| Background and AimsThe intestinal tract is exposed to massive amounts of foreign antigen stimuli, including commensal bacteria, pathogens, and dietary components. Multiple mechanisms have been developed in the regulation of host responses to such stimulation to maintain immune homeostasis in the intestines. The intestinal mucosal surface is a natural site for the development of Th17 cells, which produce a distinct set of cytokines, including IL-17A (IL-17), IL-17F, IL-21 and IL-22. It has been shown that intestinal Thl7 cell development is stimulated by a specific species of microbiota, with segmented filamentous bacteria (SFB) being identified as one of such stimulators. Although both pro-and anti-inflammatory functions of Thl7 cells have been demonstrated in different experimental systems, the enrichment of Th17 cells in the intestines suggests a role for these cells in mucosal homeostasis and more specifically in the containment of the vast local microbiota. IL-17, a signature Th17 cell cytokine, is able to stimulate intestinal epithelial cell production of antimicrobial peptides. We and others have shown recently that Th17 cells induce mucosal IgA production in the intestines and lungs, both of which could contribute to the maintenance of intestinal homeostasis. However, the cells and environmental factors which promote Thl7 cells development in intestines are still not completely understood.Intestinal dendritic cells (DCs) form an extensive network in lamina propria and are critical in both shaping innate and adaptive immune responses to commensal microbiota, as well as instructing lymphocytes homing to the intestines, by inducing the expression of the gut-homing receptors α4β7 and CCR9 on these cells. Thus DCs play a key role in controlling intestinal inflammation and maintenance of immune homeostasis. Multiple, phenotypically distinct subsets of DCs exist in intestinal lamina propria, including CD11c+CD11b+CD8α-(mDC), CD11c+CD11b-CD8α+ (lymphoid DC), and CD11c+CDllb-B220+(pDC). Some small intestinal lamina propria DCs express CX3CR1, and CX3CR1+DCs may serve as a gateway for the uptake of microbiota by the continuous sampling of luminal content via transepithelial dendrites in certain sites within the intestines.More recently, it has been shown that subsets of LPDCs express CD 103, and that CD103+and CD 103- LPDCs play different roles in generating gut-trophic T cells and in inducing B cell IgA production, thus regulating T cell and B cell responses. Four subsets of DCs can be identified based on CD 103 and CDllb expression in the intestines; however, the specific role of most of these DC subsets in enteric bacterial antigen sampling and presentation is unknown. Moreover, it also remains unclear whether these distinct DC subsets work synergistically or have distinct functions in response to intestinal microbiota, and thus in instructing adaptive immune responses and the differentiation of different types of effector T cells. Although a series of studies show an impressive degree of flexibility or "plasticity" of DCs in response to different microbial stimuli, accumulating evidence suggests that distinct DC subpopulations may have intrinsic biases in their capacities to process and present antigens, thus stimulating qualitatively different types of immune response.The various DC subsets have been shown to differentially express TLRs, as well as to respond differently to microbial stimuli. For example, while CD8α- DCs have an overall higher phagocytic capacity, CD8α+DCs internalize apoptotic cells. In response to stimulation by TLR 3,4,7, and 9, only myeloid DC (mDC), but not plasmacytoid DC (pDC), produce IL-23, although both mDC and pDC produce IL-12. Although it has been shown that generation of Th17 cells in intestinal mucosa requires bacterial antigens, it is still not completely understood what subsets of LPDC present the bacterial antigen to induce Th17 cell development. CD103+CD11b+DCs, which express TLR5 and are considered to exert innate function through detecting flagellin via activation of TLR5 signaling, were shown recently to be able to promote intestinal Th17 cell development through production of IL-6. TLR5 has also been implicated in LPDC induction of Th17 cells in intestines.Signal regulatory protein alpha (SIRPα/CD172α) is a conserved transmembrane protein. CD172α+LPDCs are able to promote Th17 cell development. However, it is still unclear whether TLR5 mediates CD172a+LPDC induction of Th17 cells in the intestine in response to commensal bacteria, and thus controls immune homeostasis and intestinal inflammation. On the basis of above findings, we would dectect these four part:1. Different cytokine profiles of CBir1 Tg CD4 T cells in spleen and lamina propria of recipients upon mucosal challenge with CBir1 flagellin,2. TLR5+Lamina propria CD172a+DC induces Thl7 cells.Method:Part1:1. Different cytokine profiles of CBirl Tg CD4 T cells in spleen and lamina propria of recipients upon mucosal challenge with CBir1 flagellin.To investigate the T cell response to microbiota antigen, we transferred CFSE-labeled CD4+T cells from CBir1 Tg mice into TCRβxδ-/- mice, in which the intestinal IgA was dramatic decreased, and CBir1 flagellin was able to translocate cross the epithelium. The recipient mice were gavaged with recombinant CBir1 flagellin. Three days after oral CBir1 challenge, spleen cells and intestinal lamina propria lymphocytes were isolated from recipients and re-stimulated with PMA/Ionomycin for 5 hrs. Intracellular IFNy and IL-17 were measured.As DCs have been shown to instruct T cell development into different subsets, we then investigated whether DC in spleen and intestinal LP differentially induce Thl and Th17 cell development. We cultured CBir1 T cells with DC isolated from spleen or intestinal LP in the presence of full-length CBirl flagellin with TLR5 activity or CBir1 peptide without TLR5 activity. Five days late, IFNy and IL-17 production was measured by flow cytometry.2. MLN DC and lamina propria DCs take up CBir1 given orally, and stimulate T cell response.To determine whether DCs in MLN and lamina propria can take up CBir1 flagellin given mucosally, wild type or TCRβxδ-/- mice were given Alexa 647-1abeled CBir1 by gavage. Alexa 647-1abeled CBir1 flagellin behaved in a physiological manner in that Alexa-CBir1 flagellin stimulated CBir1 Tg T cell proliferation at a level similar to that of unlabeled CBirl flagellin, and the cytokine profiles were also similar. Two hrs later, MLN DC and LPDC were isolated, and the uptake of CBirl by DC was determined by flow cytometry.These CBir1-bearing LPDCs of TCRβxδ-/- mice stimulated CBir1-specific T cell proliferation, but did not stimulate OVA-specific T cells when cultured with CD4 T cells from CBir1-specific Tg mice or OVA-specific OT Ⅱ mice.Part 2:1. TLR5 and CD172α expression on intestinal lamina propria dendritic cells.DCs are known to express a variety of TLRs. Because TLR5 is the receptor for flagellin, we determined TLR5 expression in the spleen and LPDC. It has been shown that CD103+DC are a subset of mucosal DC instructing T and B cells to become gut-trophic and migrate into the intestines. CD103+DCs play a role in regulating a mucosal T cell response, whereas CD 103- DCs stimulate mainly effector T cell responses.2. CBirl flagellin stimulation results in differing cytokine production profile and antigen-presenting ability between LPDC and spleen DC.To determine whether the differing TLR5 expression in spleen DC and LPDC affected their response to CBir1 flagellin stimulation, spleen DC and LPDC were isolated from C57BL/6 mice and stimulated with full-length CBir1 flagellin or flagellated A4 bacteria which produce CBir1 flagellin. Because it has been reported that expression of TLR4 is much lower in LPDC than in spleen DC, lipopolysacchirde (LPS) was also used to stimulate spleen DC and LPDC as a control. Cytokine production was measured by an ELISA in supernatant from a 24-hr culture.To determine whether these differing cytokine profiles for spleen DC and LPDC would induce different T cell responses when presented with CBir1 flagellin, we cultured CD4 T cells from CBirl Tg mice with spleen DC and LPDC from C57BL/6 mice in the presence of CBirl flagellin, and measured cytokine production in the supernatant of a three-day culture.3. TLR5+Lamina propria CD172a+DC induces Th17 cells.To determine if CD172a+LPDCs are able to promote a microbiota antigen-specific Th17 cell response, we isolated CD172a+and CD 172a- LPDCs from intestines of wild-type B6 mice and cultured them with CBirl Tg T cells in the presence of full-length CBirl flagellin, which is able to activate TLR5 of DC and CBirl T cells, or CBirl peptide, which is able to activate CBirl T cells but will not activate TLR5 of DC, as it lacking a TLR5-binding site.To further confirm the role of TLR5 in mediating CD172α+LPDCs promotion of Th17 cell development, we isolated LPDCs from TLR5-/- mice and compared them with wild-type LPDCs in their ability to induce CBirl Tg T cell development into Th17 cells in response to microbiota antigen stimulation.ResultsPart 1:1. Different cytokine profiles of CBirl Tg CD4 T cells in spleen and lamina propria of recipients upon mucosal challenge with CBirl flagellin.1. CBirl Tg T cell response to microbiota antigen CBirl Tg T cells produced IFNy, but not IL-17, in the spleen, whereas lamina propria T cells produced both IFNy and IL-17.2. DC in spleen and intestinal LP differentially induce Thl and Th17 cell development In response to CBirl peptide, CBirl T cell only produced IFNy but not IL-17 when stimulated with both splenic DC and LPDC. Interestingly, in response to full-length CBir1 flagellin, although splenic DC induced CBirl T cell production of IFNy but not IL-17, LPDC induced CBirl T cell production of both IFNy and IL-17.2. MLN DC and lamina propria DCs take up CBirl given orally, and stimulate T cell response.1. DCs in MLN and lamina propria can take up CBirl flagellin given mucosally Subsets of both MLN DC and LPDC were Alexa-647+in TCRβxδ-/-, but not wild type mice, indicating CBirl uptake by these DCs. These CBirl-bearing LPDCs of TCRβxδ-/- mice stimulated CBirl-specific T cell proliferation, but did not stimulate OVA-specific T cells when cultured with CD4 T cells from CBirl-specific Tg mice or OVA-specific OT II mice.2. MLN DC and lamina propria DCs stimulate T cell response LPDC from CBirl-fed, wild-type B6 mice did not stimulate CBirl T cell proliferation. Interestingly, LPDC from PBS-gavaged TCRβxδ-/- mice also stimulated CBirl Tg T cell proliferation, but to a much lower degree.Part 2:1. TLR5 and CD172a expression on intestinal lamina propria dendritic cells.Consistent with a previous report, compared to spleen DC, more CD11c+LPDC expressed TLR5. Both CD11c+CD11b+and CD11c+CD11b-DC expressed TLR5, but more CD11c+CD11b+LPDC expressed TLR5 than did CD11c+CD11b-LPDC.More LPDC are CD103+than are spleen DC. Both CD103+and CD103- DC expressed CD172a+at similar levels (47.6% VS.43%). Interestingly, a majority of CD172α+DCs in the intestines also expressed TLR5 in both CD103+and CD 103- DCs. Deficiency in TLR5 did not affect intestinal LP CD 172a DC population.2. CBirl flagellin stimulation results in differing cytokine production profile and antigen-presenting ability between LPDC and spleen DC.Spleen DC produced high levels of IL-12p70 and IL-6, and low levels of IL-23 in response to LPS. However, they produced much lower amounts of IL-12p70 and IL-6 upon stimulation with CBirl flagellin or flagellated A4 bacteria. In contrast, LPDC responded poorly to LPS stimulation, but produced high levels of IL-12p70, IL-23 and IL-6 in response to CBirl flagellin or flagellated A4 bacteria.CBirl Tg T cells produced both IL-17 and IFNy in the presence of LPDC, whereas they produced only IFNy with spleen DC. Addition of anti-TGFβ antibody inhibited CBirl Tg T cell IL-17, but increased IFNy production of T cells with LPDC, but not by spleen DC.3. TLR5+Lamina propria CD172α+DC induces Th17 cells.1) CD172α+LPDCs are able to promote a microbiota antigen-specific Th17 cell responsewhile both CD172α+ and CD172α- LPDCs were able to activate CBir1 T cells to produce IFNγ in the presence either full-length CBirl flagellin or CBirl peptide, CD172α+, but not CD 172α-, LPDCs stimulated CBir1 T cell production of IL-17 in the presence of full-length CBir1 flagellin. Moreover, CD172α+LPDCs did not stimulate CBir1 T cell production of IL-17 in the presence of CBir1 peptide.2) The role of TLR5 in mediating CD172α+LPDCs promotion of Th17 cellWhen stimulated with full-length CBirl flagellin, wild-type LPDCs induced CBir1 Tg T cell production of IL-17, whereas TLR5-deficient LPDCs failed to induce CBir1 Tg T cell production of IL-17. Both wild-type and TLR5 deficient LPDCs were able to induce CBirl Tg T cell production of IFNγ. Failure of TLR5-deficient LPDC induction of Th17 cell development was not an intrinsic defect, as TLR5-deficient LPDCs were able to induce CBir1 Tg T cell production of IL-17 under Th17 polarization conditions, i.e., in the presence of TGFβ and IL-6.ConclusionsPart1:1. Different cytokine profiles of CBir1 Tg CD4 T cells in spleen and lamina propria of recipients upon mucosal challenge with CBirl flagellin.1. Intestinal environment allowed effector CD4+T cell development into both Th1 and Th17 cell pathways, whereas the spleen supported only the Th1 cell pathway, in response to mirobiota stimulation.2. Spleen DC and LPDC differentially induce Th1 and Th17 cell development in response to stimulation of full-length CBir1 flagellin.2. MLN DC and lamina propria DCs take up CBirl given orally, and stimulate T cell response.1. Endogenous CBirl flagellin in the intestinal lumen crosses the mucosal barrier and is taken up by LPDC.2. MLN DC and lamina propria DCs stimulate T cell response specially.Part 2:1. TLR5 and CD 172a expression on intestinal lamina propria dendritic cells.A majority of CD172a+DCs in the intestines expressed TLR5 in both CD103+and CD 103-DCs2.CBirl flagellin stimulation results in differing cytokine production profile and antigen-presenting ability between LPDC and spleen DC.In response to enteric flagellin stimulation, spleen DC and LPDC produce a different cytokine environment that may instruct different pathways of effector T cell development. These data also suggest that upon CBirl flagellin stimulation, LPDC, but not spleen DC, produce or have acquired TGFβ which contributes to stimulation of Thl7 cell development in the intestines.3. TLR5+Lamina propria CD172a+DC induces Th17 cells.1. CD 172a+LPDCs are able to promote a microbiota antigen-specific Thl 7 cell responseCD172α+LPDCs promote Th17 cell development in response to microbiota antigens, which is mediated by TLR5 activation. However, the fact that CD 172a- LPDCs do not induce Th17 cell development even in response to stimulation of full-length CBirl flagellin, which is able to activate TLR5 signaling, indicates that TLR5 signaling alone is not sufficient to promote Th17 cell development.2. The role of TLR5 in mediating CD 172a+LPDCs promotion of Th 17 cellTLR5 mediates CD172a+LPDCs promotion of Th17 cell development in response to microbiota antigen stimulation.Significance:These findings may mean that CD172α+populations could be the LPDC subset inducing Th17 cell responses among the CD103+CD11b+subset of LPDCs.Consistently, in our study, FACS sorted CD172α+, but not CD 172α-, LPDC induced Th17 cell differentiation in response to microbiota antigen stimulation, further confirming CD172α+LPDC induction of microbiota antigen-specific Th17 cells. Interestingly, most CD172α+LPDCs also co-expressed TLR5, and TLR5-deficient LPDC did not induce Th17 cell development. However, TLR5 deficiency did not affect development of CD172α+LPDC. Thus, our data further argue that TLR5 signaling mediates induction of Th17 cells by various LPDC subsets, including CD 172α+LPDCs. |
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