Disease activity is associated with changes in the innate immune function in patients with systemic lupus erythematosus.

OBJECTIVE: To address the relationship between systemic lupus erythematosus (SLE) disease activity and the functional parameters of the innate immunity. METHODS: We evaluated a cohort of 26 adult SLE patients and 10 sex and age-paired healthy donors. When the patients had a disease flare (baseline) and when they achieve clinical response (follow-up), we assessed the systemic lupus erythematosus disease activity index 2 K (SLEDAI 2 K) and the following parameters with flow cytometry and confocal microscopy: monocyte subsets, their expression of TLR2, phagocytic monocytes and neutrophils using the pHrodo Red E. coli BioParticles, the respiratory burst with 123-dihydrorhodamine in neutrophils, and the spontaneous and lipopolysaccharide (LPS)-induced production of neutrophil extracellular traps (NETs). We used the Wilcoxon test to compare the paired medians with interquartile range (IQR) and the Mann-Whitney U test for independent medians. To assess the effect of prednisone and SLEDAI 2 K on the mentioned parameters, we applied a generalized mixed linear model. RESULTS: Twenty-three patients (88.4%) were women. The SLEDAI 2 K was higher at baseline 8 (6-14) in comparison to that at follow-up (6 (4-8), P = 0.028). At baseline, SLE patients had a decreased percentage of intermediate monocytes, a higher expression of TLR2 in total monocytes, increased phagocytosis in monocytes and neutrophils, a decreased respiratory burst intensity, and an increased production of NETs. In the mix model, the SLEDAI 2 K was the main factor influencing these functional innate immune parameters. CONCLUSION: Disease activity regulates the innate immune function in SLE which may contribute to the clinical features and infection predisposition. Key points • This is the first cohort study addressing the effect of disease activity and prednisone use on the innate immune function of lupus patients. • Our results show that the disease activity is a key regulator of the respiratory burst, phagocytosis, and the production of neutrophil extracellular traps. • Also, we observed a differential proportion of monocyte subsets according to SLE disease activity. • We consider that our manuscript contributes to the evidence addressing the intrinsic immune abnormalities of patients with SLE regardless of the use of immunosuppressants and set the bases for new research work considering the disease activity as an element to decide the prescription and duration of antibiotic prophylaxis in SLE patients, which is of interest to all rheumatologists.

Prospective predictive performance comparison between clinical gestalt and validated COVID-19 mortality scores.

Most COVID-19 mortality scores were developed at the beginning of the pandemic and clinicians now have more experience and evidence-based interventions. Therefore, we hypothesized that the predictive performance of COVID-19 mortality scores is now lower than originally reported. We aimed to prospectively evaluate the current predictive accuracy of six COVID-19 scores and compared it with the accuracy of clinical gestalt predictions. 200 patients with COVID-19 were enrolled in a tertiary hospital in Mexico City between September and December 2020. The area under the curve (AUC) of the LOW-HARM, qSOFA, MSL-COVID-19, NUTRI-CoV, and NEWS2 scores and the AUC of clinical gestalt predictions of death (as a percentage) were determined. In total, 166 patients (106 men and 60 women aged 56±9 years) with confirmed COVID-19 were included in the analysis. The AUC of all scores was significantly lower than originally reported: LOW-HARM 0.76 (95% CI 0.69 to 0.84) vs 0.96 (95% CI 0.94 to 0.98), qSOFA 0.61 (95% CI 0.53 to 0.69) vs 0.74 (95% CI 0.65 to 0.81), MSL-COVID-19 0.64 (95% CI 0.55 to 0.73) vs 0.72 (95% CI 0.69 to 0.75), NUTRI-CoV 0.60 (95% CI 0.51 to 0.69) vs 0.79 (95% CI 0.76 to 0.82), NEWS2 0.65 (95% CI 0.56 to 0.75) vs 0.84 (95% CI 0.79 to 0.90), and neutrophil to lymphocyte ratio 0.65 (95% CI 0.57 to 0.73) vs 0.74 (95% CI 0.62 to 0.85). Clinical gestalt predictions were non-inferior to mortality scores, with an AUC of 0.68 (95% CI 0.59 to 0.77). Adjusting scores with locally derived likelihood ratios did not improve their performance; however, some scores outperformed clinical gestalt predictions when clinicians' confidence of prediction was <80%. Despite its subjective nature, clinical gestalt has relevant advantages in predicting COVID-19 clinical outcomes. The need and performance of most COVID-19 mortality scores need to be evaluated regularly.

Low-Density Granulocytes and Neutrophil Extracellular Traps as Biomarkers of Disease Activity in Adult Inflammatory Myopathies.

BACKGROUND/OBJECTIVE: Biomarkers for disease activity and damage accrual in idiopathic inflammatory myopathies (IIMs) are currently lacking. The purpose of this cross-sectional study is to analyze the relationship among low-density granulocytes (LDGs), neutrophil extracellular traps (NETs), and clinical and immunological features of patients with IIM. METHODS: We assessed disease activity, damage accrual, amount of LDGs, NETs, expression of LL-37, and serum cytokines in 65 adult patients with IIM. Differences between groups and correlations were assessed by Kruskal-Wallis, Mann-Whitney U, and Spearman ρ tests. The association between LDGs, NETs, disease activity, calcinosis, and cutaneous ulcers was assessed by logistic regression. To address the capacity of LDGs and NETs to diagnose disease activity, we used receiving operating characteristic curves. RESULTS: Low-density granulocytes were higher in patients with active disease, ulcers, calcinosis, and anti-MDA5 antibodies, which correlated with serum levels of IL-17A and IL-18. Neutrophil extracellular traps were higher in patients with calcinosis, elevated titers of antinuclear antibodies, and positive anti-PM/Scl75 tests. The combination of a high proportion of both total LDGs and NETs was associated with the presence of calcinosis and cutaneous ulcers. LL-37 was higher in NETs originating from LDGs. Normal-density neutrophils were elevated in patients with active dermatomyositis. CONCLUSIONS: Low-density granulocytes and NETs containing LL-37 are increased in patients with IIM and active disease, and correlate with proinflammatory cytokines. Both total and CD10+ LDGs are potential biomarkers for disease activity and, in combination with NETs, have the potential to detect patients who are at risk for cutaneous ulcers and calcinosis.

Neutrophil Extracellular Traps Contribute to COVID-19 Hyperinflammation and Humoral Autoimmunity.

The coronavirus disease 2019 (COVID-19) is related to enhanced production of NETs, and autoimmune/autoinflammatory phenomena. We evaluated the proportion of low-density granulocytes (LDG) by flow cytometry, and their capacity to produce NETs was compared with that of conventional neutrophils. NETs and their protein cargo were quantified by confocal microscopy and ELISA. Antinuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and the degradation capacity of NETs were addressed in serum. MILLIPLEX assay was used to assess the cytokine levels in macrophages' supernatant and serum. We found a higher proportion of LDG in severe and critical COVID-19 which correlated with severity and inflammatory markers. Severe/critical COVID-19 patients had higher plasmatic NE, LL-37 and HMGB1-DNA complexes, whilst ISG-15-DNA complexes were lower in severe patients. Sera from severe/critical COVID-19 patients had lower degradation capacity of NETs, which was reverted after adding hrDNase. Anti-NET antibodies were found in COVID-19, which correlated with ANA and ANCA positivity. NET stimuli enhanced the secretion of cytokines in macrophages. This study unveils the role of COVID-19 NETs as inducers of pro-inflammatory and autoimmune responses. The deficient degradation capacity of NETs may contribute to the accumulation of these structures and anti-NET antibodies are related to the presence of autoantibodies.

Conformational changes in myeloperoxidase induced by ubiquitin and NETs containing free ISG15 from systemic lupus erythematosus patients promote a pro-inflammatory cytokine response in CD4+ T cells.

BACKGROUND: Neutrophil extracellular traps (NETs) from patients with systemic lupus erythematosus (SLE) are characterized by lower ubiquitylation and myeloperoxidase (MPO) as a substrate. The structural and functional effect of such modification and if there are additional post-translational modifications (PTMs) are unknown. METHODS: To assess the expression and functional role of PTMs in NETs of patients with SLE; reactivation, proliferation and cytokine production was evaluated by flow cytometry using co-cultures with dendritic cells (DC) and CD4+ from SLE patients and healthy controls. The impact of ubiquitylation on MPO was assessed by molecular dynamics. The expression of ISG15 in NETs was evaluated by immunofluorescence and Western Blot. RESULTS: Fifteen patients with SLE and ten healthy controls were included. In the co-cultures of CD4+ lymphocytes with DC stimulated with ubiquitylated MPO or recombinant MPO, a higher expression of IFNγ and IL-17A was found in CD4+ from SLE patients (p < 0.05). Furthermore, with DC stimulated with ubiquitylated MPO a trend towards increased expression of CD25 and Ki67 was found in lupus CD4+ lymphocytes, while the opposite was documented in controls (p < 0.05). Through molecular dynamics we found the K129-K488-K505 residues of MPO as susceptible to ubiquitylation. Ubiquitylation affects the hydration status of the HEME group depending on the residue to which it is conjugated. R239 was found near by the HEME group when the ubiquitin was in K488-K505. In addition, we found greater expression of ISG15 in the SLE NETs vs controls (p < 0.05), colocalization with H2B (r = 0.81) only in SLE samples and increased production of IFNγ in PBMCs stimulated with lupus NETs compared to healthy controls NETs. CONCLUSION: The ubiquitylated MPO has a differential effect on the induction of reactivation of CD4+ lymphocytes in patients with SLE, which may be related to structural changes by ubiquitylation at the catalytic site of MPO. Besides a lower ubiquitylation pattern, NETs of patients with SLE are characterized by the expression of ISG15, and the induction of IFNγ by Th1 cells.

TLR expression in peripheral monocyte subsets of patients with idiopathic inflammatory myopathies: association with clinical and immunological features.

BACKGROUND: Monocytes and toll-like receptors (TLR) have been found in the inflammatory infiltrate of muscle biopsies in patients with idiopathic inflammatory myopathies (IIM), suggesting an important role of these cells in the pathogenesis of myositis. The monocyte subsets, their TLR expression in peripheral blood and their relationship with the clinical characteristics of patients with IIM has not been addressed. METHODS: We recruited 45 patients with IIM diagnosis and 15 age and sex-adjusted healthy controls. We assessed the disease activity and damage, performed a nailfold capillaroscopy and registered the cardio-pulmonary parameters from the medical charts. Monocyte subsets, their expression of TLR2 and TLR4 and the serum Th1/Th2/Th17 cytokines levels were evaluated by flow cytometry. We expressed quantitative variables as medians and interquartile ranges (IQR) or minimum and maximum (min-max). Differences between groups were assessed with Mann-Whitney U and the Kruskal-Wallis tests. Correlation between quantitative variables was assessed with Spearman Rho. RESULTS: Twenty-nine patients were women (64.4%) and 32 (71.1%) had dermatomyositis. In comparison to healthy controls, patients with active IIM had a higher percentage of intermediate monocytes and lower amounts of classical monocytes. Patients with IIM had a higher expression of TLR4 in all their monocyte subsets, regardless of disease activity and prednisone treatment. Serum IL-6 correlated with the TLR2 expression in every monocyte subset and the expression of TLR2 in intermediate monocytes was higher among patients with dysphagia. Subjects with nailfold capillaroscopy abnormalities had a higher amount of TLR2+ classical and non-classical monocytes and those with interstitial lung disease (ILD) had a higher percentage of TLR4+ non-classical monocytes. The classical and intermediate monocytes from patients with anti Mi2 antibodies had a higher expression of TLR4. The percentage of intermediate monocytes and the expression of TLR4 in all monocyte subsets showed a good diagnostic capacity in patients with IIM. CONCLUSION: Patients with IIM have a differential pool of monocyte subsets with an enhanced expression of TLR2 and TLR4, which correlates with disease activity and distinctive clinical features including dysphagia, ILD, vasculopathy, and pro-inflammatory cytokines. These immunological features might be useful as a potential diagnostic tool as well as novel disease activity biomarkers in IIM.

The role of low density granulocytes and NETosis in the pathogenesis of adult-onset Still's Disease.

OBJECTIVES: To analyse the potential contribution of low-density granulocytes (LDGs) and NETosis, as well as the differential protein cargo of neutrophil extracellular traps (NETs), as physiopathogenic mechanisms of adult-onset Still's disease (AOSD). METHODS: We recruited 30 patients with AOSD according to the Yamaguchi diagnostic criteria. LDGs were addressed by multiparametric flow cytometry as those CD14-, CD15+, CD10+ cells in the peripheral blood mononuclear cells fraction. NETs were quantified by ELISA, immunofluorescence and fluorescence spectrometry. The expression of LL-37 and high mobility group box 1 (HMGB-1) in NETs was measured by immunofluorescence and confocal microscopy. Additionally, normal density neutrophils from healthy controls were stimulated with serum from patients with AOSD and NET induction was assessed by immunofluorescence. RESULTS: Patients with active disease as well as those with arthritis, cutaneous manifestations and fever had a higher amount of NETs and LDGs. Serum NETs from AOSD patients correlated with the number of swollen joints (r=0.41, p=0.032), absolute number of monocytes (r=0.529, p=0.005). The spontaneous NETs from patients with cutaneous manifestations and fever had higher cargo of HMGB-1 compared with patients in remission. CONCLUSIONS: LDGs and NETs are increased in patients with active AOSD and correlate with particular clinical features. Patients with cutaneous lesions and fever present a higher cargo of HMGB1 in their spontaneous NETs.