Mast Cell Phenotype Research Articles

Differential mast cell numbers and characteristics in human tuberculosis pulmonary lesions

Tuberculosis (TB) is still a major worldwide health threat and primarily a lung disease. The innate immune response against Mycobacterium tuberculosis (Mtb) is orchestrated by dendritic cells, macrophages, neutrophils, natural killer cells and apparently mast cells (MCs). MCs are located at mucosal sites including the lungs and contribute in host-defence against pathogens, but little is known about their role during Mtb infection. This study investigates the location and characteristics of MCs in TB lesions to assess their contribution to TB pathology. To this purpose, number, location and phenotype of MCs was studied in 11 necropsies of pulmonary TB and 3 necropsies of non-TB infected lungs that were used as controls. MCs were localised at pneumonic areas, in the granuloma periphery and particularly abundant in fibrotic tissue. Furthermore, MCs displayed intracellular Mtb and IL-17A and TGF-β immunostaining. These findings were validated by analysing, post-mortem lung tissue microarrays from 44 individuals with pulmonary TB and 25 control subjects. In affected lungs, increased numbers of MCs expressing intracellularly both tryptase and chymase were found at fibrotic sites. Altogether, our data suggest that MCs are recruited at the inflammatory site and that actively produce immune mediators such as proteases and TGF-β that may be contributing to late fibrosis in TB lesions.

Mast Cells: A New Frontier for Cancer Immunotherapy.

Mast cells are unique tissue-resident immune cells of the myeloid lineage that have long been implicated in the pathogenesis of allergic and autoimmune disorders. More recently, mast cells have been recognized as key orchestrators of anti-tumor immunity, modulators of the cancer stroma, and have also been implicated in cancer cell intrinsic properties. As such, mast cells are an underrecognized but very promising target for cancer immunotherapy. In this review, we discuss the role of mast cells in shaping cancer and its microenvironment, the interaction between mast cells and cancer therapies, and strategies to target mast cells to improve cancer outcomes. Specifically, we address (1) decreasing cell numbers through c-KIT inhibition, (2) modulating mast cell activation and phenotype (through mast cell stabilizers, FcεR1 signaling pathway activators/inhibitors, antibodies targeting inhibitory receptors and ligands, toll like receptor agonists), and (3) altering secreted mast cell mediators and their downstream effects. Finally, we discuss the importance of translational research using patient samples to advance the field of mast cell targeting to optimally improve patient outcomes. As we aim to expand the successes of existing cancer immunotherapies, focused clinical and translational studies targeting mast cells in different cancer contexts are now warranted.

Culturing cells with mast cell phenotype and function: Comparison of peripheral blood and bone marrow as a source.

BackgroundStudies on the mechanisms that govern mast cell (MC) functions are hindered by the difficulties in isolating sufficient numbers of these tissue-resident cells. Therefore, many research groups use cultured human MCs obtained out of progenitor cells. However, these culture methods significantly differ regarding primary source material, culture durations and conditions. Consequently, the finally obtained cells are likely to exhibit morphological, phenotypical and/or functional heterogeneity. ObjectiveTo compare the phenotype and functionality of cells cultured from peripheral blood and bone marrow progenitor cells from patients with suspected clonal MC disease. These cells are designated as PBCMCs and BMCMCs, respectively. MethodsTwenty paired PBCMCs and BMCMCs cultures starting from CD34+ progenitor cells were compared. Cells were cultured for 4 weeks. Phenotyping included Giemsa and CD117 staining and flow cytometric staining for CD117, CD203c, FcεRI, MRGPRX2, CD300a, CD32, CD63 and CD25. Functional assessment included measurement of the up-regulation of CD63 after cross-linking of the high affinity receptor for IgE (FcεRI) with anti-FcεRI and ligation of MRGPRX2 with substance P. ResultsPBCMCs and BMCMCs are phenotypically comparable. Functionally, after activation with anti-FcεRI and substance P, PBCMCs and BMCMCs show similar up-regulation of the lysosomal degranulation marker CD63. However, the yield of PBCMCs is higher than BMCMs and peripheral blood cultures are purer than bone marrow cultures. ConclusionPBCMCs are an attractive alternative to the more difficult to obtain BMCMCs for the exploration of the complex mechanisms that govern IgE- and MRGPRX2-dependent MC activation and degranulation. Unlike BMCMCs, PBCMCs are easily accessible and enable repetitive analyses.

Sesquiterpene-Loaded Co-Polymer Hybrid Nanoparticle Effects on Human Mast Cell Surface Receptor Expression, Granule Contents, and Degranulation.

Biodegradable polymeric nanoparticles (NPs) such as poly(lactic-co-glycolic acid) (PLGA) and polyvinyl alcohol (PVA) have been used as drug delivery systems for natural and synthetic compounds and are designed to control the loading and release of biodegradable materials to target cells, tissues, and organs. Eremophilane-type sesquiterpenes have anti-inflammatory properties but are lipophilic, cytotoxic, and not biocompatible with many cells. To determine whether biodegradable PLGA/PVA could improve the biocompatibility of sesquiterpenes, sesquiterpene-loaded NPs were synthesized and their effects on human mast cells (LAD2), the major effector cells of allergic inflammation, were determined. NPs composed of PLGA/PVA and two types of sesquiterpenes (fukinone, PLGA/PVA-21 and 10βH-8α,12-epidioxyeremophil-7(11)-en-8β-ol, PLGA/PVA-22) were produced using a microfluidic synthesis method. The NPs’ size distribution and morphology were evaluated by dynamic light scattering and cryogenic transmission electron microscopy (TEM). PLGA/PVA-21 and PLGA/PVA-22 were 60 to 70 nm and were readily internalized by LAD2 as shown by flow cytometry, fluorescence microscopy, and TEM. While unencapsulated sesquiterpenes decreased LAD2 cell viability by 20%, PLGA/PVA-21 and PLGA/PVA-22 did not alter LAD2 viability, showing that encapsulation improved the biocompatibility of the sesquiterpenes. PLGA/PVA-21 and PLGA/PVA-22 decreased the expression of genes encoding the subunits of the high affinity immunoglobulin E receptor (FcεR1α, FcεR1β, FcεR1γ) and the stem cell factor receptor (Kit,), suggesting that hybrid NPs could alter mast cell responses to antigens and shift their maturation. Similarly, PLGA/PVA-21 and PLGA/PVA-22 inhibited tryptase expression but had no effect on chymase expression, thereby promoting a shift to the tryptase-positive phenotype (MCT). Lastly, PLGA/PVA-21 and PLGA/PVA-22 inhibited mast cell degranulation when the LAD2 cells were activated by IgE crosslinking and FcεRI. Overall, our results suggest that PLGA/PVA-21 and PLGA/PVA-22 alter human mast cell phenotype and activation without modifying viability, making them a more biocompatible approach than treating cells with sesquiterpenes alone.

Mast Cell-Derived SAMD14 Is a Novel Regulator of the Human Prostate Tumor Microenvironment.

Mast cells (MCs) are important cellular components of the tumor microenvironment and are significantly associated with poor patient outcomes in prostate cancer and other solid cancers. The promotion of tumor progression partly involves heterotypic interactions between MCs and cancer-associated fibroblasts (CAFs), which combine to potentiate a pro-tumor extracellular matrix and promote epithelial cell invasion and migration. Thus far, the interactions between MCs and CAFs remain poorly understood. To identify molecular changes that may alter resident MC function in the prostate tumor microenvironment, we profiled the transcriptome of human prostate MCs isolated from patient-matched non-tumor and tumor-associated regions of fresh radical prostatectomy tissue. Transcriptomic profiling revealed a distinct gene expression profile of MCs isolated from prostate tumor regions, including the downregulation of SAMD14, a putative tumor suppressor gene. Proteomic profiling revealed that overexpression of SAMD14 in HMC-1 altered the secretion of proteins associated with immune regulation and extracellular matrix processes. To assess MC biological function within a model of the prostate tumor microenvironment, HMC-1-SAMD14+ conditioned media was added to co-cultures of primary prostatic CAFs and prostate epithelium. HMC-1-SAMD14+ secretions were shown to reduce the deposition and alignment of matrix produced by CAFs and suppress pro-tumorigenic prostate epithelial morphology. Overall, our data present the first profile of human MCs derived from prostate cancer patient specimens and identifies MC-derived SAMD14 as an important mediator of MC phenotype and function within the prostate tumor microenvironment.

Differential mast cell phenotypes in benign versus cancer tissues and prostate cancer oncologic outcomes

We reported previously that high numbers of mast cells in benign (extra-tumoral) regions of the prostate are associated with worse outcomes after radical prostatectomy including biochemical recurrence and the development of metastases. Herein, with a cohort of 384 men, we performed mast cell subtyping and report that higher minimum number of the tryptase-only (MCT ) subset of extra-tumoral mast cells is associated with increased risk of biochemical recurrence (comparing highest to lowest tertiles: HR 2.32, 95% CI 1.37-3.93; P-trend = 0.002), metastases (HR 3.62, 95% CI 1.75-7.47; P-trend 0.001), and death from prostate cancer (HR 2.87, 95% CI 1.19-6.95; P-trend = 0.02). Preliminary RNA sequencing and comparison of benign versus cancer tissue mast cells revealed differential expression of additional site-specific genes. We further demonstrate that the genes CXCR4 and TFE3 are more highly expressed in tumor-infiltrating mast cells as well as other tumor-infiltrating immune cells and in tumor cells, respectively, and represent an altered tumor microenvironment. KIT variants were also differentially expressed in benign versus cancer tissue mast cells, with KIT variant 1 (GNNK+ ) mast cells identified as more prevalent in extra-tumoral regions of the prostate. Finally, using an established mouse model, we found that mast cells do not infiltrate Hi-Myc tumors, providing a model to specifically examine the role of extra-tumoral mast cells in tumorigenesis. Hi-Myc mice crossed to mast cell knockout (Wsh) mice and aged to 1 year revealed a higher degree of pre-invasive lesions and invasive cancer in wild-type mice versus heterozygous and knockout mice. This suggests a dosage effect where higher numbers of extra-tumoral mast cells resulted in higher cancer invasion. Overall, our studies provide further evidence for a role of extra-tumoral mast cells in driving adverse prostate cancer outcomes. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Protease profile of normal and neoplastic mast cells in the human bone marrow with special emphasis on systemic mastocytosis

Mast cells (MC) are immune cells that produce a variety of mediators, such as proteases, that are important in the body’s immune responses. MC proteases have pronounced multifunctionality and in many respects determine the biological characteristics of the organ-specific MC population. Although, increased numbers of MC are one of the objective mastocytosis signs, a detailed assessment of the proteases biogenesis and excretion mechanisms in the bone marrow (BM) has not yet been carried out. Here, we performed an analysis of the expression of proteases in patients with various forms of systemic mastocytosis. We presented data on intracellular protease co-localization in human BM MCs and discussed their implication in secretory pathways of MCs in the development of the disease. Systemic mastocytosis, depending on the course, is featured by the formation of definite profiles of specific proteases in various forms of atypical mast cells. Intragranular accumulation of tryptase, chymase and carboxypeptidases in the hypochromic phenotype of atypical mast cells is characterized. Characterization of MC proteases expression during mastocytosis can be used to refine the MC classification, help in a prognosis, and increase the effectiveness of targeted therapy.

Dermal Extracellular Matrix-Derived Hydrogels as an In Vitro Substrate to Study Mast Cell Maturation.

Mast cells (MCs) are pro-inflammatory tissue-resident immune cells that play a key role in inflammation. MCs circulate in peripheral blood as progenitors and undergo terminal differentiation in the tissue microenvironment where they can remain for many years. This in situ maturation results in tissue- and species-specific MC phenotypes, culminating in significant variability in response to environmental stimuli. There are many challenges associated with studying mature tissue-derived MCs, particularly in humans. In cases where cultured MCs are able to differentiate in two-dimensional in vitro cultures, there remains an inability for full maturation. Extracellular matrix (ECM) scaffolds provide for a more physiologically relevant environment for cells in vitro and have been shown to modulate the response of other immune cells such as T cells, monocytes, and macrophages. To improve current in vitro testing platforms of MCs and to assess future use of ECM scaffolds for MC regulation, we studied the in vitro response of human MCs cultured on decellularized porcine dermis hydrogels (dermis extracellular matrix hydrogel [dECM-H]). This study investigated the effect of dECM-H on cellular metabolic activity, cell viability, and receptor expression compared to collagen type I hydrogel (Collagen-H). Human MCs showed different metabolic activity when cultured in the dECM-H and also upregulated immunoglobulin E (IgE) receptors associated with MC maturation/activation compared to collagen type I. These results suggest an overall benefit in the long-term culture of human MCs in the dECM-H compared to Collagen-H providing important steps toward a model that is more representative of in vivo conditions. Graphical abstract [Formula: see text] Impact statement Mast cells (MCs) are difficult to culture in vitro as current culture conditions and substrates fail to promote similar phenotypic features observed in vivo. Extracellular matrix (ECM)-based biomaterials offer three-dimensional, tissue-specific environments that more closely resemble in vivo conditions. Our study explores the use of dermal ECM hydrogels for MC culture and shows significant upregulation of metabolic activity, cell viability, and gene expression of markers associated with MC maturation or activation compared to collagen type I-hydrogel and tissue culture plastic controls at 7 days. These results are among the first to describe MC behavior in response to ECM hydrogels.

The impact of TLR7 agonist R848 treatment on mast cell phenotype and activity

Bearing in mind that mast cell contribution to viral clearance is still not fully understood, in this study, we evaluated the effect of Toll-like receptor (TLR)7 viral single-stranded ribonucleic acid (ssRNA) mimic ligand, namely resiquimod (R)848, on mast cell phenotype and activity. We demonstrated that rat peritoneal mast cells exhibit surface and intracellular expression of ssRNA-specific TLR7 molecule, and that mimic ligand switches the self-expression of this receptor. We also detected other proteins associated with the cellular antiviral response: interferon-alpha receptor 1 (IFNAR1), interferon-gamma receptor 1 (IFNGR1), and major histocompatibility complex I (MHC I). Moreover, we showed that R848 caused the decrease of all molecule’s expression after prolonged incubation. Interestingly, we found that R848 induced the increase of high-affinity IgE receptor (FcεRI) expression. Finally, we documented that TLR7 ligand-stimulated mast cells synthesize/release interferon (IFN)-α and -β, tumor necrosis factor (TNF), and chemokines CCL3, CXCL8, as well as pro-inflammatory lipid mediators. Our findings confirm that mast cells may respond to TLR7 ligand by altering their phenotype and synthesizing mediators and could serve as active participants in the antiviral immune response.

Perinatal androgens organize sex differences in mast cells and attenuate anaphylaxis severity into adulthood

Mast cell (MC)-associated diseases, including allergy/anaphylaxis and neuroinflammatory pain disorders, exhibit a sex bias, with females at increase risk. While much attention has been directed toward adult sex hormones as drivers of sex differences, that female sex bias in MC-associated diseases is evident in prepubertal children, suggesting early-life origins of sex differences which have yet to be explored. Utilizing rodent models of MC-mediated anaphylaxis, our data here reveal that, 1) compared with females, males exhibit significantly reduced severity of MC-mediated anaphylactic responses that emerge prior to puberty and persist into adulthood, 2) reduced severity of MC-mediated anaphylaxis in males is linked with the naturally high level of perinatal androgens and can be recapitulated in females by perinatal exposure to testosterone proprionate, 3) perinatal androgen exposure guides bone marrow MC progenitors toward a masculinized tissue MC phenotype characterized by decreased concentration of prestored MC granule mediators (e.g., histamine, serotonin, and proteases) and reduced mediator release upon degranulation, and 4) engraftment of MC-deficient Kit W-sh/W-sh mice with adult male, female, or perinatally androgenized female MCs results in MC-mediated anaphylaxis response that reflects the MC sex and not host sex. Together, these data present evidence that sex differences in MC phenotype and resulting disease severity are established in early life by perinatal androgens. Thus, factors affecting levels of perinatal androgens could have a significant impact on MC development and MC-associated disease risk across the life span.

Modulated MAPK signaling during mast cell differentiation promotes an impaired inflammatory phenotype

Abstract Mast cells are innate immune cells derived from hematopoietic stem cells. Mechanisms controlling IL-3-directed mast cell differentiation are incompletely defined. MAPK contributes to IL-3R-mediated signaling, yet specific contributions to maturation of functional mast cells are undetermined. We aimed to evaluate the role of the MAPK signaling nodes—JNK, ERK, and p38—in mast cell differentiation, and explore contributions to epigenetic modifier expression. Bone marrow-derived mast cell (BMMC) cultures were established from C57BL/6 mice and differentiated for 7 weeks in the presence or absence of MAPK inhibitors (JNK, JNK-IN-8; ERK, SCH772984; p38, GW856553X). Following inhibitor withdrawal (5 days), phenotypic and functional characteristics of IgE-sensitized BMMCs were assessed using flow cytometry, ELISA, and β-hexosaminidase release assays. Cells differentiated under JNK inhibition showed a significant impairment in the secretion of IL-6 (p<0.05) and IL-13 (p<0.05) and a non-significant impairment in TNF and CCL1 24 hrs post allergen challenge. Degranulation was also significantly decreased (by 25%, p<0.05) in JNK-inhibitor-cultured BMMCs. Expression of c-kit and level of sensitization by IgE was unaffected by impaired JNK signaling. Treatment with p38 and ERK inhibitors did not affect receptor expression, cytokine secretion, or degranulation. Interrogation of MAPK-dependent modifiers of histone acetylation by qPCR and western blot showed that JNK-mediated contributions to mast cell functional responses were independent of HDACs and HATs examined. These results position JNK as a critical contributor to normal mast cell differentiation and potential target in modulating mast cell phenotype.

Inhibition of JNK Signaling During Mast Cell Differentiation Promotes Altered Cytokine Secretion and Degranulation Profiles

Mast cells are large granulated leukocytes active in both innate and acquired immunity. These cells differentiate from hematopoietic stem cells, found in the medullary cavity of long trabecular bones. Increased inflammatory activity and numbers of mast cells contribute to allergic disease, which is of increasing concern worldwide. C‐jun N‐terminal kinase (JNK) is a major signaling node in the map kinase pathway that has been shown to contribute to IL‐3 signaling, known to promote murine mast cell differentiation. The aim of this research is to determine the role of JNK signaling in mast cell differentiation. Primary bone marrow‐derived mast cell (BMMC) cultures were established from C57BL/6 mice and differentiated for 7 weeks in the presence or absence of a JNK inhibitor (JNK‐IN‐8). Following removal of the inhibitor for 5 days, phenotypic and functional characteristics of BMMCs were probed using flow cytometry, ELISA, and β‐hexominidase release assays. Flow cytometric analysis of surface expression of the mast cell marker c‐kit, and levels of IgE bound to FcɛRI, a measure of mast cell sensitization, were unaffected by impaired JNK signaling compared to cells grown in the absence of inhibitor over mast cell differentiation, suggesting a normal mast cell phenotype with the potential to be activated via the allergen‐bound IgE‐dependent pathway. Cells differentiated under JNK inhibition showed a significant impairment in the secretion of IL‐6 (p<0.05) and IL‐13 (p<0.05) and a non‐significant impairment in TNF and CCL1 secretion at 24 hrs post‐activation compared to cells grown in the absence of inhibitor. Degranulation was also significantly decreased (by 25%; p<0.05) in BMMCs grown under JNK inhibition. To probe an epigenetic mechanistic role for histone acetylation changes in JNK‐impaired differentiation, expression of HDAC and HAT enzymes were considered by qPCR and western blotting, but were not modulated by JNK. These results suggest that JNK plays an important role in the development of a fully functional mast cell in the context of IgE‐mediated allergic inflammation and may warrant consideration as a strategy to regulate mast cell development in associated pathologies.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada (NSERC); Canada Foundation for Innovation (CFI); Government of Ontario and Brock University.

Beyond IgE: Alternative Mast Cell Activation Across Different Disease States.

Mast cells are often regarded through the lens of IgE-dependent reactions as a cell specialized only for anti-parasitic and type I hypersensitive responses. However, recently many researchers have begun to appreciate the expansive repertoire of stimuli that mast cells can respond to. After the characterization of the interleukin (IL)-33/suppression of tumorigenicity 2 (ST2) axis of mast cell activation—a pathway that is independent of the adaptive immune system—researchers are revisiting other stimuli to induce mast cell activation and/or subsequent degranulation independent of IgE. This discovery also underscores that mast cells act as important mediators in maintaining body wide homeostasis, especially through barrier defense, and can thus be the source of disease as well. Particularly in the gut, inflammatory bowel diseases (Crohn’s disease, ulcerative colitis, etc.) are characterized with enhanced mast cell activity in the context of autoimmune disease. Mast cells show phenotypic differences based on tissue residency, which could manifest as different receptor expression profiles, allowing for unique mast cell responses (both IgE and non-IgE mediated) across varying tissues as well. This variety in receptor expression suggests mast cells respond differently, such as in the gut where immunosuppressive IL-10 stimulates the development of food allergy or in the lungs where transforming growth factor-β1 (TGF-β1) can enhance mast cell IL-6 production. Such differences in receptor expression illustrate the truly diverse effector capabilities of mast cells, and careful consideration must be given toward the phenotype of mast cells observed in vitro. Given mast cells’ ubiquitous tissue presence and their capability to respond to a broad spectrum of non-IgE stimuli, it is expected that mast cells may also contribute to the progression of autoimmune disorders and other disease states such as metastatic cancer through promoting chronic inflammation in the local tissue microenvironment and ultimately polarizing toward a unique Th17 immune response. Furthermore, these interconnected, atypical activation pathways may crosstalk with IgE-mediated signaling differently across disorders such as parasitism, food allergies, and autoimmune disorders of the gut. In this review, we summarize recent research into familiar and novel pathways of mast cells activation and draw connections to clinical human disease.

Generation of a Human Allergic Mast Cell Phenotype from CD133+ Stem Cells.

Cultured human mast cells are a useful tool for research into innate immune responses as well as allergic mechanisms. Mast cells cultured from peripheral blood can provide information on immune mechanisms of known, selected individuals. With the method presented here, eight million mast cells can be cultured from ca. one million stem cells purified from one unit (450mL) of human peripheral blood. Culture with IgE and IL4 optimizes an allergic phenotype of the mast cells.

Mast Cell Biology at Molecular Level: a Comprehensive Review.

Mast cells (MCs) are portions of the innate and adaptive immune system derived from bone marrow (BM) progenitors that are rich in cytoplasmic granules. MC maturation, phenotype, and function are determined by their microenvironment. MCs accumulate at inflammatory sites associated with atopy, wound healing, and malignancies. They interact with the external environment and are predominantly located in close proximity of blood vessels and sensory nerves. MCs are key initiators and modulators of allergic, anaphylactic, and other inflammatory reactions, by induction of vasodilation, promoting of vascular permeability, recruitment of inflammatory cells, facilitation of adaptive immune responses, and modulation of angiogenesis, and fibrosis. They express a wide range of receptors, e.g., for IgE (FcεRI), IgG (FcγR), stem cell factor (SCF) (KIT receptor or CD117), complement (including C5aR), and cytokines, that upon activation trigger various signaling pathways. The final consequence of such ligand receptor-based activation of MCs is the release of a broad array of mediators which are classified in three categories. While some mediators are preformed and remain stored in granules such as heparin, histamine, and enzymes mainly chymase and tryptase, others are de novo synthesized only after activation including LTB4, LTD4, PDG2, and PAF, and the cytokines IL-10, IL-8, IL-5, IL-3, IL-1, GM-CSF, TGF-β, VEGF, and TNF-α. Depending on the stimulus, MCs calibrate their pattern of mediator release, modulate the amplification of allergic inflammation, and are involved in the resolution of the immune responses. Here, we review recent findings and reports that help to understand the MC biology, pathology, and physiology of diseases with MC involvement.

Perinatal androgens drive sex differences in mast cell phenotype and severity of mast cell disease

Abstract Highly prevalent and debilitating mast cell (MC)-associated immune disorders, such as allergy and irritable bowel syndrome exhibit a female sex bias in childhood and adulthood. The mechanisms underlying sex differences in MC disorders are unknown. Previously, we found adult female mice exhibited greater serum histamine and more severe MC-associated pathophysiology that coincided with an increased capacity to synthesize and store immune mediators, compared with males. Further, recent data showed sex differences in the MC exist prior to puberty, suggesting a mechanism independent of adult sex hormones. Here, we investigated the role of perinatal androgens, a key early life component of sexual differentiation, in sex differences in MC phenotype and MC disease. Testosterone proprionate (100 μg sc qd) or vehicle was given to pregnant female mice E16-P0 and directly to pups PN1-7. IgE-mediated anaphylaxis was performed in gonadectomized control male (CM), control female (CF) and perinatally androgenized female (AF) offspring at 8 wk of age. Serum histamine levels (CM 768 ± 106, CF 1406 ± 200, AF 676 ± 110 ng/mL) and hypothermia responses (Δ temp: CM −4.87 ± 0.45, CF − 7.28 ± 0.48, AF − 4.8 ± 0.39 °C) were ~2x lower in AF than CF mice, while AF and CM mice had similar responses. Further, bone marrow MC from AF mice had lower histamine content (AF 1305 ± 82: CF 2082 ± 94 ng/106cells) and 47% lower histamine release to IgE-FcɛRI stimulus compared to CF mice. Together, we demonstrate that sex differences in MC phenotype and disease susceptibility are established early in life by perinatal androgens. These findings provide insight into the mechanisms of sex differences in MC-associated immune disorders that could lead to new therapeutic targets for MC diseases.

Dermal Fibroblasts Control Mast Cell reactivity to commensal bacteria

Abstract Background Our data suggests, and current literature supports, that when mast cells (MCs) leave the blood and interact with dermal fibroblasts (DFs), they assume a bacterial tolerant phenotype in order to prevent unnecessary inflammation during the encounter with the beneficial commensal microbiome. We hypothesize that the mechanisms that induce the MC tolerant phenotype in human skin is due to dermal fibroblasts interactions. Methods We mimicked the “in vivo” setting by conditioning human cord blood MC (hMC) with human dermal fibroblast (hDF) and, then exposed them to commensal bacteria supernatant. Human MC transcriptome data from the RNA-seq FANTOM5 data collection on MCs freshly isolated from human skin (FANTOM5 study Motakis_et_al_2013) was used to investigate the innate immune phenotype of MCs in the skin. We determined the genes that make hDFs unique by comparing their gene expression profiles with lung and cardiac fibroblasts using RNA-seq analysis and challenged hMCs with the products of these genes. Results We have discovered that hDF conditioned with hMCs, show a decreased expression of interleukins upon commensal stimulation; specifically, multiple cytokines/chemokine such as pro-inflammatory cytokine GM-SCF, IL-8, MCP-1 and anti-inflammatory cytokines IL-10, IL-13 are down-regulated by at least 95% and these cytokines can be controlled by the product of hDF unique genes. Conclusion The human dermal MC reactivity is controlled by hDF’s unique genes that play an important role in regulating the hMC NF-Kb pathway.

Increased intratumoral mast cells foster immune suppression and gastric cancer progression through TNF-\u03b1-PD-L1 pathway

BackgroundMast cells are prominent components of solid tumors and exhibit distinct phenotypes in different tumor microenvironments. However, the nature, regulation, function, and clinical relevance of mast cells in human gastric cancer (GC) are presently unknown.MethodsFlow cytometry analyses were performed to examine level and phenotype of mast cells in samples from 114 patients with GC. Multivariate analysis of prognostic factors for overall survival was performed using the Cox proportional hazards model. Kaplan-Meier plots for patient survival were performed using the log-rank test. Mast cells, T cells and tumor cells were isolated or generated, stimulated and/or cultured for in vitro and in vivo function assays.ResultsPatients with GC showed a significantly higher mast cell infiltration in tumors. Mast cell levels increased with tumor progression and independently predicted reduced overall survival. These tumor-infiltrating mast cells accumulated in tumors by CXCL12-CXCR4 chemotaxis. Intratumoral mast cells expressed higher immunosuppressive molecule programmed death-ligand 1 (PD-L1), and mast cells induced by tumors strongly express PD-L1 proteins in both time-dependent and dose-dependent manners. Significant correlations were found between the levels of PD-L1+ mast cells and pro-inflammatory cytokine TNF-α in GC tumors, and tumor-derived TNF-α activated NF-κB signaling pathway to induce mast cell expression of PD-L1. The tumor-infiltrating and tumor-conditioned mast cells effectively suppressed normal T-cell immunity through PD-L1 in vitro, and tumor-conditioned mast cells contributed to the suppression of T-cell immunity and the growth of human GC tumors in vivo; the effect could be reversed by blocking PD-L1 on these mast cells.ConclusionThus, our results illuminate novel immunosuppressive and protumorigenic roles of mast cells in GC, and also present a novel mechanism in which PD-L1 expressing mast cells link the proinflammatory response to immune tolerance in the GC tumor milieu.

Insulin-induced lipid body accumulation is accompanied by lipid remodelling in model mast cells

ABSTRACT Mast cell lipid bodies are key to initiation, maintenance and resolution of inflammatory responses in tissue. Mast cell lines, primary bone marrow-derived mast cells and peripheral blood basophils present a ‘steatotic’ phenotype in response to chronic insulin exposure, where cells become loaded with lipid bodies. Here we show this state is associated with reduced histamine release, but increased capacity to release bioactive lipids. We describe the overall lipid phenotype of mast cells in this insulin-induced steatotic state and the consequences for critical cellular lipid classes involved in stages of inflammation. We show significant insulin-induced shifts in specific lipid classes, especially arachidonic acid derivatives, MUFA and PUFA, the EPA/DHA ratio, and in cardiolipins, especially those conjugated to certain DHA and EPAs. Functionally, insulin exposure markedly alters the FcεRI-induced release of Series 4 leukotriene LTC4, Series 2 prostaglandin PGD2, Resolvin-D1, Resolvin-D2 and Resolvin-1, reflecting the expanded precursor pools and impact on both the pro-inflammation and pro-resolution bioactive lipids that are released during mast cell activation. Chronic hyperinsulinemia is a feature of obesity and progression to Type 2 Diabetes, these data suggest that mast cell release of key lipid mediators is altered in patients with metabolic syndrome.

Aged human skin accumulates mast cells with altered functionality that localize to macrophages and vasoactive intestinal peptide-positive nerve fibres.

SummaryBackgroundSkin health declines with age and this is partially attributed to immunosenescence. Mast cells (MCs) are innate immune cells that coordinate tissue immune responses integral to skin homeostasis and disease.ObjectivesTo understand how MCs contribute to human skin ageing, we investigated how intrinsic ageing impacts MC phenotype and MC relationships with other immune cells and skin structures.MethodsIn photoprotected skin biopsies from young (≤ 30 years) and aged (≥ 75 years) individuals, immunostaining and spatial morphometry were performed to identify changes in MC phenotype, number, distribution and interaction with the vasculature and nerve fibres. Quantitative polymerase chain reaction was used to measure changes in gene expression related to immune cell activity and neuropeptide signalling.ResultsSkin MCs, macrophages and CD8+ T cells increased in number in intrinsically aged vs. young skin by 40%, 44% and 90%, respectively (P < 0·05), while CD4+ T cells and neutrophils were unchanged. In aged skin, MCs were more numerous in the papillary dermis and showed a reduced incidence of degranulation (50% lower than in young, P < 0·01), a conserved tryptase–chymase phenotype and coexpression of granzyme B. In aged skin, MCs increased their association with macrophages (~ 48% vs. ~27%, P < 0·05) and nerve fibres (~29% vs. 16%, P < 0·001), while reducing their interactions with blood vessels (~34% vs. 45%, P < 0·001). Additionally, we observed modulation of gene expression of vasoactive intestinal peptide (VIP; increased) and substance P (decreased) with age; this was associated with an increased frequency of VIP + nerve fibres (around three times higher in aged skin, P < 0·05), which were strongly associated with MCs (~19% in aged vs. 8% in young, P < 0·05).ConclusionsIn photoprotected skin we observed an accumulation of MCs with increasing age. These MCs have both altered functionality and distribution within the skin, which supports a role for these cells in altered tissue homeostasis during ageing.