BACKGROUND AND PURPOSEPhagocyte function is critical for host defense against infections. Defects in phagocytic function lead to several primary immunodeficiencies characterized by early onset of recurrent and severe infections. In this work, we further investigated the effects of BAY 41-2272, a soluble guanylate cyclase (sGC) agonist, on the activation of human peripheral blood monocytes (PBM) and THP-1 cells. EXPERIMENTAL APPROACHTHP-1 cells and PBM viability was evaluated by methylthiazoletetrazolium assay; reactive oxygen species production by lucigenin chemiluminescence; gene and protein expression of NAPDH oxidase components by qRT-PCR and Western blot analysis, respectively; phagocytosis and microbicidal activity by co-incubation, respectively, with zymosan and Escherichia coli; and cytokine release by ELISA. KEY RESULTSBAY 41-2272, compared with the untreated group, increased spreading of monocytes by at least 35%, superoxide production by at least 50%, and gp91 PHOX and p67 PHOX gene expression 20 to 40 times, in both PBM and THP-1 cells. BAY 41-2272 also augmented phagocytosis of zymosan particles threefold compared with control, doubled microbicidal activity against E. coli and enhanced the release of TNF-a and IL-12p70 by both PBM and THP-1 cells. Finally, by inhibiting sGC with ODQ, we showed that BAY 41-2272-induced superoxide production and phagocytosis is not dependent exclusively on sGC activation. CONCLUSIONS AND IMPLICATIONSIn addition to its ability to induce vasorelaxation and its potential application for therapy of vascular diseases, BAY 41-2272 was shown to activate human mononuclear phagocytes. Hence, it is a novel pro-inflammatory drug that may be useful for controlling infections in the immunocompromised host. AbbreviationsBAY 41-2272, 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo [3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine; BHI, brain heart infusion broth; CFU, colony-forming unit; CGD, chronic granulomatous disease; DMSO, dimethyl sulfoxide; EPEC, enteropathogenic Escherichia coli; FHS, fresh human serum; IL-12p70, IL-12 subunit p70; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ODQ, 1H-(1,2,4) oxadiazole (4,3-alpha) quinoxaline-1-one; PBM, peripheral blood monocytes; PBMC, peripheral blood monocyte cell; PMA, phorbol myristate acetate; ROS, reactive oxygen species; sGC, soluble guanylyl cyclase BJP British Journal of Pharmacology

Purpose About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. Methods Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. Results VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. Conclusions Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination. Graphical abstract

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BackgroundCD40 ligand (CD40L) deficiency predisposes to opportunistic infections including those caused by fungi and intracellular bacteria. Studies of CD40L-deficient patients reveal the critical role of CD40L-CD40 interaction for the function of T, B, and dendritic cells. However, the consequences of CD40L deficiency on macrophage function remain poorly understood.ObjectivesTo analzye the role of CD40L-CD40 interaction in macrophages immune response.MethodsMDMs were challenged with Paracoccidioides brasiliensis (Pb18, a highly virulent isolate) and the microbicidal activity determined by counting colony forming units (CFU). MDMs oxidative burst was measured by luminol-dependent chemiluminescence (LDCL). Supernatants of macrophages treated or untreated with rhIFN-γ or sCD40L were harvested 48 hours after P. brasiliensis or M. tuberculosis incubation and the Cytokine levels were evaluated by luminex. The analysis of M. tuberculosis (H37Rv strain) phagocytosis and proliferation control by MDMs were carried out by CFU counting. Macrophage transcriptome profiles from three CD40L deficient patients and three healthy controls were analyzed by RNAseq. All the experiments were performed before and after IFN-gamma in vitro treatment.ResultsHere we show that macrophages from CD40L-deficient patients exhibit defective fungicidal activity and reduced oxidative burst, which improved in the presence of IFN-γ but not soluble CD40L (sCD40L). In contrast, both IFN-γ and sCD40L ameliorate impaired production of multiple inflammatory cytokines. Comparing the gene expression signature of macrophages from CD40L-deficient patients to that of controls, we identified 109 differentially expressed genes (DEGs). In vitro treatment of macrophages with IFN-γ decreased the number of DEGs to only 11, and similarly affected the expression of 528 non-DEGs (140 genes down-regulated and 388 up-regulated) comparing patients and healthy controls. Finally, the addition of IFN-γ reversed defective control of M. tuberculosis proliferation by patient macrophages.ConclusionsOur findings demonstrate that absence of CD40L impairs macrophage development and function. In addition, the improvement of macrophage immune responses by IFN-γ suggests this cytokine as a potential new therapeutic option for patients with CD40L deficiency.Disclosure of InterestNone declared

Introduction: Yellow Fever (YF) vaccine is a golden standard immunobiological in terms of effectiveness and safety. Even though, rare cases of neurological or viscerotropic adverse events following immunization (YEL-AD) occurs few days after vaccination due to Human Inborn Errors of Innate Immunity (HIEII). Previous works have characterized YEL-AD cases presented mutations within IFNAR1 gene, or autoantibodies anti-IFNα, impairing type I interferon (IFN) response, the main antiviral pathway and innate hub. From the immunological point of view, it is known that YEL-AD lead to normal humoral responses, therefore cellular innate functional investigations are mandatory to understand the consequences of HIEII and help to formulate therapeutic strategies. Here, we report an analysis of innate cells, innate immune mediator's profile, and transcriptomics in cases from a national-based, YEL-AVD phase IV study.Objective: To investigate the response against YF vaccine in YEL-AVD cases focusing on innate immunologic parameters: Natural killer (NK) cells and monocyte phenotypes, the production of the immune mediator's, and transcriptomic profile.Methodology: Here, five subjects had blood samples collected 1-2 years after YEL-AD using the Brazilian vaccine (17DD) (CAAE 60575716.2.0000.5262). Peripheral Blood Mononuclear Cells (PBMC) from YEL-AD cases and ten controls at the same time post-vaccination were used to perform in vitro stimulation with attenuated YF virus, followed by immunophenotyping, Luminex assay, and RNA sequencing.Results: At this moment, we have four cases of YEL-AD viscerotropic disease, one presenting IFNAR1 deficiency, and two presenting anti-IFNα autoantibodies, and one case of neurological disease. The YEL-AD cases presented constitutive disturbances compared with controls: high percentage of NKbright (mean: 7.04 vs 2.26, p=0.04), low NKT cells (0.61 vs 2.41, p=0.03), and no detectable production of CXCL10. After YF viral stimulation the YEL-AD cases presented higher frequencies of cytotoxic NKdim cells (2.18 vs 0.60, p=0.057), and non-classical monocytes (3.03 vs 1.21, p= 0.018), accompanied by an abnormal high IL1β response (9.25 vs 0.42, p= 0.02) when compared to healthy vaccinated controls. Moreover, transcriptomic analysis after YV in vitro stimulation demonstrated that YEL-AD cases present abnormal expression of genes related with IFN pathway, chemokine signaling and antigen processing and presentation. Conclusion:Our results showed that YEL-AD has severe phenotypic innate defects. First, the remarkable HIEII in IFN pathway contribute to a deficient antiviral response, reflected in the high viral loads observed in the YEL-AD. Further, the high inflammatory environment reflected in the levels of IL1β, may contribute to NK cytotoxicity, monocytes with proinflammatory profile. Hence, despite the low sample size, here we observed a functional imbalance between inborn deficient IFN antiviral response and the abnormal inflammatory cellular profile observed in YEL_AD cases, which could lead to a non-effec...

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