Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and severe economic loss in the swine industry has been caused by the infection. Therefore, the development of an effective vaccine against the bacteria is necessary. ApxII toxin, among several virulence factors expressed by the bacteria, is considered to be a promising vaccine candidate because ApxII toxin not only accompanies cytotoxic and hemolytic activities, but is also expressed in all 15 serotypes of bacteria except serotypes 10 and 14. In this study, we identified the peptide ligand capable of targeting the ligand-conjugated ApxIIA #5 fragment antigen to nasopharynx-associated lymphoid tissue. It was found that nasal immunization with ligand-conjugated ApxIIA #5 induced efficient mucosal and systemic immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. More importantly, the nasal immunization induced protective immunity against nasal challenge infection of the bacteria, which was confirmed by histopathological studies and bacterial clearance after challenge infection. Collectively, we confirmed that the ligand capable of targeting the ligand-conjugated antigen to nasopharynx-associated lymphoid tissue can be used as an effective nasal vaccine adjuvant to induce protective immunity against A. pleuropneumoniae infection.

Interactions between microbes and epithelial cells in the gastrointestinal tract are closely associated with regulation of intestinal mucosal immune responses. Recent studies have highlighted the modulation of mucosal immunity by microbe-derived molecules such as ATP and short-chain fatty acids. In this study, we undertook to characterize the expression of the ATP-gated P2X7 receptor (P2X7R) on M cells and its role in gastrointestinal mucosal immune regulation because it was poorly characterized in Peyer's patches, although purinergic signaling via P2X7R and luminal ATP have been considered to play an important role in the gastrointestinal tract. Here, we present the first report on the expression of P2X7R on M cells and characterize the role of P2X7R in immune enhancement by ATP or LL-37.

Abstract Intestinal epithelial cells which are exposed to microbes tightly restrict the influx of luminal antigens. They also contribute to establish the tolerogenic immune microenvironment through the induction of retinoic acid, TGF-β, and IL-25 production. However, intestinal contents are continuously introduced into Peyer’s patches through M cells which are specialized epithelial cells to take up the luminal antigens. Consequently, Peyer’s patches constitute specialized immune microenvironment. Although the mechanism for luminal antigen uptake into M cells is not clearly defined, many studies suggest the role of apical proteins of M cells as a mediator for antigen influx. Here, we report the expression of C5aR, TLR 1/2, ATP-gated P2X7 receptor (P2X7R), and formyl peptide receptor 2 (FPR-2) on apical area of M cells. In addition, we found that receptor itself and/or crosstalk among these receptors were able to regulate not only modulation of the Peyer’s patch microenvironment but also transcytosis of luminal antigens. Especially, when LL-37, one of ligands for P2X7R and FPR-2, was applied to oral mucosal vaccine model, it promoted the induction of antigen-specific immune response through M cell antigen-targeting and modulation of Peyer’s patch microenvironment. Collectively, we conclude that interaction between luminal antigens and apical receptors on M cells initiate the signaling for immune induction and that these receptors could be used as new targets for oral vaccine delivery.

Ophiocordyceps sinensis is a natural fungus that has been valued as a health food and traditional Chinese medicine for centuries. The fungus is parasitic and colonizes insect larva. Naturally occurring O. sinensis thrives at high altitude in cold and grassy alpine meadows on the Himalayan mountain ranges. Wild O. sinensis is becoming increasingly rare in its natural habitats, and its price is out of reach for clinical practice. For these reasons, development of a standardized alternative is a great focus of research to allow the use of O. sinensis as a medicine. To develop an alternative for wild O. sinensis, a refined standardized extract, CBG-CS-2, was produced by artificial fermentation and extraction of the mycelial strain Paecilomyces hepiali CBG-CS-1, which originated from wild O. sinensis. In this study, we analyzed the in vivo immune-modulating effect of CBG-CS-2 in mice. Oral administration of CBG-CS-2 supported splenocyte stimulation and enhanced Th1-type cytokine expression from the splenocytes. Importantly, the same treatment significantly enhanced the natural killer cell activity of the splenocytes. Finally, oral administration of CBG-CS-2 enhanced the potential for inflammatory responses. Together, these findings indicate that the mycelial culture extract prepared from O. sinensis exhibited immune-modulating activity and suggest its possible use in the treatment of diseases caused by abnormal immune function.

It is known that formyl peptide receptor-2 (FPR-2) acts as innate immune sensor through recognition of peptides derived from pathogens such as H. pylori and HIV, mitochondrial peptides, amyloidogenic peptide including serum amyloid A, and inflammatory response-associated peptides such as LL-37. However, the role of FPR-2-expressing cells in mucosal immune inductive area, Peyer’s patch (PP), is still unraveled yet. In this study, we identified the expression of FPR-2 in both M cells located in follicle-associated epithelium of PP and follicular dendritic cells (FDCs) localized in germinal center. In addition, the level of Cxcl13 and TGF-β gene transcripts in FDCs, which are closely associated with recruitment and differentiation of IgA-secreting B cells was substantially increased by treatment of cathelicidin LL-37, one of FPR-2 ligands. Moreover, enhanced immune induction was verified in vivo mouse system through monitoring the antigen-specific mucosal immunity after oral administration of LL-37-conjugated antigen. Treatment of LL-37 has also shown the immune modulatory effects such as recruitment of CD11c+ cells and IL-17-secreting cells together with activation of FPR-2-expressing cells. Collectively, this study suggests that FPR-2-expressing cells are associated with regulation of mucosal immune induction and LL-37 could be used as a modulator for the cells.

It was characterized that the innate lymphoid cells (ILCs) are a group of immune cells closely associated with immune functions such as lymphoid organogenesis, tissue remodeling, antimicrobial immunity, and inflammation through cytokine-mediated cell regulation. ILC subsets can be categorized into three groups based on cytokine-secreting patterns. Group 1 ILCs secrete IFN-γ. Group 2 produces type 2 cytokines dependent on GATA-3. Group 3 represents all RORγt+ ILC subtypes secreting IL-17 and/or IL-22. In mucosal immunity, RORγt+ ILCs play an essential role in early protection against enteric pathogen infections, although its regulation by mucosal environment is unknown. In this study, we suggest that butyrate, one of commensal microbe-derived metabolites, as a modulator of PP ILCs, because it is known that butyrate treatment closely associated with the protection of bacterial infection or DSS-induced colitis. In order to understand the mechanism underlying the proposed role of butyrate, we monitored the expression pattern of each transcription factors and cytokines in PP ILCs after oral administration of sodium butyrate in SPF and antibiotics-treated mice. To understand the regulating activity of PP ILCs induced by butyrate in mucosal immunity, we try to understand the regulation of DSS colitis in the same mice. Collectively, this study suggests that regulation of ILCs by butyrate in gastrointestinal tract was closely associated with maintaining mucosal homeostasis.

Application of vaccine materials through oral mucosal route confers great economical advantage in animal farming industry due to much less vaccination cost compared with that of injection-based vaccination. In particular, oral administration of recombinant protein antigen against foot-and-mouth disease virus (FMDV) is an ideal strategy because it is safe from FMDV transmission during vaccine production and can induce antigen-specific immune response in mucosal compartments, where FMDV infection has been initiated, which is hardly achievable through parenteral immunization. Given that effective delivery of vaccine materials into immune inductive sites is prerequisite for effective oral mucosal vaccination, M cell-targeting strategy is crucial in successful vaccination since M cells are main gateway for luminal antigen influx into mucosal lymphoid tissue. Here, we applied previously identified M cell-targeting ligand Co1 to VP1 of FMDV in order to test the possible oral mucosal vaccination against FMDV infection. M cell-targeting ligand Co1-conjugated VP1 interacted efficiently with M cells of Peyer's patch. In addition, oral administration of ligand-conjugated VP1 enhanced the induction of VP1-specific IgG and IgA responses in systemic and mucosal compartments, respectively, in comparison with those from oral administration of VP1 alone. In addition, the enhanced VP1-specific immune response was found to be due to antigen-specific Th2-type cytokine production. Collectively, it is suggested that the M cell-targeting strategy could be applied to develop efficient oral mucosal vaccine against FMDV infection.

Oral mucosal vaccine confers feasibility and efficacy in vaccination. However, it imposes restrictions such as difficulty in antigen delivery and poor immunogenic environment. Consequently, many studies have been concentrated to develop effective mucosal adjuvants. We determined mucosal adjuvant activity of cathelicidin LL-37 based on the previous report of its activity in parenteral vaccination, although it has not been studied in mucosal system. In oral mucosal vaccination using EGFP protein, LL-37-conjugated antigen effectively evoked the antigen-specific systemic and mucosal immunities. Notably, when the LL-37 was applied to pathogenic antigen, an EDIII of dengue virus, antibody responses with high neutralization activity was induced. Further studies on chemokine profiling induced by LL-37 in Peyer’s patch lymphocytes were well accordance with the increase in the number of germinal centers in Peyer’s patch and mesenteric lymph node by chemotactic effect of LL-37. In addition, oral priming with LL-37-conjugated antigen induced Th1- and Th17-skewed immune responses through CD11c+ CD70+ cell activation in Peyer’s patch. More interestingly, we found the expression of formyl peptide receptor-2, one of the receptors for LL-37, in subepithelial dome of Peyer’s patch and M cells. Collectively, we conclude that LL-37 can play a role as mucosal adjuvant through enhanced antigen delivery, dendritic cell maturation, and chemotactic effect by FPR-2 on M cells and subepithelial dome.