IntroductionSeveral studies have reported an up-regulation of interleukin (IL)-36 in the serum of patients with systemic lupus erythematosus (SLE). Here, we sought to define the mechanisms whereby IL-36 may contribute to the over-activation of type I Interferon (IFN) responses observed in SLE.MethodsWe carried out single-cell (sc)RNA-seq in healthy peripheral blood mononuclear cells treated with IL-36 (n=5 donors). We compared the genes and transcriptional networks that were induced by IL-36 with those that were upregulated in a published SLE scRNA-seq dataset (n=33 cases and 11 controls). In follow-up studies, we validated the effects of IL-36 on monocytes by real-time PCR (n=9 donors) and flow-cytometry (n=6).ResultsClassical monocytes were the immune population most affected by IL-36 treatment (n=203 Differentially Expressed Genes). In these cells, IL-36 upregulated transcriptional networks (regulons) driven by IRF7, a key activator of type I IFN responses. A similar upregulation of IRF7 regulons was observed in the monocytes of SLE cases, where measurements of IL-36 and IRF7 activity were significantly correlated (r=0.35, P = 0.02). Experimental follow-up studies in human monocytes showed that IL-36 downregulates multiple RNAse genes (RNASE1, RNASE6, RNASET2). IL-36 treatment of monocytes also increased the percentage of apoptotic cells (45% vs 37% in untreated cells; P = 0.001), which are a critical source of self-nucleic acids.ConclusionWe find that IL-36 promotes monocyte apoptosis while downregulating self-nucleic acid clearance. Thus, IL-36 contributes to the accumulation of self-nucleic acids, a key driver of type I IFN responses in SLE.

Lupus nephritis (LN) is a life-threatening disorder characterized by a relapsing-remitting disease course necessitating immunosuppression to control disease activity. Urinary exosome microRNAs (miRNAs) were previously tested as biomarkers for fibrosis and inflammatory pathways in different kidney diseases, including LN. Small RNA sequencing was conducted on urinary exosome miRNAs in 93 patients with ISN/RPS class III/IV ± V LN from a trial with the anti-CD40 mAB BI655064*. Univariable and multivariable regression models were used to identify associations between miRNAs and spot urine protein/urine creatinine ratio (UP/UC, log-transformed), and eGFR, at time of randomization into the study. MiRNAs with high correlation (P < 0.01) in the univariable analysis are listed in Table 1 for UP/UC and in Table 2 for eGFR. A multivariable model selection procedure, taking also into account the correlation between individual miRNAs, revealed an association between miR-143-3p, miR-510-5p, miR-429, miR-509-5p, miR-10b-5p, miR-9-3p, miR-509-3p, miR-411-5p, miR-574-5p, and miR-3182 and UP/UC (model R2 = 0.613). Likewise, miR-17-5p, miR-101-3p, miR-146b-5p, miR-3173-3p, miR-545-3p, miR-6790-3p were associated with eGFR (model R2 = 0.453). Distinct miRNAs are known to play key functions during the disease progression of renal diseases (Tables 1 and 2). Urinary exosome miRNAs might serve as mechanistic supportive biomarkers associated with proteinuria and eGFR in LN and might have the potential to be used as biomarkers to monitor treatment effects in clinical trials. *Arthritis Rheumatol. 2023 Nov;75(11):1983-1993.

Smoking is a leading risk factor of chronic obstructive pulmonary disease (COPD), that is characterized by chronic lung inflammation, tissue remodeling and emphysema. Although inflammation is critical to COPD pathogenesis, the cellular and molecular basis underlying smoking-induced lung inflammation and pathology remains unclear. Using murine smoke models and single-cell RNA-sequencing, we show that smoking establishes a self-amplifying inflammatory loop characterized by an influx of molecularly heterogeneous neutrophil subsets and excessive recruitment of monocyte-derived alveolar macrophages (MoAM). In contrast to tissue-resident AM, MoAM are absent in homeostasis and characterized by a pro-inflammatory gene signature. Moreover, MoAM represent 46% of AM in emphysematous mice and express markers causally linked to emphysema. We also demonstrate the presence of pro-inflammatory and tissue remodeling associated MoAM orthologs in humans that are significantly increased in emphysematous COPD patients. Inhibition of the IRAK4 kinase depletes a rare inflammatory neutrophil subset, diminishes MoAM recruitment, and alleviates inflammation in the lung of cigarette smoke-exposed mice. This study extends our understanding of the molecular signaling circuits and cellular dynamics in smoking-induced lung inflammation and pathology, highlights the functional consequence of monocyte and neutrophil recruitment, identifies MoAM as key drivers of the inflammatory process, and supports their contribution to pathological tissue remodeling.

BackgroundPalmoplantar pustulosis (PPP) is an inflammatory skin disorder that mostly affects smokers and manifests with painful pustular eruptions on the palms and soles. While the disease can present with concurrent plaque psoriasis, TNF and IL-17/IL-23 inhibitors show limited efficacy. There is therefore a pressing need to uncover PPP disease drivers and therapeutic targets.ObjectivesTo identify genetic determinants of PPP and investigate whether cigarette smoking contributes to disease pathogenesis.MethodsWe performed a genome-wide association meta-analysis of three North-European cohorts (n=1,456 PPP cases and 402,050 controls). We then used the scGWAS program to investigate the cell-type specificity of the association signals. We also undertook genetic correlation analyses to examine the similarities between PPP and other immune-mediated diseases. Finally, we applied Mendelian randomization to analyze the causal relationship between cigarette smoking and PPP.ResultsWe found that PPP is not associated with the main genetic determinants of plaque psoriasis. Conversely, we identified genome-wide significant associations with the FCGR3A/FCGR3B and CCHCR1 loci. We also observed 13 suggestive (P<5X10-6) susceptibility regions, including the IL4/IL13 interval. Accordingly, we demonstrated a significant genetic correlation between PPP and Th2-mediated diseases like atopic dermatitis and ulcerative colitis. We also found that genes mapping to PPP-associated intervals were preferentially expressed in dendritic cells and often implicated in T-cell activation pathways. Finally, we undertook a Mendelian randomization analysis, which supported a causal role of cigarette smoking in PPP.ConclusionsThe first genome-wide association study of PPP points to a pathogenic role for deregulated Th2 responses and cigarette smoking.

BackgroundFibrostenotic disease is a common complication in Crohn’s disease (CD) patients hallmarked by transmural extracellular matrix (ECM) accumulation in the intestinal wall. The prevention and medical therapy of fibrostenotic CD is an unmet high clinical need. Although targeting IL36R signaling is a promising therapy option, downstream mediators of IL36 during inflammation and fibrosis have been incompletely understood. Candidate molecules include matrix metalloproteinases which mediate ECM turnover and are thereby potential targets for anti-fibrotic treatment. Here, we have focused on understanding the role of MMP13 during intestinal fibrosis.MethodsWe performed bulk RNA sequencing of paired colon biopsies taken from non-stenotic and stenotic areas of patients with CD. Corresponding tissue samples from healthy controls and CD patients with stenosis were used for immunofluorescent (IF) staining. MMP13 gene expression was analyzed in cDNA of intestinal biopsies from healthy controls and in subpopulations of patients with CD in the IBDome cohort. In addition, gene regulation on RNA and protein level was studied in colon tissue and primary intestinal fibroblasts from mice upon IL36R activation or blockade. Finally, in vivo studies were performed with MMP13 deficient mice and littermate controls in an experimental model of intestinal fibrosis. Ex vivo tissue analysis included Masson’s Trichrome and Sirius Red staining as well as evaluation of immune cells, fibroblasts and collagen VI by IF analysis.ResultsBulk RNA sequencing revealed high upregulation of MMP13 in colon biopsies from stenotic areas, as compared to non-stenotic regions of patients with CD. IF analysis confirmed higher levels of MMP13 in stenotic tissue sections of CD patients and demonstrated αSMA+ and Pdpn+ fibroblasts as a major source. Mechanistic experiments demonstrated that MMP13 expression was regulated by IL36R signaling. Finally, MMP13 deficient mice, as compared to littermate controls, developed less fibrosis in the chronic DSS model and showed reduced numbers of αSMA+ fibroblasts. These findings are consistent with a model suggesting a molecular axis involving IL36R activation in gut resident fibroblasts and MMP13 expression during the pathogenesis of intestinal fibrosis.ConclusionTargeting IL36R-inducible MMP13 could evolve as a promising approach to interfere with the development and progression of intestinal fibrosis.

Palmoplantar pustulosis (PPP) is a severe pustular eruption that affects the palms and/or soles, with detrimental effects on quality of life. The disease is notoriously difficult to treat because its immune and genetic determinants remain poorly defined (Twelves et al., 2019). Although mutations of the IL36RN and myeloperoxidase MPO genes have been convincingly associated with generalized pustular psoriasis, they are rarely found in patients with PPP (Haskamp et al., 2020; Twelves et al., 2019; Vergnano et al., 2020).

Background: Palmoplantar pustulosis (PPP) is a severe inflammatory skin disorder characterized by eruptions of painful, neutrophil-filled pustules on the palms and soles. Although PPP has a profound effect on quality of life, it remains poorly understood and notoriously difficult to treat. Objective: We sought to investigate the immune pathways that underlie the pathogenesis of PPP. Methods: We applied bulk and single-cell RNA sequencing (RNA-Seq) methods to the analysis of skin biopsy samples and peripheral blood mononuclear cells. We validated our results by flow cytometry and immune fluorescence microscopy Results: Bulk RNA-Seq of patient skin detected an unexpected signature of T-cell activation, with a significant overexpression of several T(H)2 genes typically upregulated in atopic dermatitis. To further explore these findings, we carried out single-cell RNA-Seq in peripheral blood mononuclear cells of healthy and affected individuals. Memory CD4(+) T cells of PPP patients were skewed toward a T(H)17 phenotype, a phenomenon that was particularly significant among cutaneous lymphocyte-associated antigen-positive skin-homing cells. We also identified a subset of memory CD4(+) T cells that expressed both T(H)17 (KLRB1/ CD161) and T(H)2 (GATA3) markers, with pseudotime analysis suggesting that the population was the result of T(H)17 to T(H)2 plasticity. Interestingly, the GATA3(+)/CD161(+) cells were overrepresented among the peripheral blood mononuclear cells of affected individuals, both in the single-cell RNA-Seq data set and in independent flow cytometry experiments. Dual-positive cells were also detected in patient skin by immune fluorescence microscopy. Conclusions: PPP is associated with complex T-cell activation patterns and may explain why biologic drugs that target individual T helper cell populations have shown limited therapeutic efficacy.

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-β1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation.

Background: The IL-36 pathway plays a key role in the pathogenesis of generalized pustular psoriasis (GPP). In a proof-of-concept clinical trial, treatment with spesolimab, an anti-IL-36 receptor antibody, resulted in rapid skin and pustular clearance in patients presenting with GPP flares. Objective: We sought to compare the molecular profiles of lesional and nonlesional skin from patients with GPP or palmoplantar pustulosis (PPP) with skin from healthy volunteers, and to investigate the molecular changes after spesolimab treatment in the skin and blood of patients with GPP flares. Methods: Pre- and post-treatment skin and blood samples were collected from patients with GPP who participated in a single-arm, phase I study (n = 7). Skin biopsies from patients with PPP (n = 8) and healthy volunteers (n = 16) were obtained for comparison at baseline. Biomarkers were assessed by RNA-sequencing, histopathology, and immunohistochemistry. Results: In GPP and PPP lesions, 1287 transcripts were commonly upregulated or downregulated. Selected transcripts from the IL-36 signaling pathway were upregulated in untreated GPP and PPP lesions. In patients with GPP, IL-36 pathway-related signatures, T(H)1/T(H)17 and innate inflammation signaling, neutrophilic mediators, and keratinocyte-driven inflammation pathways were downregulated by spesolimab as early as week 1. Spesolimab also decreased related serum biomarkers and cell populations in the skin lesions from patients with GPP, including CD3(+) T, CD11c(+), and IL-36 gamma(+) cells and lipocalin-2-expressing cells. Conclusions: In patients with GPP, spesolimab showed rapid modulation of commonly dysregulated molecular pathways in GPP and PPP, which may be associated with improved clinical outcomes.

Palmar plantar pustulosis (PPP) is a debilitating skin disorder that presents with painful pustules on the palms and soles. Disease drivers are poorly understood, as the rarity of PPP has hindered the ascertainment of substantial patient resources. Thus, the treatment of this condition remains an unmet clinical need. Here, we sought to illuminate the genetic determinants of PPP by carrying out the first exome-wide association study for this disease. We ascertained three independent resources, including a total of 1,066 cases and 317,369 controls, originating from the UK (APRICOT and PLUM studies), Finland (FinnGen project) and Norway (HUNT study). Following quality controls, we carried out a meta-analysis of 39,579 common SNPs across the three cohorts. We identified an exome-wide significant association within the class I Major Histocompatibility Complex, where the lead SNP mapped to the CCHCR1 gene (rs130071, P=4.6 x 10-7). We also observed suggestive associations at the CCDC92/DNAH10 (P=1.5 x10-5), IL4 (P=3.3 x10-5) and OAS3 (P=3.5 x10-5) loci. Interestingly, an analysis of RNA-seq profiles generated from PPP patient skin revealed that OAS3 was also up-regulated in lesional compared to non-lesional samples (fold-change 2.4; FDR=1.6 x 10-4). These findings shed new lights on the genetic determinants of PPP and warrant further investigations of the disease associated genes uncovered by our analysis.

GPP is a rare, severe, clinically heterogeneous disease characterized by acute, life-threatening flares, and widespread systemic effects. Historically, GPP has been considered a variant of PV; however, evidence indicates that these are distinct diseases, potentially requiring different treatment. We aimed to better understand the differences between GPP and PV by comparing molecular profiles of lesional skin (LS) and nonlesional skin (NLS) from patients with GPP (N=7) or PV (N=17) with skin from healthy volunteers (HV, N=10). Global transcriptome-wide RNA sequences from skin biopsies were analyzed to identify differentially expressed genes (DEGs; absolute fold change >1.5 and Benjamini–Hochberg false discovery rate <0.05) relative to HV skin. NLS from patients with PV showed 541 DEGs. NLS from patients with GPP showed 3683 DEGs (6% shared with PV NLS), demonstrating considerable nonfocal widespread skin involvement in GPP but not PV. There were more DEGs in GPP LS (6874: 3510 upregulated, 3364 downregulated) than in PV LS (3643: 1709 upregulated, 1934 downregulated). In GPP LS, 4115 DEGs (60%) did not overlap with DEGs found in PV LS; among DEGs that did overlap (40%), 1379 were upregulated in both diseases, but 789 (57%) of these showed higher dysregulation in GPP (p<0.05). The largest differences were seen in genes involved in neutrophil-associated inflammation (CXCL1 [566 vs 31], CXCL8 [1965 vs 67], CD177 [85 vs 12], CCL20 [476 vs 32]) or connected with the Th1 axis (IL1B [298 vs 9], IL36A [2339 vs 199]). These results add to a growing body of data supporting classification of GPP as a disease separate to PV, based on genetics, transcription, and clinical features.

The identification of disease alleles underlying human autoinflammatory diseases can provide important insights into the mechanisms that maintain neutrophil homeostasis. Here, we focused our attention on generalized pustular psoriasis (GPP), a potentially life-threatening disorder presenting with cutaneous and systemic neutrophilia. Following the whole-exome sequencing of 19 unrelated affected individuals, we identified a subject harboring a homozygous splice-site mutation (c.2031-2A>C) in MPO. This encodes myeloperoxidase, an essential component of neutrophil azurophil granules. MPO screening in conditions phenotypically related to GPP uncovered further disease alleles in one subject with acral pustular psoriasis (c.2031-2A>C;c.2031-2A>C) and in two individuals with acute generalized exanthematous pustulosis (c.1705C>T;c.2031-2A>C and c.1552_1565del;c.1552_1565del). A subsequent analysis of UK Biobank data demonstrated that the c.2031-2A>C and c.1705C>T (p.Arg569Trp) disease alleles were also associated with increased neutrophil abundance in the general population (p = 5.1 × 10-6 and p = 3.6 × 10-5, respectively). The same applied to three further deleterious variants that had been genotyped in the cohort, with two alleles (c.995C>T [p.Ala332Val] and c.752T>C [p.Met251Thr]) yielding p values < 10-10. Finally, treatment of healthy neutrophils with an MPO inhibitor (4-Aminobenzoic acid hydrazide) increased cell viability and delayed apoptosis, highlighting a mechanism whereby MPO mutations affect granulocyte numbers. These findings identify MPO as a genetic determinant of pustular skin disease and neutrophil abundance. Given the recent interest in the development of MPO antagonists for the treatment of neurodegenerative disease, our results also suggest that the pro-inflammatory effects of these agents should be closely monitored.

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air–liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.

Palmoplantar pustulosis (PPP) is a chronic, inflammatory skin disease, with high disease burden, that is often refractory to treatment. There is a high unmet clinical need for the treatment of patients with PPP. The objectives of this study were to evaluate the safety and efficacy of spesolimab, a novel anti-interleukin-36 receptor antibody, in patients with PPP. This was a phase IIa, multicenter, double-blind, randomized, placebo-controlled pilot study comparing 900 mg spesolimab (n = 19), 300 mg spesolimab (n = 19), and placebo (n = 21) administered intravenously every 4 weeks until week 12 in patients with PPP. The primary efficacy endpoint was the achievement of Palmoplantar Pustulosis Area and Severity Index 50 (PPP ASI50) at week 16, defined as achieving an ≥ 50% decrease from baseline PPP ASI. At week 16, 31.6% of patients in both spesolimab dose groups achieved PPP ASI50 versus 23.8% receiving placebo (risk difference 0.078; 95% confidence interval –0.190, 0.338). Thus, the primary endpoint was not met. Spesolimab was well tolerated with no clinically relevant treatment-emergent safety signals observed. PPP severity declined over time in all treatment groups after the start of treatment, with a faster decline in the spesolimab arms than in the placebo arm, indicating a potential treatment effect for spesolimab. Limitations to the study included a small sample size and lower overall disease severity than expected at baseline. It is possible that the primary efficacy endpoint may have coincided with natural disease resolution in some patients. Further effects of the efficacy of spesolimab in PPP are being explored in a phase IIb trial.

In a prospective study the relevance of magnetic resonance (MR) for the diagnoses of tumors of the parotid gland has been evaluated. Due to the excellent soft tissue contrast and also to the possibility of imaging in various planes and sections and to the high resolution achieved by surface coils, it was possible to visualize the lesions and the surrounding anatomy in great detail. MR combines the advantages of ultrasound and computed tomography and is indicated if a tumor cannot be defined sharply by using ultrasound or CT.

Background: Short non-coding microRNAs (miRNAs) are involved in various cellular processes during disease progression of Crohn's disease (CD) and remarkably stable in feces, which make them attractive biomarker candidates for reflecting intestinal inflammatory processes. Here we investigated the potential of fecal miRNAs as noninvasive and translational CD biomarkers. Methods: MiRNAs were screened in feces of 52 patients with CD and 15 healthy controls using RNA sequencing and the results were confirmed by PCR. The relationship between fecal miRNA levels and the clinical CD activity index (CDAI) or CD endoscopic index of severity (CDEIS) was explored, respectively. Additionally, fecal miRNAs were investigated in dextran sodium sulfate, adoptive T-cell transfer, and Helicobacter typhlonius/stress-induced murine colitis models using the NanoString platform. Results: Nine miRNAs (miR-15a-5p, miR-16-5p, miR-128-3p, miR-142-5p, miR-24-3p, miR-27a-3p, miR-223-3p, miR-223-5p, and miR3074-5p) were significantly (adj. P < 0.05, >3-fold) increased whereas 8 miRNAs (miR-10a-5p, miR-10b-5p, miR-141-3p, miR-192-5p, miR200a-3p, miR-375, miR-378a-3p, and let-7g-5p) were significantly decreased in CD. MiR-192-5p, miR-375, and miR-141-3p correlated (P < 0.05) with both CDAI and CDEIS whereas miR-15a-5p correlated only with CDEIS. Deregulated expression of miR-223-3p, miR-16-5p, miR-15a-5p, miR-24-3p, and miR-200a-3p was also observed in murine models. The identified altered fecal miRNA levels reflect pathophysiological mechanisms in CD, such as Th1 and Th17 inflammation, autophagy, and fibrotic processes. Conclusions: Our translational study assessed global fecal miRNA changes of patients with CD and relevant preclinical models. These fecal miRNAs show promise as translational and clinically useful noninvasive biomarkers for mechanistic investigation of intestinal pathophysiology, including monitoring of disease progression.