Macrophage Polarization and Function in Cystic Fibrosis

Abstract

If Diversity is the rule of 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- nflammatory 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+ M2s and 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 switched off are not yet clear. Macrophages play crucial roles in initiation and resolution of pulmonary inflammation, however, surprisingly little research in CF had been dedicated to macrophages. Defective phagocytosis by macrophages and its association with cystic fibrosis transmembrane conductance regulator (CFTR) gene had been reported in CF studies. Nevertheless, the influences of the mutated CFTR gene on macrophage polarization and thereby function had not previously been studied in CF. The model described above was utilized with blood samples from adults and children with CF. M2 polarization was significantly repressed in patients with CF. The number of cells expressing the human M2 marker CD209 was significantly low in CF [median (25th-75th%): healthy (n=9) 59(55-82)%; CF children (n=14) 41(30-52)% (p<0.01); CF adults (n=13) 46(25-60)% (p<0.01)]. Endocytosis was also decreased in both children and adults with CF (P<0.001). Inhibiting CFTR function with CFTRInh-172 and GlyH-101 in healthy cells recreated the CF macrophage phenotype, with a decreased number of cells expressing CD209 and decreased endocytosis. Following IL-13 treatment, both CF M0 cells and CFTRInh-172 inhibited M0 cells showed decreased surface expression of IL-13Rα1 compared to M0s from healthy volunteers indicating the inability to respond to IL-13 being associated with CFTR dysfunction. Furthermore, during acute pulmonary exacerbation (APE), but not in clinically stable CF, a greater proportion of M0 and IL-13 treated macrophages displayed surface expression of M1 marker, CD80 in both adults and children indicating both M0 and M2s during APE being prone to M1 polarization. Taken together, these data report a CFTR-dependent defective M2, but not M1, polarization of macrophages in CF which might be the possible underlying mechanism for exaggerated neutrophilic responses and impaired resolution of inflammation in CF.In summary, two novel models had been developed during this Ph.D. study. The in vitro human macrophage polarization and characterization model described the differentiation, phenotypic and functional characterization of human M0, M1 and M2 macrophages as well their plasticity nature. Knowing the functional states of pathogenic macrophages has a huge impact on understanding the disease pathogenesis and developing novel therapeutic targets. The CF macrophage model was developed with CFTR inhibitors that recreated CF macrophage phenotypes in vitro. Using this model a previously unidentified CFTR-dependent defect of M2 macrophage polarization in CF had been reported.