IκBζ−/− mice exhibit severe and chronic head and cervical inflammation, especially in aged mice17,22. Thus, they are widely used as an animal model of Sjögren syndrome22. One of the most common symptoms of Sjögren syndrome is mucosal dryness, which disrupts the mucosal barrier function. Because of this impaired host barrier function, mice with IκBζ deficiency may be more susceptible to infection by pathogenic organisms18. We hypothesised that the persistent invasion of exogenous microorganisms could be a trigger for the chronic inflammation. Furthermore, failure in innate and adaptive immunity could contribute to the chronic inflammation in IκBζ−/− mice.
IκBζ plays a critical role in eradicating pathogens such as Streptococcus pneumoniae and Legionella pneumophila23. Individuals with a single nucleotide polymorphism in the IκBζ allele are more likely to succumb to pneumococcal infection6, and IκBζ regulates the expression of IL-6 in human monocytes in response to D39, a wild-type strain of Streptococcus pneumoniae23. In the current study, we found that mice with IκBζ deficiency developed chronic inflammation but failed to mount specific immune responses, including LPS-specific Ab production and Salmonella-specific T cell responses.
In addition to adaptive immunity induced by the RASV vaccination, innate immunity associated with IκBζ could affect on the protection against Salmonella. To confirm this, we performed Salmonella infection in bone marrow derived macrophages obtained from WT and IκBζ−/− mice, and found that higher bacterial colonies were detected in IκBζ−/− mice as compared to WT mice (Supplementary Fig. 7). This means that macrophage can play a role in Salmonella suppression in an IκBζ-dependent manner at early stage of infection.
To clarify the role of IκBζ in the induction of acquired immunity, we used a live attenuated recombinant Salmonella vaccine strain (RASV) to induce Salmonella-specific T cell and B cell responses. Typically, Salmonella invades via the gastro/oral route through contaminated food or water, causing salmonellosis accompanied by fever and diarrhoea14,15. Salmonella organisms possess a variety of antigenic molecules such as LPS, flagellin, and lipoprotein, which can also bind to TLR to trigger the innate immune responses24,25,26. Previously, we showed that Myd88 deficiency in mice mediated hypergammaglobulinemia with increased LPS-specific IgG Ab after oral administration of RASV15. Furthermore, we showed that WT mice which were adoptively transferred with sera from RASV-vaccinated Myd88−/− mice were partly protected against pathogenic Salmonella infection14. Contrary to Myd88, it was suggested that IκBζ is a critical regulator of TLR-mediated class switch recombination (CSR) in B cells, and mice with IκBζ deficiency have impaired type 1 T cell-independent Ab responses19; thus, we presumed that the LPS-specific response might be reduced after RASV administration in IκBζ−/− mice. Indeed, the levels of LPS-specific Ab responses in RASV-vaccinated IκBζ−/− mice were significantly lower than those in RASV immunised WT mice. However, the levels of total IgG were markedly increased in RASV-vaccinated IκBζ−/− mice compared to those in RASV-vaccinated WT mice. Thus, we confirmed that the LPS-specific Ab response, which represents the type 1 T cells-independent Ab response, was impaired in IκBζ−/− mice after RASV vaccination. To check how much Salmonella-specific IgG was in the total IgG of RASV-vaccinated IκBζ−/− mice, we conducted ELISA using whole cells of RASV as an antigen. The levels of anti-RASV IgG were higher in the serum of RASV-vaccinated IκBζ−/− compared with those in the serum of RASV-vaccinated WT mice (Supplementary Fig. 3). These data suggest that T cell-dependent and type 2 T cell-independent antibody production responses may occur in IκBζ deficiency after RASV vaccination. However, the higher levels of total antibody and RASV-specific antibody did not confer a protective effect against lethal Salmonella challenge.
The type 1 helper T cell (Th1) response is strongly induced in the host after Salmonella infection21. Th1 cells secrete interferon-γ, which activates macrophages and monocytes and makes them more potent in capturing and digesting Salmonella21,27,28. In the current study, we found that naïve CD4+ T cells from IκBζ−/− mice differentiated less to Th1 cells under IL-12-dependent differentiation conditions than WT CD4+ T cells. Furthermore, the secretion of IFN-γ by CD4+ T cells from RASV-vaccinated IκBζ−/− mice was lower than that by cells from RASV-vaccinated WT mice after restimulation with heat-killed RASV. In a previous study, it was shown that IκBζ regulated the production and secretion of IFNγ in human NK cells after stimulation with IL-12 and IL-1827,29. Likewise, it was suggested that the production of IFN-γ was decreased by IκBζ deficiency in a macrophage cell line after stimulation with IL-1β and IL-1830. These findings showed that IκBζ binds to the NF-κB binding site of the IFN-γ promoter to switch on IFN-γ mRNA transcription29. Collectively, the T cell-intrinsic activation of the IκBζ molecule after stimulation with IL-1 may be necessary for the differentiation of naïve CD4+ T cells into IFN-γ-secreting Th1 cells and play an essential role as a transcription factor to trigger IFN-γ production.
In addition, IκBζ mediates mucosal barrier function through secretion of inflammatory cytokines in epithelial cells7,22,23,31,32. Thus, it was supposed that the weaker barrier function of IκBζ−/− mice allowed the invasion of commensal bacteria, which might also influence the occurrence of chronic inflammation. In our study, we administered RASV via the oral route, and disseminated RASV via a leaky gut might induce systemic infection. In addition, we found that higher levels of IFN-γ-positive cells are found in non-vaccinated IκBζ−/− mice as compared to those in non-vaccinated WT mice. We presume that the impaired mucosal barrier function of IκBζ−/− mice could lead higher level of IFN-γ producing CD4+ T cell against some commensal bacteria which genetically close with Salmonella organism.
Nramp1 is a well-known factor for Fe2+-dependent reactive oxygen species production by phagocytes, and the 129 Sv mice having Nramp1G169 are more resistant to Salmonella than C57BL/6 mice having homozygous mutation of Nramp1G169D allele33,34,35,36. Thus, in the current study, we confirmed that both of mixed-background 129Sv-ICR IκBζ−/− mice and littermate WT control mice have susceptible Nramp1G169D allele (Supplementary Fig. 8).
In the current study, we hypothesised that IκBζ−/− mice could be protected from pathogenic Salmonella infection through immunisation. However, the oral administration of RASV to IκBζ−/− mice did not result in the production of high levels of LPS-specific antibody, and the mice were more susceptible to challenge with a lethal strain of Salmonella because of impaired T cell and B cell responses. Therefore, we presume that the reduced expression or the defective function of IκBζ in some populations might be associated with their higher susceptibility to specific pathogenic infections and that they can also be infected by the administration of an attenuated strain of bacteria which is given for vaccination. Thus, the administration of live attenuated vaccine strains should be avoided or re-considered for individuals with insufficient induction of adaptive immune responses due to an IκBζ hypomorphic mutation. Overall, we suggest that IκBζ is critical to mediate vaccine-induced adaptive immunity as well as innate immunity.