Mucosal immunity works as the body's frontline of defense against invasion of pathogens. Against variant virus infections, mucosal S-IgA confers cross-protection, and thus may provide an overall higher protective activity than systemic IgG (23
). Furthermore, mucosal S-IgA is known as a noninflammatory Ab (21
). Although heterosubtypic protection from influenza is not only mediated by cytotoxic T cells (31
), mucosal S-IgA and respiratory B cells induced by nasal vaccination and natural infection (7
) are crucial in protecting against IAV infection. In addition, humoral and protective immunity associated with antihemagglutinin specific IgA elicited by primary infection or vaccination is important for the prevention of secondary infection with different virus subtypes (41
). Therefore, we consider that induction of mucosal S-IgA is critical for immune protection against IAV infection.
In the present study, using the IAV-infected mice model, we demonstrated that CAM boosted the induction of sufficient mucosal S-IgA for protection against IAV infection in the respiratory tract ( and ). As shown in the results in and our recent animal (45
) and human (3
) studies, OSV treatment attenuated S-IgA production in the airway. In contrast, CAM enhanced respiratory S-IgA responses in OSV-treated mice and naïve mice. However, no significant differences were seen between the OSV- and OSV-CAM-treated mice with regard to the percentages of CD11c+
DCs on NPs and MeLNs at day 6 (data not shown). The findings of reinforcement of neutralizing activities of S-IgA by CAM () were supported by the results of reinfection experiments. The lung inflammation of IAV-infected OSV-CAM-treated mice was definitely mild in reinfection compared with that of IAV-infected OSV-treated mice (). These results support boosting of protective immunity by CAM in IAV-infected mice.
Previous studies indicated that AID is essential for CSR, which specifically occurs in activated B cells (4
), and that Iμ-Cα transcript is a hallmark for active IgA CSR in vivo
). To study the mechanisms of CAM-enhanced S-IgA production, we investigated the expression of AID and Iμ-Cα molecules involved in the induction of mucosal IgA CSR by B cells of the MeLNs, lung, and NPs. The results showed higher expression of AID and Iμ-Cα molecules in B cells from the mucosal lymph nodes in the CAM- and OSV-CAM-treated groups, but surprisingly low expression in the OSV-treated group, compared with the MC vehicle-treated group. These findings indicate that CAM induces T-cell-independent IgA CSR at the respiratory effector sites. In this regard, previous studies described a T-cell-independent IgA CSR pathway, in addition to the classical T-cell-dependent CSR in the intestinal mucosa (6
), and we have also recently reported the upregulation of T-cell-independent μ- to α-isotype CSR on B-1 B cells of oral-nasopharyngeal effector tissues following the use of naïve cholera toxin as a nasal adjuvant, with T-cell-independent Ag (16
). Furthermore, the present study showed that OSV inhibited the induction of T-cell-independent IgA CSR in respiratory effector sites, whereas OSV-CAM restored the suppressed induction. These findings suggest that the combination of CAM and OSV enhances anti-IAV S-IgA production, whereas OSV suppresses such processes.
It has been reported that both intestinal DCs and epithelial cells induce CD40-independent IgA CSR through BAFF and APRIL molecules (12
). In addition, we have also reported that APRIL-expressing DCs in oral-nasopharyngeal mucosal effector tissues induced IgA CSR on mucosal B-1 B cells following the use of naïve cholera toxin with T-cell-independent Ag as a nasal adjuvant (16
). We also studied the cellular and molecular mechanisms of T-cell-independent IgA CSR in the MeLNs, lung, and NPs in CAM-treated mice by focusing on DCs from these mucosal lymph nodes of mice treated with CAM and their potential roles in the induction of T-cell-independent μ- to α-isotype CSR. The results showed that CAM increased the expression of BAFF molecules on DCs of the mucosal lymphoid tissues, compared with the other treatment groups (). However, CAM did not increase the expression of APRIL on DCs of the mucosal lymph nodes. These results suggest that BAFF-expressing DCs play a key role in IgA CSR by B cells in respiratory effector tissues. In this regard, a recent study reported that intestinal DCs activated through Toll-like receptor 5 signaling produce retinoic acid and induce IgA production by peritoneal B cells (42
). Thus, it is possible that CAM stimulates virus-specific S-IgA responses through the production of retinoic acid by activated DCs in the MeLNs, lung, and NPs.
Although it is important to identify the receptors for the BAFF molecule on B cells that are responsible for the induction of IgA CSR, stable expression of BAFF-R, TACI, and BMCA molecules was noted on B cells from the MeLNs, lung, and NPs in IAV-infected mice treated with CAM, OSV, OSV-CAM, or MC, and no significant differences in BAFF-R, TACI, and BCMA expression were evident among these groups. Taken together, the results indicate that CAM stimulates DCs in the mucosal lymphoid tissues and plays an important role in IgA CSR by upregulation of expression of BAFF molecule. However, data in the experiments to clarify whether the effect of CAM on upregulation of BAFF on DCs is a direct or indirect effect suggest that CAM indirectly enhances BAFF expression on mucosal DCs (). Further screening studies for the target molecule(s) of CAM are currently in progress.
In summary, using IAV-infected weanling mice, the present study demonstrated that orally administered CAM enhances mucosal IAV-specific S-IgA immune responses and has neutralization activities. Thus, based on the production of mucosal S-IgA Ab, we consider that the combination of CAM-OSV treatment for IAV could potentially prevent complications and aggravation of the flu symptoms at an early stage of infection.