The modulation of pulmonary group 2 innate lymphoid cell function in asthma: from inflammatory mediators to environmental and metabolic factors.

A dysregulated type 2 immune response is one of the fundamental causes of allergic asthma. Although Th2 cells are undoubtedly central to the pathogenesis of allergic asthma, the discovery of group 2 innate lymphoid cells (ILC2s) has added another layer of complexity to the etiology of this chronic disease. Through their inherent innate type 2 responses, ILC2s not only contribute to the initiation of airway inflammation but also orchestrate the recruitment and activation of other members of innate and adaptive immunity, further amplifying the inflammatory response. Moreover, ILC2s exhibit substantial cytokine plasticity, as evidenced by their ability to produce type 1- or type 17-associated cytokines under appropriate conditions, underscoring their potential contribution to nonallergic, neutrophilic asthma. Thus, understanding the mechanisms of ILC2 functions is pertinent. In this review, we present an overview of the current knowledge on ILC2s in asthma and the regulatory factors that modulate lung ILC2 functions in various experimental mouse models of asthma and in humans.

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.

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.

Immunomodulatory Role of Staphylococcus aureus in Atopic Dermatitis.

Staphylococcus aureus is a gram-positive bacterium commonly found on humans, and it constitutes the skin microbiota. Presence of S. aureus in healthy individuals usually does not pose any threat, as the human body is equipped with many mechanisms to prevent pathogen invasion and infection. However, colonization of S. aureus has been correlated with many healthcare-associated infections, and has been found in people with atopic diseases. In atopic dermatitis, constant fluctuations due to inflammation of the epidermal and mucosal barriers can cause structural changes and allow foreign antigens and pathogens to bypass the first line of defense of the innate system. As they persist, S. aureus can secrete various virulence factors to enhance their survival by host invasion and evasion mechanisms. In response, epithelial cells can release damage-associated molecular patterns, or alarmins such as TSLP, IL-25, IL-33, and chemokines, to recruit innate and adaptive immune cells to cause inflammation. Until recently, IL-36 had been found to play an important role in modulating atopic dermatitis. Secretion of IL-36 from keratinocytes can activate a Th2 independent pathway to trigger symptoms of allergic reaction resulting in clinical manifestations. This mini review aims to summarize the immunomodulatory roles of S. aureus virulence factors and how they contribute to the pathogenesis of atopic diseases.

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.

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.

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.

Protein Palmitoylation by ZDHHC13 Protects Skin against Microbial-Driven Dermatitis.

Atopic dermatitis is a complex chronic inflammatory skin disorder that results from intimate interactions among genetic predisposition, host environment, skin barrier defects, and immunological factors. However, a clear genetic roadmap leading to atopic dermatitis remains to be fully explored. From a genome-wide mutagenesis screen, deficiency of ZDHHC13, a palmitoylacyl transferase, has previously been associated with skin and multitissue inflammatory phenotypes. Here, we report that ZDHHC13 is required for skin barrier integrity and that deficiency of ZDHHC13 renders mice susceptible to environmental bacteria, resulting in persistent skin inflammation and an atopic dermatitis-like disease. This phenotype is ameliorated in a germ-free environment and is also attenuated by antibiotic treatment, but not by deletion of the Rag1 gene, suggesting that a microbial factor triggers inflammation rather than intrinsic adaptive immunity. Furthermore, skin from ZDHHC13-deficient mice has both elevated levels of IL-33 and type 2 innate lymphoid cells, reinforcing the role of innate immunity in the development of atopic dermatitis. In summary, our study suggests that loss of ZDHHC13 in skin impairs the integrity of multiple barrier functions and leads to a dermatitis lesion in response to microbial encounters.

Differential Analysis of the Secretome of WRL68 Cells Infected with the Chikungunya Virus.

The Chikungunya virus (CHIKV) is an arthropod borne virus. In the last 50 years, it has been the cause of numerous outbreaks in tropical and temperate regions, worldwide. There is limited understanding regarding the underlying molecular mechanisms involved in CHIKV replication and how the virus interacts with its host. In the present study, comparative proteomics was used to identify secreted host proteins that changed in abundance in response to early CHIKV infection. Two-dimensional gel electrophoresis was used to analyse and compare the secretome profiles of WRL-68 cells infected with CHIKV against mock control WRL-68 cells. The analysis identified 25 regulated proteins in CHIKV infected cells. STRING network analysis was then used to predict biological processes that may be affected by these proteins. The processes predicted to be affected include signal transduction, cellular component and extracellular matrix (ECM) organization, regulation of cytokine stimulus and immune response. These results provide an initial view of CHIKV may affect the secretome of infected cells during early infection. The results presented here will compliment earlier results from the study of late host response. However, functional characterization will be necessary to further enhance our understanding of the roles played by these proteins in the early stages of CHIKV infection in humans.

Differential proteome analysis of chikungunya virus infection on host cells.

BACKGROUND: Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach. METHODOLOGY AND PRINCIPAL FINDINGS: The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE). Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP) and cell cycle regulation. CONCLUSION/SIGNIFICANCE: This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1) regulation (in favour of virus survival, replication and transmission). While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.