Fluoxetine restrains allergic inflammation by targeting an FcɛRI-ATP positive feedback loop in mast cells.

There is a clinical need for new treatment options addressing allergic disease. Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants that have anti-inflammatory properties. We tested the effects of the SSRI fluoxetine on IgE-induced function of mast cells, which are critical effectors of allergic inflammation. We showed that fluoxetine treatment of murine or human mast cells reduced IgE-mediated degranulation, cytokine production, and inflammatory lipid secretion, as well as signaling mediated by the mast cell activator ATP. In a mouse model of systemic anaphylaxis, fluoxetine reduced hypothermia and cytokine production. Fluoxetine was also effective in a model of allergic airway inflammation, where it reduced bronchial responsiveness and inflammation. These data show that fluoxetine suppresses mast cell activation by impeding an FcɛRI-ATP positive feedback loop and support the potential repurposing of this SSRI for use in allergic disease.

Resveratrol Protects against Skin Inflammation through Inhibition of Mast Cell, Sphingosine Kinase-1, Stat3 and NF-κB p65 Signaling Activation in Mice.

Inflammation is pathogenic to skin diseases, including atopic dermatitis (AD) and eczema. Treatment for AD remains mostly symptomatic with newer but costly options, tainted with adverse side effects. There is an unmet need for safe therapeutic and preventative strategies for AD. Resveratrol (R) is a natural compound known for its anti-inflammatory properties. However, animal and human R studies have yielded contrasting results. Mast cells (MCs) are innate immune skin-resident cells that initiate the development of inflammation and progression to overt disease. R's effects on MCs are also controversial. Using a human-like mouse model of AD development consisting of a single topical application of antigen ovalbumin (O) for 7 days, we previously established that the activation of MCs by a bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) initiated substantial skin remodeling compared to controls. Here, we show that daily R application normalized O-mediated epidermal thickening, ameliorated cell infiltration, and inhibited skin MC activation and chemokine expression. We unraveled R's multiple mechanisms of action, including decreased activation of the S1P-producing enzyme, sphingosine kinase 1 (SphK1), and of transcription factors Signal Transducer and Activator of Transcription 3 (Stat3) and NF-κBp65, involved in chemokine production. Thus, R may be poised for protection against MC-driven pathogenic skin inflammation.

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.

The mast cell/S1P axis is not linked to pre-lesional male skin remodeling in a mouse model of eczema.

Atopic dermatitis (AD, eczema) is an inflammatory skin condition whose histopathology involves remodeling. Few preclinical AD studies are performed using male mice. The histopathological mechanisms underlying AD development were investigated here in male mice at a pre-lesional stage using a human AD-like mouse model. Hypodermal cellular infiltration without thickening of skin layers was observed after one epicutaneous exposure to antigen ovalbumin (OVA), compared to controls. In contrast to our previous report using female mice, OVA treatment did not activate skin mast cells (MC) or elevate sphingosine-1-phosphate (S1P) levels while increasing systemic but not local levels of CCL2, CCL3 and CCL5 chemokines. In contrast to the pathogenic AD mechanisms we recently uncovered in female, S1P-mediated skin MC activation with subsequent local chemokine production is not observed in male mice, supporting sex differences in pre-lesional stages of AD. We are proposing that differential involvement of the MC/S1P axis in early pathogenic skin changes contributes to the well documented yet still incompletely understood sex-dimorphic susceptibility to AD in humans.

Osthole, a Natural Plant Derivative Inhibits MRGPRX2 Induced Mast Cell Responses.

Mast cells are tissue-resident innate immune cells known for their prominent role in mediating allergic reactions. MAS-related G-protein coupled receptor-X2 (MRGPRX2) is a promiscuous G-protein coupled receptor (GPCR) expressed on mast cells that is activated by several ligands that share cationic and amphipathic properties. Interestingly, MRGPRX2 ligands include certain FDA-approved drugs, antimicrobial peptides, and neuropeptides. Consequently, this receptor has been implicated in causing mast cell-dependent pseudo-allergic reactions to these drugs and chronic inflammation associated with asthma, urticaria and rosacea in humans. In the current study we examined the role of osthole, a natural plant coumarin, in regulating mast cell responses when activated by the MRGPRX2 ligands, including compound 48/80, the neuropeptide substance P, and the cathelicidin LL-37. We demonstrate that osthole attenuates both the early (Ca2+ mobilization and degranulation) and delayed events (chemokine/cytokine production) of mast cell activation via MRGPRX2 in vitro. Osthole also inhibits MrgprB2- (mouse ortholog of human MRGPRX2) dependent inflammation in in vivo mouse models of pseudo-allergy. Molecular docking analysis suggests that osthole does not compete with the MRGPRX2 ligands for interaction with the receptor, but rather regulates MRGPRX2 activation via allosteric modifications. Furthermore, flow cytometry and confocal microscopy experiments reveal that osthole reduces both surface and intracellular expression levels of MRGPRX2 in mast cells. Collectively, our data demonstrate that osthole inhibits MRGPRX2/MrgprB2-induced mast cell responses and provides a rationale for the use of this natural compound as a safer alternative treatment for pseudo-allergic reactions in humans.

Lactic acid suppresses IgE-mediated mast cell function in vitro and in vivo.

Mast cells have functional plasticity affected by their tissue microenvironment, which greatly impacts their inflammatory responses. Because lactic acid (LA) is abundant in inflamed tissues and tumors, we investigated how it affects mast cell function. Using IgE-mediated activation as a model system, we found that LA suppressed inflammatory cytokine production and degranulation in mouse peritoneal mast cells, data that were confirmed with human skin mast cells. In mouse peritoneal mast cells, LA-mediated cytokine suppression was dependent on pH- and monocarboxylic transporter-1 expression. Additionally, LA reduced IgE-induced Syk, Btk, and ERK phosphorylation, key signals eliciting inflammation. In vivo, LA injection reduced IgE-mediated hypothermia in mice undergoing passive systemic anaphylaxis. Our data suggest that LA may serve as a feedback inhibitor that limits mast cell-mediated inflammation.

The Use of Human and Mouse Mast Cell and Basophil Cultures to Assess Type 2 Inflammation.

Mast cells and basophils are important innate immune cells involved in resistance to parasitic infection and are critical orchestrators of allergic disease. The relative ease with which they are cultured from mouse or human tissues allows one to work with primary cells that maintain a differentiated and functional phenotype. In this chapter, we describe the methods by which mouse mast cells and basophils can be cultured from bone marrow. We also provide methods for isolating and expanding mouse peritoneal mast cells and human skin mast cells.

Methods for Analyzing Sphingosine-1-Phosphate Signaling in Human and Mouse Primary Mast Cells.

Mast cells produce a potently bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) constitutively and upon activation. The ligation of S1P to its type 2 receptor on mast cells triggers a novel downstream signaling pathway that we discovered links activation of transcription factor signal transducer and activator of transcription 3 to mast cell-derived chemokine release in both humans and mice. In this chapter, we describe the methods used to study S1P signaling in human and mouse primary mast cells.

TGF-ß1 Suppresses IL-33-Induced Mast Cell Function.

TGF-ß1 is involved in many pathological conditions, including autoimmune disorders, cancer, and cardiovascular and allergic diseases. We have previously found that TGF-ß1 can suppress IgE-mediated mast cell activation of human and mouse mast cells. IL-33 is a member of the IL-1 family capable of inducing mast cell responses and enhancing IgE-mediated activation. In this study, we investigated the effects of TGF-ß on IL-33-mediated mast cell activation. Bone marrow-derived mast cells cultured in TGF-ß1, ß2, or ß3 showed reduced IL-33-mediated production of TNF, IL-6, IL-13, and MCP-1 in a concentration-dependent manner. TGF-ß1 inhibited IL-33-mediated Akt and ERK phosphorylation as well as NF-κB- and AP-1-mediated transcription. These effects were functionally important, as TGF-ß1 injection suppressed IL-33-induced systemic cytokines in vivo and inhibited IL-33-mediated cytokine release from human mast cells. TGF-ß1 also suppressed the combined effects of IL-33 and IgE-mediated activation on mouse and human mast cells. The role of IL-33 in the pathogenesis of allergic diseases is incompletely understood. These findings, consistent with our previously reported effects of TGF-ß1 on IgE-mediated activation, demonstrate that TGF-ß1 can provide broad inhibitory signals to activated mast cells.

Lactic Acid Suppresses IL-33-Mediated Mast Cell Inflammatory Responses via Hypoxia-Inducible Factor-1α-Dependent miR-155 Suppression.

Lactic acid (LA) is present in tumors, asthma, and wound healing, environments with elevated IL-33 and mast cell infiltration. Although IL-33 is a potent mast cell activator, how LA affects IL-33-mediated mast cell function is unknown. To investigate this, mouse bone marrow-derived mast cells were cultured with or without LA and activated with IL-33. LA reduced IL-33-mediated cytokine and chemokine production. Using inhibitors for monocarboxylate transporters (MCT) or replacing LA with sodium lactate revealed that LA effects are MCT-1- and pH-dependent. LA selectively altered IL-33 signaling, suppressing TGF-ß-activated kinase-1, JNK, ERK, and NF-κB phosphorylation, but not p38 phosphorylation. LA effects in other contexts have been linked to hypoxia-inducible factor (HIF)-1α, which was enhanced in bone marrow-derived mast cells treated with LA. Because HIF-1α has been shown to regulate the microRNA miR-155 in other systems, LA effects on miR-155-5p and miR-155-3p species were measured. In fact, LA selectively suppressed miR-155-5p in an HIF-1α-dependent manner. Moreover, overexpressing miR-155-5p, but not miR-155-3p, abolished LA effects on IL-33-induced cytokine production. These in vitro effects of reducing cytokines were consistent in vivo, because LA injected i.p. into C57BL/6 mice suppressed IL-33-induced plasma cytokine levels. Lastly, IL-33 effects on primary human mast cells were suppressed by LA in an MCT-dependent manner. Our data demonstrate that LA, present in inflammatory and malignant microenvironments, can alter mast cell behavior to suppress inflammation.

Dexamethasone rapidly suppresses IL-33-stimulated mast cell function by blocking transcription factor activity.

Mast cells are critical effectors of allergic disease and can be activated by IL-33, a proinflammatory member of the IL-1 cytokine family. IL-33 worsens the pathology of mast cell-mediated diseases, but therapies to antagonize IL-33 are still forthcoming. Because steroids are the mainstay of allergic disease treatment and are well known to suppress mast cell activation by other stimuli, we examined the effects of the steroid dexamethasone on IL-33-mediated mast cell function. We found that dexamethasone potently and rapidly suppressed cytokine production elicited by IL-33 from murine bone marrow-derived and peritoneal mast cells. IL-33 enhances IgE-mediated mast cell cytokine production, an activity that was also antagonized by dexamethasone. These effects were consistent in human mast cells. We additionally observed that IL-33 augmented migration of IgE-sensitized mast cells toward antigen. This enhancing effect was similarly reversed by dexamethasone. Simultaneous addition of dexamethasone with IL-33 had no effect on the phosphorylation of MAP kinases or NFκB p65 subunit; however, dexamethasone antagonized AP-1- and NFκB-mediated transcriptional activity. Intraperitoneal administration of dexamethasone completely abrogated IL-33-mediated peritoneal neutrophil recruitment and prevented plasma IL-6 elevation. These data demonstrate that steroid therapy may be an effective means of antagonizing the effects of IL-33 on mast cells in vitro and in vivo, acting partly by suppressing IL-33-induced NFκB and AP-1 activity.

IL-10-Induced miR-155 Targets SOCS1 To Enhance IgE-Mediated Mast Cell Function.

IL-10 is an important regulatory cytokine that modulates a wide range of immune cells. Whereas it is best known for its ability to suppress immune responses, IL-10 has been found to be pathogenic in several human and animal studies of immune-mediated diseases. There is a considerable gap in our understanding of the molecular mechanisms behind the stimulatory effects of IL-10 during allergic inflammation. IL-10 treatment has been shown to suppress mast cell TNF production. In this study, we report that whereas TNF secretion was reduced, IL-10 surprisingly enhanced IgE-mediated protease and cytokine production both in vitro and in vivo. This stimulatory effect was consistent in mouse and human skin mast cells. IL-10 enhanced activation of the key FcεRI signaling proteins Stat5, JNK, and ERK. We demonstrate that IL-10 effects are dependent on Stat3 activation, eliciting miR-155 expression, with a resulting loss of suppressor of cytokine signaling-1. The importance of miR-155 was demonstrated by the inability of IL-10 to enhance anaphylaxis in miR-155-deficient mice. Taken together, our results reveal an IL-10-induced, Stat3-miR-155 signaling pathway that can promote mast cell responses.

The induction of microRNA-16 in colon cancer cells by protein arginine deiminase inhibition causes a p53-dependent cell cycle arrest.

Protein Arginine Deiminases (PADs) catalyze the post-translational conversion of peptidyl-Arginine to peptidyl-Citrulline in a calcium-dependent, irreversible reaction. Evidence is emerging that PADs play a role in carcinogenesis. To determine the cancer-associated functional implications of PADs, we designed a small molecule PAD inhibitor (called Chor-amidine or Cl-amidine), and tested the impact of this drug on the cell cycle. Data derived from experiments in colon cancer cells indicate that Cl-amidine causes a G1 arrest, and that this was p53-dependent. In a separate set of experiments, we found that Cl-amidine caused a significant increase in microRNA-16 (miRNA-16), and that this increase was also p53-dependent. Because miRNA-16 is a putative tumor suppressor miRNA, and others have found that miRNA-16 suppresses proliferation, we hypothesized that the p53-dependent G1 arrest associated with PAD inhibition was, in turn, dependent on miRNA-16 expression. Results are consistent with this hypothesis. As well, we found the G1 arrest is at least in part due to the ability of Cl-amidine-mediated expression of miRNA-16 to suppress its' G1-associated targets: cyclins D1, D2, D3, E1, and cdk6. Our study sheds light into the mechanisms by which PAD inhibition can protect against or treat colon cancer.

D-amino acid based protein arginine deiminase inhibitors: Synthesis, pharmacokinetics, and in cellulo efficacy.

The protein arginine deiminases (PADs) are known to play a crucial role in the onset and progression of multiple inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and cancer. However, it is not known how each of the five PAD isozymes contributes to disease pathogenesis. As such, potent, selective, and bioavailable PAD inhibitors will be useful chemical probes to elucidate the specific roles of each isozyme. Since D-amino amino acids often possess enhanced in cellulo stability, and perhaps unique selectivities, we synthesized a series of D-amino acid analogs of our pan-PAD inhibitor Cl-amidine, hypothesizing that this change would provide inhibitors with enhanced pharmacokinetic properties. Herein, we demonstrate that d-Cl-amidine and d-o-F-amidine are potent and highly selective inhibitors of PAD1. The pharmacokinetic properties of d-Cl-amidine were moderately improved over those of l-Cl-amidine, and this compound exhibited similar cell killing in a PAD1 expressing, triple-negative MDA-MB-231 breast cancer cell line. These inhibitors represent an important step in our efforts to develop stable, bioavailable, and highly selective inhibitors for all of the PAD isozymes.

Suppression of colitis in mice by Cl-amidine: a novel peptidylarginine deiminase inhibitor.

Inflammatory bowel diseases (IBDs), mainly Crohn's disease and ulcerative colitis, are dynamic, chronic inflammatory conditions that are associated with an increased colon cancer risk. Inflammatory cell apoptosis is a key mechanism for regulating IBD. Peptidylarginine deiminases (PADs) catalyze the posttranslational conversion of peptidylarginine to peptidylcitrulline in a calcium-dependent, irreversible reaction and mediate the effects of proinflammatory cytokines. Because PAD levels are elevated in mouse and human colitis, we hypothesized that a novel small-molecule inhibitor of the PADs, i.e., chloramidine (Cl-amidine), could suppress colitis in a dextran sulfate sodium mouse model. Results are consistent with this hypothesis, as demonstrated by the finding that Cl-amidine treatment, both prophylactic and after the onset of disease, reduced the clinical signs and symptoms of colitis, without any indication of toxic side effects. Interestingly, Cl-amidine drives apoptosis of inflammatory cells in vitro and in vivo, providing a mechanism by which Cl-amidine suppresses colitis. In total, these data help validate the PADs as therapeutic targets for the treatment of IBD and further suggest Cl-amidine as a candidate therapy for this disease.