Mast Cell Reconstitution Research Articles

Stem cell factor/mast cell/CCL2/monocyte/macrophage axis promotes Coxsackievirus B3 myocarditis and cardiac fibrosis by increasing Ly6Chigh monocyte influx and fibrogenic mediators production.

Mast cells (MCs), central players in allergy and parasitic infections, play key roles in inflammation and fibrosis. Here, the impact of MCs on the progression of Coxsackievirus B3 (CVB3)-induced viral myocarditis (VMC) and fibrosis was investigated using MC-deficient KitW-sh mice. Viral titres, cellular infiltrates and heart pathologies were evaluated and compared with wild-type (WT) mice during acute CVB3 infection of C57BL/6 mice. CVB3 infection induced an increased accumulation and degranulation of MCs in the hearts of mice during acute infection. MC-deficient KitW-sh mice had slightly higher viral titres, decreased VMC and cardiac fibrosis and improved cardiac dysfunction compared to WT mice via decreasing cardiac influx of Ly6Chigh monocytes/macrophages (Mo/Mφ). While bone marrow-derived MC reconstitution decreased viral titre and worsened improved survival and VMC severity in Wsh mice. MC-fibroblasts co-culture revealed a cardiac MC-fibroblasts crosstalk during early infection: fibroblasts trigger MC degranulation and secretion of CCL2 and tumour necrosis factor alpha (TNF-α) via producing early stem cell factor (SCF); while MCs-fibrogenic mediators (TNF-α) stimulate fibroblasts to increase CCL2, α-smooth muscle actin (SMA), collagen and transforming growth factor beta（TGFβ)expression, thus aggravating cardiac fibrosis. MCs and fibroblast-derived CCL2s are both essential for cardiac Ly6Chigh Mo/Mφ influx. Administration of recombinant mouse SCF to CVB3-infected mice aggravates VMC via accelerating MCs accumulation and cardiac influx of Ly6Chi Mo/Mφ. Collectively, our data highlight an early MC-fibroblast crosstalk and SCF/MC/CCL2/Mo/Mφ axis as important mechanisms required for triggering VMC and myocardial fibrosis. This finding indicates critical roles of MCs in initiating and modulating cardiac innate response to CVB3 and has an implication in developing new and more effective treatments for VMC.

Mucosal Serotonin Reuptake Transporter Expression in Irritable Bowel Syndrome Is Modulated by Gut Microbiota Via Mast Cell–Prostaglandin E2

Increased colonic serotonin (5-HT) level and decreased serotonin reuptake transporter (SERT) expression in irritable bowel syndrome (IBS) may contribute to diarrhea and visceral hypersensitivity. We investigated whether mucosal SERT is modulated by gut microbiota via a mast cell-prostaglandin E2 (PGE2) pathway. C57Bl/6 mice received intracolonic infusion of fecal supernatant (FS) from healthy controls or patients with diarrhea-predominant irritable bowel syndrome (IBS-D). The role of mast cells was studied in mast cell-deficient mice. Colonic organoids and/or mast cells were used for invitro experiments. SERT expression was measured by quantitative polymerase chain reaction and Western blot. Visceromotor responses tocolorectal distension and colonic transit were assessed. Intracolonic infusion of IBS-D FS in mice caused an increase in mucosal 5-HT compared with healthy control FS, accompanied by ∼50% reduction in SERT expression. Mast cell stabilizers, cyclooxygenase-2 inhibitors, and PGE2 receptor antagonist prevented SERT downregulation. Intracolonic infusion of IBS-D FS failed to reduce SERT expression in mast cell-deficient (W/Wv) mice. This response was restored by mast cell reconstitution. The downregulation of SERT expression evoked by IBS FS was prevented by lipopolysaccharide (LPS) antagonist LPS from Rhodobacter sphaeroides and a bacterial trypsin inhibitor. Invitro LPS treatment caused increased cyclooxygenase-2 expression and PGE2 release from cultured mouse mast cells. Intracolonic infusion of IBS-D FS in mice reduced colonic transit, increased fecal water content, and increased visceromotor responses to colorectal distension. Ondansetron prevented these changes. Fecal LPS acting in concert with trypsin in patients with IBS-D stimulates mucosal mast cells to release PGE2, which downregulates mucosal SERT, resulting in increased mucosal 5-HT. This may contribute to diarrhea and abdominal pain common in IBS.

High FODMAP diet causes barrier loss via lipopolysaccharide-mediated mast cell activation.

Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) are carbohydrates thought to contribute to the symptoms of IBS. A diet in high in FODMAPs (HFM) induces gastrointestinal symptoms in patients with irritable bowel syndrome (IBS), and a diet low in FODMAPs (LFM) improves symptoms in up to 60% of patients with IBS. However, the mechanism by which FODMAPs affect IBS symptoms is unclear. We showed that mice fed on a HFM diet have mast cell activation and colonic barrier loss. Using mast cell–deficient mice with and without mast cell reconstitution, we showed that HFM-mediated colonic barrier loss is dependent on TLR4-dependent mast cell activation. In in vitro studies, we demonstrated that IBS fecal supernatant stimulates mast cells significantly more compared with fecal supernatant from healthy controls. This effect of IBS fecal supernatant on mast cell stimulation is ameliorated in the absence of the TLR4 receptor and after a LFM diet. We found that a LFM diet improves colonic barrier function and reduces mast cell activation while decreasing fecal LPS levels. Our findings indicate that a HFM diet causes mast cell activation via LPS, which in turn leads to colonic barrier loss, and a LFM diet reverses these pathophysiologic mucosal changes.

Immunoglobulin E Mediates Pulmonary Vascular Remodeling by Upregulating Mast Cell Secretion of VEGFC

Pulmonary vascular remodeling (PVR) is a hallmark pathological change of pulmonary hypertension (PH) and plays a very important role in the formation and development of PH. However, no effective therapy has been developed to date to reverse PVR. The present study evaluated the expression and function of immunoglobulin E (IgE) on experimental PH models. Single-cell RNA sequencing on lung tissues from mice subjected to hypoxia-induced PH revealed the existence of active B cells in the lung. A significant increase in IgE expression was observed in the lung tissues of the experimental PH model and PAH patients. Treatment with IgE neutralizing antibodies attenuated hypoxia-induced PH and pulmonary vascular remodeling in mice. VEGFC derived from IgE-stimulated mast cells promoted the proliferation and migration of pulmonary arterial endothelial cells (PAECs) as well as endothelial to mesenchymal transition, and aggravated hypoxia-induced PH in a mast cell-reconstitution model. IgE levels are elevated in PH and promote pulmonary vascular remodeling by VEGFC derived from activated mast cells. Funding Information: This work was supported by Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences [grant number: 2018-12M-1-001 toC.W.]; the National Natural Science Foundation of China [grant numbers:81622008, 91739107 to J.W.]; the National Natural Science Foundation of China [grant numbers:82000063 to Y.L.]; Thousand Young Talents Program of China to J.W.; the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences [grant number:2018JB31001 to C.W.] and State Key Laboratory Special Fund [grant number: 2060204]. Declaration of Interests: The authors have declared no conflicts of interest Ethics Approval Statement: All participants provided written informed consent for the use of the lung tissue for research before enrollment (Approval NO.:2017-877). The study was approved bythe Institutional Ethics Committee of Peking Union Medical College, and conducted according to the Declaration of Helsinki principles. All animal experiments were performed following the protocols approved by the Animal Research Committee of Institute of Laboratory Animals, Chinese Academy of Medical Sciences and Peking Union Medical College (ACUC-A01-2018-005).

Skin Mast Cell-Driven Ceramides Drive Early Apoptosis in Pre-Symptomatic Eczema in Mice.

Atopic dermatitis (AD or eczema) is the most common chronic inflammatory skin disorder worldwide. Ceramides (Cer) maintain skin barrier functions, which are disrupted in lesional skin of AD patients. However, Cer status during the pre-lesional phase of AD is not well defined. Using a variation of human AD-like preclinical model consisting of a 7-day topical exposure to ovalbumin (OVA), or control, we observed elevation of Cer C16 and C24. Skin mRNA quantification of enzymes involved in Cer metabolism [Cer synthases (CerS) and ceramidases (Asah1/Asah2)], which revealed augmented CerS 4, 5 and 6 and Asah1. Given the overall pro-apoptotic nature of Cer, local apoptosis was assessed, then quantified using novel morphometric measurements of cleaved caspase (Casp)-3-restricted immunofluorescence signal in skin samples. Apoptosis was induced in response to OVA. Because apoptosis may occur downstream of endoplasmic reticulum (ER) stress, we measured markers of ER stress-induced apoptosis and found elevated skin-associated CHOP protein upon OVA treatment. We previously substantiated the importance of mast cells (MC) in initiating early skin inflammation. OVA-induced Cer increase and local apoptosis were prevented in MC-deficient mice; however, they were restored following MC reconstitution. We propose that the MC/Cer axis is an essential pathogenic feature of pre-lesional AD, whose targeting may prevent disease development.

Prostaglandin E2, Produced by Mast Cells in Colon Tissues From Patients With Irritable Bowel Syndrome, Contributes to Visceral Hypersensitivity in Mice

Visceral hypersensitivity is common in patients with irritable bowel syndrome (IBS). We investigated whether inflammatory molecules, such as histamine and proteases, activate prostaglandin-endoperoxide synthase 2 (also called COX2) to increase the synthesis of prostaglandin E2 (PGE2) by mast cells, which activates the receptor PTGER2 (also called EP2) in the dorsal root ganglia to promote visceral hypersensitivity. We used an enzyme-linked immunosorbent assay to measure levels of spontaneous release of molecules from mast cells in colonic mucosa from patients with IBS with diarrhea (IBS-D; 18 women and 5 men; aged 28-60 years), healthy individuals (controls, n= 24), mice, and rats. We measured visceromotor responses to colorectal distension in rodents after intracolonic administration of colon biopsy supernatants, histamine, PGE2, a small interfering RNA against EP2, or an agonist of F2R like trypsin receptor 1 (F2RL1, also called protease-activated receptor 2 [PAR2]). We investigated the role of COX2, produced by mast cells, in mediation of visceral hypersensitivity using mice with the Y385F substitution in Ptgs2 (Ptgs2Y385F mice), mast cell-deficient (W/WV) mice, and W/WV mice given injections of mast cells derived from wild-type or Ptgs2Y385F mice. Colon biopsies from patients with IBS-D had increased levels of PGE2, based on enzyme-linked immunosorbent assay, and COX2 messenger RNA and protein, compared with control biopsies. Immunohistochemistry showed that most of the COX2 was in mast cells. Intracolonic infusions of rats with IBS-D biopsy supernatants generated a 3- to 4-fold increase in visceromotor responses to colorectal distension; this was associated with significant increases in PGE2, histamine, and tryptase in the colonic mucosa. These increases were prevented by a mast cell stabilizer, COX2 inhibitor, or knockdown of EP2. Intracolonic administration of supernatants from biopsies of patients with IBS-D failed to induce visceral hypersensitivity or increase the level of PGE2 in W/WV and Ptgs2Y385Fmice. Reconstitution of mast cells in W/WV mice restored the visceral hypersensitivity response. Abnormal synthesis of PGE2 by colonic mast cells appears to induce visceral hypersensitivity in patients with IBS-D.

A role for glia maturation factor dependent activation of mast cells and microglia in MPTP induced dopamine loss and behavioural deficits in mice

The molecular mechanism mediating degeneration of nigrostriatal dopaminergic neurons in Parkinson’s disease (PD) is not yet fully understood. Previously, we have shown the contribution of glia maturation factor (GMF), a proinflammatory protein in dopaminergic neurodegeneration mediated by activation of mast cells (MCs). In this study, methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal neurodegeneration and astro-glial activations were determined by western blot and immunofluorescence techniques in wild type (WT) mice, MC-deficient (MC-KO) mice and GMF-deficient (GMF-KO) mice, with or without MC reconstitution before MPTP administration. We show that GMF-KO in the MCs reduces the synergistic effects of MC and Calpain1 (calcium-activated cysteine protease enzyme)-dependent dopaminergic neuronal loss that reduces motor behavioral impairments in MPTP-treated mouse. Administration of MPTP increase in calpain-mediated proteolysis in nigral dopaminergic neurons further resulting in motor decline in mice. We found that MPTP administered WT mice exhibits oxidative stress due to significant increases in the levels of malondialdehyde, superoxide dismutase and reduction in the levels of reduced glutathione and glutathione peroxidase activity as compared with both MC-KO and GMF-KO mice. The number of TH-positive neurons in the ventral tegmental area, substantia nigra and the fibers in the striatum were significantly reduced while granulocyte macrophage colony-stimulating factor (GM-CSF), MC-Tryptase, GFAP, IBA1, Calpain1 and intracellular adhesion molecule 1 expression were significantly increased in WT mice. Similarly, tyrosine hydroxylase, dopamine transporters and vesicular monoamine transporters 2 proteins expression were significantly reduced in the SN of MPTP treated WT mice. The motor behavior as analyzed by rotarod and hang test was significantly reduced in WT mice as compared with both the MC-KO and GMF-KO mice. We conclude that GMF-dependent MC activation enhances the detrimental effect of astro-glial activation-mediated oxidative stress and neuroinflammation in the midbrain, and its inhibition may slowdown the progression of PD.

Functional Inactivation of Mast Cells Enhances Subcutaneous Adipose Tissue Browning in Mice

Adipose tissue browning and systemic energy expenditure provide a defense mechanism against obesity and associated metabolic diseases. In high-cholesterol Western diet-fed mice, mast cell (MC) inactivation ameliorates obesity and insulin resistance and improves the metabolic rate, but a direct role of adipose tissue MCs in thermogenesis and browning remains unproven. Here, we report that adrenoceptor agonist norepinephrine-stimulated metabolic rate and subcutaneous adipose tissue (SAT) browning are enhanced in MC-deficient Kitw-sh/w-sh mice and MC-stabilized wild-type mice on a chow diet. MC reconstitution to SAT in Kitw-sh/w-sh mice blocks these changes. Mechanistic studies demonstrate that MC inactivation elevates SAT platelet-derived growth factor receptor A (PDGFRα+) adipocyte precursor proliferation and accelerates beige adipocyte differentiation. Using the tryptophan hydroxylase 1 (TPH1) inhibitor and TPH1-deficient MCs, we show that MC-derived serotonin inhibits SAT browning and systemic energy expenditure. Functional inactivation of MCs or inhibition of MC serotonin synthesis in SAT promotes adipocyte browning and systemic energy metabolism in mice.

263-LB: Functional Inactivation of Mast Cell Enhances Subcutaneous Adipose Tissue Browning through Decreasing Serotonin Release in Mice

Introduction: Therapeutic targeting of brown adipocyte-mediated thermogenesis to increase energy expenditure may offer a viable alternative approach to combat obesity and metabolic disease. Several immune signaling regulated beige fat thermogenic circuits were established in adipose tissues. Genetic deficiency and pharmacological stabilization of Mast Cells (MCs) increased metabolic rate and UCP1 expression in BAT in western diet-fed mice in concert with their obesity and insulin resistance improvement. Serotonin is an important regulator of energy balance. Yet, it remains unknown whether MCs play a direct role in energy homeostasis or the above-mentioned changes in energy expenditure are merely a consequence of mouse body weight reduction. Methods and Results: We compared metabolic rate differences between WT and MC-deficient Kitw-sh/w-sh mice. In the context of mice on a chew diet, norepinephrine (NE)-stimulated metabolic rate is increased in MC-deficient Kitw-sh/w-sh mice and MC-stabilized wide-type (WT) mice. Histological staining, qPCR and western blot showed UCP1 and other thermogenic genes also increased in these mice. Such functional inactivation of MCs enhances thermogenesis and browning in subcutaneous adipose tissues (SAT), but not in brown (BAT) and epididymal adipose tissues (EAT). MC reconstitution to SAT blocks the aforesaid changes in Kitw-sh/w-sh mice. Mechanistic studies demonstrate that functional inactivation of MCs not only elevates the numbers and proliferation of PDGFRα+ bipotential adipocyte precursors but also accelerates beige adipocyte differentiation in SAT. Using tryptophan hydroxylase 1 inhibitor and Tph1-/- mast cells, we show that MC-derived serotonin inhibits SAT beige adipocyte biogenesis and systemic energy expenditure. Conclusions: Together, functional inactivation of MCs or inhibition of MC serotonin synthesis in SAT promotes adipocyte browning and systemic energy metabolism in mice. Disclosure X. Zhang: None. Funding American Heart Association

Mast Cells Augment Neuroinflammation and Neurodegeneration

Activated glial cells, and neurons express several proinflammatory mediators that are implicated in neuroinflammation and neurodegeneration in neurodegenerative diseases including Parkinson's disease (PD), Alzheimer's disease (AD) and Multiple sclerosis (MS). Mast cells are immune and inflammatory cells that are implicated in neuroinflammation by releasing several prestored and newly synthesized multifunctional cytokines, chemokines and neurotoxic mediators. Protease activated receptor‐2 (PAR‐2) expression is increased in neuroinflammatory conditions. PD is characterized by neurodegeneration of dopaminergic neurons in the brain. Here, we have investigated how mast cells can contribute to neuroinflammation, and to the pathogenesis of PD. We demonstrate that inflammatory mediators such as interleukin‐1beta (IL‐1β), tumor necrosis factor‐alpha (TNF‐α) and the chemokine (C‐C motif) ligand 2 (CCL2) were lower in the brains of mast cell deficient (MC‐KO) mice and glia maturation factor‐deficient (GMF‐KO) mice when compared to wild type mice brains after acute 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) administration. Further, we found that reconstitution of mast cells in MC‐KO mice increases neuroinflammation in the MPTP‐injected mice brains as compared to the MC‐KO brains without mast cell reconstitution. Our immunofluorescence staining revealed that the PAR‐2 expression is increased in MPTP‐injected mice brains as compared to control wild type mice brains without MPTP administration. We also show significantly increased PAR‐2 expression in the cultured primary mouse astrocytes and neurons incubated with 1‐methyl‐4‐phenylpyridinium (MPP+), inflammatory protein GMF, mouse mast cell protease‐6 (MMCP‐6), and MMCP‐7. We conclude that mast cells augment neuroinflammation and neurodegeneration in neurodegenerative diseases including PD and inhibition of mast cell activation could be a therapeutic option in neuroinflammatory and neurodegenerative disorders.Support or Funding InformationThis work was supported by Veteran Affairs Merit Award I01BX002477 and National Institutes of Health Grants AG048205 & NS073670 to Prof. Asgar ZaheerThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mast Cells in Cardiac Fibrosis: New Insights Suggest Opportunities for Intervention.

Mast cells (MC) are innate immune cells present in virtually all body tissues with key roles in allergic disease and host defense. MCs recognize damage-associated molecular patterns (DAMPs) through expression of multiple receptors including Toll-like receptors and the IL-33 receptor ST2. MCs can be activated to degranulate and release pre-formed mediators, to synthesize and secrete cytokines and chemokines without degranulation, and/or to produce lipid mediators. MC numbers are generally increased at sites of fibrosis. They are potent, resident, effector cells producing mediators that regulate the fibrotic process. The nature of the secretory products produced by MCs depend on micro-environmental signals and can be both pro- and anti-fibrotic. MCs have been repeatedly implicated in the pathogenesis of cardiac fibrosis and in angiogenic responses in hypoxic tissues, but these findings are controversial. Several rodent studies have indicated a protective role for MCs. MC-deficient mice have been reported to have poorer outcomes after coronary artery ligation and increased cardiac function upon MC reconstitution. In contrast, MCs have also been implicated as key drivers of fibrosis. MC stabilization during a hypertensive rat model and an atrial fibrillation mouse model rescued associated fibrosis. Discrepancies in the literature could be related to problems with mouse models of MC deficiency. To further complicate the issue, mice generally have a much lower density of MCs in their cardiac tissue than humans, and as such comparing MC deficient and MC containing mouse models is not necessarily reflective of the role of MCs in human disease. In this review, we will evaluate the literature regarding the role of MCs in cardiac fibrosis with an emphasis on what is known about MC biology, in this context. MCs have been well-studied in allergic disease and multiple pharmacological tools are available to regulate their function. We will identify potential opportunities to manipulate human MC function and the impact of their mediators with a view to preventing or reducing harmful fibrosis. Important therapeutic opportunities could arise from increased understanding of the impact of such potent, resident immune cells, with the ability to profoundly alter long term fibrotic processes.

Frontline Science: Mast cells regulate neutrophil homeostasis by influencing macrophage clearance activity.

The receptor tyrosine kinase cKit and its ligand stem cell factor are essential for mast cells (MC) development and survival. Strains with mutations affecting the Kit gene display a profound MC deficiency in all tissues and have been extensively used to investigate the role of MC in both physiologic and pathologic conditions. However, these mice present a variety of abnormalities in other immune cell populations that can affect the interpretation of MC-related responses. C57BL/6 KitW-sh are characterized by an aberrant extramedullary myelopoiesis and systemic neutrophilia. MC deficiency in KitW-sh mice can be selectively repaired by engraftment with in vitro-differentiated MC to validate MC-specific functions. Nevertheless, the impact of MC reconstitution on other immune populations has never been evaluated in detail. Here, we specifically investigated the neutrophil compartment in primary and secondary lymphoid organs of C57BL/6 KitW-sh mice before and after MC reconstitution. We found that, albeit not apparently affecting neutrophils phenotype or maturation, MC reconstitution of KitW-sh mice restored the number of neutrophils at a level similar to that of wild-type C57BL/6 mice. In vitro and ex vivo experiments indicated that MC can influence neutrophil clearance by increasing macrophages' phagocytic activity. Furthermore, the G-CSF/IL-17 axis was also influenced by the presence or absence of MC in KitW-sh mice. These data suggest that MC play a role in the control of neutrophil homeostasis and that this aspect should be taken into account in the interpretation of results obtained using KitW-sh mice.

Mouse Bone Marrow-Derived Mast Cells Induce Angiogenesis by Tissue Engineering in Rats: Histological Evidence.

Objective Therapeutic angiogenesis is employed to induce vascular network formation and improve functional recovery in ischemia. The aim of this study is to find an appropriate method to recover local ischemic conditions. Materials and Methods In this experimental survey, 20 male Wistar rats weighing approximately 200-250 g were randomly divided into four experimental groups respectively: ischemia group in which the femoral artery was transected; phosphate buffer solution group (PBS) in which the femoral artery transected location was immersed with PBS; chitosan (CHIT) group in which the transected location was immersed in a 50 µL CHIT solution; and mast cell transplanted group in which the transected location was immersed with a mixture of 50 µL CHIT and 50 µL PBS that contained 1×106 mast cells. Results On day 14 after surgery, mean numbers of blood vessels of different sizes in the CHIT/mast cell group significantly increased compared to the other experimental groups (P<0.05). ConclusionOur data suggest that mast cell reconstitution could offer a new approach for therapeutic angiogenesis in cases of peripheral arterial diseases.

Perivascular mast cells promote neointimal elastin deposition and suppress chronic vein graft restenosis in hyperlipidaemic mice

Aims: Mast cells are versatile innate immune cells and are reported to promote vascular inflammation and neointimal lesion formation, thereby contributing to the development of vascular stenosis and atherosclerosis. However, it is not clear whether mast cells also regulate vascular matrix remodelling in established neointima. This study addressed the hypothesis that perivascular mast cells regulate neointimal matrix remodelling using a mouse vein graft model. Methods: The impact of mast cells on neointimal remodelling was investigated using mast cell-deficient animals in both normolipidaemic (KitW-sh/W-sh) and hyperlipidaemic (apoE-/-KitW-sh/W-sh) conditions. The effect of perivascular mast cells on vascular matrix remodelling, including collagen and elastin deposition, was investigated using a local mast cell reconstitution method that selectively repopulated mast cells around the carotid artery (where the vein graft was inserted) in KitW-sh/W-sh mice. Results: In normolipidaemic vein grafts (KitW-sh/W-sh vs. the wild type control C57BL/6J), collagen synthesis was not affected by mast cell deficiency at 4 weeks. In contrast, neointimal elastin was reduced by 6.5-fold in mast cell-deficient KitW-sh/W-sh mice, which was prevented by perivascular mast cell reconstitution. Mast cell deficiency induced a similar reduction in neointimal elastin in hyperlipidaemic mice (apoE-/-KitW-sh/W-sh vs. apoE-/-), with a significant increase in cell proliferation and neointimal area at 4 week. Conclusion: Mast cells appear to promote elastin deposition in vein grafts and this may lead to further suppression of cell proliferation and neointimal thickening under hyperlipidaemic conditions.

Abstract 377: Mast Cell Serotonin Controls Subcutaneous Adipose Tissue Browning and Systemic Energy Expenditure in Mice

Adipose tissue browning and systemic energy expenditure provide a defense mechanism against obesity and associated metabolic diseases. In western diet-fed mice, inactivation of mast cells (MCs) ameliorates obesity and insulin resistance along with improved metabolic rate. Yet, a direct role of MCs in adipose tissue thermogenesis and browning remains unclear. Here we report that, in the context of mice on a chew diet, norepinephrine (NE)-stimulated metabolic rate is increased in MC-deficient Kit w-sh/w-sh mice and MC-stabilized wide-type (WT) mice. Such functional inactivation of MCs enhances thermogenesis and browning in subcutaneous adipose tissues (SAT), but not in brown (BAT) and epididymal adipose tissues (EAT). MC reconstitution to SAT blocks the aforesaid changes in Kit w-sh/w-sh mice. Mechanistic studies demonstrate that functional inactivation of MCs not only elevates the numbers of PDGFRα + bipotential adipocyte precursors but also accelerates beige adipocyte differentiation in SAT. Using tryptophan hydroxylase 1 inhibitor, we show that MC-derived serotonin inhibits SAT beige adipocyte biogenesis and systemic energy expenditure. Together, functional inactivation of MCs or inhibition of MC serotonin synthesis in SAT promotes adipocyte browning and systemic energy metabolism in mice.

Evidence for the Role of Mast Cells in Cystitis-Associated Lower Urinary Tract Dysfunction: A Multidisciplinary Approach to the Study of Chronic Pelvic Pain Research Network Animal Model Study.

Bladder inflammation frequently causes cystitis pain and lower urinary tract dysfunction (LUTD) such as urinary frequency and urgency. Although mast cells have been identified to play a critical role in bladder inflammation and pain, the role of mast cells in cystitis-associated LUTD has not been demonstrated. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain and LUTD. In this study we investigated the role of mast cells in LUTD using a transgenic autoimmune cystitis model (URO-OVA) that reproduces many clinical correlates of IC/BPS. URO-OVA mice express the membrane form of the model antigen ovalbumin (OVA) as a self-antigen on the urothelium and develop bladder inflammation upon introduction of OVA-specific T cells. To investigate the role of mast cells, we crossed URO-OVA mice with mast cell-deficient KitW-sh mice to generate URO-OVA/KitW-sh mice that retained urothelial OVA expression but lacked endogenous mast cells. We compared URO-OVA mice with URO-OVA/KitW-sh mice with and without mast cell reconstitution in response to cystitis induction. URO-OVA mice developed profound bladder inflammation with increased mast cell counts and LUTD, including increased total number of voids, decreased mean volume voided per micturition, and decreased maximum volume voided per micturition, after cystitis induction. In contrast, similarly cystitis-induced URO-OVA/KitW-sh mice developed reduced bladder inflammation with no mast cells and LUTD detected. However, after mast cell reconstitution URO-OVA/KitW-sh mice restored the ability to develop bladder inflammation and LUTD following cystitis induction. We further treated URO-OVA mice with cromolyn, a mast cell membrane stabilizer, and found that cromolyn treatment reversed bladder inflammation and LUTD in the animal model. Our results provide direct evidence for the role of mast cells in cystitis-associated LUTD, supporting the use of mast cell inhibitors for treatment of certain forms of IC/BPS.

Meningeal mast cell-T cell crosstalk regulates T cell encephalitogenicity

GM-CSF is a cytokine produced by T helper (Th) cells that plays an essential role in orchestrating neuroinflammation in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. Yet where and how Th cells acquire GM-CSF expression is unknown. In this study we identify mast cells in the meninges, tripartite tissues surrounding the brain and spinal cord, as important contributors to antigen-specific Th cell accumulation and GM-CSF expression. In the absence of mast cells, Th cells do not accumulate in the meninges nor produce GM-CSF. Mast cell-T cell co-culture experiments and selective mast cell reconstitution of the meninges of mast cell-deficient mice reveal that resident meningeal mast cells are an early source of caspase-1-dependent IL-1β that licenses Th cells to produce GM-CSF and become encephalitogenic. We also provide evidence of mast cell-T cell co-localization in the meninges and CNS of recently diagnosed acute MS patients indicating similar interactions may occur in human demyelinating disease.

Subcutaneous Allergic Sensitization to Protease Allergen Is Dependent on Mast Cells but Not IL-33: Distinct Mechanisms between Subcutaneous and Intranasal Routes.

Protease activity of papain, a plant-derived occupational allergen homologous to mite major allergens, is essential to IgE/IgG1 production and lung eosinophilia induced by intranasal papain administration in mice, and IL-33 contributes to these responses. In this work, we investigate skin and Ab responses induced by s.c. papain administration into ear lobes and responses induced by subsequent airway challenge with papain. Subcutaneous papain injection induced swelling associated with increased epidermal thickness, dermal inflammation, serum IgE/IgG1 responses, and Th2 cytokine production in draining lymph node cells restimulated in vitro. These responses were markedly less upon s.c. administration of protease inhibitor-treated papain. Results obtained by using mast cell-deficient mice and reconstitution of tissue mast cells suggested the contribution of mast cells to papain-specific IgE/IgG1 responses and eosinophil infiltration. The responses were equivalent between wild-type and IL-33(-/-) mice. After the subsequent airway challenge, the s.c. presensitized wild-type mice showed more severe lung eosinophilia than those without the presensitization. The presensitized IL-33(-/-) mice showed modest lung eosinophilia, which was absent without the presensitization, but its severity and IgE boost by the airway challenge were markedly less than the presensitized wild-type mice, in which protease activity of inhaled papain contributed to the responses. The results suggest that mechanisms for the protease-dependent sensitization differ between skin and airway and that cooperation of mast cell-dependent, IL-33-independent initial sensitization via skin and protease-induced, IL-33-mediated mechanism in re-exposure via airway to protease allergens maximizes the magnitude of the transition from skin inflammation to asthma in natural history of progression of allergic diseases.

Perivascular mast cells regulate vein graft neointimal formation and remodeling.

Objective. Emerging evidence suggests an important role for mast cells in vein graft failure. This study addressed the hypothesis that perivascular mast cells regulate in situ vascular inflammatory and proliferative responses and subsequent vein graft neointimal lesion formation, using an optimized local mast cell reconstitution method.Methods and Results. Neointimal hyperplasia was induced by insertion of a vein graft into the right carotid artery in wild type and mast cell deficient KitW−sh/W−sh mice. In some experiments, mast cells were reconstituted systemically (tail vein injection of bone marrow-derived mast cells) or locally (directly into the right neck area) prior to vein grafting. Vein graft neointimal lesion formation was significantly (P < 0.05) reduced in KitW−sh/W−sh mice. Mast cell deficiency reduced the number of proliferating cells, and inhibited L-selectin, CCL2, M-CSF and MIP-3α expression in the vein grafts. Local but not systemic mast cell reconstitution restored a perivascular mast cell population that subsequently promoted neointimal formation in mast cell deficient mice.Conclusion. Our data demonstrate that perivascular mast cells play a key role in promoting neointima formation by inducing local acute inflammatory and proliferative responses. These results suggest that ex vivo intraoperative targeting of mast cells may have therapeutic potential for the prevention of pathological vein graft remodeling.

Mast cells play an important role in chlamydia pneumoniae lung infection by facilitating immune cell recruitment into the airway.

Mast cells are known as central players in allergy and anaphylaxis, and they play a pivotal role in host defense against certain pathogens. Chlamydia pneumoniae is an important human pathogen, but it is unclear what role mast cells play during C. pneumoniae infection. We infected C57BL/6 (wild-type [WT]) and mast cell-deficient mice (Kit(W-sh/W-sh) [Wsh]) with C. pneumoniae. Wsh mice showed improved survival compared with WT mice, with fewer cells in Wsh bronchoalveolar lavage fluid (BALF), despite similar levels of cytokines and chemokines. We also found a more rapid clearance of bacteria from the lungs of Wsh mice compared with WT mice. Cromolyn, a mast cell stabilizer, reduced BALF cells and bacterial burden similar to the levels seen in Wsh mice; conversely, Compound 48/80, a mast cell degranulator, increased the number of BALF cells and bacterial burden. Histology showed that WT lungs had diffuse inflammation, whereas Wsh mice had patchy accumulations of neutrophils and perivascular accumulations of lymphocytes. Infected Wsh mice had reduced amounts of matrix metalloprotease-9 in BALF and were resistant to epithelial integral membrane protein degradation, suggesting that barrier integrity remains intact in Wsh mice. Mast cell reconstitution in Wsh mice led to enhanced bacterial growth and normal epithelial integral membrane protein degradation, highlighting the specific role of mast cells in this model. These data suggest that mast cells play a detrimental role during C. pneumoniae infection by facilitating immune cell infiltration into the airspace and providing a more favorable replicative environment for C. pneumoniae.