Development of gene expression signatures for colonic lamina propria DCs of CD300a deficient mice
ABSTRACT: To further development of our gene expression approach to CD300a deficiency on dendritic cells (DCs) in colonic lamina propria, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish CD300a deficiency on DCs in colonic lamina propria from those of WT mice. Colonic lamina propria DCs were obtained by cell sorter from WT and CD300a deficient mice raised under SPF and GF condition. Expression of Ifnb1 was significantly higher in CD300a deficient DCs, quantified in the same RNA samples by real-time PCR. Gene expression in WT and CD300a colonic lamina propria DCs raised under SPF and GF conditions were measured. Colonic lamina propria cells were obtained from 5 mice in each conditions. Takara-Bio
Project description:Mononuclear phagocytes are a heterogeneous population of leukocytes essential for immune homeostasis that develop tissue-specific functions due to unique transcriptional programs driven by local microenvironmental cues. Single cell RNA sequencing (scRNA-seq) of colonic myeloid cells from specific pathogen free (SPF) and germ-free (GF) C57BL/6 mice revealed extensive heterogeneity of both colon macrophages (MPs) and dendritic cells (DCs). Modeling of developmental pathways combined with inference of gene regulatory networks indicate two major trajectories from common CCR2+ precursors resulting in colon MP populations with unique transcription factors and downstream target genes. Compared to SPF mice, GF mice had decreased numbers of total colon MPs, as well as selective proportional decreases of two major CD11c+CD206intCD121b+ and CD11c-CD206hiCD121b- colon MP populations, whereas DC numbers and proportions were not different. Importantly, these two major colon MP populations were clearly distinct from other colon MP populations regarding their gene expression profile, localization within the lamina propria (LP) and ability to phagocytose macromolecules from the blood. These data uncover the diversity of intestinal myeloid cell populations at the molecular level and highlight the importance of microbiota on the unique developmental as well as anatomical and functional fates of colon MPs.
Project description:We report that Klebsiella pneumoniae promote Th1 cell induction in colon. To examine the influence of Klebsiella on colonic epithelial cells (ECs) and lamina propria CD11c+ dendritic cells (DCs), we performed RNA seq on them. Germ free mice were orally inoculated with Kp-2H7 or BAA-2552 and total RNA was isolated from colonic ECs and DCs 1 week after inoculation. Furthermore, we examined the involvement of TLRs in induction of Th1 cells using Myd88 KO, Trif KO, Myd88/Trif DKO mice. These deficient germ free mice were orally inoculated with Kp-2H7 and total RNA was isolated from colonic ECs 3 weeks after inoculation. Overall design: The gene expression of colonic ECs and DCs isolated from germ free mice, and GF mice inoculated with Kp-2H7 or BAA-2552, and colonic ECs isolated from GF Myd88 KO, Trif KO or Myd88/Trif DKO mice inoculated with Kp-2H7.
Project description:BACKGROUND & AIMS:Heme oxygenase-1 (HO-1) and its metabolic by-product, carbon monoxide (CO), protect against intestinal inflammation in experimental models of colitis, but little is known about their intestinal immune mechanisms. We investigated the interactions among CO, HO-1, and the enteric microbiota in mice and zebrafish. METHODS:Germ-free, wild-type, and interleukin (Il)10(-/-) mice and germ-free zebrafish embryos were colonized with specific pathogen-free (SPF) microbiota. Germ-free or SPF-raised wild-type and Il10(-/-) mice were given intraperitoneal injections of cobalt(III) protoporphyrin IX chloride (CoPP), which up-regulates HO-1, the CO-releasing molecule Alfama-186, or saline (control). Colitis was induced in wild-type mice housed in SPF conditions by infection with Salmonella typhimurium. RESULTS:In colons of germ-free, wild-type mice, SPF microbiota induced production of HO-1 via activation of nuclear factor erythroid 2-related factor 2-, IL-10-, and Toll-like receptor-dependent pathways; similar observations were made in zebrafish. SPF microbiota did not induce HO-1 in colons of germ-free Il10(-/-) mice. Administration of CoPP to Il10(-/-) mice before transition from germ-free to SPF conditions reduced their development of colitis. In Il10(-/-) mice, CO and CoPP reduced levels of enteric bacterial genomic DNA in mesenteric lymph nodes. In mice with S typhimurium-induced enterocolitis, CoPP reduced the numbers of live S typhimurium recovered from the lamina propria, mesenteric lymph nodes, spleen, and liver. Knockdown of HO-1 in mouse macrophages impaired their bactericidal activity against E coli, E faecalis, and S typhimurium, whereas exposure to CO or overexpression of HO-1 increased their bactericidal activity. HO-1 induction and CO increased acidification of phagolysosomes. CONCLUSIONS:Colonic HO-1 prevents colonic inflammation in mice. HO-1 is induced by the enteric microbiota and its homeostatic function is mediated, in part, by promoting bactericidal activities of macrophages.
Project description:Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), an intracellular pattern recognition receptor, induces autophagy on detection of muramyl dipeptide (MDP), a component of microbial cell walls. The role of bacteria and NOD2 signaling toward ischemia/reperfusion (I/R)-induced intestinal injury response is unknown. Herein, we report that I/R-induced intestinal injury in germ-free (GF) C57BL/6 wild-type (WT) mice is worse than in conventionally derived mice. More important, microbiota-mediated protection against I/R-induced intestinal injury is abrogated in conventionally derived Nod2(-/-) mice and GF Nod2(-/-) mice. Also, WT mice raised in specific pathogen-free (SPF) conditions fared better against I/R-induced injury than SPF Nod2(-/-) mice. Moreover, SPF WT mice i.p. administered 10 mg/kg MDP were protected against injury compared with mice administered the inactive enantiomer, l-MDP, an effect lost in Nod2(-/-) mice. However, MDP administration failed to protect GF mice from I/R-induced intestinal injury compared with control, a phenomenon correlating with undetectable Nod2 mRNA level in the epithelium of GF mice. More important, the autophagy-inducer rapamycin protected Nod2(-/-) mice against I/R-induced injury and increased the levels of LC3(+) puncta in injured tissue of Nod2(-/-) mice. These findings demonstrate that NOD2 protects against I/R and promotes wound healing, likely through the induction of the autophagy response.
Project description:CX(3)CR1(+) and CD103(+) dendritic cells (DCs) in intestinal lamina propria play a key role in mucosal immunity. However, the origin and the developmental pathways that regulate their differentiation in the lamina propria remain unclear. We showed that monocytes gave rise exclusively to CD103(-)CX(3)CR1(+) lamina propria DCs under the control of macrophage-colony-stimulating factor receptor (M-CSFR) and Fms-like thyrosine kinase 3 (Flt3) ligands. In contrast, common DC progenitors (CDP) and pre-DCs, which give rise to lymphoid organ DCs but not to monocytes, differentiated exclusively into CD103(+)CX(3)CR1(-) lamina propria DCs under the control of Flt3 and granulocyte-macrophage-colony-stimulating factor receptor (GM-CSFR) ligands. CD103(+)CX(3)CR1(-) DCs but not CD103(-)CX(3)CR1(+) DCs in the lamina propria constitutively expressed CCR7 and were the first DCs to transport pathogenic Salmonella from the intestinal tract to the mesenteric lymph nodes. Altogether, these results underline the diverse origin of the lamina propria DC network and identify mucosal DCs that arise from pre-DCs as key sentinels of the gut immune system.
Project description:Exposure to microbes during early childhood is associated with protection from immune-mediated diseases such as inflammatory bowel disease (IBD) and asthma. Here, we show that in germ-free (GF) mice, invariant natural killer T (iNKT) cells accumulate in the colonic lamina propria and lung, resulting in increased morbidity in models of IBD and allergic asthma as compared with that of specific pathogen-free mice. This was associated with increased intestinal and pulmonary expression of the chemokine ligand CXCL16, which was associated with increased mucosal iNKT cells. Colonization of neonatal-but not adult-GF mice with a conventional microbiota protected the animals from mucosal iNKT accumulation and related pathology. These results indicate that age-sensitive contact with commensal microbes is critical for establishing mucosal iNKT cell tolerance to later environmental exposures.
Project description:The IL-33 receptor ST2 is differentially expressed by colonic lamina propria Treg cells Microarray of sort-purified Foxp3+ Treg cells from colonic lamina propria over mesenteric lymph node
Project description:Analyses of gene expression between CD103+ lung DCs, CD24+ lung DCs and CD64+ lung macrophages in Specific pathogen free (SPF) and germ free (GF) mice. Hypothesis tested was that the IgA class switching function of lung DCs is regulated by genes differentially expressed in GF mice compared to SPF mice. Results obtained provide important insights into the mechanisms of IgA induction by lung DCs Overall design: Total RNA was obtained from flow sorted, lung CD103+ DCs, CD24+ lung DCs and CD64+ lung macrophages from SPF and GF mice
Project description:A crosstalk between commensals, gut immune cells, and colonic epithelia is required for a proper function of intestinal mucosal barrier. Here we investigated the importance of two distinct intestinal dendritic cell (DC) subsets in controlling intestinal inflammation. We show that Clec9A-diphtheria toxin receptor (DTR) mice after depletion of CD103(+)CD11b(-) DCs developed severe, low-dose dextran sodium sulfate (DSS)-induced colitis, whereas the lack of CD103(+)CD11b(+) DCs in Clec4a4-DTR mice did not exacerbate intestinal inflammation. The CD103(+)CD11b(-) DC subset has gained a functional specialization that able them to repress inflammation via several epithelial interferon-? (IFN-?)-induced proteins. Among others, we identified that epithelial IDO1 and interleukin-18-binding protein (IL-18bp) were strongly modulated by CD103(+)CD11b(-) DCs. Through its preferential property to express IL-12 and IL-15, this particular DC subset can induce lymphocytes in colonic lamina propria and in epithelia to secrete IFN-? that then can trigger a reversible early anti-inflammatory response in intestinal epithelial cells.