Neutrophils are broadly classified into conventional neutrophils (PMNs) and low-density granulocytes (LDGs). LDGs are better than PMNs at generating neutrophil extracellular traps (NETs), which may contribute to the pathology of autoimmune diseases. We hypothesized that LDGs and PMNs differ in their levels of unrestrained NE that supports NET generation. Here, we show that individuals with psoriasis contain elevated levels of LDGs and that in contrast to PMNs, the LDGs display higher staining for NE and lower staining for its inhibitor SLPI. The heterogeneity between blood-derived LDGs and PMNs was somewhat reminiscent of the differences in the NE and SLPI staining patterns observed in psoriasis skin-infiltrating neutrophils. Distinctive staining for NE and SLPI in LDGs and PMNs did not result from differences in their protein levels nor manifested in higher total proteolytic activity of NE in LDGs; rather, it likely depended on different cytosolic sequestration of these proteins. The disparate profile of NE and SLPI in LDGs and PMNs coincided with altered migratory responses of these cells to cutaneous chemoattractants. Collectively, differential NE and SLPI staining identifies common attributes of both circulating and skin-infiltrating neutrophils, which may guide neutrophil migration to distinct skin regions and determine the localization of LDGs-mediated cutaneous pathology.

Renal involvement in systemic lupus erythematosus (SLE) is associated with production of antibodies to double stranded DNA, deposition of immune complexes and organ damage. These processes have been linked with abnormalities in B- and T-cell memory compartments. The aim of the study was to analyze subsets of peripheral memory B-cells and T-cells in lupus nephritis (LN) patients. We used multicolor flow cytometry to analyze major memory subsets of peripheral blood B-cells (defined by CD27, IgD and CD21) and T-cells (CD45RA, CD45RO, CCR7) in 32 patients with active or inactive LN, and 23 control subjects. Lupus nephritis patients were characterized by increased percentage of immature/early-transitional B-cells (CD27-IgD+CD21-), higher frequency of activated switched memory (SM, CD27+IgD-CD21-) and exhausted memory B-cells (CD27-IgD-), and decrease in non-switched memory (NSM, CD27+IgD+) B-cells. CD21low subsets (immature and activated B-cells) were particularly expanded in patients with active disease. In both groups of LN patients we observed decline in the absolute count of NSM B-cells. It was paralleled by lymphopenia in naïve CD4+ T-cell compartment and increase in the frequency of effector memory T-cells, and these changes were more pronounced in active LN. B-cell memory compartment in LN is deficient in NSM cells and during active disease it is further skewed towards SM and exhausted memory phenotypes, most likely as a cause of chronic antigenic stimulation. Parallel changes in T-helper cell subsets suggest a similar mechanism of SLE-related lymphopenia for both B-cell and T-cell compartment.

Background. Certain mediators, such as soluble growth factors and cytokines, among others, are implicated in the immunopathogenesis of systemic sclerosis (SSc). Objectives. This study aimed to examine the association between serum levels of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), interferon alpha (IFN-& alpha;), and basic fibroblast growth factor (bFGF) and the clinical presentation and course of SSc. Materials and methods. This longitudinal, observational study included 43 patients with SSc and 24 healthy subjects. Serum concentrations of VEGF, IL-8, IFN-& alpha;, and bFGF were measured at baseline in patients previously treated for SSc. Medical history of patients was analyzed retrospectively at the time of cytokine measurement to infer clinical correlations, and during follow-up for a median of 5 years, assessing the incidence ofdeath or cancer. Results. The bFGF and IFN-& alpha; concentrations differed between SSc patients and controls (p < 0.01). In turn, organ involvement and SSc phenotypes did not impact studied cytokine concentrations, similar to systemic steroid and/or immunosuppressant use at enrollment. However, we have documented a positive correlation between the current oral steroid dose and serum levels of IL-8 and bFGF. Furthermore, patients with a VEGF level >_95.7 pg/mL and IFN-& alpha; level >_3.6 pg/mL required cyclophosphamide therapy more often, currently or in the past (approx. 3-fold and 4-fold, respectively). Substantially elevated VEGF and IFN-& alpha; concentrations at baseline were associated with higher cancer occurrence (n = 4) during follow-up, while elevated circulating IL-8 level was associated with an increased risk of death (n = 9). Conclusions.The SSc group was characterized by higher serum concentrations ofbFGF and IFN-& alpha; compared to healthy controls. Patients treated with cyclophosphamide or receiving higher systemic steroid doses, thus suffering from a more severe disease type, had increased cytokine levels. Elevated circulating IFN-& alpha; and VEGF levels might be correlated with cancer, whereas raised IL-8 levels may be associated with an increased risk of death. However, further research is needed to verify our findings.

The objective of this study was to investigate the mechanisms of T helper type 17 (Th17) expansion in lupus nephritis (LN) patients, and to determine whether or not it is associated with impaired function of regulatory T cells (T_reg). Major effector subsets of peripheral blood CD4⁺ T cells were assessed by flow cytometry in 33 LN patients with different activity of the disease and 19 healthy controls. The percentage of circulating Th17 cells was increased in LN (median = 1·2% of CD4⁺ compared to 0·6% in the control group, P < 0·01), while T_reg cells remained unchanged (12·3 versus 12·1% in controls), resulting in a significantly lower T_reg/Th17 ratio. Th17 expansion in the patient group was not related to LN activity, renal histology or blood and urine inflammatory biomarkers, but has been associated with a higher cumulative dose of cyclophosphamide. T_reg cells in LN displayed mainly effector memory phenotype and expressed higher levels of transforming growth factor (TGF)‐β; however, their suppressant activity in lymphocyte proliferation assay was diminished compared to controls (~fourfold, P < 0·05). Co‐culture of T_reg and conventional CD4⁺ T cells resulted in marked suppression of the Th1 subset in both of the groups studied, but also in a potent expansion of Th17 cells, which in LN was twofold higher, as in controls (P < 0·05). In conclusion, our results demonstrate that Th17 expansion in LN is not increased during disease exacerbation, but is related to chronic immunosuppressive therapy. This immune signature is probably linked to the abnormal function of T_reg cells, which were less suppressive in LN patients and even facilitated differentiation of Th17 cells.

Background: Lupus nephritis (LN) is an inflammation of the kidneys that is related to systemic lupus erythematosus (SLE). This study aimed to evaluate the differences in clinical and laboratory characteristics between LN and non-LN SLE patients. Methods: We conducted a retrospective analysis of medical records collected from SLE patients treated at the University Hospital in Kraków, Poland, from 2012 to 2022. All patients met the 2019 European League Against Rheumatism and the American College of Rheumatology (EULAR/ACR) criteria for SLE. Results: Among 921 SLE patients, LN was documented in 331 (35.94%). LN patients were younger at SLE diagnosis (29 vs. 37 years; p < 0.001) and had a male proportion that was 2.09 times higher than the non-LN group (16.62% vs. 7.97%; p < 0.001). They were more often diagnosed with serositis and hematological or neurological involvement (p < 0.001 for all). Hypertension and hypercholesterolemia occurred more frequently in these patients (p < 0.001 for both). LN patients exhibited a higher frequency of anti-dsDNA, anti-histone, and anti-nucleosome antibodies (p < 0.001 for all). Conversely, the non-LN group had a 1.24-fold (95% CI: 1.03–1.50; p = 0.021) increase in the odds ratio of having positive anti-cardiolipin IgM antibody results. LN patients were more frequently treated with immunosuppressants. The risk factors for experiencing at least three LN flares included female sex, younger age at the onset of LN or SLE, LN occurring later than SLE onset, the presence of anti-nucleosome or anti-dsDNA antibodies, and certain SLE manifestations such as myalgia, arthritis, proteinuria > 3.5 g/day, and pathological urinary casts in the urine sediment. Conclusions: LN patients differ from non-LN patients in the age of SLE diagnosis, treatment modalities, and autoantibody profile and have more frequent, severe manifestations of SLE. However, we still need more prospective studies to understand the diversity of LN and its progression in SLE patients.

Summary: In vasculitis disorders, inflammation affects blood vessels. Granulomatosis with polyangiitis (GPA) is a chronic systemic vasculitis distinguished by the presence of anti-proteinase-3 autoantibodies (anti-PR3). In this study we analyzed the molecular signature of human umbilical endothelial cells (HUVECs) in response to neutrophil-derived extracellular vesicles (EVs). EVs were obtained from anti-PR3-activated neutrophils, purified and characterized by flow cytometry, nanoparticle tracking and miRNA screening. HUVECs were stimulated with EVs and miRNA/mRNA expression was measured. Cell culture media proteins were identified by antibody microarrays and selected cytokines were measured. Comparison of differentially expressed miRNAs/mRNAs between non-stimulated and EV-stimulated HUVECs revealed two regulatory patterns. Significant up-regulation of 14 mRNA transcripts (including CXCL8, DKK1, IL1RL1, ANGPT-2, THBS1 and VCAM-1) was accompanied by 11 miRNAs silencing (including miR-661, miR-664a-3p, miR-377-3p, miR-30d-5p). Significant down-regulation was observed for nine mRNA transcripts (including FASLG, CASP8, STAT3, GATA3, IRAK1 and IL6) and accompanied by up-regulation of 10 miRNAs (including miR-223-3p, miR-142-3p, miR-211-5p). Stimulated HUVECs released IL-8, Dickkopf-related protein 1 (DKK-1), soluble interleukin (IL)-1 like receptor-1 (ST2), growth differentiation factor 15 (GDF-15), angiopoietin-2, endoglin, thrombospondin-1 and vascular adhesion molecule-1 (VCAM-1). Moreover, transfection of HUVECs with mimics of highly expressed in EVs miR-223-3p or miR-142-3p, stimulated production of IL-8, ST2 and endoglin. Cytokines released by HUVECs were also elevated in blood of patients with GPA. The most increased were IL-8, DKK-1, ST2, angiopoietin-2 and IL-33. In-vitro stimulation of HUVECs by neutrophil-derived EVs recapitulates contribution of endothelium in autoimmune vasculitis. Proinflammatory phenotype of released cytokines corresponds with the regulatory network of miRNAs/mRNAs comprising both EVs miRNA and endothelial cell transcripts.