Cooperation of immune regulators Tollip and surfactant protein A inhibits influenza A virus infection in mice.

BACKGROUND: Influenza A virus (IAV) infection is a significant risk factor for respiratory diseases, but the host defense mechanisms against IAV remain to be defined. Immune regulators such as surfactant protein A (SP-A) and Toll-interacting protein (Tollip) have been shown to be involved in IAV infection, but whether SP-A and Tollip cooperate in more effective host defense against IAV infection has not been investigated. METHODS: Wild-type (WT), Tollip knockout (KO), SP-A KO, and Tollip/SP-A double KO (dKO) mice were infected with IAV for four days. Lung macrophages were isolated for bulk RNA sequencing. Precision-cut lung slices (PCLS) from WT and dKO mice were pre-treated with SP-A and then infected with IAV for 48 h. RESULTS: Viral load was significantly increased in bronchoalveolar lavage (BAL) fluid of dKO mice compared to all other strains of mice. dKO mice had significantly less recruitment of neutrophils into the lung compared to Tollip KO mice. SP-A treatment of PCLS enhanced expression of TNF and reduced viral load in dKO mouse lung tissue. Pathway analysis of bulk RNA sequencing data suggests that macrophages from IAV-infected dKO mice reduced expression of genes involved in neutrophil recruitment, IL-17 signaling, and Toll-like receptor signaling. CONCLUSIONS: Our data suggests that both Tollip and SP-A are essential for the lung to exert more effective innate defense against IAV infection.

Tollip deficiency exaggerates airway type 2 inflammation in mice exposed to allergen and influenza A virus: role of the ATP/IL-33 signaling axis.

Toll-interacting protein (Tollip) is a negative regulator of the pro-inflammatory response to viruses, including influenza A virus (IAV). Genetic variation of Tollip has been associated with reduced airway epithelial Tollip expression and poor lung function in patients with asthma. Whether Tollip deficiency exaggerates type 2 inflammation (e.g., eosinophils) and viral infection in asthma remains unclear. We sought to address this critical, but unanswered question by using a Tollip deficient mouse asthma model with IAV infection. Further, we determined the underlying mechanisms by focusing on the role of the ATP/IL-33 signaling axis. Wild-type and Tollip KO mice were intranasally exposed to house dust mite (HDM) and IAV with or without inhibitors for IL-33 (i.e., soluble ST2, an IL-33 decoy receptor) and ATP signaling (i.e., an antagonist of the ATP receptor P2Y13). Tollip deficiency amplified airway type 2 inflammation (eosinophils, IL-5, IL-13 and mucins), and the release of ATP and IL-33. Blocking ATP receptor P2Y13 decreased IL-33 release during IAV infection in HDM-challenged Tollip KO mice. Furthermore, soluble ST2 attenuated airway eosinophilic inflammation in Tollip KO mice treated with HDM and IAV. HDM challenges decreased lung viral load in wild-type mice, but Tollip deficiency reduced the protective effects of HDM challenges on viral load. Our data suggests that during IAV infection, Tollip deficiency amplified type 2 inflammation and delayed viral clearance, in part by promoting ATP signaling and subsequent IL-33 release. Our findings may provide several therapeutic targets, including ATP and IL-33 signaling inhibition for attenuating excessive airway type 2 inflammation in human subjects with Tollip deficiency and IAV infection.

Parkin Promotes Airway Inflammatory Response to Interferon Gamma.

PURPOSE: Increased type 2 interferon (i.e., IFN-γ) signaling has been shown to be involved in airway inflammation in a subset of asthma patients who often show high levels of airway neutrophilic inflammation and poor response to corticosteroid treatment. How IFN-γ mediates airway inflammation in a mitochondrial dysfunction setting (e.g., Parkin up-regulation) remains poorly understood. The goal of this study was to determine the role of Parkin, an E3 ubiquitin ligase, in IFN-γ-mediated airway inflammation and the regulation of Parkin by IFN-γ. METHODS: A mouse model of IFN-γ treatment in wild-type and Parkin knockout mice, and cultured human primary airway epithelial cells with or without Parkin gene deficiency were used. RESULTS: Parkin was found to be necessary for the production of neutrophil chemokines (i.e., LIX and IL-8) and airway neutrophilic inflammation following IFN-γ treatment. Mechanistically, Parkin was induced by IFN-γ treatment both in vivo and in vitro, which was associated with less expression of a Parkin transcriptional repressor Thap11. Overexpression of Thap11 inhibited Parkin expression in IFN-γ-stimulated airway epithelial cells. CONCLUSIONS: Our data suggest a novel mechanism by which IFN-γ induces airway neutrophilic inflammation through the Thap11/Parkin axis. Inhibition of Parkin expression or activity may provide a new therapeutic target for the treatment of excessive neutrophilic inflammation in an IFN-γ-high environment.

Desert particulate matter from Afghanistan increases airway obstruction in human distal lungs exposed to type 2 cytokine IL-13.

Introduction: Deployment related asthma-like symptoms including distal airway obstruction have been described in U.S. military personnel who served in Iraq and Afghanistan. The mechanisms responsible for the development of distal airway obstruction in deployers exposed to desert particulate matter (PM) is not well understood. We sought to determine if respiratory exposure to PM from Afghanistan (PMa) increases human distal airway hyperresponsiveness (AHR) with or without exposures to IL-13, a type 2 cytokine. We further tested whether mitochondrial dysfunction, such as ATP signaling and oxidative stress, may contribute to PMa- mediated AHR. Methods: Precision-cut lung slices from donors without a history of lung disease, tobacco smoking, or vaping were pre-treated with IL-13 for 24 h. This was followed by exposure to PMa or PM from California (PMc, control for PMa) for up to 72 h. The role of hydrogen peroxide and ATP in AHR was assessed using the antioxidant enzyme catalase or an ATP receptor P2Y13 antagonist MRS2211. AHR in response to methacholine challenges as well as cytokine IL-8 production were measured. Results: PMa alone, but not PMc alone, trended to increase AHR. Importantly, the combination of PMa and IL-13 significantly amplified AHR compared to control or PMc+IL-13. PMa alone and in combination with IL-13 increased IL-8 as compared to the control. PMa increased H2O2 and ATP. MRS211 and catalase reduced AHR in PCLS exposed to both PMa and IL-13. Discussion: Our data suggests that PMa in a type 2 inflammation-high lung increased AHR in part through oxidative stress and ATP signaling.

Single cell RNA-sequencing of human precision-cut lung slices: A novel approach to study the effect of vaping and viral infection on lung health.

Vaping is an increasing health threat in the US and worldwide. The damaging impact of vaping on the human distal lung has been highlighted by the recent epidemic of electronic cigarette or vaping use-associated lung injury (EVALI). The pathogenesis of EVALI remains incompletely understood, due to a paucity of models that recapitulate the structural and functional complexity of the human distal lung and the still poorly defined culprit exposures to vaping products and respiratory viral infections. Our aim was to establish the feasibility of using single cell RNA-sequencing (scRNA-seq) technology in human precision-cut lung slices (PCLS) as a more physiologically relevant model to better understand how vaping regulates the antiviral and pro-inflammatory response to influenza A virus infection. Normal healthy donor PCLS were treated with vaping extract and influenza A viruses for scRNA-seq analysis. Vaping extract augmented host antiviral and pro-inflammatory responses in structural cells such as lung epithelial cells and fibroblasts, as well as in immune cells such as macrophages and monocytes. Our findings suggest that human distal lung slice model is useful to study the heterogeneous responses of immune and structural cells under EVALI conditions, such as vaping and respiratory viral infection.

High-fat diet and palmitic acid amplify airway type 2 inflammation.

Introduction: Metabolic dysfunction such as elevated levels of saturated fatty acids (SFA) may play a role in obese asthma, but its contribution to airway inflammation remains unclear. We sought to determine the role of high-fat diet (HFD) and palmitic acid (PA), a major form of SFA, in regulating type 2 inflammation. Methods: Airway samples from asthma patients with or without obesity, mouse models and human airway epithelial cell culture were utilized to test if SFA amplify type 2 inflammation. Results: Asthma patients with obesity had higher levels of airway PA than asthma patients without obesity. HFD increased the levels of PA in mice, and subsequently enhanced IL-13-induced airway eosinophilic inflammation. PA treatment amplified airway eosinophilic inflammation in mice that were previously exposed to IL-13 or house dust mite. IL-13 alone or in combination with PA increased dipeptidyl peptidase 4 (DPP4) release (soluble DPP4) and/or activity in mouse airways and human airway epithelial cells. Inhibition of DPP4 activity by linagliptin in mice pre-exposed to IL-13 or both IL-13 and PA increased airway eosinophilic and neutrophilic inflammation. Discussion: Our results demonstrated the exaggerating effect of obesity or PA on airway type 2 inflammation. Up-regulation of soluble DPP4 by IL-13 and/or PA may serve as a mechanism to prevent excessive type 2 inflammation. Soluble DPP4 may have the therapeutic potential in asthma patients with obesity who have an endotype with mixed airway eosinophilic and neutrophilic inflammation.

Parkin promotes airway inflammatory response to interferon gamma.

Background: Increased type 2 interferon (i.e., IFN-γ) signaling has been shown to be involved in airway inflammation in a subset of asthma patients who often show high levels of airway neutrophilic inflammation and poor response to corticosteroid treatment. How IFN-γ mediates airway inflammation in a mitochondrial dysfunction setting (e.g., Parkin up-regulation) remains poorly understood. The goal of this study was to determine the role of Parkin, an E3 ubiquitin ligase, in IFN-γ-mediated airway inflammation and the regulation of Parkin by IFN-γ. Results: Using a mouse model of IFN-γ treatment in wild-type and Parkin knockout mice, and cultured human primary airway epithelial cells with or without Parkin gene deficiency, we found that Parkin was necessary for the production of neutrophil chemokines (i.e., KC and IL-8) and airway neutrophilic inflammation. Mechanistically, Parkin was induced by IFN-γ treatment both in vivo and in vitro, which was associated with less expression of a Parkin transcriptional repressor Thap11. Overexpression of Thap11 inhibited Parkin expression in IFN-γ-stimulated airway epithelial cells. Conclusions: Our data suggests a novel mechanism by which IFN-γ induces airway neutrophilic inflammation through the Thap11/Parkin axis. Inhibition of Parkin expression or activity may provide a new therapeutic target for the treatment of excessive neutrophilic inflammation in an IFN-γ high environment.

Electronic Cigarette Exposure Increases the Severity of Influenza a Virus Infection via TRAIL Dysregulation in Human Precision-Cut Lung Slices.

The use of electronic nicotine dispensing systems (ENDS), also known as electronic cigarettes (ECs), is common among adolescents and young adults with limited knowledge about the detrimental effects on lung health such as respiratory viral infections and underlying mechanisms. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a protein of the TNF family involved in cell apoptosis, is upregulated in COPD patients and during influenza A virus (IAV) infections, but its role in viral infection during EC exposures remains unclear. This study was aimed to investigate the effect of ECs on viral infection and TRAIL release in a human lung precision-cut lung slices (PCLS) model, and the role of TRAIL in regulating IAV infection. PCLS prepared from lungs of nonsmoker healthy human donors were exposed to EC juice (E-juice) and IAV for up to 3 days during which viral load, TRAIL, lactate dehydrogenase (LDH), and TNF-α in the tissue and supernatants were determined. TRAIL neutralizing antibody and recombinant TRAIL were utilized to determine the contribution of TRAIL to viral infection during EC exposures. E-juice increased viral load, TRAIL, TNF-α release and cytotoxicity in IAV-infected PCLS. TRAIL neutralizing antibody increased tissue viral load but reduced viral release into supernatants. Conversely, recombinant TRAIL decreased tissue viral load but increased viral release into supernatants. Further, recombinant TRAIL enhanced the expression of interferon-ß and interferon-λ induced by E-juice exposure in IAV-infected PCLS. Our results suggest that EC exposure in human distal lungs amplifies viral infection and TRAIL release, and that TRAIL may serve as a mechanism to regulate viral infection. Appropriate levels of TRAIL may be important to control IAV infection in EC users.

Bacterial DNA amplifies neutrophilic inflammation in IL-17-exposed airways.

Background: Neutrophilic asthma (NA) is associated with increased airway interleukin (IL)-17 and abnormal bacterial community such as dominance of nontypeable Haemophilus influenzae (NTHi), particularly during asthma exacerbations. Bacteria release various products including DNA, but whether they cooperate with IL-17 in exaggerating neutrophilic inflammation is unclear. We sought to investigate the role of bacteria-derived DNA in airway neutrophilic inflammation related to IL-17-high asthma and underlying mechanisms (e.g. Toll-like receptor 9 (TLR9)/IL-36γ signalling axis). Methods: Bacterial DNA, IL-8 and IL-36γ were measured in bronchoalveolar lavage fluid (BALF) of people with asthma and healthy subjects. The role of co-exposure to IL-17 and bacterial DNA or live bacteria in neutrophilic inflammation, and the contribution of the TLR9/IL-36γ signalling axis, were determined in cultured primary human airway epithelial cells and alveolar macrophages, and mouse models. Results: Bacterial DNA levels were increased in asthma BALF, which positively correlated with IL-8 and neutrophil levels. Moreover, IL-36γ increased in BALF of NA patients. Bacterial DNA or NTHi infection under an IL-17-high setting amplified IL-8 production and mouse lung neutrophilic inflammation. DNase I treatment in IL-17-exposed and NTHi-infected mouse lungs reduced neutrophilic inflammation. Mechanistically, bacterial DNA-mediated amplification of neutrophilic inflammation is in part dependent on the TLR9/IL-36γ signalling axis. Conclusions: Bacterial DNA amplifies airway neutrophilic inflammation in an IL-17-high setting partly through the TLR9 and IL-36γ signalling axis. Our novel findings may offer several potential therapeutic targets including TLR9 antagonists, IL-36γ neutralising antibodies and DNase I to reduce asthma severity associated with exaggerated airway neutrophilic inflammation.

Role of Myeloid Cell-Specific TLR9 in Mitochondrial DNA-Induced Lung Inflammation in Mice.

Mitochondrial dysfunction is common in various pathological conditions including obesity. Release of mitochondrial DNA (mtDNA) during mitochondrial dysfunction has been shown to play a role in driving the pro-inflammatory response in leukocytes including macrophages. However, the mechanisms by which mtDNA induces leukocyte inflammatory responses in vivo are still unclear. Moreover, how mtDNA is released in an obese setting has not been well understood. By using a mouse model of TLR9 deficiency in myeloid cells (e.g., macrophages), we found that TLR9 signaling in myeloid cells was critical to mtDNA-mediated pro-inflammatory responses such as neutrophil influx and chemokine production. mtDNA release by lung macrophages was enhanced by exposure to palmitic acid (PA), a major saturated fatty acid related to obesity. Moreover, TLR9 contributed to PA-mediated mtDNA release and inflammatory responses. Pathway analysis of RNA-sequencing data in TLR9-sufficient lung macrophages revealed the up-regulation of axon guidance molecule genes and down-regulation of metabolic pathway genes by PA. However, in TLR9-deficient lung macrophages, PA down-regulated axon guidance molecule genes, but up-regulated metabolic pathway genes. Our results suggest that mtDNA utilizes TLR9 signaling in leukocytes to promote lung inflammatory responses in hosts with increased PA. Moreover, TLR9 signaling is involved in the regulation of axon guidance and metabolic pathways in lung macrophages exposed to PA.

Airway epithelial immunoproteasome subunit LMP7 protects against rhinovirus infection.

Immunoproteasomes (IP) serve as an important modulator of immune responses to pathogens and other pathological factors. LMP7/ß5i, one of the IP subunits, plays a critical role in autoimmune diseases by downregulating inflammation. Rhinovirus (RV) infection is a major risk factor in the exacerbations of respiratory inflammatory diseases, but whether LMP7 regulates RV-mediated inflammation in the lung particularly in the airway epithelium, the first line of defense against RV infection, remains unclear. In this study, we determined whether airway epithelial LMP7 promotes the resolution of RV-mediated lung inflammation. Inducible airway epithelial-specific LMP7-deficient (conditional knockout, CKO) mice were generated to reveal the in vivo anti-inflammatory and antiviral functions of LMP7. By using LMP7-deficient primary human airway epithelial cells generated by CRISPR-Cas9, we confirmed that airway epithelial LMP7 decreased pro-inflammatory cytokines and viral load during RV infection. Additionally, airway epithelial LMP7 enhanced the expression of a negative immune regulator A20/TNFAIP3 during viral infection that may contribute to the anti-inflammatory function of LMP7. We also discovered that induction of LMP7 by a low dose of polyinosinic:polycytidylic acid (PI:C) reduced RV-mediated inflammation in our CKO mice infected with RV. Our findings suggest that airway epithelial LMP7 has anti-inflammatory and antiviral functions that is critical to the resolution of RV-mediated lung inflammation. Induction of airway epithelial LMP7 may open a novel avenue for therapeutic intervention against RV infection.

Electronic cigarette vapor exposure exaggerates the pro-inflammatory response during influenza A viral infection in human distal airway epithelium.

Electronic cigarettes or vaping products have been marketed as a safer alternative to smoking, but very little is known about the health effects in the human lung, particularly in the distal airways, a key site of airway obstruction and destruction in chronic obstructive pulmonary disease that is often exacerbated by viral infections. The aim of this study was to investigate the effects of electronic cigarette vapor (e-vapor) on human distal airway epithelial responses to influenza A virus (IAV) infection. We isolated primary small airway epithelial cells (SAECs) from donor lungs free of lung disease, and cultured them at air-liquid interface (ALI). To measure markers of epithelial injury such as integrity of epithelial barrier structure and function, we selected a regimen of non-toxic, barrier preserving e-vapor exposure of cultured cells to 15 puffs of e-vapor from a commercially available e-cigarette once per day for 3 days, prior to IAV infection. After 72 h of infection, media and cell lysates were collected to measure cytokines involved in inflammatory and antiviral responses. Pre-exposure to e-vapor with IAV infection, compared to IAV infection alone, significantly increased inflammatory and antiviral mediators including IL-8, CXCL10, IFN-beta, and MX1. Our results suggest that e-vapor exposure amplifies human distal airway pro-inflammatory response to IAV infection, independently of the severity of cell injury during viral infection.

Human Bronchial Epithelial Cell Culture Models for Cigarette Smoke and Vaping Studies.

Despite the continuing public health efforts to stop or reduce smoking, cigarette smoke use remains popular in the youth and adult population. A recent surge in the use of electronic cigarette and vaping products has created another major health challenge in public health. There is an urgent need to use physiologically relevant models to study the health effect of smoking or vaping in human subjects. Airway diseases such as bronchitis (Landman et al., CMAJ 191:E1321-E1331, 2019; Goniewicz, et al. Harm Reduct J 17:91, 2020; Xie et al., JAMA Netw Open 3:e2020816, 2020) have been described in people who smoke, vape, or both. Here, we will describe methods to collect, expand, and culture human airway epithelial cells from endobronchial brushings and expose these cells cultured at the air-liquid interface to cigarette smoke or electronic cigarette vapor.

Tollip Inhibits IL-33 Release and Inflammation in Influenza A Virus-Infected Mouse Airways.

Respiratory influenza A virus (IAV) infection continues to pose significant challenges in healthcare of human diseases including asthma. IAV infection in mice was shown to increase IL-33, a key cytokine in driving airway inflammation in asthma, but how IL-33 is regulated during viral infection remains unclear. We previously found that a genetic mutation in Toll-interacting protein (Tollip) was linked to less airway epithelial Tollip expression, increased neutrophil chemokines, and lower lung function in asthma patients. As Tollip is involved in maintaining mitochondrial function, and mitochondrial stress may contribute to extracellular ATP release and IL-33 secretion, we hypothesized that Tollip downregulates IL-33 secretion via inhibiting ATP release during IAV infection. Wild-type and Tollip knockout (KO) mice were infected with IAV and treated with either an ATP converter apyrase or an IL-33 decoy receptor soluble ST2 (sST2). KO mice significantly lost more body weight and had increased extracellular ATP, IL-33 release, and neutrophilic inflammation. Apyrase treatment reduced extracellular ATP levels, IL-33 release, and neutrophilic inflammation in Tollip KO mice. Excessive lung neutrophilic inflammation in IAV-infected Tollip KO mice was reduced by sST2, which was coupled with less IL-33 release. Our data suggest that Tollip inhibits IAV infection, potentially by inhibiting extracellular ATP release and reducing IL-33 activation and lung inflammation. In addition, sST2 may serve as a potential therapeutic approach to mitigate respiratory viral infection in human subjects with Tollip deficiency.

Tollip interaction with STAT3: a novel mechanism to regulate human airway epithelial responses to type 2 cytokines.

BACKGROUND: Toll-interacting protein (Tollip) is one of the key negative regulators in host innate immunity. Genetic variation of Tollip has been associated with less Tollip expression and poor lung function in asthmatic patients, but little is known about the role of Tollip in human airway type 2 inflammatory response, a prominent feature in allergic asthma. OBJECTIVE: Our goal was to determine the role and underlying mechanisms of Tollip in human airway epithelial responses such as eotaxin to type 2 cytokine IL-13. METHODS: Tollip deficient primary human airway epithelial cells from 4 healthy donors were generated by the gene knockdown approach and stimulated with IL-13 to measure activation of transcription factor STAT3, and eotaxin-3, an eosinophilic chemokine. RESULTS: Following IL-13 treatment, Tollip deficient cells had significantly higher levels of STAT3 activation and eotaxin-3 than the scrambled control counterpart, which was reduced by a STAT3 inhibitor. Interaction between Tollip and STAT3 proteins was identified by co-immunoprecipitation. CONCLUSION: Our results, for the first time, suggest that Tollip inhibits excessive eotaxin-3 induction by IL-13, in part through the interaction and inhibition of STAT3. These findings lend evidence to the potential of a STAT3 inhibitor as a therapeutic target, especially for type 2 inflammation-high asthmatics with Tollip deficiency.

Parkin, an E3 ubiquitin ligase, enhances airway mitochondrial DNA release and inflammation.

INTRODUCTION: Parkin (Park2), an E3 ubiquitin ligase, is critical to maintain mitochondrial function by regulating mitochondrial biogenesis and degradation (mitophagy), but recent evidence suggests the involvement of Parkin in promoting inflammation. In the present study, we determined if Parkin regulates airway mitochondrial DNA (mtDNA) release and inflammatory responses to type 2 cytokine interleukin (IL)-13 and allergens. METHODS: We measured Parkin mRNA expression in brushed bronchial epithelial cells and mtDNA release in the paired bronchoalveolar lavage fluid (BALF) from normal subjects and asthmatics. Parkin-deficient primary human tracheobronchial epithelial (HTBE) cells generated using the CRISPR-Cas9 system were stimulated with IL-13. To determine the in vivo function of Parkin, Parkin knockout (PKO) and wild-type (WT) mice were treated with IL-13 or allergen (house dust mite, HDM) in the presence or absence of mtDNA isolated from normal mouse lungs. RESULTS: Parkin mRNA expression in asthmatic airway epithelium was upregulated, which positively correlated with the levels of released mtDNA in BALF. IL-13-stimulated HTBE cells increased Parkin expression. Moreover, IL-13 induced mtDNA release in Parkin-sufficient, but not in Parkin-deficient HTBE cells. PKO (vs WT) mice attenuated airway mtDNA release and inflammation following IL-13 or HDM treatments. mtDNA amplified airway inflammation in mice treated with IL-13 or HDM. Notably, Parkin also mediated mtDNA-induced exacerbation of airway inflammation. CONCLUSION: Our research findings suggest that Parkin promotes mtDNA release and inflammation in airways, thus improving our understanding of the complex role of Parkin and mitochondrial dysfunction in asthma pathogenesis.

Interleukin 1 Receptor-Like 1 (IL1RL1) Promotes Airway Bacterial and Viral Infection and Inflammation.

Interleukin 1 receptor-like 1 (IL1RL1), also known as suppression of tumorigenicity 2 (ST2), is the receptor for interleukin 33 (IL-33) and has been increasingly studied in type 2 inflammation. An increase in airway IL-33/ST2 signaling in asthma has been associated with eosinophilic inflammation, but little is known about the role of ST2 in neutrophilic inflammation. Airway Mycoplasma pneumoniae and human rhinovirus (HRV) infections are linked to neutrophilic inflammation during acute exacerbations of asthma. However, whether ST2 contributes to M. pneumoniae- and HRV-mediated airway inflammation is poorly understood. The current study sought to determine the functions of ST2 during airway M. pneumoniae or HRV infection. In cultured normal human primary airway epithelial cells, ST2 overexpression (OE) increased the production of neutrophilic chemoattractant IL-8 in the absence or presence of M. pneumoniae or HRV1B infection. ST2 OE also enhanced HRV1B-induced IP-10, a chemokine involved in asthma exacerbations. In the M. pneumoniae-infected mouse model, ST2 deficiency, in contrast to sufficiency, significantly reduced the levels of neutrophils following acute (≤24 h) infection, while in the HRV1B-infected mouse model, ST2 deficiency significantly reduced the levels of proinflammatory cytokines KC, IP-10, and IL-33 in bronchoalveolar lavage (BAL) fluid. Overall, ST2 overexpression in human epithelial cells and ST2 sufficiency in mice increased the M. pneumoniae and HRV loads in cell supernatants and BAL fluid. After pathogen infection, ST2-deficient mice showed a higher level of the host defense protein lactotransferrin in BAL fluid. Our data suggest that ST2 promotes proinflammatory responses (e.g., neutrophils) to airway bacterial and viral infection and that blocking ST2 signaling may broadly attenuate airway infection and inflammation.