Inflammatory, metabolic, and sex-dependent gene-regulatory dynamics of microglia and macrophages in neonatal hippocampus after hypoxia-ischemia

Summary Neonatal hypoxia-ischemia (HI) is a major cause of perinatal death and long-term disabilities worldwide. Post-ischemic neuroinflammation plays a pivotal role in HI pathophysiology. In the present study, we investigated the temporal dynamics of microglia (CX3CR1GFP/+) and infiltrating macrophages (CCR2RFP/+) in the hippocampi of mice subjected to HI at postnatal day 9. Using inflammatory pathway and transcription factor (TF) analyses, we identified a distinct post-ischemic response in CCR2RFP/+ cells characterized by differential gene expression in sensome, homeostatic, matrisome, lipid metabolic, and inflammatory molecular signatures. Three days after injury, transcriptomic signatures of CX3CR1GFP/+ and CCR2RFP/+ cells isolated from hippocampi showed a partial convergence. Interestingly, microglia-specific genes in CX3CR1GFP/+ cells showed a sexual dimorphism, where expression returned to control levels in males but not in females during the experimental time frame. These results highlight the importance of further investigations on metabolic rewiring to pave the way for future interventions in asphyxiated neonates.


INTRODUCTION
Neonatal hypoxic-ischemic (HI) injury is a complex and severe condition caused by an impaired flow of oxygenated blood to the infant brain and has an incidence of 1-3 cases per 1,000 living births. 1,2To date, therapeutic hypothermia is the only available treatment proven to offer neuroprotection, although about 40% of the treated babies still present long-term disabilities as a consequence of acute neuronal death and chronic neuroinflammation, resulting in neurodevelopmental abnormalities. 3,4][10][11] Microglia are the primary immune cells in the brain.They represent 12%-15% of all cells in the central nervous system (CNS) 12 and differ from other brain cells by origin, function, morphology, and gene expression patterns. 13,14Contrarily, monocytes derive from bone marrow hematopoietic stem cells, which differentiate into common myeloid progenitors, 15 and are recruited from the circulation into the CNS to participate in the inflammatory response. 8,10,16Both macrophages and microglia are sentinels in their specific environment and have phagocytic functions. 17They have the capacity to recognize injured cells and immune mediators, to produce signaling molecules and modulate inflammation. 18As neuroinflammation is critical for the delayed cell death and the progression of HI brain injury, 19 understanding the inflammatory dynamics between resident and infiltrating immune cells is paramount to developing future therapeutic approaches.Normal brain development and function Temporal and cell-type-specific signatures in resident microglia and blood-derived macrophages Based on the histological outcomes, very few CCR2 RFP+ cells reached the hippocampi seven days after HI.Thus, the following system biology approach was implemented at day 1 (D1) and day 3 (D3) post-injury to gather further insights into the temporal molecular differences between the two cell types in the hippocampi.
RNA-sequencing of the sorted cell populations one day and three days after injury revealed a total of 39,501 different coding transcripts.HI resulted in rapid and drastic alterations of the microglia transcriptome.Unsupervised clustering analyses were applied to visualize the inter-and intracellular differences in the data.Uninjured controls cluster together as expected.In contrast, GFP samples show two very distinct clusters diverging more at D1 than D3 compared with controls.Similarly, infiltrating macrophages D1 after HI depict higher diversity Figure 1.Quantification of resident microglia and infiltrating macrophages in the hippocampus after HI (A) Project design for cell quantification and transcriptomic analysis (created with BioRender.com).(B) Infiltrating macrophages (RFP) and microglia (GFP) in immunofluorescence staining, with scale bar, 250 mm upper quadrant and 50 mm lower quadrant.(C and D) Quantification of CX3CR1 GFP+ cell (C) and CCR2 RFP+ cells (D) in the hippocampus after HI (1 day: n = 6 females, n = 8 males; 3 days: n = 7 females, n = 6 males; 7 days: n = 3 females, n = 4 males).Data are analyzed with two-way ANOVA and Sida ´k-corrected post-hoc t test and presented as mean G SEM and *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Legend: control = Ctrl, injured = HI, male = M, female = F. See also Figure S1.compared with the other groups.Interestingly, RFP and GFP D3 clusters are remarkably close, suggesting a phenotypic adaptation by infiltrating macrophages after brain entry.Ultimately, macrophage and microglia gene programs tend to converge over time (Figure 2A).The number of differentially expressed genes were compared within and between groups confirming the unique temporal gene pattern for microglia and infiltrating macrophages (Figure 2B).
To better characterize the temporal pattern of these two cells and to understand how their function and roles change during post-ischemic inflammation, we focused on differentially expressed genes (DEGs, q < 0.05) (Figure 2C) and explored the significant motifs regulating the temporal and cell-type-specific differences using the ISMARA algorithm (Figures 2E, 2G-2J). 25n GFP samples, various genes involved in inflammatory (Ccl6) and apoptotic (Aen) signaling and organelle synthesis (Rsl1d1) were among the 50 most significantly dysregulated genes D1 after HI.After three days, upregulation of genes involved in cell cycle (Cdca3), lipid metabolism (Apoe, Pltp, Hpgd, Lipa), reorganization of synaptic activity (C1qa, Syngr1), or vessel remodeling (Igf1) was observed (Figure 2D).In accordance, enriched transcription factor (TF) binding motifs involved in cell cycle (Max/Mycn); microglia immune and inflammatory response mediation (Ahr); immune cell activation and regulation (Atf4, Creb1); and stress response, autophagy, and synapse stability modulation (Hsf1) were activated D1 post-injury.Also, motifs involved in the regulation of angiogenesis (Foxp1/j2) and the TGF-b/Smad1 axes (Smad1) were activated D3 after the insult (Figure 2E).
To specifically compare the characteristic features of the two different cell types after HI from an upstream regulatory gene perspective, we focused on the transcription binding motifs that would drive transcription differences between time points and cell types.ISMARA activity analysis showed 180 significant regulators between microglia and infiltrating macrophages D1 post-HI, whereas this had decreased to 18 at D3 (Figure 2H).
Among the top 10 enriched TF binding motifs, Jun, known to be involved in myeloid cell activation, was upregulated in RFP samples at both time points.One day after HI, RFP cells show activation of motifs involved in cell matrix regulation (Etsf1), cell activation (Fos), and monocyte chemotaxis (Snap/Zeb1/Snai1/2).In contrast, E2f7, which is involved in cell-cycle progression, was suppressed (Figure 2I).Three days post-HI, motifs regulating lipid homeostasis (Nr2f6) were active in RFP cells, whereas motifs involved in microglia activation and inflammatory response (Mef2a/d) were active in GFP cells (Figure 2J).
In conclusion, infiltrating macrophage differentiation and their altered signal responses are orchestrated by cis-linked transcriptional and epigenomic programs that induce gene expression signatures that allow macrophage transition toward an activated microglial phenotype.
Thus, matrisome-associated gene analysis suggests an active role of infiltrating macrophages cells in BBB permeability and immune cells activation.Concomitantly, microglia have reduced ability to maintain BBB integrity and vascular remodeling.

The temporal dynamics of inflammatory and lipid gene expression differ between cell type
Considering the predominant difference among cells acutely after HI and to further investigate the inflammatory profile of resident microglia and infiltrating macrophages, we conducted pathway analyses D1 after HI.
Pathways related to immune cell and sphingolipid signaling were upregulated in RFP cells suggesting that infiltrating macrophages are highly responsive to the post-ischemic microenvironment as soon as they reach the site of injury.GFP cells on the other hand showed significant upregulation in only a few distinct pathways involved in fatty acid degradation, oxidative phosphorylation, and the tricarboxylic acid cycle (TCA) that are associated with microglia activation phenotype (Figure 4A). 29These results are in line with the metabolic rewiring of activated microglia/macrophages in response to cytoskeleton rearrangement and production and release of inflammatory molecules.
To further define this outcome, we investigated the gene regulatory pattern of lipid metabolism (Figures 4B and 4C) to assess its correlation with immune cell activation states.We identified downregulation of cholesterol recycling and eliminating genes (Fdft1, Dhcr24, Hmgcs1 and Cyp46a1; Figure S3) and upregulation of cholesterol catabolism genes (Ch25h) concurrent to elevated levels of a direct transcriptional regulator  of the gene encoding cholesterol-25-hydroxylase that also is a mitigator of microglia inflammatory response (Atf3, Figure S4) 30 in GFP groups, whereas cholesterol efflux transcripts (Abca1 and Abcg1) were upregulated primarily in GFP cells D3 after injury (Figures 4C and 4D).Increased expression of genes related to regulation of cholesterol metabolism in macrophages (Npc1,2 and Soat1,2), cholesterol synthesis (Hmgcr and Srebf1,2), and cholesterol transport and efflux (LXR1a: Nr1h3) was observed in RFP groups at both time points (Figures 4F and S3).
In conclusion, the HI-induced neuroinflammatory cascade is accompanied by aberrant lipid metabolism with several forms of fatty acid and cholesterol dysregulation in both cell types.However, after reaching the brain, infiltrating macrophages respond rapidly and appear to be the driving force behind the post-ischemic inflammatory response and distinct cholesterogenic and lipidogenic gene expression patterns.[33] Cell-type and sex-specific patterns of inflammasome activation Fatty acid synthesis, b-oxidation, and oxidative phosphorylation have been reported to characterize pro-and anti-inflammatory states. 34,35To increase our understanding of the neuroinflammatory contribution of the CX3CR1 GFP+ and CCR2 RFP+ populations, we selected a panel of proinflammatory (105 transcripts) and anti-inflammatory (92 transcripts) markers (Figure S5). 36Our results suggest that RFP cells have a prominent role in orchestrating the inflammation as early as D1 after injury, and the expression of both pro-and anti-inflammatory genes is reduced over time to a level comparable with that of the GFP groups.Specific analysis of the activating genes D1 post-HI revealed the upregulation of the majority of transcripts for both pro-and anti-inflammatory states in RFP cells (Figure 5A).We observed a higher expression of cytokines and chemokines in GFP compared with RFP and control cells, whereas MMPs and pattern recognition receptors (PRRs) genes displayed more pronounced upregulation in RFP compared with GFP groups (Figures 5B and S6).
One day after injury, an upregulation of chemokine ligands that attract monocytes to the injury site and promote differentiation into macrophages (CCL3, CCL4, CCL8, CCL12, and CXCL14) was observed in the GFP group.In contrast, RFP cells displayed upregulation of chemokine receptors (CCR1, CCR2, CCR3, CCR5, CXCR4, XCR1) and chemokine ligands that attract neutrophils or control migration and adhesion of monocytes (CXCL1, CXCL2, CXCL3).Very few cytokines were upregulated at the one-or three-day time points: specifically, D1 after injury the GFP group had elevated expression of MIF1 and CSF1, whereas the RFP group had higher expression of IL6, IL18, and IL10 (Figure S6A).Similarly, analysis of PRRs (Figure S6B) and sensome (Figure S6C) components revealed increased gene expression primarily in the RFP samples D1 after HI.These findings suggest that peripheral macrophages are highly receptive to the environment at the injury site soon after insult and orchestrate the inflammatory cascade.In GFP samples, specifically D1 after HI, a clear downregulation of both PRR and other sensome markers was evident (Figure 5C).
Of interest, expression profiles were significantly different between males and females at the three-day time point (Figures 5D and 5E).When examining the genes differentially expressed in the two sexes, we identified significant upregulation of Cx3cr1 and Siglech, considered to be microglia homeostatic genes, in both cell types of males.
In conclusion, this corroborates that infiltrated macrophages are fast responders to microglia signaling, whereas there was a stronger reduction in microglia activation over time in males.

Microglial homeostasis genes are differentially regulated in males and females
7][38][39] As confirmed by the heatmap (Figure 6A), expression of microglia-homeostatic genes is minimal in RFP samples.One day post-HI, GFP cells showed a clear downregulation of homeostatic genes, as expected after an insult.However, a distinction between male and female samples was observed at D3. Specifically, although female samples showed a prolonged downregulation of homeostatic genes, in males these reverted to control levels displaying sex-specific clustering (Figure 6B).This clear pattern was further confirmed by significant upregulation of 24 homeostatic genes in males (p < 0.05) (Figure 6C).
To determine whether microglia ''maturation'' was linked to post-injury response, we calculated the microglial developmental index (MDI). 40Notably, the analysis confirmed clear differences in GFP MDI between males and females in the injured hemisphere but not between male and female controls (Figure 6D).
Collectively, our findings indicate a sexual dimorphism in microglia that may induce different neuroinflammatory cascades after HI.

DISCUSSION
Neuroinflammation is a major contributor to neonatal brain injury and is mediated by several different cell types, both local and peripheral, releasing a plethora of signaling molecules upon activation.Microglia and infiltrating macrophages are the main players in the CNS orchestrating the inflammatory cascade after HI and good targets for intervention; however, their signaling with one another and with other brain cells remain poorly understood.(D-F) Expression of differentially expressed genes involved in fatty acid and cholesterol processes, with color legend at the bottom of (D) and functional classification at the bottom of (E).The genes are divided by expression pattern into mainly genes expressed in microglia (D), genes also expressed in macrophages (E) or genes expressed mainly in infiltrating macrophages (F).Data are analyzed with one-way ANOVA with Sida ´k-corrected post-hoc t test and presented as mean G SEM and *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.See also Figures S3 and S4.2][43] To scrutinize the dynamic nature of these two cell types after a post-ischemic insult, we investigated the transcriptional motif network in combination with the metabolic gene regulatory program and the ECM during the inflammatory response.
The present study meticulously characterizes both cell types over a critical time window, (1) showing that infiltrating macrophages are the drivers of the post-ischemic inflammation, (2) indicating a transcription factor, ECM, and metabolic gene program that partially converges infiltrating cells and activated microglia three days after HI, and (3) suggesting a faster return to homeostatic microglia phenotype in males.
Our findings show a peak of infiltrating CCR2 RFP+ cells one day post-HI characterized by upregulation of inflammatory genes.Furthermore, the investigation of pro-and anti-inflammatory signaling molecules proposes infiltrating cells having a predominant role in orchestrating the inflammatory cascade.Differences between transcriptomic signatures of CX3CR1 GFP+ and CCR2 RFP+ cells diminished over time, suggesting an adaptation of infiltrating macrophages to the brain microenvironment.These results are consistent with other studies showing that peripheral macrophages infiltrating the brain, e.g., after an LPS/HI insult, become ''inflammatory microglia''-like cells over time, downregulating CCR2 and upregulating CX3CR1 expression. 44,45he main mechanism of modulation in these highly dynamic cell types occurs via regulatory and chromatin remodeling programs directed by sophisticated TF networks.Our results reveal cell-type-specific transcriptional motifs corresponding to TFs, indicating regulation of our identified inflammatory, matrix remodeling, and lipid/cholesterol gene programs (summarized in Figure 7).
The ECM is dynamically remodeled throughout all phases of cell proliferation, differentiation, migration as well as during injury and inflammation and is now considered to be involved in the neuroinflammatory cascade by directly modulating the expression of cyto-and chemokines. 46In the present study, we report the downregulation of context-dependent structural ECM components, such as collagens and proteoglycans, as well as vascular remodeling (Cst3) and upregulation of transcripts involved in ECM degradation (Mmps11/12 and Adam10/15/33).Alterations in ECM composition play critical roles in the preservation of BBB integrity as well as in the regulation of the cellular responses that further mediate tissue healing. 47,48Recent findings reported infiltrating macrophages to have an active role in the modulation of the ECM configuration. 49In agreement, our data displayed upregulation of several genes giving rise to matrisome or matricellular proteins in infiltrating macrophages involved in BBB permeability (Anxa1, Mmp9, Timp1), inflammatory response (Vcan, Anxa2, s100a10), and secretion of cytokines (Srgn), specifically tumor necrosis factor (TNF), modulating the inflammatory cascade. 50Of interest, TNF-a is known to regulate cholesterol by upregulating Abca1, 51 and is also the best-known substrate for Adam17 elevated one day after injury in CCR RFP+ cells, and was recently shown to boost cholesterol efflux in macrophages modulating their immune functions. 52Given the bidirectional dialog between microglia/macrophages and ECM, comprehending the matrix in the frame of metabolic rewiring is crucial to pave the way for successful immune-cell-based interventions. 53merging evidence suggests a link between ECM and lipid metabolism, which regulates multiple biological processes including synaptic formation and axon myelination. 54,55The major component of myelin sheets and CNS cell membranes is cholesterol.Unlike the adult brain, the immature brain has no stable pool of cholesterol, which is instead synthesized ex novo and accumulated to comply with the biophysical need of the developing brain. 56Currently, this aspect is poorly investigated in neonatal HI.Among the top DEGs, we identified Lipa, acknowledged for its role in breaking down lipids such as cholesteryl esters and triglycerides to produce cholesterol and fatty acids, and Apoe that facilitates the transfer of cholesterol and phospholipids between cells 34,57 and is known to be elevated after injury in neonates. 58Accordingly, our data reveal several components of the key regulatory pathways that control lipid metabolism.In resident microglia, there seems to be neither recycling nor elimination of cholesterol (Fdft1, Lipa, Cyp46a1) one day after HI but rather an increase in cholesterol catabolism (Ch25h, oxysterol synthesis) and efflux (Abca1, Abcg1, Apoe and ApoC1) primarily at three days post-injury, whereas infiltrating macrophages seem to have ex novo cholesterol synthesis (Hmgcr, Srebf2) and the consequent efflux regulation (Nr1h3, Abca1, Abcg1 and Apoe).These infiltrating cells also show increased expression of Soat1-2, a significant modifier (cholesterol esterification) of macrophage cholesterol metabolism, 59 and Npc1-2, involved in cholesterol transport and recently reported to influence phagocytic activity. 60In accordance, we see increased gene expression of vimentin (Vim) and the neonatal glycoprotein Tnfaip6.Extracellular vimentin was recently suggested to have a role not only in migration but also in the phagocytosis of activated macrophages in response to TNF-a. 61ost of the cholesterol-metabolism-regulating genes we observed are downstream targets of the liver X receptors (LXRs, e.g., Nr1h3, an LXRa, found one day post-HI in macrophages).LXRs are activated by oxysterol catabolites in the presence of oxygen, and their expression is reduced during hypoxia and hypoperfusion. 62,63However, upon reperfusion oxysterol production is supposedly restored (as we observe by the upregulation of Ch25h in microglia) and the following activation of LXRs results in increased expression of ABC transporters (e.g., Abca1 Figure 5. Inflammasome gene regulation depends on post-HI timing, sex, and cell type (A) Expression pattern of significantly expressed (p.adj<0.05,DESeq2) pro-inflammatory and anti-inflammatory subtype markers in microglia (GFP) and infiltrating macrophage (RFP) one day post-HI (hypoxia-ischemia).Heatmap displays Z-transformed RPKM-normalized RNA expression.(B) Average gene expression analysis of top 50 differentially expressed genes (DESeq2 test) comparing resident microglia (GFP), infiltrating macrophages (RFP) groups, and controls (CTRL).(C) Circos plot showing differential regulation status (p.adj<0.05,DESeq2) of genes belonging to sensomes, PRR (pattern recognition receptor), metalloproteases, cytokines, and chemokines families in each of the six pairwise comparative analysis.Ribbons represent association of genes with corresponding gene families.Red color tile denotes upregulation, and blue color tile denotes downregulation of the first term in relation to the second term of the paired comparison.(D and E) Expression pattern and gender-wise regulation status of significantly expressed (p.adj<0.05,DESeq2) genes belonging to chemokines, cytokine, metalloproteases, and sensome families in microglia (D) and infiltrating macrophages (E) three days post-HI.Legend: day1 = D1, day3 = D3, control = CTRL, resident microglia = GFP, infiltrating macrophages = RFP.See also Figures S5 and S6. and Abcg1).Macrophages can modulate the inflammatory response by activating LXRs that are able to trans-repress nuclear factor kB (NF-kB) signaling. 64Whether microglia have the same or similar ability in the neonatal brain remains to be elucidated; 25-hydroxycholesterol (25-HC), an LXR activator and the catabolite secreted by activated microglia, might modulate lipid metabolism and secretion of ApoE 57   both paracrine and autocrine effects after HI.Pharmacological and genetic modulation of cholesterol esterification enzymes 65,66 might be promising targets to evaluate in neonatal HI.Concomitant microglial upregulation of Atf3 (direct regulator of Ch25h) might inhibit/dampen the pro-inflammatory (e.g., DAMP/PAMP/TLR) activation as demonstrated in several other disease models e.g., through histone acetylation 67 or by resolving pro-inflammatory response in activated metabolic rewired macrophages, 68 but detrimental if lost. 69Therefore, Atf3 might serve as a good biomarker and a good candidate for further investigation e.g., as an indirect small molecule target.One of the debris-clearing outcomes is lipid-laden inflammatory microglia and macrophages with cholesterol as the major component. 70,71Their lipid droplets are generated via the uptake of lipids by scavenger receptors including CD36, 72 highly expressed after neonatal brain injury 73 and in accordance with our finding after HI.CD36 is a fatty acid translocase and known to partner with TLR2 during inflammation. 72Our observation of increased gene regulation of vimentin accompanied by lipid and inflammatory gene regulation can also be interpreted as a response to counteract CD36 expression and lipid uptake.The intermediate filament version of vimentin not only acts as a key regulator of inflammation in activated macrophages, by the activation of the inflammasome partnering with the MIF-NLRP3 axis, 74 but also seems to be involved in CD36 regulation affecting inflammation-mediated lipid uptake. 75nother fatty acid transporter gene Mfsd2a, which we see downregulated in microglia probably as a consequence of hypoxia, and previously demonstrated only in endothelial cells of the BBB, 76  responses caused by HI.As a putative mechanism to compensate, we see both microglia and macrophages upregulating genes corresponding to the DHA binding proteins, FABPs, responsible for shuttling the esterified DHA from the plasma membrane (where it is stored) to the organelles for the conversion into pro-resolving lipid mediators to counteract the HI-induced inflammatory response.
Interestingly, DHA is a natural direct ligand of Nr4A2 77 that is known to modulate inflammatory and metabolic processes. 31,330][81] In fact, our data display an increase of Cd36, Nr1h3, Nr4a1/2, and Pparg primarily in macrophages, suggesting an interplay between these receptors in modulating the cell phenotype (e.g., skewing toward anti-inflammatory macrophages) and the following inflammatory cascade (e.g., regulating the recruitment of macrophages by controlling the production of adhesion molecules or the secretion of chemokines). 82,83hile fatty acid synthesis is characteristic for pro-inflammatory cells to promote the formation of pro-inflammatory cytokines, the anti-inflammatory macrophages are characterized by fatty acid oxidation and oxidative phosphorylation. 35Our results highlighted the upregulation of metabolic pathways linked to the TCA cycle and oxidative phosphorylation, explicitly in microglia.5][86] In contrast, the anti-inflammatory populations were shown to maintain normal TCA cycle activity and oxidative phosphorylation. 84,87Interestingly, we detected inferred Atf4 activity in microglia.9][90] This indicates another important checkpoint to take in consideration for modulation in neonatal HI.
In contrast to previous studies that reported that endogenous signals after brain ischemia induced a pro-inflammatory phenotype, 8,91 we did not observe that neither pro-nor anti-inflammatory signatures prevailed at any of the time points.We found that the majority of inflammatory markers were upregulated in both cell types at the one-day time point rather than three days post-injury, indicating a quick response.Specifically, microglia exhibited predominant upregulation of chemokine ligands attracting infiltrating macrophages, which in turn overexpressed chemokines receptors, PRRs, and signatures characteristic for sensome.Furthermore, upregulation of the majority of inflammatory transcripts was observed in CCR2 RFP+ cells, suggesting a leading role of infiltrating macrophages in the post-ischemic inflammation.
Our findings did not indicate any general sex-dependent pattern in pro-or anti-inflammatory signatures; however, we did identify specific genes differently expressed between sexes three days after injury for both CX3CR1 GFP+ and CCR2 RFP+ populations as previously reported. 92, 24 Among these markers, upregulation of some bona fide microglia homeostasis genes (Cx3cr1 and Siglech) was observed in males.Further investigations of homeostatic hallmarks displayed similarities between controls and microglia three days after HI in males.As microglia shift toward an inflammatory phenotype after brain injury, homeostatic genes are downregulated. 36Nevertheless, this rapid return to homeostasis in males raises the question about different microglia maturation states among sexes 40 and the consequent contribution of other cells in the inflammatory cascade.
In the past decade, several studies have described microglial sexual dimorphism concerning cell numbers, morphology, and gene expression in naive animals. 22,23,93However, we identified no difference in CX3CR1 GFP+ cell densities between sexes up to seven days after injury.Thus, the recruitment of peripheral immune cells and their contribution in the inflammatory cascade might be the direct consequence of microglia maturation state in the developing brain rather than its number.Previous reports have demonstrated that microglia reach the mature phenotype by the end of the second postnatal week 37,94 ; thus injuries in the immature brain might affect these cells and their gene expression signature.This outcome might imply either an increased signaling from other brain and peripheral cells to complement the lack of microglia activation or a limited response due to a shorter window of resident cell activation.
Taken together, our results provide a comprehensive map of cell-type-specific signature dynamics comprising components of lipid/ cholesterol metabolism, PRRs, DAMPs, TREMs, NF-kB signaling, inferred TFs, and nuclear receptors after HI.As such, this study paves the way for the identification of modulators of microglia and macrophage activation, expanding the venue for future metabolic reprogramming of macrophages/microglia in the frame of extracellular matrix and to identify potentially new targets for the treatment of HI.
In summary, the present study comprehensively maps response signatures of resident microglia and infiltrating macrophages in the acute/ subacute phase of neonatal hippocampal injury and reveals a sexual dimorphism in the immune response following HI.Specifically, we find distinct DNA motifs associated to transcription factors that modulate genes related to inflammation, sensome, matrisome, and lipid/cholesterol metabolism, and we highlight their response dynamics.These findings present a repository and valuable insights, offering a foundation for further exploration into the specific roles played by microglia and infiltrating macrophages in the inflammatory cascade.This knowledge contributes to identifying the critical time frame for interventions, thereby optimizing therapeutic strategies for neonates affected by asphyxia.

Limitations of the study
We have not extended the study to evaluate microglia and macrophages in chronic settings after HI due to the limited number of infiltrating cells after seven days.This could be followed-up by single-cell RNA-sequencing.The present study aimed to identify the differences between the two cell types during the acute phase of injury in view of future interventions.However, chronic analysis could be beneficial to detect the signatures of microglia-like macrophages that infiltrated the brain after insult and to evaluate possible variations between males and females.Additional sex differences might be detected with increased number of animals.Although mouse and human neonatal brain have many serum (Jackson Immuno Research) and 0.1% Triton X-100 (Sigma) in TBS 1X, sections were incubated with primary antibodies at 4ºC for 24 -72 h, depending on the antibody.The following primary antibodies were used: rabbit anti Tmem119 1:500 (Abcam); rabbit anti ATF3 1:250 (HPA001562, Atlas antibodies); goat anti APOE 1:1000 (Sigma-Aldrich); mouse anti RFP 1:500 (Rockland); goat anti GFP 1:2000 (Abcam); rabbit anti GFP 1:2000 (Clontech).Sections were then incubated for 2h at RT with appropriate fluorescent secondary antibodies.The following secondary antibodies were used: AlexaFlour-488 donkey anti goat IgG (Molecular probes/Life Technologies; 1:1000); AlexaFlour-488 donkey anti rabbit IgG (Molecular probes/Life Technologies; 1:1000); AlexaFlour-555 donkey anti mouse IgG (Biotium, 1:1000); AlexaFlour-633 donkey anti rabbit IgG (Biotium, 1:1000); CF-633 donkey anti goat IgG (Biotium, 1:1000).Hoechst 33342 (Molecular Probes/Life Technologies) was used as a nuclear counterstain.Sections were mounted onto slides and were coverslipped using ProLong Gold anti-fade reagent (Molecular probes/Life Technologies).

Microscopy and cell quantifications
The analysis of resident microglia and blood-derived macrophages was performed by quantifying CX3CR1 GFP+ and CCR2 RFP+ cells in the hippocampus.For quantification of immune-positive cells, consecutive 3mm z-stack images of the whole thickness of each section were acquired using a Zeiss LSM700 confocal microscope.A blinded offline analysis was performed using Zen Black Software version 3.8 (Carl Zeiss).andall immuno-positive cells were counted in 3-4 sections per animal spaced 400 mm apart.The cell density calculated on regional volumes according to the Cavalieri principle, with the formula: where V is the total volume, SA is the sum of area measurements, P is the inverse of the sampling fraction of the region (in this case 1/10, i.e., P=10) and T is the section thickness.
Tmem119, ApoE and Aft3 image representations were acquired in sequential scans performed at 1 mm section intervals using a 403 objective lens and 1 airy pinhole setting.

Sample preparation for FACS
Animals were sacrificed 1 day (1d; males: n=3 control and n=3 HI; females: n=3 control and n=3 HI) and 3 days (3d; males: n=4 control and n=3 HI; females: n=4 control and n=5 HI) after HI for FACS analysis.Animals were deeply anesthetized with isoflurane and perfused with cold phosphate-buffered saline (pH 7.4) (Life technologies) and brains were removed.The hippocampi from the ipsilateral and contralateral hemispheres were dissected, separately homogenized, and incubated for 20 min at 37 C in a mixture of papain (5.0 U/mL Worthington Biochemical Corporation, to a final concentration of 100 U/l) and DNase (5.0 U/mL, Sigma-Aldrich, to a final concentration of 10 U/l) in 15 mM HEPES buffer consisting of HBSS, 2mM EDTA, and 0.5% bovine serum albumin.Cells were pipetted up and down 20 times and filtered through a cell strainer (40 mm mesh, FALCON) followed by addition of 10% FBS in PBS with 2mM EDTA to a final volume of 10ml and the strainer covered with parafilm was centrifuged in 50mL tubes at 600 g at 4 C for 10 min.After the supernatant was removed, microglia and macrophages were purified with anti-CD11b magnetic beads and columns (Militenyi Biotec).The purified resident microglia and infiltrating macrophages were incubated with brilliant violet 421-conjugated anti-CD11b antibody (1:1000; BioLegend) on ice for 10min.Samples were washed twice with 0.5% FBS in PBS with 2mM EDTA to remove any traces of remaining antibody followed by centrifugation at 600 g at 4 C for 5min and 10 min.Prior to flow cytometry, cells were recovered in 500 ml of 0.5% FBS in PBS with 2mM EDTA.

Sample processing and RNA sequencing
CX3CR1 GFP+ /CD11b + and CCR2 RFP+ /CD11b + cells obtained by FACS using MoFloä XPD (Beckman Coulter), were further processed for total RNA (RNeasy Micro Kit, Qiagen) followed by pre-RNA Sequencing procedures.Libraries were prepared using SMARTer Ultra Low Input RNA for Illumina and sequenced on HiSeq 2000. 99Reads were aligned using STAR 2.4.2a with default settings 100 against Mus musculus reference genome (assembly mm10, without unplaced contigs).Non-uniquely aligned reads were discarded from the alignment.Reads per kilobase of gene per million mapped reads (RPKM) and read counts were generated using the python script rpkmforgenes (available at https://github.com/danielramskold/S3_species-specific_sequencing) 101 with settings -readcount -fulltranscript -mRNAnorm -rmnameoverlap -bothendceil and RefSeq gene annotation.Gene level read count data was used to find significantly expressed genes between any pair of groups.Differential gene expression analysis was performed using R/Bioconductor package DESeq2 v1.26.0. 102Gender information of the samples was added in the differential expression model as covariate to cancel out any possible variance.Genes with adjusted p-value <0.05 were considered significantly expressed.Heatmaps visualizing gene expression pattern were generated using R package ComplexHeatmap v2.2.0. 103Volcano were made using R package ggplot2 v3.3.2. 104Uniform Manifold Approximation and Projection (UMAP) clustering of the samples was performed using R package umap v0.2.6.0.

QUANTIFICATION AND STATISTICAL ANALYSIS
All statistical tests except for the test on RPKM values were carried out using GraphPad Prism6 (GraphPad Inc), and were presented as mean G SEM. Statistical tests on RPKM values were described above.Two-way ANOVA followed by Sida ´k-corrected post-hoc t-test was used to analyze the neuropathological score, cell density and body weight.One-way ANOVA followed by Sida ´k-corrected post-hoc t-test was

Figure 2 .
Figure 2. Temporal molecular differences of infiltrating macrophages and resident microglia in the hippocampus (A) Sample distribution based on UMAP dimensionality reduction.Samples in the same group were manually circled.(B) Venn diagram showing unique and commonly regulated (p.adj <0.05, DESeq2) genes between the three pairwise analyses.(C) Bar graph showing number of statistically significant differentially regulated genes (p.adj <0.05) in each pairwise analysis.(D) MA plot visualizes differential gene regulation results of microglia (GFP) day1 vs. day3 post-HI (hypoxia-ischemia).Red and blue dots denote more (p.adj<0.05, DESeq2) and less (p.adj <0.05, DESeq2) expressed genes, respectively, in microglia day1 compared with day3 post-HI.(E) Temporal differences applying the ISMARA algorithm identifying transcription factor binding motifs in microglia.(F) MA plot visualizes differential gene regulation results of macrophages (RFP) day1 vs. day3 post-HI.Red and blue dots denote upregulated (p.adj <0.05, DESeq2) and downregulated (p.adj <0.05, DESeq2) genes, respectively, in macrophages day1 compared with day3.(G) Temporal differences applying the ISMARA algorithm identifying transcription factor binding motifs in microglia.(H) Venn diagram showing regulated (p.adj <0.05, Mann-Whitney U-test with Benjamini-Hochman correction) motifs between cell type and time point.(I) Cell-type-specific differences applying the ISMARA algorithm identifying transcription factor binding motifs 1 day after HI. (J) Cell-type-specific differences applying the ISMARA algorithm identifying transcription factor binding motifs 3 days after HI.Legend: day1 = D1, day3 = D3, control = Ctrl, resident microglia = GFP, infiltrating macrophages = RFP.

Figure 3 .
Figure 3. Matrisome gene dynamics in microglia and infiltrating macrophages (A) Matrisome classes.(B) Bubble plots visualizing expression level of genes associated with corresponding matrisome groups (related to Figure 2).RPKM-normalized expression values among the samples were Z score scaled, and average Z scores per groups are visualized.Bubble size is proportional to the absolute value of Z score.Legend: day1 = D1, day3 = D3, resident microglia = GFP, infiltrating macrophages = RFP.See also Figure S2.

Figure 4 .
Figure 4. Inflammation and lipid regulation (A) Bubble plot showing the gene set enrichment analysis results of infiltrating macrophages (GFP) vs. resident microglia (RFP) day1 post-HI (hypoxia-ischemia), where red bubbles denote upregulated pathways and blue bubbles denote downregulated pathways in macrophages, and the bubble size is proportional to negative log10 scaled adjusted p values of enrichment (p.adj <0.05, piano GSA).(B) Bar graph representing differentially expressed number of genes in six comparisons.(C) Heatmap showing differential gene regulation status.Log2 scaled fold change values of genes in different pairwise comparisons are visualized, and row annotation represents the differential comparisons.White cells denote non-significant regulation (p.adj >0.05, DESeq2).(D-F)Expression of differentially expressed genes involved in fatty acid and cholesterol processes, with color legend at the bottom of (D) and functional classification at the bottom of (E).The genes are divided by expression pattern into mainly genes expressed in microglia (D), genes also expressed in macrophages (E) or genes expressed mainly in infiltrating macrophages (F).Data are analyzed with one-way ANOVA with Sida ´k-corrected post-hoc t test and presented as mean G SEM and *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.See also FiguresS3 and S4.

Figure 7 .
Figure 7. Schematics representing activated microglia and infiltrating macrophages converging over time Upon hypoxia-ischemia (HI), peripheral macrophages are recruited to the injured site by resident cells.Cell-type-specific TF binding motifs and corresponding TFs (transcription factors) regulate inflammatory, ECM (extracellular matrix), and lipid/cholesterol genes during the injury cascade.In microglia: Myc/Max/Mycn (E-box*) and chromatin remodeler Zbtb33/Chd2 (Kaiso*) motifs are found near differentially expressed genes Aen and Npm1 (apoptosis and DNA damage response), respectively, Hsf1 modulates the NF-kB pathway, and its motif is found near Ccl4 (inflammation and macrophage chemotaxis).In infiltrating macrophages: Fos/Fosl2/Bach2/Jun and Rela/Rel/Nfkb1 motifs are found near differentially expressed genes vimentin, Il1a/b, and Tlr2 (inflammation and phagocytosis), respectively; Runx2/Bcl11a (Runt*), Stat4/3/5b, and Cepb (bZIP*) motifs near Serglycin (Srgn) and Fibronectin1 (Fn1, ECM remodeling); and Nfatc2 motif near CD36 (regulation of cholesterol and fatty acid transport).Both cell types have upregulation of specific pathways and genes involved in vessels remodeling, leukocyte recruitment, and lipid metabolism one day after injury, although by the 3rd day after HI we observe a convergence in transcript expression.* DNA-binding motif in promoter assessed by ISMARA algorithm.Created with BioRender.com.
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