Genetic association and differential expression of HLAComplexGroup lncRNAs in pemphigus.

BACKGROUND: Pemphigus is a group of bullous diseases characterized by acantholysis and skin blisters. As for other autoimmune diseases, the strongest genetic associations found so far for pemphigus foliaceus (PF) and vulgaris (PV) are with alleles of HLA genes. However, apart from protein-coding genes, the MHC region includes a set of poorly explored long non-coding RNA (lncRNA) genes, the HLA complex group (HCG). OBJECTIVES: To investigate if HCG lncRNA alleles are associated with pemphigus susceptibility. METHODS AND RESULTS: We analyzed SNPs in 13 HCG lncRNA genes, both in PV (Germany: 241 patients; 1,188 controls) and endemic PF (Brazil: 227 patients; 194 controls), applying multivariate logistic regression. We found 55 associations with PV (pcorr < 0.01) and nine with endemic PF (pcorr < 0.05), the majority located in TSBP1-AS1 (which includes HCG23) and HCG27 lncRNA genes, independently of HLA alleles previously associated with pemphigus. The association of TSBP1-AS1 rs3129949*A allele was further replicated in sporadic PF (p = 0.027, OR = 0.054; 75 patients and 150 controls, all from Germany). Next, we evaluated the expression levels of TSBP1-AS1, TSBP1, HCG23, and HCG27 in blood mononuclear cells of Brazilian patients and controls. HCG27 was upregulated in endemic PF (p = 0.035, log2 FC = 1.3), while TSBP1-AS1 was downregulated in PV (p = 0.029, log2 FC = -1.29). The same expression patterns were also seen in cultured keratinocytes stimulated with IgG antibodies from patients and controls from Germany. TSBP1 mRNA levels were also decreased in endemic PF blood cells (p = 0.042, log2 FC = -2.14). TSBP1-AS1 and HCG27 were also observed downregulated in CD19+ cells of endemic PF (p < 0.01, log2 FC = -0.226 and -0.46 respectively). CONCLUSIONS: HCG lncRNAs are associated with susceptibility to pemphigus, being TSBP1-AS1 and HCG27 also differentially expressed in distinct cell populations. These results suggest a role for HCG lncRNAs in pemphigus autoimmunity.

Neutrophil Extracellular Traps: A Perspective of Neuroinflammation and Complement Activation in Alzheimer's Disease.

Complement system (CS) components are associated with Alzheimer's disease (AD), the commonest cause of dementia in the world. Neutrophils can be attracted to amyloid-ß plaques by several pro-inflammatory factors, including the complement anaphylatoxin C5a. They may release neutrophil extracellular traps (NETs), which are chromatin nets associated with myeloperoxidase, elastase, and other enzymes. Some CS molecules, such as C5a, C1q, and CR1, are associated with increased neutrophil recruitment and NETs release. However, the relationship between CS molecules and NETs in AD is poorly understood. In this work, we detected higher NET concentrations in plasma and serum of Brazilian AD patients, than in elderly controls (medians = 2.78 [2.07-6.19] vs. 2.23 [0.33-4.14] ng/mL, p = 0.0005). We discussed these results within the context of our former findings on complement and AD and the context of the literature on complement and NET release, suggesting both as possible therapeutic targets to prevent the progress of the disease.

MASPs at the crossroad between the complement and the coagulation cascades - the case for COVID-19.

Components of the complement system and atypical parameters of coagulation were reported in COVID-19 patients, as well as the exacerbation of the inflammation and coagulation activity. Mannose binding lectin (MBL)- associated serine proteases (MASPs) play an important role in viral recognition and subsequent activation of the lectin pathway of the complement system and blood coagulation, connecting both processes. Genetic variants of MASP1 and MASP2 genes are further associated with different levels and functional efficiency of their encoded proteins, modulating susceptibility and severity to diseases. Our review highlights the possible role of MASPs in SARS-COV-2 binding and activation of the lectin pathway and blood coagulation cascades, as well as their associations with comorbidities of COVID-19. MASP-1 and/or MASP-2 present an increased expression in patients with COVID-19 risk factors: diabetes, arterial hypertension and cardiovascular disease, chronic kidney disease, chronic obstructive pulmonary disease, and cerebrovascular disease. Based also on the positive results of COVID-19 patients with anti-MASP-2 antibody, we propose the use of MASPs as a possible biomarker of the progression of COVID-19 and the investigation of new treatment strategies taking into consideration the dual role of MASPs, including MASP inhibitors as promising therapeutic targets against COVID-19.

Genetic variability of immune-related lncRNAs: polymorphisms in LINC-PINT and LY86-AS1 are associated with pemphigus foliaceus susceptibility.

Pemphigus foliaceus (PF) is an autoimmune blistering disease of the skin, clinically characterized by erosions and, histopathologically, by acantholysis. PF is endemic in the Brazilian Central-Western region. Numerous single nucleotide polymorphisms (SNPs) have been shown to affect the susceptibility for PF, including SNPs at long non-coding RNA (lncRNA) genes, which are known to participate in many physiological and pathogenic processes, such as autoimmunity. Here, we investigated whether the genetic variation of immune-related lncRNA genes affects the risk for endemic and sporadic forms of PF. We analysed 692 novel SNPs for PF from 135 immune-related lncRNA genes in 227 endemic PF patients and 194 controls. The SNPs were genotyped by Illumina microarray and analysed by applying logistic regression at additive model, with correction for sex and population structure. Six associated SNPs were also evaluated in an independent German cohort of 76 sporadic PF patients and 150 controls. Further, we measured the expression levels of two associated lncRNA genes (LINC-PINT and LY86-AS1) by quantitative PCR, stratified by genotypes, in peripheral blood mononuclear cells of healthy subjects. We found 27 SNPs in 11 lncRNA genes associated with endemic PF (p < .05 without overlapping with protein-coding genes). Among them, the LINC-PINT SNP rs10228040*A (OR = 1.47, p = .012) was also associated with increased susceptibility for sporadic PF (OR = 2.28, p = .002). Moreover, the A+ carriers of LY86-AS1*rs12192707 mark lowest LY86-AS1 RNA levels, which might be associated with a decreasing autoimmune response. Our results suggest a critical role of lncRNA variants in immunopathogenesis of both PF endemic and sporadic forms.

Identification of novel therapeutic targets for blocking acantholysis in pemphigus.

BACKGROUND AND PURPOSE: Pemphigus is caused by autoantibodies against desmoglein (Dsg) 1, Dsg3, and/or non-Dsg antigens. Pemphigus vulgaris (PV) is the most common manifestation of pemphigus, with painful erosions on mucous membranes. In most cases, blistering also occurs on the skin, leading to areas of extensive denudation. Despite improvements in pemphigus treatment, time to achieve remission is long, severe adverse events are frequent and 20% of patients do not respond adequately. Current clinical developments focus exclusively on modulating B cell function or autoantibody half-life. However, topical modulation of PV autoantibody-induced blistering is an attractive target because it could promptly relieve symptoms. EXPERIMENTAL APPROACH: To address this issue, we performed an unbiased screening in a complex biological system using 141 low MW inhibitors from a chemical library. Specifically, we evaluated PV IgG-induced Dsg3 internalization in HaCaT keratinocytes. Validation of the 20 identified compounds was performed using keratinocyte fragmentation assays, as well as a human skin organ culture (HSOC) model. KEY RESULTS: Overall, this approach led to the identification of four molecules involved in PV IgG-induced skin pathology: MEK1, TrkA, PI3Kα, and VEGFR2. CONCLUSION AND IMPLICATIONS: This unbiased screening revealed novel mechanisms by which PV autoantibodies induce blistering in keratinocytes and identified new treatment targets for this severe and potentially life-threatening skin disease.

Hepatitis B Virus Infection Among Leprosy Patients: A Case for Polymorphisms Compromising Activation of the Lectin Pathway and Complement Receptors.

Thousands of leprosy patients not only suffer from physical deformities, but also either have or have had hepatitis B virus (HBV) coinfection. Polymorphisms of the complement system modulate susceptibility to leprosy, but genetic susceptibility to past or present HBV infection is unknown. We used sequencing and multiplex sequence-specific PCR to genotype 72 polymorphisms of seven genes (MBL2, FCN1, FCN2, FCN3, MASP1, MASP2, C3) encoding components of the lectin pathway, and two genes encoding complement receptors (CR1, VSIG4) in 190 patients, of which 74 were positive for HBsAg and/or anti-HBc (HBV+, 93.2% with a resolved infection) and 116 lepromatous patients, and 408 HBV-blood donors. In addition, we tested for levels of proteins of the lectin pathway. We found no difference between serum concentrations of mannan-binding lectin (MBL), MBL-associated serine proteins (MASP-1, MASP-2, MASP-3, MAp44), ficolin-3 (FCN-3), soluble complement receptor 1 (sCR1) and MBL mediated C4 activation, measured by ELISA or TRIFMA in up to 167 HBV+ and HBV- patients. Haplotypes lowering protein levels or encoding dysfunctional proteins increased susceptibility to HBV infection: MBL2*LYQC (OR = 3.4, p = 0.02), MASP1*AC_CC (OR = 4.0, p = 0.015) and MASP2*1C2-l (OR = 5.4, p = 0.03). Conversely, FCN1*3C2 haplotype, associated with higher gene expression, was protective (OR = 0.56, P = 0.033). Other haplotypes associated with HBV susceptibility were: MASP2*2B1-i (OR = 19.25, P = 0.003), CR1*3A (OR = 2.65, P = 0.011) and VSIG4*TGGRCG (OR = 12.55, P = 0.014). Some polymorphisms in ficolin genes associated with lower protein levels increased susceptibility to leprosy/HBV infection: FCN*1 (OR = 1.66, P = 0.029), FCN2*GGGCAC (OR = 6.73, P = 0.008), and FCN3*del_del_C (OR = 12.54, P = 0.037), and to lepromatous disease/HBV infection: FCN2*TA (OR = 2.5, P = 0.009), whereas FCN2*MAG was associated with increased FCN-2 expression and resistance against coinfection (OR = 0.29, P = 0.026). These associations were independent of demographic factors and did not increase susceptibility to leprosy per se, except MASP2*1C2-l. Associations for FCN2, FCN3, MASP1, MASP2, and VSIG4 variants were also independent of each other. In conclusion, polymorphisms compromising activation of the lectin pathway of complement increase susceptibility to HBV infection, with ficolin polymorphisms playing a major role in modulating the susceptibility among leprosy patients.

Condemned or Not to Die? Gene Polymorphisms Associated With Cell Death in Pemphigus Foliaceus.

Pemphigus foliaceus (PF) is an autoimmune blistering skin disease that occurs sporadically across the globe and is endemic in Brazil. Keratinocyte adhesion loss (acantholysis) is associated with high levels of anti-desmoglein 1 IgG autoantibodies, but the role of cell death is poorly understood in PF. Current evidence disqualifies apoptosis as the major cell death mechanism and no other process has yet been investigated. To approach the role of variation in genes responsible for cell death pathways in pemphigus susceptibility, we systematically investigated the frequencies of 1,167 polymorphisms from genes encoding products of all 12 well-established cell death cascades (intrinsic and extrinsic apoptosis, necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic, NETotic, lysosome-dependent, autophagy-dependent, and immunogenic). By multivariate logistic regression, we compared allelic and genotypic frequencies of 227 PF patients and 194 controls obtained by microarray hybridization. We found 10 variants associated with PF (p < 0.005), belonging to six cell death pathways: apoptosis (TNF, TRAF2, CD36, and PAK2), immunogenic cell death (EIF2AK3, CD47, and SIRPA), necroptosis (TNF and TRAF2), necrosis (RAPGEF3), parthanatos (HK1), and pyroptosis (PRKN). Five polymorphisms were associated with susceptibility: TNF rs1800630*A (OR = 1.9, p = 0.0003), CD36 rs4112274*T (OR = 2.14, p = 0.0015), CD47 rs12695175*G (OR = 1.77, p = 0.0043), SIRPA rs6075340*A/A (OR = 2.75, p = 0.0009), and HK1 rs7072268*T (OR = 1.48, p = 0.0045). Other five variants were associated with protection: TRAF2 rs10781522*G (OR = 0.64, p = 0.0014), PAK2 rs9325377*A/A (OR = 0.48, p = 0.0023), EIF2AK3 rs10167879*T (OR = 0.48, p = 0.0007), RAPGEF3 rs10747521*A/A (OR = 0.42, p = 0.0040), and PRKN rs9355950*C (OR = 0.57, p = 0.0004). Through functional annotation, we found that all associated alleles, with the exception of PRKN rs9355950*C, were previously associated with differential gene expression levels in healthy individuals (mostly in skin and peripheral blood). Further functional validation of these genetic associations may contribute to the understanding of PF etiology and to the development of new drugs and therapeutic regimens for the disease.

Sparking Fire Under the Skin? Answers From the Association of Complement Genes With Pemphigus Foliaceus.

Skin blisters of pemphigus foliaceus (PF) present concomitant deposition of autoantibodies and components of the complement system (CS), whose gene polymorphisms are associated with susceptibility to different autoimmune diseases. To investigate these in PF, we evaluated 992 single-nucleotide polymorphisms (SNPs) of 44 CS genes, genotyped through microarray hybridization in 229 PF patients and 194 controls. After excluding SNPs with minor allele frequency <1%, out of Hardy-Weinberg equilibrium in controls or in strong linkage disequilibrium (r2 ≥ 0.8), 201 SNPs remained for logistic regression. Polymorphisms of 11 genes were associated with PF. MASP1 encodes a crucial serine protease of the lectin pathway (rs13094773: OR = 0.5, p = 0.0316; rs850309: OR = 0.23, p = 0.03; rs3864098: OR = 1.53, p = 0.0383; rs698104: OR = 1.52, p = 0.0424; rs72549154: OR = 0.55, p = 0.0453). C9 (rs187875: OR = 1.46, p = 0.0189; rs700218: OR = 0.12, p = 0.0471) and C8A (rs11206934: OR = 4.02, p = 0.0323) encode proteins of the membrane attack complex (MAC) and C5AR1 (rs10404456: OR = 1.43, p = 0.0155), a potent anaphylatoxin-receptor. Two encode complement regulators: MAC-blocking CD59 (rs1047581: OR = 0.62, p = 0.0152) and alternative pathway-blocking CFH (rs34388368: OR = 2.57, p = 0.0195). One encodes opsonin: C3 (rs4807895: OR = 2.52, p = 0.0239), whereas four encode receptors for C3 fragments: CR1 (haplotype with rs6656401: OR = 1.37, p = 0.0382), CR2 (rs2182911: OR = 0.23, p = 0.0263), ITGAM (CR3, rs12928810: OR = 0.66, p = 0.0435), and ITGAX (CR4, rs11574637: OR = 0.63, p = 0.0056). Associations reinforced former findings, regarding differential gene expression, serum levels, C3, and MAC deposition on lesions. Deregulation of previously barely noticed processes, e.g., the lectin and alternative pathways and opsonization-mediated phagocytosis, also modulate PF susceptibility. The results open new crucial avenues for understanding disease etiology and may improve PF treatment through additional therapeutic targets.