IκBζ facilitates protective immunity against Salmonella infection via Th1 differentiation and IgG production

Abstract

Inhibitor of kappa B (IκB)-ζ transcription is rapidly induced by stimulation with TLR ligands and IL-1. Despite high IκBζ expression in inflammation sites, the association of IκBζ with host defence via systemic immune responses against bacterial infection remains unclear. Oral immunisation with a recombinant attenuated Salmonella vaccine (RASV) strain did not protect IκBζ-deficient mice against a lethal Salmonella challenge. IκBζ-deficient mice failed to produce Salmonella LPS-specific IgG, especially IgG2a, although inflammatory cytokine production and immune cell infiltration into the liver increased after oral RASV administration. Moreover, IκBζ-deficient mice exhibited enhanced splenic germinal centre reactions followed by increased total IgG production, despite IκBζ-deficient B cells having an intrinsic antibody class switching defect. IκBζ-deficient CD4⁺ T cells poorly differentiated into Th1 cells. IFN-γ production by CD4⁺ T cells from IκBζ-deficient mice immunised with RASV significantly decreased after restimulation with heat-killed RASV in vitro, suggesting that IκBζ-deficient mice failed to mount protective immune responses against Salmonella infection because of insufficient Th1 and IgG production. Therefore, IκBζ is crucial in protecting against Salmonella infection by inducing Th1 differentiation followed by IgG production.

Figure 1

IκBζ^−/− mice exhibited enhanced susceptibility after challenge with lethal Salmonella infection even following pre-vaccination. (A) Wild-type (WT) and IκBζ^−/− mice were orally challenged with 10⁷ CFU of a lethal wild-type Salmonella strain (UK-1) per mouse (n = 11/group). ns; not significant (log-rank test). Experiment came to the end at 25 days of post infection. Survived mice was sacrificed. (B) Wild-type and IκBζ^−/− mice were immunised by oral administration with 10⁹ CFU of a recombinant attenuated Salmonella vaccine strain (RASV) per mouse twice at 2-week intervals. Mice were challenged with UK-1 at 10⁷ CFU per mouse 14 d after the final RASV oral immunisation. The survival of mice was monitored following challenge (n = 13 for WT/RASV/UK-1, n = 12 for IκBζ^−/−/RASV/UK-1; ^***P < 0.001, based on log-rank test). (C,D) Wild-type and IκBζ^−/− mice were immunised with oral administration of 10⁹ CFU of RASV per mouse twice at 2-week intervals. (C) Liver tissues and (D) spleens from the mice were homogenised to determine the CFU of RASV at unimmunised, 14 d (D14) after the first administration with RASV, and 14 d after the second administration (D28). Data are representative of three independent experiments. ns, not significant; ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 based on ANOVA with Bonferroni’s multiple comparison test.

Figure 2

IκBζ^−/− mice exhibited enhanced inflammation after oral administration of a RASV strain. Wild-type and IκBζ^−/− mice were immunised with oral administration of 10⁹ CFU of RASV per mouse twice at 2-week intervals. (A,B) Inflammatory cytokines in the sera were analysed 14 d after the second immunisation (D28). Levels of TNF-α and IL-6 in the serum obtained from WT/PBS, IκBζ^−/−/PBS, WT/RASV and IκBζ^−/−/RASV were measured by cytokine bead array (CBA). ^**P < 0.01 and ^***P < 0.001 based on ANOVA with Bonferroni’s multiple comparison test. (C) Histopathology of the livers of wild type (WT) and IκBζ knock-out (IκBζ^−/−) mouse on day 14 and day 28. Note the mononuclear inflammatory cell foci (arrows) in the liver parenchyma which were evident in the livers of both types on day 14 but only in the IκBζ^−/− mouse liver on day 28. H&E. Bars mean 100 μm. (D) The number of immune cell infiltration lesions was counted from histological liver examination images. Data are representative of three independent experiments. ND, not detected.

Figure 3

Salmonella LPS-specific IgG responses were significantly decreased in IκBζ^−/− mice following oral administration of a RASV strain. Wild-type and IκBζ^−/− mice were immunised orally with 10⁹ CFU of a RASV strain per mouse twice at 2-week intervals (n > 3 per group). (A,B) The level of Salmonella LPS-specific Ab in serum was measured at 14 d after the second immunisation. To titrate the levels of LPS-specific IgG and IgG2a, LPS-specific IgG and IgG2a antibodies were measured from the serum of WT/PBS, IκBζ^−/−/PBS, WT/RASV and IκBζ^−/−/RASV mice 14 d after the second immunisation. ^**P < 0.01 and ^***P < 0.001 based on ANOVA with Bonferroni’s multiple comparison test. (C,D) The number of LPS-specific Ab-secreting cells isolated from spleen (C) and mesenteric lymph node (D) was measured by ELISPOT assay 14 d after the second immunisation with the RASV strain. (E) Splenic resting B cells were cultured in the presence of 12.5 µg/ml LPS plus 25 nM of retinoic acid (RA) and/or 0.2 ng/ml of TGF-β for 7 d to induce Ab class switching. Total IgG levels were measured by ELISA in the culture supernatant. Data are representative of three independent experiments. ^**P < 0.01 and ^***P < 0.001 based on unpaired t-test.

Figure 4

Germinal centre reaction after oral administration of a RASV strain in vivo. Wild-type and IκBζ^−/− mice were orally immunised with 10⁹ CFU of a RASV strain per mouse twice at 2-week intervals. (A) Representative H&E image of the spleen from an unimmunized mouse, 14 d after the first RASV oral administration (D14) and 14 d after the second RASV oral administration (D28). (B) Splenic follicle size was measured from histologic images. (C) Fourteen days after the second oral immunisation with RASV, the total IgG Ab level was measured in the serum. Data are representative of three independent experiments. ns, not significant; ^*P < 0.05 and ^***P < 0.001 based on ANOVA with Bonferroni’s multiple comparison test.

Figure 5

IFN-γ producing Th1 responses were impaired in IκBζ^−/− mice. (A) Naïve CD4⁺ T cells were cultured for 5 d with 2 µg/ml anti-CD3 and anti-CD28 antibody as induced to differentiate into Th1 cell with 10 ng/ml of IL-12. Under Th1 differentiation conditions, the intracellular IFN-γ on CD4⁺ T cells were analysed. ^*P < 0.05 and ^**P < 0.01 based on unpaired t-test. (B,C) Splenocytes isolated from RASV-immunised wild-type and IκBζ^−/− mice were re-stimulated by co-culturing with 10 heat-killed RASV per cell for 3 d. IFN-γ produced by CD4⁺ T cells were measured. (B) Representative intracellular staining results of IFN-γ synthesised by CD4⁺ T cells. (C) The percentages of IFN-γ-producing CD4⁺ T cells are shown. Data are representative of three independent experiments. ^*P < 0.05 and ^**P < 0.01 based on ANOVA with Bonferroni’s multiple comparison test.