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Refereed/Peer-reviewed Background: Mast cells (MCs) are tissue-resident immune cells that mediate IgE-dependent allergic responses. Downstream of FcεRI, an intricate network of receptor-specific signaling pathways and adaptor proteins govern MC function. The 14-3-3 family of serine-threonine phosphorylation–dependent adapter proteins are known to organize intracellular signaling. However, the role of 14-3-3 in IgE-dependent activation remains poorly defined. Objective: We sought to determine whether 14-3-3 proteins are required for IgE-dependent MC activation and whether 14-3-3 is a viable target for the treatment of MC-mediated inflammatory diseases. Methods: Genetic manipulation of 14-3-3ζ expression in human and mouse MCs was performed and IgE-dependent mediator release assessed. Pharmacologic inhibitors of 14-3-3 and 14-3-3ζ knockout mice were used to assess 14-3-3ζ function in a MC-dependent in vivo passive cutaneous anaphylaxis (PCA) model of allergic inflammation. Expression and function of 14-3-3ζ were assessed in human nasal polyp tissue MCs. Results: IgE-dependent mediator release from human MCs was decreased by 14-3-3ζ knockdown and increased by 14-3-3ζ overexpression. Deletion of the 14-3-3ζ gene decreased IgE-dependent activation of mouse MCs in vitro and PCA responses in vivo. Furthermore, the 14-3-3 inhibitor, RB-11, which impairs dimerization of 14-3-3, inhibited cultured MC and polyp tissue MC activation and signaling downstream of the FcεRI receptor and dose-dependently attenuated PCA responses. Conclusion: IgE/FcεRI-mediated MC activation is positively regulated by 14-3-3ζ. We identify a critical role for this p-Ser/Thr–binding protein in the regulation of MC FcεRI signaling and IgE-dependent immune responses and show that this pathway may be amenable to pharmacologic targeting.

Background Chronic obstructive pulmonary disease (COPD) exacerbations are heterogenous and profoundly impact the disease trajectory. Bioactive lipid lysophosphatidic acid (LPA) has been implicated in airway inflammation but the significance of LPA in COPD exacerbation is not known. The aim of the study was to investigate the utility of serum LPA species (LPA16:0, 18:0, 18:1, 18:2, 20:4) as biomarkers of COPD exacerbation. Patients and methods LPA species were measured in the baseline placebo sera of a COPD randomized controlled trial. Tertile levels of each LPA were used to assign patients into biomarker high, medium, and low subgroups. Exacerbation rate and risk were compared among the LPA subgroups. Results The levels of LPA species were intercorrelated (rho 0.29–0.91). Patients with low and medium levels of LPA (LPA16:0, 20:4) had significantly higher exacerbation rate compared to the respective LPA-high patients [estimated rate per patient per year (95% CI)]: LPA16:0-low = 1.2 (0.8–1.9) (p = 0.019), LPA16:0-medium = 1.3 (0.8–2.0) (p = 0.013), LPA16:0-high = 0.5 (0.2–0.9); LPA20:4-low = 1.4 (0.9–2.1) (p = 0.0033), LPA20:4-medium = 1.2 (0.8–1.8) (p = 0.0089), LPA20:4-high = 0.4 (0.2–0.8). These patients also had earlier time to first exacerbation (hazard ratio (95% CI): LPA16:0-low = 2.6 (1.1–6.0) (p = 0.028), LPA16:0-medium = 2.7 (1.2–6.3) (p = 0.020); LPA20.4-low = 2.8 (1.2–6.6) (p = 0.017), LPA20:4-medium = 2.7 (1.2–6.4) (p = 0.021). Accordingly, these patients had a significant increased exacerbation risk compared to the respective LPA-high subgroups [odd ratio (95% CI)]: LPA16:0-low = 3.1 (1.1–8.8) (p = 0.030), LPA16:0-medium = 3.0 (1.1–8.3) (p = 0.031); LPA20:4-low = 3.8 (1.3–10.9) (p = 0.012), LPA20:4-medium = 3.3 (1.2–9.5) (p = 0.025). For the other LPA species (LPA18:0, 18:1, 18:2), the results were mixed; patients with low and medium levels of LPA18:0 and 18:2 had increased exacerbation rate, but only LPA18:0-low patients had significant increase in exacerbation risk and earlier time to first exacerbation compared to the LPA18:0-high subgroup. Conclusions The study provided evidence of association between systemic LPA levels and exacerbation in COPD. Patients with low and medium levels of specific LPA species (LPA16:0, 20:4) had increased exacerbation rate, risk, and earlier time to first exacerbation. These non-invasive biomarkers may aid in identifying high risk patients with dysregulated LPA pathway to inform risk management and drug development.

IntroductionCOPD exacerbations are heterogeneous and can be triggered by bacterial, viral, or non-infectious insults. Exacerbations are also heterogeneous in neutrophilic or eosinophilic inflammatory responses. A non-invasive peripheral biomarker of COPD exacerbations characterised by bacterial/neutrophilic inflammation is lacking. G-CSF is a key cytokine elevated during bacterial infection and mediates survival, proliferation, differentiation, and function of neutrophils.ObjectiveWe hypothesized that high peripheral G-CSF would be indicative of COPD exacerbations with a neutrophilic and bacterial phenotype associated with microbial dysbiosis.MethodsSerum G-CSF was measured during hospitalised exacerbation and after 30 days of recovery in 37 subjects. In a second cohort, serum and sputum cytokines were measured in 59 COPD patients during stable disease, at exacerbation, and 2- and 6-weeks following exacerbations.ResultsSerum G-CSF is increased during exacerbation in a subset of patients. These exacerbations were enriched for bacterial and not viral or type-2 biologies. The median serum G-CSF levels was 1.6-fold higher in bacterial exacerbation compared to non-bacterial exacerbation (22 pg·mL−1versus 13 pg·mL−1, p=0.0007). Serum G-CSF classified bacterial exacerbations with an AUROC=0.76. Exacerbations with a ≥2-fold increase in serum G-CSF were characterised by neutrophilic inflammation, with increased sputum and blood neutrophils and high sputum IL-1β, IL-6, and SAA. These exacerbations were preceded by dysbiosis, with decreased microbiome diversity and enrichment of respiratory pathogens such as Haemophilus and Moraxella. Furthermore, serum G-CSF at exacerbation classified neutrophilic-dysbiotic exacerbations (AUROC=0.75).ConclusionsHigh serum G-CSF enriches for COPD exacerbations characterised by neutrophilic inflammation with underlying bacterial dysbiosis.

The recent outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has led to a worldwide pandemic. One week after initial symptoms develop, a subset of patients progresses to severe disease, with high mortality and limited treatment options. To design novel interventions aimed at preventing spread of the virus and reducing progression to severe disease, detailed knowledge of the cell types and regulating factors driving cellular entry is urgently needed. Here we assess the expression patterns in genes required for COVID-19 entry into cells and replication, and their regulation by genetic, epigenetic and environmental factors, throughout the respiratory tract using samples collected from the upper (nasal) and lower airways (bronchi). Matched samples from the upper and lower airways show a clear increased expression of these genes in the nose compared to the bronchi and parenchyma. Cellular deconvolution indicates a clear association of these genes with the proportion of secretory epithelial cells. Smoking status was found to increase the majority of COVID-19 related genes including ACE2 and TMPRSS2 but only in the lower airways, which was associated with a significant increase in the predicted proportion of goblet cells in bronchial samples of current smokers. Both acute and second hand smoke were found to increase ACE2 expression in the bronchus. Inhaled corticosteroids decrease ACE2 expression in the lower airways. No significant effect of genetics on ACE2 expression was observed, but a strong association of DNA- methylation with ACE2 and TMPRSS2- mRNA expression was identified in the bronchus.

Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (βc) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit βc, a property that has made βc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of βc has precluded testing of inhibitors of human βc in mouse models. To overcome this problem, we developed a human βc receptor transgenic mouse strain with a hematopoietic cell‒specific expression of human βc instead of mouse βc. Human βc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3–/– mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking anti–human βc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human βc receptor transgenic mouse as a unique platform to test the inhibitors of βc in vivo. Refereed/Peer-reviewed

Chronic obstructive pulmonary disease (COPD) is a heterogeneous inflammatory airway disease. The epithelial-derived IL-33 and its receptor ST2 have been implicated in airway inflammation and infection. We aimed to determine whether astegolimab, a selective ST2 IgG2 monoclonal antibody, reduces exacerbations in COPD.COPD-ST2OP was a single-centre, randomised, double-blinded, placebo-controlled phase 2a trial in moderate-to-very severe COPD. Participants were randomly assigned (1:1) with a web-based system to received 490 mg subcutaneous astegolimab or subcutaneous placebo, every 4 weeks for 44 weeks. The primary endpoint was exacerbation rate assessed for 48 weeks assessed with a negative binomial count model in the intention-to-treat population, with prespecified subgroup analysis by baseline blood eosinophil count. The model was the number of exacerbations over the 48-week treatment period, with treatment group as a covariate. Safety was assessed in the whole study population until week 60. Secondary endpoints included Saint George's Respiratory Questionnaire for COPD (SGRQ-C), FEVThe exacerbation rate at 48 weeks in the intention-to-treat analysis was not significantly different between the astegolimab group (2·18 [95% CI 1·59 to 2·78]) and the placebo group (2·81 [2·05 to 3·58]; rate ratio 0·78 [95% CI 0·53 to 1·14]; p=0·19]). In the prespecified analysis stratifying patients by blood eosinophil count, patients with 170 or fewer cells per μL had 0·69 exacerbations (0·39 to 1·21), whereas those with more than 170 cells per μL had 0·83 exacerbations (0·49 to 1·40). For the secondary outcomes, the mean difference between the SGRQ-C in the astegolimab group versus placebo group was -3·3 (95% CI -6·4 to -0·2; p=0·039), and mean difference in FEVIn patients with moderate-to-very severe COPD, astegolimab did not significantly reduce exacerbation rate, but did improve health status compared with placebo.Funded by Genentech and National Institute for Health Research Biomedical Research Centres.

Refereed/Peer-reviewed

Lysophospholipids are bioactive signaling molecules derived from cell membrane glycerophospholipids or sphingolipids and are highly regulated under normal physiological conditions. Lysophosphatidic acids (LPAs) are a class of lysophospholipids that act on G-protein-coupled receptors to exert a variety of cellular functions. Dysregulation of phospholipase activity and consequently LPA synthesis in serum have been linked to inflammation, such as seen in chronic obstructive pulmonary disease (COPD). The accurate measurement of phospholipids is critical for evaluating their dysregulation in disease. In this study, we optimized experimental parameters for the sensitive measurement of LPAs. We validated the method based on matrix, linearity, accuracy, precision, and stability. An investigation into sample extraction processes emphasized that the common practice of including low concentration of hydrochloric acid in the extraction buffer causes an overestimation of lipid recovery. The liquid chromatography gradient was optimized to separate various lysophospholipid classes. After optimization, detection limits of LPA were sufficiently sensitive for subsequent analysis, ranging from 2 to 8 nM. The validated workflow was applied to a cohort of healthy donor and COPD patient sera. Eight LPA species were identified, and five unique species of LPA were quantified. Most LPA species increased significantly in COPD patients compared to healthy donors. The correlation between LPAs and other demographic parameters was further investigated in a sample set of over 200 baseline patient sera from a COPD clinical trial. For the first time, LPAs other than the two most abundant and readily detectable moieties are quantified in COPD patients using validated methods, opening the door to downstream biomarker evaluation in respiratory disease.

Janus Kinases (JAKs) mediate activity of many asthma-relevant cytokines. GDC-0214, an inhaled small molecule JAK1 inhibitor, has previously been shown to reduce fractional exhaled nitric oxide (FeNO) in patients with mild asthma, but required an excessive number of inhalations.To assess whether GDC-4379, a new inhaled JAK inhibitor, reduces FeNO and peripheral biomarkers of inflammation.This study assessed the activity of GDC-4379 in a double-blind, randomized, placebo-controlled, Phase 1 study in patients with mild asthma. Participants included adults (18-65y) with a diagnosis of asthma for ≥6 months, forced expiratory volume in 1 s (FEVsub1/sub)gt; 70% predicted, FeNOgt;40 ppb, using as-needed short-acting beta-agonist medication only. Four sequential, 14-day, ascending-dose cohorts (10 mg QD, 30 mg QD, 40 mg BID, and 80 mg QD) of 12 participants each were randomized 2:1 to GDC-4379 or placebo. The primary activity outcome was percent change from baseline (CFB) in FeNO to Day 14 compared to the pooled placebo group. Safety, tolerability, pharmacokinetics, and pharmacodynamic biomarkers, including blood eosinophils, serum CCL17, and serum CCL18, were also assessed.Of 48 enrolled participants, the mean age was 25 years and 54% were female. Median (range) FeNO at baseline was 79 (41-222) ppb. GDC-4379 treatment led to dose-dependent reductions in FeNO. Compared to placebo, mean (95% CI) percent CFB in FeNO to Day 14 was: -6 (-43, 32) at 10 mg QD, -26 (-53, 2) at 30 mg QD, -55 (-78, -32) at 40 mg BID and -52 (-72, -32) at 80 mg QD. Dose-dependent reductions in blood eosinophils and serum CCL17 were also observed. Higher plasma drug concentrations corresponded with greater FeNO reductions. No serious AEs occurred. The majority of AEs were mild to moderate. The most common AEs were headache and oropharyngeal pain. Minor changes in neutrophils were noted at 80 mg QD, but were not considered clinically meaningful.In patients with mild asthma, 14-day treatment with GDC-4379 reduced FeNO levels and peripheral biomarkers of inflammation. Treatment was well tolerated without any major safety concerns.ACTRN12619000227190.

Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (β

Vascular leakage, or edema, is a serious complication of acute allergic reactions. Vascular leakage is triggered by the release of histamine and serotonin from granules within tissue-resident mast cells. Here, we show that expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates mast cell-mediated vascular leakage. In myeloid precursors, the granulocyte–macrophage progenitors (GMPs), loss of NSP4 results in the decrease of cellular levels of histamine, serotonin and heparin/heparan sulfate. Mast cells that are derived from NSP4-deficient GMPs have abnormal secretory granule morphology and a sustained reduction in histamine and serotonin levels. Consequently, in passive cutaneous anaphylaxis and acute arthritis models, mast cell-mediated vascular leakage in the skin and joints is substantially reduced in NSP4-deficient mice. Our findings reveal that NSP4 is required for the proper storage of vasoactive amines in mast cell granules, which impacts mast cell-dependent vascular leakage in mouse models of immune complex-mediated diseases., AhYoung, Eckard et al. show that the expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates the levels of histamine and serotonin in mast cell granules. This study reveals an important physiological function of NSP4 in mast cell-mediated vascular leakage in mice, establishing NSP4 as a potential therapeutic target for mast cell-dependent immune disorders.

Introduction: Autotaxin-Lysophosphatidic acid (ATX-LPA) pathway has been associated with inflammatory lung conditions including COPD as it has pleiotropic roles in cell proliferation, lymphocyte homing and fibrosis. ATX functions primarily as lysophospholipase D to generate bioactive lipid LPA detected in blood and inflamed sites. Aims: We assessed if systemic LPA levels were associated with exacerbation frequency and severity in COPD patients. Methods: Mass spectrometry assays for LPA species (LPAs) were developed in-house using a custom lipid extraction method, and demonstrated good reproducibility and recovery. LPAs were measured in baseline serum samples from the placebo arm (n=136) of a COPD randomized controlled trial (NCT02546700). The relationship between LPA levels, baseline demographics, and other biomarkers were assessed using Mann-Whitney or Spearman method. Tertile levels of each LPA species were used to assign patients into biomarker high, mid, and low subgroups, and association with on-study exacerbation assessed by a Quasi-Poisson model. Results: Levels of LPA species were correlated with each other (Spearman rho 0.35 – 0.92), and were influenced by age, gender, region, and exacerbation history. After adjusting for these confounders, patients with the lowest tertile of LPAs had two times higher exacerbation rate in the 6-month follow-up, compared to patients with the highest tertile of LPAs (p Conclusions: These findings provide preliminary support that systemic LPAs are detectable in COPD and associate with exacerbation rate.

BackgroundThe recent outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has led to a worldwide pandemic. A subset of COVID-19 patients progresses to severe disease, with high mortality and limited treatment options. Detailed knowledge of the expression regulation of genes required for viral entry into respiratory epithelial cells is urgently needed.MethodsHere we assess the expression patterns of genes required for SARS-CoV-2 entry into cells, and their regulation by genetic, epigenetic and environmental factors, throughout the respiratory tract using samples collected from the upper (nasal) and lower airways (bronchi).FindingsGenes encoding viral receptors and activating protease are increased in the nose compared to the bronchi in matched samples and associated with the proportion of secretory epithelial cells in cellular deconvolution analyses. Current or ex-smoking was found to increase expression of these genes only in lower airways, which was associated with a significant increase in the predicted proportion of goblet cells. Both acute and second hand smoke exposure were found to increase ACE2 expression while inhaled corticosteroids decrease ACE2 expression in the lower airways. A strong association of DNA- methylation with ACE2 and TMPRSS2- mRNA expression was identified.InterpretationGenes associated with SARS-CoV-2 viral entry into cells are high in upper airways, but strongly increased in lower airways by smoke exposure. In contrast, ICS decreases ACE2 expression, indicating that inhaled corticosteroids are unlikely to increase the risk for more severe COVID-19 disease.FundingThis work was supported by a Seed Network grant from the Chan Zuckerberg Initiative to M.C.N. and by the European Union’s H2020 Research and Innovation Program under grant agreement no. 874656 (discovAIR) to M.C.N. U BIOPRED was supported by an Innovative Medicines Initiative Joint Undertaking (No. 115010), resources from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution (www.imi.europa.eu). Longfonds Junior Fellowship. We acknowledge the contribution of the whole U-BIOPRED team as listed in the Appendix S1.’ SDB, FM and RFS would like to thank the Helmholtz Association, Germany, for support.” NIH K08HL146943; Parker B. Francis Fellowship; ATS Foundation/Boehringer Ingelheim Pharmaceuticals Inc. Research Fellowship in IPF. RCR is part funded by Cancer Research UK Cambridge Centre and the Cambridge NIHR Biomedical Research Centre. BAP was funded by programme support from Cancer Research UK. The CRUKPAP Study was supported by the CRUK Cambridge Cancer Centre, by the NIHR Cambridge Biomedical Research Centre and by the Cambridge Bioresource. PIAMA was supported by The Netherlands Organization for Health Research and Development; The Netherlands Organization for Scientific Research; The Netherlands Lung Foundation (with methylation studies supported by AF 4.1.14.001); The Netherlands Ministry of Spatial Planning, Housing, and the Environment; and The Netherlands Ministry of Health, Welfare, and Sport. Dr. Qi is supported by a grant from the China Scholarship Council.

Background The receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) is implicated in COPD. Although these receptors share common ligands and signalling pathways, it is not known whether they act in concert to drive pathological processes in COPD. We examined the impact of RAGE and/or TLR4 gene deficiency in a mouse model of COPD and also determined whether expression of these receptors correlates with airway neutrophilia and airway hyperresponsiveness (AHR) in COPD patients. Methods We measured airway inflammation and AHR in wild-type, RAGE(-/-), TLR4(-/-)and TLR4(-/-)RAGE(-/-)mice following acute exposure to cigarette smoke (CS). We also examined the impact of smoking status onAGER(encodes RAGE) andTLR4bronchial gene expression in patients with and without COPD. Finally, we determined whether expression of these receptors correlates with airway neutrophilia and AHR in COPD patients. Results RAGE(-/-)mice were protected against CS-induced neutrophilia and AHR. In contrast, TLR4(-/-)mice were not protected against CS-induced neutrophilia and had more severe CS-induced AHR. TLR4(-/-)RAGE(-/-)mice were not protected against CS-induced neutrophilia but were partially protected against CS-induced mediator release and AHR. Current smoking was associated with significantly lowerAGERandTLR4expression irrespective of COPD status, possibly reflecting negative feedback regulation. However, consistent with preclinical findings,AGERexpression correlated with higher sputum neutrophil counts and more severe AHR in COPD patients.TLR4expression did not correlate with neutrophilic inflammation or AHR. Conclusions Inhibition of RAGE but not TLR4 signalling may protect against airway neutrophilia and AHR in COPD.

Protective high-affinity antibody responses depend on competitive selection of B cells carrying somatically mutated B-cell receptors by follicular helper T (T FH ) cells in germinal centres. The rapid T-B-cell interactions that occur during this process are reminiscent of neural synaptic transmission pathways. Here we show that a proportion of human T FH cells contain dense-core granules marked by chromogranin B, which are normally found in neuronal presynaptic terminals storing catecholamines such as dopamine. T FH cells produce high amounts of dopamine and release it upon cognate interaction with B cells. Dopamine causes rapid translocation of intracellular ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) to the B-cell surface, which enhances accumulation of CD40L and chromogranin B granules at the human T FH cell synapse and increases the synapse area. Mathematical modelling suggests that faster dopamine-induced T-B-cell interactions increase total germinal centre output and accelerate it by days. Delivery of neurotransmitters across the T-B-cell synapse may be advantageous in the face of infection. Refereed/Peer-reviewed

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent, submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages, reduces tumor cell proliferation and angiogenesis, and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33, an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11, which is required for the growth of gastric cancers in mice. Accordingly, ablation of the cognate IL-33 receptor St2 limits tumor growth, and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2, Ccl3, and Il6. Conversely, genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore, tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer., Mast cells within the tumor microenvironment have controversial roles. Here, the authors show, using genetic mouse models, that in gastric cancer, mast cells at the periphery of the tumors are activated via cancer cell produced-IL33 and promote tumorigenesis by recruiting macrophages within the tumors.

Chronic mucus hypersecretion (CMH) is a common feature in chronic obstructive pulmonary disease (COPD) and is associated with worse prognosis and quality of life. This study aimed to identify microRNA (miRNA)–mRNA regulatory networks underlying CMH.The expression profiles of miRNA and mRNA in bronchial biopsies from 63 COPD patients were associated with CMH using linear regression. Potential mRNA targets of each CMH-associated miRNA were identified using Pearson correlations. Gene set enrichment analysis (GSEA) and STRING (search tool for the retrieval of interacting genes/proteins) analysis were used to identify key genes and pathways.20 miRNAs and 539 mRNAs were differentially expressed with CMH in COPD. The expression of 10 miRNAs was significantly correlated with the expression of one or more mRNAs. Of these, miR-134-5p, miR-146a-5p and the let-7 family had the highest representation of CMH-associated mRNAs among their negatively correlated predicted targets. KRAS and EDN1 were identified as key regulators of CMH and were negatively correlated predicted targets of miR-134-5p and let-7a-5p, let-7d-5p, and let-7f-5p, respectively. GSEA suggested involvement of MUC5AC-related genes and several other relevant gene sets in CMH. The lower expression of miR-134-5p was confirmed in primary airway fibroblasts from COPD patients with CMH.We identified miR-134-5p, miR-146a-5p and let-7 family, along with their potential target genes including KRAS and EDN1, as potential key miRNA–mRNA networks regulating CMH in COPD.

Mast cells (MCs) are versatile immune cells capable of rapidly responding to a diverse range of extracellular cues. Here, we mapped the genomic and transcriptomic changes in human MCs upon diverse stimuli. Our analyses revealed broad H3K4me3 domains and enhancers associated with activation. Notably, the rise of intracellular calcium concentration upon immunoglobulin E (IgE)-mediated crosslinking of the high-affinity IgE receptor (FcεRI) resulted in genome-wide reorganization of the chromatin landscape and was associated with a specific chromatin signature, which we term Ca2+-dependent open chromatin (COC) domains. Examination of differentially expressed genes revealed potential effectors of MC function, and we provide evidence for fibrinogen-like protein 2 (FGL2) as an MC mediator with potential relevance in chronic spontaneous urticaria. Disease-associated single-nucleotide polymorphisms mapped onto cis-regulatory regions of human MCs suggest that MC function may impact a broad range of pathologies. The datasets presented here constitute a resource for the further study of MC function. Cildir et al. map the genomic and transcriptomic changes in human mast cells upon diverse stimuli, defining chromatin changes associated with activation and Ca2+ flux and revealing potential effectors of mast cell function. Disease-associated SNPs mapped onto cis-regulatory regions of mast cells suggest that mast cell function may impact a broad range of pathologies. Refereed/Peer-reviewed

The β common ([βc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use βc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the βc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease. This review will focus on the emerging biological roles for the βc cytokines, our progress toward understanding the mechanisms of receptor assembly and signaling, and the application of this knowledge to develop exciting new therapeutic approaches against human disease. Refereed/Peer-reviewed

Topical application of biologically active vitamin D [1,25-dihydroxyvitamin D (1,25(OH) 2 D)], or low-calcemic analogues, curb skin inflammation through mechanisms that involve migratory dendritic cells (DCs) and regulatory T (T Reg ) cells. 1,25(OH) 2 D also promotes immunoregulation by mast cells, and inhibits the development of T helper type-9 (Th9) cells that secrete interleukin-9 (IL-9). Here, we investigated the ability of topical 1,25(OH) 2 D to suppress contact dermatitis through an IL-9-dependent process, examining mast cells and IL-9-secreting T cells. Contact dermatitis was modelled in adult BALB/c female mice by initiating a “biphasic ear swelling response” following a single application of 2,4-dinitrofluorobenzene (DNFB). Topical 1,25(OH) 2 D (125 ng) applied to ear pinnae prior to (but not after) DNFB sensitisation suppressed the efferent phase of the ear swelling response. This dose of 1,25(OH) 2 D did not cause hypercalcemia. At the peak of the efferent ear swelling response, proportions of T Reg (CD3 + Foxp3+) cells and numbers of mast cells were increased in ear skin of 1,25(OH) 2 D-treated mice. Topical 1,25(OH) 2 D increased the proportion of Foxp3 + IL-9 + T Reg cells and the capacity of T Reg cells to secrete IL-9 ex vivo. However, the proportion of the IL-9 + cells of the total T Reg cell population was small ( < 1%), and the amount of IL-9 secreted by T Reg cells from mice treated with IL-9 was low ( < 50 pg/ml). Furthermore, injection of anti-IL-9 neutralising antibody (100 µg, intraperitoneally) prior to sensitisation did not significantly reverse the suppressive effects of 1,25(OH) 2 D. In conclusion, topically applied 1,25(OH) 2 D suppressed the efferent phase of a biphasic cutaneous ear swelling response through mechanism(s) that may be dependent on mast cells and T Reg cells; however, the role of IL-9 in mediating these responses is uncertain. More studies are needed to further characterise the mechanisms by which topical 1,25(OH) 2 D modulates cell-mediated immune responses central to its suppressive effects upon contact dermatitis. Refereed/Peer-reviewed

International audience; Contact hypersensitivity (CHS) is a common skin disease induced by epicutaneous sensitization to haptens. Conflicting results have been obtained regarding pathogenic versus protective roles of mast cells (MCs) in CHS, and this has been attributed in part to the limitations of certain models for studying MC functions in vivo. Here we describe a fluorescent imaging approach that enables in vivo selective labeling and tracking of MC secretory granules by real-time intravital 2-photon microscopy in living mice, and permits the identification of such MCs as a potential source of cytokines in different disease models. We show using this method that dermal MCs release their granules progressively into the surrounding microenvironment, but also represent an initial source of the antiinflammatory cytokine IL-10, during the early phase of severe CHS reactions. Finally, using 3 different types of MC-deficient mice, as well as mice in which IL-10 is ablated specifically in MCs, we show that IL-10 production by MCs can significantly limit the inflammation and tissue pathology observed in severe CHS reactions.

Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine(C-C motif) lig and 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke Refereed/Peer-reviewed

Alterations to dendritic cell (DC) progenitors in the bone marrow (BM) may contribute to long-lasting systemic immunosuppression (>28 d) following exposure of the skin of mice to erythemal UV radiation (UVR). DCs differentiated in vitro from the BM of mice 3 d after UVR (8 kJ/m2) have a reduced capacity to initiate immunity (both skin and airways) when adoptively transferred into naive mice. Studies in IL-10−/− mice suggested that UV-induced IL-10 was not significantly involved. To investigate the immune capabilities of peripheral tissue DCs generated in vivo from the BM of UV-irradiated mice, chimeric mice were established. Sixteen weeks after reconstitution, contact hypersensitivity responses were significantly reduced in mice reconstituted with BM from UV-irradiated mice (UV-chimeric). When the dorsal skin of UV-chimeric mice was challenged with innate inflammatory agents, the hypertrophy induced in the draining lymph nodes was minimal and significantly less than that measured in control-chimeric mice challenged with the same inflammatory agent. When DCs were differentiated from the BM of UV-chimeric mice using FLT3 ligand or GM-CSF + IL-4, the cells maintained a reduced priming ability. The diminished responses in UV-chimeric mice were not due to different numerical or proportional reconstitution of BM or the hematopoietic cells in blood, lymph nodes, and skin. Erythemal UVR may imprint a long-lasting epigenetic effect on DC progenitors in the BM and alter the function of their terminally differentiated progeny.

Neutrophil granulocytes traffic into sites of organ injury in which they may not only participate in tissue repair and pathogen clearance but may also contribute to collateral cell damage through the release of noxious mediators. The dynamics and mechanisms of neutrophil migration in the extravascular space toward loci of tissue damage are not well understood. Here, we have used intravital multi-photon microscopy to dissect the behavior of neutrophils in response to tissue injury in the dermis of mice. We found that, following confined physical injury, initially rare scouting neutrophils migrated in a directional manner toward the damage focus. This was followed by the attraction of waves of additional neutrophils, and finally stabilization of the neutrophil cluster around the injury. Although neutrophil migration in the steady state and during the scouting phase depended on pertussis toxin-sensitive signals, the amplification phase was sensitive to interference with the cyclic adenosine diphosphate ribose pathway. We finally demonstrated that neutrophil scouts also transit through the non-inflamed dermis, suggesting immunosurveillance function by these cells. Together, our data unravel a three-step cascade of events that mediates the specific accumulation of neutrophils at sites of sterile tissue injury in the interstitial space. Refereed/Peer-reviewed

Mast cell production of interleukin-10 (IL-10) can limit the skin pathology induced by chronic low-dose ultraviolet (UV)-B irradiation. Although the mechanism that promotes mast cell IL-10 production in this setting is unknown, exposure of the skin to UVB irradiation induces increased production of the immune modifying agent 1alpha,25-dihydroxyvitamin D(3) (1alpha,25[OH](2)D(3)). We now show that 1alpha,25(OH)(2)D(3) can up-regulate IL-10 mRNA expression and induce IL-10 secretion in mouse mast cells in vitro. To investigate the roles of 1alpha,25(OH)(2)D(3) and mast cell vitamin D receptor (VDR) expression in chronically UVB-irradiated skin in vivo, we engrafted the skin of genetically mast cell-deficient WBB6F(1)-Kit(W/W-v) mice with bone marrow-derived cultured mast cells derived from C57BL/6 wild-type or VDR(-/-) mice. Optimal mast cell-dependent suppression of the inflammation, local production of proinflammatory cytokines, epidermal hyperplasia, and epidermal ulceration associated with chronic UVB irradiation of the skin in Kit(W/W-v) mice required expression of VDR by the adoptively transferred mast cells. Our findings suggest that 1alpha,25(OH)(2)D(3)/VDR-dependent induction of IL-10 production by cutaneous mast cells can contribute to the mast cell's ability to suppress inflammation and skin pathology at sites of chronic UVB irradiation.

We used mast cell-engrafted genetically mast cell-deficient C57BL/6-Kit(W-sh/W-sh) mice to investigate the roles of mast cells and mast cell-derived tumor necrosis factor in two models of severe bacterial infection. In these mice, we confirmed findings derived from studies of mast cell-deficient WBB6F(1)-Kit(W/W-v) mice indicating that mast cells can promote survival in cecal ligation and puncture (CLP) of moderate severity. However, we found that the beneficial role of mast cells in this setting can occur independently of mast cell-derived tumor necrosis factor. By contrast, using mast cell-engrafted C57BL/6-Kit(W-sh/W-sh) mice, we found that mast cell-derived tumor necrosis factor can increase mortality during severe CLP and can also enhance bacterial growth and hasten death after intraperitoneal inoculation of Salmonella typhimurium. In WBB6F(1)-Kit(W-sh/W-sh) mice, mast cells enhanced survival during moderately severe CLP but did not significantly change the survival observed in severe CLP. Our findings in three types of genetically mast cell-deficient mice thus support the hypothesis that, depending on the circumstances (including mouse strain background, the nature of the mutation resulting in a mast cell deficiency, and type and severity of infection), mast cells can have either no detectable effect or opposite effects on survival during bacterial infections, eg, promoting survival during moderately severe CLP associated with low mortality but, in C57BL/6-Kit(W-sh/W-sh) mice, increasing mortality during severe CLP or infection with S. typhimurium. Refereed/Peer-reviewed

Mast cells can promote inflammation and other tissue changes in IgE-associated allergic disorders, as well as in certain innate and adaptive immune responses that are thought to be independent of IgE. However, mast cells can also have anti-inflammatory and immunosuppressive functions. Here, we review the evidence that mast cells can have negative, as well as positive, immunomodulatory roles in vivo, and we propose that mast cells can both enhance and later suppress certain features of an immune response.

Detailing the inflammatory mechanisms of biomaterial-implant induced foreign body responses (FBR) has implications for revealing targetable pathways that may reduce leukocyte activation and fibrotic encapsulation of the implant. We have adapted a model of poly(methylmethacrylate) (PMMA) bead injection to perform an assessment of the mechanistic role of the ASC-dependent inflammasome in this process. We first demonstrate that ASC−/− mice subjected to PMMA bead injections had reduced cell infiltration and altered collagen deposition, suggesting a role for the inflammasome in the FBR. We next investigated the NLRP3 and AIM2 sensors because of their known contributions in recognising damaged and apoptotic cells. We found that NLRP3 was dispensable for the fibrotic encapsulation; however AIM2 expression influenced leukocyte infiltration and controlled collagen deposition, suggesting a previously unexplored link between AIM2 and biomaterial-induced FBR.

Cross-linkage of the high-affinity immunoglobulin E (IgE) receptor (FcɛRI) on mast cells by antigen ligation has a critical role in the pathology of IgE-dependent allergic disorders, such as anaphylaxis and asthma. Restraint of intracellular signal transduction pathways that promote release of mast cell-derived pro-inflammatory mediators is necessary to dampen activation and restore homoeostasis. Here we show that the ligase Nedd4-2 and the adaptor Ndfip1 (Nedd4 family interacting protein 1) limit the intensity and duration of IgE-FcɛRI-induced positive signal transduction by ubiquitinating phosphorylated Syk, a tyrosine kinase that is indispensable for downstream FcɛRI signalosome activity. Importantly, loss of Nedd4-2 or Ndfip1 in mast cells results in exacerbated and prolonged IgE-mediated cutaneous anaphylaxis in vivo. Our findings reveal an important negative regulatory function for Nedd4-2 and Ndfip1 in IgE-dependent mast cell activity., Aberrant activation of the IgE receptor on mast cells leads to allergic responses. Here, the authors identify an E3 ligase and adaptor protein that can reduce IgE signalling by targeting phosphorylated-Syk for degradation.

Observations of increased numbers of mast cells at sites of chronic inflammation have been reported for over a hundred years. Light and electron microscopic evidence of mast cell activation at such sites, taken together with the known functions of the diverse mediators, cytokines, and growth factors that can be secreted by appropriately activated mast cells, have suggested a wide range of possible functions for mast cells in promoting (or suppressing) many features of chronic inflammation. Similarly, these and other lines of evidence have implicated mast cells in a variety of adaptive or pathological responses that are associated with persistent inflammation at the affected sites. Definitively characterizing the importance of mast cells in chronic inflammation in humans is difficult. However, mice that genetically lack mast cells, especially those which can undergo engraftment with wildtype or genetically altered mast cells, provide a means to investigate the importance of mast cells and specific mast cell functions or products in diverse models of chronic inflammation. Such work has confirmed that mast cells can significantly influence multiple features of chronic inflammatory responses, through diverse effects that can either promote or, perhaps more surprisingly, suppress aspects of these responses.

▪ Abstract This review focuses on recent progress in our understanding of how mast cells can contribute to the initiation, development, expression, and regulation of acquired immune responses, both those associated with IgE and those that are apparently expressed independently of this class of Ig. We emphasize findings derived from in vivo studies in mice, particularly those employing genetic approaches to influence mast cell numbers and/or to alter or delete components of pathways that can regulate mast cell development, signaling, or function. We advance the hypothesis that mast cells not only can function as proinflammatory effector cells and drivers of tissue remodeling in established acquired immune responses, but also may contribute to the initiation and regulation of such responses. That is, we propose that mast cells can also function as immunoregulatory cells. Finally, we show that the notion that mast cells have primarily two functional configurations, off (or resting) or on (or activated for extensive mediator release), markedly oversimplifies reality. Instead, we propose that mast cells are “tunable,” by both genetic and environmental factors, such that, depending on the circumstances, the cell can be positioned phenotypically to express a wide spectrum of variation in the types, kinetics, and/or magnitude of its secretory functions.

Cutaneous exposure to ultraviolet (UV) A (320–400 nm) results in the formation of damaging reactive oxygen intermediates, which are implicated as mediators of DNA damage, apoptosis, and photoaging. S100A8 is a low-molecular-weight calcium-binding protein, highly sensitive to oxidation. In this study, UVA-induced S100A8 expression by keratinocytes was investigated. UVA (50–100 kJ per m2) strongly induced S100A8 in differentiated keratinocytes in the epidermis of BALB/c mice. Similarly, S100A8 mRNA and monomeric protein were significantly upregulated in PAM212 cells (a murine keratinocyte cell line) in response to 10 kJ per m2 UVA 24 h after irradiation. Although S100A9 associates with S100A8 in neutrophils and abnormally differentiated keratinocytes (human psoriasis), in this study it was not coinduced with keratinocyte S100A8. Dorsal application of 4-hydroxy-tempo (a superoxide dis-mutase-mimicking agent) to mice concentration-dependently reduced UVA-induced S100A8 expression. Incubation of PAM212 cells with superoxide dismutase and catalase during UVA irradiation also abrogated S100A8 induction. These results suggest that UVA-induced S100A8 is expressed by keratinocytes in response to generation of reactive oxygen intermediates.

Atopic dermatitis is a common pruritic and inflammatory skin disorder with unknown etiology. Most commonly occurring during early childhood, atopic dermatitis is associated with eczematous lesions and lichenification, in which the epidermis becomes hypertrophied resulting in thickening of the skin. In this study, we report an atopic dermatitis-like pathophysiology results in a murine model following the expression of the high-risk human papillomavirus (HPV) 16 oncoprotein E7 in keratinocytes under the keratin 14 promoter. We show that HPV16 E7 expression in the skin is associated with skin thickening, acanthosis and light spongiosis. Locally, HPV16 E7-expressing skin secreted high levels of thymic stromal lymphopoietin (TSLP) and contained increased numbers of innate lymphoid cells (ILCs). High levels of circulating immunoglobulin E were associated with increased susceptibility to skin allergy in a model of cutaneous challenge, and to airway bronchiolar inflammation, enhanced airway goblet cell metaplasia and mucus production in a model of atopic march. Surprisingly, skin pathology occurred independently of T cells and mast cells. Thus, our findings suggest that the expression of a single HPV oncogene in the skin can drive the onset of atopic dermatitis-like pathology through the induction of TSLP and type 2 ILC infiltration. Refereed/Peer-reviewed

There is a direct correlation between dermal mast cell prevalence in dorsal skin of different mouse strains and susceptibility to UVB-induced systemic immunosuppression; highly UV-susceptible C57BL/6 mice have a high dermal mast cell prevalence while BALB/c mice, which require considerable UV radiation for 50% immunosuppression, have a low mast cell prevalence. There is also a functional link between the prevalence of dermal mast cells and susceptibility to UVB- and cis-urocanic acid (UCA)-induced systemic immunosuppression. Mast cell-depleted mice are unresponsive to UVB or cis-UCA for systemic immunosuppression unless they are previously reconstituted at the irradiated or cis-UCA-administered site with bone marrow-derived mast cell precursors. cis-UCA does not stimulate mast cell degranulation directly. Instead, in support of studies showing that neither UVB nor cis-UCA was immunosuppressive in capsaicin-treated, neuropeptide-depleted mice, cis-UCA-stimulated neuropeptide release from sensory c-fibers which, in turn, could efficiently degranulate mast cells. Studies in mice suggested that histamine, and not tumor necrosis factor alpha (TNF-alpha), was the product from mast cells that stimulated downstream immunosuppression. Histamine receptor antagonists reduced by approximately 60% UVB and cis-UCA-induced systemic immunosuppression. Indomethacin administration to mice had a similar effect which was not cumulative with the histamine receptor antagonists. Histamine can stimulate keratinocyte prostanoid production. We propose that both histamine and prostaglandin E(2) are important in downstream immunosuppression; both are regulatory molecules supporting the development of T helper 2 cells and reduced expression of type 1 immune responses such as a contact hypersensitivity reaction.

Human Papillomavirus (HPV) 16 E7 protein promotes the transformation of HPV infected epithelium to malignancy. Here, we use a murine model in which the E7 protein of HPV16 is expressed as a transgene in epithelium to show that mast cells are recruited to the basal layer of E7-expressing epithelium, and that this recruitment is dependent on the epithelial hyperproliferation induced by E7 by inactivating Rb dependent cell cycle regulation. E7 induced epithelial hyperplasia is associated with increased epidermal secretion of CCL2 and CCL5 chemokines, which attract mast cells to the skin. Mast cells in E7 transgenic skin, in contrast to those in non-transgenic skin, exhibit degranulation. Notably, we found that resident mast cells in E7 transgenic skin cause local immune suppression as evidenced by tolerance of E7 transgenic skin grafts when mast cells are present compared to the rejection of mast cell-deficient E7 grafts in otherwise competent hosts. Thus, our findings suggest that mast cells, recruited towards CCL2 and CCL5 expressed by epithelium induced to proliferate by E7, may contribute to an immunosuppressive environment that enables the persistence of HPV E7 protein induced pre-cancerous lesions., Author Summary Worldwide, around 50% of sexually active women are believed to become infected by Human papillomavirus type 16, the major cause of cervical cancer, and 2% will remain infected and therefore at lifetime risk of developing cancer. Why some women remain infected is unknown. Here we used a mouse engineered to express the HPV16 protein (E7) in skin, which drives development of pre-cancer lesions. This protein induces skin thickening, and the thickened skin releases molecules that attract mast cells. We show further that these cells locally suppress the function of the immune effector cells that can reject E7 expressing skin. We believe that targeting mast cells or impairing their attraction to the HPV infected tissue might therefore reduce the risk of cervical cancer for women infected with HPV16 by enabling them to clear their chronic infection.

Previous studies using an antibody to cis-urocanic acid and mast-cell-depleted mice implicated both cis-urocanic acid and mast cells in the mechanisms by which ultraviolet B light suppresses systemic contact hypersensitivity responses in mice. In the absence of a direct stimulatory effect of cis-urocanic acid on connective tissue mast cells, an indirect association was investigated. A blister induced in the rat hind footpad was used to examine the effects of slowly perfused cis-urocanic acid on cutaneous blood flow. cis-Urocanic acid but not trans-urocanic acid increased microvascular flow by a mechanism largely dependent on the combined activity of the neuropeptides, substance P and calcitonin gene-related peptide. Perfusion of cis-urocanic acid over the base of blisters induced in sensory-neuropeptide-depleted rats did not have any stimulatory effect above that seen with perfusion of cis-urocanic acid together with neuropeptide receptor antagonists in control rats. There was a small direct effect of cis-urocanic acid on microvascular blood flow. As both substance P and calcitonin gene-related peptide could directly degranulate connective tissue mast cells, this study suggests that cis-urocanic acid indirectly activates mast cells via its effects on peripheral terminals of unmyelinated primary afferent sensory nerves. cis-Urocanic-acid-induced neuropeptides may also contribute to ultraviolet-B-induced cutaneous inflammation and alterations to Langerhans cell activity.

1. The development into tumours of skin cells transformed by ultraviolet (UV) B radiation of wavelengths 290-320 nm is enhanced by the ability of UVB to suppress an immune response that would otherwise destroy them. Ultraviolet B-induced immunomodulation may be by multiple mechanisms, but generally manifests in an antigen-presenting cell defect and an altered cytokine environment in the draining lymph nodes. 2. Immune responses to microbial or self-antigens may be dysfunctional by similar mechanisms following UVB exposure. 3. Earliest-acting intermediates in the initiation of UVB-induced immunosuppression are the UVB absorbers (photoreceptors) of the skin, notably DNA resulting in immunoregulatory cytokine production, and trans-urocanic acid (UCA), which, upon isomerization to its cis isomer, signals downstream immunosuppressive events. 4. In mice, dermal mast cells are critical to UVB-induced systemic immunomodulation. In mice, there is a functional link as well as a linear relationship between the prevalence of histamine-staining dermal mast cells and the log of the dose of UVB required for 50% immunosuppression. Studies with histamine receptor antagonists support histamine as the main' product of mast cells involved. Histamine acts in large part via a prostanoid-dependent pathway. 5. Approximately 50% of humans and greater than 90% of patients with non-melanoma skin cancer are UVB susceptible for suppression of a contact hypersensitivity response. Neither cytokine polymorphisms nor UVB-induced levels of cis-UCA in irradiated skin have been linked to UVB susceptibility. Patients with basal cell carcinomas (BCC) have an increased dermal mast cell prevalence in non-sun-exposed buttock skin. We propose that mast cells function in humans, as in mice, by initiating immunosuppression and, thereby, allowing a permissive environment for BCC development.

Ultraviolet B radiation (280–320 nm) can initiate skin cancer as well as suppress the immune system, thereby preventing the rejection of ultraviolet-B-induced tumors. Recently we reported that there was not only a correlation but also a functional link between dermal mast cell prevalence and susceptibility to ultraviolet-B-induced systemic immunosuppression in multiple strains of mice. In this study, we investigated whether increased dermal mast cell prevalence is a significant predisposing factor for basal cell carcinoma development in humans. In 21 Danes with a history of basal cell carcinoma and 20 control subjects of similar age, sex, skin phototype, and recreational sun exposure over the past 12 mo, dermal mast cell prevalence was quantified on non-sun-exposed buttock skin. We investigated this skin site in order to avoid any changes in mast cell prevalence caused by sun exposure and assumed that the prevalence of mast cells in buttock skin correlated with that at sun-exposed sites at critical times in the development of basal cell carcinomas. Patients with a history of basal cell carcinoma had a significantly higher median dermal mast cell prevalence than control subjects (p = 0.01, Mann–Whitney U). No correlation was observed between dermal mast cell prevalence and age of basal cell carcinoma patients and control subjects. These results suggest that increased dermal mast cell prevalence is a predisposing factor for basal cell carcinoma development in humans. We hypothesize that mast cells function in humans, as in mice, by initiating immunosuppression and thereby allowing a permissive environment for basal cell carcinoma development.

Many studies have implicated cis-urocanic acid (cis-UCA) in UVB-induced immunomodulation. The strongest evidence came from studies in mice whereby a cis-UCA antibody blocked UVB-induced suppression of delayed-type hypersensitivity responses. Furthermore, in several studies, the cis-UCA antibody at least partially reversed UVB suppression of contact hypersensitivity responses. Previous reports suggested that cis-UCA was immunomodulatory through its effects on keratinocytes, Langerhans cells, fibroblasts, T lymphocytes, natural killer cells and monocytes/macrophages. As dermal mast cells were recently demonstrated to be critical to UVB-induced systemic suppression of certain delayed-type and contact hypersensitivity responses, we investigated whether they were involved in the processes by which cis-UCA was immunomodulatory. Not only was there a correlation between dermal mast cell prevalence and the degree of susceptibility of different strains of mice to the immunomodulatory effects of cis-UCA, there was also a functional link. Mast cell-depleted Wf/Wf mice were rendered susceptible to immunomodulation by cis-UCA injected subcutaneously only after their dorsal skin had been reconstituted with bone marrow-derived mast cell precursors. These studies suggest that mast cells are critical to the processes by which cis-UCA suppresses systemic contact hypersensitivity responses to the hapten, trinitrochlorobenzene, in mice.

Differences in dermal mast cell prevalence for adult mice of different strains have been reported previously. In this study, the dermal mast cell prevalence for BALB/c and C57BL/6 mice at 6 weeks of age was similar but as BALB/c mice matured from 6 to 10 weeks of age, their dermal mast cell prevalence halved. In contrast, there was no significant difference in the dermal mast cell prevalence of 6- and 10-week-old C57BL/6 mice. These differences determined the degree of susceptibility of BALB/c and C57BL/6 mice of different ages to UVB (UV radiation of wavelength 280-320 nm)-induced systemic immunosuppression. Expression of the receptor for stem cell factor, Kit protein, was examined on mast cells under conditions in which the dermal mast cell prevalence varied. A significant correlation was observed between Kit expression by mast cells from adult BALB/c, DBA/2 and C57BL/6 mice and dermal mast cell prevalence. In BALB/c mice, mast cell Kit expression decreased as the mice matured from 6 to 10 weeks of age and correlated with the reduction in dermal mast cell numbers. Kit levels on dermal mast cells from C57BL/6 mice were consistently higher than on mast cells from BALB/c mice although significant reductions in Kit were also measured with ageing from 6 to 10 weeks. We hypothesize that regardless of the extent of Kit expression, the dermal mast cell populations were maximally expanded in C57BL/6 mice. We suggest that BALB/c mice of 6 and 10 weeks of age are useful hosts in which to quantitatively evaluate mast cell involvement in a range of functional assays involving skin.