Xiaoyang Hua

Analysis of NLRP3 in the Development of Allergic Airway Disease in Mice

The contribution of NLRP3, a member of the nucleotide-binding domain leucine-rich repeat–containing (NLR) family, to the development of allergic airway disease is currently controversial. In this study, we used multiple allergic asthma models to examine the physiologic role of NLRP3. We found no significant differences in airway eosinophilia, histopathologic condition, mucus production, and airway hyperresponsiveness between wild-type and Nlrp3−/− mice in either acute (alum-dependent) or chronic (alum-independent) OVA models. In addition to the OVA model, we did not detect a role for NLRP3 in the development of allergic airway disease induced by either acute or chronic house dust mite Ag exposure. Although we did not observe significant phenotypic differences in any of the models tested, we did note a significant reduction of IL-13 and IL-33 in Nlrp3−/− mice compared with wild-type controls in the chronic OVA model without added alum. In all of the allergic airway disease models, the NLRP3 inflammasome-associated cytokines IL-1β and IL-18 in the lung were below the level of detection. In sum, this report surveyed four different allergic asthma models and found a modest and selected role for NLRP3 in the alum-free OVA model. However, this difference did not greatly alter the clinical outcome of the disease. This finding suggests that the role of NLRP3 in allergic asthma must be re-evaluated.

Modulation of murine dendritic cell function by adenine nucleotides and adenosine: Involvement of the A2B receptor

Adenosine triphosphate has previously been shown to induce semi‐mature human monocyte‐derived dendritic cells (DC). These are characterized by the up‐regulation of co‐stimulatory molecules, the inhibition of IL‐12 and the up‐regulation of some genes involved in immune tolerance, such as thrombospondin‐1 and indoleamine 2,3‐dioxygenase. The actions of adenosine triphosphate are mediated by the P2Y11 receptor; since there is no functional P2Y11 gene in the murine genome, we investigated the action of adenine nucleotides on murine DC. Adenosine 5′‐(3‐thiotriphosphate) and adenosine inhibited the production of IL‐12p70 by bone marrow‐derived DC (BMDC). These inhibitions were relieved by 8‐p‐sulfophenyltheophylline, an adenosine receptor antagonist. The use of selective ligands and A  2B–/– BMDC indicated the involvement of the A2B receptor. A microarray experiment, confirmed by quantitative PCR, showed that, in presence of LPS, 5′‐(N‐ethylcarboxamido) adenosine (NECA, the most potent A2B receptor agonist) regulated the expression of several genes: arginase I and II, thrombospondin‐1 and vascular endothelial growth factor were up‐regulated whereas CCL2 and CCL12 were down‐regulated. We further showed that NECA, in combination with LPS, increased the arginase I enzymatic activity. In conclusion, the described actions of adenine nucleotides on BMDC are mediated by their degradation product, adenosine, acting on the A2B receptor, and will possibly lead to an impairment of Th1 response or tolerance.

Retinoid-Related Orphan Receptor γ Controls Immunoglobulin Production and Th1/Th2 Cytokine Balance in the Adaptive Immune Response to Allergen

The retinoid-related orphan receptors (ROR) comprise a distinct subfamily of nuclear receptors with the capacity to act as both repressors and activators of transcription. RORγ, the most recently identified member of the ROR family, has been shown to be important for the development of normal lymphocyte compartments as well as organogenesis of some lymphoid organs. In this report, we examine the capacity of RORγ-deficient mice to develop an adaptive immune response to Ag using OVA-induced inflammation in mice as a model for allergic airway disease. In sham-treated mice lacking RORγ, low-grade pulmonary inflammation was observed and characterized by the perivascular accumulation of B and T lymphocytes, increased numbers of inflammatory cells in the lung lavage fluid, and polyclonal Ig activation. Following sensitization and challenge, the capacity of these animals to develop the allergic phenotype was severely impaired as evidenced by attenuated eosinophilic pulmonary inflammation, reduced numbers of CD4+ lymphocytes, and lower Th2 cytokines/chemokine protein and mRNA expression in the lungs. IFN-γ and IL-10 production was markedly greater in splenocytes from RORγ-deficient mice following in vitro restimulation with OVA compared with wild-type splenocytes, and a shift toward a Th1 immune response was observed in sensitized/challenged RORγ-deficient animals in vivo. These data reveal a critical role for RORγ in the regulation of Ig production and Th1/Th2 balance in adaptive immunity.

Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

Ferritin is a protein principally known for its role in iron storage. We have previously shown that ferritin can bind high-molecular-weight kininogen (HK). Upon proteolytic cleavage by the protease kallikrein, HK releases the proinflammatory peptide bradykinin (BK) and other biologically active products, such as two-chain high-molecular-weight kininogen, HKa. At inflammatory sites, HK is oxidized, which renders it a poor substrate for kallikrein. However, oxidized HK remains a good substrate for elastase and tryptase, thereby providing an alternative cleavage mechanism for HK during inflammation. Here we report that ferritin can retard the cleavage of both native HK and oxidized HK by elastase and tryptase. Initial rates of cleavage were reduced 45-75% in the presence of ferritin. Ferritin is not a substrate for elastase or tryptase and does not interfere with the ability of either protease to digest a synthetic substrate, suggesting that ferritin may impede HK cleavage through direct interaction with HK. Immunoprecipitation and solid phase binding studies reveal that ferritin and HK bind directly with a Kd of 134 nM. To test whether ferritin regulates HK cleavage in vivo, we used THP-1 cells, a human monocyte/macrophage cell line that has been used to model pulmonary inflammatory cells. We observed that ferritin impedes the cleavage of HK by secretory proteases in stimulated macrophages. Furthermore, ferritin, HK, and elastase are all present in or on alveolar macrophages in a mouse model of pulmonary inflammation. Collectively, these results implicate ferritin in the modulation of HK cleavage at sites of inflammation.

The Chemokine, CCL3, and Its Receptor, CCR1, Mediate Thoracic Radiation–Induced Pulmonary Fibrosis

Patients receiving thoracic radiation often develop pulmonary injury and fibrosis. Currently, there are no effective measures to prevent or treat these conditions. We tested whether blockade of the chemokine, CC chemokine ligand (CCL) 3, and its receptors, CC chemokine receptor (CCR) 1 and CCR5, can prevent radiation-induced lung inflammation and fibrosis. C57BL/6J mice received thoracic radiation, and the interaction of CCL3 with CCR1 or CCR5 was blocked using genetic techniques, or by pharmacologic intervention. Lung inflammation was assessed by histochemical staining of lung tissue and by flow cytometry. Fibrosis was measured by hydroxyproline assays and collagen staining, and lung function was studied by invasive procedures. Irradiated mice lacking CCL3 or its receptor, CCR1, did not develop the lung inflammation, fibrosis, and decline in lung function seen in irradiated wild-type mice. Pharmacologic treatment of wild-type mice with a small molecule inhibitor of CCR1 also prevented lung inflammation and fibrosis. By contrast, mice lacking CCR5 were not protected from radiation-induced injury and fibrosis. The selective interaction of CCL3 with its receptor, CCR1, is critical for radiation-induced lung inflammation and fibrosis, and these conditions can be largely prevented by a small molecule inhibitor of CCR1.

MyD88 provides a protective role in long-term radiation-induced lung injury

Purpose: The role of innate immune regulators is investigated in injury sustained from irradiation as in the clinic for cancer treatment or from a nuclear incident. The protective benefits of flagellin signaling through Toll-like receptors (TLR) in an irradiation setting warrant study of a key intracellular adaptor of TLR signaling, namely Myeloid differentiation primary response factor 88 (MyD88). The role of MyD88 in regulating innate immunity and Nuclear factor kappa-B (NF-κB)-activated responses targets this critical factor for influencing injury and recovery as well as maintaining immune homeostasis. Materials and methods: To examine the role of MyD88, we examined immune cells and factors during acute pneumonitic and fibrotic phases in Myd88-deficient animals receiving thoracic gamma (γ)-irradiation. Results: We found that MyD88 supports survival from radiation-induced injury through the regulation of inflammatory factors that aid in recovery from irradiation. The absence of MyD88 resulted in unresolved pulmonary infiltrate and enhanced collagen deposition plus elevated type 2 helper T cell (Th2) cytokines in long-term survivors of irradiation. Conclusions: These results based only on a gene deletion model suggest that alterations of MyD88-dependent inflammatory processes impact chronic lung injury. Therefore, MyD88 may contribute to attenuating long-term radiation-induced lung injury and protecting against fibrosis.

IL-4 Amplifies the Pro-Inflammatory Effect of Adenosine in Human Mast Cells by Changing Expression Levels of Adenosine Receptors

Adenosine inhalation produces immediate bronchoconstriction in asthmatics but not in normal subjects. The bronchospastic effect of adenosine is largely mediated through adenosine-induced mast cell activation, the mechanism of which is poorly understood due to limitations in culturing human primary mast cells. Here, we show that human umbilical cord blood -derived mast cells incubated with the Th2 cytokine IL-4 develop increased sensitivity to adenosine. Potentiation of anti-IgE- induced and calcium ionophore/PMA-induced degranulation was augmented in mast cells cultured with IL-4, and this effect was reduced or abolished by pre-treatment with A2BsiRNA and selective A2B receptor antagonists, respectively. IL-4 incubation resulted in the increased expression of A2B and reduced expression of A2A adenosine receptors on human mast cells. These results suggest that Th2 cytokines in the asthmatic lung may alter adenosine receptor expression on airway mast cells to promote increased responsiveness to adenosine.

Exacerbation of allergic inflammation in mice exposed to diesel exhaust particles prior to viral infection

BackgroundViral infections and exposure to oxidant air pollutants are two of the most important inducers of asthma exacerbation. Our previous studies have demonstrated that exposure to diesel exhaust increases the susceptibility to influenza virus infections both in epithelial cells in vitro and in mice in vivo. Therefore, we examined whether in the setting of allergic asthma, exposure to oxidant air pollutants enhances the susceptibility to respiratory virus infections, which in turn leads to increased virus-induced exacerbation of asthma. Ovalbumin-sensitized (OVA) male C57BL/6 mice were instilled with diesel exhaust particles (DEP) or saline and 24 hours later infected with influenza A/PR/8. Animals were sacrificed 24 hours post-infection and analyzed for markers of lung injury, allergic inflammation, and pro-inflammatory cytokine production.ResultsExposure to DEP or infection with influenza alone had no significant effects on markers of injury or allergic inflammation. However, OVA-sensitized mice that were exposed to DEP and subsequently infected with influenza showed increased levels of eosinophils in lung lavage and tissue. In addition Th2-type cytokines, such as IL-4 and IL-13, and markers of eosinophil chemotaxis, such as CCL11 and CCR3, were increased in OVA-sensitized mice exposed to DEP prior to infection with influenza. These mice also showed increased levels of IL-1α, but not IL-10, RANTES, and MCP-1 in lung homogenates.ConclusionThese data suggest that in the setting of allergic asthma, exposure to diesel exhaust could enhance virus-induced exacerbation of allergic inflammation.

Deficiency of RAMP1 Attenuates Antigen-Induced Airway Hyperresponsiveness in Mice

Asthma is a chronic inflammatory disease affecting the lung, characterized by breathing difficulty during an attack following exposure to an environmental trigger. Calcitonin gene-related peptide (CGRP) is a neuropeptide that may have a pathological role in asthma. The CGRP receptor is comprised of two components, which include the G-protein coupled receptor, calcitonin receptor-like receptor (CLR), and receptor activity-modifying protein 1 (RAMP1). RAMPs, including RAMP1, mediate ligand specificity in addition to aiding in the localization of receptors to the cell surface. Since there has been some controversy regarding the effect of CGRP on asthma, we sought to determine the effect of CGRP signaling ablation in an animal model of asthma. Using gene-targeting techniques, we generated mice deficient for RAMP1 by excising exon 3. After determining that these mice are viable and overtly normal, we sensitized the animals to ovalbumin prior to assessing airway resistance and inflammation after methacholine challenge. We found that mice lacking RAMP1 had reduced airway resistance and inflammation compared to wildtype animals. Additionally, we found that a 50% reduction of CLR, the G-protein receptor component of the CGRP receptor, also ameliorated airway resistance and inflammation in this model of allergic asthma. Interestingly, the loss of CLR from the smooth muscle cells did not alter the airway resistance, indicating that CGRP does not act directly on the smooth muscle cells to drive airway hyperresponsiveness. Together, these data indicate that signaling through RAMP1 and CLR plays a role in mediating asthma pathology. Since RAMP1 and CLR interact to form a receptor for CGRP, our data indicate that aberrant CGRP signaling, perhaps on lung endothelial and inflammatory cells, contributes to asthma pathophysiology. Finally, since RAMP-receptor interfaces are pharmacologically tractable, it may be possible to develop compounds targeting the RAMP1/CLR interface to assist in the treatment of asthma.

Critical role of phospholipase A2 group IID in age-related susceptibility to severe acute respiratory syndrome–CoV infection

Vijay et al. show that an age-dependent increase of phospholipase A2 group IID (PLA2G2D) in the lung contributes to worse outcomes in mice infected with SARS-CoV. Mice lacking (PLA2G2D) had increased survival to lethal infection with enhanced DC migration to the dLN and augmented T cell responses. The results suggest that targeting (PLA2G2D) in elderly patients with respiratory infections could represent an attractive therapeutic strategy.