Gene Expression and Chromatin Dynamics During Macrophage Polarization in Health and Disease

• Main Author: Carvalho, Klebea
• Other Authors: Mortazavi, Ali
• Format: Other/Unknown Material
• Language: English
• Published: eScholarship, University of California 2021
• Subjects: Genetics; Immunology; Neurosciences; Alzheimer's disease; Complement cascade; Footprinting; Gene regulatory networks; Macrophage polarization; Microglia activation; Arctic
• Online Access: https://escholarship.org/uc/item/5vz0591r

The complex task of maintaining homeostasis and fighting diseases involves an intricate network of immune cells with many relevant players. This thesis is focused on the plasticity and versatility of a critical class of innate immune cells called macrophages. Most naïve macrophages, named M0s, have the ability to polarize into two main subtypes, M1s and M2s, which help maintain a balance of inflammatory and anti-inflammatory responses, respectively. An imbalance in the ratio of M1s to M2s is associated with poor prognoses for a variety of diseases. Thus, understanding the markers and the gene regulatory networks (GRNs) that underlie the M0 to M1 or M2 polarization is crucial to help modulate these cells ratios for therapeutic purposes. Here, we applied bulk and single-cell RNA-seq and ATAC-seq to a high-resolution time series of HL-60-derived M0s polarizing towards M1 or M2 over 24 hours. We identified transient M1 and M2 markers and the main transcription factors (TFs) that drive polarization. In addition, we identified a novel M2 marker, ID2. We built bulk and single-cell polarization GRNs and identified at least 30 novel TF-TF interactions during M1/M2 polarization. We further compared the strengths of using bulk and single-cell technologies to build our GRNs providing experimental and computational guidelines for building GRNs of cellular maturation in response to microenvironmental cues. We concluded that despite the great advances of single-cell analysis, a combination of bulk and single-cell techniques provided a more complete GRN. The brain resident macrophages, named microglia, do not fit into the dichotomic M1/M2 dogma of polarization. However, microglial activation and inflammation are directly linked to progression of Alzheimer’s disease (AD). Neuroinflammation, hyperphosphorylated tau, and accumulation of amyloid beta plaques in the brain are hallmarks of AD, which presents progressive dementia as its main clinical feature. Amyloid plaques can activate the complement system. Complement activation, specifically activation of complement factor C5a and its receptor C5aR1 enhances microglial inflammation, which can worsen disease pathology through local injury and neuronal death. Thus, the C5a-C5aR1 signaling pathway is a potential target for modulation of AD. In order to investigate the effects of C5a in AD progression, we observed changes in hippocampal gene expression, hippocampal-dependent memory decline, and neuronal loss in two variants of the Artic mouse model of AD: one which lacks C5aR1 (cohort ArcticC5ar1KO) and one that overexpresses C5a under the GFAP promoter (cohort ArcticC5a+). The ArcticC5aR1KO group showed decreased inflammation, reduced activity of phagocytic and lysosomal pathways, and reduced cholesterol biosynthesis compared to Arctic mice. Furthermore, C5a overexpression led to poor cognitive performance, neuronal loss, and advanced disease progression compared to control. Our results suggest that pharmacological inhibition of C5a-C5aR1 signaling is a promising therapeutic strategy to treat AD.