Inflammatory skin diseases (ISDs) exhibit varying prevalence across different ancestry background and geographical regions. Genetic research for complex ISDs has predominantly centered on European Ancestry (EurA) populations and genetic effects on immune cell responses but generally failed to consider contributions from other cell types in skin. Here, we utilized 273 genetic signals from seven different ISDs: acne, alopecia areata (AA), atopic dermatitis (AD), psoriasis, systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and vitiligo, to demonstrate enriched IL1 signaling in keratinocytes, particularly in signals with higher risk allele frequencies in the African ancestry. Using a combination of ATAC-seq, Bru-seq, and promoter capture Hi-C, we revealed potential regulatory mechanisms of the acne locus on chromosome 2q13. We further demonstrated differential responses in keratinocytes upon IL1β stimulation, including the pro-inflammatory mediators CCL5, IL36G, and CXCL8. Taken together, our findings highlight IL1 signaling in epidermal keratinocytes as a contributor to ancestry-related differences in ISDs.

Gene regulation is intricately influenced by the three-dimensional organization of the genome. In particular, chromatin can exist in loop structures that enable long-range regulatory interactions. By utilizing chromosome conformation capture techniques such as Hi-C, valuable information regarding the organization of these loop structures in 3D space can be obtained. Although functional/feature enrichment is now a common downstream analysis for various genomic platforms to provide biological context, tools specifically designed for high-throughput assays that capture chromosome conformation remain relatively limited. Here, we present Loopsim, a command-line application that performs enrichment analysis on Hi-C loop profiles against user-defined regions (available on GitHub at https://github.com/CutaneousBioinf/Loopsim). Loopsim efficiently simulates a background distribution using a distinctive sampling approach that considers loop size, intervals, loop-loop distances, and structure; it then computes statistics based on the empirical null distribution.

IntroductionInflammation is a vital immune response, tightly orchestrated through both biochemical and biophysical cues. Dysregulated inflammation contributes to chronic diseases, highlighting the need for novel therapies that modulate immune responses with minimal side effects. While several biochemical pathways of inflammation are well understood, the influence of physical properties such as substrate curvature on immune cell behavior remains underexplored. This study investigates how substrate curvature impacts macrophage cytoskeletal dynamics, gene expression, and immunophenotype through mechanosensitive pathways.MethodsGelatin-based microgels with tunable surface curvatures were fabricated via water-in-oil emulsification and crosslinked with genipin. Microgels were sorted into three size ranges, yielding high (40-50 µm), intermediate (150-250 µm), and low (350-400 µm) curvature profiles. Macrophages were seeded onto these microgels, and cytoskeletal dynamics were examined using confocal microscopy, SEM, and actin-specific staining. Gene expression of pro- and anti-inflammatory markers was quantified using qPCR. The role of actin polymerization was assessed using Latrunculin-A (Lat-A) treatment.ResultsMacrophages adhered effectively to both high- and low-curvature microgels, displaying curvature-dependent morphological changes. Confocal imaging revealed that macrophages on low-curvature microgels exhibited significantly higher F-actin density than those on high-curvature microgels. Correspondingly, qPCR analysis showed upregulation of pro-inflammatory markers (e.g., Tnf, Nos2) in high-curvature conditions, while anti-inflammatory markers (e.g., Arg1) were elevated in low-curvature conditions. Lat-A treatment reduced F-actin density and modulated gene expression patterns, confirming the cytoskeletal regulation of macrophage phenotype.DiscussionThese findings demonstrate that substrate curvature influences macrophage behavior by modulating cytoskeletal dynamics and associated immunophenotypic markers through actin-mediated transcriptional pathways. By controlling curvature, therapeutic biomaterials may direct immune responses, offering a new avenue for treating inflammatory diseases. This mechanobiological approach presents a promising strategy for precision immunomodulation in regenerative medicine.

Nonunion and delayed-union fractures pose a significant clinical challenge, often leading to prolonged morbidity and impaired quality of life. Fracture-induced hematoma and acute inflammation are crucial for establishing the healing cascade. However, aberrant inflammatory phenotypes can suppress healing and cause bone resorption. Elucidating these mechanisms is necessary to develop potent immunomodulatory therapies and prevent nonunion. Here, we report a delayed fracture healing model enabling the modulation of interfragmentary strain that mimics the etiology of hypertrophic nonunions to elucidate the role of dysregulated immune response in poor healing outcomes. High interfragmentary strain (>15%) was associated with larger callus volumes but delayed bone healing, increased inflammation, and inferior healing outcomes, while lower strain levels (<5%) corresponded to normal bone healing. In addition, we found distinct differences in the ossification, chondrification, and fibrosis patterns between high and low-strain groups, underscoring the significant impact of strain on the healing process. A comprehensive analysis of the systemic immune response revealed dynamic alterations in immune cell populations and factors, particularly within the early hours and days post-fracture. Several immune factors exhibited significant correlations with various functional healing outcomes, indicating their potential as predictive markers for assessing fracture healing progression. Our results also highlighted the significance of timely resolution of proinflammatory signals and the elevation of pro-regenerative immune cell phenotypes in promoting bone regeneration. Multivariate analysis revealed that CD25+ T-regulatory cells were influential in predicting proper bone healing, followed by CD206+ macrophages, underscoring the pivotal role of immune cell populations in the bone healing process. In conclusion, our study provides valuable insights into the intricate interplay between interfragmentary strain, immune response, and the ultimate outcomes of fracture healing. By shedding light on the underlying mechanisms that drive hypertrophic nonunion pathogenesis, our research lays the foundation for enhanced surgical management of nonunions and offers a promising avenue for developing targeted therapeutic interventions and personalized treatment strategies for individuals suffering from fracture nonunion.

The expressions of long noncoding RNAs (lncRNAs) and their roles in epidermal differentiation have been previously defined using bulk RNA sequencing. Despite their tissue-specific expression profiles, most lncRNAs are not well-annotated at the single-cell level. In this study, we evaluated the use of single-cell RNA sequencing to profile and characterize lncRNAs using data from 6 patients with psoriasis with paired uninvolved and lesional psoriatic skin. Despite their overall lower expression, we were able to detect >7000 skin-expressing lncRNAs and their cellular sources. Differential gene expression analysis revealed 137 differentially expressed lncRNAs in lesional psoriasis skin and identified 169 cell-type-specific lncRNAs. Keratinocytes had the highest number of differentially expressed lncRNA in psoriatic skin, which we validated using spatial transcriptomic data. We further showed that expression of the keratinocyte-specific lncRNA, AC020916.1, upregulated in lesional skin, is significantly correlated with expressions of genes participating in cell proliferation/epidermal differentiation, including SPRR2E and transcription factor ZFP36, particularly in the psoriatic skin. Our study highlights the potential for using single-cell RNA sequencing to profile skin-expressing lncRNA transcripts and to infer their cellular origins, providing a crucial approach that can be applied to the study of other inflammatory skin conditions.

Spatial profiling, through single-cell gene-level expression data paired with cell localization, offers unprecedented biologic insights within the intact spatial context of cells in healthy and diseased tissue, adding a novel dimension to data interpretation. This review summarizes recent developments in this field, its application to allergy and inflammation, and recent single-cell resolution platforms designed for spatial transcriptomics with a focus on data processing and analyses for efficient biologic interpretation of data. By preserving spatial context, these technologies provide critical insights into tissue architecture and cellular interactions that are unattainable with traditional transcriptomics methods, such as revealing localized inflammatory cell network in atopic dermatitis and T-cell interactions in the lung in chronic obstructive pulmonary disease. Spatial profiling offers opportunities for discovering novel biomarkers, defining compartmentalization of immune responses within tissues and individual diseases, and accelerating novel discoveries toward a greater understanding of fundamental disease mechanisms and, eventually, toward the development of future targeted therapies.

Inflammation serves as a critical defense mechanism against pathogens and tissue damage but can lead to chronic diseases, such as cardiovascular disease and diabetes, when dysregulated. Macrophages play a pivotal role in orchestrating inflammatory responses, transitioning from pro-inflammatory M1 to anti-inflammatory M2 phenotypes to resolve inflammation and promote tissue repair. Current approaches to modulate macrophage phenotype predominantly rely on biochemical cues, which may induce systemic side effects. Given the mechanosensitivity of macrophages, this study investigates biophysical cues, specifically substrate curvature, as a localized strategy to regulate macrophage phenotype and minimize systemic repercussions. We hypothesized that substrate curvature influences macrophage immunophenotype by modulating F-actin polymerization. To test this hypothesis, we fabricated spherical microgels with tunable curvatures and characterized their biophysical properties. Our findings indicate that macrophages adhere to microgel surfaces irrespective of curvature, but the curvature significantly alters F-actin dynamics. Furthermore, manipulating cytoskeletal dynamics via selective actin inhibition partially reversed curvature-induced changes in macrophage phenotype. These results underscore the pivotal role of substrate curvature in modulating macrophage behavior and immunophenotype. Overall, our study demonstrates that substrate curvature significantly influences macrophage cytoskeletal dynamics and resulting immunophenotype. This simple approach can be utilized as a localized immunomodulatory treatment for inflammatory diseases.

Background: Prurigo nodularis (PN) is a chronic neuroimmune skin disease characterized by bilaterally distributed pruritic hyperkeratotic nodules on extremities and trunk. Neuroimmune dysregulation and chronic scratching are believed to both induce and maintain the characteristic lesions. Objectives: This study sought to provide a comprehensive view of the molecular pathogenesis of PN at the single-cell level to identify and outline key pathologic processes and the cell types involved. Features that distinguish PN skin from the skin of patients with atopic dermatitis were of particular interest. We further aimed to determine the impact of the IL31RA antagonist, nemolizumab, and its specificity at the single-cell level. Methods: Single-cell RNA-sequencing of skin from 15 healthy donors and nonlesional and lesional skin from 6 patients each with PN and atopic dermatitis, combined with spatialsequencing using the 10x Visium platform. Integration with bulk RNA-sequencing data from patients treated with nemolizumab. Results: This study demonstrates that PN is an inflammatory skin disease characterized by both keratinocyte proliferation and activation of profibrotic responses. This study also demonstrates that the COL11A1+ fibroblast subset is a major contributor to fibrosis and is predominantly found in the papillary dermis of PN skin. Activation of fibrotic responses is the main distinguishing feature between PN and atopic dermatitis skin. This study further shows the broad effect of nemolizumab on PN cell types, with a prominent effect driving COL11A1+ fibroblast and keratinocyte responses toward normal. Conclusions: This study provides a high -resolution characterization of the cell types and cellular processes activated in PN skin, establishing PN as a chronic fibrotic inflammatory skin disease. It further demonstrates the broad effect of nemolizumab on pathological processes in PN skin. (J Allergy Clin Immunol 2024;153:146-60.) dermatitis skin. This study further shows the broad effect of nemolizumab on PN cell types, with a prominent effect driving COL11A1+ fibroblast and keratinocyte responses toward normal. Conclusions: This study provides a high -resolution characterization of the cell types and cellular processes activated in PN skin, establishing PN as a chronic fibrotic inflammatory skin disease. It further demonstrates the broad effect of nemolizumab on pathological processes in PN skin. (J Allergy Clin Immunol 2024;153:146-60.)

ABSTRACT Atopic dermatitis (AD) is a highly heritable and common inflammatory skin condition affecting children and adults worldwide. Multi-ancestry approaches to AD genetic association studies are poised to boost power to detect genetic signal and identify ancestry-specific loci contributing to AD risk. Here, we present a multi-ancestry GWAS meta-analysis of twelve AD cohorts from five ancestral populations totaling 56,146 cases and 602,280 controls. We report 101 genomic loci associated with AD, including 15 loci that have not been previously associated with AD or eczema. Fine-mapping, QTL colocalization, and cell-type enrichment analyses identified genes and cell types implicated in AD pathophysiology. Functional analyses in keratinocytes provide evidence for genes that could play a role in AD through epidermal barrier function. Our study provides new insights into the etiology of AD by harnessing multiple genetic and functional approaches to unveil the mechanisms by which AD-associated variants impact genes and cell types. Disclosure Statement BRG, MO, CH, KMS are employees of AbbVie. FT was an employee of AbbVie at the time of the study. JEG (University of Michigan) has received research support from AbbVie, Janssen, Almirall, Prometheus Biosciences/Merck, BMS/Celgene, Boehringer Ingelheim, Galderma, Eli Lilly, and advisor to Sanofi, Eli Lilly, Galderma, BMS, Boehringer Ingelheim. MKS, RU, MTP, QL, RW, JMK, LCT are employees of University of Michigan and have no funding to disclose. MEM, AHS, FDM, DW, JTG, HH are employees of the Children’s Hospital of Philadelphia and no funding to disclose. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

IntroductionThe utilization of large-scale claims databases has greatly improved the management, accessibility, and integration of extensive medical data. However, its potential for systematically identifying comorbidities in the context of skin diseases remains unexplored.MethodsThis study aims to assess the capability of a comprehensive claims database in identifying comorbidities linked to 14 specific skin and skin-related conditions and examining temporal changes in their association patterns. This study employed a retrospective case-control cohort design utilizing 13 million skin/skin-related patients and 2 million randomly sampled controls from Optum’s de-identified Clinformatics® Data Mart Database spanning the period from 2001 to 2018. A broad spectrum of comorbidities encompassing cancer, diabetes, respiratory, mental, immunity, gastrointestinal, and cardiovascular conditions were examined for each of the 14 skin and skin-related disorders in the study.ResultsUsing the established type-2 diabetes (T2D) and psoriasis comorbidity as example, we demonstrated the association is significant (P-values<1x10-15) and stable across years (OR=1.15-1.31). Analysis of the 2014-2018 data reveals that celiac disease, Crohn’s disease, and ulcerative colitis exhibit the strongest associations with the 14 skin/skin-related conditions. Systemic lupus erythematosus (SLE), leprosy, and hidradenitis suppurativa show the strongest associations with 30 different comorbidities. Particularly notable associations include Crohn’s disease with leprosy (odds ratio [OR]=6.60, 95% confidence interval [CI]: 3.09-14.08), primary biliary cirrhosis with SLE (OR=6.07, 95% CI: 4.93-7.46), and celiac disease with SLE (OR=6.06, 95% CI: 5.49-6.69). In addition, changes in associations were observed over time. For instance, the association between atopic dermatitis and lung cancer demonstrates a marked decrease over the past decade, with the odds ratio decreasing from 1.75 (95% CI: 1.47-2.07) to 1.02 (95% CI: 0.97-1.07). The identification of skin-associated comorbidities contributes to individualized healthcare and improved clinical management, while also enhancing our understanding of shared pathophysiology. Moreover, tracking these associations over time aids in evaluating the progression of clinical diagnosis and treatment.DiscussionThe findings highlight the potential of utilizing comprehensive claims databases in advancing research and improving patient care in dermatology.

In this article, we review recent developments in the experimental study of grain growth in nanocrystalline metallic thin films, emphasizing transmission electron microscopy-based imaging and orientation mapping techniques and highlighting useful experimental and data analytical frameworks for dynamic experiments. Studies of grain growth have fallen short of the scale required to fully characterize the coarsening process, and models still fail to fully capture the true behavior of grain growth in polycrystalline systems as they pertain to geometric, topological and crystallographic metrics. Moreover, existing grain growth studies are either coarse in time and temperature or otherwise limited in scope. Nevertheless, important observations such as the stagnation of thin film grain growth at a universal grain size distribution and the strong correlations between the grain boundary character distributions in thin film and bulk materials motivate larger-scale dynamic studies. Additionally, recent hardware and software advances have removed bottlenecks to large-scale and in situ data acquisition via (1) automated grain boundary segmentation in micrographs, (2) low thermal mass microelectromechanical systems and (3) integrated hardware-software drift correction and data management solutions. We argue that these innovations render thin films a key integrated experimental platform for the next generation of grain growth studies.

The skin varies greatly across anatomic sites, both in thickness and texture, with each site prone to different skin disorders. However, a comprehensive understanding of skin biology and cell-cell interactions has been lacking but can shed light on the underlying biological mechanisms contributing to body site variation in skin disease. To address this, we conducted scRNA-seq on 96 biopsies from 51 healthy individuals, including 7 anatomic sites: face, scalp, axilla, palmoplantar, arm, leg, and back skin. Among the 274,834 cells, we focused on epidermal function through analyses of 87,213 keratinocytes from 4 different subtypes (basal, differentiated, keratinized, and cycling), along with hair follicle keratinocytes and Merkel cells. Focusing on cell-cell interactions, we detected 36 significant (p < 0.05) signaling pathways, 11 of which were shared among the 7 anatomic sites. Interestingly, 4 and 1 of these pathways were unique to facial and palmoplantar skin, respectively. Notably, palmoplantar skin uniquely had enriched Tenascin signaling, which has a role in maintaining extracellular matrix integrity, but palmoplantar skin lacked PARs and MIF signaling, which are involved in protease activation and macrophage recruitment and retention, respectively. Accordingly, TNC was upregulated in palmoplantar skin, while genes associated with the PAR and MIF pathways (e.g., ACKR3 and EGFR) were downregulated. Facial skin uniquely showed significant activity of WNT, TGFb, and TRAIL pathways, with upregulation of BMP7, ITGB1, and TNFRSF10B. These findings demonstrate shifts in cell-cell communication across anatomic sites providing critical information to explain disease predisposition to specific regions of the skin.

Long noncoding RNAs (lncRNAs) regulate the expression of protein-coding genes and have been shown to play important roles in inflammatory skin diseases. However, we still have limited understanding of the functional impact of lncRNAs in skin, partly due to their tissue specificity and lower expression levels compared with protein-coding genes. We compiled a comprehensive list of 18,517 lncRNAs from different sources and studied their expression profiles in 834 RNA-Seq samples from multiple inflammatory skin conditions and cytokine-stimulated keratinocytes. Applying a balanced random forest to predict involvement in biological functions, we achieved a median AUROC of 0.79 in 10-fold cross-validation, identifying significant DNA binding domains (DBDs) for 39 lncRNAs. G18244, a skin-expressing lncRNA predicted for IL-4/IL-13 signaling in keratinocytes, was highly correlated in expression with F13A1, a protein-coding gene involved in macrophage regulation, and we further identified a significant DBD in F13A1 for G18244. Reflecting clinical implications, AC090198.1 (predicted for IL-17 pathway) and AC005332.6 (predicted for IFN-γ pathway) had significant negative correlation with the SCORAD metric for atopic dermatitis. We also utilized single-cell RNA and spatial sequencing data to validate cell type specificity. Our research demonstrates lncRNAs have important immunological roles and can help prioritize their impact on inflammatory skin diseases.

BACKGROUNDMultiple treatment options are available for the management of psoriasis, but clinical response varies among individual patients and no biomarkers are available to facilitate treatment selection for improved patient outcomes.OBJECTIVESWe aim to utilize retrospective data to conduct a pharmacogenetic study. This design can bypass the obstacle of patient follow-up in prospective study and increase the sample size. Successful implementation of the study can nominate new candidates and their target genes to explore the potential genetic pathways associated with drug response in the treatment of psoriasis.METHODSWe conducted a retrospective pharmacogenetic study using self-evaluated treatment response from 1,942 genotyped psoriatic patients. We examined 6,502,658 genetic markers to model their associations with responses from six treatment options using linear regression, adjusting for cohort variables and demographic features. We further utilized an integrative approach incorporating epigenomic, transcriptomic, and a longitudinal clinical cohort to provide biological implications for the top signals associated with drug response.RESULTSTwo novel markers were revealed to be associated with treatment response: rs1991820 (p = 1.30×10-6) for anti-TNF biologics; and rs62264137 (p = 2.94×10-6) for methotrexate, which also associated with cutaneous mRNA expression levels of two known psoriasis-related genes KLK7 (p = 1.0×10-12) and CD200 (p = 5.4×10-6). We demonstrate that KLK7 expression is increased in the psoriatic epidermis as shown by immunohistochemistry as well as single-cell RNA-sequencing, and we highlight its responsiveness to anti-TNF treatment. By inhibiting the expression of KLK7, we further illustrate keratinocytes have decrease in pro-inflammatory responses to TNF.CONCLUSIONSOur study implicates the genetic regulation of cytokine responses in predicting clinical drug response and supports the association between pharmacogenetic loci and anti-TNF response, as shown here for KLK7.

Multiple genome-wide association studies (GWASs) have reproducibly identified the MTMR3/HORMAD2/LIF/OSM locus to be associated with IgA nephropathy (IgAN). However, the causal variant(s), implicated gene(s), and altered mechanisms remain poorly understood. Here, we performed fine-mapping analyses based on GWAS datasets encompassing 2762 IgAN cases and 5803 control individuals, and identified rs4823074 as the candidate causal variant that intersects the MTMR3 promoter in Blymphoblastoid cells. Mendelian randomization studies suggested the risk allele may modulate disease susceptibility by affecting serum IgA levels through increased MTMR3 expression. Consistently, elevated MTMR3 expression in peripheral blood mononuclear cells was observed in patients with IgAN. Further mechanistic studies in vitro demonstrated that MTMR3 increased IgA production dependent upon its phosphatidylinositol 3-phosphate binding domain. Moreover, our study provided the in vivo functional evidence that Mtmr3-/mice exhibited defective Toll Like Receptor 9-induced IgA production, glomerular IgA deposition, as well as mesangial cell proliferation. RNA-seq and pathway analyses showed that MTMR3 deficiency resulted in an impaired intestinal immune network for IgA production. Thus, our results support the role of MTMR3 in IgAN pathogenesis by enhancing Toll Like Receptor 9-induced IgA immunity. Kidney International (2023) 104, 562-576

Transcriptome-wide association studies (TWAS) model the genetically regulated component (GRC) of gene expression in reference tissue to reveal potential causal genes in GWAS. Current TWAS focus on using control tissues, and studies that leverage different immune-activated tissues as references are limited. Here, we generated 400 RNA-seq samples in keratinocytes under seven different cytokine-stimulated conditions (IFNa, IFNg, IL-4, IL-13, IL-17A, IL-17A+TNF, and TNF) from 50 genotyped individuals. After modeling the GRC for 18,599 genes in each condition, we used MultiXcan to integrate the modeled transcriptome and conducted TWAS in seven common skin conditions: acne, alopecia areata (AA), atopic dermatitis (AD), psoriasis (PSO), systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and vitiligo (VIT). The number of TWAS significant genes for each disease ranged from 182 to 458; notably ∼57% of these signals are contributed by at least 4 conditions. Our results illustrated that IKZF4 is significantly associated with all diseases except for PSO (p-values from 4.66×10-98 to 4.11×10-9), with the IL-13-stimulated condition contributing to the association for all diseases. Among the TWAS significant results that overlap with the GWAS signals, we were also able to highlight these potential causal genes that are also up-regulated (UR) in keratinocytes (following UR p-values are based on analyses of differentially expressed genes): LCE3D for PSO with TNF as the strongest contributing cytokine (TWAS p=1.98×10-12, UR p=2.89×10-3); RAET1L for AA with TNF (TWAS p=3.74×10-20, UR p=1.38×10-57); ITGAM for SLE with TNF (TWAS p=6.25×10-48, UR p=1.66×10-27); IL23A for VIT with IFNa (TWAS p=9.97×10-35, UR p=2.54×10-89). This study reveals the causal gene candidates that exhibit cytokine-dependent regulation for common complex skin conditions, highlighting the roles of specific susceptibility regions in keratinocytes.