bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

1 The EGR2 is indispensable for tissue-specific imprinting of alveolar

2 macrophages in health and tissue repair

3

4 Jack McCowan1,2, Phoebe M. Kirkwood1,2, Frédéric Fercoq3, Wouter T’Jonck1,2, Connar M.

5 Mawer1,4, Richard Cunningham1,2, Ananda S. Mirchandani1,2, Anna Hoy1,6, Gareth-Rhys

6 Jones1,2, Carsten G. Hansen1,2, Nik Hirani1,2, Stephen J. Jenkins1,2, Sandrine Henri7, Bernard

7 Malissen7, Sarah R. Walmsley1,2, David H. Dockrell1,2, Philippa T. K. Saunders1,2, Leo M.

8 Carlin3,8, Calum C. Bain1,2*

9 10 1 University of Edinburgh Centre for Inflammation Research, Queens Medical Research 11 Institute, 47 Little France Crescent, Edinburgh BioQuarter, Edinburgh, EH16 4TJ, UK 12 2 Institute for Regeneration and Repair, University of Edinburgh, 5 Little France Drive, 13 Edinburgh BioQuarter, Edinburgh EH16 4UU, UK 14 3 Cancer Research UK Beatson Institute, Glasgow, G61 1BD, UK 15 4 Current address: Rayne Institute, University College London, 5 University St, Bloomsbury, 16 London WC1E 6JF 17 5 Institute of Immunology & Infection Research, School of Biological Sciences, University of 18 Edinburgh, Edinburgh EH9 3FL, UK 19 6 Current address: Resolution Therapeutics Limited, Centre for Regenerative Medicine, 20 Edinburgh BioQuarter, Edinburgh, EH16 4UU, UK 21 7 Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, INSERM, U1104, 22 CNRS UMR7280, 13288 Marseille, France 23 8 Institute of Cancer Sciences, University of Glasgow, Glasgow, G61 1QH, UK 24 25 *Corresponding author & Lead Contact: [email protected] 26 27 One Sentence Summary: EGR2 controls alveolar macrophage function in health and disease

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29

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1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

31 Abstract

32 Alveolar macrophages are the most abundant macrophages in the healthy lung where they

33 play key roles in homeostasis and immune surveillance against air-borne pathogens. Tissue-

34 specific differentiation and survival of alveolar macrophages relies on niche-derived factors,

35 such as colony stimulating factor 2 (CSF-2) and transforming growth factor beta (TGF-b).

36 However, the nature of the downstream molecular pathways that regulate the identity and

37 function of alveolar macrophages and their response to injury remains poorly understood.

38 Here, we identify that the transcriptional factor EGR2 is an evolutionarily conserved feature of

39 lung alveolar macrophages and show that cell-intrinsic EGR2 is indispensable for the tissue-

40 specific identity of alveolar macrophages. Mechanistically, we show that EGR2 is driven by

41 TGF-b and CSF-2 in a PPAR-g-dependent manner to control alveolar macrophage

42 differentiation. Functionally, EGR2 was dispensable for lipid handling, but crucial for the

43 effective elimination of the respiratory pathogen Streptococcus pneumoniae. Finally, we show

44 that EGR2 is required for repopulation of the alveolar niche following sterile, bleomycin-

45 induced lung injury and demonstrate that EGR2-dependent, monocyte-derived alveolar

46 macrophages are vital for effective tissue repair following injury. Collectively, we demonstrate

47 that EGR2 is an indispensable component of the transcriptional network controlling the identity

48 and function of alveolar macrophages in health and disease.

49

50

2 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

51 Introduction

52 Tissue resident macrophages play fundamental roles in protective immunity and wound repair

53 following injury, but also in the maintenance of homeostasis. The functions of macrophages

54 vary to meet the demands of the local environment, and this is reflected in the phenotypic

55 diversity detected amongst macrophages in different tissues. Indeed, although all tissue

56 macrophages possess a common ‘core’ transcriptional signature (1), there are additional

57 tissue-specific expression characteristics that enable organ- and even niche-specific

58 functions (2, 3). However, the local environmental signals and the downstream molecular

59 pathways that control this tissue-specific imprinting of macrophages in different environments

60 are incompletely understood.

61 Alveolar macrophages are the most abundant macrophage population in the healthy

62 lung, where they provide a first line of defence against airborne pathogens, as well as

63 maintaining lung homeostasis, for instance through the regulation of pulmonary surfactant.

64 However, in chronic lung pathologies such as allergic asthma, idiopathic pulmonary fibrosis

65 (IPF) and chronic obstructive pulmonary disease (COPD), alveolar macrophages display

66 aberrant activity and, in many cases, appear to perpetuate disease (4). Moreover, monocytes

67 and macrophages appear to play a particular pathogenic role in the context of severe

68 coronavirus disease 2019 (COVID-19) (5). Thus, the mechanisms governing alveolar

69 macrophage imprinting may yield important insights into how lung-specific cues regulate

70 homeostasis and susceptibility to disease.

71 Alveolar macrophages are derived from erythromyeloid progenitors (EMPs) and foetal

72 liver monocytes that seed the lung during embryonic development (6-8). However, the

73 characteristic phenotype and functional properties of alveolar macrophages do not develop

74 until the first few days of postnatal life in parallel with alveolarisation of the lung and are

75 controlled by CSF-2 (GM-CSF) (8-10) and the immunoregulatory cytokine TGF-b (11).

76 Together these cytokines induce expression of the transcription factor peroxisome proliferator-

77 activated gamma (PPAR-g) to promote survival and tissue-specific specialisation,

3 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

78 including upregulation of involved in lipid uptake and metabolism. Consequently, mice

79 in whom Csf2rb, Tgfbr2 or Pparg has been genetically ablated in myeloid cells develop

80 spontaneous pulmonary alveolar proteinosis (9-11). However, alveolar macrophages largely

81 fail to develop in the absence of CSF-2 and TGF-b receptor signalling due to their key role in

82 macrophage survival. Therefore, it remains unclear if or how these factors control the tissue-

83 specific identity and function of alveolar macrophages. Moreover, while considered the ‘master

84 transcription factor’ of alveolar macrophages, PPAR-g has been implicated in the control of

85 other tissue macrophages, including splenic red pulp macrophages (12, 13), and thus, the

86 transcriptional network responsible for conferring specificity upon alveolar macrophage

87 differentiation remains unclear. Finally, if and how additional transcriptional regulators are

88 involved in regulating these processes in the context of inflammation and repair is largely

89 unexplored.

90 Here, we have used single cell RNA sequencing (scRNA-seq) to identify the

91 transcriptional regulators expressed by alveolar macrophages. We show that expression of

92 the transcriptional factor EGR2 is a unique feature of lung alveolar macrophages compared

93 with other lung mononuclear phagocytes and macrophages resident in other tissues. Using

94 cell-specific ablation of Egr2 and mixed bone marrow chimeric mice, we show that cell-intrinsic

95 EGR2 is indispensable for the tissue-specific identity of alveolar macrophages and their ability

96 to control infection with a major respiratory pathogen, Streptococcus pneumoniae. RNA

97 sequencing (RNA-seq) shows that EGR2 controls a large proportion of the core transcriptional

98 signature of alveolar macrophages, including expression of Siglec5, Epcam and Car4.

99 Mechanistically, we show that EGR2 expression is induced by TGF-b and CSF-2-dependent

100 signalling, and acts to maintain expression of CCAAT-enhancer-binding beta (C/EBPb)

101 to control alveolar macrophage differentiation. Finally, using the bleomycin-induced model of

102 lung injury and a combination of fate mapping approaches, we show that post-injury

103 repopulation of the alveolar macrophage niche occurs via differentiation of bone marrow-

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104 derived cells in an EGR2-dependent manner and that these monocyte-derived macrophages

105 are indispensable for effective tissue repair and resetting of tissue homeostasis.

106

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107 Results

108 EGR2 expression is a selective property of alveolar macrophages

109 To begin to dissect the molecular pathways underlying the niche-specific imprinting of alveolar

110 macrophages, we performed scRNA-seq of murine lung mononuclear phagocytes from lung

111 digests to identify the unique transcriptional profile of alveolar macrophages. To this end, non-

112 granulocytic CD45+ cells from lungs of Rag1–/– mice were purified by FACS and sequenced

113 using the 10x Chromium platform (Supplementary Figure 1A). 3936 cells passed quality

114 control and were clustered using Uniform Manifold Approximation and Projection (UMAP)

115 dimensionality reduction analysis within the Seurat R package. NK cells, identified by their

116 expression of Ncr1, Nkg7 and Gzma, were excluded (Supplementary Figure 1A) and the

117 remaining myeloid cells were re-clustered to leave six clusters of mononuclear phagocytes,

118 and these were annotated using known landmark profiles (Figure 1A, B).

119 Cluster 1 represented monocytes based on their expression of Itgam (encoding CD11b), Csf1r

120 and Cd68, and could be divided into classical and non-classical monocytes based on

121 expression of Ly6c2 and Treml4 respectively (Figure 1A, B). Cluster 2 represented interstitial

122 macrophages based on their high expression of Cx3cr1, Cd68, Csf1r and H2-Aa and lack of

123 the Xcr1 and Cd209a genes which defined cDC1 (cluster 5) and cDC2 (cluster 6) respectively.

124 Alveolar macrophages (cluster 3) formed the largest population and could be defined by their

125 expression of Itgax (encoding CD11c), Siglec5 (encoding SiglecF) and Car4, and lack of

126 Cx3cr1 and Itgam. Cluster 4 was transcriptionally similar to cluster 3, but was defined by genes

127 associated with cell cycle, including Mki67, Birc5 and Tubb5, suggesting these represent

128 proliferating alveolar macrophages (Figure 1A, B). Next, we compared gene expression

129 profiles of these clusters, focussing on genes more highly expressed by alveolar macrophages

130 relative to all other mononuclear phagocytes. 722 genes fitted these criteria, including Fapb1,

131 Spp1 (encoding osteopontin) and Cidec which are known to be uniquely and highly expressed

132 by alveolar macrophages (Supplementary Table 1) (1, 3). Within this cassette of genes, we

133 turned our attention to genes encoding transcription factors/regulators, as we hypothesised

134 that these might control the tissue specific differentiation of alveolar macrophages. As

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135 expected, these included Pparg, Cebpb and Bhlhe41 which have been shown to control the

136 development and self-renewal capacity of alveolar macrophages (9, 14, 15) (Figure 1C).

137 However, this analysis also revealed transcription factors such as Id1, Klf7 and Egr2 which

138 have not previously been implicated in the control of alveolar macrophage differentiation. We

139 focussed on EGR2, which is part of a family of early growth response (EGR) transcription

140 factors, comprising EGR1-4, as Egr2 appeared to be expressed in a particularly selective

141 manner by alveolar macrophages (Figure 1D) when compared with other tissue macrophages

142 at mRNA (Figure 1E) and protein level (Figure 1F, G & Supplementary Figure 2A). In

143 contrast, while highly expressed by alveolar macrophages, Pparg was also expressed at a

144 high level by splenic red pulp macrophages (Figure 1E), consistent with previous reports (12,

145 13). Next, we performed analogous analysis of EGR2 expression across a variety of human

146 macrophage populations from scRNA-seq data sets within the Human Cell Atlas. Consistent

147 with our analysis in the mouse, this showed that EGR2 expression was confined to lung

148 macrophages, and in particular FABP4+ macrophages which correspond to airway

149 macrophages (Figure 1H), and we confirmed this at protein level, showing that CD163+HLA-

150 DR+ bronchoalveolar lavage (BAL) macrophages uniformly express EGR2 (Supplementary

151 Figure 2B). Thus, these data demonstrate that EGR2 expression is a constitutive, specific

152 and evolutionary conserved feature of alveolar macrophages.

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Figure 1

A B Cd68 Itgam Csf1r Ly6c2 Treml4 Cx3cr1 Cd63 1-Monocytes 2-InterstitialMφ 3-AlveolarMφ 4-Prolif.Alv.Mφ 5-cDC1 6-cDC2

UMAP2 H2-Aa Itgax Siglec5 Car4 Mki67 Xcr1 Cd209a

1 2 3 4 5 6 UMAP1

C D Egr2 Egr2

Expression 3 Monocytes Atf3 Irf7 Mef2a Interstitial Mφ Cebpb Irf9 Mef2c -2 0 2 Fos Junb Rara 2 Alveolar Mφ Hes1 Klf2 Zeb2 Prolif. Alv. Mφ UMAP2 Irf1 1 cDC1 Irf2 Klf13 cDC2 Irf5 Maf1 Ahr Klf6 Nfat5 Expression level 0 Atf3 Jun Rbpj Batf3 Junb Rel UMAP1 Foxp1 Maf Runx3 Id2 Mafb Tcf4 Irf1 Mef2a Irf5 Mef2c E F Baz1a Fosb Lrrfp1 Creb5 Bhlhe41 Fosl2 Lsr Cebpa 7.5 Cebpa Hmgn2 Pparg Bhlhe41 Cebpb Id1 Runx1 Lmo4 Lung Creb5 Klf7 Runx2 5.0 alveolar Mφ Ctnnb1 Lima1 Rxra Hmgn2 Baz1a Baz1b Egr2 Lmo4 Tcf7l2 2.5 Ttf2 Fosl2 Erh Lsr Pbx3 Pparg 0 Brain Cbx1 logCPM microglia Taf1 Fosb Taf6 Runx2 Atf1 Egr2 Maz Id1 Lmo4 Batf3 Cebpb Spleen red Id2 Runx1 pulp Mφ Irf8 Ctnnb1 Rxra Lima1 100 80 Tcf7l2 60 Peritoneal Nr4a1 Lrrfp1 40 F4/80hi Mφ Hes6 Klf7 20

Events (% of ) 0 φ φ φ 0102 103 104 105 EGR2-PE Microglia Alveolar M Spleen RP MPeritoneal M G H EGR2

) 2 Lung 3 **** 10 3 3 Liver × Spleen 2 2 Brain MFI ( 1 Δ EGR2 1 1 EGR2 0 Expression level 0 0 φ φ φ φ φ M M M Lung Liver Spleen Brain 0 2 4 6 + ) – cDC1cDC2 mono. ) Alv. M Int. hi FABP4 MicrogliaPeritoneal Ly6C Splenic M Kupffer cells Lung Langerhans cells DermalDermal (MHCII (MHCII 161 162 Figure 1. EGR2 expression is a selective property of alveolar macrophages

163 A. UMAP dimensionality reduction analysis of 3936 cells (non-granulocyte, myeloid cells) reveals six 164 clusters of mononuclear phagocytes, including monocytes, macrophages (Mf) and conventional 165 dendritic cells (cDC) in murine lungs. 166 167 B. Feature plots displaying expression of individual genes by clusters identified in A. 168 169 C. Heatmap showing the top 20 most differentially expressed genes by each cluster defined in A. and 170 annotated to show the upregulated transcription factors/regulators within each cluster. 171 D. Overlay UMAP plot and feature plot showing expression of Egr2 by clusters identified in A. 172 E. Heatmap showing relative expression of selected transcription factors by lung alveolar macrophages, 173 CD102+ peritoneal macrophages, brain microglia and red pulp splenic macrophages as derived from 174 the ImmGen consortium. 175 F. Representative expression of EGR2 by lung alveolar macrophages, CD102+ peritoneal macrophages, 176 brain microglia and red pulp splenic macrophages obtained from adult unmanipulated C57BL/6 mice. 177 Shaded histograms represent isotype controls. Data are from one of three independent experiments 178 with 3-4 mice per experiment. 179

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180 G. Expression of EGR2 by the indicated macrophage and myeloid cell populations shown as relative 181 mean fluorescence intensity (MFI) (MFI in Egr2fl/fl (Cre–) – MFI in Lyz2Cre/+.Egr2fl/fl (Cre+) mice). Data 182 represent 3-4 mice per tissue. Error bars represent S.D. One-way ANOVA followed by Tukey’s multiple 183 comparisons post-test. **** p<0.0001 184 H. In silico analysis of EGR2 and FABP4 expression by lung, liver, spleen and brain macrophages 185 extracted on the basis of C1QA+ expression from (16-18). 186 187 188 EGR2 is required for the phenotypic identity of alveolar macrophages

189 Previous work has suggested that EGR1 and EGR2 act in a redundant manner (19), while

190 other studies have suggested EGR transcription factors are completely dispensable for

191 macrophage differentiation (20). However, many of these studies were performed in vitro and

192 the roles of EGRs in tissue-specific macrophage differentiation has not been assessed

193 comprehensively in vivo, in part, due to the postnatal lethality of global Egr2–/– mice (21, 22).

194 To determine the role of EGR2 in alveolar macrophage development and differentiation, we

195 crossed Lyz2Cre mice (23) with Egr2fl/fl mice (24), to generate a strain in which myeloid cells,

196 including monocytes, macrophages, dendritic cells and neutrophils, lack EGR2 in a

197 constitutive manner. We performed unbiased UMAP flow cytometry analysis on lung

198 leukocytes obtained from Lyz2Cre.Egr2fl/fl mice (referred to here as Cre+) and Egr2fl/fl littermate

199 controls (referred to here as Cre– mice), focussing on ‘lineage’ negative (CD3–CD19–NK1.1–

200 Ly6G–) CD11c+ and CD11b+ cells in lung digests (Figure 2A). Surface marker analysis of cells

201 pooled from Cre– and Cre+ mice confirmed the presence of alveolar and interstitial

202 macrophages, eosinophils and subsets of dendritic cells and monocytes (Figure 2A) and this

203 was validated by manual gating (Figure 2B & Supplementary Figure 3A). Due to their

204 CD11chiCD11b– phenotype, alveolar macrophages clustered separately from the other

205 CD11b+ myeloid cells (Figure 2A-C). All myeloid cells, including alveolar macrophages, were

206 equally abundant in the lungs of Cre– and Cre+ mice. (Figure 2D). However, whereas alveolar

207 macrophages from Cre– mice expressed high levels of SiglecF, the majority of alveolar

208 macrophages obtained from Cre+ mice lacked SiglecF expression (Figure 2E), explaining

209 their distinct positioning within the alveolar macrophage cluster in the UMAP analysis. Indeed

210 only ~7% of alveolar macrophages in Cre+ mice expressed high levels of SiglecF, and further

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211 analysis showed that these expressed high levels of EGR2 (Figure 2F), suggesting that the

212 SiglecF+ cells remaining in the Cre+ mouse represent cells that have escaped Cre-mediated

213 recombination. Consistent with this, SiglecF+ cells in the Cre+ mouse expressed high levels of

214 CD11c equivalent to alveolar macrophages from Cre– mice, whereas SiglecF– alveolar

215 macrophages expressed lower levels of CD11c (Figure 2G). We did not detect differences in

216 the proliferative activity of Egr2-sufficient and -deficient alveolar macrophages (Figure 2G).

217 Importantly and consistent with the lack of EGR2 expression by other tissue resident

218 macrophages, we saw no effect on the cell number and expression of signature markers by

219 resident macrophages in other tissues, including in the spleen where macrophages share a

220 dependence on PPAR-g (12, 13) (Supplementary Figure 3B, C). Thus, these data

221 demonstrate that while EGR2 expression is dispensable for alveolar macrophages survival

222 and self-maintenance, it is indispensable for imprinting key phenotypic features of the cells in

223 the healthy lung. Figure 2

A Combined Cre– +Cre+ CD64 MerTK CD11c CD11b Ly6C CD103 30 SiglecF High

20

10 UMAP2 0 Low 0 10 20 30 UMAP1

B C Cre– Cre+ D 30 30 Alveolar Mφ 3 3 Alveolar Mφ Interstitial Mφ hi 18.6 19.7 Interstitial Mφ

20 Ly6C monocytes ) hi lo 20 – Ly6C monocytes Ly6C monocytic cells cells) 2 Eosinophils + 2 cDC1

/Cre cDC2 10 cDC1

10 + cDC2 1 1 (Cre UMAP2 UMAP2 0 0 (of CD45 Absolute number

0 10 20 30 0 10 20 30 Frequency of subset UMAP1 UMAP1 0 0

E Gated: alveolar Mφ F G 15 20

) –

**** – 3 Cre 100 Cre – 84.8 Cre + + 7.06 10 **** + + + Cre SiglecF Cre 82.8 0.389 Cre SiglecF × 15 + – Cre+ 80 10 *** Cre SiglecF Cre+ SiglecF– 60 10 of AM (%) of AM (%) + – 40 5 + Cre 5

20 Ki67

98.9 CD11c MFI ( SiglecF Events (% of max) 0 0 3 4 0 0 10 10 0 102 103 104 105 224 SiglecF-PE-CF594 EGR2-PE 225 Figure 2: EGR2 is required for the phenotypic identity of alveolar macrophages

226 A. UMAP analysis of CD3–CD19–NK1.1–Ly6G–CD11b+/CD11c+ cells pooled from adult unmanipulated 227 Cre– and Cre+ mice (left panel). Heatmap plots showing the relative expression of the indicated markers 228 by myeloid clusters. 229 B. Cluster identity confirmed by manual gating (see Supplementary Figure 3A). 230 C. Relative frequency of alveolar macrophages (red gate) of all CD45+ leukocytes in unmanipulated 231 adult Cre– and Cre+ mice.

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232 D. Relative frequency and absolute number of alveolar macrophages, cDC1, cDC2, Ly6Chi monocytes 233 and CD64+MHCII+ interstitial macrophages in lung digests from adult unmanipulated Cre+ mice 234 compared with their abundance in Cre– littermates. Symbols represent individual mice and data are 235 pooled from three independent experiments with 8 mice per group. Error bars represent S.D. 236 E. Representative expression of SiglecF by CD11chiCD11blo alveolar macrophages (from F) obtained 237 from lung digests from adult unmanipulated Cre– or Cre+ mice (left), frequency of SiglecF+ macrophages 238 in each strain (right). Symbols represent individual mice with 4 mice per group. Data are from one of at 239 least 5 independent experiments. Error bars represent S.D. Student’s t test, ****p<0.0001 (SiglecF) 240 F. Representative expression of EGR2 by SiglecF-defined alveolar macrophages. Shaded histograms 241 represent isotype controls. Data are from one of three independent experiments with 3-4 mice per 242 experiment. 243 G. Mean fluorescence intensity (MFI) of CD11c expression by SiglecF-defined CD11chiCD11blo alveolar 244 macrophages from lung digests from adult unmanipulated Cre– or Cre+ mice. Symbols represent 245 individual mice with 4 mice per group. Data are from one of at least 5 independent experiments. Error 246 bars represent S.D. One-way ANOVA followed by Tukey’s multiple comparisons post-test. *** p<0.001, 247 **** p<0.0001. 248 249 250 251 EGR2 controls the tissue-specific transcriptional programme of alveolar macrophages

252 The failure of alveolar macrophages from Cre+ mice to express SiglecF suggested that the

253 tissue-specific differentiation programme of these cells may be altered by Egr2 deficiency.

254 Hence, to ascertain the global effects of Egr2 deletion on alveolar macrophage differentiation,

255 we next performed bulk RNA-seq of CD11chiCD11blo alveolar macrophages from lung digests

256 of Cre– and Cre+ mice (using only SiglecF– macrophages from Cre+ mice to exclude

257 confounding effects of EGR2-sufficient alveolar macrophages) (Supplementary Figure 4).

258 Unbiased clustering confirmed the biological replicates from each group were highly similar

259 (Figure 3A) and differential gene expression (DEG) analysis revealed that 858 genes were

260 differentially expressed by at least 2-fold (417 and 441 genes downregulated and upregulated,

261 respectively) (Supplementary Table 2). Consistent with our flow cytometry analysis, Siglec5,

262 which encodes SiglecF, was one of the most downregulated genes in Egr2 deficient alveolar

263 macrophages (Figure 3B). Many of the most differentially expressed genes formed part of the

264 alveolar macrophage gene set identified in our scRNA-seq analysis. Moreover, approximately

265 30% of the core alveolar macrophage signature identified by the ImmGen consortium (1) was

266 altered by Egr2 deficiency (32 genes) (Figure 3B, C), including the expression of Spp1,

267 Epcam, Car4 and Fabp1, all of which were confirmed by flow cytometry or qPCR (Figure 3D,

268 E). The vast majority of these ‘signature’ genes was downregulated in Egr2-deficient

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269 macrophages compared with their Egr2-sufficient counterparts. (GO) analysis

270 revealed that the top pathways affected by Egr2 deficiency were ‘Chemotaxis’, ‘Cell

271 chemotaxis’ and ‘Immune system process’ (Supplementary Table 3). Consistent with this,

272 the expression of chemokine receptors, such as Ccr2 and Cx3cr1, was elevated in alveolar

273 macrophages from Cre+ mice compared with their Cre– counterparts (Figure 3F). Genes

274 encoding antigen presentation machinery, such as H2-Aa, H2-Eb1, Ciita and Cd74 were also

275 upregulated in alveolar macrophages from Cre+ mice. In parallel, there was significantly

276 greater expression of MHCII at the protein level in Egr2 deficient alveolar macrophages

277 (Figure 3F). Indeed, over 50 genes upregulated in Egr2 deficient alveolar macrophages were

278 genes that defined interstitial macrophages in our scRNA-seq analysis, including Cd63, Mafb,

279 Mmp12 and Msr1 (Figure 3D, Supplementary Table 2). Thus, EGR2 ablation renders

280 alveolar macrophages transcriptionally more similar to their interstitial counterparts.

281 Further phenotypic analysis revealed reduced expression of ‘core signature’ alveolar

282 macrophage markers TREM1 and CD11a at protein level in the context of Egr2 deficiency

283 (Figure 3G). EpCAM and CD11a expression have been implicated in regulating adherence to

284 and patrolling of the lung epithelium by alveolar macrophages (25). Interestingly, while we

285 found equivalent numbers of alveolar macrophages amongst tissue digests, we obtained

286 consistently higher numbers of alveolar macrophages in the bronchoalveolar lavage (BAL)

287 fluid of Cre+ mice, suggesting the EGR2-dependent differentiation programme may control the

288 ability of alveolar macrophages to adhere to and interact with cells of their niche in the airways

289 (Figure 3H).

290 291 292 293 294 295 296 297 298

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Figure 3

A C 2 'Core alveolar macrophage signature' genes fold) – Cre– 1 2 0 – Cre 2 -2 Cre– 3 vs Cre

Cre– 4 + -4 + Cre 1 Cre -6 Cre+ 2 + -8

Cre 3 Expression (Log + 11 Cre 4 Lipf Spp1 Car4 Ear1 I1lrn Clmn Htr2c Krt19 Cd69Cdh1 Cldn1 Akap5 Fabp1 Bub1bMtmr7 Aldoc rim29 Epcam Siglec5 Cpne5Zfp125Slc9a4 Clec7a Phlda1Atp10a T Acaa1b Kazald1 Slc39a2 S100a Scgb1a1 Tmem216 Atp6v0d2

Cre– Cre+ B D – + E F10 Cre Cre ) )

3 5 4

) 6 ) 2 – 4 Ear1 *** 4 **** Cre– **** Cre 10 3 10 **** + 10 × 4 10 + Alas1 Cre × Cre × Il1rn × 3 4 Cd63 2 Events 1 mRNA (

2 mRNA (

Plxdc2 (% of max) 1 2 Cd300a 1 Spp1 0 102 103 104 105 SiglecF MFI ( EpCAM MFI (

Epcam EpCAM-PE Wfdc21 0 0 Siglec5 0 0 Kazald1 Epcam 4 3 )

10 )

Rhoc 3 4 **** ) **** 3 ** 10 Tmsb10 8 10 1.0 10 × 3 × Slpi × 2 Siglec5 6 2 Events

Mapk6 MFI ( 0.5 4 (% of max) mRNA ( Slc9a4 mRNA ( 1

1 mRNA (relative) Ffar4 2 0 102 103 104 105 Spp1 Car4

Spp1 Spp1 [mRNA]-AF647 Gramd4 0 0 0 0.0 Slc37a2 Car4 Basp1 Fam198b 2 ) 6 1.5 ) 3 **** 3 4 Gm9844 ) **** 2 10 10 ×

Coro6 10 **** × Ppm1j 2 × 4 1.0 Cldn1 1 Events mRNA (

Mrgpre (% of max) mRNA ( 1 2 mRNA (relative) 0.5 Zfp125 0 102 103 104 105 Plxdc1 CD63 MFI ( Cd63 CD63-PE Fabp1 Nab2 0 0 0 Fabp1 0.0

6 10 14

F ) G H ) ) 4 4 4 – 4 – 2 3 8 8 2 )

150 ) ) ) 5 – Cre **** Cre

*** 3 3 3 3 ** ** ** Cre 1.5 ** 10 + + 10 ** 10 + Cre Cre

× **** 10 10 10 10 × 4 **** × Cre × ×

× 3 × 6 3 3 6 100 2 1.0 3 4 2 2 4 2 mRNA ( 2 mRNA ( (relative) mRNA ( 50 1 0.5 1 1 2 1 CCR2 MFI 2 1 BAL cellularity MHCII MFI ( CD11a MFI ( Ccr2 TREM1 MFI ( H2-Aa CX3CR1 MFI (

Cx3cr1 0 0 0 0 0 0 0 299 0 0 300 301 Figure 3: EGR2 controls tissue-specific transcriptional programme of alveolar macrophages

302 303 A. Heatmap of RNA-seq data showing the distance between samples from Cre– and Cre+ mice. 304 B. Heatmap showing expression of the 30 most differentially expressed genes by alveolar macrophages 305 from Cre– and Cre+ mice. Each column represents a biological replicate with four mice per group. Genes 306 highlighted in red appear in the ‘core signature’ of alveolar macrophages as defined by the ImmGen 307 Consortium (1).

308 C. Log2-fold expression of differentially expressed genes that form part of the ‘core signature’ of alveolar 309 macrophages as defined by the ImmGen Consortium (1). 310 D. Expression of Epcam, Spp1 and Cd63 from the RNA-seq dataset (left panels), representative flow 311 cytometric validation of EpCAM, Spp1 (mRNA detected by PrimeFlow technology) and CD63 312 expression (middle panels) and replicate MFI expression data of each of these markers by alveolar 313 macrophages from adult unmanipulated Cre– and Cre+ mice. Symbols represent individual mice and 314 data are from one of two independent experiments with 5 (Cre–) and 4 (Cre+) mice per group. Student’s 315 t test, **p<0.01, ***p<0.001, ****p<0.0001. 316 E. Expression of Siglec5, Car4 and Fabp1 from the RNA-seq dataset (left panels) and validation by flow 317 cytometry (SiglecF) or qPCR (Car4, Fabp1). Symbols represent individual mice and data for SiglecF is 318 from one of at least 10 independent experiments with 5 (Cre–) and 4 (Cre+) mice per group. Data for 319 Car4 and Fabp1 represents 2 (Cre–) and 4 (Cre+) mice per group. Student’s t test, ****p<0.0001. 320 F. Expression of Ccr2, Cx3cr1 and H2-Aa from the RNA-seq dataset and replicate MFI expression data 321 of CCR2, CX3CR1 and MHCII as determined by flow cytometry. Symbols represent individual mice and 322 data are from one of two independent experiments with 5 (Cre–) and 4 (Cre+) mice per group. Student’s 323 t test, **p<0.01, ***p<0.001, ****p<0.0001.

13 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

324 G. Replicate MFI data of for CD11a and TREM1 expression as determined by flow cytometry. Symbols 325 represent individual mice and data are from one of two independent experiments with 5 (Cre–) and 4 326 (Cre+) mice per group. Student’s t test, **p<0.01, ****p<0.0001. 327 H. Absolute number of CD11chiCD11b– alveolar macrophages present in the BAL of adult 328 unmanipulated Cre+ mice relative to their abundance in Cre– littermates. Symbols represent individual 329 mice and data are pooled from three independent experiments with 15 (Cre–) and 12 (Cre+) mice per 330 group. 331 In all graphs error bars represent S.D. 332 333 334 EGR2 controls distinct functional characteristics of alveolar macrophages 335 336 Individuals with mutations in EGR2 develop peripheral neuropathies due to the crucial role for

337 EGR2 in Schwann cell function (26). However, many of these individuals also frequently

338 encounter respiratory complications, including recurrent pneumonias and/or restrictive

339 pulmonary disease, and in some cases respiratory failure (26). The cause of respiratory

340 compromise in these individuals remains unexplained. To determine if alterations in alveolar

341 macrophage behaviour may contribute to this, we next tested the function of Egr2-deficient

342 alveolar macrophages. A major homeostatic function of alveolar macrophages is the

343 regulation of pulmonary surfactant, and the absence of alveolar macrophages results in the

344 development of pulmonary alveolar proteinosis (PAP) (8-10, 27, 28). However, Egr2

345 deficiency did not lead to spontaneous PAP, as there were no differences in the levels of total

346 protein in BAL fluid from Cre+ and Cre– mice at either 4 or >9 months of age, a time at which

347 PAP is detectable in Csf2rb–/– mice (28) (Figure 4A). However, these results were confounded

348 by the fact that the majority of alveolar macrophages in aged (>9 months) Cre+ mice was now

349 EGR2-sufficient, with most cells expressing high levels of SiglecF (Figure 4B, C). These

350 findings suggested that the cells that had escaped Cre recombination may have a competitive

351 advantage over their EGR2-deficient counterparts and indeed, the absolute number of

352 SiglecF+ alveolar macrophages no longer differed between aged Cre– and Cre+ mice (Figure

353 4D). These data are consistent with other studies noting age-related repopulation of the

354 alveolar niche with Cre ‘escapees’ in the Lyz2Cre mouse (11). Notably, however, this

355 preferential expansion of EGR2-sufficient ‘escapees’ did not relate to differences in the level

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356 of proliferation by EGR2-defined subsets, with identical frequencies of Ki67+ cells amongst

357 EGR2-sufficient and -deficient macrophages in young adult and aged mice (Figure 4E).

358 In an attempt to circumvent the confounding effects of these escapees, we generated

359 a second strain to delete Egr2 from macrophages by crossing Egr2fl/fl mice with mice

360 expressing ‘improved’ Cre recombinase under control of the endogenous Fgcr1 promoter

361 (Fcgr1iCre (3)). By using Fcgr1iCre.Rosa26LSL-RFP reporter mice, we confirmed that this approach

362 led to efficient Cre recombination in alveolar macrophages, as well as in other tissue

363 macrophages, but not in other leukocytes (Supplementary Figure 5A). Importantly, alveolar

364 macrophages from Fcgr1iCre.Egr2fl/fl mice phenocopied those from Lyz2Cre.Egr2fl/fl mice

365 (Supplementary Figure 5B), but the frequency of Cre escapees was markedly lower in

366 Fcgr1iCre.Egr2fl/fl mice compared with Lyz2Cre.Egr2fl/fl mice (Supplementary Figure 5C, D).

367 Despite this, we did not detect the development of proteinosis in aged Fcgr1iCre.Egr2fl/fl mice

368 compared to their littermate controls (Supplementary Figure 5E). Consistent with this, Egr2

369 deficiency had little if any effect on the expression of genes associated with lipid uptake and

370 metabolism that are characteristic of normal alveolar macrophages (9) (Figure 4F). Thus,

371 while EGR2 is indispensable for the phenotypic identity of alveolar macrophages, it seems to

372 be dispensable for regulating lipid handling.

373 We next sought to determine if EGR2-dependent differentiation controls protective

374 immune functions of alveolar macrophages. To do so, we infected Cre– (Egr2fl/fl) mice and

375 Cre+ (Lyz2Cre.Egr2fl/fl) mice with 1x104 colony forming units (CFU) Streptococcus pneumoniae,

376 based on previous work showing that wild type alveolar macrophages efficiently clear infection

377 at this dose (30, 31). This showed that the majority of Cre– mice (8/12) had cleared infection

378 at 14 hours post infection, whereas the majority of Cre+ mice (8/10) had detectable bacteria in

379 the airways at this timepoint (Figure 4G). Importantly, the failure to clear bacteria did not

380 reflect the loss of tissue resident macrophages that can occur during inflammation or infection,

381 as alveolar macrophages continued to dominate the airways in both Cre+ and Cre– mice

382 (Figure 4H). However, our RNA-seq analysis showed that expression of genes encoding

383 molecules for the recognition, opsonisation and elimination of bacteria, including Colec12,

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384 Wfdc10, C3 and Marco, the latter of which has been shown to be indispensable for immunity

385 to S. pneumoniae (32), were significantly reduced in Egr2-deficient alveolar macrophages

386 (Figure 4I). Thus, EGR2-dependent differentiation is crucial for equipping alveolar

387 macrophages with the machinery to capture and destroy pneumococci. Figure 4

A Cre– Cre+ B F Isotype control Cre– Cre+ Lipid import & storage 400 – 6 5 4 *** Cre 86.8 1.89 24.7 74.6 16.9 68.2 )

10 3

** + fold) SiglecF + 4 – 2 g/ml) 300 4 10 – Cre μ 10 × 3 SiglecF 2

200 103 0 2 vs Cre + -2 100 102 0 1 Cre -4 11.2 0.174 0.377 0.321 12.7 2.19 SiglecF-PE-CF594 EGR2 MFI ( BAL protein ( 0 2 3 4 5 -6 0 10 10 10 10 0 Expression (Log 4 >9 EGR2-PE Plin2 Olr1 Egr2 Abca1 Fabp4 Abcg1 Cd36Cidec Fabp1 Age (months) Slc27a1 Angptl4 Scarb1 Slc27a6Slc27a4

Cre– Cre– + + SiglecF + C Cre D – + – E Cre Lipid metabolism Cre Cre Cre SiglecF– 6 ** + 15 30 100 5 ) 6 78.8 80 5 Cre 10 fold) 4 – 2 80 4 10 10 × 2 4 10 20 60 103 0 of AM (%) vs Cre of AM (%) + + 40 2 + -2 10 2 5 10 0 Cre -4

20 Ki67 CD11c-BV785 20.5 19.5 AM number ( SiglecF 2 3 4 5 -6 0 10 10 10 10 0 0 0 Expression (Log 0 8weeks >9 months CD11b-APC-Fire750 Acsl1 Ech1 Gpd1 Egr2 Acox1 Soat1Soat2Ch25h Ehhadh Acaa1aAcaa1b Agpat9 Hsd17b4 Phospho1

G * H I ) 10 * – 6 8 –

5 * 1.5 1000 Cre– 100 Cre *** * Cre

) + )

+ ) 2

+ 10 Cre 3 3 Cre (%) Cre 10 80 × **** + 6 10 100 ) 10 10 3

4 ×

× 1.0 × ( ( 60 ( 1 10 10 4 × CFU 40 ( 5 2 0.5 1 2 C3 Marco 20 Wfdc10 Colec12 AM of CD45 AM number ( 0.1 0 0.1 0 0 0 0 388 389 390 Figure 4: EGR2 controls distinct functional characteristics of alveolar macrophages 391 A. Protein levels in the BAL fluid of Cre– or Cre+ mice at 4 or 9-12 months of age. Symbols represent 392 individual mice and error is S.D. Data are from 6-9 mice per group pooled from two independent cohorts 393 of aged mice. 394 B. Representative expression of SiglecF and EGR2 by CD11chiCD11blo macrophages and mean 395 fluorescent intensity (MFI) of EGR2 by SiglecF-defined CD11chiCD11blo macrophages obtained from 396 11-12 month old Cre– or Cre+ mice. Symbols represent individual mice and error is S.D. Data are from 397 5 mice per group pooled from two independent cohorts of aged mice. One-way ANOVA followed by 398 Tukey’s multiple comparisons post-test. ** p<0.01, *** p<0.001. 399 C. Frequency of SiglecF+ cells amongst CD11chiCD11blo macrophages obtained from 11-12 month old 400 Cre– or Cre+ mice. Symbols represent individual mice and error is S.D. Data are from 7 mice per group 401 pooled from three independent cohorts of aged mice. Unpaired Student’s t-test, **p<0.01 402 D. Representative expression of CD11c and CD11b by Ly6CloCD64+ macrophages obtained from 11- 403 12 month old Cre– or Cre+ mice. Symbols represent individual mice and error is s.d.. Data represent 7 404 mice per group pooled from three independent cohorts of aged mice. 405 E. Frequency of Ki67+ cells amongst SiglecF-defined CD11chiCD11blo macrophages obtained from 8 406 week old or 11-12 month old Cre– or Cre+ mice. Symbols represent individual mice and error is s.d.. 407 Data represent 7 (Cre+) or 8 (Cre–) mice per group (8 week old mice) or 5 mice per group (aged mice) 408 pooled from two independent experiments.

409 F. Log2-fold expression of genes that are implicated in lipid uptake or metabolism in alveolar 410 macrophages as defined by (9). Expression of Egr2 is included as a reference. 411 G. Bacterial levels (colony forming units, CFU) in the BAL fluid of Cre– or Cre+ mice 14hrs after infection. 412 Symbols represent individual mice and error is S.D. Data are from 10 (Cre+) or 12 (Cre–) mice per group 413 pooled from three independent experiments. Mann Whitney test, *p<0.05.

16 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

414 H. Frequency and absolute number of CD11chiCD11blo alveolar macrophages in the BAL fluid of Cre– 415 or Cre+ mice 14hrs after infection. Symbols represent individual mice and error is S.D. Data are from 416 10 (Cre+) or 11 (Cre–) mice per group pooled from three independent experiments. Mann Whitney test, 417 *p<0.05. 418 I. Expression of Marco, Wfdc10, Colec12 and C3 from the RNA-seq dataset (left panels). Each symbol 419 represents a biological replicate with four mice per group. 420

421 EGR2 expression by alveolar macrophages is dependent on TGFb and CSF2

422 Alveolar macrophages derive from foetal monocytes that seed the developing lung in the late

423 gestational period (8). To determine the point at which EGR2 is first expressed, we assessed

424 EGR2 expression by E10.5 yolk sac macrophages, by macrophages in the embryonic lung

425 (E16.5) and by CD11chiCD11blo alveolar macrophages in the neonatal and adult lung using

426 the ImmGen database. This revealed that Egr2 was absent from yolk sac macrophages and

427 macrophages in the embryonic lung at E16.5, but it was expressed by both neonatal and adult

428 alveolar macrophages (Figure 5A), suggesting that it is induced during alveolarization in the

429 neonatal period. Consistent with this, we found high expression of EGR2 at protein level by

430 neonatal (d1) CD64+ lung macrophages (sometimes referred to as ‘pre-alveolar macrophages’)

431 in Cre– mice; as expected, this expression was deleted efficiently in Cre+ mice (Figure 5B, C).

432 Importantly, Ly6Chi monocytes in the lung of d1 neonatal mice lacked any expression of EGR2

433 (Figure 5B, C), reinforcing the selectivity of EGR2 expression even at this highly dynamic

434 stage of myeloid cell development in the lung. Consistent with our analysis of mature alveolar

435 macrophages in adult mice, Egr2 deletion had no impact on the frequency and absolute

436 number of pre-alveolar macrophages (Figure 5D). However, phenotypic differences were

437 already apparent in Cre+ (Lyz2Cre/+.Egr2fl/fl) macrophages at this stage, with reduced CD11c

438 and SiglecF expression which persisted into adulthood (Figure 5E, F). In parallel, EpCAM

439 expression was absent from alveolar macrophages in the neonatal period and was

440 progressively upregulated with age in an EGR2-dependent manner (Figure 5F). CD11b

441 expression, which is downregulated in mature alveolar macrophages, was found on pre-

442 alveolar macrophages in both Cre– and Cre+ mice, and it was downregulated to the same

443 extent with age in both strains.

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444 We next set out to determine the environmental factors that drive EGR2 expression.

445 Many studies employing in vitro culture systems have described EGR2 expression as a

446 feature of ‘alternatively activated’ macrophages, dependent on IL-4R signalling (33-35).

447 Importantly, expression of EGR2 by alveolar macrophages was independent of IL-4R

448 signalling (Figure 5G & Supplementary Figure 5F), as were key EGR2-dependent

449 phenotypic traits, such as SiglecF and EpCAM expression (Supplementary Figure 5F). TGF-

450 b has recently been shown to be crucial for the development of alveolar macrophages (11)

451 and thus we next explored if the TGF-b-TGF-bR axis drives expression of EGR2. To do so,

452 we generated a new mouse line by crossing Fcgr1iCre mice to mice with LoxP sites flanking

453 the Tgfbr2 allele (Tgfbr2fl/fl). Consistent with the crucial role for TGF-bR in controlling alveolar

454 macrophage development (11), there was a paucity of alveolar macrophages in the lungs of

455 neonatal Fcgr1iCre/+.Tgfbr2fl/fl compared with Fcgr1+/+.Tgfbr2fl/fl and Fcgr1iCre/+.Tgfbr2fl/+ controls

456 (Figure 5H). Strikingly, while CD11c+CD11blo alveolar macrophages expressed high levels of

457 EGR2 in control groups, EGR2 expression was largely abolished in Fcgr1iCre/+.Tgfbr2fl/fl mice,

458 demonstrating that TGF-bR signalling is vital for EGR2 induction in vivo. As Fcgr1iCre/+.Tgfbr2fl/fl

459 developed fatal seizures between d14 and d21 of age, perhaps reflecting the indispensable

460 role for TGF-bR in controlling microglia activity (37-39), we were unable to carry out further

461 analyses using this strain.

462 Given the central role for CSF-2 in alveolar macrophage development, we also

463 assessed the role of CSF-2 in driving EGR2 expression using an in vitro culture system in

464 which Ly6Chi monocytes from mouse bone marrow were FACS-purified and cultured with

465 recombinant CSF-1 or CSF-2. This revealed that CSF-2 was also capable of driving EGR2

466 expression in this system (Figure 5I). Given that CSF-2R and TGF-bR signalling is known to

467 induce expression of PPAR-g (9, 11), we next determined if PPAR-g (encoded by Pparg) is

468 upstream of EGR2. Analysis of a published dataset comparing the transcriptional profile of

469 Pparg-sufficient and -deficient lung macrophages revealed significant downregulation (2.1-

470 fold change) of Egr2 in the context of Pparg deficiency (Figure 5j). In contrast, Pparg

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471 expression was unaffected in alveolar macrophages from Lyz2Cre.Egr2fl/fl mice (Figure 5K),

472 suggesting EGR2 is downstream of PPAR-g. Another transcription factor implicated in

473 controlling alveolar macrophage differentiation is C/EBPb (14) and EGR2 has been shown to

474 modulate C/EBPb in vitro (33). In our hands, Egr2-deficiency led to reduced expression of

475 C/EBPb at mRNA (Figure 5K) and protein level (Supplementary Figure 5G). Taken together,

476 these data support the premise that EGR2 expression by alveolar macrophages is induced by

477 TGF-b and CSF-2 in a PPAR-g-dependent manner in the neonatal period and this in turn

478 induces expression of C/EBPb to drive tissue-specific differentiation. Figure 5

+ – Live CD45 Lin hi + – + Ly6C monocytes CD64 Mφ A B Cre Cre C 6 – ) Cre

100 2 5 Cre– Adult microglia 10 Cre+ 16.8 21.1 80 Cre+ 10 × Adult Lung Alv.Mφ 104 4 60 Neonatal Lung Alv.Mφ 3 10 40 E16.5 Lung Mφ 73.2 66.2 2 E10.5 Yolk Sac Mφ 102 20 EGR2 MFI (

Ly6C-eF450 0

Events (% of max) 0 2 3 4 5 0 200 400 600 800 2 3 4 5 0 10 10 10 10 0 0 10 10 10 10 hi + Egr2 EGR2-PE Ly6C CD64 CD64-APC mono Mφ

Live CD45+ Lin– CD64+ D E Cre– Cre+ – 40 0.3 5 5 8

Cre ) )

) – 2

6 10 + ** 2 **** Cre Cre 1.0 + 10 10 4 4 10 Cre φ 10 30 × × (%)

× 6 ( M

+ 0.2

+ 3 φ 10 3

20 M 4

+ 2 2 0.5 0.1 10 (relative) CD64 0

of CD45 10

2 SiglecF MFI

CD11c-BV785 1 CD64 CD11c MFI ( 2 3 4 5 CD11b MFI ( 0 0.0 010 10 10 10 0 0 0.0 SiglecF-PE-CF594

C57BL/6 **** **** 6 –/– F 9 5 **** – G ) Il4ra 15 100 **** ³ **** ) Cre ) **** 2 ****

3 **** **** **** + Cre 10

**** 10 4 × 10 **** 80 (

*** × × 10 6 4 60 3 of AM (%) *** *** hi 40 2 5 3 **** 2 1 20 ** ****** EGR2 MFI CD11b MFI ( CD11c MFI ( SiglecF

0 0 0 EpCAM MFI (relative) 0 0 d5 3wk 5wk d5 3wk 5wk d5 3wk 5wk d5 3wk 5wk

Fcgr1iCre.Tgfbr2f/f CSF-1 CSF-2 – 3 – +/f f/f I 100 Cre ) **** H Tgfbr2 Tgfbr2 3 Cre + **** + – Cre 100 10 Cre 11.1 86.6 12.5 85.9 Cre 80 + × (%) 80 Cre 2

+ 60

– 60 Cre 40 1 40 20 EGR2 MFI (

20 **** Events (% of max) 0 0 AM of CD64 3 4 5 2.13 0.211 1.37 0.247 0 10 10 10 CSF-1 CSF-2 0 EGR2-APC 100 105 8.62 88.2 0.822 8.57

4 80 10 J 0 K 1.5 Cre–

60 ) 3 +

+ of AM (%) Cre 10 +/+

Cre + 40 **** -1 1.0 102 Pparg

0 20 / – EGR2 / – expression SiglecF-PE-CF594 2.77 0.399 80.2 10.4 0 -2 0.5 2 3 4 5 f/+ f/f f/+ f/f mRNA (relative)

0 10 10 10 10 Egr2 Pparg ( EGR2-PE -3 0.0 479 Pparg Cebpb

480 Figure 5: TGFb and CSF2 drive EGR2 expression

481 A. Normalised expression (by DESeq2) of Egr2 by the indicated populations (data obtained from the 482 Immgen Consortium). 483 B. Representative expression of Ly6C and CD64 by live CD45+CD3–CD19–Ly6G– cells from the lungs 484 of unmanipulated newborn Cre– (Egr2fl/fl) or Cre+ (Lyz2Cre/+.Egr2fl/fl) mice. Data are pooled from one of 485 two independent experiments performed.

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486 C. Histograms show representative expression of EGR2 by CD64+ ‘pre-alveolar macrophages’ and 487 Ly6Chi monocytes from the lungs of unmanipulated newborn Cre– (Egr2fl/fl) or Cre+ (Lyz2Cre/+.Egr2fl/fl) 488 mice and bar chart shows the mean fluorescent intensity (MFI) of EGR2 expression by these cells. 489 D. Frequency and absolute number of CD64+ ‘pre-alveolar macrophages’ from mice in B. Symbols 490 represent individual mice. Data are pooled from two independent experiments with 4 (Cre–) or 8 (Cre+) 491 mice per group. 492 E. FACS plots show representative expression of CD11c and SiglecF by CD64+ ‘pre-alveolar 493 macrophages’ from mice in B and bar chart shows the MFI of CD11c, SiglecF and CD11b expression 494 by these cells. Symbols represent individual mice. Data are pooled from two independent experiments 495 with 4 (Cre–) or 8 (Cre+) mice per group. Unpaired Student’s t-test, **p<0.01, ****p<0.0001 496 F. MFI of CD11c, SiglecF, CD11b and EpCAM (relative to d5 Cre– cells) expression by CD11chiCD11blo 497 alveolar macrophages obtained from unmanipulated Egr2fl/fl (Cre–) or Lyz2Cre.Egr2fl/fl (Cre+) mice at the 498 indicated ages. Symbols represent individual mice. Coloured * denote significance between d5 and 3 499 and 5 weeks within the Cre– (blue) and Cre+ (red) data. Data are pooled from two independent 500 experiments with 4-9 mice per group. Two-way ANOVA with Tukey’s multiple comparisons test, 501 ****p<0.0001 502 G. Representative expression of EGR2 by alveolar macrophages from adult WT (C57BL/6J) and Il4ra– 503 /– adult mice. Data from one experiment with 4 mice per group. 504 H. Representative expression of EGR2 and SiglecF by CD11chiCD11blo alveolar macrophages obtained 505 from lungs of neonatal (d8) Fcgr1iCre.Tgfbr2fl/fl and Cre– and Tgfbr2fl/+ littermate controls. Bar charts show 506 the mean frequencies of CD11chiCD11blo alveolar macrophages of all Ly6CloCD64+ cells (upper) and 507 EGR2+ cells amongst CD11chiCD11blo alveolar macrophages (lower). Symbols represent individual 508 mice. Data are pooled from two independent experiments with 3-7 mice per group. **** p<0.0001 (One- 509 way ANOVA followed by Tukey’s multiple comparisons post-test). 510 I. Representative expression of EGR2 (left) and MFI of EGR2 (right) by FACS-purified Ly6Chi 511 monocytes cultured in vitro with recombinant CSF-1 (20ng/ml) or CSF-2 (20ng/ml) for five days. 512 Symbols represent monocytes isolated from individual mice and horizontal lines represent the mean. 513 Data are from 6 Cre– (Egr2fl/fl) or 3 Cre+ (Lyz2Cre.Egr2fl/fl) mice per group pooled from two independents 514 experiment. Two-way ANOVA followed by Tukey’s multiple comparisons post-test, **** p<0.0001. 515 Coloured * denote significance between CSF-1 and CSF-2 within the Cre– (blue) and Cre+ (red) data. 516 J. Relative expression of Egr2 by alveolar macrophages obtained from Ppargfl/fl or ItgaxCre.Ppargfl/fl mice 517 from (9). 518 K. qPCR analysis of Pparg and Cebpb mRNA by BAL cells from unmanipulated adult Egr2fl/fl (Cre–) or 519 Lyz2Cre.Egr2fl/fl (Cre+) mice. Data represent 2 Egr2fl/fl (Cre–) or 4 Lyz2Cre.Egr2fl/fl (Cre+) mice per group. 520 521 522 Egr2 deficiency confers a competitive disadvantage on alveolar macrophages

523 Given the observation that EGR2-sufficient alveolar macrophages come to dominate the

524 Lyz2Cre.Egr2fl/fl (Cre+) mice airspace, we next set out to determine if EGR2 deletion confers an

525 intrinsic competitive disadvantage on alveolar macrophages. To this end, we generated mixed

526 bone marrow chimeric mice by reconstituting lethally irradiated WT (CD45.1+/.2+) mice with a

527 1:1 ratio of WT (CD45.1+) and either Cre– (Egr2fl/fl) or Cre+ (Lyz2Cre.Egr2fl/fl) (CD45.2+) bone

528 marrow cells (Figure 6A). 8 weeks after reconstitution, we found that Egr2 deficient (Cre+)

529 and Egr2 sufficient (Cre–) bone marrow contributed equally to blood monocytes and

530 neutrophils (data not shown), as they did to the pools of monocytes, interstitial macrophages

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531 and dendritic cell subsets in the lung (Figure 6B, C). In contrast, alveolar macrophages were

532 derived almost exclusively from WT BM in WT:Cre+ chimeric mice, whereas they were derived

533 equally from both BM sources in WT:Cre– chimeric mice (Figure 6B-D). The mixed BM

534 chimeric model also confirmed that the phenotypic and morphologic differences seen in intact

535 Lyz2Cre.Egr2fl/fl mice were due to cell intrinsic loss of EGR2, rather than effects of Egr2

536 deficiency on the lung environment (Figure 6E, F). We also used this system to confirm the

537 reduced expression of C/EBPb by alveolar macrophages deriving from Lyz2Cre.Egr2fl/fl (Cre+)

538 bone marrow (Figure 6F). Taken together, these results demonstrate that cell intrinsic EGR2

539 is indispensable for the differentiation of alveolar macrophages and repopulation of the

540 alveolar niche following radiation-induced depletion.

541 Figure 6

Non-host Ly6Clo CD103– CD64+ cells A Lung B WT:Cre– WT:Cre+ C 105 **** WT:Cre–

) + – 1.0 WT:Cre 104 WT Cre– or Cre+ 3 (CD45.1+) + 10 (CD45.2 ) 0.5

BM cells at 0 Chimerism (relative to Cre 1:1 ratio CD11c-BV785 0.0 0 103 104 105 Alv. Int. cDC1 cDC2 Ly6Chi Eos. CD11b-APC-Cy7 Mφ Mφ mono

WT:Cre– WT:Cre+ D Cre–-derived Cre+-derived CD45.1/.2+ 105 53.5 97.7 105 61.1 4.41 104 104 103 103 Analysis of chimerism 102 0 8weekspost-reconstitution CD45.1-AF488 46 2.1 0

2 3 4 5 CD11c-BV785 0 10 10 10 10 3 4 5 CD45.2-AF700 0 10 10 10 CD11b-APC-Cy7

E WT-derived Egr2f/f-derived F

8 ) ) ) 15 5 4 4 ) 3 4 WT-derived 4 * f/f

10 Egr2 -derived 10 10

10 4 ** × × 6 × × 3 – 10 WT:Cre 3

4 MFI ( 2 **** + 2 β WT:Cre **** 5 2 1 **** 1 CD11c MFI ( SiglecF MFI ( C/EBP 3 4 5 2 3 4 5 2 3 4 5 **** EpCAM MFI ( 010 10 10 010 10 10 10 010 10 10 10 0 0 0 0 542 CD11c-BV785 SiglecF-PE-CF594 EpCAM-PE WT:Cre– WT:Cre+ WT:Cre– WT:Cre+ WT:Cre– WT:Cre+ WT:Cre– WT:Cre+

543 Figure 6: Egr2 deficiency confers a competitive disadvantage on alveolar macrophages

544 A. Schematic of the generation of mixed bone marrow chimeric mice using Cre– (Egr2fl/fl) or Cre+ 545 (Lyz2Cre.Egr2fl/fl) mice as donors. 546 B. Representative expression of CD11c and CD11b by Ly6CloCD64+ macrophages amongst live 547 CD45+CD3–CD19–Ly6G–CD103– cells (upper panels) and representative expression of CD45.1 and 548 CD45.2 by CD11chiCD11blo alveolar macrophages (lower panels) from WT:Cre– or WT:Cre– chimeric 549 mice. 550 C. Contribution of Egr2fl/fl BM to the indicated lung myeloid populations in WT:Cre+ chimeric mice 551 relative to WT:Cre– mice. Chimerism was normalised to Ly6Chi blood monocytes before normalisation 552 of Cre+ to Cre–. Symbols represent individual mice. Data are from 12 (WT:Cre+) or 13 (WT:Cre–) mice 553 per group pooled from two independent experiments. Student’s t-test with Holm-Sidak correction, **** 554 p<0.0001.

21 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

555 D. Representative composition of the CD64+ macrophage compartment deriving from Cre– or Cre+ bone 556 marrow in WT:Cre– or WT:Cre– chimeric mice. 557 E. Representative expression of CD11c, SiglecF and EpCAM by WT- and Egr2fl/fl-derived alveolar 558 macrophages in WT:Cre– or WT:Cre– chimeric mice. Shaded histograms represent FMO controls. 559 F. Mean fluorescent intensity (MFI) of CD11c, SiglecF, EpCAM and C/EBPb expression by WT- and 560 Egr2fl/fl-derived alveolar macrophages in WT:Cre– or WT:Cre– chimeric mice. Symbols represent 561 individual mice. Data are from 10 mice per group from one experiment of two performed. One-way 562 ANOVA followed by Tukey’s multiple comparisons post-test, **** p<0.0001. 563 564 565 566 Bone marrow-derived monocytes replenish the alveolar macrophage niche following

567 lung injury

568 Loss of tissue resident macrophages is a frequent consequence of inflammation, including in

569 the lung (39). Thus, given that Egr2-deficient macrophages failed to replenish the alveolar

570 niche following radiation treatment, we next sought to determine if EGR2 plays a role in

571 macrophage repopulation following lung injury. The chemotherapeutic agent bleomycin is a

572 common model of chronic lung injury and self-resolving pulmonary fibrosis (40), which is

573 characterised by initial loss of alveolar macrophages during the inflammatory phase (day 7),

574 followed by repopulation during the fibrotic and resolution phases (from day 14 onwards)

575 (Figure 7A). Post-injury repopulation of macrophages can occur via two mechanisms: in situ

576 proliferation of resident cells and/or replenishment by bone marrow-derived monocytes. To

577 determine if bone marrow-derived monocytes contribute to the alveolar macrophage

578 compartment following bleomycin-induced injury, we used tissue protected bone marrow

579 chimeric mice to assess replenishment kinetics without exposing the lung to the additional

580 insult of ionising radiation (Figure 7B). Consistent with previous studies (41), we found that

581 bleomycin instillation led to progressive replacement of resident alveolar macrophages by BM-

582 derived cells, with the entire alveolar macrophage compartment being replaced at 32 weeks

583 post injury (Figure 7B). Interestingly, recently arrived, monocyte-derived alveolar

584 macrophages expressed low-intermediate levels of SiglecF, with acquisition of SiglecF

585 requiring long-term residence in the airway (Figure 7C).

586 We next interrogated this process further to determine if interstitial macrophages that

587 accumulate in the lung parenchyma during injury can subsequently mature into alveolar

22 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

588 macrophages during tissue repair (41, 42). Indeed, during the recovery phase of disease, we

589 noted the presence of cells with features of both alveolar and interstitial macrophages

590 (CD11chiCD11b+ MHCII+CD64hi) in the BAL fluid (Figure 7D), and these cells expressed

591 intermediate levels of SiglecF (Figure 7E), indicative of recent monocyte origin. To examine

592 the relationship of these intermediate cells to interstitial macrophages more directly, we

593 performed fate mapping studies using Cx3cr1Cre-ERT2/+.Rosa26LSL-RFP/+ reporter mice, in which

594 administration of tamoxifen leads to irreversible expression of RFP by CX3CR1 expressing

595 cells (43, 44) (Figure 7F). Interstitial macrophages are characterised by high expression of

596 CX3CR1 and administration of tamoxifen led to labelling of 40-50% of interstitial macrophages

597 in both healthy lung and at d21 post bleomycin administration (Figure 7F). No recombination

598 was seen in Cx3cr1Cre-ERT2/+.Rosa26LSL-RFP/+ mice in the absence of tamoxifen

599 (Supplementary Figure 6A). Although very low levels of recombination were detected in

600 control alveolar macrophages, a clear population of RFP+ cells could be detected in the BAL

601 of the recipients of bleomycin following tamoxifen treatment (Figure 7F). As monocytes are

602 poorly labelled in this system and Cx3cr1 levels did not change in bona fide resident alveolar

603 macrophages in response to bleomycin treatment (Supplementary Figure 6B), these RFP+

604 cells must represent fate-mapped, monocyte-derived CX3CR1+ cells. In line with this, RFP+

605 cells had the ‘hybrid’ alveolar/interstitial CD11chiCD11b+SiglecFint profile, supporting the idea

606 that these represent transitional cells (Figure 7F). Thus, following bleomycin-induced injury,

607 the alveolar macrophage compartment is restored by monocytes that transition through a

608 CX3CR1hi state.

609

610

23 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Figure 7

A B C

250 ****

) **** 1.0 6 PBS PBS 1wk PBS **** ** 1.0 Bleomycin BM + Bleomycin **** 10 CD45.1/.2 200 Bleo. 0.8

× cells (

φ *** 150 **** 0.6 0.5 100 *** 100 32wk 80 0.4

Lead Normalised 50 60 chimerism (%) 0.2 + 40 SiglecF expression

8 CD45.2 Events (% of max) Alveolar M 20 0 wks 0 0.0 1 3 0 (. to host-derived cells) PBS or 1 3 32 0102 103 104 105 1 3 32 Post bleomycin bleomycin (o.p.) Post bleomycin SiglecF-PE-CF594 (weeks) Post bleomycin (weeks) (weeks)

D F Cx3cr1Cre-ERT2/+.Rosa26LSL-RFP/+ Vehicle (PBS) Bleomycin (d16) PBS 5 15 10 *** Bleo. PBS or Tamoxifen 104 bleomycin (o.p.) 3 10 Vehicle (PBS) Bleomycin 10 0.46 8.41 AM (%) 5

+ 10 2 10 TAM 4 0 5 16d 10

CD11c-BV785 5d 103 CD11b 0 102 103 104 105 102 CD11b-APC-Cy7 0 0

0.83× 2 3 4 5 60 2.0 5.97× 010 10 10 10 E *** PBS SiglecF-PE-CF594 + 5 CD11b Int. Mφ 1.5 Bleo 10 – CD11b Alv. M 40 4 φ 10 CD11b+ Alv. Mφ

cells (%) 1.0 3 10 + 20 102 1.05× 0.5 RFP 0 CD11c-BV785 Events (% of max) MHCII-AF700 SiglecF-PE-CF594 EGR2-PE 0 0.0 0 102 103 104 105 Ly6Chi Int. Alv. CD11b-APC-Cy7 monos Mφ Mφ 611

612 Figure 7: Monocyte-derived, CX3CR1+ interstitial macrophages can replenish the alveolar

613 macrophage niche following injury

614 A. Absolute numbers of alveolar macrophages 1- and 3-weeks following bleomycin administration or 615 PBS vehicle control. Symbols represent individual mice and error is S.D. Data are pooled from two 616 independent experiments at each time point with 13-15 mice per group. Student’s t test with Holm-Sidak 617 correction, ***p<0.001. 618 B. Non-host chimerism of alveolar macrophages in tissue protected bone marrow chimeric mice at 1-, 619 3- or 32-weeks following administration of bleomycin or PBS vehicle control. Chimerism is normalised 620 to Ly6Chi blood monocytes. Symbols represent individual mice and error is S.D. Data are pooled from 621 two independent experiments at each time point with 13-15 mice per group. Two-way ANOVA with 622 Tukey’s multiple comparisons test. ***p<0.001, ****p<0.0001. 623 C. Expression of SiglecF by CD11chiCD11blo alveolar macrophages from the lung of mice in B. at 1 624 week and 32 weeks post bleomycin or PBS administration. Symbols represent individual mice and error 625 is S.D. Data are from one of two independent experiments at each time point with 4 mice per group. 626 Two-way ANOVA with Tukey’s multiple comparisons test. **p<0.01, ****p<0.0001. 627 D. Representative expression of CD11c and CD11b by CD11chiCD64+ cells obtained by BAL from WT 628 mice two weeks after instillation of bleomycin or vehicle control (left). Graph shows the mean frequency 629 of CD11b+ alveolar macrophages (right). Symbols represent individual mice and error is S.D. Data are 630 pooled from two independent experiments with 7 (Cre–) or 10 (Cre+) mice per group. Mann Whitney 631 test, ***p<0.001. 632 E. Representative expression of MHCII, SiglecF and EGR2 by CD11b+ interstitial macrophages and 633 CD11b-defined CD11chi alveolar macrophages from mice in D. 634 F. Experimental scheme for the induction of lung injury and tamoxifen administration in Cx3cr1Cre- 635 ERT2/+.Rosa26LSL-RFP/+ fate mapping mice. Lower graphs show the levels of recombination in Ly6Chi 636 monocytes, CD64+ interstitial macrophages and alveolar macrophages from Cx3cr1Cre-ERT2/+.Rosa26LSL- 637 RFP/+ mice administered bleomycin or vehicle control. Representative expression of CD11c, SiglecF and 638 CD11b by RFP+ (red) or RFP– (grey) cells present in the BAL fluid of Cx3cr1Cre-ERT2/+.Rosa26LSL-RFP/+ 639 mice 3 weeks after bleomycin or vehicle instillation. Graphs show the mean fluorescent intensity (MFI) 640 of CD11c and SiglecF expression by RFP+ cells. Symbols represent individual mice and error is S.D. 641 Mann Whitney test, ***p<0.001. 642

24 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

643 EGR2 is indispensable for alveolar macrophage repopulation and tissue repair

644 following lung injury

645 Given that transitional CD11b+SiglecFint cells also expressed EGR2, contrasting with its

646 restriction to SiglecFhi alveolar macrophages in health (Figure 7E), we examined whether

647 EGR2 is necessary for the replenishment of the alveolar niche during recovery from

648 bleomycin-induced injury. To do this, we administered bleomycin to Lyz2Cre.Egr2fl/fl mice and

649 their Cre– littermates and assessed macrophage dynamics in total lung digests. The

650 inflammatory phase of this disorder (day 7) was associated with accumulation of CD11b+

651 macrophages and this occurred to the same extent in Cre– and Cre+ mice (Figure 8A, B).

652 Consistent with recent reports (45), the CD11b+CD64+ interstitial macrophage population was

653 heterogeneous during the fibrotic phase of disease (d14-d21), with MHCII+ and

654 MHCIIloCD36+Lyve1+ subsets. This pattern was identical in between Cre– and Cre+ groups

655 (Supplementary Figure 7A, C), as were the numbers of Ly6Chi monocytes and neutrophils

656 (Supplementary Figure 7A, B). We did however detect a significant reduction in eosinophils

657 in the lung of Lyz2Cre.Egr2fl/fl mice compared with Cre– littermates, despite eosinophils lacking

658 EGR2 expression (Supplementary Figure 7D, E).

659 A reduction in alveolar macrophages was observed in both groups on day 7 after

660 administration of bleomycin. Although this began to be restored by day 14 in Cre– (Egr2fl/fl)

661 control mice, this did not occur in Cre+ (Lyz2Cre.Egr2fl/fl) mice and indeed, the alveolar

662 macrophage compartment remained significantly reduced in Cre+ mice compared with Cre–

663 littermates even after 6 weeks (Figure 8A-C), suggesting EGR2 is indispensable for the

664 repopulation of the alveolar macrophage niche following bleomycin-induced injury. The lack

665 of repopulation in Cre+ mice did not appear to reflect an inability of Egr2 deficient macrophages

666 to proliferate, as the proportion of Ki67+ proliferating cells was equivalent in Cre– and Cre+ mice

667 (Figure 8D). Equally, this also did not reflect a lack of chemoattractants in the airways to

668 recruit monocyte-derived cells, as both CCL2 and CCL7 were actually elevated in Cre+ mice

669 compared with Cre– littermates (Figure 8E). Similarly, CSF2 levels were elevated in the BAL

670 fluid of Cre+ mice, ruling out the possibility that lack of appropriate growth factors is responsible

25 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

671 for defective alveolar macrophage differentiation in the absence of EGR2 (Figure 8E). Instead,

672 these data suggested that Egr2 deficiency led to an intrinsic inability of bone marrow-derived

673 cells to repopulate the macrophage niche. To test this directly, we crossed Cx3cr1Cre-

674 ERT2/+.Rosa26LSL-RFP/+ mice with Egr2fl/fl mice to allow for temporal RFP labelling of CX3CR1-

675 expressing cells and Egr2 deficiency in the same animal. We administered tamoxifen during

676 the period of alveolar macrophage reconstitution (d16 to d21) and assessed the presence of

677 RFP-labelled cells amongst alveolar macrophages. Compared with tamoxifen-treated controls

678 (Cx3cr1Cre-ERT2/+.Rosa26LSL-RFP/+.Egr2+/+ or Cx3cr1Cre-ERT2/+.Rosa26LSL-RFP/+.Egr2fl/+ mice), we

679 found a marked reduction in the frequency of RFP+ alveolar macrophages in the BAL of

680 tamoxifen treated Cx3cr1Cre-ERT2/+.Rosa26LSL-RFP/+.Egr2fl/fl mice during lung repair (Figure 8F),

681 demonstrating that EGR2 controls the post-injury repopulation of the alveolar macrophage

682 compartment by CX3CR1+ cells.

683 To determine the consequence of the failure of Egr2 deficient cells to reconstitute the

684 alveolar niche, we assessed the fibrotic response and subsequent repair processes in

685 Lyz2Cre.Egr2fl/fl mice. Notably, we did not detect differences in the degree of fibrosis or

686 expression of key genes associated with fibrosis, including Col3a1 and Pdgfrb between

687 untreated Lyz2Cre.Egr2fl/fl mice and their Egr2fl/fl littermate controls at day 21, a time considered

688 ‘peak’ fibrosis (Figure 8H, Supplementary Figure 8A, B). However, analysis at 6 weeks post

689 bleomycin showed that whereas the Cre– mice had largely repaired their lungs, Cre+ mice had

690 defective repair evidenced by persistent fibrosis and architectural damage (Figure 8G, H,

691 Supplementary Figure 8A). This was paralleled by elevated numbers of macrophages in the

692 lung parenchyma (Figure 8I, Supplementary Figure 8A) and parenchymal macrophage

693 persistence correlated with the degree of fibrosis (Supplementary Figure 8C). Furthermore,

694 homeostasis failed to be restored in the airways. Flow cytometric analysis of BAL fluid

695 revealed that CD45+ leukocytes comprised only 10% of all events in Cre+ mice compared with

696 ~60% in their Cre– littermates (Figure 8J). The vast majority of the CD45– fraction failed to

697 express signature markers for cells of epithelial, endothelial or fibroblast origin, suggesting

698 this may represent cellular debris, which could also be found amongst lung digests

26 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

699 (Supplementary Figure 9A, B). This was paralleled by elevated BAL fluid protein levels and

700 turbidity in the Cre+ mice compared with Cre– controls, suggesting that the inability to replenish

701 the alveolar macrophage niche following injury was associated with the development of

702 alveolar proteinosis (Figure 8K). Thus, loss of EGR2-dependent, monocyte-derived alveolar

703 macrophages leads to defective tissue repair, persistent cellular damage and failed restoration

704 of lung homeostasis. Figure 8

Lyz2Cre/+.Egr2f/fl C A B ) 100 6 0.8 Cre– Cre– Bleomycin * **** **** 10 * + 80 + × Cre (%) Cre 0.6

Vehicle (PBS) 1week 2week 3week 6week + AM hi

60 hi

1c ** 0.4 * 1c 1

40 1 CD of CD64

CD 0.2 Cre– 20 86.7 16.5 36.1 46.4 52.7 φ M 0 per right0.0 lobe ( 45.5 12.9 81.1 62.1 51.4 ** D 25 100 ** * **** **** Cre– 5 10 +

(%) 80 20 Cre

4 + + +

10 AM (%) 60 15 hi + 3 1b

Cre 10 1 1c Ki67

1c-BV785 10 10.8 11.3 15.1 18.7 40 1 1 2 83.1

10 CD 0 of CD64 20 5 CD 16.3 87.5 86.5 82 80.4 φ of CD M 0102 103 104 105 0 0 CD11b-APC-Cy7 PBS 1 2 3 6 Post bleomycin instillation (weeks)

E F Cx3cr1Cre-ERT2/+.Egr2f/f.Rosa26LSL-RFP/+ Tamoxifen 300 300 30 – Cre Egr2+/+ Egr2f/fl +/+ +/fl + No tamoxifen 1.5 Egr2 /Egr2 ) ) ) * * Cre ** 5 ) f/fl

10 + Egr2 / *** M 20 4 + 200 200 10 2 1.0 3 10 Egr / 0.08 0.63 0.37 BAL A f/f 1c-BV785 100 100 10 2 + 2 1 10 P CSF2 (pg/ml CCL7 (pg/ml CCL2 (pg/ml 0 0.5 CD RF Egr ( 0 0 0 0 102 103 104 105 0.0 Figure 8 RFP 705

G Cre– Cre+ H I

60 **** **** Cre– ² 200 Cre– *** Cre+ Cre+ 150 * 40 * 100 20 50 Macrophages/mm Fibrotic tissue (%) 0 0 0 3 6 Paren- Fibrotic Time post bleomycin chyma area (weeks)

CD68 SHG αSMA DAPI Autof.

J Cre– Cre+ K 250K 100 **** – 2 0.8 89.8 Cre * * – 50 + Cre 200K Cre – + 80 Cre+ Cre Cre 150K 60 0.6

100K 600 events (%) A

– 1 40 50K OD 0.4

SSC- 0 20 0 102 103 104 105 CD45

CD45 BV510 0 BAL protein (mg/ml) 0 0.2 706 707 708 Figure 8: EGR2 is indispensable for the repopulation of the alveolar macrophage niche and 709 tissue repair following lung injury 710 711 A. Representative expression of CD11c and CD11b by live CD45+CD3–CD19–Ly6G–CD64+ cells from 712 the lungs of Egr2fl/fl (Cre–) and Lyz2Cre/+.Egr2fl/fl (Cre+) mice at 1, 2, 3 or 6 weeks post bleomycin or 713 vehicle controls. 714 B. Frequency of CD11chiCD11blo alveolar macrophages and CD11cvarCD11b+ cells from mice in A. 715 Symbols represent individual mice and error is the S.D. Data are pooled from at least two independent

27 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

716 experiments at each time point with 3-7 mice per group. Two-way ANOVA with Tukey’s multiple 717 comparisons test, *p<0.05. **p<0.01, p<0.001, ****p<0.0001 718 C. Absolute number of CD11chiCD11blo alveolar macrophages in lungs six weeks post bleomycin 719 instillation. Symbols represent individual mice and error is the S.D. Data are pooled from two 720 independent experiments with 6 (Cre–) or 7 (Cre+) mice per group. Mann Whitney test, *p<0.05. 721 D. Frequency of Ki67+ CD11chiCD11blo alveolar macrophages in lungs six weeks post bleomycin 722 instillation. Symbols represent individual mice and error is the S.D. Data are pooled from two 723 independent experiments with 6 (Cre–) or 7 (Cre+) mice per group. 724 E. CCL2, CCL7 and CSF2 levels in BAL fluid obtained from Egr2fl/fl (Cre–) and Lyz2Cre/+.Egr2fl/fl (Cre+) 725 mice six weeks post bleomycin instillation. Symbols represent individual mice and error is the S.D. Data 726 are pooled from two independent experiments with 6 mice per group. Mann Whitney test (CCL2, CCL7), 727 *p<0.05, unpaired Student’s t test (CSF2), **p<0.01. 728 F. Representative expression of RFP by CD11chiCD64+ alveolar macrophages present in the BAL fluid 729 of Cx3cr1Cre-ERT2/+.Rosa26LSL-RFP/+.Egr2fl/fl and their Cx3cr1Cre-ERT2/+.Rosa26LSL-RFP/+.Egr2+/+ (open circles) 730 or Cx3cr1Cre-ERT2/+.Rosa26LSL-RFP/+.Egr2fl/+ (solid circles) controls 3 weeks following instillation of 731 bleomycin or vehicle control. Graph shows the relative frequency of RFP+ alveolar macrophages 732 present in the BAL fluid. Symbols represent individual mice and error is the S.D. Data are pooled from 733 two independent experiments at each time point with 10 (Egr2+/+ [open symbols]/Egr2fl/+ [filled symbols]) 734 or 6 (Egr2fl/fl) per group. Unpaired Student’s t test, ***p<0.001. 735 G. 2-photon fluorescence imaging of lung tissue from adult Egr2fl/fl (Cre–) and Lyz2Cre/+.Egr2fl/fl (Cre+) 736 mice 6 weeks following bleomycin administration. Sections were stained with CD68, aSMA and DAPI. 737 Autofluorescence is depicted in grey and collagen was detected by second harmonic generation (SHG). 738 H. Quantification of fibrotic score of lung tissue from Egr2fl/fl (Cre–) and Lyz2Cre/+.Egr2fl/fl (Cre+) 3 or 6 739 weeks following bleomycin administration or PBS controls (from 3 week time point). See 740 Supplementary Figure 8. Symbols represent individual mice and error is the S.D. Data are pooled 741 from two independent experiments from one experiment with 6 (Cre–) or 7 (Cre+) mice per group. Two- 742 way ANOVA followed by Tukey’s multiple comparisons test, *p<0.05, **p<0.01, ***p<0.001, 743 ****p<0.0001. 744 I. Quantification of macrophage density in the parenchyma and fibrotic areas of lung tissue from Egr2fl/fl 745 (Cre–) and Lyz2Cre/+.Egr2fl/fl (Cre+) 6 weeks following bleomycin administration. See Supplementary 746 Figure 8. Symbols represent individual mice and error is the S.D. Data are pooled from two independent 747 experiments with 6 (Cre–) or 7 (Cre+) mice per group. Student’s t test with Holm-Sidak correction for 748 multiple tests, *p<0.05. 749 J. SSC-A profile and expression of CD45 by BAL obtained from Egr2fl/fl (Cre–) and Lyz2Cre/+.Egr2fl/fl (Cre+) 750 6 weeks following bleomycin administration. Graph shows the mean frequency of CD45+ cells amongst 751 all live, single events. Symbols represent individual mice and error is the S.D. Data are pooled from two 752 independent experiments with 5 (Cre–) or 7 (Cre+) mice per group. Unpaired Student’s t test, 753 ****p<0.0001. 754 K. Total protein concentration (left), turbidity (centre) and representative pictures (right) of BAL fluid 755 from Egr2fl/fl (Cre–) and Lyz2Cre/+.Egr2fl/fl (Cre+) 6 weeks following bleomycin administration. Symbols 756 represent individual mice. Data are pooled from two independent experiments from one experiment 757 with 6 (Cre–) or 7 (Cre+) mice per group. Mann Whitney test, *p<0.05. 758

28 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

759 Discussion

760 Given the multifaceted role of macrophages in tissue homeostasis, inflammation and tissue 761 repair, as well as many chronic pathologies, understanding the environmental signals and the 762 downstream molecular pathways that govern macrophage differentiation is a key objective in 763 the field of immunology. Here, we identify the transcription factor EGR2 as a selective and 764 indispensable part of the tissue-specific differentiation of lung alveolar macrophages.

765 Our transcriptomic analysis identified EGR2 as a feature of murine lung alveolar 766 macrophages, a finding consistent with previous studies using bulk transcriptomics to 767 compare macrophages from different tissues (1, 6) and a recent study using a similar scRNA- 768 seq based approach, which identified EGR2 specifically upregulated in alveolar, and not 769 interstitial, macrophages (46). Importantly, EGR2 is also expressed by alveolar macrophages 770 in rats (47) and we showed that EGR2 is a feature of alveolar macrophages in man, a finding 771 consistent with recent cross-species analysis (34). Thus, EGR2 appears to represent an 772 evolutionarily conserved transcriptional regulator of alveolar macrophages.

773 While EGR2 has been implicated in controlling monocyte to macrophage differentiation 774 in the past, these studies have often reached discrepant conclusions (19, 20, 34). This could 775 reflect the fact that most studies examining the role of EGR2 in monocyte-macrophage 776 differentiation have employed in vitro culture systems due to the postnatal lethality of global 777 Egr2 deficient mice (21, 22). By generating Lyz2Cre.Egr2fl/fl and Fcgr1iCre.Egr2fl/fl mice, we 778 circumvented this lethality and allowed for myeloid- and macrophage-specific deletion of 779 EGR2, respectively. Our analysis showed that only macrophages in the airways were affected 780 by Egr2 deletion and our transcriptional profiling and extensive phenotypic characterisation 781 demonstrated that EGR2 controls a large proportion of the alveolar macrophage ‘signature’, 782 including key phenotypic traits such as SiglecF, EpCAM and TREM1. This is consistent with 783 recent epigenetic analysis showing an overrepresentation of EGR motifs in the genes defining 784 alveolar macrophages (2, 46). Importantly, although previous work has suggested that there 785 is redundancy between EGR family members, specifically EGR1 and EGR2, we found Egr1 786 expression was unaffected by EGR2 deficiency and was unable to rescue alveolar 787 macrophage differentiation. Indeed, consistent with a recent study (46), we found EGR1 to be 788 expressed at low levels by alveolar macrophages, but higher by interstitial macrophages. Thus, 789 it is plausible that distinct EGR family members may be involved in the differentiation of 790 anatomically distinct lung macrophages.

791 Notably, if assessed simply on the basis of their unique CD11chiCD11blo profile, the 792 absolute number of alveolar macrophages was equivalent between adult Cre– (Egr2fl/fl) and 793 Cre+ (Lyz2Cre.Egr2fl/fl) mice. This could explain why a recent study by the Nagy lab using an

29 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

794 independent strain of Lyz2Cre.Egr2fl/fl mice concluded that EGR2 is not needed for macrophage 795 differentiation (29). Alternatively, this could reflect that the majority of their studies involved in 796 vitro generated macrophages obtained by culturing bone marrow cells with CSF-1 (29). Indeed, 797 we found that Egr2 deficient monocytes matured into macrophages equally well when cultured 798 in vitro with CSF-1. However, despite having been reported to drive expression of EGR family 799 members, including EGR2 (20), in our hands, CSF-1 led to poor upregulation of EGR2 in 800 maturing macrophages in vitro. Instead, we identified CSF-2 (GM-CSF) to be a potent inducer 801 of EGR2 expression in maturing macrophages in vitro, a finding consistent with the almost 802 unique dependence of alveolar macrophages on alveolar epithelial cell-derived CSF-2, and 803 not CSF-1, for their development and survival (8-10). However, TG-Fb also induced EGR2 804 and we confirmed that TGF-b is indispensable for the development of alveolar macrophages 805 (11). Indeed, TGF-b has been shown to induce EGR2 expression outwith the 806 monocyte/macrophage lineage, such as in mammary epithelial cells (49). Exactly how CSF-2 807 and TGF-b cooperate to promote alveolar macrophage differentiation is incompletely 808 understood, however they both induce expression of PPAR-g (9, 11) and Pparg deficient 809 alveolar macrophages expressed reduced EGR2 (9), suggesting EGR2 lies downstream of 810 PPAR-g. Notably, alveolar macrophages from mice with genetic ablation of Pparg, Csf2rb or 811 Tgfbr2 (as shown here) have defects in their ability to establish and self-maintain, a phenotype 812 not seen in mice with Egr2 deficiency. Thus, the EGR2-dependent programme appears to 813 represent a discrete part of alveolar macrophage differentiation, independent of cell survival 814 in health.

815 EGR2 is consistently referred to as a feature of alternative macrophage activation 816 induced by IL-4. Indeed, addition of IL-4 to in vitro macrophage cultures leads to upregulation 817 of EGR2 and deletion of STAT6, a downstream adaptor molecule in the IL-4R signalling 818 cascade, abrogates EGR2 upregulation by IL-4 treated, in vitro generated macrophages (33- 819 35). However, mature alveolar macrophages are considered relatively refractory to IL-4 (50) 820 and we found no effect of Il4ra deficiency on EGR2 expression in health and nor did we detect 821 upregulation of EGR2 by interstitial macrophages which reside in the IL-4/IL-13-rich lung 822 parenchyma during bleomycin-induced fibrosis. Thus, the IL-4–IL-4R axis is sufficient, but not 823 necessary, for inducing EGR2 expression in vivo.

824 Unlike mice deficient in Pparg, Csf2rb or Tgfbr2, mice with myeloid or macrophage 825 deletion of Egr2 did not develop spontaneous alveolar proteinosis, suggesting EGR2 does not 826 control the lipid handling and metabolic capacity of alveolar macrophages. However, Egr2- 827 deficient mice displayed functional deficiencies in the ability to control low dose S. pneumoniae 828 infection, suggesting EGR2 controls the immune protective features of alveolar macrophages. 829 Although we cannot rule out the possibility that this reflects differences in the killing capacity

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830 of Egr2-deficient alveolar macrophages, genes encoding e.g. reactive oxygen and nitrogen 831 species were unaffected by Egr2 deficiency. Instead, genes encoding key pathogen 832 recognition receptors and opsonins, were significantly downregulated in the absence of EGR2. 833 These included MARCO and the complement component C3, both of which have been shown 834 to be crucial for the effective elimination of S. pneumoniae (32, 51). Indeed, opsonisation is a 835 critical factor in optimizing bacterial clearance by alveolar macrophages in health and disease 836 (52). Thus, EGR2-dependent differentiation equips alveolar macrophages with the machinery 837 to recognise and eliminate pneumococci and this may explain the recurrent pneumonias in 838 individuals with mutations in EGR2 (26). In future work it will be important to determine if this 839 extends to other respiratory pathogens.

840 Loss of tissue resident macrophages is a common feature of inflammation or tissue 841 injury. We showed that the resident alveolar macrophage population is diminished markedly 842 following administration bleomycin, a chemotherapeutic agent routinely used to generate lung 843 fibrosis. Consistent with previous work (41, 53), we found that the principal mechanism of 844 macrophage replenishment was through recruitment of BM-derived cells which mature into 845 bona fide alveolar macrophages with time. Using Cx3cr1-based genetic fate mapping, we also 846 showed that CX3CR1+MHCII+ cells with a hybrid phenotype could be found in the airways 847 during the fibrotic phase of injury, suggesting that monocyte-derived interstitial macrophages 848 that accumulate following injury may replenish the alveolar macrophage niche. Although we 849 cannot rule out that monocytes enter the airways during this phase to give rise to alveolar 850 macrophages directly, the phenotype of the RFP+ transitional cells was more aligned with the 851 phenotype of interstitial macrophages, including high levels of MHCII. Importantly, 852 repopulation of the alveolar macrophage compartment was dependent on EGR2, with 853 constitutive deletion of EGR2 severely blunting the engraftment of monocyte-derived cells into 854 the alveolar macrophage niche. This contrasts with initial population of the developing alveolar 855 niche by foetal liver-derived monocytes, where Egr2 deficiency does not affect the 856 development of alveolar macrophages. This could indicate differential dependence of 857 developmentally distinct monocytes on EGR2, or the presence of compensatory pathways 858 during development that are not present during repopulation and further work is required to 859 fully understand this.

860 Interestingly, although previous work has suggested that monocyte-derived alveolar 861 macrophages are key pro-fibrotic cells (41, 53), fibrosis appeared to develop normally in Egr2 862 deficient mice, despite the near absence of monocyte-derived alveolar macrophages. The 863 reason for the discrepancy in our findings and those of Misharin et al. (41) is unclear, but it 864 could reflect differences in the systems used. For instance, the Misharin study exploited the 865 dependence of alveolar macrophages on Caspase-8 to impede monocyte differentiation into

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866 alveolar macrophages by using Lyz2Cre.Casp8fl/fl and ItgaxCre.Casp8fl/fl mice. However, deletion 867 of Caspase-8 also affects the ability of interstitial macrophages to repopulate following 868 depletion, meaning that Casp8 deficiency may have wider effects on lung macrophage 869 behaviour than disrupting the differentiation of monocyte-derived alveolar macrophages. In 870 contrast, EGR2 expression is restricted to alveolar macrophages and deletion does not affect 871 the reconstitution of the interstitial macrophage compartment. The location of interstitial 872 macrophages in the parenchyma adjacent to fibroblasts and their production of the fibroblast 873 mitogen PDGF-aa, suggests that interstitial macrophages are likely to be key to the fibrotic 874 process (42). Indeed, depletion of interstitial macrophages using Cx3cr1Cre-ERT2.Rosa26LSL-DTA 875 mice reduces lung fibrosis (42), although as we show here, this will also target CX3CR1+ cells 876 destined to become monocyte-derived alveolar macrophages. Nevertheless, our data show a 877 clear role for monocyte-derived macrophages in tissue repair processes, as Lyz2Cre.Egr2fl/fl 878 mice failed to repair the lung after injury, a finding consistent with an older study using non- 879 specific, clodronate-mediated depletion of lung macrophages (54) and a recent study 880 implicating ApoE-producing, monocyte-derived alveolar macrophages in lung fibrosis 881 resolution (55). These results may help explain the development of restrictive pulmonary 882 disease in individuals with mutations in EGR2 (26).

883 In summary, our results demonstrate that EGR2 is an evolutionarily conserved 884 transcriptional regulator of alveolar macrophage differentiation, loss of which leads to major 885 phenotypic, transcriptional and functional deficiencies. By identifying EGR2 as a 886 transcriptional regulator, we have begun to dissect how common factors such as CSF2 and 887 TGFb confer specificity during macrophage differentiation. Importantly, given that recent 888 studies using human systems have proposed that alveolar macrophage maintenance in 889 humans requires monocyte input (56, 57), EGR2 may play a particularly important role in 890 alveolar macrophage differentiation in man. Thus, further work is required to fully understand 891 the molecular pathways downstream of EGR2 and whether this is conserved between mouse 892 and humans, and if EGR2 plays distinct roles in different pathological settings.

893 894

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895 Materials and Methods

896 Experimental Animals 897 Mice were bred and maintained in specific pathogen free (SPF) facilities at the University of 898 Edinburgh or University of Glasgow, UK. All experimental mice were age matched and both 899 sexes were used throughout the study. The mice used in each experiment is documented in 900 the appropriate figure legend. Experiments performed at UK establishments were permitted 901 under licence by the UK Home Office and were approved by the University of Edinburgh 902 Animal Welfare and Ethical Review Body. Genotyping was performed by Transnetyx using 903 real-time PCR. 904 Strain Source Identifier C57BL/6J CD45.1 University of Edinburgh C57BL/6J CD45.2+ University of Edinburgh C57BL/6J CD45.1/.2+ University of Edinburgh Rag1–/– University of Glasgow, UK Il4ra–/– Prof. Rick Maizels, University of Glasgow, UK

Cre fl/fl Lyz2 .Egr2 Generated for this study. • Lyz2Cre mice (23) • Egr2fl/fl mice (24)

Fcgr1Cre (B6-Fcgr1tm2-Ciphe) Prof. Bernard Malissen, Dr Sandrine Henri, (3) CIML and CIPHE, France Fcgr1Cre.Egr2fl/fl Generated for this study. Cx3cr1tm2.1(cre/ERT2)Jung Jackson Laboratories (JAX) Stock ID: 020940 Rosa26LSL-tdRFP (Gt(Rosa)26Sortm1Hjf) Elaine Dzierzak, University of Edinburgh (58) Cx3cr1Cre-ERT2.Egr2fl/fl.Rosa26LSL-RFP Generated for this study. Tgfbr2fl/fl Jackson Laboratories (JAX) Stock ID: 012603 Fcgr1Cre.Tgfbr2fl/fl Generated for this study. 905

906 Human cells. BAL fluid was obtained from patients attending the Edinburgh Lung Fibrosis 907 Clinic. Ethical permission was granted from the NHS Lothian Research ethics board (LREC 908 07/S1102/20 06/S0703/53). BAL fluid cells were stained for flow cytometric analysis with 909 antibodies listed in Supplementary Table 4.

910 Tamoxifen-based fate mapping. For induction of Cre activity in Cx3cr1Cre-ERT2/+ mice, 911 tamoxifen was dissolved in sesame oil overnight at 50mg/ml in a glass vial and administered 912 by oral gavage at 5mg per day for five consecutive days. In bleomycin experiments, tamoxifen 913 was administered from d16 post bleomycin administration for 5 days. Fresh tamoxifen was 914 prepared for each experiment. 915 916 Bleomycin lung injury. Bleomycin sulphate (Cayman chemicals) was prepared by first 917 dissolving in sterile DMSO (Sigma) and further in sterile PBS at 0.66µg/ml. 8-12-week-old 918 Lyz2Cre.Egr2fl/fl and Egr2fl/fl littermate controls were anaesthetised with isofluorane and 919 administered 50µl bleomycin (33ng) or vehicle control (DMSO/PBS) by oropharyngeal 920 aspiration.

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921 Streptococcus pneumoniae infection. Lyz2Cre/+.Egr2fl/fl mice and Egr2fl/fl littermate control 922 male mice (8–14-week-old) were anaesthetised ketamine/medetomidine and inoculated 923 intratracheally with 50µl of PBS containing 104 CFU S. pneumoniae (capsular type 2 strain 924 D39). 100µl of inoculum was plated on blood agar to determine exact dose. Mice were culled 925 14 h later and BAL fluid collected by lavage performed using sterile PBS. 100µl of lavage fluid 926 was cultured for bacterial growth for 24 h. The remaining lavage fluid was centrifuged at 400g 927 for 5 mins and the resulting cells counted and prepared for flow cytometric analysis.

928 BM chimeric mice. To generate WT:Lyz2Cre.Egr2fl/fl mixed chimeras, CD45.1+CD45.2+ WT 929 mice were lethally irradiated with two doses of 5 Gy 1 hour apart before being reconstituted 930 immediately WT (CD45.1+) and Lyz2Cre/+.Egr2fl/fl or Egr2fl/fl (CD45.2+) bone marrow at a ratio 931 of 1:1.Chimerism was assessed at 8 weeks after reconstitution. 932 933 Processing of tissues. Mice were sacrificed by overdose with sodium pentobarbitone 934 followed by exsanguination. Mice were then gently perfused with PBS through the heart. In 935 lung injury/fibrosis experiments, the right lobe was tied off, excised and stored in RPMI with 936 10% FCS on ice before being prepared for enzymatic digestion (see below). The left lung lobe 937 was inflated with 600µl 4% PFA through an intra-tracheal canula. The trachea was tied off with 938 thread and the lung and heart carefully excised and stored in 4% PFA overnight. Fixed lung 939 tissue was moved to 70% ethanol before being processed for histological assessment. Right 940 lung lobes were chopped finely and digested in pre-warmed RPMI1640 with ‘collagenase 941 cocktail’ (0.625mg ml–1 collagenase D (Roche), 0.425mg ml–1 collagenase V (Sigma-Aldrich), 942 1mg ml–1 Dispase (ThermoFisher), and 30 U ml–1 DNase (Roche Diagnostics GmbH)) for 25 943 minutes in a shaking incubator at 37°C before being passed through a 100µm strainer followed 944 by centrifugation at 300g for 5 mins. Cell suspensions were then incubated in 2mls Red Blood 945 Cell Lysing Buffer Hybri-Max (Sigma-Aldrich) for 2mins at room temperature to lyse 946 erythrocytes. Cell suspensions were then washed in FACS buffer (2% FCS/2mM EDTA/PBS) 947 followed by centrifugation at 300g for 5 mins. Cells were resuspended in 5mls of FACS buffer, 948 counted and kept on ice until staining for flow cytometry. In some experiments BAL fluid was 949 obtained by lavaging the lungs with 0.8ml DPBS/2mM EDTA via an intra-tracheal catheter. 950 This was repeated three times, with the first wash kept separate for analysis of BAL cytokines, 951 turbidity and protein concentration. To obtain splenic leukocytes, spleens were chopped and 952 digested in HBSS with 1mg/ml collagenase D for 45 mins in a shaking incubator at 37°C before 953 being passed through a 100µm strainer followed by centrifugation at 400g for 5 mins. Tissue 954 preparations were washed in FACS buffer (2% FCS/2mM EDTA/PBS) followed by 955 centrifugation at 300g for 5 mins. Erythrocytes were lysed using red blood cell lysis buffer 956 (Sigma-Aldrich). To obtain liver leukocytes, livers were perfused through the inferior vena cava 957 with sterile PBS and liver tissue excised. Livers were then chopped finely and digested in pre- 958 warmed collagenase ‘cocktail’ (5ml/liver) for 30 minutes in a shaking incubator at 37°C before 959 being passed through an 100μm filter. Cells were washed twice in 50ml ice cold RPMI followed 960 by centrifugation at 300g for 5 mins (59). Supernatants were discarded and erythrocytes were 961 lysed. Epidermal and dermal leukocytes were isolated as described previously (61). To obtain 962 peritoneal leukocytes, the peritoneal cavity was lavaged with RPMI containing 2mM EDTA 963 and 10mM HEPES (both ThermoFisher) as described previously (62). Cells were 964 resuspended in FACS buffer, counted and kept on ice until staining for flow cytometry. 965

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966 Flow cytometry. For analysis of unfixed cells, an equal number of cells were first incubated 967 with 0.025 μg anti-CD16/32 (2.4G2; Biolegend) for 10mins on ice to block Fc receptors and 968 then stained with a combination of the antibodies detailed in Supplementary Table 4. Where 969 appropriate, cells were subsequently stained with streptavidin-conjugated BV650 (Biolegend). 970 Dead cells were excluded using DAPI or 7-AAD (Biolegend) added 2mins before acquisition. 971 When assessing intracellular markers, cells were first washed in PBS and then incubated with 972 Zombie NIR fixable viability dye (Biolegend) for 10mins at room temperature protected from 973 light before following the approach detailed above. Following the final wash step, cells were 974 subsequently fixed and permeabilized using FoxP3/Transcription Factor Staining Buffer Set 975 (eBioscience), and intracellular staining performed using antibodies detailed in Supplementary 976 Table 4. Fluorescence-minus-one (FMO) controls confirmed gating strategies, while discrete 977 populations within lineage+ cells were confirmed by omission of the corresponding population- 978 specific antibody. Samples were acquired using a FACS LSRFortessa or AriaII using 979 FACSDiva software (BD) and analyzed with FlowJo software (version 9 or 10; Tree Star). 980 Analysis was performed on single live cells determined using forward scatter height (FCS-H) 981 versus area (FSC-A) and negativity for viability dyes. mRNA was detected by flow cytometry 982 using PrimeFlow technology (ThermoFisher) using probes against Spp1 (AF647) according 983 to the manufacturer’s guidelines. For staining controls in PrimeFlow analysis, the Target Probe 984 Hybridization step was omitted with all other steps identical to samples. 985 986 Transcriptional Analysis. 987 qPCR: Real-time PCR assays for the detection of mRNAs were performed using Light Cycler 988 System (Roche) and 384-Well Reaction Plates (Roche). Primer sequences are as follows: 989 Forward Reverse Ppia 5’-ACGCCACTGTCGCTTTTC-3 5’-CTGCAAACAGCTCGAAGGA-3’ Car4 5’-CAAACCAAGGATCCTAGAAGCA-3’ 5’-GGGGACTGCTGATTCTCCTT-3’ Fabp1 5’-CCATGACTGGGGAAAAAGTC-3’ 5’-GCCTTTGAAAGTTGTCACCAT-3’

Qiagen QuantiTect Primers Col3a1 Mm_Col3a1_1_SG; Cat No. QT01055516; Lot No. 287520017 Pdgfrb Mm_Pdgfrb_1_SG; Cat No. QT00113148; Lot No. 224645185 990 991 Reactions were performed using SYBR Green System (LightCycler® 480 SYBR Green I 992 Master) according to the manufacturer protocol. 1ul of cDNA (1:50 dilution) were used per 993 sample in a total reaction volume of 10uL. The temperature profile used was as follows: pre- 994 denaturation 5 min at 95°C and then 45 cycles of denaturation for 10s at 95°C, annealing 10s 995 at 60°C, elongation 10s at 72°C. Fluorescence data collection was performed at the end of 996 each elongation step. All samples were tested in duplicates and nuclease free water was used 997 as a non-template control. The relative change was calculated using the 2-ΔΔCt method (63), 998 normalized to Ppia. 999 1000 Bulk sequencing: Alveolar macrophages were FACS-purified from lung digests from 1001 unmanipulated female Lyz2Cre.Egr2fl/fl mice or Egr2fl/fl controls. For each population, 25,000 1002 cells were sorted into 500µl RLT buffer (Qiagen) and snap frozen on dry ice. RNA was isolated 1003 using the RNeasy Plus Micro kit (Qiagen). RNA samples were quantified using Qubit 2.0 1004 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with 1005 2100 TapeStation (Agilent Technologies, Palo Alto, CA, USA). SMART-Seq v4 Ultra Low Input 1006 Kit for Sequencing was used for full-length cDNA synthesis and amplification (Clontech,

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1007 Mountain View, CA), and Illumina Nextera XT library was used for sequencing library 1008 preparation. Briefly, cDNA was fragmented and adaptor was added using Transposase, 1009 followed by limited-cycle PCR to enrich and add index to the cDNA fragments. The final library 1010 was assessed with Qubit 2.0 Fluorometer and Agilent TapeStation. The sequencing libraries 1011 were multiplexed and clustered on two lanes of a flowcell. After clustering, the flowcell were 1012 loaded on the Illumina HiSeq instrument according to manufacturer’s instructions. The 1013 samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and 1014 base calling were conducted by the HiSeq Control Software (HCS) on the HiSeq instrument. 1015 Raw sequence data (.bcl files) generated from Illumina HiSeq were be converted into fastq 1016 files and de-multiplexed using Illumina bcl2fastq v. 2.17 program. One mis-match was allowed 1017 for index sequence identification. After demultiplexing, sequence data was checked for overall 1018 quality and yield. Then, sequence reads were trimmed to remove possible adapter sequences 1019 and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped 1020 to the Mus musculus mm10 reference genome available on ENSEMBL using the STAR 1021 aligner v.2.5.2b. The STAR aligner uses a splice aligner that detects splice junctions and 1022 incorporates them to help align the entire read sequences. BAM files were generated as a 1023 result of this step. Unique gene hit counts were calculated by using featureCounts from the 1024 Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. After 1025 extraction of gene hit counts, the gene hit counts table was used for downstream differential 1026 expression analysis. Using DESeq2, a comparison of gene expression between the groups of 1027 samples was performed. The Wald test was used to generate p-values and Log2 fold changes. 1028 Genes with adjusted p-values < 0.05 and absolute log2 fold changes > 1 were called as 1029 differentially expressed genes for each comparison. 1030

1031 scRNA-seq: CD11c+ and CD11b+ cells, excluding Ly6G+ and SiglecF+CD11b+ eosinophils, –/– 1032 were FACS-purified from unmanipulated an adult Rag1 mouse. Single cells were processed 1033 through the Chromium Single Cell Platform using the Chromium Single Cell 3' Library and Gel 1034 Bead Kit V2 and the Chromium Single Cell A Chip Kit (both 10X Genomics) as per the 1035 manufacturer’s protocol (64). Briefly, single myeloid cells were purified by FACS into PBS/2% 1036 FBS, washed twice and cell number measured using a Bio-Rad TC20 Automated Cell Counter 1037 (BioRad). Approximately 10,000 cells were loaded to each lane of a 10X chip and partitioned 1038 into Gel Beads in Emulsion containing distinct barcodes in the Chromium instrument, where 1039 cell lysis and barcoded reverse transcription of RNA occurred, followed by amplification, 1040 fragmentation and 5' adaptor and sample index attachment. Libraries were sequenced on an 1041 Illumina HiSeq 4000. For analysis, Illumina BCL sequencing files were demultiplexed using 1042 10x Cell Ranger (version 2.1.1; https://www.10xgenomics.com; ‘cellranger_mkfastq’). 1043 Resultant FASTQ files were fed into ‘cellranger_count’ with the transcriptome ‘refdata- 1044 cellranger-mm10-1.2.0’ to perform genome alignment, filtering, barcode counting and UMI 1045 counting. Downstream QC, clustering and gene expression analysis was performed using the 1046 Seurat R package (V3; R version 4.0.2) following the standard pre-processing workflow (65). 1047 Cells were filtered on QC covariates used to identify nonviable cells or doublets: number of 1048 unique genes per cell (nFeatureRNA >200 & <4000); percentage mitochondrial genes (<20%). 1049 Data for resultant 3936 cells were normalized and scaled prior to PCA analysis. Unsupervised 1050 clustering based on the first 20 principal components of the most variably expressed genes 1051 was performed using a KNN graph-based approach and resultant clusters visualised using 1052 the Uniform Manifold Approximation and Projection (UMAP) method. Differential gene 1053 expression analysis was used to identify genes expressed by each cell cluster relative to all

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1054 others, using the nonparametric Wilcoxon rank-sum test and p-value threshold of <0.05. 1055 Canonical cell phenotypes were assigned to individual clusters based on the expression of 1056 known landmark gene expression profiles.

1057 Publicly available datasets were downloaded from the COVID-19 Cell Atlas (13-15) to perform 1058 in silico analysis of EGR2 expression in human tissue macrophages. Data were pre-processed 1059 and merged using the Seurat R package (V3; R version 4.0.2) following standard methods. 1060 Macrophages were extracted based on the expression of C1QA > 0 to compare expression of 1061 EGR2 in different human tissue settings. 1062 1063 BAL fluid analysis. The first BAL wash was centrifuged at 400g for 5mins and supernatant 1064 removed and stored at -80ºC until analysis. Total protein concentrations in BAL fluid were 1065 measured by BCA Protein Assay according to the manufacturer’s instructions (ThermoFisher). 1066 Turbidity was determined following gentle mixing by diluting 25ul of sample with 75ul DPBS 1067 and measuring the optical density of 600nm and multiplying by the dilution factor. BAL 1068 cytokines were measured using 50ul undiluted sample and the Cytokine & Chemokine 26- 1069 Plex ProcartaPlex (Panel 1) assay according to manufacturer’s guidelines (ThermoFisher). 1070 1071 Lung histology. Formalin-inflated lungs were fixed overnight in 4% buffered formalin and 1072 stored in 70% ethanol. Paraffin-embedded sections of mouse lungs were stained with 1073 Masson’s trichome as per the manufacturer’s guidelines. 1074 1075 Immunofluorescence imaging. Imagin was performed as described recently (Fercoq et al., 1076 2020). Briefly, samples were permeabilized and blocked for 20min in PBS/Neutral goat serum 1077 (NGS) 10%/BSA1%/TritonX-100 (Tx100) 0.3%/Azide 0.05% at 37 °C and stained with 150 µl 1078 rabbit anti-CD68 Ab (Polyclonal, ab125212, abcam, 1/200) diluted in PBS/ NGS10%/BSA1%/ 1079 TX-100 0.3%/Azide 0.05% for 20min. Samples were washed 3 times with PBS/BSA1%/TX- 1080 100 0.1%/Azide 0.05% before adding 150 µl of a solution containing DAPI (1/10000), aSMA- 1081 Cy3 (clone 1A4, Sigma, 1/1000), anti-rabbit-AF488 (polyclonal, A-21206 ThermoFisher) 1082 diluted in PBS/ NGS10%/BSA1%/ TX-100 0.3%/Azide 0.05% for 1h. Samples were washed 1083 3 times with PBS/BSA1%/TX-100 0.1%/Azide 0.05% and 2 times in PBS. Finally slides were 1084 mounted with Vectashield (Vector Laboratories, H-1700). Images were acquired with a Zeiss 1085 LSM 880 NLO multiphoton microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 1086 32 channel Gallium arsenide phosphide (GaAsP) spectral detector using 20×/1 NA water 1087 immersion objective lens. Samples were excited with a tunable laser (680–1300 nm) set up at 1088 1000 nm and signal was collected onto a linear array of the 32 GaAsp detectors in lambda 1089 mode with a resolution of 8.9 nm over the visible spectrum. Spectral images were then 1090 unmixed with Zen software (Carl Zeiss) using references spectra acquired from unstained 1091 tissues (tissue autofluorescence and second harmonic generation) or beads labelled with Cy3- 1092 or AF488-conjugated antibodies. 1093 1094 Image analysis. Fluorescence images were analysed with QuPath (66). Full lung section was 1095 annotated using the “simple tissue detection” tool and non-pulmonary tissue (trachea, heart 1096 tissue) were manually removed from the annotation. In order to refine the analysis, “Pixel 1097 classification” was used to segment lung regions of interest. Briefly, software was trained to 1098 recognize the different regions using fluorescence (aSMA, SHG and autofluorescence) and 1099 texture (all available) features from example images. 2-3 example areas per regions of interest 1100 were annotated for each lung to train the pixel classifier. The following regions were analysed:

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1101 (1) normal lung parenchyma/alveolar tissue, (2) pathologic/fibrotic tissue and (3) collagen rich 1102 areas: perivascular/(peri)bronchial spaces + pleura were segmented to avoid false fibrotic 1103 region detection. Macrophages were detected using the “Positive cell detection” tool and were 1104 expressed as the number DAPI+ CD68+ cells/mm2 of analysed region (full section or regions 1105 of interest). Fibrosis was defined as percentage of full section with fibrotic features. All fibrosis 1106 scoring and macrophage quantification was performed in a blinded fashion. 1107 1108 Statistics. Statistics were performed using Prism 7 (GraphPad Software). The statistical test 1109 used in each experiment is detailed in the relevant figure legend. 1110 1111 Accession codes. RNA sequencing data that support the findings of this study will be 1112 deposited in National Center for Biotechnology Information Gene Expression Omnibus public 1113 database (http://www.ncbi.nlm.nih.gov/geo/) upon acceptance. 1114 1115 Data availability. Data that support the findings of this study are available from the 1116 corresponding authors upon reasonable request. 1117 1118 Further information and requests for resources and reagents should be directed to and will 1119 be fulfilled by the Lead Contact, Calum Bain ([email protected]). 1120 1121 References 1122 1123 1. E. L. Gautier, T. Shay, J. Miller, M. Greter, C. Jakubzick, S. Ivanov, J. Helft, A. Chow, K. 1124 G. Elpek, S. Gordonov, A. R. Mazloom, A. Ma'ayan, W. J. Chua, T. H. Hansen, S. J. 1125 Turley, M. Merad, G. J. Randolph, Gene-expression profiles and transcriptional 1126 regulatory pathways that underlie the identity and diversity of mouse tissue 1127 macrophages, Nature immunology 13, 1118–1128 (2012). 1128 2. Y. Lavin, D. Winter, R. Blecher-Gonen, E. David, H. Keren-Shaul, M. Merad, S. Jung, I. 1129 Amit, Tissue-resident macrophage enhancer landscapes are shaped by the local 1130 microenvironment, Cell 159, 1312–1326 (2014). 1131 3. C. L. Scott, W. T'Jonck, L. Martens, H. Todorov, D. Sichien, B. Soen, J. Bonnardel, S. 1132 De Prijck, N. Vandamme, R. Cannoodt, W. Saelens, B. Vanneste, W. Toussaint, P. De 1133 Bleser, N. Takahashi, P. Vandenabeele, S. Henri, C. Pridans, D. A. Hume, B. N. 1134 Lambrecht, P. De Baetselier, S. W. F. Milling, J. A. Van Ginderachter, B. Malissen, G. 1135 Berx, A. Beschin, Y. Saeys, M. Guilliams, The Transcription Factor ZEB2 Is Required to 1136 Maintain the Tissue-Specific Identities of Macrophages, Immunity 49, 312–325.e5 1137 (2018). 1138 4. P. P. Ogger, A. J. Byrne, Macrophage metabolic reprogramming during chronic lung 1139 disease, Mucosal Immunol 14, 282–295 (2021). 1140 5. M. Merad, J. C. Martin, Pathological inflammation in patients with COVID-19: a key role 1141 for monocytes and macrophages, Nature reviews 20, 355–362 (2020). 1142 6. E. Mass, I. Ballesteros, M. Farlik, F. Halbritter, P. Günther, L. Crozet, C. E. Jacome- 1143 Galarza, K. Händler, J. Klughammer, Y. Kobayashi, E. Gomez Perdiguero, J. L. 1144 Schultze, M. Beyer, C. Bock, F. Geissmann, Specification of tissue-resident 1145 macrophages during organogenesis, Science (New York, N.Y 353 (2016), 1146 doi:10.1126/science.aaf4238. 1147 7. E. Gomez Perdiguero, K. Klapproth, C. Schulz, K. Busch, E. Azzoni, L. Crozet, H. 1148 Garner, C. Trouillet, M. F. de Bruijn, F. Geissmann, H.-R. Rodewald, Tissue-resident 1149 macrophages originate from yolk-sac-derived erythro-myeloid progenitors, Nature 518, 1150 547–551 (2015).

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1386 1387 Acknowledgements 1388 We are grateful to Prof. Ping Wang and Dr. Su-Ling Li, QMUL, London for the kind gift of the 1389 Egr2-floxed strain and to Prof. Rick Maziels, University of Glasgow for the provision of Il4ra- 1390 deficient mice. Flow cytometry data were generated with support from the QMRI Flow 1391 Cytometry and Cell Sorting Facility, University of Edinburgh. mRNA sequencing was 1392 performed by Edinburgh Genomics, The University of Edinburgh. Edinburgh Genomics is 1393 partly supported through core grants from NERC (R8/H10/56), MRC (MR/K001744/1) and 1394 BBSRC (BB/J004243/1). Genewiz performed the bulk RNA-seq analysis. We would like to 1395 thank Beth Henderson for technical expertise in setting up 10X sequencing; Dr. Jordan 1396 Portman for initial processing of the scRNAseq raw data; the ShIELD (Sheffield, Edinburgh, 1397 Newcastle and Birmingham) consortium for access to bacterial stocks; Dr. Duncan Humphries 1398 for training and technical assistance with infection studies and Dr. Brian McHugh for advice 1399 on bacterial studies. We would also like to thank the Core Services and Advanced 1400 Technologies at the Cancer Research UK Beatson Institute, with particular thanks to the 1401 Beatson Advanced Imaging Resource (BAIR). Finally, we would like to thank the Bioresearch 1402 and Veterinary Services at the University of Edinburgh for husbandry of our mice and other 1403 technical assistance. Images from Servier Medical Art (CC BY licence 3 1404 https://creativecommons.org/licenses/by/3.0/) were used and adapted for the generation of 1405 some of the graphics. 1406 1407 This research was funded by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust 1408 and the Royal Society [Grant number 206234/Z/17/Z to C.C.B]. CGH is supported by a 1409 University of Edinburgh Chancellor’s Fellowship and CGH and RC are supported by 1410 Worldwide Cancer Research. The Lyz2Cre.Egr2fl/fl line was originally generated with funding 1411 from the Medical Research Council UK (MR/L008076/1 to S.J.J). GRJ is funded by a 1412 Wellcome Trust Clinical Career Development Fellowship (220725/Z/20/Z). SRW is funded by 1413 a Wellcome Trust Senior Clinical Fellowship (209220). PTKS received funding from the MRC 1414 (MR/N024524/1). FF and LMC are funded by Cancer Research UK core funding (A23983 & 1415 A31287), and Breast Cancer Now (2019DecPR1424).

43 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.06.442095; this version posted May 6, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

1416 For the purpose of open access, the author has applied a CC BY public copyright 1417 licence to any Author Accepted Manuscript version arising from this submission. 1418 1419 Author Contributions 1420 J. McC. performed experiments, analysed the data and edited the manuscript. P.M.K. 1421 Performed scRNA-seq analysis. F.F. Designed and performed immunofluorescence analysis 1422 of lung tissue. W. T’J. Performed experiments, analysed data and edited the manuscript. 1423 C.M.M performed experiments and analysed the data. R. C. and C. G. H provided 1424 bioinformatic analysis of ImmGen data. A.S.M. provided advice on and help with the execution 1425 of fibrosis experiments. A. H. performed analysis. G.R.J. performed histological analysis of 1426 lung sections. S.J.J. generated the Lyz2Cre.Egr2fl/fl mice. N. H. provided access to human 1427 bronchoalveolar samples. S.H. and B.M. generated and provided the Fcgr1iCre mice. S.R.W. 1428 advised on the design of fibrosis experiments and provided reagents for infection experiments. 1429 D.D. advised on the design and execution of infection experiments. P.T.K.S. advised and 1430 provided infrastructure to perform scRNA-seq analysis. L.M.C. advised and provided 1431 infrastructure to perform multi-parameter immunofluorescence analysis. C.C.B. conceived 1432 and performed experiments, analysed and interpreted the data, wrote the manuscript, 1433 obtained funding and supervised the project. 1434

1435 Declaration of Interests

1436 The authors declare no competing interests.

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