There is growing appreciation that cellular metabolic and bioenergetic pathways do not play merely passive roles in activated leukocytes. Rather, metabolism has important roles in controlling cellular activation, differentiation, survival, and effector function. Much of this work has been performed in T cells; however, there is still very little information regarding mast cell metabolic reprogramming and its effect on cellular function. Mast cells perform important barrier functions and help control type 2 immune responses. In this study we show that murine bone marrow–derived mast cells rapidly alter their metabolism in response to stimulation through the FcεRI. We also demonstrate that specific metabolic pathways appear to be differentially required for the control of mast cell function. Manipulation of metabolic pathways may represent a novel point for the manipulation of mast cell activation.
Mast cells are important leukocytes at the interface of innate and adaptive (particularly type 2) immunity (1). These cells play critical roles in the expulsion of parasitic worms, by virtue of their sensitivity to antigenic crosslinking of IgE prebound to FcεRI-bearing mast cells. Mast cells are also involved in the pathology caused by dysregulated type 2 immunity associated with many allergic or atopic diseases. In response to FcεRI crosslinking by Ag and IgE, mast cells are rapidly activated. This is a multiphasic response, including early release of granules that contain prepackaged histamine and TNF-α, and later phases that include de novo formation of lipid mediators and induction of transcription that leads to the production of multiple cytokines, chemokines, and other effector molecules (2, 3).
Although metabolism and nutrient sensing are key pathways that govern cellular homeostasis, it is now clear that, especially in leukocytes, these pathways interact significantly with cellular fate and function (4). This has been most extensively studied in T cells, which undergo dynamic and complex metabolic reprogramming in response to activation, cytokine stimulation, and other changes in their microenvironment (5). Metabolic manipulation can lead to substantial alterations not only in acute lymphocyte effector function, but also the differentiation of helper subsets and memory responses (6). Similar themes have been explored in NK, B, and dendritic cells, which also have complex differentiation and effector program changes. By contrast, there is still limited information on how metabolism is modulated in postmitotic, terminally differentiated effector cell types, such as Ag-stimulated primary murine mast cells (7–11).
In this study, we show mast cells, too, use metabolic reprogramming to influence their effector function. Activation of mast cells through the FcεR results in rapid induction of glycolysis reminiscent of immediate reprogramming induced by lymphocyte activation. Modulation of downstream metabolic pathways results in inhibition of certain, but not all, effector functions.
Materials and Methods
Bone marrow–derived mast cell culture
Bone marrow derived mast cells (BMMCs) were generated by culturing bone marrow from C57BL/6 or Nur77GFP mice for 4–6 wk in IL-3 supplemented media, after which cells were at least 90% positive for the growth factor receptor c-kit and the multichain activating receptor FcεRI.
Seahorse metabolic flux assays
For direct in-Seahorse measurement of the immediate effects of Ag stimulation (Fig. 1), BMMCs (100,000/well) were presensitized for 3 h with 1 μg/ml DNP-specific IgE (clone SPE-7; Sigma). After 30 min of basal measurements, indicated amounts of high (DNP32-human serum albumin) or low (DNP5-BSA) valency Ag were injected using the Seahorse metabolic flux analyzer and metabolic readings were taken as indicated. For Seahorse metabolic stress tests (Fig. 2), BMMCs were IgE-sensitized (1 μg/ml) overnight and stimulated with indicated amounts of high (DNP32-HSA) or low (DNP5-BSA) valency Ags for 2.5 h, prior to Seahorse metabolic flux analysis, as described (7). Basal measurements were taken for 30 min prior to sequential injections of 1 μM oligomycin, which inhibits mitochondrial ATP production and stimulates glycolysis, 0.5 μM FCCP, which uncouples the respiratory chain and stimulates maximal oxygen consumption, and 0.5 μM rotenone/antimycin A, which inhibits complex I and complex III, respectively, to inhibit mitochondrial oxygen consumption altogether. Seahorse media included 2 mM glucose and 2 mM glutamine in minimal, unbuffered, DMEM.
Mast cell functional assays
Mast cell degranulation, cytokine production and Nur77GFP induction were carried out as previously described (12). Briefly, cells were preloaded with anti-DNP IgE (1 μg/ml) overnight. The next day, cells were washed and preincubated for 30 min with the indicated inhibitors, before stimulating the cells with 50 ng/ml DNP32-HSA. For degranulation, a flow cytometry based assay was used (12). This short-term assay relies not only on PS exposure as vesicles are fused with the plasma membrane (read-out by Annexin V staining), but also on a concomitant decrease in Lysotracker staining, which also occurs when exocytic granules fuse with the plasma membrane. Cells were monitored for cell death, using an exclusion dye (Ghost Dye; Tonbo Biosciences), and only live cells were analyzed for degranulation.
Results and Discussion
We set out to measure the relative state of the major bioenergetic pathways in mast cells, before and after activation of the cells by IgE and Ag. We first wanted to determine whether Ag crosslinking triggers immediate changes in metabolic responses in mast cells. As shown by others (13) and our recent paper (12), mast cell function is linked to FcεRI signal strength in a complex fashion, through the Src family kinase Lyn. Stimulation with a high-valency Ag leads to initial strong signaling, followed by rapid downregulation, due to recruitment of inhibitory phosphatases to the FcεRI signalosome. By contrast, low valency Ag only engages the positive effects of Lyn, leading to initially weaker signaling that is not subject to the same negative feedback, resulting in more sustained signaling. Thus, when we directly stimulated IgE-sensitized BMMCs in-Seahorse with high- versus low-valency Ag, we observed that the former (DNP32) led to an immediate, robust increase in glycolysis, which peaked within 10 min and persisted for over 2 h. However, stimulation with lower valency Ag (DNP5) did not result in a significant change in glycolysis (Fig. 1A). Ag concentration was equally important in engaging this glycolytic switch (Fig. 1A). Ag crosslinking did not immediately alter the state of oxidative phosphorylation in mast cells, as the oxygen consumption rate (OCR) was relatively unchanged despite a small decrease upon Ag injection (Fig. 1B). As shown in our recent report (12), signal strength through FcεRI can also be modulated by the receptor Tim-3, so we asked whether the Tim-3 expression could also modulate the acute changes in metabolic flux that we observed in mast cells. Thus, high Ag valency stimulated an immediate increase in glycolysis in wild-type (WT) BMMCs, which did not occur with low-valency Ag and was also reduced in Tim3-deficient BMMCs (Fig. 1C), whereas, again, the OCR was unaffected (Fig. 1D). Interestingly, the sensitivity of increased glycolysis, as read-out by extracellular acidification rate (ECAR), was also reflected in a detectable, although delayed, increase in ECAR when mast cells were treated with IgE alone, in the absence of Ag (Supplemental Fig. 1A). However, no increase in either ECAR or OCR was noted in the complete absence of Ag and IgE (Supplemental Fig. 1A, 1B).
Next, we measured both glycolysis and oxidative phosphorylation of unmanipulated mast cells, or cells that were prestimulated for 2.5 h with IgE/Ag. Thus, we subjected BMMCs from normal mice to metabolic stress tests, using the Seahorse metabolic flux analyzer. As shown in Fig. 2, mast cells that were prestimulated with IgE/Ag (green symbols) increased both their ability to carry out oxidative phosphorylation [as determined by the OCR (Fig. 2A) and spare respiratory capacity (SRC)] as well as their capacity for glycolysis, determined by the ECAR and glycolytic reserve (Fig. 2B). Thus, preactivation of mast cells results in a broader increase in the metabolic potential (as determined by the SRC and glycolytic reserve) of these cells, compared with what we observed upon acute stimulation in Fig. 1. Overall, the results in Figs. 1 and 2 indicate that a rapid glycolytic switch in mast cells is closely associated with intensity of Ag receptor signaling and costimulation, e.g., through Tim-3. Furthermore, the results in Fig. 1 suggest that the change in mitochondrial respiration shown in Fig. 2A (occurring after several hours of prestimulation) requires more complex, e.g., transcriptional, reprogramming, because it did not occur with acute in-Seahorse stimulation.
We next determined whether downstream metabolic pathways are important for mast cell effector function in response to stimulation with IgE/Ag. We took advantage of well-characterized pharmacological inhibitors of key metabolic enzymes. First we employed dichloroacetate (DCA), which inhibits pyruvate dehydrogenase kinase that itself phosphorylates and inhibits pyruvate dehydrogenase (14). Thus, DCA treatment increases the conversion of pyruvate into acetyl CoA, at the expense of lactate production, while preserving oxidative phosphorylation. As shown in Fig. 3A, DCA inhibited mast cell degranulation at non-toxic doses of this compound (Fig. 3A, 3B). Production of IL-6 by mast cells in response to IgE/Ag stimulation w as also inhibited by DCA in a similar dose range (Fig. 3D). However, these effects did not appear to be due to general inhibition of FcεRI signaling, because activation of a transgenic Nur77GFP reporter, a transcriptional readout for calcium-dependent signaling in mast cells and lymphocytes (12, 15), was not affected by doses of DCA that significantly inhibited both cytokine production and degranulation (Fig. 3C). Another compound that results in a relative shift from glycolysis to oxidative phosphorylation is 2-deoxyglucose, which inhibits hexokinase, a key early enzyme in the glycolytic pathway. Although 2-deoxyglucose was not as potent as DCA, it nevertheless led to a modest, but statistically significant, decrease in both degranulation and cytokine production upon activation of mast cells with IgE/Ag (Supplemental Fig. 2A, 2B).
To assess the functional role of mitochondrial respiration in mast cell activation, we used rotenone, an inhibitor of the mitochondrial electron transport chain (16). Thus, titrating in doses of rotenone that were not toxic to mast cells (data not shown), we observed that rotenone significantly inhibited Ag-stimulated cytokine (IL-6) production by bone marrow mast cells (Fig. 4A). In addition, IgE/Ag-induced degranulation of mast cells was also inhibited in a similar dose range (Fig. 4B). However, despite inhibition of both these functions, induction of the Nur77GFP transcriptional reporter was not inhibited by rotenone at theses doses (data not shown). Combined inhibition of both metabolic pathways further reduced the ability of mast cell to degranulate (Fig 4D) and to produce cytokines (Fig. 4C). These results suggest that mast cells require both glycolysis and oxidative phosphorylation for their immediate and late-phase responses. However, despite a role for general mitochondrial respiration, fatty acid oxidation appears to not be required for mast cell activation, because etomoxir, which inhibits this process (17), did not affect IgE/Ag-induced Nur77GFP induction (Fig. 5B), nor production of IL-6 (Fig. 5A) or degranulation (Fig. 5C).
In summary, we have demonstrated in this study that the activation of mast cells by IgE and Ag is associated with a rapid increase in glycolysis, but not necessarily in mitochondrial respiration, effects reminiscent of what has been reported for T cells (18, 19). Nonetheless, although not connected with immediate mast cell activation, oxidative phosphorylation is important for mast cell effector function, because its inhibition drastically decreases degranulation and cytokine production. Intriguingly, our data suggest these metabolic changes are actively induced by mast cells to carry out specific tasks. Our findings are intriguing in light of the observations that mast cell mitochondria translocate to the site of exocytosis during degranulation (20), and that mitochondrial STAT3 is responsible for induction of mitochondrial oxidative phosphorylation, ATP synthesis, and subsequent degranulation (21).
Notably, the mast cell metabolic reprogramming that we observed, both glycolytic and oxidative, appeared to be dispensable for upregulation of Nur77, a direct readout of activation downstream of FcεRI. This suggests that mast cells use these metabolic pathways to modulate and amplify the cellular immune response, but they are not obligatory for activation. In T cells, translation of IFN-γ, for instance, is directly influenced by the glycolytic machinery (7). Because mast cells are terminally differentiated, they may use these metabolic pathways to further shape their cellular responses to accommodate changes in the environment. Importantly, barrier surfaces are metabolically distinct, having decreased oxygen tension as well as lower nutrient levels, especially at sites of inflammation. Thus, metabolic intervention may represent a strategy to modulate mast cell effector function while preserving their differentiation status and cellularity at barrier sites.
This work was supported by Public Health Service Grant R56AI067544 (to L.P.K.) and Sidney Kimmel Foundation for Cancer Research Grant SKF-015-039 (to G.M.D.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
bone marrow–derived mast cell
extracellular acidification rate
human serum albumin
oxygen consumption rate
spare respiratory capacity
The authors have no financial conflicts of interest.