Yask Gupta

Genetic identification and functional validation of FcγRIV as key molecule in autoantibody-induced tissue injury†

Autoantibody‐mediated diseases are clinically heterogeneous and often fail conventional therapeutic strategies. Gene expression profiling has helped to identify new molecular pathways in these diseases, although their potential as treatment targets largely remains to be functionally validated. Based on weighted gene co‐expression network analysis, we determined the transcriptional network in experimental epidermolysis bullosa acquisita (EBA), a paradigm of an antibody‐mediated organ‐specific autoimmune disease characterized by autoantibodies directed against type VII collagen. We identified 33 distinct and differentially expressed modules, including Fcγ receptor (FcγR) IV and components of the neutrophil‐associated enzyme system in autoantibody transfer‐induced EBA. Validation experiments, including functional analysis, demonstrated that FcγRIV expression on neutrophils crucially contributes to autoantibody‐induced tissue injury in the transfer model of EBA. Mice lacking the common γ‐chain of activating FcγRs, deficient in FcγRIV or treated with FcγRIV function blocking antibody, but not mice deficient in FcγRI, FcγRIIB, FcγRIII or both FcγRI and FcγRIII, were effectively protected from EBA. Skin disease was restored in γ‐chain‐deficient mice locally reconstituted with neutrophils from wild‐type, but not from γ‐chain‐deficient, mice. Our findings both genetically and functionally identify a novel disease‐related molecule, FcγRIV, in an autoantibody‐mediated disorder, which may be of importance for the development of novel targeted therapies. Copyright

Genetic control of psoriasis is relatively distinct from that of metabolic syndrome and coronary artery disease.

Psoriasis is a common chronic inflammatory skin disease, associated with significant comorbidity, for example, metabolic syndrome (MetS) and coronary heart disease (CHD). This association implies that the risk to develop these diseases is commonly controlled or that the presence of one disease favours manifestation of the other. Therefore, we assessed the catalogue of genome‐wide association studies (GWAS) to analyse whether psoriasis, MetS and CHD share susceptibility loci. Interestingly, genetic control of psoriasis is almost completely independent from both MetS and CHD. In contrast, MetS and CHD share 10 common loci. Like by GWAS analysis, psoriasis susceptibility genes showed close clustering in Ingenuity Pathway Analysis, while genes conferring susceptibility to MetS and CHD were interlinked separately. These findings lead to the hypothesis that the clinically observed co‐occurrence of psoriasis with MetS and CHD may be due to a common environmental factor, for example, diet, which is known as a risk factor for all of these diseases.

Apolipoprotein E (APOE) genotype regulates body weight and fatty acid utilization-Studies in gene-targeted replacement mice

SCOPE Of the three human apolipoprotein E (APOE) alleles, the ε3 allele is most common, which may be a result of adaptive evolution. In this study, we investigated whether the APOE genotype affects body weight and energy metabolism through regulation of fatty acid utilization. METHODS AND RESULTS Targeted replacement mice expressing the human APOE3 were significantly heavier on low- and high-fat diets compared to APOE4 mice. Particularly on high-fat feeding, food intake and dietary energy yields as well as fat mass were increased in APOE3 mice. Fatty acid mobilization determined as activation of adipose tissue lipase and fasting plasma nonesterified fatty acid levels were significantly lower in APOE3 than APOE4 mice. APOE4 mice, in contrast, exhibited higher expression of proteins involved in fatty acid oxidation in skeletal muscle. CONCLUSION Our data suggest that APOE3 is associated with the potential to more efficiently harvest dietary energy and to deposit fat in adipose tissue, while APOE4 carriers tend to increase fatty acid mobilization and utilization as fuel substrates especially under high-fat intake. The different handling of dietary energy may have contributed to the evolution and worldwide distribution of the ε3 allele.

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. Copyright

The Leukotriene B4 and its Receptor BLT1 Act as Critical Drivers of Neutrophil Recruitment in Murine Bullous Pemphigoid-Like Epidermolysis Bullosa Acquisita

Recruitment of neutrophils and eosinophils into the skin is a hallmark of pemphigoid diseases. The molecular cues regulating granulocyte recruitment into the skin and the individual contributions of neutrophils and eosinophils to pemphigoid diseases are, however, poorly understood. The lipid mediator leukotriene B4 (LTB4) is a potent granulocyte chemoattractant and is abundant in the skin blister fluid of bullous pemphigoid (BP) patients, but its pathogenic significance is unknown. Using mouse models of BP-like epidermolysis bullosa acquisita and of BP, we show that LTB4 and its receptor BLT1 act as critical drivers of neutrophil entry into the skin upon antibody deposition at the dermal-epidermal junction. Mice deficient in 5-lipoxygenase, a key enzyme in LTB4 biosynthesis, or in BLT1 exhibited dramatic resistance to neutrophil recruitment and, consequently, skin inflammation. Accordingly, liquid chromatography-mass spectrometry, used to comprehensively profile lipid mediator generation in the first 48 hours after antibody deposition, showed a pronounced parallel increase in LTB4 and in neutrophils in the skin. Subsequent mechanistic studies in BP-like epidermolysis bullosa acquisita uncovered that neutrophils are necessary for skin inflammation, whereas eosinophils are dispensable, thus identifying neutrophils as major culprits of blister formation. Our results highlight LTB4/BLT1 as absolutely critical drivers of murine pemphigoid disease-like skin inflammation.

Combining genetic mapping with genome-wide expression in experimental autoimmune encephalomyelitis highlights a gene network enriched for T cell functions and candidate genes regulating autoimmunity

The experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system commonly used to study multiple sclerosis (MS). We combined clinical EAE phenotypes with genome-wide expression profiling in spleens from 150 backcross rats between susceptible DA and resistant PVG rat strains during the chronic EAE phase. This enabled correlation of transcripts with genotypes, other transcripts and clinical EAE phenotypes and implicated potential genetic causes and pathways in EAE. We detected 2285 expression quantitative trait loci (eQTLs). Sixty out of 599 cis-eQTLs overlapped well-known EAE QTLs and constitute positional candidate genes, including Ifit1 (Eae7), Atg7 (Eae20-22), Klrc3 (eEae22) and Mfsd4 (Eae17). A trans-eQTL that overlaps Eae23a regulated a large number of small RNAs and implicates a master regulator of transcription. We defined several disease-correlated networks enriched for pathways involved in cell-mediated immunity. They include C-type lectins, G protein coupled receptors, mitogen-activated protein kinases, transmembrane proteins, suppressors of transcription (Jundp2 and Nr1d1) and STAT transcription factors (Stat4) involved in interferon signaling. The most significant network was enriched for T cell functions, similar to genetic findings in MS, and revealed both established and novel gene interactions. Transcripts in the network have been associated with T cell proliferation and differentiation, the TCR signaling and regulation of regulatory T cells. A number of network genes and their family members have been associated with MS and/or other autoimmune diseases. Combining disease and genome-wide expression phenotypes provides a link between disease risk genes and distinct molecular pathways that are dysregulated during chronic autoimmune inflammation.

Radiosensitive Hematopoietic Cells Determine the Extent of Skin Inflammation in Experimental Epidermolysis Bullosa Acquisita

Animal models have enhanced our understanding of the pathogenesis of autoimmune diseases. For these models, genetically identical, inbred mice have commonly been used. Different inbred mouse strains, however, show a high variability in disease manifestation. Identifying the factors that influence this disease variability could provide unrecognized insights into pathogenesis. We established a novel Ab transfer-induced model of epidermolysis bullosa acquisita (EBA), an autoimmune disease characterized by (muco)-cutaneous blistering caused by anti-type VII collagen (COL7) autoantibodies. Blistering after anti-COL7 IgG (directed against the von Willebrand factor A–like domain 2) transfer showed clear variability among inbred mouse strains, that is, severe cutaneous blistering and inflammation in C57BL/6J and absence of skin lesions in MRL/MpJ mice. The transfer of anti-COL7 IgG into irradiated, EBA-resistant MRL/MpJ mice, rescued by transplantation with bone marrow from EBA-susceptible B6.AK-H2k mice, induced blistering. To the contrary, irradiated EBA-susceptible B6.AK-H2k mice that were rescued using MRL/MpJ bone marrow were devoid of blistering. In vitro, immune complex activation of neutrophils from C57BL/6J or MRL/MpJ mice showed an impaired reactive oxygen species release from the latter, whereas no differences were observed after PMA activation. This finding was paralleled by divergent expression profiles of immune complex–activated neutrophils from either C57BL/6J or MRL/MpJ mice. Collectively, we demonstrate that radiosensitive cells determine the varying extent of skin inflammation and blistering in the end-stage effector phase of EBA.

Improved detection of gene-microbe interactions in the mouse skin microbiota using high-resolution QTL mapping of 16S rRNA transcripts

BackgroundRecent studies highlight the utility of quantitative trait locus (QTL) mapping for determining the contribution of host genetics to interindividual variation in the microbiota. We previously demonstrated that similar to the gut microbiota, abundances of bacterial taxa in the skin are significantly influenced by host genetic variation. In this study, we analyzed the skin microbiota of mice from the 15th generation of an advanced intercross line using a novel approach of extending bacterial trait mapping to both the 16S rRNA gene copy (DNA) and transcript (RNA) levels, which reflect relative bacterial cell number and activity, respectively.ResultsRemarkably, the combination of highly recombined individuals and 53,203 informative SNPs allowed the identification of genomic intervals as small as <0.1 megabases containing single genes. Furthermore, the inclusion of 16S rRNA transcript-level mapping dramatically increased the number of significant associations detected, with five versus 21 significant SNP-bacterial trait associations based on DNA- compared to RNA-level profiling, respectively. Importantly, the genomic intervals identified contain many genes involved in skin inflammation and cancer and are further supported by the bacterial traits they influence, which in some cases have known genotoxic or probiotic capabilities.ConclusionsThese results indicate that profiling based on the relative activity levels of bacterial community members greatly enhances the capability of detecting interactions between the host and its associated microbes. Finally, the identification of several genes involved in skin cancer suggests that similar to colon carcinogenesis, the resident microbiota may play a role in skin cancer susceptibility and its potential prevention and/or treatment.

Lifespan effects of mitochondrial mutations

Somatic mitochondrial DNA (mtDNA) mutations accumulate within various tissues with age1,2, however evidence directly showing the influence of mtDNA natural variations on ageing has been limited to date. Recently, Latorre-Pellicer et al. demonstrated that polymorphisms within mtDNA affect reactive oxygen species (ROS) levels, body mass, ageing score, tumour incidence and lifespan of conplastic mice3. Here we show that similarly generated conplastic strains, which carry a nuclear Nnt mutation, do not show any alterations in these parameters, demonstrating the relevance of specific mitonuclear interactions in determining mammalian healthspan through increased production of ROS. Latorre-Pellicer et al.3 compared a conplastic mouse strain that they developed from a C57BL/6JOlaHsd nuclear genome and NZB/OlaHsd mtDNA (BL/6NZB) mice carrying various mutations in the mitochondrial genome with the original C57BL/6JOlaHsd carrying unaltered mtDNA (BL/6C57). Despite the increased levels of ROS at a young age, BL/6NZB mice showed a delayed ageing phenotype, including reduced tumour incidence culminating in an extended lifespan, which is consistent with previously published findings in invertebrates4. In this study4, it was shown that metabolic induction of mitochondrial ROS formation promotes longevity in the nematode Caenorhabditis elegans, and that quenching this ROS signal by antioxidants abrogates the increase in lifespan. C57BL/6J mice (Jackson Laboratories, JAX no. 000664) are known to harbour a mutation in a nuclear gene encoding the mitochondrially located nicotinamide nucleotide transhydrogenase (NNT) protein that renders the enzyme undetectable, resulting in reduced cytosolic antioxidant capacity and increased production of hydrogen peroxide5 as well as impaired glucose tolerance6, independent of any additional mitochondrial variation. We generated conplastic C57BL/6J-mtNZB/BnlJ (mtNZB/BlnJ)7 and C57BL/6J (mtC57BL/6J) mice similar to the design used by Latorre-Pellicer et al.3. Notably, we did not observe an extension of the median or maximum lifespan of our conplastic mtNZB/BlnJ mice (P = 0.251, log-rank test; P = 0.943, Gehan test; Fig. 1a and Extended Data Table 1a–d) despite our large cohort size (n = 155, mtC57BL/6J; n = 131, mtNZB/BlnJ) resulting in high statistical power (> 99% for the Gehan test, which places higher weight on early deaths; and as used by Latorre-Pellicer et al.3). Furthermore, we did not observe any differences in body mass, ageing score, tumour incidence (Fig. 1b–d) or spontaneous locomotor activity (Extended Data Fig. 1a, b). In addition, ROS levels, electron transport chain complex activity, and energy expenditure between our two strains showed no significant differences. An independent survival analysis with a log-rank test (which weights all subjects equally) also did not reveal any effect of the introduction of conplastic mtDNA on the lifespan of Nnt-deficient mice. Given that the published BL/6NZB line and our mtNZB/BlnJ mice harbour essentially the same mtDNA mutations (Extended Data Table 2), the simplest interpretation for the different results obtained by Latorre-Pellicer et al.3 and us is that the absence of NNT protein negates the effects of mitochondrial variation on healthspan. Together with the data from Latorre-Pellicer et al.3, our findings indicate that mtDNA mutations that increase ROS levels on a functional NNT background are associated with an increased healthspan3, whereas unaltered ROS levels prevent this effect on the progression of ageing (Fig. 1), both consistent with findings on mitohormesis4. As demonstrated in a variety of biological systems including humans8, low-dose increases in mitochondrial ROS promote health and longevity, whereas higher doses cause 3 6 12 18 24 15 20 25 30 35 40 45

A Family with Atypical Hailey Hailey Disease- Is There More to the Underlying Genetics than ATP2C1?

The autosomal dominant Hailey Hailey disease (HHD) is caused by mutations in the ATP2C1 gene encoding for human secretory pathway Ca2+/Mn2+ ATPase protein (hSPCA1) in the Golgi apparatus. Clinically, HHD presents with erosions and hyperkeratosis predominantly in the intertrigines. Here we report an exome next generation sequencing (NGS) based analysis of ATPase genes in a Greek family with 3 HHD patients presenting with clinically atypical lesions mainly localized on the neck and shoulders. By NGS of one HHD-patient and in silico SNP calling and SNP filtering we identified a SNP in the expected ATP2C1 gene and SNPs in further ATPase genes. Verification in all 3 affected family members revealed a heterozygous frameshift deletion at position 2355_2358 in exon 24 of ATP2C1 in all three patients. 7 additional SNPs in 4 ATPase genes (ATP9B, ATP11A, ATP2B3 and ATP13A5) were identified. The SNPs rs138177421 in the ATP9B gene and rs2280268 in the ATP13A5 gene were detected in all 3 affected, but not in 2 non affected family members. The SNPs in the ATP2B3 and ATP11A gene as well as further SNPs in the ATP13A5 gene could not be confirmed in all affected family members. One may speculate that besides the level of functional hSPCA1 protein, levels of other ATPase proteins may influence expressivity of the disease and might also contribute, as in this case, to atypical presentations.