We established the method to isolate lamina propria cells from small intestine and reported that innate immune cells in lamina propria are divided into 4 subsets based on the difference in CD11c/CD11b expression patterns:CD11chighCD11blow dendritic cells (DCs), CD11chighCD11bhighDCs, CD11cintCD11bint macrophages (MFs), and CD11cintCD11bhigh eosinophils (Uematsu S, et al. Nat Immunol. 2008.).
１）Analysis of CD11chighCD11bhigh DCs in small intestinal lamina propria
Toll-like receptors (TLRs) recognize distinct microbial components and induce innate immune responses. TLR5 is triggered by bacterial flagellin. Here we generated Tlr5-/- mice and assessed TLR5 function in vivo. Unlike other TLRs, TLR5 was not expressed on conventional dendritic cells or macrophages. In contrast, TLR5 was expressed mainly on intestinal CD11c+ lamina propria cells (LPCs). CD11c+ LPCs detected pathogenic bacteria and secreted proinflammatory cytokines in a TLR5-dependent way. However, CD11c+ LPCs do not express TLR4 and did not secrete proinflammatory cytokines after exposure to a commensal bacterium. Notably, transport of pathogenic Salmonella typhimurium from the intestinal tract to mesenteric lymph nodes was impaired in Tlr5-/- mice. These data suggest that CD11c+ LPCs, via TLR5, detect and are used by pathogenic bacteria in the intestinal lumen (Uematsu S, et al. Nat Immunol. 2006.). We further identify a subset of CD11chighCD11bhigh LPDCs that expressed TLR5 in the small intestine. When stimulated by the TLR5 ligand flagellin, TLR5+ LPDCs induced the differentiation of naive B cells into immunoglobulin (Ig) A-producing plasma cells by a mechanism independent of gut-associated lymphoid tissue. In addition, by a mechanism dependent on TLR5 stimulation, these LPDCs promoted the differentiation of antigen-specific Th17 cells and Th1cells. Unlike spleen DCs, the LPDCs specifically produced retinoic acid, which, in a dose-dependent way, supported the generation and retention of IgA-producing cells in the LP and positively regulated the differentiation Th17 cells. Our findings demonstrate unique properties of LPDCs and the importance of TLR5 for adaptive immunity in the intestine (Uematsu S, et al. Nat Immunol. 2008.).
2）Analysis of CD11chighCD11blow DCs in small intestinal lamina propria
CD103+ DCs are the major conventional DC population in the intestinal LP. Our previous report showed that low density cells in the LP could be classified into four subsets based on the difference in CD11c/CD11b expression patterns: CD11chighCD11blow DCs, CD11chighCD11bhigh DCs, CD11cintCD11bint MFs, and CD11cintCD11bhigh eosinophils. The CD11chighCD11bhigh DCs, which are CD103+, specifically express TLR5 and induce the differentiation of naive B cells into IgA+ plasma cells. These DCs also mediate the differentiation of Ag-specific Th17 and Th1 cells in response to flagellin. We found that small intestine CD103+ DCs of the LP (LPDCs) could be divided into a small subset of CD8α+ cells and a larger subset of CD8α- cells. Flow cytometry analysis revealed that CD103+CD8α+ and CD103+CD8α- LPDCs were equivalent to CD11chighCD11blow DCs and CD11chighCD11bhigh DCs, respectively. We analyzed a novel subset of CD8α+ LPDCs to elucidate their immunological function. CD103+CD8α+ LPDCs expressed TLR3, TLR7, and TLR9 and produced IL-6 and IL-12p40, but not TNF-α, IL-10, or IL-23, following TLR ligand stimulation. CD103+CD8α+ LPDCs did not express the gene encoding retinoic acid-converting enzyme Raldh2 and were not involved in T cell-independent IgA synthesis or Foxp3+ regulatory T cell induction. Furthermore, CD103+CD8α+ LPDCs induced Ag-specific IgG in serum, a Th1 response, and CTL activity in vivo. Accordingly, CD103+CD8α+ LPDCs exhibit a different function from CD103+CD8α- LPDCs in active immunity. This is the first analysis, to our knowledge, of CD8α+ DCs in the LP of the small intestine (Fujimoto K, et al. J. Immunol. 2011.).
3）Analysis of CD11cintCD11bint MFs and CD11cintCD11bhigh epsinophils in small intestinal lamina propria
CD11cintCD11bint MFs express CX3CR1. We are analyzing on the MFs about expression patterns of TLRs, cytokine production, regulatory function and roles in inflammation and infection.
It is well known that large numbers of eosinophils reside in small intestinal lamina propira. However, the function of intestinal eosinophils is yet to be elucidated. We are analyzing on the eosinophils about the activation mechanism and roles in inflammation, parasite infection and food allergy.
4）Role of TLR3 in acute radiation syndrome
High-dose ionizing radiation induces severe DNA damage in the epithelial stem cells in small intestinal crypts and causes gastrointestinal syndrome (GIS). Although the tumour suppressor p53 is a primary factor inducing death of crypt cells with DNA damage, its essential role in maintaining genome stability means inhibiting p53 to prevent GIS is not a viable strategy. Here we show that the innate immune receptor Toll-like receptor 3 (TLR3) is critical for the pathogenesis of GIS. Tlr3−/− mice show substantial resistance to GIS owing to significantly reduced radiation-induced crypt cell death. Despite showing reduced crypt cell death, p53-dependent crypt cell death is not impaired in Tlr3−/− mice. p53-dependent crypt cell death causes leakage of cellular RNA, which induces extensive cell death via TLR3. An inhibitor of TLR3–RNA binding ameliorates GIS by reducing crypt cell death. Thus, we propose blocking TLR3 activation as a novel approach to treat GIS (Takemura N, et al. Nat Commun. 2014.).