Multiple Modes of Action Mediate the Therapeutic Effect of Intravenous IgG in Experimental Epidermolysis Bullosa Acquisita.

Substitution of IgG in antibody deficiency or application of high-dose intravenous IgG in patients with autoimmunity is a well-established treatment. However, data on the mode of action of intravenous IgG are controversial and may differ for distinct diseases. In this study, we investigated the impact and molecular mechanism of high-dose IgG (hd-IgG) treatment in murine autoantibody‒induced skin inflammation, namely, epidermolysis bullosa acquisita. Epidermolysis bullosa acquisita is caused by antibodies directed against type VII collagen and is mediated by complement activation, the release of ROS, and proteases by myeloid cells. In murine experimental epidermolysis bullosa acquisita, the disease can be induced by injection of anti‒type VII collagen IgG. In this study, we substantiate that treatment with hd-IgG improves clinical disease manifestation. Mechanistically, hd-IgG reduced the amount of anti‒type VII collagen in the skin and sera, which is indicative of an FcRn-dependent mode of action. Furthermore, in a nonreceptor-mediated fashion, hd-IgG showed antioxidative properties by scavenging extracellular ROS. Hd-IgG also impaired complement activation and served as a substrate for proteases, both key events during epidermolysis bullosa acquisita pathogenesis. Collectively, the nonreceptor-mediated anti-inflammatory properties of hd-IgG may explain the therapeutic benefit of intravenous IgG treatment in skin autoimmunity.

NLRP12: The Monarch of the Gut.

Innate pattern recognition receptors have been implicated in the obesity-associated imbalance of gut microbiota. In this issue of Cell Host & Microbe, Truax et al. (2018) report that NLRP12 prevents high-fat-diet-induced obesity by maintaining beneficial short-chain fatty acid-producing microbiota.

Association of PTPN22 Single Nucleotide Polymorphisms with Celiac Disease.

OBJECTIVES: Celiac disease is a chronic autoimmune disease in which gene-environment interactions cause the immune system to unfavorably react to naturally gluten-containing foods. PTPN22 plays a crucial role in regulating the function of various cells of the immune system, particularly T cells. Polymorphisms of the PTPN22 gene have been associated with many autoimmune diseases. The present genetic association study was conducted to investigate the possible associations between PTPNTT single nucleotide polymorphisms (SNPs) and celiac disease in an Iranian population. MATERIALS AND METHODS: The study population consisted of 45 patients with celiac disease and 93 healthy controls. The study genotyped five SNPs of the PTPN22 gene: rs12760457, rs1310182, rs1217414, rs33996649, and rs2476601. RESULTS AND CONCLUSIONS: Control and patient groups did not differ on the genotype distribution of four of five investigated SNPs in the PTPN22 gene, for example, rs12760457, rs2476601, rs1217414, and rs33996649. The only investigated PTPN22 variant, which could be associated with CD, was rs1310182. A significant increase in the carriage of the T allele of rs1310182 in CD patients was observed (OR (95% CI) = 11.42 (5.41, 24.1), p value < 0.0001). The TT genotype of this SNP was significantly associated with celiac disease. Our study suggests that the rs1310182 SNP of PTPN22 gene may be a predisposing factor of celiac disease in the Iranian population. Further studies are required to investigate the issue in other racial and ethnic subgroups.

The retinoid-related orphan receptor alpha is essential for the end-stage effector phase of experimental epidermolysis bullosa acquisita.

Genetic studies have added to the understanding of complex diseases. Here, we used a combined genetic approach for risk-loci identification in a prototypic, organ-specific, autoimmune disease, namely experimental epidermolysis bullosa acquisita (EBA), in which autoantibodies to type VII collagen (COL7) and neutrophil activation cause mucocutaneous blisters. Anti-COL7 IgG induced moderate blistering in most inbred mouse strains, while some showed severe disease or were completely protected. Using publicly available genotyping data, we identified haplotype blocks that control blistering and confirmed two haplotype blocks in outbred mice. To identify the blistering-associated genes, haplotype blocks encoding genes that are differentially expressed in EBA-affected skin were considered. This procedure identified nine genes, including retinoid-related orphan receptor alpha (RORα), known to be involved in neurological development and function. After anti-COL7 IgG injection, RORα+/- mice showed reduced blistering and homozygous mice were completely resistant to EBA induction. Furthermore, pharmacological RORα inhibition dose-dependently blocked reactive oxygen species (ROS) release from activated neutrophils but did not affect migration or phagocytosis. Thus, forward genomics combined with multiple validation steps identifies RORα to be essential to drive inflammation in experimental EBA.

Caspase-1-independent IL-1 release mediates blister formation in autoantibody-induced tissue injury through modulation of endothelial adhesion molecules.

Although reports documented aberrant cytokine expression in autoimmune bullous dermatoses (AIBDs), cytokine-targeting therapies have not been established in these disorders. We showed previously that IL-6 treatment protected against tissue destruction in experimental epidermolysis bullosa acquisita (EBA), an AIBD caused by autoantibodies to type VII collagen (COL7). The anti-inflammatory effects of IL-6 were mediated by induction of IL-1ra, and prophylactic IL-1ra administration prevented blistering. In this article, we demonstrate elevated serum concentrations of IL-1ß in both mice with experimental EBA induced by injection of anti-COL7 IgG and in EBA patients. Increased IL-1α and IL-1ß expression also was observed in the skin of anti-COL7 IgG-injected wild-type mice compared with the significantly less diseased IL-1R-deficient or wild-type mice treated with the IL-1R antagonist anakinra or anti-IL-1ß. These findings suggested that IL-1 contributed to recruitment of inflammatory cells into the skin. Accordingly, the expression of ICAM-1 was decreased in IL-1R-deficient and anakinra-treated mice injected with anti-COL7. This effect appeared to be specifically attributable to IL-1 because anakinra blocked the upregulation of different endothelial adhesion molecules on IL-1-stimulated, but not on TNF-α-stimulated, cultured endothelial cells. Interestingly, injection of caspase-1/11-deficient mice with anti-COL7 IgG led to the same extent of skin lesions as in wild-type mice. Collectively, our data suggest that IL-1, independently of caspase-1, contributes to the pathogenesis of EBA. Because anti-IL-1ß in a prophylactic setting and anakinra in a quasi-therapeutic setting (i.e., when skin lesions had already developed) improved experimental EBA, IL-1 appears to be a potential therapeutic target for EBA and related AIBDs.

Population genetic structure and polymorphism analysis of gene encoding apical membrane antigen-1 (AMA-1) of Iranian Plasmodium vivax wild isolates.

Plasmodium vivax apical membrane antigen-1 (PvAMA-1) is a major candidate antigen for human malaria vaccine. In the present study, polymorphism of pvama-1 among Iranian isolates was investigated to generate useful information on this vaccine candidate antigen, which is required for the rational design of a vaccine against P. vivax. Blood samples were collected from P. vivax-infected Iranian patients during 2009-2010. Of 99 collected isolates, 37 were analyzed for almost the entire pvama-1 gene using sequencing. The overall nucleotide diversity (π) was 0.00826 ± 0.0004 and the majority of polymorphic sites were identified in domain I (DI) of the pvama-1 gene. Neutrality analysis using Tajima's D, Fu and Li's D* and F* and McDonald Kreitman tests showed a significant positive departure from neutral substitution patterns, indicating a possible balancing selection across the entire ectodomain and DI sequences of pvama-1 gene. However, no evidence was found for the balancing selection in DII and DIII regions of Iranian PvAMA-1. Also, 29 haplotypes with different frequencies were identified and the overall haplotype diversity was 0.982 ± 0.012. Epitope mapping prediction of PvAMA-1 showed the potential B-cell epitopes across DI-DIII overlap with E145K, P210S, R249H, G253E, K352E, R438H and N445D mutations; however, no mutation has been found in intrinsically unstructured/disordered regions. The fixation index (Fst) estimation between Iran and the closest geographical sites such as India (0.0707) showed a slight geographical genetic differentiation; however, the Fst estimation between Iran and Thailand (0.1253) suggested a moderate geographical isolation. In summary, genetic investigation in pvama-1 among Iranian P. vivax isolates indicates that this antigen showed limited antigenic diversity and most of the detected mutations are located outside B-cell epitopes. Therefore, the present results have significant implications in understanding the nature of P. vivax population circulating in Iran as well as in providing useful information for malaria vaccine development based on this antigen.

Recombinant IL-6 treatment protects mice from organ specific autoimmune disease by IL-6 classical signalling-dependent IL-1ra induction.

Cytokines are key regulators of physiological inflammatory responses, while aberrant cytokine expression contributes to pathogenesis of autoimmune diseases. We noted increased IL-6 levels in human and murine epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune bullous dermatoses (AIBD) induced by autoantibodies to type VII collagen (COL7). In contrast to rheumatoid arthritis, blockade of IL-6 led to strikingly enhanced experimental EBA, while treatment with recombinant IL-6 was protective. This was due to classical IL-6 signalling and independent of IL-6 trans-signalling, as treatment of mice with sgp130Fc had no impact on EBA manifestation. Induction of EBA in mice led to increased IL-1ra levels in skin and serum, while blockade of IL-6 completely inhibited IL-1ra expression induced by autoantibodies to COL7. In line, treatment of mice with EBA with recombinant IL-6 induced IL-1ra concentrations exceeding those of untreated animals with EBA, and IL-1ra (anakinra) administration significantly impaired experimental EBA induction. We here identified a novel anti-inflammatory pathway in an organ-specific autoimmune disease. Modulation of this IL-1ra pathway by classical IL-6 signalling demonstrates anti-inflammatory and protective activities of IL-6 in vivo.

Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method.

Many ligase-independent cloning methods have been developed to overcome problems of standard restriction cloning such as low transformation efficiency and high background of vector with no insert. Most of these methods are still enzyme based, require time-consuming incubation and multiple purification steps, and/or might have a low robustness in handling. Thus, with the aim to establish a robust enzyme/ligase-free method, we developed the phosphorothioate-based ligase-independent gene cloning (PLICing) method, which is based on a chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution. After optimization of polymerase chain reaction (PCR) and DNA cleavage conditions, PLICing performs competitively with all commercialized methods in terms of handling and transformation efficiency. In addition, PLICing is absolutely sequence independent and surpasses other concepts regarding cloning efficiency given that none of the 240 analyzed clones showed any religation event for three different model genes. A developed fast PLICing protocol does not require any purification step and can be completed within 10 min. Due to its robustness, reliability, and simplicity, PLICing should prove to be a true alternative to other well-established cloning techniques.