Distinct Roles for tissue-resident versus tissue-recruited macrophages in antiviral immunity Restricted; Files Only
Eddins, Devon (Fall 2021)
Abstract
Tissue-resident macrophages (TRMΦ) are important immune sentinels responsible for maintaining tissue and immune homeostasis within their specific niche. Recently, the origins of TRMΦ have undergone intense scrutiny where now most TRMΦ are thought to originate early during embryonic development independent of hematopoietic stem cells (HSCs). We previously characterized two distinct subsets of mouse peritoneal cavity MΦ (Large and Small Peritoneal MΦ; LPM and SPM, respectively) whose origins and relationship to both fetal and adult long-term (LT)-HSCs have not been fully investigated. Here we employ highly purified LT-HSC transplantation and in vivo lineage tracing to show a dual ontogeny for LPM and SPM, where the initial wave of peritoneal MΦ is seeded from yolk sac-derived precursors, which later require LT-HSCs for regeneration. In contrast, transplanted fetal and adult LT-HSCs are not able to regenerate brain-resident microglia following lethal irradiation. Thus, we demonstrate that LT-HSCs retain the potential to develop into TRMΦ, but their requirement is tissue-specific in the peritoneum and brain.
In late 2019, severe acute respiratory syndrome coronavirus 2 emerged from Wuhan, China spurring the Coronavirus Disease-19 (COVID-19) pandemic that has resulted in over 219 million confirmed cases and nearly 4.6 million deaths worldwide. In the United States, there have been ~48 million cases and over 779,000 deaths where troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African-American (AA) patients in some areas, yet targeted studies within this demographic are scant. We performed multi-omics single-cell analyses of immune profiles from matching airways and blood samples of Black/AA patients during acute infection that revealed low viral load, yet pronounced and persistent pulmonary neutrophilia with advanced features of cytokine release syndrome and acute respiratory distress syndrome (ARDS), including exacerbated production of IL-8, IL-1β, IL-6, and CCL3/4 along with elevated levels of neutrophil elastase and myeloperoxidase. Notably, recruited IFITM2+ neutrophils are transcriptionally reprogrammed by pulmonary TNF and IL-1β into long-lived and hyper-inflammatory subsets with increased degranulation in the airways. Circulating S100A12+/IFITM2+ mature neutrophils are recruited via the IL-8/CXCR2 axis, which emerges as a potential therapeutic target to reduce pathogenic neutrophilia and constrain ARDS in severe COVID-19 disease.
Table of Contents
Table of Contents
Dissertation Abstract..................................................................................................... i
Dedication..................................................................................................................... iii
Table of Contents.......................................................................................................... iv
Acknowledgements....................................................................................................... vii
Chapter 1. Introduction
1.1. Macrophages.......................................................................................................... 1
1.2. Ontogeny of tissue-resident macrophages............................................................. 2
1.3. Supplementation vs. supplanting of TRMΦ.......................................................... 7
1.4. Animal models for studying TRMΦ...................................................................... 8
1.5. Pulmonary Macrophages...................................................................................... 11
1.6. Role of MΦ ontogeny in function........................................................................ 15
1.7. Respiratory infections.......................................................................................... 18
1.8. Emergence of SARS-CoV-2................................................................................ 20
1.9. Summary ............................................................................................................. 21
Chapter 2. Requirement of LT-HSC for TRMΦ regeneration is tissue-specific
2.1. Introduction......................................................................................................... 23
2.2. Results................................................................................................................. 25
2.3. Discussion............................................................................................................ 28
2.4. Methods............................................................................................................... 33
2.5. Tables and Figures
2.5.1. Table 1: FACS staining reagents used in this study................................. 40
2.5.2. Table 2: Fluidigm plate design for single-cell HT-qPCR assay............... 41
2.5.3. Fig. 1: Tissue-resident peritoneal and brain MΦ develop and take residence during early fetal development 42
2.5.4. Fig. 2: Tissue-resident SPM, LPM, and microglia emerge before the development of fetal LT-HSCs 43
2.5.5. Fig. 3: Transplanted LT-HSC from fetal and adult origin fully regenerate tissue-resident peritoneal MΦ but not brain microglia.................................................................................................................. 45
2.5.6. Fig. 4: Tissue-resident peritoneal MΦ derived from LT-HSC transplants are functionally comparable to their host-derived counterpart................................................................................................ 47
2.5.7. Fig. 5: Representative gating strategy–PerC and frequency of eGFP labeled PerC MΦ in Runx1 lineage tracing mice at age of sacrifice................................................................................................ 49
2.5.8. Fig. 6: Representative gating strategy (Brain) and brain engrafting cells in transplanted mice 51
2.5.9. Fig.7: LT-HSC adoptive transplantation strategy and representative blood RFP chimerism kinetics of transplant recipient mice........................................................................................................... 53
2.6. Acknowledgements............................................................................................. 55
Chapter 3. Innate immune responses in COVID-19 pathogenesis
3.1. Inactivation of SARS-CoV-2 and COVID-19 patient samples
3.1.1. Introduction............................................................................................... 56
3.1.2. Results....................................................................................................... 57
3.1.3. Discussion................................................................................................. 61
3.1.4. Methods.................................................................................................... 63
3.1.5. Figures
3.1.5.1. Fig. 1: Fixation with commercially-available fixatives promote complete inactivation of SARS-CoV-2-infected cells amenable to flow cytometric analyses.............................................. 72
3.1.5.2. Fig 2: UVC irradiation exposure for 30 minutes inactivates SARS-CoV-2 with minimal effects on antibody/protein detection assays................................................................................. 73
3.1.5.3. Fig 3: Metabolite extraction solvent (Solution A) completely inactivates SARS-CoV-2 and maintains sample quality for downstream metabolomics assays..................................................... 75
3.1.5.4. Fig. 4: Heat inactivation during cDNA synthesis completely inactivates SARS-CoV-2 in scRNA-seq emulsions 77
3.1.5.5. Fig. 5: UVC inactivation abolishes microbial growth in plaque assay cultures. 78
3.1.6. Acknowledgements................................................................................... 79
3.2. Pathogenic neutrophilia in severe COVID-19 disease
3.2.1. Introduction .............................................................................................. 81
3.2.2. Results....................................................................................................... 82
3.2.3. Discussion................................................................................................. 90
3.2.4. Methods.................................................................................................... 95
3.2.5. Tables and Figures
3.2.5.1. Table 1: Demographic and clinical data from the 35 Black/African American individuals enrolled in our studies 104
3.2.5.2. Table 2: Clinical characteristics of the 35 Black/African American individuals enrolled in our studies 105
3.2.5.3. Table 3: High-dimensional 30-parameter, including intracellular cytokine staining, and 17-parameter flow cytometry panels used for airway and blood cells............................................. 107
3.2.5.4. Table 4: Mesoscale U-PLEX biomarker group 1 human and myeloperoxidase (MPO) assays 109
3.2.5.5. Table 5: Panel of oligo-conjugated antibodies used in multi-omics scRNA-seq assays to measure surface protein markers on cells from airways and blood....................................................... 110
3.2.5.6. Table 6: Sequencing depth of independent samples......................... 112
3.2.5.7. Table 7: Mitochondrial gene thresholds and total ADT UMIs of individual samples 114
3.2.5.8. Fig. 1 Experimental design for the systems immunology approach (integrated multi-omics single-cell assays) to study COVID-19 patient samples............................................................... 115
3.2.5.9. Fig. 2 Exacerbated neutrophilia in the airways and matching blood of severe COVID-19 patients 116
3.2.5.10. Fig: 3 Cytokine release syndrome is dominated by IL-8 and IL-1β with pronounced myeloperoxidase content and activity in the lung microenvironment.............................................. 117
3.2.5.11. Fig. 4 Multi-omic single-cell RNA-seq reveals emergency granulopoiesis in the circulation and abundant heterogeneous populations of mature neutrophils in the airways with distinct inflammatory states 119
3.2.5.12. Fig.5 Mature neutrophils are continuously recruited from circulation and progress toward a hyperinflammatory state........................................................................................................... 121
3.2.5.13. Fig. 6 TNF and IL-1β drive inflammatory reprogramming in neutrophils recruited to the lung 123
3.2.5.14. Fig. 7 Viral transcripts (scRNA-seq) and viral load in the airways of severe patients 125
3.2.5.15. Fig. 8 Representative gating strategy for Hi-D FACS data.............. 126
3.2.5.16. Fig. 9 Additional cytokine assessment in blood and lungs............... 127
3.2.5.17. Fig. 10 Multi-omic scRNA-seq analysis of leukocytes in the blood and lung 128
3.2.5.18. Fig. 11 Gene signature for immature neutrophils is lacking in healthy donors and lungs of COVID-19 patients 130
3.2.5.19. Fig. 12 Cellular signatures during SARS-CoV-2 pathogenesis........ 132
3.2.6. Acknowledgements................................................................................... 134
4. Chapter 4. Conclusion
4.1 Distinct roles of LT-HSC-independent TRMΦ versus LT-HSC-dependent MDMΦ 136
4.2 Innate immune regulation and dysfunction in COVID-19 pathogenesis............. 139
Literature Cited................................................................................................................. 142
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