Takachika Hiroi

CCR7 is critically important for migration of dendritic cells in intestinal lamina propria to mesenteric lymph nodes

Although dendritic cells (DCs) located in the small intestinal lamina propria (LP-DCs) migrate to mesenteric lymph nodes (MLNs) constitutively, it is unclear which chemokines regulate their trafficking to MLNs. In this study we report that LP-DCs in unperturbed mice require CCR7 to migrate to MLNs. In vitro, LP-DCs expressing CCR7 migrated toward CCL21, although the LP-DCs appeared morphologically and phenotypically immature. In MLNs, DCs bearing the unique LP-DC phenotype (CD11chighCD8αintCD11blowαLlowβ7high and CD11chighCD8α−CD11bhighαLlowβ7high) were abundant in wild-type mice, but were markedly fewer in CCL19-, CCL21-Ser-deficient plt/plt mice and were almost absent in CCR7-deficient mice, indicating the critical importance of CCR7 in LP-DC trafficking to MLNs. Interestingly, CCR7+ DCs in MLNs with the unique LP-DC phenotype had numerous vacuoles containing cellular debris in the cytoplasm, although MLN-DCs themselves were poorly phagocytic, suggesting that the debris was derived from the LP, where the LP-DCs ingested apoptotic intestinal epithelial cells (IECs). Consistent with this, LP-DCs ingested IECs vigorously in vitro. By presenting IEC-associated Ag, the LP-DCs also induce T cells to produce IL-4 and IL-10. Collectively, these results strongly suggest that LP-DCs with unique immunomodulatory activities migrate to MLNs in a CCR7-dependent manner to engage in the presentation of IEC-associated Ags acquired in the LP.

IgA Class Switch Occurs in the Organized Nasopharynx- and Gut-Associated Lymphoid Tissue, but Not in the Diffuse Lamina Propria of Airways and Gut

Secretory IgA plays a crucial role in the host immune response as a first line of defense. A recent demonstration of in situ IgA class switching in intestinal lamina propria provided an opportunity to reconsider the model for the homing of IgA-committed B cells characterized by distinctive trafficking patterns to effector sites. Those effector sites depend on the organized mucosa-associated lymphoid tissues as their site of induction. In this report we show the preferential presence of IgM+B220+ and IgA+B220+ cells belonging to pre- and post-IgA isotype class-switched cells in the organized mucosa-associated lymphoid tissues, such as nasopharynx-associated lymphoid tissues, isolated lymphoid follicles, and Peyer’s patches, and the defect of those populations in the diffuse effector tissues, such as the nasal passage and intestinal lamina propria. Consistent with these findings, the expressions of a series of IgA isotype class switch recombination-related molecules, including activation-induced cytidine deaminase, Iα-Cμ circle transcripts, and Iα-Cμ circle transcripts, were selectively detected in these organized mucosa-associated lymphoid structures, but not in the diffuse mucosal effector sites. Taken together, these findings suggest that IgA isotype class switching occurs only in the organized mucosa-associated lymphoid organs (e.g., nasopharynx-associated lymphoid tissues, isolated lymphoid follicles, and Peyer’s patches), but not in the diffuse effector tissues of the upper respiratory and gastrointestinal tracts.

Intracellularly Expressed TLR2s and TLR4s Contribution to an Immunosilent Environment at the Ocular Mucosal Epithelium

Epithelial cells are key players in the first line of defense offered by the mucosal immune system against invading pathogens. In the present study we sought to determine whether human corneal epithelial cells expressing Toll-like receptors (TLRs) function as pattern-recognition receptors in the innate immune system and, if so, whether these TLRs act as a first line of defense in ocular mucosal immunity. Incubation of human primary corneal epithelial cells and the human corneal epithelial cell line (HCE-T) with peptidoglycan or LPS did not lead to activation, at the level of DNA transcription, of NF-κB or the secretion of inflammation-associated molecules such as IL-6, IL-8, and human β-defensin-2. However, when incubated with IL-1α to activate NF-κB, the production by these cells of such inflammatory mediators was enhanced. Human corneal epithelial cells were observed to express both TLR2- and TLR4-specific mRNA as well as their corresponding proteins intracellularly, but not at the cell surface. However, even when LPS was artificially introduced into the cytoplasm, it did not lead to the activation of epithelial cells. Taken together, our results demonstrate that the intracellular expression of TLR2 and TLR4 in human corneal epithelial cells fails to elicit innate immune responses and therefore, perhaps purposely, contributes to an immunosilent environment at the ocular mucosal epithelium.

Role of Gut-Associated Lymphoreticular Tissues in Antigen-Specific Intestinal IgA Immunity

This study assessed the roles of the postnatal lymphotoxin-β receptor (LTβR)-mediated signals in the gut-associated lymphoreticular tissues of mice for subsequent regulation of Ag-specific intestinal IgA responses. Blockade of LTβR-dependent events by postnatal administration of the fusion protein of LTβR and IgG Fc (LTβR-Ig) reduced both the size and numbers of Peyer’s patches (PP) without influencing the PP microarchitecture. Interestingly, inhibition of LTβR-dependent signaling revealed significant reductions in the formation of follicular dendritic cell clusters in mesenteric lymph nodes (MLN). Furthermore, these postnatal signaling events controlled the development of isolated lymphoid follicles (ILF) because treatment with LTβR-Ig eliminated the formation of ILF. LTβR-Ig-treated mice with altered microarchitecture of MLN and lacking ILF were still able to produce significant Ag-specific mucosal IgA responses after oral immunization; however, the levels were significantly lower than those seen in control mice. These results imply the importance of ILF for Ag-specific intestinal immunity. However, mice treated with both TNFR55-Ig and LTβR-Ig in utero, which lack PP and MLN, but retain intact ILF, failed to induce Ag-specific IgA responses after oral immunization. These findings demonstrate that ILF are not essential for induction of intestinal IgA Ab responses to orally administered Ag. Furthermore, the induction of intestinal IgA Ab responses requires the proper maintenance of the MLN microarchitecture, including a follicular dendritic cell network.

Prenatal Blockage of Lymphotoxin β Receptor and TNF Receptor p55 Signaling Cascade Resulted in the Acceleration of Tissue Genesis for Isolated Lymphoid Follicles in the Large Intestine

Signaling by lymphotoxin (LT) and TNF is essential for the organogenesis of secondary lymphoid tissues in systemic and mucosal compartments. In this study, we demonstrated that the progeny of mice treated with fusion protein of LTβR and IgGFc (LTβR-Ig) or LTβR-Ig plus TNFR55-Ig (double Ig) showed significantly increased numbers of isolated lymphoid follicles (ILF) in the large intestine. Interestingly, double Ig treatment accelerated the maturation of large intestinal ILF. Three-week-old progeny of double Ig-treated mice showed increased numbers of ILF in the large intestine, but not in the small intestine. Furthermore, alteration of intestinal microflora by feeding of antibiotic water did not affect the increased numbers of ILF in the large intestine of double Ig-treated mice. Most interestingly, mice that developed numerous ILF also had increased levels of activation-induced cytidine deaminase expression and numbers of IgA-expressing cells in the lamina propria of the large intestine. Taken together, these results suggest that ILF formation in the large intestine is accelerated by blockage of LTβR and TNFR55 signals in utero, and ILF, like colonic patches, might play a role in the induction of IgA response in the large intestine.

Autocrine IL-15 Mediates Intestinal Epithelial Cell Death Via the Activation of Neighboring Intraepithelial NK Cells

Intestinal intraepithelial lymphocytes (IELs), which reside between the basolateral faces of intestinal epithelial cells (IECs), provide a first-line defense against pathogens via their cytotoxic activity. Although IEC-derived IL-7 and IL-15 are key regulatory cytokines for the development and activation of IELs, we report here that IL-15 but not IL-7 mediates the reciprocal interaction between IELs and IECs, an important interaction for the regulation of appropriate mucosal immunohomeostasis. IL-15-treated IELs induced cell death in IECs via the cytotoxic activity in vitro. Among the different subsets of IL-15-treated IELs, CD4−CD8−TCR− IELs, which express NK marker (DX5 or NK1.1), showed the most potent syngenic IEC killing activity. These intraepithelial NK cells expressed Ly-49 molecules, NKG2 receptors, and perforin. These results suggest the possibility that the cell death program of IECs could be regulated by self-produced IL-15 through the activation of intraepithelial NK cells.

Therapeutic effects of roxithromycin in interleukin-10-deficient colitis

Background A limited number of therapeutic strategies are currently available to treat patients with inflammatory bowel disease (IBD). Interleukin‐10 (IL‐10)—deficient mice, well characterized as an experimental model of IBD, develop severe chronic colitis because of aberrant Th1 responses. Roxithromycin (RXM), a macrolide antibiotic, has received attention because it offers not only antibacterial but also immunosuppressive effects. We examined the immunosuppressive effect of RXM on the development of IBD. Methods To test the efficacy of short‐term administration of RXM, elder IL‐10‐deficient mice (16–20 weeks old) with established colitis were orally treated for 10 days with RXM (20 mg/kg per day). To test the long‐term preventive effects of RXM, for 20 weeks young adult IL‐10‐deficient mice (4–5 weeks old) also were administered RXM orally (20 mg/kg per day). Results The short‐term treatment‐oriented administration of RXM reduced the degree of inflammatory change and lowered serum amyloid A in IL‐10‐deficient mice with severe colitis. Mononuclear cells from the lamina propria of RXM‐treated large intestines showed lower production of IFN‐&ggr; than did those from diseased mice that were untreated. Long‐term prevention‐oriented administration of RXM suppressed the development of severe colitis and decreased production of IFN‐&ggr; and IL‐12. In addition to its expected immunosuppressive effect, RXM treatment also decreased the level of Bacteroides vulgatus, a Gram‐negative anaerobe. Conclusions The anti‐inflammatory changes observed in IL‐10‐deficient mice resulted from the efficacy of RXM as an immunosuppressant as well as from its efficacy as an antibiotic. According to our findings, RXM would seem to have significant potential as a preventive and/or therapeutic agent for IBD. (Inflamm Bowel Dis 2007)

HIV mucosal vaccine: nasal immunization with rBCG-V3J1 induces a long-term V3J1 peptide-specific neutralizing immunity in Th1- and Th2-deficient condition

Abstract In the vaccine strategy against HIV, the bacillus Calmette–Guerin (BCG), a live attenuated strain of Mycobacterium bovis, is considered to be one of the potential vectors for mucosal delivery of antigens. We analyzed the induction of antigen-specific antibodies by nasal immunization with recombinant BCG vector-based vaccine (rBCG-V3J1) that secretes the V3 principal neutralizing epitope of HIV [Proc. Natl. Acad. Sci. U. S. A. 92 (1063) 1995]. C57BL/6 mice were nasally immunized with the rBCG-V3J1 (10 μg) four times by weekly intervals. After 1 week of the final immunization, V3J1-specific IgG was seen in the serum. However, antigen-specific secretary and serum IgA antibodies were not induced. High levels of antigen-specific serum IgG responses were maintained for 6 months following the nasal immunization. Antigen-specific IgG-producing cells were detected in mononuclear cells isolated from spleen, nasal cavity, and salivary gland of the nasally vaccinized mice. Furthermore, antigen-specific IgG antibodies induced by nasal immunization with rBCG-V3J1 possessed the ability to neutralize HIV in vitro. Collectively, nasal immunization together with rBCG-V3J1 is considered to be a powerful vaccination regimen for the induction of effective V3J1-specific immune responses. We are currently investigating antigen-specific CTL activity and cytokine production following nasal vaccination with rBCG-V3J1.

NALT-genesis is induced by Id2 gene-dependent CD3−CD4+CD45+ cells

Abstract CD3 − CD4 + CD45 + cells are thought to be an inducer cell population for Peyers patch (PP) and lymph node development. Nasopharyngeal-associated lymphoid tissue (NALT) is an organized lymphoid structure found on both sides of the nasopharyngeal duct dorsal to the cartilaginous soft palate. Although NALT is considered to be functionally analogous to PP in the intestinal tract, the association of CD3 − CD4 + CD45 + cells with the initiation of NALT development has not yet been clarified. To examine this issue further, we isolated CD3 − CD4 + CD45 + cells from the fetal intestines of wild-type mice. Id2 −/− mice genetically deficient in NALT were adoptively transferred with purified CD3 − CD4 + CD45 + cells or fetal liver cells. After the adoptive transfer, we employed immunohistochemical analysis to examine the expression of PNAd, an important addressin for the recruitment of lymphocytes, in the nasal tissue of Id2 −/− mice transferred with fetal liver cells. Though NALT-like structures were indeed induced in Id2 −/− mice adoptively transferred with CD3 − CD4 + CD45 + cells or fetal liver cells, those structures were not observed to express PNAd. These findings indicate that CD3 − CD4 + CD45 + cells and Id2 genes are key to the initiation of NALT. However, they also suggest that an additional element is required for the induction of PNAd.