John J. Ryan

Introduction: Allergic diseases exhibit considerable sex-based variations, with greater incidence of allergic asthma, atopic dermatitis, food allergy, and chronic urticaria among adult females. This female dominance may be explained by differences in inflammatory signaling. Methods: We used primary mouse mast cells, neutrophils, and eosinophils as well as two mouse models of IL-33-induced inflammation to measure male and female responses to IL-33. Results: We show that the allergy-associated cytokine IL-33 elicits stronger responses from female mast cells and neutrophils compared to males, both in vitro and in vivo . The greater female response was not due to increased expression or sensitivity of the IL-33 receptor in female cells. Instead, we noted female-selective activation of the JNK pathway in response to IL-33. Male and female mast cells both required the ERK, p38, NF-kB, and AP-1 pathways to produce inflammatory cytokines in response to IL-33. However, inhibiting the JNK pathway selectively reduced IL-33-mediated cytokine production in female mast cells. A mast cell-dependent model of IL-33-induced peritonitis showed that gonadectomy ablated sexual dimorphism in the neutrophil influx response. However, plasma IL-13 levels remained higher in females even among mice lacking sex organs. Discussion: These data show that IL-33 induces female-dominant mast cell and neutrophil responses, partly via female-selective JNK signaling.

Allergic diseases are common, affecting more than one-third of the global population and varying widely in severity. A consistent theme in asthma, food allergy, atopic dermatitis, and chronic urticaria is a female-dominant prevalence in adulthood. Some of these begin with male predominance in childhood and shift with puberty. A smaller subset of allergic diseases, including eosinophilic esophagitis and vernal keratoconjunctivitis, has a clear male prevalence. In fact, nearly all allergic diseases exhibit sexual dimorphism in either prevalence or severity. These striking disparities are being unraveled to reveal mechanisms that might be clinically targeted. In this review, we discuss the current state of knowledge about sexual dimorphism for these important inflammatory disorders. These insights are foundational for optimizing personalized medicine.

Background: Patient engagement leads to improved disease outcomes; however, an unmet need exists for understanding and disseminating the benefits of patient engagement and shared decision making (SDM) in pulmonary arterial hypertension (PAH). Purpose: To develop an expert consensus on strategies for patient engagement and SDM in PAH. Methodology: United States (US)-based physicians ( n = 5), Advanced Practice Providers ( n = 5), and Registered Nurses (RNs; n = 3) who are responsible for managing patients with PAH were recruited to a double-blinded modified-Delphi panel (two survey rounds and a virtual consensus meeting). Consensus was defined as ≥80% of panelists rating their agreement or disagreement using a 9-point Likert scale. Results: Engaged patients actively participate in SDM by asking questions, providing feedback, and following up routinely, which in turn leads to improved disease management in PAH. Factors that motivate engagement include provider encouragement and social support. Factors that discourage engagement include frustration due to symptom severity and medication side effects, poor health literacy, and financial burden. Communicating the importance of risk assessments and resources to address poor health literacy are areas that require improvement. Creating an action plan that involves patients and family members and educating and encouraging patients to research and understand PAH are essential components of SDM. Conclusions: Consensus was reached on the importance of SDM and strategies to empower patients to be more engaged in their PAH management. Implications: The identified areas of improvement may motivate greater involvement of patients, and their caregivers, in the management of PAH to advocate for their goals and preferences.

Introduction: Allergic disease is a common and symptomatically heterogeneous group of inflammatory disorders marked by overactive Th2 and mast cell (MC) responses along with eosinophil infiltration. Treatment options require continual assessment due to breakthrough symptoms on standard regimens. One approach to improved therapy is drug repurposing. Our lab previously showed that cholesterol-lowering statin drugs can suppress IgE-mediated mast cell function by inhibiting protein isoprenylation, a pathway using cholesterol biosynthesis intermediates. Additionally, mast cells are activated by the alarmin IL-33, released by epithelial cells after contact with cellular stressors. We hypothesized that IL-33-mediated mast cell function can be inhibited by disrupting isoprenylation via statins or the dual farnesyltransferase (FT) geranylgeranyltransferase-1 inhibitor, FGTI-2734. Methods: We used IL-33 to stimulate mast cells and eosinophils in vitro and inhibited their function using simvastatin and FGTI-2734. Using primary mast cells and eosinophils, we measured cytokine production by ELISA and qPCR. Flow cytometry and western blots were used to measure phosphorylation of IL-33 signaling components, and eosinophil migration. Human mast cells were assessed by ELISA for cytokine inhibition. Lastly, a murine model of IL-33 induced peritonitis was used to assess the effects of isoprenylation inhibition on eosinophil and neutrophil influx. Results: We show simvastatin and FGTI-2734 suppressed IL-33-mediated cytokine protein and mRNA production in primary murine mast cells from the C57BL/6 strain. Simvastatin effects were lost on mast cells from the 129/SvJ strain and were inconsistent among primary human mast cells. In contrast, FGTI-2734 inhibited IL-33-induced cytokine production by mast cells on the 129/SvJ strain and among human donors. Simvastatin and FGTI-2734 also inhibited IL-33-induced cytokine production and chemokine-induced migration of C57BL/6 primary eosinophils. Simvastatin and FGTI-2734 had no effect on expression of the IL-33 receptor, ST2, suggesting that inhibition occurs at a step in IL-33 signaling. Importantly, FGTI-2734 significantly reduced eosinophil and neutrophil influx in a model of IL-33-induced peritonitis, whereas simvastatin had no effect. Discussion: These findings indicate that targeting FT and GGT-1 is a viable target in IL-33-induced inflammation.

BACKGROUND: Peripheral microvascular dysfunction is a hallmark feature of both heart failure with reduced ejection fraction (HFrEF) and preserved ejection fraction (HFpEF) pathophysiology, due partly to impairments in nitric oxide signaling secondary to tetrahydrobiopterin (BH ₄ ) deficiency and oxidative stress. METHODS: Using a randomized, double-blind, placebo-controlled crossover design, this study examined the impact of enteral BH ₄ (10 mg/kg), an antioxidant cocktail (AOx), and coadministration of these 2 agents (BH ₄ +AOx) on microvascular function in patients with HFrEF (n=14, 64±10 years) and HFpEF (n=19, 74±9 years). Passive limb movement was utilized to assess locomotor muscle microvascular function, and biomarkers of inflammation and oxidative damage were measured. RESULTS: Compared with placebo, the peak change in leg blood flow was not statistically different after AOx administration (HFrEF, P =0.60; HFpEF, P =0.61), but improved following BH ₄ ( P =0.033) and BH ₄ +AOx ( P =0.019) in both HFrEF (placebo: 234±31; BH ₄ : 357±45; BH ₄ +AOx: 355±49 mL/min) and HFpEF (placebo: 269±33; BH ₄ : 367±47; BH ₄ +AOx: 394±65 mL/min). The total hyperemic response to passive limb movement (leg blood flow area under the curve) was not statistically different across treatments in patients with HFrEF ( P =0.29), but increased following BH ₄ ( P =0.016) and BH ₄ +AOx ( P =0.040) in the HFpEF group. CRP (C-reactive protein) was lower following BH ₄ ( P =0.007) and BH ₄ +AOx ( P =0.007) in HFpEF (placebo: 4268±547; BH ₄ : 2721±391; BH ₄ +AOx: 2779±376 ng/mL), but was not statistically different in HFrEF ( P =0.39). CONCLUSIONS: Together, these results provide new evidence for the efficacy of acute BH ₄ administration to improve some aspects of locomotor muscle microvascular function in patients with HFrEF and HFpEF, with no apparent benefit of AOx administration, alone or in combination with BH ₄ , in either group. These findings lend further conceptual support for the nitric oxide pathway as a modifiable target in the treatment of heart failure.

IL-33 is an inflammatory cytokine that promotes allergic disease by activating group 2 innate lymphoid cells, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. Homeostatic control of IL-33 signaling is poorly understood. Because the IL-33 receptor, ST2, acts via cascades used by the TLR family, similar feedback mechanisms may exist. MicroRNA (miR)-146a is induced by LPS-mediated TLR4 signaling and serves as a feedback inhibitor. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow–derived mast cells (BMMCs). BMMCs transfected with a miR-146a antagonist or derived from miR-146a knockout mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33–ST2 signaling. In vivo, miR-146a expression in plasma exosomes was elevated after i.p. injection of IL-33 in wild-type but not mast cell–deficient KitW-sh/W-sh mice. Finally, KitW-sh/W-sh mice acutely reconstituted with miR-146a knockout BMMCs prior to IL-33 challenge had elevated plasma IL-6 levels compared with littermates receiving wild-type BMMCs. These results support the hypothesis that miR-146a is a feedback regulator of IL-33–mediated mast cell functions associated with allergic disease.

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.

IgE-mediated mast cell activation is a driving force in allergic disease in need of novel interventions. Statins, long used to lower serum cholesterol, have been shown in multiple large-cohort studies to reduce asthma severity. We previously found that statins inhibit IgE-induced mast cell function, but these effects varied widely among mouse strains and human donors, likely due to the upregulation of the statin target, 3-hydroxy-3-methylgutaryl-CoA reductase. Statin inhibition of mast cell function appeared to be mediated not by cholesterol reduction but by suppressing protein isoprenylation events that use cholesterol pathway intermediates. Therefore, we sought to circumvent statin resistance by targeting isoprenylation. Using genetic depletion of the isoprenylation enzymes farnesyltransferase and geranylgeranyl transferase 1 or their substrate K-Ras, we show a significant reduction in FcεRI-mediated degranulation and cytokine production. Furthermore, similar effects were observed with pharmacological inhibition with the dual farnesyltransferase and geranylgeranyl transferase 1 inhibitor FGTI-2734. Our data indicate that both transferases must be inhibited to reduce mast cell function and that K-Ras is a critical isoprenylation target. Importantly, FGTI-2734 was effective in vivo, suppressing mast cell–dependent anaphylaxis, allergic pulmonary inflammation, and airway hyperresponsiveness. Collectively, these findings suggest that K-Ras is among the isoprenylation substrates critical for FcεRI-induced mast cell function and reveal isoprenylation as a new means of targeting allergic disease.