Fasting-Refeeding Impacts Immune Cell Dynamics and Mucosal Immune Responses.

Nutritional status potentially influences immune responses; however, how nutritional signals regulate cellular dynamics and functionality remains obscure. Herein, we report that temporary fasting drastically reduces the number of lymphocytes by ∼50% in Peyer's patches (PPs), the inductive site of the gut immune response. Subsequent refeeding seemingly restored the number of lymphocytes, but whose cellular composition was conspicuously altered. A large portion of germinal center and IgA+ B cells were lost via apoptosis during fasting. Meanwhile, naive B cells migrated from PPs to the bone marrow during fasting and then back to PPs during refeeding when stromal cells sensed nutritional signals and upregulated CXCL13 expression to recruit naive B cells. Furthermore, temporal fasting before oral immunization with ovalbumin abolished the induction of antigen-specific IgA, failed to induce oral tolerance, and eventually exacerbated food antigen-induced diarrhea. Thus, nutritional signals are critical in maintaining gut immune homeostasis.

Epithelial-stromal interaction via Notch signaling is essential for the full maturation of gut-associated lymphoid tissues.

Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj-a gene encoding a transcription factor implicated in Notch signaling-in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE.

Expression pattern changes and function of RANKL during mouse lymph node microarchitecture development.

Receptor activator of nuclear factor kappa-B ligand (RANKL) expression was examined during the development of mouse fetal peripheral lymphoid organs. A shift in the expression pattern was detected during the transition from lymphoid tissue inducer (LTi) cells to lymphoid tissue organizer (LTo) cells in the lymph node (LN) anlagen but not in the Peyer's patch anlagen. In order to understand the functional impact of these changes in the fetal expression of RANKL, the RANKL function was blocked by a blocking antibody. Excess anti-RANKL antibody was administered to pregnant mice between 13.5 and 16.5 dpc and was found to completely block LN anlagen development, suggesting that RANKL function during this period is critical for LN development. In addition, small amounts of anti-RANKL antibodies were injected directly into the amniotic space at 13.5 dpc, resulting in perturbed B-cell follicle formation and high endothelial venule differentiation after birth. These results suggest that RANKL expression on LTi cells during the early phase of LN development is critical for the development LN microarchitecture.

Runx1/Cbfß2 complexes are required for lymphoid tissue inducer cell differentiation at two developmental stages.

Hematopoietic lymphoid tissue inducer (LTi) cells are essential for the development of secondary lymphoid tissues including lymph nodes and Peyer's patches. Two transcription factors, the helix-loop-helix inhibitor Id2 and the retinoic acid-related orphan receptor γt (Rorγt), have been shown to be crucial for LTi cell development. However, it remains unclear how the specification of multipotent hematopoietic progenitor cells toward the LTi lineage is programmed. In this study, we report impaired lymphoid tissue organogenesis in mice in which the function of Runx1/Cbfß transcription factor complexes was attenuated by the loss of either the distal promoter-derived Runx1 or Cbfß2 variant protein. We found that LTi progenitors in fetal liver, defined previously as a lineage marker-negative α4ß7 integrin (α4ß7)(+) IL-7R α-chain (IL-7Rα)(+) population, can be subdivided into Rorγt-expressing IL-7Rα(high) cells and nonexpressing IL-7Rα(mid) cells. Whereas Id2 and Rorγt are required to direct α4ß7(+)IL-7Rα(mid) cells to become α4ß7(+)IL-7Rα(high) cells, Runx1/Cbfß2 complexes are necessary for the emergence of α4ß7(+)IL-7Rα(mid) cells. In addition, the loss of Cbfß2, but not P1-Runx1, resulted in an inefficient upregulation of Rorγt in residual α4ß7(+)IL-7Rα(+) LTi cells at anlagen. Our results thus revealed that Runx1/Cbfß2 complexes regulate the differentiation of LTi cells at two stages: an early specification of hematopoietic progenitors toward the LTi lineage and a subsequent activation of Rorγt expression at anlagen.

Epithelium-Intrinsic MicroRNAs Contribute to Mucosal Immune Homeostasis by Promoting M-Cell Maturation.

M cells in the follicle-associated epithelium (FAE) of Peyer's patches (PPs) serve as a main portal for external antigens and function as a sentinel in mucosal immune responses. The scarcity of these cells has hampered identification of M cell-specific molecules. Recent efforts have begun to provide insight into antigen transcytosis and differentiation of M cells; however, the molecular mechanisms underlying these processes are not fully elucidated. Small non-coding RNAs including microRNA (miRNA) have been reported to regulate gene expression and control various biological processes such as cellular differentiation and function. To evaluate the expression of miRNAs in FAE, including M cells, we previously performed microarray analysis comparing intestinal villous epithelium (VE) and PP FAE. Here we confirmed FAE specific miRNA expression levels by quantitative PCR. To gain insight into miRNA function, we generated mice with intestinal epithelial cell-specific deletion of Dicer1 (DicerΔIEC) and analyzed intestinal phenotypes, including M-cell differentiation, morphology and function. DicerΔIEC mice had a marked decrease in M cells compared to control floxed Dicer mice, suggesting an essential role of miRNAs in maturation of these cells. Furthermore, transmission electron microscopic analysis revealed that depletion of miRNA caused the loss of endosomal structures in M cells. In addition, antigen uptake by M cells was impaired in DicerΔIEC mice. These results suggest that miRNAs play a significant role in M cell differentiation and help secure mucosal immune homeostasis.