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BACKGROUND: Hidradenitis suppurativa (HS) is an inflammatory chronic skin disorder of unknown etiology characterized by inflamed abscess-like nodules and boils resulting in sinus tract formation, tissue scarring and massive infiltration of neutrophils. Multiple lines of evidence have highlighted the potential association between alterations in the Notch pathway and HS pathogenesis, but the mechanisms remain incompletely characterized. OBJECTIVE: Herein, we aim to elucidate the role of neutrophil extracellular traps in Notch-γ-secretase, signaling. METHODS: Twenty-six HS lesional tissues, primary HS macrophages and skin fibroblasts were interrogated by quantitative PCR, western blot, and Elisa analyses. γ-Secretase, and TACE activities were measured in HS skin lesions, macrophages and skin fibroblasts. Immunofluorescence and RNAscope analyzes were performed in HS and control skin. RESULTS: A prominent presence of Notch ligands, DLL4 and JAG2 were detected at the protein and mRNA levels in HS skin lesion when compared to control. Levels of DLL4, JAG1, cit-H3-DNA and γ-secretase activity correlated with HS disease severity. Additionally, significantly elevated levels of Notch ligands and γ-secretase activity were found in dissected sinus tracts when compared to the rest of HS tissue. Immunofluorescence microscopy in HS skin lesions showed activation of Notch 1 signaling in macrophages and skin fibroblasts. Neutrophil extracellular traps (NETs) purified from HS patients displayed elevated levels of DLL4. HS-NETs activated the Notch pathway in macrophages and dermal fibroblasts isolated from HS patients. HS skin fibroblasts displayed elevated levels of CD90 and DPP4 in association with increased migratory capacity and Notch activation. Inhibition of Notch decreased migratory capacity and pro-fibrotic markers in HS fibroblasts. CONCLUSION: These data support a pathogenic connection between NETs, Notch- γ-secretase activation and the release of pro-fibrotic molecules that promote dysregulation of macrophages and skin fibroblasts in HS. Unveiling the relevance of these molecular events not only expands our understanding of HS but also opens new venues for the development of targeted therapies to address the fibrotic complications of advanced stages of HS.

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Dysregulation in the phenotype and function of neutrophils may play important roles in the initiation and perpetuation of autoimmune responses, including conditions affecting the skin. Neutrophils can have local and systemic effects on innate and adaptive immune cells as well as on resident cells in the skin, including keratinocytes (KCs). Aberrant formation/clearance of neutrophil extracellular traps (NETs) in systemic autoimmunity and chronic inflammatory diseases have been associated with the externalization of modified autoantigens in peripheral blood and tissues. NETs can impact the function of many cells, including macrophages, lymphocytes, dendritic cells, fibroblasts, and KCs. Emerging evidence has unveiled the pathogenic key roles of neutrophils in systemic lupus erythematosus, idiopathic inflammatory myopathies, psoriasis, hidradenitis suppurativa, and other chronic inflammatory conditions. As such, neutrophil-targeting strategies represent promising therapeutic options for these diseases.

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OBJECTIVE: Neutrophil extracellular traps (NETs) are extracellular lattices composed of nucleic material bound to neutrophil granule proteins. NETs may play pathogenic roles in the development and severity of autoimmune diseases such as systemic lupus erythematosus (SLE), at least in part, through induction of type I interferon (IFN) responses via externalization of oxidized immunostimulatory DNA. A distinct subset of SLE proinflammatory neutrophils (low-density granulocytes [LDGs]) displays enhanced ability to form proinflammatory NETs that damage the vasculature. We undertook this study to assess whether NET-bound RNA can contribute to inflammatory responses in endothelial cells (ECs) and the pathways that mediate this effect. METHODS: Expression of newly synthesized and total RNA was quantified in NETs from healthy controls and lupus patients. The ability of ECs to take up NET-bound RNA and downstream induction of type I IFN responses were quantified. RNAs present in NETs were sequenced and specific small RNAs were tested for induction of endothelial type I IFN pathways. RESULTS: NETs extruded RNA that was internalized by ECs, and this was enhanced when NET-bound nucleic acids were oxidized, particularly in lupus LDG-derived NETs. Internalization of NET-bound RNA by ECs was dependent on endosomal Toll-like receptors (TLRs) and the actin cytoskeleton and induced type I IFN-stimulated genes (ISGs). This ISG induction was dependent on NET-associated microRNA let-7b, a small RNA expressed at higher levels in LDG-derived NETs, which acted as a TLR-7 agonist. CONCLUSION: These findings highlight underappreciated roles for small RNAs externalized in NETs in the induction of proinflammatory responses in vascular cells, with implications for lupus vasculopathy.

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PURPOSE OF REVIEW: To provide an update on state-of-the-art evidence on the role of immunometabolism reprogramming in the pathogenesis of systemic lupus erythematosus (SLE). RECENT FINDINGS: Mitochondrial dysfunction and enhanced oxidative stress, along with specific defects in other metabolic pathways, can promote dysregulation of innate and adaptive immune responses in SLE. These abnormalities appear to be driven by genetic and epigenetic factors, modulated by stochastic events. In addition to extensive descriptions of abnormalities in immunometabolism of lupus lymphocytes, recent studies support the critical role of dysregulation of metabolic pathways in innate immune cells including neutrophils, macrophages and dendritic cells, in SLE pathogenesis. Recent abnormalities described in lipid metabolism have been associated with SLE disease activity and related damage. Promising therapeutic strategies that target these metabolic abnormalities have recently been described in SLE. SUMMARY: Fundamental new insights regarding the role of mitochondrial dysfunction in innate immune dysregulation in SLE pathogenesis have recently emerged. Defects in specific molecular pathways pertinent to immunometabolism in SLE have been described. New insights in translational medicine and promising therapeutic targets have been proposed based on these recent findings.

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BACKGROUND/OBJECTIVE: The aim of this cross-sectional study was to explore which factors affect the impact of musculoskeletal ultrasound (MUS) on the treatment proposal among rheumatologists with different degree of experience. METHODS: Sixteen clinical vignettes summarized data from rheumatoid arthritis (RA) outpatients; vignettes included clinical evaluation and a blank section for a first treatment proposal; MUS information was then added, based on German Ultrasound score, followed by a blank section for treatment re-consideration, if applicable. During a 6 months period, each vignette was concomitantly presented to six trainees and six senior rheumatologists (SR); three SR had ≥15 years of experience. Participants were blinded to colleagues' responses. Appropriated statistics were used. RESULTS: Vignettes included data from female patients, who had a mean ± SD age of 43.3 ± 9 years, 7.6 ± 3.5 years of disease duration and comorbidities (68.8%). MUS induced treatment modification in 24% of evaluations, with similar percentage among SR and trainees. Within SR, more experienced rheumatologists (≥15 years) never translated MUS findings in a different treatment proposal, compared to 34% of those with lesser experience, p ≤ 0.0001. There were 60 clinical scenarios each, with remission and moderate disease activity, and 36 clinical scenarios each, with low and high disease activity. MUS-induced treatment modifications were more frequent in scenarios with low and moderate disease activity, compared to remission and high disease activity, p = 0.008. CONCLUSIONS: Physician's experience and disease activity level affect the impact of MUS on the treatment decision in RA outpatients. RA patients with intermediate disease activity may benefit from MUS incorporation to standard assessments.

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The primary aim of this study was to assess demographic, clinical, and serological factors associated with remission in systemic lupus erythematosus (SLE). A retrospective cohort study was performed. We examined relevant features in patients with SLE with a follow-up of at least 8 years from active disease (SLE Disease Activity Index-2000 [SLEDAI-2K] ≥6). The primary outcome was to assess various remission states in SLE according to disease activity and treatment. Differences between groups were assessed by Student's t test and chi-square test for continuous and categorical variables, respectively. Multivariate Cox proportional hazard analysis was used to assess association between variables, and we performed a Kaplan-Meier analysis with log rank test to evaluate time to remission. One hundred twenty-four patients fulfilled our inclusion criteria: 116 (93.54%) were women with a mean age of 30.23 ± 8.52 years. Twenty-four patients (19.35%), 25 patients (20.16%), and 16 patients (12.9%) achieved complete remission, clinical remission on corticosteroids, and clinical remission off corticosteroids, respectively. SLEDAI-2K at 3rd month of follow-up (HR = 0.85, 95% CI = 0.73-0.98, p = 0.029) and total number of disease flares (HR = 0.73, 95% CI = 0.56-0.95, p = 0.024) were associated with complete remission, and total number of disease flares (HR = 0.80, 95% CI = 0.67-0.95, p = 0.011) was associated with clinical remission on corticosteroids. Our findings are clinically relevant to encourage an intensive immunosuppressive treatment and close monitoring early after active disease.

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Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). At our Institution, patients transplanted using G-CSF-primed bone marrow (G-BM), have a lower incidence of GVHD when compared to other sources. The objective of this study was to analyze and compare T cell subsets and cytokines in donor G-BM and steady-state BM (SS-BM). A prospective study was performed in 48 donor samples. Mononuclear cells were isolated by gradient density. T cell subsets and cytokine production in supernatants were analyzed by multiparametric flow cytometry. Six and 16 patients developed acute and chronic GVHD, respectively. Patients who developed GVHD were characterized by a predominant pro-inflammatory response (IL-17A (10.02 vs 0.43pg/mL, p=0.006), TNF-α (54.57 vs 0.81pg/mL, p=0.001)), in contrast to a deficient suppressor profile (IL-10 (7.87 vs 41.37pg/mL, p=0.003)) and Tregs (0.95% vs 1.52%, p=0.004). G-BM showed an enhanced suppressive phenotype (increased Th2 and Tregs) in comparison to SS-BM. GVHD is associated with an imbalance between pro-inflammatory and suppressor immune responses. G-BM showed a more favorable immunologic profile characterized by diminished pro-inflammatory cytokine production, which was associated with a lower frequency of GVHD in our cohort.

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Cytotoxic T-Lymphocye Antigen 4 (CTLA-4) or CD152 is an inhibitory molecule that plays a critical role in maintenance of tolerance to self-antigens. CTLA-4 is structurally as well as functionally related to CD28, since it shares 31% of homology and binds the B7 family molecules CD80 and CD86 with higher affinity. Nevertheless, CTLA-4 has opposing effects on T cell activation and current evidence shows that its inhibitory role goes beyond the ligand-binding interaction. CTLA-4 competes with CD28 in binding to B7, interacts within the immunological synapsis elements and with clathrin adaptor proteins and tyrosine phosphatases through its cytoplasmic domain to regulate cell trafficking and to set the activation threshold within T cells. Moreover, we have learned from the knock out model that CTLA-4 plays a key role in regulatory T cells and in central tolerance. Because of its importance in maintenance of peripheral tolerance, CTLA-4 has been implicated in several autoimmune diseases, such as systemic lupus erythematosus. Multiple single-nucleotide polymorphisms have been located to human Ctla-4 gene, and their association with autoimmune disease is still a matter of controversy. Despite the promising results of abatacept or CTLA-4-Ig in rheumatoid arthritis and murine lupus nephritis, more clinical randomized trials and standardization of outcomes are needed to prove its efficacy and safety in human lupus nephritis.

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OBJECTIVE: To analyze whether the expression and modulation of T cell receptor (TCR) signaling is dependent on Casitas B lineage lymphoma b (Cbl-b) in T cells from patients with systemic lupus erythematosus (SLE) upon stimulation with a tolerogenic substance. METHODS: Peripheral blood mononuclear cells were obtained from 20 patients with SLE (active disease or in remission) and 20 healthy controls. Levels of Cbl-b expression were measured using reverse transcription-polymerase chain reaction and Western blotting in peripheral CD4+ T cells from SLE patients and healthy controls upon anergy induction. Cell proliferation was measured using the carboxyfluorescein diacetate succinimidyl ester dilution method. Cytokine production was analyzed by luminometry, and surface expression of activation markers was assessed by flow cytometry. Transfection assays were performed to induce overexpression of Cbl-b, and phosphorylation of TCR-associated kinases was evaluated. RESULTS: CD4+ T cells from SLE patients displayed resistance to anergy (as evidenced by increased cell proliferation, interleukin-2 production, and expression of activation and costimulatory markers), and this was associated with altered Cbl-b expression. Upon ionomycin treatment, primary T cells showed enhanced MAPK activity and decreased Akt phosphorylation, which was representative of the anergic state. In T cells from lupus patients, Cbl-b overexpression led to increased expression of phosphorylated MAPK, thus indicating the reversibility of anergy resistance. CONCLUSION: These findings suggest that abnormal peripheral tolerance in SLE is caused by a deficiency in Cbl-b, and that this ubiquitin ligase plays a key role in regulating TCR signaling during the induction of peripheral tolerance.

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Follicular helper T cells (T(FH)) have been implicated as a lineage that provides sufficient help to B cells in order to become professional antibody producers. This T helper subset is characterized by a distinctive cell-surface phenotype (CD4(+)CD57(+)CXCR5(+)) and cytokine profile (IL-21, IL-6, and IL-27) as well as transcriptional program (BCL-6, ICOS, and PD-1). Evidence supports the concept that T(FH) subset development, as well as for other lineages, is dependent on microenvironment cues that modulate a particular transcriptional program, susceptible to plasticity. Recently, it has been shown that BCL-6 and IL-21 act as master regulators for the development and function of T(FH) cells. Moreover, costimulation via ICOS, as well as signaling proteins such as SAP constitute required elements of the regulatory network that modulates T(FH) functions. T(FH) dysregulation has been implicated in the development of autoimmune pathology, such as SLE. Indeed, the Sanroque mice associated to the mutation of Roquin, a ubiquitin ligase, essential for the regulation of ICOS and germinal center responses, constitutes a model that shares features with human SLE. Recently, the expansion of "circulating T(FH) cells" (CD4(+)CXCR5(+)ICOS(high)PD1(high)) has been described for a subset of SLE patients that share T(FH) dependent features of disease with Sanroque mice, such as glomerulonephritis and cytopenias.

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