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Background Airway abnormalities and lung tissue citrullination are found in both rheumatoid arthritis (RA) patients and individuals at-risk for disease development. This suggests the possibility that the lung could be a site of autoimmunity generation in RA, perhaps in response to microbiota changes. We therefore sought to test whether the RA lung microbiome contains distinct taxonomic features associated with local and/or systemic autoimmunity. Methods 16S rRNA gene high-throughput sequencing was utilized to compare the bacterial community composition of bronchoalveolar lavage fluid (BAL) in patients with early, disease-modifying anti-rheumatic drugs (DMARD)-naïve RA, patients with lung sarcoidosis, and healthy control subjects. Samples were further assessed for the presence and levels of anti-citrullinated peptide antibodies (including fine specificities) in both BAL and serum. Results The BAL microbiota of RA patients was significantly less diverse and abundant when compared to healthy controls, but similar to sarcoidosis patients. This distal airway dysbiosis was attributed to the reduced presence of several genus (i.e., Actynomyces and Burkhordelia ) as well as reported periodontopathic taxa, including Treponema , Prevotella , and Porphyromonas . While multiple clades correlated with local and systemic levels of autoantibodies, the genus Pseudonocardia and various related OTUs were the only taxa overrepresented in RA BAL and correlated with higher disease activity and erosions. Conclusions Distal airway dysbiosis is present in untreated early RA and similar to that detected in sarcoidosis lung inflammation. This community perturbation, which correlates with local and systemic autoimmune/inflammatory changes, may potentially drive initiation of RA in a proportion of cases.

Vimentin has been implicated in pulmonary sarcoidosis as a T-cell autoantigen, particularly in the context of , the Vα2.3/Vβ22 T-cell receptor (TCR), and Löfgren's syndrome. As vimentin is a known antigenic target in B-cell-mediated autoimmunity, we investigated humoral anti-vimentin responses in pulmonary sarcoidosis and their relationship with . Sarcoid and healthy control (HC) lung biopsies were analyzed by multi-color confocal microscopy for B-cells, T-cells, proliferation, and vimentin, and compared to tonsillectomy tissue. Bronchoalveolar lavage fluid (BALF) and serum from 48 sarcoidosis patients and 15 healthy volunteers were typed for and titrated for antibodies to full-length vimentin, vimentin truncations, and total IgG and IgA by ELISA. Presence of extracellular vimentin in BALF was determined by mass spectrometry and T-cell populations measured by flow cytometry. Sarcoid lung samples, especially from HLA-DRB1*03 patients, contained vimentin-rich tertiary lymphoid structures and corresponding BALF was highly enriched for both IgG and IgA anti-vimentin antibody (AVA) titers. Furthermore, sarcoidosis patient BALF AVA concentrations (expressed as arbitrary units per milligram of total immunoglobulin isotype) correlated with the percentage of CD4 T-cells expressing the Vα2.3/Vβ22 TCR. BALF antibody reactivity to the vimentin N-terminus was most prominent in HCs, whereas reactivity to the C-terminus (Vim ) was enriched in the sarcoid lung. Specifically, HLA-DRB1*03 patient BALF contained higher concentrations of anti-Vim antibodies than BALF from both HCs and HLA-DRB1*03 patients. Consistent with the lung as a site of AVA production, the concentration of AVAs in BALF was dramatically higher than in matched serum samples. Overall, there was a poor correlation between BALF and serum AVA concentrations. Together, these studies reveal the presence of linked recognition of vimentin by both T- and B-cells in HLA-DRB1*03 sarcoidosis patients, associated with a selective humoral immune response to the vimentin C-terminus.

Background To address the reactivity and affinity against histidyl-transfer RNA synthetase (HisRS) autoantigen of anti-Jo1 autoantibodies from serum and bronchoalveolar lavage fluid (BALF) in patients with idiopathic inflammatory myopathies/anti-synthetase syndrome (IIM/ASSD). To investigate the associations between the reactivity profile and clinical data over time. Methods Samples and clinical data were obtained from (i) 25 anti-Jo1 + patients (19 sera with 16 longitudinal samples and 6 BALF/matching sera at diagnosis), (ii) 29 anti-Jo1 − patients (25 sera and 4 BALF/matching sera at diagnosis), and (iii) 27 age/gender-matched healthy controls (24 sera and 3 BALF/matching sera). Reactivity towards HisRS full-length (HisRS-FL), three HisRS domains (WHEP, antigen binding domain (ABD), and catalytic domain (CD)), and the HisRS splice variant (SV) was tested. Anti-Jo1 IgG reactivity was evaluated by ELISA and western blot using IgG purified from serum by affinity chromatography. In paired serum-BALF, anti-Jo1 IgG and IgA reactivity was analyzed by ELISA. Autoantibody affinity was measured by surface plasmon resonance using IgG purified from sera. Correlations between autoantibody reactivity and clinical data were evaluated at diagnosis and longitudinally. Results Anti-Jo1 IgG from serum and BALF bound HisRS-FL, WHEP, and SV with high reactivity at the time of diagnosis and recognized both conformation-dependent and conformation-independent HisRS epitopes. Anti-HisRS-FL IgG displayed high affinity early in the disease. At the time of IIM/ASSD diagnosis, the highest autoantibody levels against HisRS-FL were found in patients ever developing interstitial lung disease (ILD) and arthritis, but with less skin involvement. Moreover, the reactivity of anti-WHEP IgG in BALF correlated with poor pulmonary function. Levels of autoantibodies against HisRS-FL, HisRS domains, and HisRS splice variant generally decreased over time. With some exceptions, longitudinal anti-HisRS-FL antibody levels changed in line with ILD activity. Conclusion High levels and high-affinity anti-Jo1 autoantibodies towards HisRS-FL were found early in disease in sera and BALF. In combination with the correlation of anti-HisRS-FL antibody levels with ILD and ILD activity in longitudinal samples as well as of anti-WHEP IgG in BALF with poor pulmonary function, this supports the previously raised hypothesis that the lung might have a role in the immune reaction in anti-Jo1-positive patients.

Background Sarcoidosis is diagnosed by a combination of typical clinical and radiological findings together with biopsy proof of non-caseating epithelioid cell granulomas in affected tissues and/or the cell distribution in bronchoalveolar lavage fluid (BALF). We aimed at investigating the usefulness of measuring the proportion of T-cell receptor (TCR) CD4+ Vα2.3+ T-cells in BALF as an additive marker to CD4/CD8-ratio to confirm the diagnosis. Methods From a register consisting of 749 sarcoidosis patients [Löfgren’s syndrome (LS) n  = 274, non-LS n  = 475] with information on Vα2.3+ T-cells, an expansion of CD4+ Vα2.3+ T-cells (CD4+ Vα2.3+ T cells > 10.5% in BALF) was seen in 268 (36%). Controls were healthy volunteers ( n  = 69) and patients with other pulmonary conditions ( n  = 39), investigated because of suspicion of sarcoidosis. Results A proportion of CD4+ Vα2.3+ T-cells in BALF > 10.5% was highly specific for sarcoidosis, with a specificity of 97% and with a sensitivity of 36% ( p  < 0.0001). Receiver operating characteristic (ROC) curves show that testing for CD4+ Vα2.3+ T-cells in BALF was a more useable test in individuals with LS [area under the curve (AUC) 0.82, p  < 0.0001] compared to the whole patient group (AUC 0.64, p  < 0.0001). Conclusion In this study, we show that an increased proportion of CD4+ Vα2.3+ T-cells in BALF is highly specific for sarcoidosis. This suggests that this T-cell subset could be used as an additional tool to the CD4/CD8-ratio to support the sarcoidosis diagnosis, particularly in patients with LS but also in patients with non-LS.

Cigarette smoke causes both acute and chronic changes of the immune system. Excluding recent smoking is therefore important in clinical studies with chronic inflammation as primary focus. In this context, it is common to ask the study subjects to refrain from smoking within a certain time frame prior to sampling. The duration of the smoking cessation is typically from midnight the evening before, i.e. 8 hours from sampling. As it has been shown that a proportion of current smokers underestimates or denies smoking, objective assessment of recent smoking status is of great importance. Our aim was to extend the use of exhaled carbon monoxide (CO(breath)), a well-established method for separating smokers from non-smokers, to assessment of recent smoking status. The time course of CO(breath) decline was investigated by hourly measurements during one day on non-symptomatic smokers and non-smokers (6+7), as well as by measurements on three separate occasions on non-smokers (n = 29), smokers with normal lung function (n = 38) and smokers with chronic obstructive pulmonary disease (n = 19) participating in a clinical study. We used regression analysis to model the decay, and receiver operator characteristics analysis for evaluation of model performance. The decline was described as a mono-exponential decay (r(2) = 0.7) with a half-life of 4.5 hours. CO decline rate depends on initial CO levels, and by necessity a generic cut-off is therefore crude as initial CO(breath) varies a lot between individuals. However, a cut-off level of 12 ppm could classify recent smokers from smokers having refrained from smoking during the past 8 hours with a specificity of 94% and a sensitivity of 90%. We hereby describe a method for classifying recent smokers from smokers having refrained from smoking for >8 hours that is easy to implement in a clinical setting.

Findings from molecular studies suggesting that several infectious agents cause sarcoidosis are intriguing yet conflicting and likely biased due to their cross-sectional design. As done in other inflammatory diseases to overcome this issue, prospectively-collected register data could be used, but reverse causation is a threat when the onset of disease is difficult to establish. We investigated the association between infectious diseases and sarcoidosis to understand if they are etiologically related. We conducted a nested case–control study (2009–2013) using incident sarcoidosis cases from the Swedish National Patient Register (n = 4075) and matched general population controls (n = 40,688). Infectious disease was defined using inpatient/outpatient visits and/or antimicrobial dispensations starting 3 years before diagnosis/matching. Adjusted odds ratios (aOR) of sarcoidosis were estimated using conditional logistic regression and tested for robustness assuming the presence of reverse causation bias. The aOR of sarcoidosis associated with history of infectious disease was 1.19 (95% confidence interval [CI] 1.09, 1.29; 21% vs. 16% exposed cases and controls, respectively). Upper respiratory and ocular infections conferred the highest OR. Findings were similar when we altered the infection definition or varied the infection-sarcoidosis latency period (1–7 years). In bias analyses assuming one in 10 infections occurred because of preclinical sarcoidosis, the observed association was completely attenuated (aOR 1.02; 95% CI 0.90, 1.15). Our findings, likely induced by reverse causation due to preclinical sarcoidosis, do not support the hypothesis that common symptomatic infectious diseases are etiologically linked to sarcoidosis. Caution for reverse causation bias is required when the real disease onset is unknown.

BackgroundEarly identification of patients at risk for progressive sarcoidosis may improve intervention. High bronchoalveolar lavage fluid (BALF) lymphocytes and peripheral blood (PB) lymphopenia are associated with worse prognosis. The mechanisms behind are not disentangled, and to date, it is not possible to predict disease course with certainty.ObjectivesInsight into the frequency of T regulatory cells (Tregs), proliferating CD4+ and CD8+ T cells in BALF and PB in clinically well-characterised patients, may provide clues to mechanisms behind differences in disease course.MethodsNineteen treatment-naïve patients with newly diagnosed sarcoidosis were assessed with BAL and PB samples at diagnosis. From the majority, repeated PB samples were collected over a year after diagnosis. The patients were followed for a median of 3 years and clinical parameters were used to classify patients into resolving, chronic progressive and chronic stable disease. Lymphocyte counts, frequency of Tregs defined as forkhead box protein 3+ (FoxP3+) CD4+T cells, and proliferating CD4+ and CD8+ T cells assessed with Ki-67 were analysed.ResultsEleven patients disclosed a chronic stable, and eight a progressive disease course, no one resolved during the study period. In PB, lower number of lymphocytes associated with chronic progressive disease, an increased frequency of Ki-67+CD4+ and CD8+ T cells, and a tendency towards higher percentage of FoxP3+CD4+ T cells compared with chronic stable patients.ConclusionA reduction of PB lymphocytes despite increased proliferation of CD4+and CD8+ T cells was observed in patients with chronic active compared with chronic stable sarcoidosis, indicating an increased PB lymphocyte turn-over in patients with deteriorating disease. Measurement of PB Tregs, Ki-67+CD4+ and Ki-67+CD8+ T cells may help in predicting sarcoidosis disease course.

Pulmonary sarcoidosis has unknown etiology, a difficult diagnostic procedure and no curative treatment. Extracellular vesicles including exosomes are nano-sized entities released from all cell types. Previous studies of exosomes from bronchoalveolar lavage fluid (BALF) of sarcoidosis patients have revealed pro-inflammatory components and abilities, but cell sources and mechanisms have not been identified. In the current study, we found that BALF exosomes from sarcoidosis patients, but not from healthy individuals, induced a dose-dependent elevation of intracellular IL-1β in monocytes. Analyses of supernatants showed that patient exosomes also induced release of IL-1β, IL-6 and TNF from both PBMCs and enriched monocytes, suggesting that the observed effect is direct on monocytes. The potently chemotactic chemokine CCL2 was induced by exosomes from a subgroup of patients, and in a blocking assay the exosome-induced CCL2 was reduced for 13 out of 19 patients by the asthma drug Montelukast, a cysteinyl leukotriene receptor antagonist. Further, reactive oxygen species generation by PBMCs was induced to a higher degree by patient exosomes compared to healthy exosomes. These findings add to an emerging picture of exosomes as mediators and disseminators of inflammation, and open for further investigations of the link between CCL2 and exosomal leukotrienes in sarcoidosis.

Sarcoidosis is a multisystem disease of unknown cause. Löfgren's syndrome (LS) is a characteristic subgroup of sarcoidosis that is associated with a good prognosis in sarcoidosis. However, little is known about its genetic architecture or its broader phenotype, non-LS sarcoidosis. To address the genetic architecture of sarcoidosis phenotypes, LS and non-LS. An association study in a white Swedish cohort of 384 LS, 664 non-LS, and 2,086 control subjects, totaling 3,134 subjects using a fine-mapping genotyping platform was conducted. Replication was performed in four independent cohorts, three of white European descent (Germany, n = 4,975; the Netherlands, n = 613; and Czech Republic, n = 521), and one of black African descent (United States, n = 1,657), totaling 7,766 subjects. A total of 727 LS-associated variants expanding throughout the extended major histocompatibility complex (MHC) region and 68 non-LS-associated variants located in the MHC class II region were identified and confirmed. A shared overlap between LS and non-LS defined by 17 variants located in the MHC class II region was found. Outside the MHC region, two LS-associated loci, in ADCY3 and between CSMD1 and MCPH1, were observed and replicated. Comprehensive and integrative analyses of genetics, transcription, and pathway modeling on LS and non-LS indicates that these sarcoidosis phenotypes have different genetic susceptibility, genomic distributions, and cellular activities, suggesting distinct molecular mechanisms in pathways related to immune response with a common region.