Asthma is one of the most significant clinical problems in the developed world. Over 25 million people in the U.S. have asthma, with estimated health care and lost work productivity costs in the billions of dollars/year. Unfortunately, asthma prevalence is rapidly growing in developing countries such as China and India, presumably due to increased exposure to industrial pollution and progressive urbanization, making this a world-wide epidemic.
While some children with asthma “grow out of it”, a significant percentage of adolescents or young adults do not. Chronic asthma is a debilitating disease. Excess airway fibrosis and scarring are among the most significant sequela. This progressive change in airway anatomy and function is called remodeling, and results in reduced airflow despite increased respiratory effort. Remodeling is driven by cytokine producing, infiltrating immune cells, particularly eosinophils (EOS) that migrate into the lung from the peripheral blood in response to chronic allergic stimulation. Once there, EOS secrete both pro-fibrotic cytokines, especially TGF-β1 and pro-survival cytokines including GM-CSF, IL-3 and IL-5. TGF-β1 activates resident fibroblasts (Fb) and induces the production of extracellular matrix (ECM). GM-CSF and IL-5 prevent apoptosis, facilitating the accumulation of long-lived EOS in the inflamed lung.
A large number of clinical studies have shown that ablation of EOS, typically with inhaled corticosteroids, can prevent or mitigate airway remodeling. However, long-term steroid use has significant morbidity and better therapies are urgently needed. Therefore, we have focused on two main aspects of asthma biology:
- How are the production of pro-survival and pro-fibrotic cytokines regulated by EOS, and
- What are the signaling cascades and physiologic outputs, both in EOS as well as fibroblasts, that these cytokines engage to alter cell function?
Our ultimate goals are to identify proteins that can be targeted to reduce EOS numbers (induce apoptosis), prevent cytokine production, or modulate downstream signaling events. If exploitable, each endpoint should reduce airway damage and remodeling.
We have shown that Pin1 is highly expressed by airway Fb and EOS and that PPIase activity is rapidly enhanced by physiologic concentrations of GM-CSF, IL-5 or TGF-β1. Cytokine treatment induced Pin1 binding to Smad3, Smad6, multiple PI-3-K isoforms and Akt, suggesting an important role in the initiation and proximal propagation of TGF-β1 signals. Indeed, Pin1 KO or chemical inhibition blocked TGF-β1 mediated Smad3, Akt and JNK phosphorylation, disrupted Smad6 trafficking, and prevented Smad3 nuclear translocation and target gene activation. In vivo, Pin1 KO markedly attenuated pulmonary fibrosis in models of asthma or pulmonary (bleomycin) injury. Consistent with animal studies, Pin1 is overexpressed in airway inflammatory cells of patients with allergic disease. We are currently exploring how Pin1 mediates these effects by defining the interaction site(s) in target proteins, characterizing changes in gene expression in the absence of Pin1 as well as generating conditional KO mice for interrogation of the tissue specific function of Pin1 in pulmonary and other disease models.
In peripheral blood, EOS have a life-span of approximately 3 days before undergoing apoptosis. IL-5 or GM-CSF prevents cell death both in vitro and in vivo. Thus EOS are an excellent, primary cell model to study cell death and the production and signaling of factors that can modulate it. Pharmacologic inhibition of Pin1 accelerated EOS death despite pro-survival cytokine signaling. We showed that IL-5 or GM-CSF induces Pin1 activation and binding to the pro-apoptotic Bcl-2 homolog, Bax at Thr167-Pro168. Binding was preceded by and dependent on Thr167 phosphorylation by Erk. Mutation of Thr167 to alanine prevented Pin1 interactions and rendered transduced EOS insensitive to GM-CSF or IL-5. Conversely, productive interactions prevented Bax activation and EOS death. We are currently defining how Pin1 affects other components of the intrinsic and extrinsic apoptotic pathways with a particular focus on Fas and FADD.