PPARγ activation following apoptotic cell instillation promotes resolution of lung inflammation and fibrosis
Pulmonary fibrosis is a potentially fatal disease characterized by continuous alveolar epithelial injury and dysregulated repair, leading to myofibroblast accumulation and excessive deposition of extracellular matrix and connective tissue. Previous studies have indicated that efficient alveolar epithelial cell repair is critical for normal healing without fibrosis. Because apoptotic cell recognition and clearance result in growth factor release and activation of signaling molecules involved in maintaining the epithelium and endothelium, the maintenance of alveolar integrity has been proposed to be critically dependent on intact mechanisms of apoptotic cell clearance.
The feasibility of cellular therapy based on the immunomodulatory properties of apoptotic cells has already been evaluated in different experimental models of acute and chronic inflammation, to restore or induce immune tolerance. These beneficial effects have been attributed to the release of anti-inflammatory cytokines, such as TGF-β and IL-10, by macrophages upon apoptotic cell recognition and clearance. However, TGF-β1 production in alternatively activated macrophages has been linked to the development of pulmonary fibrosis. Several studies have demonstrated that PPARγ plays important roles in regulating processes related to fibrogenesis in vitro and in vivo. Thus, important questions are raised regarding whether PPARγ activation is enhanced by in vivo exposure to apoptotic cells over the course of lung injury, thereby leading to altered alveolar macrophage programming and, ultimately, hastening the resolution of both lung inflammation and fibrosis. Inflammatory responses have been associated with the initial stage of idiopathic pulmonary fibrosis.
We first characterized changes in PPARγ expression and activation following apoptotic cell instillation over the course of bleomycin-induced lung injury. We show that PPARγ mRNA and protein levels in alveolar macrophages and lung tissue were immediately enhanced following instillation of apoptotic cells relative to bleomycin with or without viable cells. Moreover, the levels of PPARγ mRNA and protein following early exposure to apoptotic cells increased gradually for up to 21 days after bleomycin treatment. However, the presence of viable Jurkat cells had no effect. These data demonstrate the specificity of PPARγ induction for apoptotic cell recognition systems. Based on our results, alveolar macrophages are a primary source of PPARγ following in vivo exposure to apoptotic cells. Moreover, immunohistochemistry and double immunofluorescence staining studies indicate that apoptotic cell-induced PPARγ expression was observed mainly in macrophages (Figure 1) and, to some extent, fibroblasts on days 7 and 14 after bleomycin treatment.
Concomitant with PPARγ expression, immediate and prolonged enhancement of PPARγ activity following apoptotic cell instillation was demonstrated in bleomycin-stimulated lung tissue. In support of this, enhanced expression of direct PPARγ targets, including CD36, MMR, and Arg1, in alveolar macrophages and lung tissue following apoptotic cell instillation was greater than that observed following bleomycin with or without viable cells. Interestingly, all of these genes are known to be up-regulated by PPARγ activation and are characteristic of alternative macrophage programming.
Next, using a pharmacological approach involving a PPARγ antagonist GW9662, we investigated the role of enhanced PPARγ activation following apoptotic cell instillation in altering inflammatory and fibrogenic programs in alveolar macrophages and the lung. Co-administration of the PPARγ antagonist, GW9662, reversed the enhanced efferocytosis, and the reduced pro-inflammatory cytokine expression, neutrophil recruitment, myeloperoxidase activity, hydroxyproline contents, and fibrosis markers, including type 1 collagen α2, and α-smooth muscle actin, in the lung by apoptotic cell instillation. In addition, inhibition of PPARγ activity reversed the expression of transforming growth factor beta (TGF-β), interleukin (IL)-10, and hepatocyte growth factor (HGF). A mechanism diagram summarizing and integrating the effects of enhanced PPARγ activity following apoptotic cell instillation in bleomycin-induced lung fibrosis was shown in Figure 2. Our study supports the concept that administration of apoptotic cells may be a potential tool with great therapeutic efficacy due to induction of PPARγ shifting alternatively programmed alveolar macrophages associated with enhanced efferocytic ability and coordinated regulation of TGF-β, IL-10, and HGF in favor of resolving inflammation and preventing excessive, fibrotic outcomes.
Figure 1. PPARγ-positive staining in alveolar macrophages and lung fibroblasts after apoptotic cell instillation.
Figure 2. A mechanism diagram summarizing and integrating the effects of enhanced PPARγ activity following apoptotic cell instillation in bleomycin-induced lung fibrosis.
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PPARγ activation following apoptotic cell instillation promotes resolution of lung inflammation and fibrosis via regulation of efferocytosis and pro-resolving cytokines. Mucosal Immunol 8:1031-1046(2015.9).
Apoptotic cell instillation after bleomycin attenuates lung injury through hepatocyte growth factor induction. Eur. Respir. J. 40, 424-435 (2012).
Coordinated induction of cyclooxygenase-2/prostaglandin E2 and hepatocyte growth factor by apoptotic cells prevents lung fibrosis. J. Leukoc. Biol. 94, 1037-1049 (2013).