Liraglutide pretreatment attenuates sepsis-induced acute lung injury.

There are no effective targeted therapies to treat acute respiratory distress syndrome (ARDS). Recently, the commonly used diabetes and obesity medications, glucagon-like peptide-1 (GLP-1) receptor agonists, have been found to have anti-inflammatory properties. We, therefore, hypothesized that liraglutide pretreatment would attenuate murine sepsis-induced acute lung injury (ALI). We used a two-hit model of ALI (sepsis+hyperoxia). Sepsis was induced by intraperitoneal injection of cecal slurry (CS; 2.4 mg/g) or 5% dextrose (control) followed by hyperoxia [HO; fraction of inspired oxygen ([Formula: see text]) = 0.95] or room air (control; [Formula: see text] = 0.21). Mice were pretreated twice daily with subcutaneous injections of liraglutide (0.1 mg/kg) or saline for 3 days before initiation of CS+HO. At 24-h post CS+HO, physiological dysfunction was measured by weight loss, severity of illness score, and survival. Animals were euthanized, and bronchoalveolar lavage (BAL) fluid, lung, and spleen tissues were collected. Bacterial burden was assessed in the lung and spleen. Lung inflammation was assessed by BAL inflammatory cell numbers, cytokine concentrations, lung tissue myeloperoxidase activity, and cytokine expression. Disruption of the alveolar-capillary barrier was measured by lung wet-to-dry weight ratios, BAL protein, and epithelial injury markers (receptor for advanced glycation end products and sulfated glycosaminoglycans). Histological evidence of lung injury was quantified using a five-point score with four parameters: inflammation, edema, septal thickening, and red blood cells (RBCs) in the alveolar space. Compared with saline treatment, liraglutide improved sepsis-induced physiological dysfunction and reduced lung inflammation, alveolar-capillary barrier disruption, and lung injury. GLP-1 receptor activation may hold promise as a novel treatment strategy for sepsis-induced ARDS. Additional studies are needed to better elucidate its mechanism of action.NEW & NOTEWORTHY In this study, pretreatment with liraglutide, a commonly used diabetes medication and glucagon-like peptide-1 (GLP-1) receptor agonist, attenuated sepsis-induced acute lung injury in a two-hit mouse model (sepsis + hyperoxia). Septic mice who received the drug were less sick, lived longer, and displayed reduced lung inflammation, edema, and injury. These therapeutic effects were not dependent on weight loss. GLP-1 receptor activation may hold promise as a new treatment strategy for sepsis-induced acute respiratory distress syndrome.

COX Inhibition Increases Alternaria-Induced Pulmonary Group 2 Innate Lymphoid Cell Responses and IL-33 Release in Mice.

The cyclooxygenase (COX) metabolic pathway regulates immune responses and inflammation. The effect of the COX pathway on innate pulmonary inflammation induced by protease-containing fungal allergens, such as Alternaria alternata, is not fully defined. In this study, we tested the hypothesis that COX inhibition augments Alternaria-induced pulmonary group 2 innate lymphoid cell (ILC2) responses and IL-33 release. Mice were treated with the COX inhibitors indomethacin, flurbiprofen, or vehicle and challenged intranasally with Alternaria extract for four consecutive days to induce innate lung inflammation. We found that indomethacin and flurbiprofen significantly increased the numbers of ILC2 and IL-5 and IL-13 expression by ILC2 in the lung. Indomethacin also increased ILC2 proliferation, the percentages of eosinophils, and mucus production in the lung. Both indomethacin and flurbiprofen augmented the release of IL-33 in bronchoalveolar lavage fluid after Alternaria challenge, suggesting that more IL-33 was available for ILC2 activation and that a COX product(s) inhibited IL-33 release. This is supported by the in vitro finding that the COX product PGE2 and the PGI2 analogs cicaprost decreased Alternaria extract-induced IL-33 release by human bronchial epithelial cells. Although contrasting effects of PGD2, PGE2, and PGI2 on ILC2 responses have been previously reported, the overall effect of the COX pathway on ILC2 function is inhibitory in Alternaria-induced innate airway inflammation.

Glucagon-like peptide-1 receptor agonist inhibits aeroallergen-induced activation of ILC2 and neutrophilic airway inflammation in obese mice.

BACKGROUND: Obesity is a risk factor for the development of asthma. However, pharmacologic therapeutic strategies that specifically target obese asthmatics have not been identified. We hypothesize that glucagon-like peptide-1 receptor agonist (GLP-1RA) treatment inhibits aeroallergen-induced early innate airway inflammation in a mouse model of asthma in the setting of obesity. METHODS: SWR (lean) and TALLYHO (obese) mice were challenged intranasally with Alternaria alternata extract (Alt-Ext) or PBS for 4 consecutive days concurrent with GLP-1RA or vehicle treatment. RESULTS: TALLYHO mice had greater Alt-Ext-induced airway neutrophilia and lung protein expression of IL-5, IL-13, CCL11, CXCL1, and CXCL5, in addition to ICAM-1 expression on lung epithelial cells compared with SWR mice, and all endpoints were reduced by GLP-1RA treatment. Alt-Ext significantly increased BALF IL-33 in both TALLYHO and SWR mice compared to PBS challenge, but there was no difference in the BALF IL-33 levels between these two strains. However, TALLYHO, but not SWR, mice had significantly higher airway TSLP in BALF following Alt-Ext challenge compared to PBS, and BALF TSLP was significantly greater in TALLYHO mice compared to SWR mice following airway Alt-Ext challenge. GLP-1RA treatment significantly decreased the Alt-Ext-induced TSLP and IL-33 release in TALLYHO mice. While TSLP or ST2 inhibition with a neutralizing antibody decreased airway eosinophils, they did not reduce airway neutrophils in TALLYHO mice. CONCLUSIONS: These results suggest that GLP-1RA treatment may be a novel pharmacologic therapeutic strategy for obese persons with asthma by inhibiting aeroallergen-induced neutrophilia, a feature not seen with either TSLP or ST2 inhibition.

TSLP and IL-33 reciprocally promote each other's lung protein expression and ILC2 receptor expression to enhance innate type-2 airway inflammation.

BACKGROUND: The epithelial cell-derived danger signal mediators thymic stromal lymphopoietin (TSLP) and IL-33 are consistently associated with adaptive Th2 immune responses in asthma. In addition, TSLP and IL-33 synergistically promoted group 2 innate lymphoid cell (ILC2) activation to induce innate allergic inflammation. However, the mechanism of this synergistic ILC2 activation is unknown. METHODS: BALB/c WT and TSLP receptor-deficient (TSLPR-/- ) mice were challenged intranasally with Alternaria extract (Alt-Ext) or PBS for 4 consecutive days to evaluate innate airway allergic inflammation. WT mice pre-administered with rTSLP or vehicle, TSLPR-/- mice, and IL-33 receptor-deficient (ST2-/- ) mice were challenged intranasally with Alt-Ext or vehicle once or twice to evaluate IL-33 release and TSLP expression in the lung. TSLPR and ST2 expression on lung ILC2 were measured by flow cytometry after treatment of rTSLP, rIL-33, rTSLP + rIL-33, or vehicle. RESULTS: Thymic stromal lymphopoietin receptor deficient mice had significantly decreased the number of lung ILC2 expressing IL-5 and IL-13 following Alt-Ext-challenge compared to WT mice. Further, eosinophilia, protein level of lung IL-4, IL-5, and IL-13, and airway mucus score were also significantly decreased in TSLPR-/- mice compared to WT mice. Endogenous and exogenous TSLP increased Alt-Ext-induced IL-33 release into BALF, and ST2 deficiency decreased Alt-Ext-induced TSLP expression in the lung. Further, rTSLP and rIL-33 treatment reciprocally increased each other's receptor expression on lung ILC2 in vivo and in vitro. CONCLUSION: Thymic stromal lymphopoietin and IL-33 signaling reciprocally enhanced each other's protein release and expression in the lung following Alt-Ext-challenge and each other's receptor expression on lung ILC2 to enhance ILC2 activation.

The PGI2 Analog Cicaprost Inhibits IL-33-Induced Th2 Responses, IL-2 Production, and CD25 Expression in Mouse CD4+ T Cells.

IL-33 has pleiotropic functions in immune responses and promotes the development of allergic diseases and asthma. IL-33 induces Th2 differentiation and enhances type 2 cytokine production by CD4+ T cells. However, the regulation of IL-33-driven type 2 cytokine responses is not fully defined. In this study, we investigated the effect of PGI2, a lipid mediator formed in the cyclooxygenase pathway of arachidonic acid metabolism, on naive CD4+ T cell activation, proliferation, and differentiation by IL-33. Using wild-type and PGI2 receptor (IP) knockout mice, we found that the PGI2 analog cicaprost dose-dependently inhibited IL-33-driven IL-4, IL-5, and IL-13 production by CD4+ T cells in an IP-specific manner. In addition, cicaprost inhibited IL-33-driven IL-2 production and CD25 expression by CD4+ T cells. Furthermore, IP knockout mice had increased IL-5 and IL-13 responses of CD4+ T cells to Alternaria sensitization and challenge in mouse lungs. Because IL-33 is critical for Alternaria-induced type 2 responses, these data suggest that PGI2 not only inhibits IL-33-stimulated CD4+ Th2 cell responses in vitro but also suppresses IL-33-induced Th2 responses caused by protease-containing allergens in vivo.

Reduction in the Doses of Direct Oral Anticoagulants and Risk of Ischemic Stroke Events: A Hospital Survey.

Inappropriately reduced doses (IRDs) of direct oral anticoagulants (DOACs) are common in clinical practice. We performed a retrospective review using electronic medical records of St. Marianna University School of Medicine Hospital (a 1200-bed teaching hospital in Japan) to address the prevalence of IRDs and patient-related factors that result in IRDs. We also surveyed DOAC-treated patients who were hospitalized due to a stroke during the 5-year study period to analyze the association between stroke events and IRDs. We found that one in five patients who were newly prescribed a DOAC was treated with IRDs. Patients treated with edoxaban received the most IRDs (64%, 7/11), followed by those treated with dabigatran (50%, 1/2), apixaban (32%, 19/61), and rivaroxaban (27%, 12/44). Our analysis showed that the renal function (measured as serum creatinine and creatinine clearance values) and age are possible factors influencing dose reduction. The HAS-BLED score and antiplatelet use were not associated with IRD prescription. An analysis of the 5-year hospital records revealed 20 stroke cases despite ongoing treatments with DOACs, and IRDs were noted in three of these cases. In all three cases, the patients had been on an IRD of rivaroxaban. To prevent IRDs of DOACs, we suggest that a clinical protocol be incorporated into formularies to support the prescription process.

The Innate Immune Protein S100A9 Protects from T-Helper Cell Type 2-mediated Allergic Airway Inflammation.

Calprotectin is a heterodimer of the proteins S100A8 and S100A9, and it is an abundant innate immune protein associated with inflammation. In humans, calprotectin transcription and protein abundance are associated with asthma and disease severity. However, mechanistic studies in experimental asthma models have been inconclusive, identifying both protective and pathogenic effects of calprotectin. To clarify the role of calprotectin in asthma, calprotectin-deficient S100A9-/- and wild-type (WT) C57BL/6 mice were compared in a murine model of allergic airway inflammation. Mice were intranasally challenged with extracts of the clinically relevant allergen, Alternaria alternata (Alt Ext), or PBS every third day over 9 days. On Day 10, BAL fluid and lung tissue homogenates were harvested and allergic airway inflammation was assessed. Alt Ext challenge induced release of S100A8/S100A9 to the alveolar space and increased protein expression in the alveolar epithelium of WT mice. Compared with WT mice, S100A9-/- mice displayed significantly enhanced allergic airway inflammation, including production of IL-13, CCL11, CCL24, serum IgE, eosinophil recruitment, and airway resistance and elastance. In response to Alt Ext, S100A9-/- mice accumulated significantly more IL-13+IL-5+CD4+ T-helper type 2 cells. S100A9-/- mice also accumulated a significantly lower proportion of CD4+ T regulatory (Treg) cells in the lung that had significantly lower expression of CD25. Calprotectin enhanced WT Treg cell suppressive activity in vitro. Therefore, this study identifies a role for the innate immune protein, S100A9, in protection from CD4+ T-helper type 2 cell hyperinflammation in response to Alt Ext. This protection is mediated, at least in part, by CD4+ Treg cell function.

Glucagon-like peptide 1 signaling inhibits allergen-induced lung IL-33 release and reduces group 2 innate lymphoid cell cytokine production in vivo.

BACKGROUND: IL-33 is one of the most consistently associated gene candidates for asthma identified by using a genome-wide association study. Studies in mice and in human cells have confirmed the importance of IL-33 in inducing type 2 cytokine production from both group 2 innate lymphoid cells (ILC2s) and TH2 cells. However, there are no pharmacologic agents known to inhibit IL-33 release from airway cells. OBJECTIVE: We sought to determine the effect of glucagon-like peptide 1 receptor (GLP-1R) signaling on aeroallergen-induced airway IL-33 production and release and on innate type 2 airway inflammation. METHODS: BALB/c mice were challenged intranasally with Alternaria extract for 4 consecutive days. GLP-1R agonist or vehicle was administered starting either 2 days before the first Alternaria extract challenge or 1 day after the first Alternaria extract challenge. RESULTS: GLP-1R agonist treatment starting 2 days before the first Alternaria extract challenge decreased IL-33 release in the bronchoalveolar lavage fluid and dual oxidase 1 (Duox1) mRNA expression 1 hour after the first Alternaria extract challenge and IL-33 expression in lung epithelial cells 24 hours after the last Alternaria extract challenge. Furthermore, GLP-1R agonist significantly decreased the number of ILC2s expressing IL-5 and IL-13, lung protein expression of type 2 cytokines and chemokines, the number of perivascular eosinophils, mucus production, and airway responsiveness compared with vehicle treatment. GLP-1R agonist treatment starting 1 day after the first Alternaria extract challenge also significantly decreased eosinophilia and type 2 cytokine and chemokine expression in the airway after 4 days of Alternaria extract challenge. CONCLUSION: These results reveal that GLP-1R signaling might be a therapy to reduce IL-33 release and inhibit the ILC2 response to protease-containing aeroallergens, such as Alternaria.

Endogenous PGI2 signaling through IP inhibits neutrophilic lung inflammation in LPS-induced acute lung injury mice model.

Endogenous prostaglandin I2 (PGI2) has inhibitory effects on immune responses against pathogens or allergens; however, the immunomodulatory activity of endogenous PGI2 signaling in endotoxin-induced inflammation is unknown. To test the hypothesis that endogenous PGI2 down-regulates endotoxin-induced lung inflammation, C57BL/6 wild type (WT) and PGI2 receptor (IP) KO mice were challenged intranasally with LPS. Urine 6-keto-PGF1α, a stable metabolite of PGI2, was significantly increased following the LPS-challenge, suggesting that endogenous PGI2 signaling modulates the host response to LPS-challenge. IPKO mice had a significant increase in neutrophils in the BAL fluid as well as increased proteins of KC, LIX, and TNF-α in lung homogenates compared with WT mice. In contrast, IL-10 was decreased in LPS-challenged IPKO mice compared with WT mice. The PGI2 analog cicaprost significantly decreased LPS-induced KC, and TNF-α, but increased IL-10 and AREG in bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMMs) compared with vehicle-treatment. These results indicated that endogenous PGI2 signaling attenuated neutrophilic lung inflammation through the reduced inflammatory cytokine and chemokine and enhanced IL-10.

Prostaglandin I2 Suppresses Proinflammatory Chemokine Expression, CD4 T Cell Activation, and STAT6-Independent Allergic Lung Inflammation.

Allergic airway diseases are immune disorders associated with heightened type 2 immune responses and IL-5 and IL-13 production at the site of inflammation. We have previously reported that cyclooxygenase (COX) inhibition by indomethacin augmented allergic airway inflammation in a STAT6-independent manner. However, the key COX product(s) responsible for restraining indomethacin-mediated STAT6-independent allergic inflammation is unknown. In this study, using the mouse model of OVA-induced allergic airway inflammation, we identified that PGI2 receptor (IP) signaling was critical for indomethacin-induced, STAT6-independent proallergic effects. We demonstrated that IP deficiency increased inflammatory cell infiltration, eosinophilia, and IL-5 and IL-13 expression in the lung in a STAT6-independent manner. The augmented STAT6-independent allergic inflammation correlated with enhanced primary immune responses to allergic sensitization and elevated production of multiple inflammatory chemokines (CCL11, CCL17, CCL22, and CXCL12) in the lung after allergen challenge. We also showed that the PGI2 analogue cicaprost inhibited CD4 T cell proliferation and IL-5 and IL-13 expression in vitro, and IP deficiency diminished the stimulatory effect of indomethacin on STAT6-independent IL-5 and IL-13 responses in vivo. The inhibitory effects of PGI2 and the IP signaling pathway on CD4 T cell activation, inflammatory chemokine production, and allergic sensitization and airway inflammation suggest that PGI2 and its analogue iloprost, both Food and Drug Administration-approved drugs, may be useful in treating allergic diseases and asthma. In addition, inhibiting PGI2 signaling by drugs that either block PGI2 production or restrain IP signaling may augment STAT6-independent pathways of allergic inflammation.

Prostaglandin I2 Signaling and Inhibition of Group 2 Innate Lymphoid Cell Responses.

RATIONALE: Group 2 innate lymphoid cells (ILC2s) robustly produce IL-5 and IL-13, cytokines central to the asthma phenotype; however, the effect of prostaglandin (PG) I2 on ILC2 function is unknown. OBJECTIVES: To determine the effect of PGI2 on mouse and human ILC2 cytokine expression in vitro and the effect of endogenous PGI2 and the PGI2 analog cicaprost on lung ILC2s in vivo. METHODS: Flow-sorted bone marrow ILC2s of wild-type (WT) and PGI2 receptor-deficient (IP(-/-)) mice were cultured with IL-33 and treated with the PGI2 analog cicaprost. WT and IP(-/-) mice were challenged intranasally with Alternaria alternata extract for 4 consecutive days to induce ILC2 responses, and these were quantified. Prior to A. alternata extract, challenged WT mice were treated with cicaprost. Human flow-sorted peripheral blood ILC2s were cultured with IL-33 and IL-2 and treated with the PGI2 analog cicaprost. MEASUREMENT AND MAIN RESULTS: We demonstrate that PGI2 inhibits IL-5 and IL-13 protein expression by IL-33-stimulated ILC2s purified from mouse bone marrow in a manner that was dependent on signaling through the PGI2 receptor IP. In a mouse model of 4 consecutive days of airway challenge with an extract of A. alternata, a fungal aeroallergen associated with severe asthma exacerbations, endogenous PGI2 signaling significantly inhibited lung IL-5 and IL-13 protein expression, and reduced the number of lung IL-5- and IL-13-expressing ILC2s, as well as the mean fluorescence intensity of IL-5 and IL-13 staining. In addition, exogenous administration of a PGI2 analog inhibited Alternaria extract-induced lung IL-5 and IL-13 protein expression, and reduced the number of lung IL-5- and IL-13-expressing ILC2s and the mean fluorescence intensity of IL-5 and IL-13 staining. Finally, a PGI2 analog inhibited IL-5 and IL-13 expression by human ILC2s that were stimulated with IL-2 and IL-33. CONCLUSIONS: These results suggest that PGI2 may be a potential therapy to reduce the ILC2 response to protease-containing aeroallergens, such as Alternaria.

Respiratory syncytial virus infection activates IL-13-producing group 2 innate lymphoid cells through thymic stromal lymphopoietin.

BACKGROUND: Respiratory syncytial virus (RSV) is a major health care burden with a particularly high worldwide morbidity and mortality rate among infants. Data suggest that severe RSV-associated illness is in part caused by immunopathology associated with a robust type 2 response. OBJECTIVE: We sought to determine the capacity of RSV infection to stimulate group 2 innate lymphoid cells (ILC2s) and the associated mechanism in a murine model. METHODS: Wild-type (WT) BALB/c, thymic stromal lymphopoietin receptor (TSLPR) knockout (KO), or WT mice receiving an anti-TSLP neutralizing antibody were infected with the RSV strain 01/2-20. During the first 4 to 6 days of infection, lungs were collected for evaluation of viral load, protein concentration, airway mucus, airway reactivity, or ILC2 numbers. Results were confirmed with 2 additional RSV clinical isolates, 12/11-19 and 12/12-6, with known human pathogenic potential. RESULTS: RSV induced a 3-fold increase in the number of IL-13-producing ILC2s at day 4 after infection, with a concurrent increase in total lung IL-13 levels. Both thymic stromal lymphopoietin (TSLP) and IL-33 levels were increased 12 hours after infection. TSLPR KO mice did not mount an IL-13-producing ILC2 response to RSV infection. Additionally, neutralization of TSLP significantly attenuated the RSV-induced IL-13-producing ILC2 response. TSLPR KO mice displayed reduced lung IL-13 protein levels, decreased airway mucus and reactivity, attenuated weight loss, and similar viral loads as WT mice. Both 12/11-19 and 12/12-6 similarly induced IL-13-producing ILC2s through a TSLP-dependent mechanism. CONCLUSION: These data demonstrate that multiple pathogenic strains of RSV induce IL-13-producing ILC2 proliferation and activation through a TSLP-dependent mechanism in a murine model and suggest the potential therapeutic targeting of TSLP during severe RSV infection.

The histone deacetylase inhibitor trichostatin A suppresses murine innate allergic inflammation by blocking group 2 innate lymphoid cell (ILC2) activation.

BACKGROUND: Group 2 innate lymphoid cells (ILC2) are an important source of the type 2 cytokines interleukin (IL)-5 and IL-13 that are critical to the allergic airway phenotype. Previous studies reported that histone deacetylase (HDAC) inhibition by trichostatin A (TSA) downregulated adaptive allergic immune responses; however, the effect of HDAC inhibition on the early innate allergic immune response is unknown. Therefore, we investigated the effect of TSA on innate airway inflammation mediated by ILC2 activation. METHODS: BALB/c mice were challenged intranasally with Alternaria extract, exogenous recombinant mouse IL-33 (rmIL-33) or the respective vehicles for four consecutive days following TSA or vehicle treatment. Bronchoalveolar lavage (BAL) fluids and lungs were harvested 24 h after the last challenge. RESULTS: We found that TSA treatment significantly decreased the number of ILC2 expressing IL-5 and IL-13 in the lungs challenged with Alternaria extract or rmIL-33 compared with vehicle treatment (p<0.05). TSA treatment significantly decreased protein expression of IL-5, IL-13, CCL11 and CCL24 in the lung homogenates from Alternaria extract-challenged mice or rmIL-33-challenged mice compared with vehicle treatment (p<0.05). Further, TSA treatment significantly decreased the number of perivascular eosinophils and mucus production in the large airways that are critical components of the asthma phenotype (p<0.05). TSA did not change early IL-33 release in the BAL fluids; however, TSA decreased lung IL-33 expression from epithelial cells 24 h after last Alternaria extract challenge compared with vehicle treatment (p<0.05). CONCLUSIONS: These results reveal that TSA reduces allergen-induced ILC2 activation and the early innate immune responses to an inhaled protease-containing aeroallergen.

STAT4 deficiency fails to induce lung Th2 or Th17 immunity following primary or secondary respiratory syncytial virus (RSV) challenge but enhances the lung RSV-specific CD8+ T cell immune response to secondary challenge.

UNLABELLED: Immune-mediated lung injury is a hallmark of lower respiratory tract illness caused by respiratory syncytial virus (RSV). STAT4 plays a critical role in CD4+ Th1 lineage differentiation and gamma interferon (IFN-γ) protein expression by CD4+ T cells. As CD4+ Th1 differentiation is associated with negative regulation of CD4+ Th2 and Th17 differentiation, we hypothesized that RSV infection of STAT4-/- mice would result in enhanced lung Th2 and Th17 inflammation and impaired lung Th1 inflammation compared to wild-type (WT) mice. We performed primary and secondary RSV challenges in WT and STAT4-/- mice and used STAT1-/- mice as a positive control for the development of RSV-specific lung Th2 and Th17 inflammation during primary challenge. Primary RSV challenge of STAT4-/- mice resulted in decreased T-bet and IFN-γ expression levels in CD4+ T cells compared to those of WT mice. Lung Th2 and Th17 inflammation did not develop in primary RSV-challenged STAT4-/- mice. Decreased IFN-γ expression by NK cells, CD4+ T cells, and CD8+ T cells was associated with attenuated weight loss and enhanced viral clearance with primary challenge in STAT4-/- mice compared to WT mice. Following secondary challenge, WT and STAT4-/- mice also did not develop lung Th2 or Th17 inflammation. In contrast to primary challenge, secondary RSV challenge of STAT4-/- mice resulted in enhanced weight loss, an increased lung IFN-γ expression level, and an increased lung RSV-specific CD8+ T cell response compared to those of WT mice. These data demonstrate that STAT4 regulates the RSV-specific CD8+ T cell response to secondary infection but does not independently regulate lung Th2 or Th17 immune responses to RSV challenge. IMPORTANCE: STAT4 is a protein critical for both innate and adaptive immune responses to viral infection. Our results show that STAT4 regulates the immune response to primary and secondary challenge with RSV but does not restrain RSV-induced lung Th2 or Th17 immune responses. These findings suggest that STAT4 expression may influence lung immunity and severity of illness following primary and secondary RSV infections.

Cyclooxygenase inhibition abrogates aeroallergen-induced immune tolerance by suppressing prostaglandin I2 receptor signaling.

BACKGROUND: The prevalence of allergic diseases has doubled in developed countries in the past several decades. Cyclooxygenase (COX)-inhibiting drugs augmented allergic diseases in mice by increasing allergic sensitization and memory immune responses. However, whether COX inhibition can promote allergic airway diseases by inhibiting immune tolerance is not known. OBJECTIVE: To determine the role of the COX pathway and prostaglandin I2 (PGI2) signaling through the PGI2 receptor (IP) in aeroallergen-induced immune tolerance. METHODS: Wild-type (WT) BALB/c mice and IP knockout mice were aerosolized with ovalbumin (OVA) to induce immune tolerance prior to immune sensitization with an intraperitoneal injection of OVA/alum. The COX inhibitor indomethacin or vehicle was administered in drinking water to inhibit enzyme activity during the sensitization phase. Two weeks after sensitization, the mice were challenged with OVA aerosols. Mouse bronchoalveolar lavage fluid was harvested for cell counts and TH2 cytokine measurements. RESULTS: WT mice treated with indomethacin had greater numbers of total cells, eosinophils, and lymphocytes, and increased IL-5 and IL-13 protein expression in BAL fluid compared to vehicle-treated mice. Similarly, IP knockout mice had augmented inflammation and TH2 cytokine responses compared to WT mice. In contrast, the PGI2 analog cicaprost attenuated the anti-tolerance effect of COX inhibition. CONCLUSION: COX inhibition abrogated immune tolerance by suppressing PGI2 IP signaling, suggesting that PGI2 signaling promotes immune tolerance and that clinical use of COX-inhibiting drugs may increase the risk of developing allergic diseases.

Allergic airway inflammation decreases lung bacterial burden following acute Klebsiella pneumoniae infection in a neutrophil- and CCL8-dependent manner.

The Th17 cytokines interleukin-17A (IL-17A), IL-17F, and IL-22 are critical for the lung immune response to a variety of bacterial pathogens, including Klebsiella pneumoniae. Th2 cytokine expression in the airways is a characteristic feature of asthma and allergic airway inflammation. The Th2 cytokines IL-4 and IL-13 diminish ex vivo and in vivo IL-17A protein expression by Th17 cells. To determine the effect of IL-4 and IL-13 on IL-17-dependent lung immune responses to acute bacterial infection, we developed a combined model in which allergic airway inflammation and lung IL-4 and IL-13 expression were induced by ovalbumin sensitization and challenge prior to acute lung infection with K. pneumoniae. We hypothesized that preexisting allergic airway inflammation decreases lung IL-17A expression and airway neutrophil recruitment in response to acute K. pneumoniae infection and thereby increases the lung K. pneumoniae burden. As hypothesized, we found that allergic airway inflammation decreased the number of K. pneumoniae-induced airway neutrophils and lung IL-17A, IL-17F, and IL-22 expression. Despite the marked reduction in postinfection airway neutrophilia and lung expression of Th17 cytokines, allergic airway inflammation significantly decreased the lung K. pneumoniae burden and postinfection mortality. We showed that the decreased lung K. pneumoniae burden was independent of IL-4, IL-5, and IL-17A and partially dependent on IL-13 and STAT6. Additionally, we demonstrated that the decreased lung K. pneumoniae burden associated with allergic airway inflammation was both neutrophil and CCL8 dependent. These findings suggest a novel role for CCL8 in lung antibacterial immunity against K. pneumoniae and suggest new mechanisms of orchestrating lung antibacterial immunity.

Deficiency of gp91phox inhibits allergic airway inflammation.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a multienzyme complex, is the major source for production of reactive oxygen species (ROS). ROS are increased in allergic diseases, such as asthma, but the role of ROS in disease pathogenesis remains uncertain. We hypothesized that mice unable to generate ROS via the NADPH oxidase pathway would have decreased allergic airway inflammation. To test this hypothesis, we studied gp91phox(-/-) mice in a model of allergic airway inflammation after sensitization and challenge with ovalbumin. Serum, bronchoalveolar lavage fluid, and lungs were then examined for evidence of allergic inflammation. We found that mice lacking a functional NADPH oxidase complex had significantly decreased ROS production and allergic airway inflammation, compared with wild-type (WT) control animals. To determine the mechanism by which allergic inflammation was inhibited by gp91phox deficiency, we cultured bone marrow-derived dendritic cells from WT and gp91phox(-/-) mice and activated them with LPS. IL-12 expression was significantly increased in the gp91phox(-/-) bone marrow-derived dendritic cells, suggesting that the cytokine profile produced in the absence of gp91phox enhanced the conditions leading to T helper (Th) type 1 differentiation, while inhibiting Th2 polarization. Splenocytes from sensitized gp91phox(-/-) animals produced significantly less IL-13 in response to ovalbumin challenge in vitro compared with splenocytes from sensitized WT mice, suggesting that NADPH oxidase promotes allergic sensitization. In contrast, inflammatory cytokines produced by T cells cultured from WT and gp91phox(-/-) mice under Th0, Th1, Th2, and Th17 conditions were not significantly different. This study demonstrates the importance of NADPH oxidase activity and ROS production in a murine model of asthma.