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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-β–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.

TGF-β1 is involved in many pathological conditions, including autoimmune disorders, cancer, and cardiovascular and allergic diseases. We have previously found that TGF-β1 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-β on IL-33–mediated mast cell activation. Bone marrow–derived mast cells cultured in TGF-β1, β2, or β3 showed reduced IL-33–mediated production of TNF, IL-6, IL-13, and MCP-1 in a concentration-dependent manner. TGF-β1 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-β1 injection suppressed IL-33–induced systemic cytokines in vivo and inhibited IL-33–mediated cytokine release from human mast cells. TGF-β1 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-β1 on IgE-mediated activation, demonstrate that TGF-β1 can provide broad inhibitory signals to activated mast cells.

Mast cell (MC)– and basophil-associated inflammatory diseases are a considerable burden to society. A significant portion of patients have symptoms despite standard-of-care therapy. Statins, used to lower serum cholesterol, have immune-modulating activities. We tested the in vitro and in vivo effects of statins on IgE-mediated MC and basophil activation. Fluvastatin showed the most significant inhibitory effects of the six statins tested, suppressing IgE-induced cytokine secretion among mouse MCs and basophils. The effects of fluvastatin were reversed by mevalonic acid or geranylgeranyl pyrophosphatase, and mimicked by geranylgeranyl transferase inhibition. Fluvastatin selectively suppressed key FcεRI signaling pathways, including Akt and ERK. Although MCs and basophils from the C57BL/6J mouse strain were responsive to fluvastatin, those from 129/SvImJ mice were completely resistant. Resistance correlated with fluvastatin-induced upregulation of the statin target HMG-CoA reductase. Human MC cultures from eight donors showed a wide range of fluvastatin responsiveness. These data demonstrate that fluvastatin is a potent suppressor of IgE-mediated MC activation, acting at least partly via blockade of geranyl lipid production downstream of HMG-CoA reductase. Importantly, consideration of statin use for treating MC–associated disease needs to incorporate genetic background effects, which can yield drug resistance.

Lactic acid (LA) is elevated in tumors, asthma, and wound healing, environments that also include mast cells and IL-33. However, how LA affects mast cell function is unknown. Therefore we evaluated how LA modifies the IL-33-mediated mast cell response. When bone marrow derived mast cells were cultured with LA, we noted reduced IL-33-mediated cytokine production, an effect that was both monocarboxylate transporter (MCT)1- and pH-dependent. LA selectively decreased IL-33-induced TAK1, JNK, ERK, and NFkB phosphorylation, but not p38 activation. Additionally, LA increased HIF-1α expression. Since HIF-1α has been shown to regulate the pro-inflammatory microRNA miR-155, we examined miR-155 expression. miR-155-5p was reduced by LA, an effect that was reversed by HIF-1α antagonism. More importantly, miR-155-5p overexpression abolished the suppressive effects of LA. Additionally, the negative regulator SOCS1, a known miR-155 target, was elevated by LA addition. This suggests that LA employs HIF-1a to suppress miR-155, indirectly increasing SOCS-1 and limiting cytokine production. These data were recapitulated in vivo, since C57BL/6 mice injected with LA showed less IL-33-induced plasma cytokine levels than control mice. Lastly, LA suppressed IL-33-mediated human skin mast cell activation, an effect that was also MCT1-dependent. Our data demonstrate that lactic acid, present in inflammatory and malignant microenvironments, alters mast cell function to suppress inflammation.

TGFβ is involved in many pathological conditions, including autoimmune disorders, cancer, and cardiovascular and allergic diseases. We have previously found that TGFβ suppresses IgE-mediated mast cell activation in human and mouse mast cells in vitro. IL-33 is a recently discovered member of the IL-1 family that functions as an “alarmin” stimulating mast cell responses and enhancing IgE-mediated activation. How TGFβ affects IL-33 signaling is now known. We find that mouse bone marrow-derived mast cells cultured in TGFβ -1, -2, or -3 showed reduced IL-33-mediated production of TNF, IL-6, IL-13 and MCP-1, in a concentration-dependent manner. Furthermore, TGFβ also reduced expression of the IL-33 receptor ST2, as well as IL-33-mediated TAK1, IKB and ERK phosphorylation. TGF-β1 injection suppressed IL-33-mediated production of systemic inflammatory cytokines in vivo. The role of IL-33 in the pathogenesis of allergic diseases is incompletely understood. These findings, consistent with our previously reported effects of TGFβ on IgE-mediated activation, demonstrate that TGFβ can provide broad and substantial inhibitory signals to activated mast cells. These data suggest possible therapeutic strategies to maintain mast cell homeostasis in autoimmune and allergic diseases.

Mast cells are best known for their role in inflammatory and allergic disease. Mast cell leukemia is a rare subtype of acute myelogenous leukemia, most of which express the c-Kit mutation, D816V, that yields factor-independent growth. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase inhibitors to lower serum cholesterol, have also been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that Fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and P815 mastocytoma cells in a dose- and time-dependent manner. Apoptosis was evidenced by DNA fragmentation and caspase activation. Importantly, the mastocytoma line P815, which expresses mutant c-Kit, was readily killed by Fluvastatin, with an IC50 of 5–10μM and apoptosis peaking at 72 hours. An up-regulation of the mutated c-Kit receptor was seen at 12 hours, followed by mitochondrial membrane instability and Caspase-9 activation within 24–28 hours. Finally, preliminary evidence of autophagy was detected during fluvastatin treatment. Interestingly, autophagy appeared to be cytoprotective in primary mast cells but cytotoxic in P815 mastocytoma cells. These preliminary data support the theory that statin family drugs induce mast cell autophagy and apoptosis, lending new therapeutic approaches for suppressing growth of both normal and transformed mast cells.