ObjectivesGenetic variations in TNFAIP3 (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis.MethodsCRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A KI cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926.ResultsGenetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes.ConclusionsWe propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation.

The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) regulates nuclear-factor-kappa-B (NF-κB) activation downstream of surface receptors with immunoreceptor tyrosine-based activation motifs (ITAMs), such as the B-cell or Tcell receptor and has thus emerged as a therapeutic target for autoimmune diseases. However, recent reports demonstrate the development of lethal autoimmune inflammation due to the excessive production of interferon gamma (IFN-ɣ) and defective differentiation of regulatory T-cells in genetically modified mice deficient in MALT1 paracaspase activity. To address this issue, we explored the effects of pharmacological MALT1 inhibition on the balance between T-effector and regulatory T-cells. Here we demonstrate that allosteric inhibition of MALT1 suppressed Th1, Th17 and Th1/Th17 effector responses, and inhibited T-cell dependent B-cell proliferation and antibody production. Allosteric MALT1 inhibition did not interfere with the suppressive function of human T-regulatory cells, although it impaired de novo differentiation of regulatory T-cells from naïve T-cells. Treatment with an allosteric MALT1 inhibitor alleviated the cytokine storm, including IFN-ɣ, in a mouse model of acute Tcell activation, and long-term treatment did not lead to an increase in IFN-ɣ producing CD4 cells or tissue inflammation. Together, our data demonstrate that the effects of allosteric inhibition of MALT1 differ from those seen in mice with proteolytically inactive MALT1, and thus we believe that MALT1 is a viable target for B and T-cell driven autoimmune diseases.

BackgroundIL-6 trans-signalling (IL-6TS) is emerging as a pathogenic mechanism in chronic respiratory diseases, however the drivers of IL-6TS in the airways and the phenotypic characteristic of patients with increased IL-6TS pathway activation remain poorly understood.ObjectiveOur aim was to identify and characterize COPD patients with increased airway IL-6TS and to elucidate the biological drivers of IL-6TS pathway activation.MethodsWe used an IL-6TS-specific sputum biomarker profile (sIL-6R, IL-6, IL-1β, IL-8, MIP-1β) to stratify sputum data from patients with COPD (n=74; BEAT-COPD) by hierarchical clustering. The IL-6TS signature was related to clinical characteristics and sputum microbiome profiles. The induction of neutrophil extracellular trap formation (NETosis) and IL-6TS by Haemophilus influenzae were studied in human neutrophils.ResultsHierarchical clustering revealed an IL-6TS-high subset (n=24) of COPD patients, which shared phenotypic traits with an IL-6TS-high subset previously identified in asthma. The subset was characterized by increased sputum cell counts (p=0.0001), persistent sputum neutrophilia (p=0.0004), reduced quality of life (CRQ total score; p=0.008), and increased levels of pro-inflammatory mediators and MMPs in sputum. IL-6TS-high COPD patients showed an increase in Proteobacteria, with Haemophilus as the dominating genus. NETosis induced by H. influenzae was identified as a potential mechanism for increased soluble IL-6 receptor (sIL-6R) levels. This was supported by a significant positive correlation between sIL-6R and NETosis markers in bronchoalveolar lavage fluid from COPD patients.ConclusionIL-6TS pathway activation due to chronic colonization with Haemophilus may be an important disease driver in a subset of COPD patients.

SummaryThere is a current lack of effective mucosal vaccines against major gastroenteric pathogens and particularly against Helicobacter pylori, which causes a chronic infection that can lead to peptic ulcers and gastric cancer in a subpopulation of infected individuals. Mucosal CD4 + T-cell responses have been shown to be essential for vaccine-induced protection against H. pylori infection. The current study addresses the influence of the adjuvant and site of mucosal immunization on early CD4 + T-cell priming to H. pylori antigens. The vaccine formulation consisted of H. pylori lysate antigens and mucosal adjuvants, cholera toxin (CT) or a detoxified double-mutant heat-labile enterotoxin from Escherichia coli (dmLT), which were administered by either the sublingual or intragastric route. We report that in vitro, adjuvants CT and dmLT induce up-regulation of proinflammatory gene expression in purified dendritic cells and enhance the H. pylori-specific CD4 + T-cell response including interleukin-17A (IL-

Bleomycin hydrolase (BLMH) is a well-conserved cysteine protease widely expressed in several mammalian tissues. In skin, which contains high levels of BLMH, this protease is involved in the degradation of citrullinated filaggrin monomers into free amino acids important for skin hydration. Interestingly, the expression and activity of BLMH is reduced in patients with atopic dermatitis (AD) and psoriasis, and BLMH knockout mice acquire tail dermatitis. Apart from its already known function, we have discovered a novel role of BLMH in the regulation of inflammatory chemokines and wound healing. We show that lowered BLMH levels in keratinocytes result in increased release of the pro-inflammatory chemokines CXCL8 and GROα, which are upregulated in skin from AD patients compared to healthy individuals. Conditioned media from keratinocytes expressing low levels of BLMH increased chemotaxis by neutrophils and caused a delayed wound healing in the presence of low-level TNFα. This defective wound healing was improved by blocking the shared receptor of CXCL8 and GROα, namely CXCR2, using a specific receptor antagonist. Collectively, our results present a novel function of BLMH in regulating the secretion of chemokines involved in inflammation and wound healing in human keratinocytes.