Macrophage Polarization and Function in Cystic Fibrosis

Abstract

If "Diversity is the rule of nature", macrophages, one of the innate immune cells of our body, have proven to be a true apostle. At the early stage of infection, they protect the host by exhibiting the pro-inflammatory phenotype (M1). Upon clearance of microbial threats, macrophages engage themselves in the repair process of the damaged tissue by showing anti-inflammatory or wound healing (M2) attributes. While incomplete microbial clearance leads to recurrent infections, defects in macrophage-mediated tissue repair mechanism result in immunopathology. The classical (M1) and alternatively (M2) polarized macrophages are the two extreme ends of a spectrum of in vivo macrophages phenotypes that dictate the nature, duration and severity of inflammation. Murine models of chronic inflammatory and autoimmune diseases showed the necessity of an adequate balance between macrophage subsets to maintain homeostasis, while imbalance is likely to lead exaggerated inflammation. In humans, an association between defective macrophage function and disease severity has been reported in many diseases including asthma, cystic fibrosis (CF), COPD, and atherosclerosis. Unfortunately, human M1 and M2 macrophages have not been well characterized. Firstly, the lack of homologs for certain murine genes in humans makes murine markers of limited use in humans. Secondly, there was no consensus method for in vitro differentiation of human M1 and M2 macrophages. Therefore, the first part of this thesis aimed to develop a novel method to differentiate and characterize human M1 and M2 macrophages. Initial studies with THP-1 cell line derived macrophage-like cells demonstrated that they didn't fully represent human monocyte-derived macrophages (MDMs). MDMs were therefore chosen as starting materials for the rest of the study. MDMs were considered as uncommitted "M0" macrophages. A number of inducers were employed to polarize M0 macrophages into either M1s or M2s. LPS treated M1 macrophages showed CD64 + CD80 + phenotype, whereas, IFN- induced M1s exhibited CD64 ++ CD80 -phenotype. These M1s secreted pro-inflammatory cytokines including TNF-, IL-1, IL-8 and were highly phagocytic. On the contrary, IL-4/IL-13 induced M2 macrophages were identified as CD11b + CD209 + cell population and were endocytic. Once polarized, macrophages then were left in cytokine-deficient medium to assess the persistence of polarized phenotype over time. In cytokine-free condition, previously polarized macrophages reverted to M0 state by 12 days.Treatment with IL-13 on previously polarized M1 macrophages resulted in a switch to CD209 + M2sand vice versa indicating the plasticity nature of human macrophages.Excessive neutrophilic pulmonary inflammation is the hallmark of cystic fibrosis (CF).However, factors that trigger such dysregulated neutrophilic inflammation and why this is not