Dissertation

Clearance of Dying Cells Fuels Cardiac Macrophages for Wound Healing

Lantz, Connor William

2024

Overview

While largely appreciated for their antimicrobial and repair functions, macrophages have emerged as indispensable for the development, homeostasis, and regeneration of tissue, including regeneration of the neonatal heart. Upon activation, mammalian neonatal macrophages express and secrete factors that coordinate angiogenesis, resolve inflammation, and ultimately induce cardiomyocyte proliferation. This is contrary to adult macrophages, which are incapable of inducing significant levels of cardiac regeneration. The underlying mechanisms by which pro-regenerative macrophages are activated and regulated remain vague.Therefore, we compared the macrophage response after cardiac injury in newborns to adults. Single-cell RNA sequencing revealed accumulation of tissue-resident C1q+ TLF+ neonatal macrophages that were selectively polarized for apoptotic cell recognition (or efferocytosis). Genetic ablation of the apoptotic cell receptor MerTK in neonatal macrophages prevented cardiac regeneration, leading to impaired cardiac function and fibrotic scarring. Loss of MerTK greatly remodeled macrophage gene expression required for cellular metabolism, arachidonic acid metabolism, and biosynthesis of the eicosanoid thromboxane, the latter which unexpectedly activated parenchymal cell proliferation. Thromboxane receptors were discovered on neighboring cardiomyocytes and were required for a cellular metabolic shift which was coupled to mitogenic signaling. These data are consistent with a fundamental age-defined macrophage response in which lipid mitogens produced during efferocytosis support organ regeneration.Given our findings that efferocytosis is a key regulator of neonatal cardiac regeneration, we further investigated how efferocytosis triggers transcriptional and metabolic reprogramming in macrophages. Our current understanding of the transcriptional remodeling by efferocytosis is largely informed from analysis of bulk phagocyte populations; however, this precludes the heterogeneity between individual macrophages during apoptotic cell uptake. Moreover, phagocytes may contain so called “passenger” transcripts that originate from engulfed apoptotic bodies, thus obscuring the true transcriptional reprogramming of the phagocyte. To define the transcriptional diversity during efferocytosis, we utilized single-cell mRNA sequencing after co-cultivating macrophages with apoptotic cells. Importantly, transcriptomic analyses were performed after validating the disappearance of apoptotic cell-derived RNA sequences. Our findings reveal new heterogeneity of macrophage efferocytosis at a single-cell resolution, particularly evident between large peritoneal macrophages and small peritoneal macrophages. Ablation of MerTK, blunted efferocytosis signatures, disrupted macrophage metabolism, and led to the escalation of cell death-associated transcriptional signatures in large peritoneal macrophages. Taken together, our results newly elucidate the heterogenous transcriptional response of macrophages after exposure to apoptotic cells.Lastly, in vitro macrophage polarization is dependent on alterations in metabolite usage, however the in vivo relevance of this is vague and important to understanding divergence in immune function. Utilizing an optimized low-input metabolomic profiling platform in parallel with ex vivo isotope tracing, we assessed the metabolic network and transcriptome of sorted CCR2hi versus CCR2lo macrophages after cardiac injury. The data revealed a striking divergence and unexpected metabolite preference between cell subsets that was distinct from paradigms reported in vitro. Inflammatory CCR2hi macrophages prioritized glycolysis and glutamine metabolism as opposed to CCR2lo macrophages, which favored fatty acid oxidation and nucleotide metabolism, supportive of cell proliferation. CCR2lo macrophages notably also increased catabolism of polyamines, in conjunction with signatures of efferocytosis and resolution of inflammation. Genetic blockade of efferocytosis through MerTK blunted both mitochondrial metabolism and polyamine accumulation, leading to heightened inflammation and impaired cardiac repair. These findings not only reveal an atlas of in vivo metabolic preferences in macrophage subsets that are distinct from in vitro metabolism, but also identify how efferocytosis regulates polyamine metabolism to directly remodel macrophage metabolism for inflammation resolution and cardiac repair.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biology / Cellular biology / Immunology
• ISBN: 9798382761350