Shuhe Wang

A GABAergic system in airway epithelium is essential for mucus overproduction in asthma

γ-Aminobutyric acid (GABA) is an important neurotransmitter that, through the subtype A GABA receptor (GABAAR), induces inhibition in the adult brain. Here we show that an excitatory, rather than inhibitory, GABAergic system exists in airway epithelial cells. Both GABAARs and the GABA synthetic enzyme glutamic acid decarboxylase (GAD) are expressed in pulmonary epithelial cells. Activation of GABAARs depolarized these cells. The expression of GAD in the cytosol and GABAARs in the apical membranes of airway epithelial cells increased markedly when mice were sensitized and then challenged with ovalbumin, an approach for inducing allergic asthmatic reactions. Similarly, GAD and GABAARs in airway epithelial cells of humans with asthma increased after allergen inhalation challenge. Intranasal application of selective GABAAR inhibitors suppressed the hyperplasia of goblet cells and the overproduction of mucus induced by ovalbumin or interleukin-13 in mice. These findings show that a previously unknown epithelial GABAergic system has an essential role in asthma.

IFN‐γ knockout mice show Th2‐associated delayed‐type hypersensitivity and the inflammatory cells fail to localize and control chlamydial infection

Delayed‐type hypersensitivity (DTH) has been demonstrated to be a Th1 type immune response which is important in the host defense against infection with intracellular bacteria, including Chlamydia. In the present study, we surprisingly observed that C. trachomatis mouse pneumonitis MoPn‐infected IFN‐γ gene knockout (KO) mice mounted strong DTH responses following foopad challenge with inactivated organisms. The DTH responses in IFN‐γ KO mice were associated with Th2 cytokine production and partially blocked by anti‐IL‐4 monoclonal antibodies. In addition, the inflammatory cells in IFN‐γ KO mice failed to target the cellular sites of chlamydial inclusions in infected tissues and failed to clear the infection. The data, in conjunction with previous studies, suggest that different types (Th1 and Th2 associated) of DTH responses may function differently in host defense against chlamydial infection and that the functional differences in DTH responses may account for the dual role that DTH is speculated to play in chlamydial protective immunity and immunopathology. Moreover, the data suggest that the IFN‐γ KO mouse is a useful model system for studying chlamydial pathogenesis.

IL-17/Th17 Promotes Type 1 T Cell Immunity against Pulmonary Intracellular Bacterial Infection through Modulating Dendritic Cell Function

Although their contribution to host defense against extracellular infections has been well defined, IL-17 and Th17 are generally thought to have limited impact on intracellular infections. In this study, we investigated the role and mechanisms of IL-17/Th17 in host defense against Chlamydia muridarum, an obligate intracellular bacterium, lung infection. Our data showed rapid increase in IL-17 production and expansion of Th17 cells following C. muridarum infection and significant detrimental impact of in vivo IL-17 neutralization by anti-IL-17 mAb on disease course, immune response, and dendritic cell (DC) function. Specifically, IL-17-neutralized mice exhibited significantly greater body weight loss, higher organism growth, and much more severe pathological changes in the lung compared with sham-treated control mice. Immunological analysis showed that IL-17 neutralization significantly reduced Chlamydia-specific Th1 responses, but increased Th2 responses. Interestingly, the DC isolated from IL-17-neutralized mice showed lower CD40 and MHC II expression and IL-12 production, but higher IL-10 production compared with those from sham-treated mice. In two DC-T cell coculture systems, DC isolated from IL-17-neutralized mice induced higher IL-4, but lower IFN-γ production by Ag-specific T cells than those from sham-treated mice in cell priming and reaction settings. Adoptive transfer of DC isolated from IL-17-neutralized mice, unlike those from sham-treated mice, failed to protect the recipients against challenge infection. These findings provide in vivo evidence that IL-17/Th17 plays an important role in host defense against intracellular bacterial infection, and suggest that IL-17/Th17 can promote type 1 T cell immunity through modulating DC function.

Type I IFNs Enhance Susceptibility to Chlamydia muridarum Lung Infection by Enhancing Apoptosis of Local Macrophages

Type I IFNs (IFNIs) have pleiotropic functions in regulating host innate and adaptive immune responses to pathogens. To elucidate the role of IFNIs in host resistance to chlamydial infection in vivo, we compared IFN-α/β receptor knockout (IFNAR−/−) and wild-type control mice in susceptibility to Chlamydia trachomatis mouse pneumonitis (Chlamydia muridarum) lung infection. We found that the IFNAR−/− mice were significantly more resistant to C. muridarum infection showing less bacterial burden and bodyweight loss, and milder pathological changes. However, IFN-γ response, which is believed to be critical in host defense against chlamydial infection, was similar between the wild-type and IFNAR−/− mice. More importantly, TUNEL analysis showed less macrophage apoptosis in IFNAR−/− mice, which was consistent with lower expressions of IFNI-induced apoptotic factors, TRAIL, Daxx, and PKR. Furthermore, depletion of lung macrophages with dichloromethylene diphosphonate-liposome significantly increased the susceptibility of the IFNAR−/− mice to C. muridarum, confirming the importance of macrophages. Overall, the data indicate that IFNIs play a promoting role in C. muridarum lung infection, largely through increase of local macrophage apoptosis.

IL-10 deficiency prevents IL-5 overproduction and eosinophilic inflammation in a murine model of asthma-like reaction

Eosinophilic inflammation and bronchial mucus secretion are among the characteristic pathological changes in asthmatic reaction, which is mediated by Th2 type responses. Although it belongs to Th2 cytokines especially in the mouse, IL‐10 is often considered an inhibitory cytokine for both Th1 and Th2 cells. In the present study, using a murine asthma model induced by ovalbumin (OVA), we demonstrated that endogenous IL‐10 is critical for the development of asthma‐like responses. Specifically, in comparison with wild‐type controls, IL‐10 gene knockout (KO) mice showed significantly reduced IL‐5 production, eosinophilic inflammation and mucus production without notable changes in IL‐4 and IgE responses following i.  p. sensitization and subsequent intranasal challenge with OVA. In addition, Th1‐related cytokine (IFN‐γ and IL‐12) production in IL‐10 KO mice was significantly higher than that in wild‐type mice. The results suggest that endogenous IL‐10 plays an important role in promoting pulmonary eosinophilic inflammatory reaction and mucus production during asthmatic reaction. The data also argue that IL‐10 may be more influential in the development of IL‐5‐producing Th2 cells which differ from typical Th2 cells producing both IL‐4 and IL‐5.

Natural killer T cells contribute to airway eosinophilic inflammation induced by ragweed through enhanced IL-4 and eotaxin production

Although NKT cells have been found to be capable of modulating immune responses in several model systems, the role of NKT cells in allergy remains unclear. Using CD1 gene knockout (KO) mice, which lack NKT cells, we examined the function of NKT cells in the development of allergic inflammation induced by a common airborne human allergen, ragweed. The data showed that airway eosinophilia and mucus overproduction induced by ragweed were significantly reduced in CD1 KO mice, which was correlated with significantly lower allergen‐driven IL‐4 production and lower eotaxin responses in the airways of CD1 KO mice. Moreover, both ragweed‐specific and total serum IgE levels in CD1 KO mice were significantly lower than those in control BALB/c mice. The reduced allergic reaction in CD1 KO mice is not due to intrinsic deficiency because they showed normal levels of immune cells and function. In addition, in vivo stimulation of NKT cells using their natural ligand, α‐galactosylceramide, enhanced ragweed‐induced airway eosinophilia, IL‐4, and eotaxin production in control, but not CD1 KO mice. These data provide in vivo evidence for the involvementof NKT cells in the allergic mechanisms responsible for allergen‐driven cytokine and chemokine production and airway inflammation.

NK T Cell Activation Promotes Chlamydia trachomatis Infection In Vivo

We used two approaches to examine the role of NK T cells (NKT) in an intracellular bacterial (Chlamydia trachomatis mouse pneumonitis (C. muridarum)) infection. One is to use CD1 gene knockout (KO) mice, which lack NKT, and the other is to activate NKT using α-galactosylceramide (α-GalCer), a natural ligand of these cells. The data showed a promoting effect of NKT activation on Chlamydia lung infection. Specifically, CD1 KO mice exhibited significantly lower levels of body weight loss, less severe pathological change and lower chlamydial in vivo growth than wild-type mice. Immunological analysis showed that CD1 KO mice exhibited significantly lower C. muridarum-specific IL-4 and serum IgE Ab responses as well as more pronounced delayed-type hypersensitivity response compared with wild-type controls. In line with the finding in KO mice, the in vivo stimulation of NKT using α-GalCer enhanced chlamydial growth in vivo, which were correlated with reduced delayed-type hypersensitivity response and increased C. muridarum-driven IL-4/IgE production. Moreover, neutralization of IL-4 activity in the α-GalCer-treated BALB/c mice significantly reduced the promoting effect of α-GalCer treatment on chlamydial growth in vivo. These data provide in vivo evidence for the involvement of NKT in a bacterial pathogenesis and its role in promoting Th2 responses during infection.

Distinct NKT Cell Subsets Are Induced by Different Chlamydia Species Leading to Differential Adaptive Immunity and Host Resistance to the Infections

We investigated the role of NKT cells in immunity to Chlamydia pneumoniae and Chlamydia muridarum infections using a combination of knockout mice and specific cellular activation approaches. The NKT-deficient mice showed exacerbated susceptibility to C. pneumoniae infection, but more resistance to C. muridarum infection. Activation of NKT reduced C. pneumoniae in vivo growth, but enhanced C. muridarum infection. Cellular analysis of invariant NKT cells revealed distinct cytokine patterns following C. pneumoniae and C. muridarum infections, i.e., predominant IFN-γ in the former, while predominant IL-4 in the latter. The cytokine patterns of CD4+ and CD8+ T cells matched those of NKT cells. Our data provide in vivo evidence for a functionally diverse role of NKT cells in immune response to two intracellular bacterial pathogens. These results suggest that distinct NKT subsets are induced by even biologically closely related pathogens, thus leading to differential adaptive immune response and infection outcomes.

IL-12-Dependent Vascular Cell Adhesion Molecule-1 Expression Contributes to Airway Eosinophilic Inflammation in a Mouse Model of Asthma-Like Reaction

Bronchial-alveolar eosinophilic inflammation is among the characteristic pathological changes in asthma, which has been shown to be correlated with type 2 cytokine and chemokine production. Exogenous IL-12 has been found to be inhibitory for pulmonary eosinophilia in reported studies. Using a murine asthma-like model induced by OVA, we found in the present study that IL-12 gene knockout (KO) mice showed substantially reduced airway recruitment of eosinophils compared with wild-type control mice following OVA sensitization/challenge, although the levels of circulating eosinophils were comparable in these two groups of mice. Cytokine analysis showed Ag-driven Th1 (IFN-γ) and Th2 (IL-4, IL-5, IL-10, and IL-13) cytokine production by CD4 T cells from local draining lymph nodes and spleen. Similarly, local eotaxin production was comparable in wild-type and IL-12 KO mice. In contrast, immunohistochemical analysis showed that the expression of VCAM-1 on the lung endothelium of IL-12 KO mice was dramatically less than that in wild-type mice. Furthermore, administration of rIL-12 at the stage of sensitization and challenge with OVA restored airway eosinophilia and VCAM-1 expression in IL-12 KO mice. The results suggest that endogenous IL-12 contributes to the recruitment of eosinophils into airways observed in asthma, possibly via enhancement of the expression of VCAM-1 on local vascular endothelial cells.

Mycobacterial infection inhibits established allergic inflammatory responses via alteration of cytokine production and vascular cell adhesion molecule-1 expression

Our previous studies, as well as those of others, have demonstrated that local or systemic Mycobacterium bovis bacille Calmette–Gue´rin (BCG) infection can inhibit de novo allergen‐induced asthma‐like reactions, but the effect of this infection on established allergic responses is unknown. The aim of this study was therefore to examine the effect of mycobacterial infection on established allergy in a murine model of asthma‐like reaction. Mice were sensitized with ovalbumin (OVA) in alum followed by infection with BCG and subsequent intranasal challenge with the same allergen. In some experiments, mice were sensitized with OVA followed by intranasal challenge with OVA and then given BCG infection with subsequent rechallenge with OVA. Mice without BCG infection but treated with OVA in the same manner, were used as a control. The mice were examined for immunoglobulin E (IgE) response and eosinophilic inflammation, mucus production, cytokine/chemokine patterns and adhesion molecule expression in the lung. The results showed that postallergen BCG infection suppressed the established airway eosinophilia and mucus overproduction, but not IgE responses. The inhibition of asthma‐like reactions by BCG infection was correlated with a shift of allergen‐driven cytokine production pattern and, more interestingly, with a dramatic decrease of vascular cell adhesion molecule‐1 (VCAM‐1) expression in the lung. These findings suggest that intracellular bacterial infection can inhibit established allergic responses via alteration of local cytokine production and the expression of adhesion molecules.