Pulmonary IL-33 orchestrates innate immune cells to mediate respiratory syncytial virus-evoked airway hyperreactivity and eosinophilia.

BACKGROUND: Respiratory syncytial virus (RSV) infection is epidemiologically linked to asthma. During RSV infection, IL-33 is elevated and promotes immune cell activation, leading to the development of asthma. However, which immune cells are responsible for triggering airway hyperreactivity (AHR), inflammation and eosinophilia remained to be clarified. We aimed to elucidate the individual roles of IL-33-activated innate immune cells, including ILC2s and ST2+ myeloid cells, in RSV infection-triggered pathophysiology. METHODS: The role of IL-33/ILC2 axis in RSV-induced AHR inflammation and eosinophilia were evaluated in the IL-33-deficient and YetCre-13 Rosa-DTA mice. Myeloid-specific, IL-33-deficient or ST2-deficient mice were employed to examine the role of IL-33 and ST2 signaling in myeloid cells. RESULTS: We found that IL-33-activated ILC2s were crucial for the development of AHR and airway inflammation, during RSV infection. ILC2-derived IL-13 was sufficient for RSV-driven AHR, since reconstitution of wild-type ILC2 rescued RSV-driven AHR in IL-13-deficient mice. Meanwhile, myeloid cell-derived IL-33 was required for airway inflammation, ST2+ myeloid cells contributed to exacerbation of airway inflammation, suggesting the importance of IL-33 signaling in these cells. Local and peripheral eosinophilia is linked to both ILC2 and myeloid IL-33 signaling. CONCLUSIONS: This study highlights the importance of IL-33-activated ILC2s in mediating RSV-triggered AHR and eosinophilia. In addition, IL-33 signaling in myeloid cells is crucial for airway inflammation.

Toll-like receptor 9-dependent interferon production prevents group 2 innate lymphoid cell-driven airway hyperreactivity.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are important mediators of allergic asthma. Bacterial components, such as unmethylated CpG DNA, a Toll-like receptor (TLR) 9 agonist, are known to possess beneficial immunomodulatory effects in patients with T cell-mediated chronic asthma. However, their roles in regulating ILC2s remain unclear. OBJECTIVE: We sought to determine the role of TLR9 activation in regulating ILC2 function and to evaluate the therapeutic utility of an immunomodulatory microparticle containing natural TLR9 ligand (MIS416). METHODS: We evaluated the immunomodulatory effects of CpG A in IL-33-induced airway hyperreactivity (AHR) and airway inflammation. The roles of interferons were examined in vivo and in vitro by using signal transducer and activator of transcription 1 (Stat1)-/- mice and neutralizing antibodies against IFN-γ and IFN-α/ß receptor subunit 1, and their cellular sources were identified. The therapeutic utility of MIS416 was investigated in the Alternaria alternata model of allergic asthma and in humanized NSG mice. RESULTS: We show that TLR9 activation by CpG A suppresses IL-33-mediated AHR and airway inflammation through inhibition of ILC2s. Activation of TLR9 leads to production of IFN-α, which drives IFN-γ production by natural killer cells. Importantly, IFN-γ is essential for TLR9-driven suppression, and IFN-α cannot compensate for impaired IFN-γ signaling. We further show that IFN-γ directly inhibits ILC2 function through a STAT1-dependent mechanism. Finally, we demonstrate the therapeutic potential of MIS416 in A alternata-induced airway inflammation and validated these findings in human subjects. CONCLUSION: TLR9 activation alleviates ILC2-driven AHR and airway inflammation through direct suppression of cell function. Microparticle-based delivery of TLR9 ligands might serve as a therapeutic strategy for asthma treatment.

Regulation of type 2 innate lymphoid cell-dependent airway hyperreactivity by butyrate.

BACKGROUND: Allergic asthma is characterized by airway hyperreactivity (AHR) and inflammation driven by aberrant TH2 responses. Type 2 innate lymphoid cells (ILC2s) are a critical source of the TH2 cytokines IL-5 and IL-13, which promote acute asthma exacerbation. Short-chain fatty acids (SCFAs) have been shown to attenuate T cell-mediated allergic airway inflammation. However, their role in regulation of ILC2-driven AHR and lung inflammation remains unknown. OBJECTIVE: We investigated the immunomodulatory role of SCFAs in regulation of ILC2-induced AHR and airway inflammation and delineated the mechanism involved. METHODS: We assessed the role of SCFAs in regulating survival, proliferation, and cytokine production in lung sorted ILC2s. The SCFA butyrate was administered through drinking water or intranasally in BALB/c mice to evaluate its role in the ILC2-driven inflammatory response in IL-33 and Alternaria alternata models of allergic inflammation. We further confirmed our findings in human ILC2s. RESULTS: We show that butyrate, but not acetate or propionate, inhibited IL-13 and IL-5 production by murine ILC2s. Systemic and local administration of butyrate significantly ameliorated ILC2-driven AHR and airway inflammation. We further demonstrate that butyrate inhibited ILC2 proliferation and GATA3 expression but did not induce cell apoptosis, likely through histone deacetylase (HDAC) inhibition, because trichostatin A, a pan-HDAC inhibitor, exerted similar effects on ILC2s. Importantly, cotreatment with trichostatin A and butyrate did not result in an additive effect. Finally, we show that butyrate reduces cytokine production in human ILC2s. CONCLUSION: Our findings identify butyrate as a critical regulator of ILC2 proliferation and function through its HDAC inhibitory activity and can serve as a potential therapeutic target for asthma.

Serine-/Cysteine-Based sp2-Iminoglycolipids as Novel TLR4 Agonists: Evaluation of Their Adjuvancy and Immunotherapeutic Properties in a Murine Model of Asthma.

Glycolipids with TLR4 agonistic properties can serve either as therapeutic agents or as vaccine adjuvants by stimulating the development of proinflammatory responses. Translating them to the clinical setting is hampered by synthetic difficulties, the lack of stability in biological media, and/or a suboptimal profile of balanced immune mediator secretion. Here, we show that replacement of the sugar fragment by an sp2-iminosugar moiety in a prototypic TLR4 agonist, CCL-34, yields iminoglycolipid analogues that retain or improve their biological activity in vitro and in vivo and can be accessed through scalable protocols with total stereoselectivity. Their adjuvant potential is manifested in their ability to induce the secretion of proinflammatory cytokines, prime the maturation of dendritic cells, and promote the proliferation of CD8+ T cells, pertaining to a Th1-biased profile. Additionally, their therapeutic potential for the treatment of asthma, a Th2-dominated inflammatory pathology, has been confirmed in an ovalbumin-induced airway hyperreactivity mouse model.

Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.

The association between neutrophil extracellular traps (NETs) and response to inhaled corticosteroids (ICS) in asthma is unclear. To better understand this relationship, we analyzed the blood transcriptomes from children with controlled and uncontrolled asthma in the Taiwanese Consortium of Childhood Asthma Study using weighted gene coexpression network analysis and pathway enrichment methods. We identified 298 uncontrolled asthma-specific differentially expressed genes and one gene module associated with neutrophil-mediated immunity, highlighting a potential role for neutrophils in uncontrolled asthma. We also found that NET abundance was associated with nonresponse to ICS in patients. In a neutrophilic airway inflammation murine model, steroid treatment could not suppress neutrophilic inflammation and airway hyperreactivity. However, NET disruption with deoxyribonuclease I (DNase I) efficiently inhibited airway hyperreactivity and inflammation. Using neutrophil-specific transcriptomic profiles, we found that CCL4L2 was associated with ICS nonresponse in asthma, which was validated in human and murine lung tissue. CCL4L2 expression was also negatively correlated with pulmonary function change after ICS treatment. In summary, steroids fail to suppress neutrophilic airway inflammation, highlighting the potential need to use alternative therapies such as leukotriene receptor antagonists or DNase I that target the neutrophil-associated phenotype. Furthermore, these results highlight CCL4L2 as a potential therapeutic target for individuals with asthma refractory to ICS.

The ketone body ß-hydroxybutyrate mitigates ILC2-driven airway inflammation by regulating mast cell function.

Ketone bodies are increasingly understood to have regulatory effects on immune cell function, with ß-hydroxybutyrate (BHB) exerting a predominantly anti-inflammatory response. Dietary strategies to increase endogenous ketone body availability such as the ketogenic diet (KD) have recently been shown to alleviate inflammation of the respiratory tract. However, the role of BHB has not been addressed. Here, we observe that BHB suppresses group 2 innate lymphoid cell (ILC2)-mediated airway inflammation. Central to this are mast cells, which support ILC2 proliferation through interleukin-2 (IL-2). Suppression of the mast cell/IL-2 axis by BHB attenuates ILC2 proliferation and the ensuing type 2 cytokine response and immunopathology. Mechanistically, BHB directly inhibits mast cell function in part through GPR109A activation. Similar effects are achieved with either the KD or 1,3-butanediol. Our data reveal the protective role of BHB in ILC2-driven airway inflammation, which underscores the potential therapeutic value of ketone body supplementation for the management of asthma.

Identification of a PD-L1+Tim-1+ iNKT subset that protects against fine particulate matter-induced airway inflammation.

Although air pollutants such as fine particulate matter (PM2.5) are associated with acute and chronic lung inflammation, the etiology of PM2.5-induced airway inflammation remains poorly understood. Here we report that PM2.5 triggered airway hyperreactivity (AHR) and neutrophilic inflammation with concomitant increases in Th1 and Th17 responses and epithelial cell apoptosis. We found that γδ T cells promoted neutrophilic inflammation and AHR through IL-17A. Unexpectedly, we found that invariant natural killer T (iNKT) cells played a protective role in PM2.5-induced pulmonary inflammation. Specifically, PM2.5 activated a suppressive CD4- iNKT cell subset that coexpressed Tim-1 and programmed cell death ligand 1 (PD-L1). Activation of this suppressive subset was mediated by Tim-1 recognition of phosphatidylserine on apoptotic cells. The suppressive iNKT subset inhibited γδ T cell expansion and intrinsic IL-17A production, and the inhibitory effects of iNKT cells on the cytokine-producing capacity of γδ T cells were mediated in part by PD-1/PD-L1 signaling. Taken together, our findings underscore a pathogenic role for IL-17A-producing γδ T cells in PM2.5-elicited inflammation and identify PD-L1+Tim-1+CD4- iNKT cells as a protective subset that prevents PM2.5-induced AHR and neutrophilia by inhibiting γδ T cell function.

HIF-2α is indispensable for regulatory T cell function.

Hypoxia-inducible factor 1α (HIF-1α) and HIF-2α are master transcription factors that regulate cellular responses to hypoxia, but the exact function in regulatory T (Treg) cells is controversial. Here, we show that Treg cell development is normal in mice with Foxp3-specific knockout (KO) of HIF-1α or HIF-2α. However, HIF-2α-KO (but not HIF-1α-KO) Treg cells are functionally defective in suppressing effector T cell-induced colitis and inhibiting airway hypersensitivity. HIF-2α-KO Treg cells have enhanced reprogramming into IL-17-secreting cells. We show crosstalk between HIF-2α and HIF-1α, and that HIF-2α represses HIF-1α expression. HIF-1α is upregulated in HIF-2α-KO Treg cells and further deletion of HIF-1α restores the inhibitory function of HIF-2α-KO Treg cells. Mice with Foxp3-conditional KO of HIF-2α are resistant to growth of MC38 colon adenocarcinoma and metastases of B16F10 melanoma. Together, these results indicate that targeting HIF-2α to destabilize Treg cells might be an approach for regulating the functional activity of Treg cells.

α-Lactosylceramide Protects Against iNKT-Mediated Murine Airway Hyperreactivity and Liver Injury Through Competitive Inhibition of Cd1d Binding.

Invariant natural killer T (iNKT) cells, which are activated by T cell receptor (TCR)-dependent recognition of lipid-based antigens presented by the CD1d molecule, have been shown to participate in the pathogenesis of many diseases, including asthma and liver injury. Previous studies have shown the inhibition of iNKT cell activation using lipid antagonists can attenuate iNKT cell-induced disease pathogenesis. Hence, the development of iNKT cell-targeted glycolipids can facilitate the discovery of new therapeutics. In this study, we synthesized and evaluated α-lactosylceramide (α-LacCer), an α-galactosylceramide (α-GalCer) analog with lactose substitution for the galactose head and a shortened acyl chain in the ceramide tail, toward iNKT cell activation. We demonstrated that α-LacCer was a weak inducer for both mouse and human iNKT cell activation and cytokine production, and the iNKT induction by α-LacCer was CD1d-dependent. However, when co-administered with α-GalCer, α-LacCer inhibited α-GalCer-induced IL-4 and IFN-γ production from iNKT cells. Consequently, α-LacCer also ameliorated both α-GalCer and GSL-1-induced airway hyperreactivity and α-GalCer-induced neutrophilia when co-administered in vivo. Furthermore, we were able to inhibit the increases of ConA-induced AST, ALT and IFN-γ serum levels through α-LacCer pre-treatment, suggesting α-LacCer could protect against ConA-induced liver injury. Mechanistically, we discerned that α-LacCer suppressed α-GalCer-stimulated cytokine production through competing for CD1d binding. Since iNKT cells play a critical role in the development of AHR and liver injury, the inhibition of iNKT cell activation by α-LacCer present a possible new approach in treating iNKT cell-mediated diseases.