I[Kappa]B NS differentially affects in vivo CD4+ and CD8+ T cell activation and plays a detrimental role in innate immunity to Listeria monocytogenes infection in mice

Abstract

The inducible transcription factor NF-κB is involved in the regulation of a plethora of immunological processes and thus a tight regulation of NF-κB has to be ensured. This is governed by classical (e.g. IκBα, IκBβ, IκBɛ) and atypical (e.g. Bcl-3, IκBζ, IκBNS) inhibitory κB proteins. The protein IκBNS can act as activator as well as suppressor of NF-κB mediated gene expression in the nucleus. Most scientific work to elucidate the role of IκBNS in the regulation of immune responses done so far was performed under in vitro or ex vivo conditions, while the impact of IκBNS in controlling in vivo immune responses during systemic infections still remains elusive. The first part of this thesis addressed the role of IκBNS during antigen-specific activation of CD4+ and CD8+ T effector cells. For this a model system based on the adoptive transfer of ovalbumin (OVA)-specific T cells in combination with OVA-expressing Listeria monocytogenes as antigen-specific in vivo stimulus was used. This experimental approach revealed that the antigen-specific activation of CD4+ T cells following in vivo pathogen encounter strongly relies on IκBNS. The differentiation into Th1 effector cells was affected as indicated by a significantly reduced proliferation, marked changes in expression of activation markers and reduced IFNγ and IL2 production in CD4+ T cells lacking IκBNS. The pathogen-specific in vivo activation of CD8+ T cells was less affected by IκBNS-deficiency, and here especially proliferation and secretion of IFNγ does not depend on IκBNS. Since time-dependent differences were observed with respect to the expression of CD25, PD1 and TNFα between both genotypes, a slightly delayed activation program in CD8+ T cells lacking IκBNS might be reasonable. Despite the aforementioned differences between both genotypes, IκBNS-deficiency did not affect the capacity of CD8+ T cells to establish in vivo cytotoxic T cell responses. The second part of the thesis focused on the impact of IκBNS on innate immune responses towards LM infection. IκBNS-proficient (WT) mice succumbed within few days post high-dose LM infection while IκBNS-deficient (KO) mice were completely protected. Histological examination revealed a mitigated immunopathology 4 days post LM infection in liver and spleen samples of KO compared to WT mice, which was however not the consequence of an improved pathogen clearance but rather due to an overall blunted inflammatory immune response induced in mice lacking IκBNS. This hypothesis was further supported by comprehensive genome-wide transcriptome analyses of LM infected livers, which confirmed reduced numbers and reduced expression levels of inflammation-associated genes in KO compared to WT mice. In-depth flow cytometric analysis of the immune cell composition in spleen and liver in concert with their cell-specific IκBNS-promoter activity during the course of LM infection revealed that IκBNS expression in inflammatory monocytes might represent one of the key factors responsible for inducing hyperinflammation during high-dose LM infection. This was further supported by data obtained from infection experiments done in conditional KO mice lacking IκBNS specifically in monocytes, macrophages and neutrophils. In summary, IκBNS was proven to be detrimental during systemic LM infection by promoting a transcriptional program resulting in severe hyperinflammation, which ultimately results in fatal disease course.