Post-translational modification pathways such as SUMOylation are integral to all cellular processes and tissue homeostasis. We investigated the possible involvement of SUMOylation in the epithelial signalling in Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD). Initially in a murine model of IBD, induced by dextran–sulfate–sodium (DSS mice), we observed inflammation accompanied by a lowering of global SUMOylation of colonic epithelium. The observed SUMOylation alteration was due to a decrease in the sole SUMO E2 enzyme (Ubc9). Mass-spectrometric analysis revealed the existence of a distinct SUMOylome (SUMO-conjugated proteome) in DSS mice with alteration of key cellular regulators, including master kinase Akt1. Knocking-down of Ubc9 in epithelial cells resulted in dramatic activation of inflammatory gene expression, a phenomenon that acted via reduction in Akt1 and its SUMOylated form. Importantly, a strong decrease in Ubc9 and Akt1 was also seen in endoscopic biopsy samples (N = 66) of human CD and UC patients. Furthermore, patients with maximum disease indices were always accompanied by severely lowered Ubc9 or SUMOylated-Akt1. Mucosal tissues with severely compromised Ubc9 function displayed higher levels of pro-inflammatory cytokines and compromised wound-healing markers. Thus, our results reveal an important and previously undescribed role for the SUMOylation pathway involving Ubc9 and Akt1 in modulation of epithelial inflammatory signalling in IBD.

Summary Activation of B and T lymphocytes leads to major remodelling of the metabolic landscape of the cells enabling their post‐activation functions. However, naive B and T lymphocytes also show metabolic differences, and the genesis, nature and functional significance of these differences are not yet well understood. Here we show that resting B‐cells appeared to have lower energy demands than resting T‐cells as they consumed lower levels of glucose and fatty acids and produced less ATP. Resting B‐cells are more dependent on OXPHOS, while T‐cells show more dependence on aerobic glycolysis. However, despite an apparently higher energy demand, T lineage cells showed lower rates of protein synthesis than equivalent B lineage stages. These metabolic differences between the two lineages were established early during lineage differentiation, and were functionally significant. Higher levels of protein synthesis in B‐cells were associated with increased synthesis of MHC class II molecules and other proteins associated with antigen internalization, transport and presentation. The combination of higher energy demand and lower protein synthesis in T‐cells was consistent with their higher ATP‐dependent motility. Our data provide an integrated perspective of the metabolic differences and their functional implications between the B and T lymphocyte lineages.

Lymphotoxin-beta receptor (LTβR) present on stromal cells engages the noncanonical NF-κB pathway to mediate RelB-dependent expressions of homeostatic chemokines, which direct steady-state ingress of naïve lymphocytes to secondary lymphoid organs (SLOs). In this pathway, NIK promotes partial proteolysis of p100 into p52 that induces nuclear translocation of the RelB NF-κB heterodimers. Microbial infections often deplete homeostatic chemokines; it is thought that infection-inflicted destruction of stromal cells results in the downregulation of these chemokines. Whether inflammation also regulates these processes remains unclear. We show that TNF accumulated upon non-infectious immunization of mice similarly downregulates the expressions of these chemokines and consequently diminishes the ingress of naïve lymphocytes in inflamed SLOs. Mechanistically, TNF inactivated NIK in LTβR-stimulated cells and induced the synthesis of mRNA encoding p100; these together potently accumulated unprocessed p100, which attenuated the RelB activity as inhibitory IκBδ. Finally, a lack of p100 alleviated these TNF-mediated inhibitions in inflamed SLOs of immunized mice. In sum, we reveal that an inhibitory TNF-p100 pathway modulates the adaptive compartment during immune responses.

SummaryActivated T‐cells make both interleukin‐2 (IL2) and its high‐affinity receptor component CD25. Regulatory CD4 T‐cells (Treg cells) do not make IL2, and the IL2‐CD25 circuit is considered a paracrine circuit crucial in their generation and maintenance. Yet, all T‐cells are capable of making IL2 at some stage during differentiation, making a cell‐intrinsic autocrine circuit additionally possible. When we re‐visited experiments with mixed bone marrow chimeras using a wide range of ratios of wild‐type (WT) and IL2−/− genotype progenitors, we found that, as expected, thymic Treg cells were almost equivalent between WT and IL2−/− genotypes at ratios with WT prominence. However, at WT‐limiting ratios, the IL2−/− genotype showed lower thymic Treg frequencies, indicating a role for cell‐intrinsic autocrine IL2 in thymic Treg generation under IL2‐limiting conditions. Further, peripheral IL2−/− naive CD4 T‐cells showed poor conversion to inducible Tregs (pTregs) both in vivo and in vitro, again indicating a significant role for cell‐intrinsic autocrine IL2 in their generation. Peripherally, the IL2−/− genotype was less prominent at all WT:IL2−/− ratios among both thymic Tregs (tTregs) and pTregs, adoptively transferred IL2−/− Tregs showed poorer survival than WT Tregs did, and RNA‐seq analysis of WT and IL2−/− Tregs showed interesting differences in the T‐cell receptor and transforming growth factor‐beta‐bone morphogenetic protein‐JNK pathways between them, suggesting a non‐titrating role for cell‐intrinsic autocrine IL2 in Treg programming. These data indicate that cell‐intrinsic autocrine IL2 plays significant roles in Treg generation and maintenance.

The studies described here provide an analysis of the pathogenesis of Blau syndrome and thereby the function of NOD2 as seen through the lens of its dysfunction resulting from Blau-associated NOD2 mutations in its nucleotidebinding domain (NBD). As such, this analysis also sheds light on the role of NOD2 risk polymorphisms in the LRR domain occurring in Crohn's disease. The main finding was that Blau NOD2 mutations precipitate a loss of canonical NOD2 signaling via RIPK2 and that this loss has two consequences: first, it results in defective NOD2 ligand (MDP)-mediated NF-kB activation and second, it disrupts NOD2-mediated cross-regulation whereby NOD2 downregulates concomitant innate (TLR) responses. Strong evidence is also presented favoring the view that NOD2-mediated cross-regulation is under mechanistic control by IRF4 and that failure to up-regulate this factor because of faulty NOD2 signaling is the proximal cause of defective cross-regulation and the latter's effect on Blau syndrome inflammation. Overall, these studies highlight the role of NOD2 as a regulatory factor and thus provide additional insight into its function in inflammatory disease. Mutations in the nucleotide binding domain of the CARD15 (NOD2) gene underlie the granulomatous inflammation characterizing Blau syndrome (BS). In studies probing the mechanism of this inflammation we show here that NOD2 plasmids expressing various Blau mutations in HEK293 cells result in reduced NOD2 activation of RIPK2 and correspondingly reduced NOD2 activation of NF-kB. These in vitro studies of NOD2 signaling were accompanied by in vivo studies showing that BS-NOD2 also exhibit defects in cross-regulation of innate responses underlying inflammation. Thus, whereas over-expressed intact NOD2 suppresses TNBS-colitis, over-expressed BS-NOD2 does not; in addition, whereas administration of NOD2 ligand (muramyl dipeptide, MDP) suppresses DSS-colitis in Wild Type (WT) mice it fails to do so in homozygous or heterozygous mice bearing a NOD2 Blau mutation. Similarly, mice bearing a Blau mutation exhibit enhanced anti-collagen antibody-induced arthritis. The Frontiers in Immunology frontiersin.org 01