Transcriptomic phenotyping of the macrophage response to filarial nematode infection

Abstract

Helminths are paraphyletic group of parasitic metazoans that collectively consist of the nematodes, trematodes, cestodes and platyhleminthes. Helminth infections are characterized, amongst other things, by IL-4 and IL-13 production from CD4* Th2 cells. IL-4 and IL-13 induce alternatively activated macrophages (AAMΦ) via their action on the IL-4Rα receptor subunit. AAMΦ are also implicated in numerous other diseases such as fibrosis, allergy, cancer and diabetes.STAT6 drives IL-4Rα-dependent transcription in AAMΦ, leading to the induction of many genes including Arg -1, YM1 and RELMα. IL-4 also induces macrophage proliferation, a phenotype observed in vivo, but not in vitro. In vitro studies have shown that IL-4 upregulates macrophage mitochondrial metabolism in a process dependent upon the Iigand-dependent transcription factor PPARγ (peroxisome proliferator activator receptor gamma). In spite of the far -reaching effects of IL-4 on macrophage biology and physiology, we are unaware of the true role(s) of AAMΦ during helminth infection.In this thesis I present two transcriptomic analyses of macrophage responses to filarial nematode infection. In the first case we define IL-4Rα-dependent components of the macrophage response to nematode infection. Using RNA-Seq, WT and IL-4Rα-/- mice implanted with the parasitic nematode Brugia malayi are compared against inflammatory - like thioglycollate-elicited macrophages. This has allowed us to infer an anti-inflammatory phenotype for nematode -elicited macrophages (NeMΦ) in vivo. We also identify an unanticipated role for NeMΦ in the production of complement components in response to B. malayi challenge. During this project I develop a method to map transcription start site (TSS) usage with RNA-Seq data, this algorithm has been developed into the software 'TSS-Predictor' and is presented in chapter 3. Using TSS-Predictor we define TSS usage in our RNA -Seq dataset, and a subsequent analysis of transcription factor binding sites in AAMΦ-associated promoters confirms a role for PPARs in AAMΦ in vivo. Furthermore, we find that PPARδ, but not PPARγ is expressed during helminth infection. A targeted lipid analysis of macrophage-derived eicosanoids identified prostacyclin (PGI2) as a candidate ligand to mediate PPARδ-dependent transcription in NeMΦ during helminth infection in vivo.Finally, we integrate flow cytometry and a microarray gene expression profiling in a time series of macrophages elicited in response to the parasitic nematode Litomosoides sigmodontis. We capitalize on the discordant kinetics of alternative activation and proliferation in this model to define gene expression signatures associated with proliferation and AAMΦ. Based upon this analysis we present a working model for IL-4-dependent macrophage proliferation in vivo and characterise candidate receptors mediating this process. We also demonstrate in vivo that AAMΦ increase mitochondrial mass in response to L. sigmodontis infection.