No abstract

IntroductionThe role of CD3−CD56+ natural killer (NK) cells in granulomatosis with polyangiitis (GPA) is poorly understood. Recently, it has been shown that peripheral blood NK cells can kill renal microvascular endothelial cells, suggesting a pathogenic role of NK cells in this disease. So far, subset distribution, phenotype, and function of peripheral blood NK cells in relation to GPA disease activity have not been elucidated. Moreover, it is not known whether NK cells infiltrate GPA tissue lesions.MethodsParaffin sections of GPA granulomas and controls were stained with anti-CD56 and anti-CD3 antibodies. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. NK cell degranulation was analyzed using cocultures of patient PBMCs with target cells and surface expression of CD107a. Clinical data were extracted from medical records. Statistical analysis was performed in an exploratory way.ResultsCD56+ cells were not detectable in active granulomatous GPA lesions but were found frequently in granulomas from tuberculosis and sarcoidosis patients. In GPA, the proportion of NK cells among peripheral blood lymphocytes correlated negatively with the Birmingham Vasculitis Activity Score (BVAS) (n = 28). Accordingly, NK cell percentages correlated positively with the duration of remission (n = 28) and were significantly higher in inactive GPA (BVAS = 0, n = 17) than in active GPA, healthy controls (n = 29), and inactive control diseases (n = 12). The highest NK cell percentages were found in patients with long-term remission and tapered immunosuppressive therapy. NK cell percentages >18.5 % of peripheral blood lymphocytes (n = 12/28) determined GPA inactivity with a specificity of 100 %. The differentiation into CD56dim and CD56bright NK cell subsets was unchanged in GPA (n = 28), irrespective of disease activity. Similar surface expression of the activating NK cell-receptors (NKp30, NKp46, and NKG2D) was determined. Like in healthy controls, GPA NK cells degranulated in the presence of NK cell receptor ligand bearing epithelial and lymphatic target cells.ConclusionsNK cells were not detectable in GPA granulomas. Peripheral blood NK cell percentages positively correlate with the suppression of GPA activity and could serve as a biomarker for GPA activity. Peripheral blood NK cells in GPA patients are mature NK cells with preserved immune recognition.

BackgroundRituximab has broad and increasing application in rheumatic diseases. It is known from lymphoma studies that natural killer (NK) cells can lyse rituximab-coated transformed B cells. However, the role of NK cells in mediating rituximab-induced depletion of non-malignant B cells is unknown. The purpose of this study was to provide fundamental data on rituximab-mediated effects on NK cells in PBMCs without tumor cells, in order to simulate effects that could be relevant in patients with rheumatic disease.MethodsFreshly isolated peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured overnight with therapeutic antibodies. NK cells were isolated using a commercial kit or depleted from PBMCs using anti-CD56 and anti-CD16 monoclonal antibodies and magnetic beads. Cells were analyzed by multicolor flow cytometry. Cytotoxicity assays were performed using 51Cr-labeled K562 target cells.ResultsAddition of rituximab to PBMCs resulted in depletion of B cells, which was dependent on NK cells and serum factors. The extent of B cell depletion correlated with the percentage of NK cells. Following incubation with rituximab, NK cells within PBMCs were activated, degranulated and downregulated the low affinitiy Fc-γ-receptor CD16 (FcγRIIIA). The co-activating receptor CD137 (41BB) was upregulated on a fraction of NK cells. NK cell function was altered in some donors in whom we observed rituximab-dependent reduction in NK cell cytotoxicity towards K562 tumor cells.ConclusionsNK cells mediate rituximab-induced B cell depletion. Rituximab induces altered NK cell phenotype and function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1101-3) contains supplementary material, which is available to authorized users.

Background A dysregulated glucose metabolism in synovial fibroblasts (SF) has been associated with their aggressive phenotype in rheumatoid arthritis (RA). Even though T helper (Th) cells are key effector cells in the propagation and exacerbation of synovitis in RA, little is known about their influence on the metabolism of SF. Thus, this study investigates the effect of Th cells on the glucose metabolism and phenotype of SF and how this is influenced by the blockade of cytokines, janus kinases (JAKs) and glycolysis. Methods SF from patients with RA or osteoarthritis (OA) were cultured in the presence of a stable glucose isotopomer ([U-13C]-glucose) and stimulated with the conditioned media of activated Th cells (ThCM). Glucose consumption and lactate production were measured by proton nuclear magnetic resonance (1H NMR) spectroscopy. Cytokine secretion was quantified by ELISA. The expression of glycolytic enzymes was analysed by PCR, western blot and immunofluorescence. JAKs were blocked using either baricitinib or tofacitinib and glycolysis by using either 3-bromopyruvate or FX11. Results Quiescent RASF produced significantly higher levels of lactate, interleukin (IL)-6 and matrix metalloproteinase (MMP) 3 than OASF. Stimulation by ThCM clearly changed the metabolic profile of both RASF and OASF by inducing a shift towards aerobic glycolysis with strongly increased lactate production together with a rise in IL-6 and MMP3 secretion. Interestingly, chronic stimulation of OASF by ThCM triggered an inflammatory phenotype with significantly increased glycolytic activity compared to unstimulated, singly stimulated or re-stimulated OASF. Finally, in contrast to cytokine-neutralizing biologics, inhibition of JAKs or glycolytic enzymes both significantly reduced lactate production and cytokine secretion by Th cell-stimulated SF. Conclusions Soluble mediators released by Th cells drive SF towards a glycolytic and pro-inflammatory phenotype. Targeting of JAKs or glycolytic enzymes both potently modulate SF’s glucose metabolism and decrease the release of IL-6 and MMP3. Thus, manipulation of glycolytic pathways could represent a new therapeutic strategy to decrease the pro-inflammatory phenotype of SF.

BackgroundThe role of natural killer (NK) cells in granulomatosis with polyangiitis (GPA) is poorly understood. We recently reported that peripheral blood NK cell percentages correlate with the suppression of GPA activity (cohort I). The purpose of the current study was to further characterize NK cell subsets, phenotype and function in a second GPA cohort (cohort II).MethodsPeripheral blood lymphocyte subsets were analyzed at a clinical diagnostic laboratory. Clinical data were extracted from medical records and patients were grouped according to their activity state (remission vs. active/non-remission). Separate analysis (cohort II, n = 22) and combined analysis (cohorts I and II, n = 34/57) of NK cell counts/percentages was performed. NK cell subsets and phenotypes were analyzed by multicolor flow cytometry. Cytotoxicity assays were performed using 51Cr-labeled K562 target cells.ResultsIn cohort II, NK cell counts were lower than the lower limit of normal in active GPA, despite normal percentages due to lymphopenia. NK cell counts, but not other lymphocyte counts, were significantly higher in remission. Combined analysis of cohorts I and II confirmed decreased NK cell counts in active GPA and increased percentages in long-term remission. Follow-up measurements of six patients revealed increasing NK cell percentages during successful induction therapy. Multicolor analysis from cohort II revealed that in active GPA, the CD56dim subset was responsible for decreased NK cell counts, expressed more frequently CD69, downregulated the Fc-receptor CD16 and upregulated the adhesion molecule CD54, the chemokine receptor CCR5 and the activating receptor NKG2C. In remission, these markers were unaltered or marginally altered. All other receptors investigated (NKp30, NKp44, NKp46, NKG2D, DNAM1, 2B4, CRACC, 41BB) remained unchanged. Natural cytotoxicity was not detectable in most patients with active GPA, but was restored in remission.ConclusionsNK cell numbers correlate inversely with GPA activity. Reduced CD56dim NK cells in active GPA have an activated phenotype, which intriguingly is associated with profound deficiency in cytotoxicity. These data suggest a function for NK cells in the pathogenesis and/or modulation of inflammation in GPA. NK cell numbers, phenotype (CD16, CD69, NKG2C) or overall natural cytotoxicity are promising candidates to serve as clinical biomarkers to determine GPA activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1098-7) contains supplementary material, which is available to authorized users.

Objective Findings from recent transcriptome analyses of the synovium of patients with rheumatoid arthritis (RA) have revealed that 15‐fold expanded HLA–DR+CD90+ synovial fibroblasts potentially act as key mediators of inflammation. The reasons for the expansion of HLA–DR+CD90+ synovial fibroblasts are unclear, but genetic signatures indicate that interferon‐γ (IFNγ) plays a central role in the generation of this fibroblast subset. The present study was undertaken to investigate the generation, function and therapeutically intended blockage of HLA–DR+CD90+ synovial fibroblasts. Methods We combined functional assays using primary human materials and focused bioinformatic analyses of mass cytometry and transcriptomics patient data sets. Results We detected enriched and activated Fcγ receptor type IIIa–positive (CD16+) NK cells in the synovial tissue from patients with active RA. Soluble immune complexes were recognized by CD16 in a newly described reporter cell model, a mechanism that could be contributing to the activation of natural killer (NK) cells in RA. In vitro, NK cell–derived IFNγ induced HLA–DR on CD90+ synovial fibroblasts, leading to an inflammatory, cytokine‐secreting HLA–DR+CD90+ phenotype. HLA–DR+CD90+ synovial fibroblasts consecutively activated CD4+ T cells upon receptor crosslinking via superantigens. HLA–DR+CD90+ synovial fibroblasts also activated CD4+ T cells in the absence of superantigens, an effect that was initiated by NK cell–derived IFNγ and that was 4 times stronger in patients with RA compared to patients with osteoarthritis. Finally, JAK inhibition in synovial fibroblasts prevented HLA–DR induction and blocked proinflammatory signals to T cells. Conclusion The HLA–DR+CD90+ phenotype represents an activation state of synovial fibroblasts during the process of inflammation in RA that can be induced by IFNγ, likely generated from infiltrating leukocytes such as activated NK cells. The induction of these proinflammatory, interleukin‐6–producing, and likely antigen‐presenting synovial fibroblasts can be targeted by JAK inhibition.

ObjectiveIn the last few years, anti-CD20 antibody rituximab profoundly changed the therapeutic landscape of granulomatosis with polyangiitis (GPA). Here, we investigated whether natural killer (NK) cells may play a role in rituximab’s mechanism of action in GPA.MethodsB cell depletion, NK cell degranulation, and the expression of CD69 and CD16 on NK cells were measured in a series of in vitro experiments using peripheral blood mononuclear cells (PBMCs). In vivo activation of NK cells was investigated in patients receiving rituximab infusions. Cells were analyzed by seven-color flow cytometry.ResultsNK cells from GPA patients were activated by immobilized rituximab. Also soluble rituximab activated NK cells, provided that B cells were present. NK cells degranulated and expressed the activation marker CD69 while CD16 expression was decreased. This activation of NK cells by soluble rituximab was accompanied by a reduction of B cells. The next-generation anti-CD20 antibody obinutuzumab showed stronger effects compared to rituximab on both the reduction of B cells and the activation of NK cells. Finally, we found that rituximab led to the activation of NK cells in vivo, provided that B cells were not depleted due to prior rituximab infusions.ConclusionB cell-bound rituximab activates NK cells in GPA. While NK cells therefore participate in rituximab’s mechanism of action in humans, their potential may be more efficiently exploited, e.g., by Fc engineering of therapeutic antibodies.

In response to tissue injury, fibroblasts differentiate into professional repair cells called myofibroblasts, which orchestrate many aspects of the normal tissue repair programme including synthesis, deposition and contraction of extracellular matrix proteins, leading to wound closure. Successful tissue repair responses involve termination of myofibroblast activities in order to prevent pathologic fibrotic scarring. Here, we discuss the cellular and molecular mechanisms limiting myofibroblast activities during physiological tissue repair, including myofibroblast deactivation, apoptosis, reprogramming and immune clearance of senescent myofibroblasts. In addition, we summarize pathological mechanisms leading to myofibroblast persistence and survival, a hallmark of fibrotic diseases. Finally, we discuss emerging anti-fibrotic therapies aimed at targeting myofibroblast fate such as senolytics, gene therapy, cellular immunotherapy and CAR-T cells.