Konstantinos Ritis

Neutrophils interact with and activate fibroblasts through the release of neutrophil extracellular traps (NETs). We investigated the role of NETs‐fibroblast crosstalk in the cutaneous wound healing of type 2 diabetes (T2D). Neutrophils/NETs, serum, and primary human skin fibroblasts (HSFs) were obtained from individuals with T2D and age/sex‐matched controls. NET‐stimulation studies were performed on neutrophils/HSFs, with and without specific inhibitors, while HSF healing capacity was assessed using a scratch wound healing assay. T2D HSFs display a profibrotic phenotype, showing increased CCN2/CTGF, α‐smooth muscle actin, and collagen release, albeit with impaired healing capacity, elevated type I collagen C‐terminal telopeptide, and collagen degradation associated with increased (∼3.5‐fold) matrix metalloproteinase‐9 (MMP‐9) in T2D neutrophils/NETs. IL‐8 induced the expression of MMP‐9 in neutrophils/NETs. Moreover, T2D neutrophils/NETs exhibited increased IL‐8 content, which acted in an autocrine/paracrine fashion to further augment its production by neutrophils/HSFs. The findings were validated in normoglycemic individuals during a hyperglycemic clamp with concomitant lipid infusion and further corroborated immunohistochemically in diabetic plantar ulcer biopsies. This novel, vicious circle of NETs/interleukin‐8/MMP‐9/HSFs was hindered by IL‐8 or MMP‐9 blockade via specific inhibitors or by dismantling the NET‐scaffold with DNase I, suggesting candidate therapeutic targets in wound healing impairment of T2D.

Introduction: Crohn’s disease (CD) is characterized by chronic inflammation and intestinal fibrosis leading to lifelong complications. However, the disease pathogenesis remains elusive, and the therapeutic options are limited. Here, we investigated the interaction between neutrophils and intestinal fibroblasts in the development of CD immunofibrosis, a disease mechanism predisposing to inflammatory and fibrotic complications.Methods: Peripheral neutrophils, enriched neutrophil extracellular traps (eNETs), serum, primary intestinal fibroblasts (PIFs) and intestinal biopsies from CD, ulcerative colitis (UC) patients, and healthy individuals (HI), were studied. Transcriptome analysis of neutrophils, multi-cytokine profiling and cell-based functional assays at mRNA/protein level were performed.Results: Compared to UC, PIFs from CD patients, independently to the presence of strictures, displayed a distinct pro-fibrotic phenotype characterized by negative Krüppellike Factor-2 (KLF2) and increased cellular communication network factor-2 (CCN2) expression leading to collagen production. In both UC and CD, PIFs-derived IL-8 acted as a culprit chemoattractant for neutrophils in the intestine, where CD neutrophils were accumulated close to fibrotic lesions. Functionally, only CD neutrophils via eNETs induced a CD-like phenotype in HI PIFs, suggesting their fibrotic plasticity. High IFNa in serum and IFΝ-responsive signature in peripheral neutrophils were observed in CD, distinguishing it from UC. Moreover, CD serum stimulated the release of fibrogenic eNETs from neutrophils in an IFNa-dependent manner, suggesting the priming role of IFNa in circulating neutrophils. Inhibition of eNETs or JAK signaling in neutrophils or PIFs prevented the neutrophil-mediated fibrotic effect on PIFs. Furthermore, both serum IFNa levels and mRNA levels of key IFN signaling components in neutrophils were wellcorrelated with CD severity.Conclusions: This study reveals the important role of the IFNa/neutrophil/fibroblast axis in CD immunofibrosis, suggesting candidate biomarkers and putative therapeutic targets.

This study investigated the interaction between neutrophils and intestinal fibroblasts in Crohn’s disease (CD) immunofibrosis. Peripheral neutrophils, enriched-neutrophil extracellular traps (eNETs), serum, primary intestinal fibroblasts (PIFs) and intestinal biopsies were studied. Neutrophils’ RNA-sequencing, multi-cytokine profiling and cell-based functional assays at mRNA/protein level were performed. Compared to ulcerative colitis (UC), PIFs from CD patients displayed a distinct fibrotic phenotype characterized by negative Krüppel-like Factor-2 (KLF2) and increased cellular communication network factor-2 (CCN2) expression leading to collagen production. PIFs-derived IL-8 appears as a culprit chemoattractant of neutrophils in the intestine, where CD neutrophils were accumulated close to fibrotic lesions. Functionally, only CD neutrophils via eNETs can induce a CD-like phenotype in HI PIFs. High serum IFNα and IFΝ-responsive signature in neutrophils were observed in CD, distinguishing it from UC. Moreover, CD serum can stimulate the release of fibrogenic eNETs in an IFNα-dependent manner, suggesting the priming role of IFNα in circulating neutrophils. Inhibition of eNETs or JAK signaling in neutrophils or PIFs prevented the neutrophil-mediated fibrotic effect on PIFs. Furthermore, serum IFNα and transcripts of key IFN-signaling components in neutrophils were well-correlated with CD severity. This study reveals the role of IFNα/neutrophil/fibroblast axis in CD immunofibrosis, suggesting candidate biomarkers and therapeutic targets.

Background and Aims: Several studies have suggested that mutations in MEFV, the gene responsible for familial Mediterranean fever (FMF), are frequently detected in inflammatory bowel disease (IBD) patients. We aimed to provide further evidence regarding a potential correlation between MEFV gene mutations and IBD by identifying all relevant studies and analyzing their results. Methods: EMBASE, PubMed/MEDLINE, and Google Scholar were used to identify all studies that published until January 2021 and reported MEFV mutation patterns in patients with ulcerative colitis (UC), Crohn’s disease (CD) and indeterminate colitis (IC) with or without a control group. The Newcastle-Ottawa quality assessment scale was used to appraise the quality of the included studies. Results: Thirteen observational studies, including 937 patients and 977 controls, were analyzed. MEFV mutation rate in IBD patients was 0.238 (95%CI: 0.209-0.270; I 2 =95%); MEFV mutated alleles were more frequent in IBD patients when compared with controls (p=0.03 for UC, p=0.01 for CD and IC). Subgroup analysis indicated that MEFV mutations were increased in patients with IC when compared with UC and CD (I 2 =91%, p<0.001). Patients with extra-intestinal manifestations and pancolitis had 2.57 (95%CI 1.07-6.14; p=0.03) and 2.02 (95%CI: 1.01-4.04, P=0.049) odds ratios to carry MEFV mutant genotypes, respectively. Exon 10 mutations had the most serious impact. No source of heterogeneity was detected. Conclusions: MEFV mutations are common in IBD and are linked with the presence of extra-intestinal manifestations and pancolitis. Further research to assess the clinical significance and evolutionary significance of MEFV mutations in IBD patients is warranted.

COVID-19-related severe respiratory failure (SRF) leads to mechanical ventilation increasing the in-hospital mortality substantially. Abundancy of lung fibroblasts (LFs) in injured lung tissue has been associated with the progression of respiratory failure in COVID-19. Aiming to reduce mortality in patients with SRF (PaO2/FiO2<100 mmHg) and considering the multi-mechanistic nature of severe COVID-19 pathogenesis, we applied a combined rescue treatment (COMBI) on top of standard-of-care (SOC: dexamethasone and heparin) comprised inhaled DNase to dissolve thrombogenic neutrophil extracellular traps, plus agents against cytokine-mediated hyperinflammation, such as anti-IL-6 receptor tocilizumab and selective JAK1/2 inhibitor baricitinib. COMBI (n=22) was compared with SOC (n= 26), and with two previously and consecutively used therapeutic approaches, including either IL-1 receptor antagonist anakinra (ANA, n=19), or tocilizumab (TOCI, n=11), on top of SOC. In parallel, evaluation of immunothrombosis was assessed in vitro in human LFs, treated with the applied therapeutic agents upon stimulation with COVID-19 plasma. COMBI was associated with lower in-hospital mortality (p=0.014) and intubation rate (p=0.013), shorter duration of hospitalization (p=0.019), and prolonged overall survival after a median follow-up of 110±4 days (p=0.003). In vitro, COVID-19 plasma markedly induced tissue factor/thrombin pathway in LFs, while this effect was inhibited by the immunomodulatory agents of COMBI providing a mechanistic explanation for the clinical observations. These results suggest the design of randomized trials using combined immunomodulatory therapies in COVID-19-associated SRF targeting multiple interconnected pathways of immunothrombosis.

Innate immunity and chronic inflammation are involved in atherosclerosis and atherothrombosis, leading to target organ damage in essential hypertension (EH). However, the role of neutrophils in EH is still elusive. We investigated the association between angiotensin II (Ang II) and neutrophil extracellular traps (NETs) in pathogenesis of EH. Plasma samples, kidney biopsies, and surgical specimens of abdominal aortic aneurysms (AAAs) from patients with EH were used. Cell-based assays, NETs/human aortic endothelial cell cocultures, and in situ studies were performed. Increased plasma levels of NETs and tissue factor (TF) activity were detected in untreated, newly diagnosed patients with EH. Stimulation of control neutrophils with plasma from patients with untreated EH generated TF-enriched NETs promoting endothelial collagen production. Ang II induced NETosis in vitro via an ROS/peptidylarginine deiminase type 4 and autophagy-dependent pathway. Circulating NETs and thrombin generation levels were reduced substantially in patients with EH starting treatment with Ang II receptor blockers, whereas their plasma was unable to trigger procoagulant NETs. Moreover, TF-bearing NETotic neutrophils/remnants accumulated in sites of interstitial renal fibrosis and in the subendothelial layer of AAAs. These data reveal the important pathogenic role of an Ang II/ROS/NET/TF axis in EH, linking thromboinflammation with endothelial dysfunction and fibrosis.

Ankylosing spondylitis (AS) is an inflammatory disease characterized by excessive bone formation. We investigated the presence of neutrophil extracellular traps (NETs) in AS and how they are involved in the osteogenic capacity of bone marrow mesenchymal stem cells (MSCs) through interleukin‐17A (IL‐17A). Peripheral neutrophils and sera were obtained from patients with active AS and healthy controls. NET formation and neutrophil/NET‐associated proteins were studied using immunofluorescence, immunoblotting, qPCR and ELISA. In vitro co‐culture systems of AS NET structures and MSCs isolated from controls were deployed to examine the role of NETs in the differentiation of MSCs towards osteogenic cells. Analysis was performed using specific staining and qPCR. Neutrophils from patients with AS were characterized by enhanced formation of NETs carrying bioactive IL‐17A and IL‐1β. IL‐17A‐enriched AS NETs mediated the differentiation of MSCs towards bone‐forming cells. The neutrophil expression of IL‐17A was positively regulated by IL‐1β. Blocking IL‐1β signaling on neutrophils with anakinra or dismantling NETs using DNase‐I disrupted osteogenesis driven by IL‐17A‐bearing NETs. These findings propose a novel role of neutrophils in AS‐related inflammation, linking IL‐17A‐decorated NETs with the differentiation of MSCs towards bone‐forming cells. Moreover, IL‐1β triggers the expression of IL‐17A on NETs offering an additional therapeutic target in AS. This article is protected by copyright. All rights reserved