Intranasal Chitosan-DNA Immunization Stimulated Intestinal DCs To Drive IgA Class Switching Of B Cells In The Mesenteric Lymph Node

Secretory Immunoglobulin A?SIgA?is the most abundant antibody isotype in mucosal secretions.SIgA blocks attachment of pathogens or microbial toxins to epithelial cells and promotes phagocytosis of Ags through Fc?RI.IgA even excludes out bacteria which trespass the epithelial barrier via pIgR.It also regulates inflammatory response of epithelial cells as well as subepithelial dendritic cells?DC?and promotes the maintenance of appropriate bacterial communities in intestinal segments.In general,high-affinity IgA neutralizes microbial toxins and invasive pathogens,whereas low-affinity IgA confines commensals in the intestinal lumen.Given that most pathogens first infect mucosal surfaces,there is increasing interest in the establishment of protective mucosal immunity,achieved by vaccination via mucosal routes.To solve the necessity to amplify the immune responses to a wide array of antigens that are poorly immunogenic at the mucosal sites,chitosan?cationic polysaccharide?-based mucosal carriers and adjutants have been developed with unique advantages,such as ability of mucosal adhesion and opening tight junctions,good tolerability,biocompatibility and excellent immune stimulation which guarantee the great potential in capitalizing on their application in human clinical trial.To enhance SIgA induction and mucosal vaccine protection effect,we previously developed a DNA vaccine?pVP1?against Coxsackievirus B3?CVB3?and encapsulated DNA with chitosan to generate a nanoparticle vaccine.Intranasal immunization of chitosan-DNA significantly increased intestinal SIgA production which was associated with vaccine-enhanced protection.Concerning the mucosal immunity-activating mechanism of chitosan,some research attributed to macrophage-modulatory effect of chitosan.Recent studies indicate that chitosan engage the STING-cGAS pathway to trigger antigen specific Th1 and IgG2c responses.However,how chitosan-formulated DNA engages intestinal DC subsets to activate Ig A class switching of intestinal B cells remains largely unexplored.Intestinal SIgA generation is potentiated by class switch recombination?CSR?of the intestinal B cells,regulated by both T cell-dependent?TD?and T cell-independent?TI?pathways.In IgA inductive sites,Peyer's patches?PPs?,DCs capture antigen and migrate into the perifollicular area of PP or the draining mesenteric lymph nodes?MLNs?.They present antigen to CD4⁺T cells which release TGF?1,IL-4 and trigger B cells to undergo CSR from C?to C?through a CD40-dependent pathway.T-cell-independent IgA production occurs mainly in intestinal lamina propria?LP?and requires B-1 cells sensing polysaccharides Ags.IgA expression also requires B-cell-activating factor?BAFF?and IL-6 from DCs.Generation of high-affinity IgA antibodies is orchestrated by intestinal DCs.Intestinal DCs comprise conventional DCs?cDCs?and plasmacytoid DCs?pDCs?.Two cDCs subsets are present:CD103⁺CD11b^-which support Th17 cell differentiation in MLN and CD103⁺CD11b⁺which induce the differentiation of CD4⁺Foxp3⁺Treg.Both could produce cytokines affecting IgA CSR of B cells.Whether CD103⁺DCs or pDCs contribute to intestinal IgA CSR after chitosan-DNA immunization needs to be explored.In the present study,to clarify the intestinal DC-involved mechanism by chitosan-DNA to promote intestinal IgA CSR,we exploite flow cytometry and DC-B-T co-culture to investigate which intestinal DC subset is selectively activated by chitosan-DNA,and how this subset affect intestinal IgA CSR in vivo.We show here that intranasal immunization with chitosan-DNA induced the recruitment of CD103⁺CD11b^-DCs into the MLN which were responsible for the priming of CD4⁺Th17 cells in the MLN and are indispensable for chitosan-mediated antigen-specific B cell class switching to IgA via Th-dependent mechanism.Part one:chitosan-DNA immunization significantly increased IgA class switching of MLN B-2 B cells1.chitosan-DNA immunization increased the induction of CVB3 specific IgAGroups of male BALB/c mice were intranasally immunized with DNA or chitosan-DNA comprising 50ug DNA for 4 times biweekly.We first examined B cell Ab responses in the fecal extracts.There was a pronounced elevated IgA production in the intestine at wk4 which reached a peak at wk8 after final immunization.SIgA level induced by chitosan-DNA was substantially higher than that by“naked”DNA?p<0.05?.These results confirmed that chitosan-DNA enhanced intestinal IgA production after i.n.immunization.2.Nasal chitosan-DNA selectively induced IgA⁺B2 response in the MLNNext we examined and compared B cell responses in various intestinal lymphoid organs?MLN,LP and PP?and the spleen using flow cytometry.Compared with DNA,chitosan-DNA remarkably increased frequency and numbers of IgA⁺B and IgG⁺B cells in the MLN and LP at wk8 after chitosan-DNA immunization?p<0.5?.To assess the Ab response in greater depth,we assessed by ELISPOT assay the Ig isotype of CVB3 specific IgA-secreting cells in the spleen and MLN of mice.Data has been not available yet.To exclude the unspecific B1 response,we evaluated the mean fluorescence intensity?MFI?levels of IgA expression by CD5⁺B220⁺B1-B cells and CD5^-B220⁺B2cells.We found that a there was a marked increase of B2,but not B1 cells in the MLN and LP.Although 2%of B1-B could secrete IgA,B2-B cells constituted the majority of the IgA⁺B cells and IgA expression was highest in chitosan-DNA-treated mice.3.chitosan-DNA immunization enhanced expression of B cells?-germinal transcripts in MLNIn addition to detect IgA⁺B cells,we detected the mRNA expression of AID?Activation-induced cytidine deaminase,an essential enzyme for CSR?,and?-GT??-germinal transcript,precursor transcripts of C?-C??by MLN mononuclear cells of mice by RT-PCR.The expression of?-GT and Aicda?AID gene?were significantly higher in chitosan-DNA-treated mice than DNA-treated mice,indicating higher levels of IgA CSR were induced in the MLN after nasal chitosan-DNA immunizaiton.Part two:Intranasal immunization with chitosan-DNA selectively recruited CD103⁺DC and pDC into the MLN1.chitosan-DNA immunization increased migratory DCs and CD103⁺DC infiltration into the MLNUnder steady state or under infection,migratory DCs?mDCs?in the gut move from the small intestine to MLNs to present antigens to T cells.Therefore,we first quantified total DCs,cDCs,and migratory DCs in the MLN,PP and LP 24 hrs after chitosan-DNA immunization,using the gating strategy described.We found that 1)numbers of CD11c⁺MHCII⁺total DCs were significantly elevated in MLNs of chitosan-DNA-treated mice?p<0.001?.2)Numbers of resident CD11c⁺MHCII^int rDC were unchanged in various mucosal lymphoid tissues after immunization.3)Frequencies and numbers of migratory CD11c⁺MHCII^hi mDC were significantly elevated in MLNs of chitosan-DNA-treated mice?p<0.001?.Significantly elevated levels of CD80 and CD86 expression by m DCs were confirmed.4)chitosan-DNA immunization led to a significantly increased frequencies?59%to 75%/CD11c⁺DC,p<0.05?and numbers?1.5×10⁴ to 3.6×10⁴,p<0.05?of CD103⁺CD11b^-DC subsets as well as 5)CD103⁺DCs in MLN.2.chitosan-DNA immunization significantly increased pDC accumulation in the MLNBy two cytometry gating schedules for p DC(CD45⁺CD11^lowMHC II^low and CD11c^-PDCA1⁺),we confirmed that,24 hrs after chitosan-DNA immunization,a significant increase in p DC frequency?0.38%to 0.66%/MLN,p<0.05?and numbers?4×10³ to 9.8×10³,p<0.05?were observed in the in the MLN compared with DNA-immunized mice.In contrast,pDC numbers after chitosan-DNA immunization were comparable to those of DNA-treated mice in the spleen.Part Three:MLN CD103⁺DC facilitated T-dependent IgA CSR by secreting IL-6 and inducing intestinal Th17 differentiation under chitosan-DNA immunizaiton1.CD103⁺DC mediated B cell SIgA CSR was Th cell dependentTo examine the function of MLN CD103⁺DCs for IgA B2 cell CSR,MLN B220⁺B cells were co-cultured with CD103⁺CD11c⁺DCs from MLNs of mice given nasal chitosan-DNA or DNA in the presence of IL-4 and BAFF cytokines.After stimulation for 4 days,VP1 peptide and CD4⁺Th cells was added and continued culture until day 7when CVB3-specific Ig A level was detected by ELISA.It was found that under IgA-CSR-inducing conditions,CD103⁺DC could increase IgA production by B cells only in presence of CD4⁺T cells?p<0.05?,suggesting that intestinal CD103⁺DC-mediated intestinal B SIgA CSR was T-cell dependent.2.chitosan-DNA-primed CD103⁺DC induced Th17 differentiation which was closely associated with CD103⁺DC-mediated Ig A CSRTo examine the Th response in the MLN after chitosan-DNA i.n.immunization,we adopted FACS analysis on IL-17 expression by CD3⁺T cells and furtherabT andgdT cells in the MLNs.The assay revealed that compared with DNA,chitosan-DNA immunization significantly increased of LPabT numbers and IL-17⁺Th17 response in the MLN but not in the spleen.Then we speculate that in the MLN,CD103⁺DCs which carry VP1 antigens may directly influence local Th cells response and those further regulate B cell CSR.To confirm that,in the in vitro CD103⁺DC-B-Th cell co-culture system,we did found an elevated IL-17A level in the supernatant?p<0.05?.Then we conducted CD103⁺DC?pre-treated with chitosan-DNA for 12 hrs?-CD4⁺Th co-culture assay.24 hrs incubation with chitosan-DNA-pretreated CD103⁺DC significantly promoted the expression of ROR?t in CD4⁺T?0.64%to 1.38%?,and induction of IL-17A⁺Th17?0.27%to 0.34%?.These data indicated that chitosan-DNA-primed CD103⁺DC could induce Th17 differentiation in the MLN which favored B cell IgA CSR.3.CD103⁺DC directly provided B-stimulating factors BAFF and IL-6We next examined T-cell-independent mechanism adopted by CD103⁺DC,which is mediated by DC-derived cytokines including BAFF,APRIL,IL-6,TGF?,iNOS and RA.In this context,in the CD103⁺DC-B-Th co-culture system,on chitosan-DNA stimulation,expression of IL-6 and BAFF were elevated in CD103⁺DCs?p<0.05?,while expression of iNOS and TGF?were unaffected.Taken together,these findings showed that CD103⁺DCs were selectively activated and accumulated into the MLN by chitosan-DNA i.n.immunization.And CD103⁺DCs in MLNs play key roles in the induction of B-2 B cell IgA CSR both in stimulating Th17 response and in providing BAFF and IL-6 cytokines.Part four:pDC supported MLN B Ig A CSR via mAPRIL-TACI axis in a T-cell-independent manner1.pDC promoted MLN B IgA CSR in a T cell-indepentent mannerTo clarify the mechanism of pDC to facilitate MLN B cell IgA CSR,B220+B cells were co-cultured CD11c⁺PDCA-1^hipDCs from MLNs of mice given nasal chitosan-DNA or DNA in the presence of IL-4 and BAFF cytokines for 7 days.Compared to MLN CD103⁺DC,pDC alone could significantly promote Ig A production by MLN B cells.It indicated that after chitosan-DNA i.n.immunization,MLN B cells could undergo pDC-supported IgA CSR in T cell-independent manner.2.Nasal chitosan-DNA increased mAPRIL expression by pDCsIt has been reported that intestinal DCs induce TI IgA CSR through APRIL and BAFF molecules.We next examined whether pDC expressed these molecules.Although CD103⁺DCs expressed no mAPRIL,pDCs expressed a high level of mAPRIL at steady state;Significantly higher frequencies of APRIL-expressing pDCs as well as higher MFI levels of APRIL expression by pDCs were noted in chitosan-DNA-treated mice than those in DNA-treated mice.3.Nasal chitosan-DNA increased TACI expression by MLN B cellsReceptor for BAFF and APRIL include BAFF-R,transmembrane activator and calcium modulator cyclophilin ligand interactor?TACI?and B cell maturation antigen?BCMA?.By RT-PCR assay,we found that chitosan or chitosan-DNA significantly up-regulated expression of APRIL but not BAFF-R and BCMA,on MLN B cells as compared to DNA immunization.4.MLN pDCs induced B cell IgA CSR via activating mAPRIL-TACI axisTo further examine whether MLN pDCs directly induce TI IgA CSR in B cells,CD11c^-PDCA1⁺pDCs isolated from MLNs of mice nasally immunized with DNA or chitosan-DNA were primed with DNA or chitosan-DNA for 12hrs,then cultured with MLN B220⁺B cells in the presence of BAFF and IL-4.7 days after incubation,CVB3-specific IgA level was detected.Higher IgA production was seen in cultures containing pDCs from MLNs of mice nasally treated with chitosan-DNA when compared with those cultures incubated with DCs from mice given DNA only.To clarify if this pDC-mediated TI IgA CSR was dependent on APRIL and TACI,both expressions of which were significantly elevated by chitosan-DNA immunization,anti-APRIL mAb or anti-TACI mAb was added into the pDC-B co-culture system.It was found that the elevated production of Ig A facilitated by pDC incubation was significantly blocked by either addition of anti-APRIL or by anti-TACI m Ab.To further confirm the critical role for membrane APRIL,p DC and B cells were co-cultured in a transwell system.And transwell blockade of pDC contact with B cells totally blocked the IgA production.Collectively,these results demonstrate that pDCs contribute to B cell IgA CSR in the MLNs via activating mAPRIL-TACI axis after chitosan-DNA i.n.immunization.These results suggest that mAPRIL-up-regulating pDCs following nasal chitosan-DNA immunization most likely migrate into the MLNs for the induction of IgA CSR probably via a T-cell-independent way?a redundant help to CD103⁺DCs-mediated B IgA CSR?.Part five:chitosan-DNA reduced jmjd3 expression enabling epigenetic regulation of M1-to-M2 polarization to promote B cells IgA CSRIntestinal macrophages may also affect B cell CSR via antigen presentation,inflammatory effect and altering their polarization phenotype.DNA immunization usually induces M1 activation while IL-4/IL-13 or Ag-Ab complex would trigger M2a or M2b activation.Chitosan-DNA intranasal immunization may drive B cell SIgA CSR through epigenetic regulation of macrophage phenotype in the MLN.1.Chitosan-DNA drived macrophage M1-to-M2 transition and suppressed Th1 inductionTo see the effect of chitosan-DNA on macrophage polarization,bone marrow-derived macrophages?BMDM?were in vitro stimulated with chitosan,DNA and chitosan-DNA for 24 hours,the phenotypes were assessed by RT-PCR and ELISA and compared with those of classical cultured M0,M1,M2a,M2b,and M2c cells.DNA induced macrophage M1 polarization?IL-12,and IL-23?.Compared with that,chitosan-DNA significantly inhibited IL-12 expression by DNA-primed macrophage?p<0.05?indicating the DNA-drived M1 macrophage was transitioned to a M2-tending phenotype.To confirm this effect,chitosan-DNA-primed?2hrs?BMDMs were cultured with Th0 cells for 24 hours.The levels of IL-12 and IFN?in the supernatant were significantly reduced?p<0.001?,suggesting that chitosan-DNA-triggered M2macrophages suppressed Th1 responses.2.Chitosan-DNA impaired macrophage IL-12 production by inhibiting NF-?B and MAPK activationDNA activated macrophages production of IL-12 is dependent on NF-?B and MAPK signaling activation.To study the effect of chitosan-DNA on NF-?B and MAPK signalling,BMDMs were primed with DNA or chitosan-DNA and the phosphorylation of ERK,p38,JNK and I?B?was detected.DNA incubation significantly induced phosphorylation of the above molecules which were profoundly inhibited by chitosan-DNA.3.Chitosan-DNA inhibited expression of demethylase Jmjd3 in DNA-primed macrophagesMacrophage polarization can be epigenetically regulated by chromosome histone methylation,which is controlled by a demethylase,Jmjd3.To see the possible regulation of jmjd3 expression by chitosan-DNA,BMDMs were incubated with DNA and chitosan-DNA for 0-60 hrs,RT-PCR and western blotting assay all showed that DNA priming up-regulated of Jmjd3 expression which was significantly inhibited by chitosan-DNA from 12 hrs?p<0.05?.How does chitosan-DAN-down-regulated Jmjd3modulate macrophage M1-to M2 polarization needs further investigation.Taken together,in the present study,we found that intranasal immunization with chitosan-formulated DNA resulted in a pronounced switching of CVB3-specific B cells to IgA in the MLN of mice.In the MLN but not the spleen,chitosan-DNA drives accumulation and activation of two kinds of intestinal DC subsets,CD103⁺DCs and pDC.The nasal adjuvant effect of chitosan formulation in Ag-specific Ig A responses was uniquely regulated by TD IgA CSR by CD103⁺DC-stimulated Th17 response and by CD103⁺DC-derived BAFF and IL-6 in the MLNs.While MLN pDCs induced IgA generation from B cells by expressing mAPRIL.We have elucidated a novel cellular and molecular mechanism for chitosan adjuvanticity for the induction of TD-Ag-specific IgA in the intestinal tract.It has yet to be shown that chitosan serves a similar function in the oral-nasopharyngeal tract.This will be elucidated in our future studies.