Sara Reiss

IL-13 Regulates Th17 Secretion of IL-17A in an IL-10–Dependent Manner

IL-13 is a central mediator of airway hyperresponsiveness and mucus expression, both hallmarks of asthma. IL-13 is found in the sputum of patients with asthma; therefore, IL-13 is an attractive drug target for treating asthma. We have shown previously that IL-13 inhibits Th17 cell production of IL-17A and IL-21 in vitro. Th17 cells are associated with autoimmune diseases, host immune responses, and severe asthma. In this study, we extend our in vitro findings and determine that IL-13 increases IL-10 production from Th17-polarized cells and that IL-13–induced IL-10 production negatively regulates the secretion of IL-17A and IL-21. To determine if IL-13 negatively regulates lung IL-17A expression via an IL-10–dependent mechanism in vivo, we used a model of respiratory syncytial virus (RSV) strain A2 infection in STAT1 knockout (KO) mice that increases lung IL-17A and IL-13 expression, cytokines not produced during RSV infection in wild-type mice. To test the hypothesis that IL-13 negatively regulates lung IL-17A expression, we created STAT1/IL-13 double KO (DKO) mice. We found that RSV-infected STAT1/IL-13 DKO mice had significantly greater lung IL-17A expression compared with that of STAT1 KO mice and that increased IL-17A expression was abrogated by anti-IL-10 Ab treatment. RSV-infected STAT1/IL-13 DKO mice also had increased neutrophil infiltration compared with that of RSV-infected STAT1 KO mice. Neutralizing IL-10 increased the infiltration of inflammatory cells into the lungs of STAT1 KO mice but not STAT1/IL-13 DKO mice. These findings are vital to understanding the potential side effects of therapeutics targeting IL-13. Inhibiting IL-13 may decrease IL-10 production and increase IL-17A production, thus potentiating IL-17A–associated diseases.

Molecular defects in human carbamoy phosphate synthetase I: mutational spectrum, diagnostic and protein structure considerations

Deficiency of carbamoyl phosphate synthetase I (CPSI) results in hyperammonemia ranging from neonatally lethal to environmentally induced adult‐onset disease. Over 24 years, analysis of tissue and DNA samples from 205 unrelated individuals diagnosed with CPSI deficiency (CPSID) detected 192 unique CPS1 gene changes, of which 130 are reported here for the first time. Pooled with the already reported mutations, they constitute a total of 222 changes, including 136 missense, 15 nonsense, 50 changes of other types resulting in enzyme truncation, and 21 other changes causing in‐frame alterations. Only ∼10% of the mutations recur in unrelated families, predominantly affecting CpG dinucleotides, further complicating the diagnosis because of the “private” nature of such mutations. Missense changes are unevenly distributed along the gene, highlighting the existence of CPSI regions having greater functional importance than other regions. We exploit the crystal structure of the CPSI allosteric domain to rationalize the effects of mutations affecting it. Comparative modeling is used to create a structural model for the remainder of the enzyme. Missense changes are found to directly correlate, respectively, with the one‐residue evolutionary importance and inversely correlate with solvent accessibility of the mutated residue. This is the first large‐scale report of CPS1 mutations spanning a wide variety of molecular defects highlighting important regions in this protein. Hum Mutat 32:1–11, 2011.

IL-17A inhibits airway reactivity induced by respiratory syncytial virus infection during allergic airway inflammation

Background Viral infections are the most frequent cause of asthma exacerbations and are linked to increased airway reactivity (AR) and inflammation. Mice infected with respiratory syncytial virus (RSV) during ovalbumin (OVA)-induced allergic airway inflammation (OVA/RSV) had increased AR compared with OVA or RSV mice alone. Furthermore, interleukin 17A (IL-17A) was only increased in OVA/RSV mice. Objective To determine whether IL-17A increases AR and inflammation in the OVA/RSV model. Methods Wild-type (WT) BALB/c and IL-17A knockout (KO) mice underwent mock, RSV, OVA or OVA/RSV protocols. Lungs, bronchoalveolar lavage (BAL) fluid and/or mediastinal lymph nodes (MLNs) were harvested after infection. Cytokine expression was determined by ELISA in the lungs or BAL fluid. MLNs were restimulated with either OVA (323–229) peptide or RSV M2 (127–135) peptide and IL-17A protein expression was analysed. AR was determined by methacholine challenge. Results RSV increased IL-17A protein expression by OVA-specific T cells 6 days after infection. OVA/RSV mice had decreased interferon-β protein expression compared with RSV mice. OVA/RSV mice had increased IL-23p19 mRNA expression in lung homogenates compared with mock, OVA or RSV mice. Unexpectedly, IL-17A KO OVA/RSV mice had increased AR compared with WT OVA/RSV mice. Furthermore, IL-17A KO OVA/RSV mice had increased eosinophils, lymphocytes and IL-13 protein expression in BAL fluid compared with WT OVA/RSV mice. Conclusions IL-17A negatively regulated AR and airway inflammation in OVA/RSV mice. This finding is important because IL-17A has been identified as a potential therapeutic target in asthma, and inhibiting IL-17A in the setting of virally-induced asthma exacerbations may have adverse consequences.

Asthmatic Airway Neutrophilia after Allergen Challenge Is Associated with the Glutathione S-Transferase M1 Genotype

RATIONALE Asthma is a heterogeneous lung disorder characterized by airway inflammation and airway dysfunction, manifesting as hyperresponsiveness and obstruction. Glutathione S-transferase M1 (GSTM1) is a multifunctional phase II enzyme and regulator of stress-activated cellular signaling relevant to asthma pathobiology. A common homozygous deletion polymorphism of the GSTM1 gene eliminates enzyme activity. OBJECTIVES To determine the effect of GSTM1 on airway inflammation and reactivity in adults with established atopic asthma in vivo. METHODS Nineteen GSTM1 wild-type and eighteen GSTM1-null individuals with mild atopic asthma underwent methacholine and inhaled allergen challenges, and endobronchial allergen provocations through a bronchoscope. MEASUREMENTS AND MAIN RESULTS The influx of inflammatory cells, panels of cytokines and chemokines linked to asthmatic inflammation, F(2)-isoprostanes (markers of oxidative stress), and IgE were measured in bronchoalveolar lavage fluid at baseline and 24 hours after allergen instillation. Individuals with asthma with the GSTM1 wild-type genotype had greater baseline and allergen-provoked airway neutrophilia and concentrations of myeloperoxidase than GSTM1-null patients. In contrast, the eosinophilic inflammation was unaffected by GSTM1. The allergen-stimulated generation of acute-stress and proneutrophilic mediators, tumor necrosis factor-α, CXCL-8, IL-1β, and IL-6, was also greater in the GSTM1 wild-type patients. Moreover, post-allergen airway concentrations of IgE and neutrophil-generated mediators, matrix metalloproteinase-9, B-cell activating factor, transforming growth factor-β1, and elastase were higher in GSTM1 wild-type individuals with asthma. Total airway IgE correlated with B-cell activating factor concentrations. In contrast, levels of F(2)-isoprostane were comparable in both groups. Finally, GSTM1 wild-type individuals with asthma required lower threshold concentrations of allergen to produce bronchoconstriction. CONCLUSIONS The functional GSTM1 genotype promotes neutrophilic airway inflammation in humans with atopic asthma in vivo.

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.

Glutathione S-transferase P1 Ile105Val polymorphism modulates allergen-induced airway inflammation in human atopic asthmatics in vivo.

Glutathione S‐transferase P1 is a Phase II cytoprotective and detoxifying enzyme that is widely expressed in human airways. The glutathione S‐transferase P1 Ile105Val polymorphism has been linked with atopic disorders and asthma. Yet, little remains known about the regulation of allergic inflammation by glutathione S‐transferase P1 in human asthmatics.

Allergic Airway Inflammation Decreases Lung Bacterial Burden following Acute Klebsiella pneumoniae Infection in a Neutrophil- and CCL8-Dependent Manner

ABSTRACT 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.

The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

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

ABSTRACT 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.

Endogenous PGI 2 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.