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Immune regulation of NF-kappaB in Dendritic Cell subsets by the ubiquitin editing enzyme A20

Final Report Summary - A20 DC SUBSETS (Immune regulation of NF-kappaB in Dendritic Cell subsets by the ubiquitin editing enzyme A20)

The main role of the immune system is to protect our body from infection. The immune system is divided into two major branches: the innate immune system and the adaptive immune system. The innate immune system is nonspecific as to the type of organism it fights and is ready to be mobilized upon the first signs of infection, which will be within minutes or hours after infection. The dendritic cell (DC) is pivotal in this response. It senses components of bacteria, viruses, parasites, and fungi through its so-called pathogen-recognition receptors (PPRs). PPRs recognize molecular patterns found in microorganisms. Triggering the PRRs not only activates innate immunity, it also shapes the character of the ensuing adaptive immune response. DCs are seen as nature’s adjuvant and have the potential to recognize foreign antigen, process it into small peptides for presentation onto Major Histocompatibility Complex (MHC) molecules to the T cell receptor (TCR), and to provide the essential costimulatory molecules for activation of naïve CD4+ and CD8+ T cells. DCs express virtually all PRRs and PRR activation induces their functional maturation. PRR triggering leads to the production of polarizing cytokines by DCs that induce differentiation of T cells into either Th1 cells (IFNγ, IL-2 production), Th2 cells (IL-4, IL-5, IL-13 production), or Th17 cells (IL-17, IL-21, IL-22 production). DCs are heterogeneous and can be subdivided by anatomical location, origin, and function. In lungs of mice, several DC subsets can be found; CD103+ XCR1+ cDCs (cDC1), Sirpα+ CD11b+ cDCs (cDC2), pDCs, and during inflammation also monocyte-derived DCs (mo-DCs). House dust mite (HDM)-driven mouse models for allergic asthma have shown that cDC2s are essential for Th2 priming, whereas mo-DCs amplify inflammatory responses by chemokine secretion. There is contradiction about the role of cDC1s in the Th2-mediated response. DCs can be activated by PRR triggering, or by cytokines such as IL-1 and TNFα via their respective receptors. Activation of these receptors leads to the production of proinflammatory cytokines, such as IL-6, IL-1β, IL-12 and TNFα. Crucial to the regulation of these cytokines is the activation and nuclear translocation of the key transcription factor NF-κB. NF-κB proteins control the expression of multiple genes involved in immune responses. Since NF-κB is so crucial, not surprisingly its activation is tightly controlled by several mechanisms. In one of the mechanisms the ubiquitin-modifying enzyme A20/TNFAIP3 plays a crucial role. A20 down-regulates NF-κB signalling through the cooperative activity of its two ubiquitin-editing domains.
Using Next-Generation RNA-sequencing, we have compared A20 knock-out (KO), heterozygote (Hz) and control wild-type (WT) in-vitro GM-CSF cultured DCs. Strikingly, A20KO-DCs were completely separate from A20Hz-DCs, which were closely related to WT-DCs. Comparing A20KO-DCs to WT-DCs revealed more than 2000 genes, which were more than 2-fold up- or down-regulated. These included genes involved in Th17-cell differentiation (e.g. IL-6, IL-1b, IL-12), B cell activation (e.g. IL-6, APRIL, BAFF), chemokines for neutrophil attraction (e.g. CXCL3), and several growth factors (e.g. FGF-2, VEGF). Several targets were validated based on protein expression. In-vitro cultures of A20KO-DCs with naïve Th-cells induced IL-17-producing Th-cells compared to Th-cell cultures with WT-DCs, where Th2-cells were induced. Th-cell activation/differentiation was enhanced when A20KO-DCs were activated using either PPR ligands (e.g. LPS/CpG motifs) or HDM. This also strongly depended on the DC-subset used, as A20-deficient monocyte-derived DCs were superior in Th17-cell induction in-vitro compared A20-deficient cDC1/cDC2.
Next, using the mouse Cre/LoxP technology we examined the effect of A20 deficiency in several DC subsets. We have crossed A20/Tnfaip3 floxed mice to different cre-recombinase mice, each targeting other DC subsets. Cd11c-cre Tnfaip3fl/fl mice (targeting all DC subsets and alveolar macrophages) and LysM-cre Tnfaip3fl/fl mice (targeting monocyte, mo-DCs, macrophages, neutrophils, and peripheral DCs) were already extensively studied for their spontaneous activation of the immune system. However, as A20KO-DCs could directly activate B-cells, we wondered whether the autoimmune phenotype seen in Cd11c-cre Tnfaip3fl/fl mice would be caused by direct DC-B cell interaction. To evaluate this, we crossed Cd11c-cre Tnfaip3fl/fl mice to Cd40lKO mice, in which T-B cell communication cannot occur. T-B cell communication is crucial for a proper germinal center response, where B cell activation and selection occurs. Surprisingly and supporting our hypothesis, Cd11c-cre Tnfaip3fl/fl X Cd40lKO mice still exhibited spontaneous B cell activation, although to a lesser degree than Cd11c-cre Tnfaip3fl/fl mice but significantly increased compared to CD40LKO mice. Cd11c-cre Tnfaip3fl/fl X Cd40lKO mice have similar increase in plasma cells (antibody-producing cells) as Cd11c-cre Tnfaip3fl/fl mice, however for immunoglobulin class-switching T-B cell communication is crucial.
Surprisingly, the Th2-promoting chronic HDM-model did not induced Th2-responses in Cd11c-cre Tnfaip3fl/fl mice and LysM-cre Tnfaip3fl/fl mice; rather they developed strong Th17-cell induction, also seen in severe asthma patients. Resembling the severe phenotype seen in asthma patients, HDM-exposed LysM-cre Tnfaip3fl/fl mice were unresponsive for steroid treatment, in contrast to control mice. Detailed analysis showed that specific A20-deficient mo-DCs are the superior Th17-inducing cells, through their secretion of IL-6, IL-1b and TGFb. Neutrophilic inflammation is characteristic for the Th17-mediated asthma response, thus we analyzed LysM-cre Tnfaip3fl/fl X Il17raKO mice, where IL-17 signaling is abrogated. Surprisingly, after exposing these mice to our HDM-model, they still developed lung neutrophilia. Our RNA-seq data showed that A20KO-DCs have high expression of CXCL3, which also attracts neutrophils in an IL-17-independent manner.
To study the effect of A20 deficiency in lung cDC1s and skin Langerhans cells, we have used Langerin-cre Tnfaip3fl/fl mice, which did not develop spontaneous immune activation. As polymorphisms in TNFAIP3 have been found in psoriasis and Langerhans cells play an important role in this disease, I hypothized that Langerin-cre Tnfaip3fl/fl mice would develop psoriasis-like disease. However, this did not occur, even when mice were 24 weeks of age. Rather regulatory mechanisms were induced at baseline as numbers of regulatory T cells (Tregs) and IL-10+ CD8+ T cells were increased in Langerin-cre Tnfaip3fl/fl mice. To evaluate the specificity of Langerin-cre, we crossed Langerin-cre Tnfaip3fl/fl mice to ROSA-Stopfl/fl-YFP mice, showing Langerin promotor activity by YFP expression. These studies revealed that the Langerin promotor targeted indeed only lung cDC1s, however strikingly only 10-20% of the lung cDC1s were affected. We have exposed these Langerin-cre Tnfaip3fl/fl mice to our HDM model for allergic asthma and influenza infection. Langerin-cre Tnfaip3fl/fl mice were protected from Th2-associated immune responses, but were superior in clearing influenza infection compared to control mice. The decreased Th2 immune response is not caused by the increased Tregs, however, the exact mechanism is unclear at the moment.
Lastly, to examine whether cDC1 and cDC2 activation causes immune activation, without affecting mo-DCs, we crossed Tnfaip3fl/fl mice to Dngr1-cre mice, where 95% of cDC1s and 25% of cDC2s are affected. Dngr1-cre Tnfaip3fl/fl mice did not develop systemic autoimmunity as seen in Cd11c-cre Tnfaip3fl/fl mice, strikingly they showed specific phenotype in the lung and liver. They developed pulmonary hypertension, shown by increased right ventricle systolic pressure (RSVP), a hallmark of pulmonary hypertension and lung arterial remodeling and lung tertiary lymphoid organs, with activated T cells. Liver pathology resembles Primary Biliary Cholangitis, with immune infiltrates of CD8+ T cells and B cells around the portal vessels and giant cell formation.
Our data indicates that each DC subset has their specific role in maintaining immune homeostasis, but their role during disease. DC subset specific activation is crucial in the subsequent immune response, as Langerin+ cDC1s promote regulatory CD4+ and CD8+ T cells, but also are very potent in promoting anti-virus responses. In contrast, mo-DCs are efficient Th17-cell inducers and cDC1/2 promote organ specific immune responses. Further and detailed analyses are required to understand fundamental processes of DC biology but also to design DC subset specific targeting strategies. The latter could lead to possible new therapies for immune-driven diseases.