Neutrophil Extracellular Traps in Systemic Lupus Erythematosus Stimulate IgG2 Production From B Lymphocytes.

Circulating autoantibodies of IgG2 isotype predominate in Systemic Lupus Erythematosus (SLE) and concur to the development of the renal lesions characteristic of Lupus Nephritis (LN). Anti-dsDNA and anti-histones IgG2, together with anti-podocyte proteins (i.e., α-enolase) are the major autoantibodies in serum and renal glomeruli of LN patients. The mechanisms underlying autoantibody formation and isotype switching in SLE and LN are unknown. A major issue is how DNA/histones are externalized from cell nucleus, driving the autoimmune response. Neutrophil Extracellular Traps (NETs) have been recently identified as crucial players in this context, representing the main source of DNA and nucleosome proteins. A second key point is what regulates IgG2 isotype switching: in mouse models, T-bet transcription factor has been described as essential for IgG2a class switch. We hypothesized that, in SLE, NET formation is the key mechanism responsible for externalization of autoantigens (i.e., dsDNA, histones 2,3, and α-enolase) and that T-bet is upregulated by NETs, driving, in this way, immunoglobulin class switch recombination (CSR), with production of IgG2 autoantibodies. The data here presented show that NETs, purified from SLE patients, stimulate ex vivo IgG2 isotype class switch possibly through the induction of T-bet. Of note, we observed a prominent effect of NETs on the release of soluble IgG2 in SLE patients', but not in healthy donors' B cells. Our results add important knowledge on the mechanisms of IgG2 class switch in SLE and contribute to further elucidate the role of NETs in LN pathogenesis.

Neutrophil Extracellular Traps Profiles in Patients with Incident Systemic Lupus Erythematosus and Lupus Nephritis.

OBJECTIVE: Neutrophil extracellular traps (NET) expose modified antigens for autoantibodies in vasculitis. Little is known about levels and removal pathways of NET in systemic lupus erythematosus (SLE), especially in lupus nephritis (LN). We determined circulating levels and defined NET removal in large subsets of patients with incident SLE (iSLE), some of whom had new-onset nephritis. METHODS: Serum levels of NET (ELISA), DNase1/DNase1L3 (ELISA), and DNase activity (functional assay) were determined in 216 patients with iSLE [103 had incident LN (iLN)], in 50 patients with other primary glomerulonephritis, and in healthy controls. Ex vivo NET production by neutrophils purified from a random selection of patients was quantified as elastase/DNA release and by immunofluorescence techniques. RESULTS: Serum NET levels were very high in iSLE/iLN compared to all groups of controls and correlated with anti-dsDNA, C3-C4, and proteinuria; iLN had the highest levels. DNase activity was decreased in iLN compared to SLE (20% had one-half DNase activity) despite similar serum levels of DNase1/DNase1L3. In these cases, pretreatment of serum with protein A restored DNase efficiency; 1 patient was homozygous for a c.289_290delAC variant of DNASE1L3. Ex vivo NET production by neutrophils purified from LN, SLE, and normal controls was similar in all cases. CONCLUSION: Patients with iLN have increased circulating NET and reduced DNase activity, the latter being explained by the presence of inhibitory substances in circulation and/or by rare DNase1L3 mutations. Accumulation of NET derives from a multifactorial mechanism, and is associated and may contribute to disease severity in SLE, in particular to renal lesions. (Clinical trial registration: The Zeus study was registered at ClinicalTrials.gov, study number NCT02403115).

Expression of membrane-bound human leucocyte antigen-G in systemic sclerosis and systemic lupus erythematosus.

Human leucocyte antigen-G (HLA-G) is a nonclassical class I major histocompatibility complex (MHC) molecule characterized by complex immunoregulatory and tolerogenic functions. Membrane-bound HLA-G is expressed on the surface of different cell populations in both physiological and pathological conditions. Systemic sclerosis (SSc) is a multisystem autoimmune disease characterized by widespread tissue fibrosis, vascular lesions and immunological alterations. Systemic lupus erythematosus is the prototypic systemic autoimmune disease affecting virtually any organ system, such as skin, joints, central nervous system, or kidneys. In SSc and SLE patients, the membrane expression of HLA-G on monocytes (0.88 ± 1.54 and 0.43 ± 0.75, respectively), CD4+ (0.42 ± 0.78 and 0.63 ± 0.48, respectively), CD8+ (2.65 ± 3.47 and 1.29 ± 1.34, respectively) and CD4+ CD8+ double-positive cells (13.87 ± 15.97 and 3.79 ± 3.11, respectively) was significantly higher than in healthy controls (0.12 ± 0.07; 0.01 ± 0.01; 0.14 ± 0.20 and 0.32 ± 0.38, respectively) (p < 0.0001). Our results show that in SSc and SLE the membrane expression of HLA-G by different subpopulations of peripheral blood mononuclear cells (PBMC) is increased, suggesting a potential role of HLA-G molecules in the complex immunological pathogenesis of these two autoimmune disorders.

Estimate of the Prevalence of Anti-Gastric Parietal Cell Autoantibodies in Healthy Individuals Is Method Dependent.

OBJECTIVES: Anti-parietal cell antibodies (APCA) are a serologic marker of autoimmune gastritis. Their prevalence in healthy individuals is not well defined. METHODS: We evaluated APCA prevalence in 515 healthy blood-donors by rat/primate tissue indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA), and immunoblot. RESULTS: Fifty-three of 515 (10.3%) subjects were positive for APCA by at least one method: 18 only by ELISA, 10 by rodent tissue IIF, and one by primate tissue IIF; 18 were positive by ELISA and primate tissue IIF, and one by ELISA and rodent tissue IIF. Two were positive by both IIF methods, and three were triple positive. APCA positivity was confirmed by immunoblot in 100% of ELISA positive, in 95.8% of positive primate tissue IIF, and in 50% of positive rat tissue IIF. CONCLUSIONS: A great discrepancy in APCA prevalence detected by different methods in this cohort was apparent. Thus, the results on APCA prevalence in healthy individuals are likely method-dependent.

Unexpected Elevated Free Thyroid Hormones in Pregnancy.

BACKGROUND: The use of thyrotropin and free thyroid hormone assays to evaluate thyroid function is widespread, but in some situations the results are inconsistent with the patient's thyroid status. SUMMARY: A 35-year-old woman with a known diagnosis of chronic autoimmune thyroiditis was referred to the authors' clinic at week 26 of her second pregnancy. The patient was clinically euthyroid. Consistent with this, her serum thyrotropin (TSH) was normal (0.79 mIU/L), but she had elevated free thyroid hormones-free triiodothyronine (fT3) and free thyroxine (fT4)-as determined by a one-step chemiluminescent assay. The patient was taking levothyroxine replacement therapy (125 µg/day), and the dose was confirmed. Previous blood tests showed concordance between TSH and free thyroid hormone values. The patient was followed up throughout gestation and at 12 months postpartum. During gestation, her free thyroid hormones remained high using one-step methods, while the total thyroid hormone concentration values were within the reference range, in agreement with the TSH values. Postpartum fT4 and fT3 values returned progressively to normality, in agreement with the TSH values. The presence of circulating thyroid hormone autoantibodies (THAb) was hypothesized, which are known to interfere, although to a variable extent, with thyroid hormone one-step assays. Using stored frozen sera, this hypothesis was confirmed indirectly by measuring normal levels of fT3 and fT4 with a two-step method, and directly by demonstrating THAb against the two hormones. CONCLUSION: Despite their relative rarity, circulating THAb may be suspected when laboratory data are not consistent and contrast with the clinical picture. To the authors' knowledge, no previous case of transient appearance of THAb in pregnancy has been described.