Oral and non-oral lichen planus show genetic heterogeneity and differential risk for autoimmune disease and oral cancer.

Lichen planus (LP) is a T-cell-mediated inflammatory disease affecting squamous epithelia in many parts of the body, most often the skin and oral mucosa. Cutaneous LP is usually transient and oral LP (OLP) is most often chronic, so we performed a large-scale genetic and epidemiological study of LP to address whether the oral and non-oral subgroups have shared or distinct underlying pathologies and their overlap with autoimmune disease. Using lifelong records covering diagnoses, procedures, and clinic identity from 473,580 individuals in the FinnGen study, genome-wide association analyses were conducted on carefully constructed subcategories of OLP (n = 3,323) and non-oral LP (n = 4,356) and on the combined group. We identified 15 genome-wide significant associations in FinnGen and an additional 12 when meta-analyzed with UKBB (27 independent associations at 25 distinct genomic locations), most of which are shared between oral and non-oral LP. Many associations coincide with known autoimmune disease loci, consistent with the epidemiologic enrichment of LP with hypothyroidism and other autoimmune diseases. Notably, a third of the FinnGen associations demonstrate significant differences between OLP and non-OLP. We also observed a 13.6-fold risk for tongue cancer and an elevated risk for other oral cancers in OLP, in agreement with earlier reports that connect LP with higher cancer incidence. In addition to a large-scale dissection of LP genetics and comorbidities, our study demonstrates the use of comprehensive, multidimensional health registry data to address outstanding clinical questions and reveal underlying biological mechanisms in common but understudied diseases.

Atypical Spindle Cell/Pleomorphic Lipomatous Tumor With Sarcomatous Transformation: Clinicopathologic and Molecular Analysis of 4 Cases.

Atypical spindle cell/pleomorphic lipomatous tumor (ASPLT) is a recently described adipocytic tumor predominantly affecting the subcutaneous soft tissues of adults. Previous studies have shown that ASPLT follows a benign clinical course with a 4% to 12% local recurrence rate and no risk of dedifferentiation. Herein, we describe the clinicopathologic and molecular findings of 4 cases of ASPLT showing unequivocal sarcomatous transformation. Three patients were male and one was female, aged 65, 70, 74, and 78 years. Two cases presented as mass-forming lesions, while 1 case was incidentally discovered. The tumors measured 30, 55, 80, and 110 mm and occurred in the chest wall (n = 2) or arm (n = 2); all were subcutaneous. Microscopically, they showed a biphasic appearance comprising a low-grade ASPLT component and a high-grade sarcomatous component. The low-grade components showed features in the spectrum of either atypical pleomorphic lipomatous tumor (n = 2) or atypical spindle cell lipomatous tumor (n = 2). The high-grade components displayed leiomyosarcoma-like (n = 2), pleomorphic liposarcoma-like (n = 1) or undifferentiated sarcoma-like (n = 1) morphology. On immunohistochemistry, tumors were negative for MDM2 and showed loss of RB1 expression. In addition, the leiomyosarcoma-like areas seen in 2 cases were positive for smooth muscle actin and H-caldesmon. Single-nucleotide polymorphism array, performed in 3 cases, showed deletions of TP53, RB1, and flanking genes in both components. In contrast, the sarcomatous components showed more complex genomic profiles with rare segmental gains and recurrent loss of PTEN (n = 3), ATM (n = 2), and CDKN2A/B (n = 2) among other genes. Whole exome sequencing identified a TP53 variant in one case and an ATRX variant in another, each occurring in both tumor components. Limited clinical follow-up showed no recurrence or metastasis after 1 to 13 months (median, 7.5 months) postsurgical excision. Altogether, our data support that ASPLT can rarely develop sarcomatous transformation and offer insights into the molecular mechanisms underlying this event.

Hydrogen Production Using a Nickel Catalyst Combining Redox Activity and Pendent Base Effects.

With the mounting need for clean and renewable energy, catalysts for hydrogen production based on earth abundant elements are of great interest. Herein, we describe the synthesis, characterization, and catalytic activity of two nickel complexes based on the pyridinediimine ligand that possess basic nitrogen moieties of pyridine and imidazole that could potentially serve as pendent bases to enhance catalysis. Although these ligands have previously been reported to be complexed to some metal ions, they have not been applied to nickel. The nickel complex with the pendent pyridines was found to be the most active of the two, catalyzing proton reduction electrochemically with an overpotential of 490 mV. The appearance of a wave that preceded the Ni(I/0) redox couple in the presence of protons suggests that protonation of a dissociated pyridine was likely. Further evidence of this was provided with density functional theory calculations, and a mechanism of hydrogen production is proposed. Furthermore, in a light-driven system containing Ru(bpy)32+ and ascorbic acid, TON of 1400 were obtained.

Genetic vulnerability to Crohn's disease reveals a spatially resolved epithelial restitution program.

Effective tissue repair requires coordinated intercellular communication to sense damage, remodel the tissue, and restore function. Here, we dissected the healing response in the intestinal mucosa by mapping intercellular communication at single-cell resolution and integrating with spatial transcriptomics. We demonstrated that a risk variant for Crohn's disease, hepatocyte growth factor activator (HGFAC) Arg509His (R509H), disrupted a damage-sensing pathway connecting the coagulation cascade to growth factors that drive the differentiation of wound-associated epithelial (WAE) cells and production of a localized retinoic acid (RA) gradient to promote fibroblast-mediated tissue remodeling. Specifically, we showed that HGFAC R509H was activated by thrombin protease activity but exhibited impaired proteolytic activation of the growth factor macrophage-stimulating protein (MSP). In Hgfac R509H mice, reduced MSP activation in response to wounding of the colon resulted in impaired WAE cell induction and delayed healing. Through integration of single-cell transcriptomics and spatial transcriptomics, we demonstrated that WAE cells generated RA in a spatially restricted region of the wound site and that mucosal fibroblasts responded to this signal by producing extracellular matrix and growth factors. We further dissected this WAE cell-fibroblast signaling circuit in vitro using a genetically tractable organoid coculture model. Collectively, these studies exploited a genetic perturbation associated with human disease to disrupt a fundamental biological process and then reconstructed a spatially resolved mechanistic model of tissue healing.

Revisiting Coley's Toxins: Immunogenic Cardiolipins from Streptococcus pyogenes.

Coley's toxins, an early and enigmatic form of cancer (immuno)therapy, were based on preparations of Streptococcus pyogenes. As part of a program to explore bacterial metabolites with immunomodulatory potential, S. pyogenes metabolites were assayed in a cell-based immune assay, and a single membrane lipid, 18:1/18:0/18:1/18:0 cardiolipin, was identified. Its activity was profiled in additional cellular assays, which showed it to be an agonist of a TLR2-TLR1 signaling pathway with a 6 µM EC50 and robust TNF-α induction. A synthetic analog with switched acyl chains had no measurable activity in immune assays. The identification of a single immunogenic cardiolipin with a restricted structure-activity profile has implications for immune regulation, cancer immunotherapy, and poststreptococcal autoimmune diseases.

Constitutively active autophagy in macrophages dampens inflammation through metabolic and post-transcriptional regulation of cytokine production.

Autophagy is an essential cellular process that is deeply integrated with innate immune signaling; however, studies that examine the impact of autophagic modulation in the context of inflammatory conditions are lacking. Here, using mice with a constitutively active variant of the autophagy gene Beclin1, we show that increased autophagy dampens cytokine production during a model of macrophage activation syndrome and in adherent-invasive Escherichia coli (AIEC) infection. Moreover, loss of functional autophagy through conditional deletion of Beclin1 in myeloid cells significantly enhances innate immunity in these contexts. We further analyzed primary macrophages from these animals with a combination of transcriptomics and proteomics to identify mechanistic targets downstream of autophagy. Our study reveals glutamine/glutathione metabolism and the RNF128/TBK1 axis as independent regulators of inflammation. Altogether, our work highlights increased autophagic flux as a potential approach to reduce inflammation and defines independent mechanistic cascades involved in this control.

Identification of host regulators of Mycobacterium tuberculosis phenotypes uncovers a role for the MMGT1-GPR156 lipid droplet axis in persistence.

The ability of Mycobacterium tuberculosis (Mtb) to establish latency affects disease and response to treatment. The host factors that influence the establishment of latency remain elusive. We engineered a multi-fluorescent Mtb strain that reports survival, active replication, and stressed non-replication states and determined the host transcriptome of the infected macrophages in these states. Additionally, we conducted a genome-wide CRISPR screen to identify host factors that modulated the phenotypic state of Mtb. We validated hits in a phenotype-specific manner and prioritized membrane magnesium transporter 1 (MMGT1) for a detailed mechanistic investigation. Mtb infection of MMGT1-deficient macrophages promoted a switch to persistence, upregulated lipid metabolism genes, and accumulated lipid droplets during infection. Targeting triacylglycerol synthesis reduced both droplet formation and Mtb persistence. The orphan G protein-coupled receptor GPR156 is a key inducer of droplet accumulation in ΔMMGT1 cells. Our work uncovers the role of MMGT1-GPR156-lipid droplets in the induction of Mtb persistence.

HLA-II immunopeptidome profiling and deep learning reveal features of antigenicity to inform antigen discovery.

CD4+ T cell responses are exquisitely antigen specific and directed toward peptide epitopes displayed by human leukocyte antigen class II (HLA-II) on antigen-presenting cells. Underrepresentation of diverse alleles in ligand databases and an incomplete understanding of factors affecting antigen presentation in vivo have limited progress in defining principles of peptide immunogenicity. Here, we employed monoallelic immunopeptidomics to identify 358,024 HLA-II binders, with a particular focus on HLA-DQ and HLA-DP. We uncovered peptide-binding patterns across a spectrum of binding affinities and enrichment of structural antigen features. These aspects underpinned the development of context-aware predictor of T cell antigens (CAPTAn), a deep learning model that predicts peptide antigens based on their affinity to HLA-II and full sequence of their source proteins. CAPTAn was instrumental in discovering prevalent T cell epitopes from bacteria in the human microbiome and a pan-variant epitope from SARS-CoV-2. Together CAPTAn and associated datasets present a resource for antigen discovery and the unraveling genetic associations of HLA alleles with immunopathologies.

The landscape of immune dysregulation in Crohn's disease revealed through single-cell transcriptomic profiling in the ileum and colon.

Crohn's disease (CD) is a chronic gastrointestinal disease that is increasing in prevalence worldwide. CD is multifactorial, involving the complex interplay of genetic, immune, and environmental factors, necessitating a system-level understanding of its etiology. To characterize cell-type-specific transcriptional heterogeneity in active CD, we profiled 720,633 cells from the terminal ileum and colon of 71 donors with varying inflammation status. Our integrated datasets revealed organ- and compartment-specific responses to acute and chronic inflammation; most immune changes were in cell composition, whereas transcriptional changes dominated among epithelial and stromal cells. These changes correlated with endoscopic inflammation, but small and large intestines exhibited distinct responses, which were particularly apparent when focusing on IBD risk genes. Finally, we mapped markers of disease-associated myofibroblast activation and identified CHMP1A, TBX3, and RNF168 as regulators of fibrotic complications. Altogether, our results provide a roadmap for understanding cell-type- and organ-specific differences in CD and potential directions for therapeutic development.

The CD4+ T cell response to a commensal-derived epitope transitions from a tolerant to an inflammatory state in Crohn's disease.

Reciprocal interactions between host T helper cells and gut microbiota enforce local immunological tolerance and modulate extra-intestinal immunity. However, our understanding of antigen-specific tolerance to the microbiome is limited. Here, we developed a systematic approach to predict HLA class-II-specific epitopes using the humanized bacteria-originated T cell antigen (hBOTA) algorithm. We identified a diverse set of microbiome epitopes spanning all major taxa that are compatible with presentation by multiple HLA-II alleles. In particular, we uncovered an immunodominant epitope from the TonB-dependent receptor SusC that was universally recognized and ubiquitous among Bacteroidales. In healthy human subjects, SusC-reactive T cell responses were characterized by IL-10-dominant cytokine profiles, whereas in patients with active Crohn's disease, responses were associated with elevated IL-17A. Our results highlight the potential of targeted antigen discovery within the microbiome to reveal principles of tolerance and functional transitions during inflammation.

Large-scale sequencing identifies multiple genes and rare variants associated with Crohn's disease susceptibility.

Genome-wide association studies (GWASs) have identified hundreds of loci associated with Crohn's disease (CD). However, as with all complex diseases, robust identification of the genes dysregulated by noncoding variants typically driving GWAS discoveries has been challenging. Here, to complement GWASs and better define actionable biological targets, we analyzed sequence data from more than 30,000 patients with CD and 80,000 population controls. We directly implicate ten genes in general onset CD for the first time to our knowledge via association to coding variation, four of which lie within established CD GWAS loci. In nine instances, a single coding variant is significantly associated, and in the tenth, ATG4C, we see additionally a significantly increased burden of very rare coding variants in CD cases. In addition to reiterating the central role of innate and adaptive immune cells as well as autophagy in CD pathogenesis, these newly associated genes highlight the emerging role of mesenchymal cells in the development and maintenance of intestinal inflammation.

Akkermansia muciniphila phospholipid induces homeostatic immune responses.

Multiple studies have established associations between human gut bacteria and host physiology, but determining the molecular mechanisms underlying these associations has been challenging1-3. Akkermansia muciniphila has been robustly associated with positive systemic effects on host metabolism, favourable outcomes to checkpoint blockade in cancer immunotherapy and homeostatic immunity4-7. Here we report the identification of a lipid from A. muciniphila's cell membrane that recapitulates the immunomodulatory activity of A. muciniphila in cell-based assays8. The isolated immunogen, a diacyl phosphatidylethanolamine with two branched chains (a15:0-i15:0 PE), was characterized through both spectroscopic analysis and chemical synthesis. The immunogenic activity of a15:0-i15:0 PE has a highly restricted structure-activity relationship, and its immune signalling requires an unexpected toll-like receptor TLR2-TLR1 heterodimer9,10. Certain features of the phospholipid's activity are worth noting: it is significantly less potent than known natural and synthetic TLR2 agonists; it preferentially induces some inflammatory cytokines but not others; and, at low doses (1% of EC50) it resets activation thresholds and responses for immune signalling. Identifying both the molecule and an equipotent synthetic analogue, its non-canonical TLR2-TLR1 signalling pathway, its immunomodulatory selectivity and its low-dose immunoregulatory effects provide a molecular mechanism for a model of A. muciniphila's ability to set immunological tone and its varied roles in health and disease.

pH sensing controls tissue inflammation by modulating cellular metabolism and endo-lysosomal function of immune cells.

Extracellular acidification occurs in inflamed tissue and the tumor microenvironment; however, a systematic study on how pH sensing contributes to tissue homeostasis is lacking. In the present study, we examine cell type-specific roles of the pH sensor G protein-coupled receptor 65 (GPR65) and its inflammatory disease-associated Ile231Leu-coding variant in inflammation control. GPR65 Ile231Leu knock-in mice are highly susceptible to both bacterial infection-induced and T cell-driven colitis. Mechanistically, GPR65 Ile231Leu elicits a cytokine imbalance through impaired helper type 17 T cell (TH17 cell) and TH22 cell differentiation and interleukin (IL)-22 production in association with altered cellular metabolism controlled through the cAMP-CREB-DGAT1 axis. In dendritic cells, GPR65 Ile231Leu elevates IL-12 and IL-23 release at acidic pH and alters endo-lysosomal fusion and degradation capacity, resulting in enhanced antigen presentation. The present study highlights GPR65 Ile231Leu as a multistep risk factor in intestinal inflammation and illuminates a mechanism by which pH sensing controls inflammatory circuits and tissue homeostasis.

Lyme Disease, Borrelia burgdorferi, and Lipid Immunogens.

The human immune system detects potentially pathogenic microbes with receptors that respond to microbial metabolites. While the overall immune signaling pathway is known in considerable detail, the initial molecular signals, the microbially produced immunogens, for important diseases like Lyme disease (LD) are often not well-defined. The immunogens for LD are produced by the spirochete Borrelia burgdorferi, and a galactoglycerolipid (1) has been identified as a key trigger for the inflammatory immune response that characterizes LD. This report corrects the original structural assignment of 1 to 3, a change of an α-galactopyranose to an α-galactofuranose headgroup. The seemingly small change has important implications for the diagnosis, prevention, and treatment of LD.

Colon stroma mediates an inflammation-driven fibroblastic response controlling matrix remodeling and healing.

Chronic inflammation is often associated with the development of tissue fibrosis, but how mesenchymal cell responses dictate pathological fibrosis versus resolution and healing remains unclear. Defining stromal heterogeneity and identifying molecular circuits driving extracellular matrix deposition and remodeling stands to illuminate the relationship between inflammation, fibrosis, and healing. We performed single-cell RNA-sequencing of colon-derived stromal cells and identified distinct classes of fibroblasts with gene signatures that are differentially regulated by chronic inflammation, including IL-11-producing inflammatory fibroblasts. We further identify a transcriptional program associated with trans-differentiation of mucosa-associated fibroblasts and define a functional gene signature associated with matrix deposition and remodeling in the inflamed colon. Our analysis supports a critical role for the metalloprotease Adamdec1 at the interface between tissue remodeling and healing during colitis, demonstrating its requirement for colon epithelial integrity. These findings provide mechanistic insight into how inflammation perturbs stromal cell behaviors to drive fibroblastic responses controlling mucosal matrix remodeling and healing.

SAC1 regulates autophagosomal phosphatidylinositol-4-phosphate for xenophagy-directed bacterial clearance.

Phosphoinositides are important molecules in lipid signaling, membrane identity, and trafficking that are spatiotemporally controlled by factors from both mammalian cells and intracellular pathogens. Here, using small interfering RNA (siRNA) directed against phosphoinositide kinases and phosphatases, we screen for regulators of the host innate defense response to intracellular bacterial replication. We identify SAC1, a transmembrane phosphoinositide phosphatase, as an essential regulator of xenophagy. Depletion or inactivation of SAC1 compromises fusion between Salmonella-containing autophagosomes and lysosomes, leading to increased bacterial replication. Mechanistically, the loss of SAC1 results in aberrant accumulation of phosphatidylinositol-4-phosphate [PI(4)P] on Salmonella-containing autophagosomes, thus facilitating recruitment of SteA, a PI(4)P-binding Salmonella effector protein, which impedes lysosomal fusion. Replication of Salmonella lacking SteA is suppressed by SAC-1-deficient cells, however, demonstrating bacterial adaptation to xenophagy. Our findings uncover a paradigm in which a host protein regulates the level of its substrate and impairs the function of a bacterial effector during xenophagy.

MS-Based HLA-II Peptidomics Combined With Multiomics Will Aid the Development of Future Immunotherapies.

Immunotherapies have emerged to treat diseases by selectively modulating a patient's immune response. Although the roles of T and B cells in adaptive immunity have been well studied, it remains difficult to select targets for immunotherapeutic strategies. Because human leukocyte antigen class II (HLA-II) peptides activate CD4+ T cells and regulate B cell activation, proliferation, and differentiation, these peptide antigens represent a class of potential immunotherapy targets and biomarkers. To better understand the molecular basis of how HLA-II antigen presentation is involved in disease progression and treatment, systematic HLA-II peptidomics combined with multiomic analyses of diverse cell types in healthy and diseased states is required. For this reason, MS-based innovations that facilitate investigations into the interplay between disease pathologies and the presentation of HLA-II peptides to CD4+ T cells will aid in the development of patient-focused immunotherapies.

Exploring Social Ecological Determinants of Physical Activity Among Adult Survivors of Childhood Cancer.

Purpose: Adult survivors of childhood cancer (ASCCs) are at high risk for cardiovascular disease from chemotherapy- and radiation therapy-related cardiotoxicity. Physical activity (PA) can reduce this risk, but the majority of ASCCs do not engage in sufficient PA. The purpose of this study was to identify barriers, facilitators, and resources for PA among ASCCs using the ecological model of physical activity (EMPA) as a theoretical framework. Methods: A concept elicitation survey was distributed independently to ASCCs (diagnosed with cancer before the age of 18, and currently 18-39 years old) and parents/legal guardians of an ASCC. The survey consisted of open-ended questions asking about barriers, facilitators, and resources for PA. Content analysis of open-ended questions categorized responses into levels of the EMPA and identified key themes. Results: Seventeen ASCCs and eight parents of ASCCs completed the survey. The majority of barriers, facilitators, and resources reported were at the individual and microsystem level of the EMPA. Six themes emerged, suggesting that ASCC's PA was related to proximity/access, social support, equipment, time/schedule, finances, and health-related barriers. Conclusion: This is the first study to examine barriers, facilitators, and resources of PA among ASCCs using the EMPA. Findings from this study provide a multilevel perspective on the influences of PA among ASCCs, and can be used for future, in-depth qualitative studies and quantitative survey development, and as a foundational step toward supportive efforts in increasing PA among ASCCs.

A missense variant in SLC39A8 confers risk for Crohn's disease by disrupting manganese homeostasis and intestinal barrier integrity.

Common genetic variants interact with environmental factors to impact risk of heritable diseases. A notable example of this is a single-nucleotide variant in the Solute Carrier Family 39 Member 8 (SLC39A8) gene encoding the missense variant A391T, which is associated with a variety of traits ranging from Parkinson's disease and neuropsychiatric disease to cardiovascular and metabolic diseases and Crohn's disease. The remarkable extent of pleiotropy exhibited by SLC39A8 A391T raises key questions regarding how a single coding variant can contribute to this diversity of clinical outcomes and what is the mechanistic basis for this pleiotropy. Here, we generate a murine model for the Slc39a8 A391T allele and demonstrate that these mice exhibit Mn deficiency in the colon associated with impaired intestinal barrier function and epithelial glycocalyx disruption. Consequently, Slc39a8 A391T mice exhibit increased sensitivity to epithelial injury and pathological inflammation in the colon. Taken together, our results link a genetic variant with a dietary trace element to shed light on a tissue-specific mechanism of disease risk based on impaired intestinal barrier integrity.

The Human and Mouse Enteric Nervous System at Single-Cell Resolution.

The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease.