Role of A2B adenosine receptor signaling in adenosine-dependent pulmonary inflammation and injury.

Adenosine has been implicated in the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease. In vitro studies suggest that activation of the A2B adenosine receptor (A2BAR) results in proinflammatory and profibrotic effects relevant to the progression of lung diseases; however, in vivo data supporting these observations are lacking. Adenosine deaminase-deficient (ADA-deficient) mice develop pulmonary inflammation and injury that are dependent on increased lung adenosine levels. To investigate the role of the A2BAR in vivo, ADA-deficient mice were treated with the selective A2BAR antagonist CVT-6883, and pulmonary inflammation, fibrosis, and airspace integrity were assessed. Untreated and vehicle-treated ADA-deficient mice developed pulmonary inflammation, fibrosis, and enlargement of alveolar airspaces; conversely, CVT-6883-treated ADA-deficient mice showed less pulmonary inflammation, fibrosis, and alveolar airspace enlargement. A2BAR antagonism significantly reduced elevations in proinflammatory cytokines and chemokines as well as mediators of fibrosis and airway destruction. In addition, treatment with CVT-6883 attenuated pulmonary inflammation and fibrosis in wild-type mice subjected to bleomycin-induced lung injury. These findings suggest that A2BAR signaling influences pathways critical for pulmonary inflammation and injury in vivo. Thus in chronic lung diseases associated with increased adenosine, antagonism of A2BAR-mediated responses may prove to be a beneficial therapy

Negative Regulation of Pulmonary Th17 Responses by C3a Anaphylatoxin during Allergic Inflammation in Mice

<div><p>Activation of complement is one of the earliest immune responses to exogenous threats, resulting in various cleavage products including anaphylatoxin C3a. In addition to its contribution to host defense, C3a has been shown to mediate Th2 responses in animal models of asthma. However, the role of C3a on pulmonary Th17 responses during allergic inflammation remains unclear. Here, we show that mice deficient in C3a receptor (C3aR) exhibited (i) higher percentages of endogenous IL-17-producing CD4⁺ T cells in the lungs, (ii) higher amounts of IL-17 in the bronchoalveolar lavage fluid, and (iii) more neutrophils in the lungs than wild-type mice when challenged with intranasal allergens. Moreover, adoptive transfer experiments showed that the frequencies of antigen-specific IL-17-producing CD4⁺ T cells were significantly higher in the lungs and bronchial lymph nodes of C3aR-deficient recipients than those of wild-types recipients. Bone-marrow reconstitution study indicated that C3aR-deficiency on hematopoietic cells was required for the increased Th17 responses. Furthermore, C3aR-deficient mice exhibited increased percentages of Foxp3⁺ regulatory T cells; however, depletion of these cells minimally affected the induction of antigen-specific Th17 cell population in the lungs. Neutralization of IL-17 significantly reduced the number of neutrophils in bronchoalveolar lavage fluid of C3aR-deficient mice. Our findings demonstrate that C3a signals negatively regulate antigen-specific Th17 responses during allergic lung inflammation and the size of Foxp3⁺ regulatory T cell population in the periphery.</p> </div

Effect of IL-17 neutralization on the lung inflammation of C3aR^−/− mice.

<p>Groups of C3aR^−/− mice were (n = 3–4) intranasally challenge with Asp/OVA every other days four times (day 0, 2, 4, 6), and i.p. injected with isotype control antibody or with IL-17 neutralizing antibody on day 0, 2, 4. Twenty-four hours after the last challenge, mice were anesthetized, mechanically ventilated, and airway responses to increasing doses of intravenous acetylcholine were measured. AHR is expressed as the percentage of changes from baseline (<i>B</i>). The BAL fluid and lungs were collected, and the amounts of IL-17 (<i>A</i>) and number of the indicated cell population (<i>C</i>) in the BAL fluid were measured. Histology of the lungs was examined by H&E and PAS staining (×20 magnification) and visualized by light microscope (<i>D</i>). Data shown are mean ± SE. *, p<0.05 in comparison with control antibody treated C3aR^−/− mice.</p

Antigen-specific pulmonary Th17 responses in C3aR-deficient mice.

<p><i>A–C</i>, Groups of C3aR^−/− and wild-type mice (n = 7–9) were i.v. injected with CD45.1⁺ OT-II T cells (5×10⁶ cells/transfer; day -1), and were intranasally injected with the mixture of <i>Aspergillus</i> proteinase plus OVA on day 0, 2, 4, 6. On day 7, lymphoid cells from the lung (<i>B</i>) and draining LNs (<i>C</i>) were restimulated with PMA and ionomycin in the presence of brefeldin A and monensin, stained with anti-CD45.1 and CD4, and the expression of IL-17, IFN-γ, or IL-4 + IL-5 by CD45.1⁺CD4⁺ donor T cells was analyzed by intracellular staining. Left panels in <i>A</i> illustrate gating strategy. Bars in <i>B</i> and <i>C</i> show the mean values. Data shown represent at least three independent experiments. *, p<0.05 or ***, p<0.001 in comparison with wild-type recipients.</p