparticles Prevents Autoimmunity

Antibodies to DNA and chromatin drive autoimmunity in systemic lupus erythematosus (SLE). Null mutations and hypomorphic variants of the secreted deoxyribonuclease DNASE1L3 are linked to familial and sporadic SLE, respectively. We report that DNASE1L3-deficient mice rapidly develop autoantibodies to DNA and chromatin, followed by an SLE-like disease. Circulating DNASE1L3 is produced by dendritic cells and macrophages, and its levels inversely correlate with anti-DNA antibody response. DNASE1L3 is uniquely capable of digesting chromatin in microparticles released from apoptotic cells. Accordingly, DNASE1L3-deficient mice and human patients have elevated DNA levels in plasma, particularly in circulating microparticles. Murine and human autoantibody clones and serum antibodies from human SLE patients bind to DNASE1L3-sensitive chromatin on the surface of microparticles. Thus, extracellular micro-particle-associated chromatin is a potential self-antigen normally digested by circulating DNASE1L3. The loss of this tolerance mechanismcan contribute to SLE, and its restoration may represent a therapeutic opportunity in the disease