Marcela T. Taruselli

Fibroblasts are dynamic structural cells that direct both beneficial tissue repair and pathological organ fibrosis through interactions with tissue-resident type 2 lymphocytes (T2Ls) and type 3/17 lymphocytes (T3Ls). The cytokines interleukin-13 (IL-13) and IL-17A, produced by T2Ls and T3Ls, respectively, are linked to both tissue inflammation and fibrosis, but how their spatial positioning influences beneficial or pathological organ remodeling remains unclear. Using mouse models of liver injury and fibrosis, three-dimensional microscopy, and spatial transcriptomics, we found an accumulation of periportal and fibrotic tract T2Ls, predominantly group 2 innate lymphoid cells (ILC2s), positioned near T3Ls and niche adventitial fibroblasts and adjacent to discrete profibrotic myofibroblasts. Unexpectedly, T2L ablation worsened both carbon tetrachloride– and bile duct ligation–induced liver fibrosis, accompanied by increased IL-17A ⁺ T3Ls, predominantly γδ T cells. In contrast, concurrent T2L and T3L ablation reduced liver fibrosis. Our work suggests a spatially associated cross-talk between liver lymphocytes and fibroblast niches that tunes liver repair but can go awry in pathological liver fibrosis.

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.

Statin drugs are widely employed in the clinic to reduce serum cholesterol. Because of their hydroxymethylglutaryl coenzyme A reductase antagonism, statins also reduce isoprenyl lipids necessary for the membrane anchorage and signaling of small G-proteins in the Ras superfamily. We previously found that statins suppress immunoglobulin E (IgE)-mediated mast cell activation, suggesting these drugs might be useful in treating allergic disease. Although IgE-induced function is critical to allergic inflammation, mast cell proliferation and survival also impact atopic disease and mast cell neoplasia. In this study, we describe fluvastatin-mediated apoptosis in primary and transformed mast cells. An IC₅₀ was achieved between 0.8 and 3.5 μM in both cell types, concentrations similar to the reported fluvastatin serum C_max value. Apoptosis was correlated with reduced stem cell factor (SCF)-mediated signal transduction, mitochondrial dysfunction, and caspase activation. Complementing these data, we found that p53 deficiency or Bcl-2 overexpression reduced fluvastatin-induced apoptosis. We also noted evidence of cytoprotective autophagy in primary mast cells treated with fluvastatin. Finally, we found that intraperitoneal fluvastatin treatment reduced peritoneal mast cell numbers in vivo. These findings offer insight into the mechanisms of mast cell survival and support the possible utility of statins in mast cell–associated allergic and neoplastic diseases. SIGNIFICANCE STATEMENT Fluvastatin, a statin drug used to lower cholesterol, induces apoptosis in primary and transformed mast cells by antagonizing protein isoprenylation, effectively inhibiting stem cell factor (SCF)-induced survival signals. This drug may be an effective means of suppressing mast cell survival.

Sepsis has a well-studied inflammatory phase, with a less-understood secondary immunosuppressive phase. Elevated blood lactate and slow lactate clearance are associated with mortality; however, regulatory roles are unknown. We hypothesized that lactic acid (LA) contributes to the late phase and is not solely a consequence of bacterial infection. No studies have examined LA effects in sepsis models in vivo or a mechanism by which it suppresses LPS-induced activation in vitro. Because mast cells can be activated systemically and contribute to sepsis, we examined LA effects on the mast cell response to LPS. LA significantly suppressed LPS-induced cytokine production and NF-κB transcriptional activity in mouse bone marrow–derived mast cells and cytokine production in peritoneal mast cells. Suppression was MCT-1 dependent and reproducible with sodium lactate or formic acid. Further, LA significantly suppressed cytokine induction following LPS-induced endotoxemia in mice. Because glycolysis is linked to inflammation and LA is a byproduct of this process, we examined changes in glucose metabolism. LA treatment reduced glucose uptake and lactate export during LPS stimulation. LA effects were mimicked by glycolytic inhibitors and reversed by increasing ATP availability. These results indicate that glycolytic suppression and ATP production are necessary and sufficient for LA effects. Our work suggests that enhancing glycolysis and ATP production could improve immune function, counteracting LA suppressive effects in the immunosuppressive phase of sepsis.

Cellular metabolism and energy sensing pathways are closely linked to inflammation, but there is little understanding of how these pathways affect mast cell function. Mast cells are major effectors of allergy and asthma, and can be activated by the alarmin IL-33, which is linked to allergic disease. Therefore, we investigated the metabolic requirements for IL-33-induced mast cell function, to identify targets for controlling inflammation. We found that IL-33 increases glycolysis, glycolytic protein expression, and oxidative phosphorylation (OX PHOS). Inhibiting OX PHOS had little effect on cytokine production, but antagonizing glycolysis with 2-deoxyglucose or oxamate suppressed inflammatory cytokine production in vitro and in vivo. ATP reversed this suppression. Glycolytic blockade suppressed IL-33 signaling, including ERK phosphorylation, NFκB transcription, and ROS production in vitro, and suppressed IL-33-induced neutrophil recruitment in vivo. To test a clinically relevant way to modulate these pathways, we examined the effects of the FDA-approved drug metformin on IL-33 activation. Metformin activates AMPK, which suppresses glycolysis in immune cells. We found that metformin suppressed cytokine production in vitro and in vivo, effects that were reversed by ATP, mimicking the actions of the glycolytic inhibitors we tested. These data suggest that glycolytic ATP production is important for IL-33-induced mast cell activation, and that targeting this pathway may be useful in allergic disease.

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.

Mast cells are tissue resident, innate immune cells with heterogenous phenotypes tuned by cytokines and other microenvironmental stimuli. Playing a protective role in parasitic, bacterial, and viral infections, mast cells are also known for their role in the pathogenesis of allergy, asthma, and autoimmune diseases. Here, we review factors controlling mast cell activation, with a focus on receptor signaling and potential therapies for allergic disease. Specifically, we will discuss our work with FcεRI and FγR signaling, IL-4, IL-10, and TGF-β1 treatment, and Stat5. We conclude with potential therapeutics for allergic disease. Much of these efforts have been influenced by the work of Bill Paul. With many mechanistic targets for mast cell activation and different classes of therapeutics being studied, there is reason to be hopeful for continued clinical progress in this area.