Single-cell transcriptomics analysis of bullous pemphigoid unveils immune-stromal crosstalk in type 2 inflammatory disease.

Bullous pemphigoid (BP) is a type 2 inflammation- and immunity-driven skin disease, yet a comprehensive understanding of the immune landscape, particularly immune-stromal crosstalk in BP, remains elusive. Herein, using single-cell RNA sequencing (scRNA-seq) and in vitro functional analyzes, we pinpoint Th2 cells, dendritic cells (DCs), and fibroblasts as crucial cell populations. The IL13-IL13RA1 ligand-receptor pair is identified as the most significant mediator of immune-stromal crosstalk in BP. Notably, fibroblasts and DCs expressing IL13RA1 respond to IL13-secreting Th2 cells, thereby amplifying Th2 cell-mediated cascade responses, which occurs through the specific upregulation of PLA2G2A in fibroblasts and CCL17 in myeloid cells, creating a positive feedback loop integral to immune-stromal crosstalk. Furthermore, PLA2G2A and CCL17 contribute to an increased titer of pathogenic anti-BP180-NC16A autoantibodies in BP patients. Our work provides a comprehensive insight into BP pathogenesis and shows a mechanism governing immune-stromal interactions, providing potential avenues for future therapeutic research.

Targeted serum proteome profiling reveals nicotinamide adenine dinucleotide phosphate (NADPH)-related biomarkers to discriminate linear IgA bullous disorder from dermatitis herpetiformis.

Linear IgA bullous dermatosis (LABD) and dermatitis herpetiformis (DH) represent the major subtypes of IgA mediated autoimmune bullous disorders. We sought to understand the disease etiology by using serum proteomics. We assessed 92 organ damage biomarkers in LAB, DH, and healthy controls using the Olink high-throughput proteomics. The positive proteomic serum biomarkers were used to correlate with clinical features and HLA type. Targeted proteomic analysis of IgA deposition bullous disorders vs. controls showed elevated biomarkers. Further clustering and enrichment analyses identified distinct clusters between LABD and DH, highlighting the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Comparative analysis revealed biomarkers with distinction between LABD and DH and validated in the skin lesion. Finally, qualitative correlation analysis with DEPs suggested six biomarkers (NBN, NCF2, CAPG, FES, BID, and PXN) have better prognosis in DH patients. These findings provide potential biomarkers to differentiate the disease subtype of IgA deposition bullous disease.

Genome-wide meta-analysis and fine-mapping prioritize potential causal variants and genes related to leprosy.

To date, genome-wide association studies (GWASs) have discovered 35 susceptible loci of leprosy; however, the cumulative effects of these loci can only partially explain the overall risk of leprosy, and the causal variants and genes within these loci remain unknown. Here, we conducted out new GWASs in two independent cohorts of 5007 cases and 4579 controls and then a meta-analysis in these newly generated and multiple previously published (2277 cases and 3159 controls) datasets were performed. Three novel and 15 previously reported risk loci were identified from these datasets, increasing the known leprosy risk loci of explained genetic heritability from 23.0 to 38.5%. A comprehensive fine-mapping analysis was conducted, and 19 causal variants and 14 causal genes were identified. Specifically, manual checking of epigenomic information from the Epimap database revealed that the causal variants were mainly located within the immune-relevant or immune-specific regulatory elements. Furthermore, by using gene-set, tissue, and cell-type enrichment analyses, we highlighted the key roles of immune-related tissues and cells and implicated the PD-1 signaling pathways in the pathogenetic mechanism of leprosy. Collectively, our study identified candidate causal variants and elucidated the potential regulatory and coding mechanisms for genes associated with leprosy.

HLA-C*15:02 and epidermal growth factor receptor inhibitor-induced erosive pustular dermatosis of the scalp.

Epidermal growth factor receptor inhibitors (EGFRIs) are widely used to treat various types of malignancies. One of the common adverse reactions is cutaneous toxicity, mostly presenting as acneiform eruptions, paronychia and xerosis. Erosive pustular dermatosis of the scalp (EPDS) is a rare cutaneous adverse reaction that develops during treatment with EGFRIs. The pathogenesis of EGFRI-induced EPDS is poorly understood. Here we present three cases of EPDS induced by EGFRIs. The proteins LTA4H (leukotriene A-4 hydrolase), METAP1 (methionine aminopeptidase 1), BID (BH3-interacting domain death agonist), SMAD1 (mothers against decapentaplegic homologue), PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A), YES1 (tyrosine-protein kinase Yes) and EGFL7 (epidermal growth factor-like protein 7) were significantly upregulated in EGFRI-stimulated peripheral blood mononuclear cell cultures, and validated in the lesions. All of the proteins colocalized with CD4+ and CD8+ T-cell expression. Next-generation-based human leucocyte antigen (HLA) typing showed all patients carried HLA-C*15:02, and modelling studies showed that afatinib and erlotinib bound well within the E/F binding pockets of HLA-C*15:02. Moreover, T cells were preferentially activated by EGFRIs in individuals carrying HLA-C*15:02. The case series revealed that EGFRI-induced EPDS may be mediated by drug-specific T cells.

Single-cell sequencing analysis reveals development and differentiation trajectory of Schwann cells manipulated by M. leprae.

BACKGROUND: M. leprae preferentially infects Schwann cells (SCs) in the peripheral nerves leading to nerve damage and irreversible disability. Knowledge of how M. leprae infects and interacts with host SCs is essential for understanding mechanisms of nerve damage and revealing potential new therapeutic strategies. METHODOLOGY/PRINCIPAL FINDINGS: We performed a time-course single-cell sequencing analysis of SCs infected with M. leprae at different time points, further analyzed the heterogeneity of SCs, subpopulations associated with M. leprae infection, developmental trajectory of SCs and validated by Western blot or flow cytometry. Different subpopulations of SCs exhibiting distinct genetic features and functional enrichments were present. We observed two subpopulations associated with M. leprae infection, a stem cell-like cell subpopulation increased significantly at 24 h but declined by 72 h after M. leprae infection, and an adipocyte-like cell subpopulation, emerged at 72 h post-infection. The results were validated and confirmed that a stem cell-like cell subpopulation was in the early stage of differentiation and could differentiate into an adipocyte-like cell subpopulation. CONCLUSIONS/SIGNIFICANCE: Our results present a systematic time-course analysis of SC heterogeneity after infection by M. leprae at single-cell resolution, provide valuable information to understand the critical biological processes underlying reprogramming and lipid metabolism during M. leprae infection of SCs, and increase understanding of the disease-causing mechanisms at play in leprosy patients as well as revealing potential new therapeutic strategies.

TAP2 Drives HLA-B∗13:01‒Linked Dapsone Hypersensitivity Syndrome Tolerance and Reactivity.

Dapsone hypersensitivity syndrome (DHS) is restricted to HLA-B∗13:01. However, the positive predictive value for HLA-B∗13:01 is only 7.8%. To explore the potential coexisting factors involved in the occurrence of DHS, we carried out a GWAS and a genome-wide DNA methylation profile analysis comparing patients with DHS with dapsone-tolerant control subjects (all carrying HLA-B∗13:01). No non-HLA SNPs associated with DHS were identified at the genome-wide level. However, the pathway of antigen processing and presentation was enriched in patients with DHS, and the gene TAP2 was identified. Expression of TAP2 and its molecular chaperone, TAP1, were validated by quantitative PCR, and in vitro functional experiments were performed. The results showed that patients with DHS have higher mRNA levels of TAP1 and TAP2 and an enhanced capacity for antigen-presenting cells activating dapsone-specific T cells compared with dapsone-tolerant controls. Activation of dapsone-specific T cells was inhibited when TAP function of antigen-presenting cells was impaired. This study shows that epigenetic regulation of TAP1 and TAP2 affects the function of antigen-presenting cells and is a critical factor that mediates the development of DHS.

IL-23/IL-23R Promote Macrophage Pyroptosis and T Helper 1/T Helper 17 Cell Differentiation in Mycobacterial Infection.

Pathogen-induced epigenetic modifications can reshape anti-infection immune processes and control the magnitude of host responses. DNA methylation profiling has identified crucial aberrant methylation changes associated with diseases, thus providing biological insights into the roles of epigenetic factors in mycobacterial infection. In this study, we performed a genome-wide methylation analysis of skin biopsies from patients with leprosy and healthy controls. T helper 17 differentiation pathway was found to be significantly associated with leprosy through functional enrichment analysis. As a key gene in this pathway, IL-23R was found to be critical to mycobacterial immunity in leprosy, according to integrated analysis with DNA methylation, RNA sequencing, and GWASs. Functional analysis revealed that IL-23/IL-23R-enhanced bacterial clearance by activating caspase-1/GSDMD-mediated pyroptosis in a manner dependent on NLRP3 through signal transducer and activator of transcription 3 signaling in macrophages. Moreover, IL23/IL-23R promoted T helper 1 and T helper 17 cell differentiation and proinflammatory cytokine secretion, thereby increasing host bactericidal activity. IL-23R knockout attenuated the effects and increased susceptibility to mycobacterial infection mentioned earlier. These findings illustrate the biological functions of IL-23/IL-23R in modulating intracellular bacterial clearance in macrophages and further support their regulatory effects in T helper cell differentiation. Our study highlights that IL-23/IL-23R might serve as potential targets for the prevention and treatment of leprosy and other mycobacterial infections.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

Detection of Hepatic Drug Metabolite-Specific T-Cell Responses Using a Human Hepatocyte, Immune Cell Coculture System.

Drug-responsive T-cells are activated with the parent compound or metabolites, often via different pathways (pharmacological interaction and hapten). An obstacle to the investigation of drug hypersensitivity is the scarcity of reactive metabolites for functional studies and the absence of coculture systems to generate metabolites in situ. Thus, the aim of this study was to utilize dapsone metabolite-responsive T-cells from hypersensitive patients, alongside primary human hepatocytes to drive metabolite formation, and subsequent drug-specific T-cell responses. Nitroso dapsone-responsive T-cell clones were generated from hypersensitive patients and characterized in terms of cross-reactivity and pathways of T-cell activation. Primary human hepatocytes, antigen-presenting cells, and T-cell cocultures were established in various formats with the liver and immune cells separated to avoid cell contact. Cultures were exposed to dapsone, and metabolite formation and T-cell activation were measured by LC-MS and proliferation assessment, respectively. Nitroso dapsone-responsive CD4+ T-cell clones from hypersensitive patients were found to proliferate and secrete cytokines in a dose-dependent manner when exposed to the drug metabolite. Clones were activated with nitroso dapsone-pulsed antigen-presenting cells, while fixation of antigen-presenting cells or omission of antigen-presenting cells from the assay abrogated the nitroso dapsone-specific T-cell response. Importantly, clones displayed no cross-reactivity with the parent drug. Nitroso dapsone glutathione conjugates were detected in the supernatant of hepatocyte immune cell cocultures, indicating that hepatocyte-derived metabolites are formed and transferred to the immune cell compartment. Similarly, nitroso dapsone-responsive clones were stimulated to proliferate with dapsone, when hepatocytes were added to the coculture system. Collectively, our study demonstrates the use of hepatocyte immune cell coculture systems to detect in situ metabolite formation and metabolite-specific T-cell responses. Similar systems should be used in future diagnostic and predictive assays to detect metabolite-specific T-cell responses when synthetic metabolites are not available.

Identification of the BTN3A3 Gene as a Molecule Implicated in Generalized Pustular Psoriasis in a Chinese Population.

The discovery of pathogenic variants provided biological insight into the role of host genetic factors in generalized pustular psoriasis (GPP). However, not all those affected by GPP carry variants in the reported genes. To comprehensively explore the molecular pathogenesis of GPP, whole-exome sequencing was performed, and two loci were identified with exome-wide significance through single variant association analysis: rs148755083 in the IL36RN gene (Pcombined = 1.19 × 10-18, OR = 8.26) and HLA-C∗06:02 within the major histocompatibility complex region (Pcombined = 8.38 × 10-12, OR = 2.98). Gene burden testing revealed that BTN3A3 correlated with GPP (Pcombined = 1.14 × 10-10, OR = 5.59). Subtype analysis showed that IL36RN and BTN3A3 were both significantly associated with GPP alone and GPP with psoriasis vulgaris, whereas a correlation with HLA-C∗06:02 was only observed in GPP with psoriasis vulgaris. Functional analysis revealed that BTN3A3 regulated cell proliferation and inflammatory balance in GPP. In particular, loss of function of BTN3A3 activated NF-κB and promoted the production of inflammatory cytokines by inhibiting IL-36Ra expression to disturb the IL-1/IL-36 inflammatory axis and enhance the TNF-α-mediated pathway. Our findings identify BTN3A3 as, to our knowledge, a previously unreported pathogenic determinant, expanding our understanding of the genetic basis of GPP.

SERPINB7 mutation causes Nagashima-type palmoplantar keratosis and its spatiotemporal expression in zebrafish.

Serine protease inhibitor B7 (SERPINB7) mutations have been reported to cause Nagashima-type palmoplantar keratosis (NPPK), but their biological effects are largely unknown. We conducted whole-exome sequencing and identified a c.796C>T (p.Arg266Ter) mutation in SERPINB7 in a Chinese pedigree, which presented as an autosomal recessive inheritance pattern. We assessed the function of SERPINB7 in homozygous and heterozygous mutation carriers, and the results suggested that the single c.796C>T mutation may alter the subcellular localization of SERPINB7. One of the homozygous mutation patients (II-3) was treated with ixekizumab and showed moderate improvement in keratinization. In addition, we analysed the spatiotemporal expression of serpinb1l1 and serpinb1l3, the zebrafish homologue of human SERPINB7, which is expressed in larvae and adults. In larvae, both serpinb1l1 and serpinb1l3 were expressed in the digestive tract. Then, we performed RT-PCR on adult fins based on similarity to the site of NPPK expression in humans and found that the genes were expressed in five fins (pectoral, pelvic, dorsal, anal and caudal) of the zebrafish distal extremity. Taken together, our results demonstrated that the single c.796C>T (p.Arg266Ter) mutation may alter the location of SERPINB7-encoded protein in the skin, while zebrafish SERPINB7 homologue was expressed in adult fins. These findings will enable us to construct knock-out models to explore the pathogenesis of palmoplantar keratosis.

Immunometabolism in the pathogenesis of vitiligo.

Vitiligo is a common depigmenting skin disorder characterized by the selective loss of melanocytes. Autoimmunity, genetic, environmental, and biochemical etiology have been proposed in vitiligo pathogenesis. However, the exact molecular mechanisms of vitiligo development and progression are unclear, particularly for immunometabolism. Sporadic studies have suggested mitochondrial dysfunction, enhanced oxidative stress, and specific defects in other metabolic pathways can promote dysregulation of innate and adaptive immune responses in vitiligo. These abnormalities appear to be driven by genetic and epigenetic factors modulated by stochastic events. In addition, glucose and lipid abnormalities in metabolism have been associated with vitiligo. Specific skin cell populations are also involved in the critical role of dysregulation of metabolic pathways, including melanocytes, keratinocytes, and tissue-resident memory T cells in vitiligo pathogenesis. Novel therapeutic treatments are also raised based on the abnormalities of immunometabolism. This review summarizes the current knowledge on immunometabolism reprogramming in the pathogenesis of vitiligo and novel treatment options.