A nasal double DNA adjuvant system induces atheroprotective IgM antibodies via dendritic cell-B-1a B cell interactions

We previously demonstrated that the dendritic cell (DC)-targeting nasal double DNA adjuvant system, which consists of a DNA plasmid expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotide 1826 (CpG ODN), elicits specific immune responses to various antigens in the mucosal and systemic compartments. Here, we investigated, using phosphorylcholine (PC)-conjugated keyhole limpet hemocyanin (PC-KLH) as an antigen, whether the nasal double DNA adjuvant system induces protective immunity to atherosclerosis in apolipoprotein E-deficient (ApoE KO) mice. Further, we assessed the molecular and cellular mechanisms in the induction of anti-PC-specific immune responses. Nasal immunization with PC-KLH plus pFL and CpG ODN enhanced induction of PC-specific IgM in plasma, peritoneal fluids, and nasal washes when compared with mice administered PC-KLH alone. Of importance, these antibodies exhibited highly specific binding to the PC molecule, and dose-dependent binding to anti-T15 idiotype (AB1-2). Twelve weeks after the last immunization, the nasal double DNA adjuvant system with PC-KLH resulted in a reduction of atherogenesis in the aortic arch of ApoE KO mice. Therefore, we next assessed immunocytological mechanism to induce these antibodies. The nasal double DNA adjuvant system with PC-KLH resulted not only in significantly increased frequencies of CD11c+ DCs in the spleen, peritoneal cavity (PEC), and nasopharyngeal-associated lymphoid tissues (NALT), but also significantly increased expression of a proliferation-inducing ligand and B-cell-activating factor by CD11c+ DCs. In addition, the double DNA adjuvant system induced significantly increased numbers of B-1 B cells in the spleen, PEC, and NALT, and increased expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor on CD5+ B220+ (B-1a) B cells. These findings demonstrated that the nasal double DNA adjuvant system with PC-KLH resulted in the induction of T15-like antibodies in the mucosal and systemic lymphoid tissues through interaction between DCs and B-1a B cells, and inhibited the progression of atherogenesis

Human salivary protein-derived peptides specific-salivary SIgA antibodies enhanced by nasal double DNA adjuvant in mice play an essential role in preventing Porphyromonas gingivalis colonization : an in-vitro study

Background: We previously showed that fimbriae-bore from Poryphyromonas gingivalis (Pg), one of the putative periodontopathogenic bacteria specifically bound to a peptide domain (stat23, prp21) shared on statherin or acidic proline-rich protein 1 (PRP1) molecule of human salivary proteins (HSPs). Here, we investigated whether the nasal administration of DNA plasmid expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotide 1826 as double DNA adjuvant (dDA) with stat23 and prpr21 induces antigen (Ag)-specific salivary secretory IgA (SIgA) antibodies (Abs) in mice. Further, we examined that stat23- and prpr21-specific salivary SIgA Abs induced by dDA have an impact on Pg-binding to human whole saliva-coated hydroxyapatite beads (wsHAPs). Material and methods: C57BL/6N mice were nasally immunized with dDA plus sta23 or/and prp21 peptide as Ag four times at weekly intervals. Saliva was collected one week after the final immunization and was subjected to Ag-specific ELISA. To examine the functional applicability of Ag-specific SIgA Abs, SIgA-enriched saliva samples were subjected to Pg binding inhibition assay to wsHAPs. Results: Significantly elevated levels of salivary SIgA Ab to stat23 or prp21 were seen in mice given nasal stat23 or prp21 with dDA compared to those in mice given Ag alone. Of interest, mice nasally given the mixture of stat23 and prp21 as double Ags plus dDA, resulted in both stat23- and prp21-specific salivary SIgA Ab responses, which are mediated through significantly increased numbers of CD11c+ dendritic cell populations and markedly elevated Th1 and Th2 cytokines production by CD4+ T cells in the mucosal inductive and effector tissues. The SIgA Ab-enriched saliva showed significantly reduced numbers of live Pg cells binding to wsHAPs as compared with those in mice given double Ags without dDA or naïve mice. Additionally, saliva from IgA-deficient mice given nasal double Ags plus dDA indicated no decrease of live Pg binding to wsHAPs. Conclusion: These findings show that HSP-derived peptides-specific salivary SIgA Abs induced by nasal administration of stat23 and prp21 peptides plus dDA, play an essential role in preventing Pg attachment and colonization on the surface of teeth, suggesting a potency that the SIgA may interrupt and mask fimbriae-binding domains in HSPs on the teeth

DNA adjuvants for potent mucosal immunity

In order to develop safe vaccines for effective mucosal immunity to major pulmonary bacterial infections, one must consider appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants. Such vaccine constructs can induce Ag-specific immune responses which provide effective protection from mucosal infections. In particular, it has been shown that adjuvant-based mucosal vaccine preparations are relatively easy to construct by simply mixing the adjuvant with the bacterial Ag, and the resulting vaccine can elicit protective immunity. We have studied DNA-based nasal adjuvants targeting mucosal dendritic cells (DCs) in order to induce Ag-specific mucosal and systemic immune responses that provide essential protection against microbial pathogens which invade our mucosal surfaces. In this review, we initially introduce a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule which is a growth factor for DCs as an effective adjuvant for mucosal immunity to pneumococcal infections. Next, we discuss the potential of adding unmethylated CpG oligodeoxynucleotide together with pFL together with a pneumococcal Ag for protection from pneumococcal infections. To do this, we have used pneumococcal surface protein A as vaccine for the restoration of mucosal immunity in aging. Further, we have also used our nasal pFL adjuvant system with phosphorylcholine-keyhole limpet hemocyanin (PC-KLH) in pneumococcal vaccine development, to successfully induce complete protection from nasal carriage by Streptococcus pneumoniae. Finally, we discuss the possibility that anti-PC antibodies induced by nasal delivery of pFL plus PC-KLH may play a protective role for prevention of atherogenesis and thus block the subsequent development of cardiovascular disease

Porphyromonas gingivalis Clearance by SIgA

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients

Oral-Nasopharyngeal Dendritic Cells Mediate T Cell-Independent IgA Class Switching on B-1 B Cells

Native cholera toxin (nCT) as a nasal adjuvant was shown to elicit increased levels of T-independent S-IgA antibody (Ab) responses through IL-5- IL-5 receptor interactions between CD4+ T cells and IgA+ B-1 B cells in murine submandibular glands (SMGs) and nasal passages (NPs). Here, we further investigate whether oral-nasopharyngeal dendritic cells (DCs) play a central role in the induction of B-1 B cell IgA class switch recombination (CSR) for the enhancement of T cell-independent (TI) mucosal S-IgA Ab responses. High expression levels of activation-induced cytidine deaminase, Iα-Cμ circulation transcripts and Iμ-Cα transcripts were seen on B-1 B cells purified from SMGs and NPs of both TCRβ−/− mice and wild-type mice given nasal trinitrophenyl (TNP)-LPS plus nCT, than in the same tissues of mice given nCT or TNP-LPS alone. Further, DCs from SMGs, NPs and NALT of mice given nasal TNP-LPS plus nCT expressed significantly higher levels of a proliferation-inducing ligand (APRIL) than those in mice given TNP-LPS or nCT alone, whereas the B-1 B cells in SMGs and NPs showed elevated levels of transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI) expression. Interestingly, high frequencies of IgA+ B-1 B cells were induced when peritoneal IgA− IgM+ B cells were stimulated with mucosal DCs from mice given nasal TNP-LPS plus nCT. Taken together, these findings show that nasal nCT plays a key role in the enhancement of mucosal DC-mediated TI IgA CSR by B-1 B cells through their interactions with APRIL and TACI

Id2-, RORγt-, and LTβR-independent initiation of lymphoid organogenesis in ocular immunity

The eye is protected by the ocular immunosurveillance system. We show that tear duct–associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/differentiation 2, retinoic acid–related orphan receptor γt, lymphotoxin (LT) α1β2–LTβR, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity

Inside the mucosal immune system.

An intricate network of innate and immune cells and their derived mediators function in unison to protect us from toxic elements and infectious microbial diseases that are encountered in our environment. This vast network operates efficiently by use of a single cell epithelium in, for example, the gastrointestinal (GI) and upper respiratory (UR) tracts, fortified by adjoining cells and lymphoid tissues that protect its integrity. Perturbations certainly occur, sometimes resulting in inflammatory diseases or infections that can be debilitating and life threatening. For example, allergies in the eyes, skin, nose, and the UR or digestive tracts are common. Likewise, genetic background and environmental microbial encounters can lead to inflammatory bowel diseases (IBDs). This mucosal immune system (MIS) in both health and disease is currently under intense investigation worldwide by scientists with diverse expertise and interests. Despite this activity, there are numerous questions remaining that will require detailed answers in order to use the MIS to our advantage. In this issue of PLOS Biology, a research article describes a multi-scale in vivo systems approach to determine precisely how the gut epithelium responds to an inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), given by the intravenous route. This article reveals a previously unknown pathway in which several cell types and their secreted mediators work in unison to prevent epithelial cell death in the mouse small intestine. The results of this interesting study illustrate how in vivo systems biology approaches can be used to unravel the complex mechanisms used to protect the host from its environment