Zika Virus Infection Induces Interleukin-1β-Mediated Inflammatory Responses by Macrophages in the Brain of an Adult Mouse Model

ABSTRACT During the 2015–2016 Zika virus (ZIKV) epidemic, ZIKV-associated neurological diseases were reported in adults, including microcephaly, Guillain-Barre syndrome, myelitis, meningoencephalitis, and fatal encephalitis. However, the mechanisms underlying the neuropathogenesis of ZIKV infection are not yet fully understood. In this study, we used an adult ZIKV infection mouse model (Ifnar1−/−) to investigate the mechanisms underlying neuroinflammation and neuropathogenesis. ZIKV infection induced the expression of proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, gamma interferon, and tumor necrosis factor alpha, in the brains of Ifnar1−/− mice. RNA-seq analysis of the infected mouse brain also revealed that genes involved in innate immune responses and cytokine-mediated signaling pathways were significantly upregulated at 6 days postinfection. Furthermore, ZIKV infection induced macrophage infiltration and activation and augmented IL-1β expression, whereas microgliosis was not observed in the brain. Using human monocyte THP-1 cells, we confirmed that ZIKV infection promotes inflammatory cell death and increases IL-1β secretion. In addition, expression of the complement component C3, which is associated with neurodegenerative diseases and known to be upregulated by proinflammatory cytokines, was induced by ZIKV infection through the IL-1β-mediated pathway. An increase in C5a produced by complement activation in the brains of ZIKV-infected mice was also verified. Taken together, our results suggest that ZIKV infection in the brain of this animal model augments IL-1β expression in infiltrating macrophages and elicits IL-1β-mediated inflammation, which can lead to the destructive consequences of neuroinflammation. IMPORTANCE Zika virus (ZIKV) associated neurological impairments are an important global health problem. Our results suggest that ZIKV infection in the mouse brain can induce IL-1β-mediated inflammation and complement activation, thereby contributing to the development of neurological disorders. Thus, our findings reveal a mechanism by which ZIKV induces neuroinflammation in the mouse brain. Although we used adult type I interferon receptor IFNAR knockout (Ifnar1−/−) mice owing to the limited mouse models of ZIKV pathogenesis, our conclusions contributed to the understanding ZIKV-associated neurological diseases to develop treatment strategies for patients with ZIKV infection based on these findings.

ZIKV belongs to the Flaviviridae family of RNA viruses and is a neurotropic flavivirus, along with Japanese encephalitis and West Nile viruses (9). The detection of ZIKV RNA in the brains and cerebrospinal fluid of adult patients with ZIKV-induced neurological disorders also suggests a neuroinvasive characteristic of ZIKV (6,(10)(11)(12)(13). ZIKV can infect human neuronal progenitor cells (NPCs), leading to cell death, abnormal growth, and brain atrophy (14)(15)(16). ZIKV infection of NPCs in the adult mouse brain results in cell death and reduced proliferation (17). In addition to NPCs, human placental macrophages (18) and glial cells (19) are also susceptible to ZIKV infection. Lum et al. demonstrated that ZIKV infects human fetal brain microglia and macrophages, which induces high levels of proinflammatory cytokines; these are implicated in ZIKV-derived neuroinflammation (20). Increasing evidence indicates that neuroinflammation is a major contributor to the pathogenesis of neurological diseases (21). Thus, an investigation to address the mechanisms underlying the neuropathogenesis of ZIKV infection would help to understand ZIKV-associated neurological diseases.
Proinflammatory cytokines are central mediators of the inflammatory response. Interleukin-1b (IL-1b) is one of the most extensively studied cytokines involved in neuroinflammation and neurodegenerative diseases (22). In the brains of patients with human immunodeficiency virus 1 encephalitis, IL-1b expression is increased in infiltrating macrophages, microglia, and astrocytes (23,24). Complement component 3 (C3) is also induced in neurodegenerative diseases and is upregulated by proinflammatory cytokines such as IL-1b, gamma interferon (IFN-g ), and tumor necrosis factor alpha (TNF-a) (25)(26)(27)(28). Simian immunodeficiency virus infection of the central nervous system (CNS) in rhesus macaques induces C3 expression in infiltrating macrophages, astrocytes, and neurons (29). Although both IL-1b and C3 can play neuroprotective roles in immune responses, uncontrolled biosynthesis and activation can cause critical brain tissue damage (25,30). Hence, whether IL-1b and C3 expression in the brain is affected by ZIKV infection needs to be investigated to explore ZIKV-associated neurological diseases.
Immune-competent adult mice are resistant to ZIKV infection, in part because ZIKV fails to effectively antagonize Stat2-dependent IFN responses in mice despite ZIKV NS5 protein binding and degrading STAT2 for the immune evasion. (31)(32)(33)(34)(35). Thus, we used adult type I IFN receptor IFNAR knockout (Ifnar1 2/2 ) mice as a ZIKV infection mouse model to examine proinflammatory responses in the CNS and the neuropathogenesis of ZIKV infection. Transcriptome sequencing (RNA-seq) analysis was performed to examine neuroinflammation in response to ZIKV infection in the brains of Ifnar1 2/2 mice. We focused on the immune cells in the CNS that are susceptible to ZIKV infection and consequently contribute to neuroinflammation in this animal model.

RESULTS
ZIKV infection in the brain induces proinflammatory responses in Ifnar1 2/2 mice. When Ifnar1 2/2 C57BL/6 mice were infected with 10 3 PFU of the PRVABC59 strain of ZIKV via a subcutaneous route, most infected mice became paralyzed on at least one hind limb at 9 days postinfection (dpi) (Fig. 1A). To our knowledge, there are two mechanisms underlying ZIKV-associated paralysis in Ifnar1 2/2 mice. First, ZIKV infection of astrocytes breaks down the blood-brain barrier of Ifnar1 2/2 mice, leading to a large influx of CD8 1 T cells that promotes paralysis (36). Second, ZIKV infection in the spinal cord of Ifnar1 2/2 mice results in motor neuron synaptic retraction and inflammation (37). To address whether infection in the brain or spinal cord accounts for paralysis in ZIKV-infected mice, we assessed the viral load at 6 dpi in the brain and spinal cord, along with that in the eyes, kidneys, testes, and ovaries, which have been reported as ZIKV-susceptible organs (Fig. 1B). High levels of viral RNA were detected in both the brain and spinal cord, but the levels in the spinal cord were higher than those in the brain. Next, we analyzed the mRNA levels of proinflammatory cytokines, including IL-1b, IL-6, IFN-g , and TNF-a, in the brain and spinal cord using reverse transcriptionquantitative PCR (RT-qPCR) at 6 dpi (Fig. 1C). Notably, the mRNA expression of proinflammatory cytokines in the brains of ZIKV-infected mice was much higher than that in the spinal cord. Upregulated proinflammatory cytokine expression in the ZIKV-infected mouse brain was confirmed using enzyme-linked immunosorbent assay (ELISA) (Fig.  1D). These results indicated that ZIKV infection of the brain induces proinflammatory responses that contribute to neuroinflammation. FIG 1 ZIKV infection of the brain elicits proinflammatory responses in Ifnar1 2/2 mice. (A) Ifnar1 2/2 mice were subcutaneously infected with 10 3 PFU of ZIKV or PBS (mock). They were monitored daily for a neurological disease score as described in Materials and Methods. (B) Ifnar1 2/2 mice (n = 5 for males and n = 3 for females) were infected with 10 3 PFU of ZIKV. Viral RNA levels in the brain, spinal cord, eyes, kidney, testes, and ovary were assessed using RT-qPCR at 6 dpi. (C) RNA extracted from the brain and spinal cord homogenates was used to assess mRNA levels of proinflammatory cytokines, including IL-1b, IL-6, IFN-g , and TNF-a, by RT-qPCR at 6 dpi. (D) The brain homogenates were used to measure protein levels of proinflammatory cytokines by ELISA at 6 dpi. Statistically significant differences between the groups were determined using multiple two-tailed t tests (A), one-way analysis of variance (ANOVA; B), and Student t test (C and D). *, P , 0.05; **, P , 0.01; ***, P , 0.001; ****, P , 0.0001. Bars indicate means 6 the standard errors of the mean (SEM).
Distinct transcriptional signatures and gene expression changes in the brain of ZIKV-infected Ifnar1 2/2 mice. To assess the effects of ZIKV infection on gene expression in the mouse brain, we performed RNA-seq on brain homogenates of ZIKVinfected Ifnar1 2/2 mice at 0, 3, and 6 dpi. Genes with an adjusted P value of ,0.05 were considered as differentially expressed genes (DEGs). We identified 930 DEGs at 6 dpi (upregulated, 546; downregulated, 384) that were differentially expressed compared to those at 0 dpi, whereas there were 56 DEGs at 3 dpi (upregulated, 19; downregulated, 37). The volcano plot for DEGs at 6 dpi versus 0 dpi showed that several highly significant DEGs associated with IFN signaling were upregulated ( Fig. 2A). According to the enrichment analysis of the biological category of gene ontology (GO), DEGs at 6 dpi were highly enriched in the cytokine-mediated signaling pathway, inflammatory responses, and neutrophil-mediated immunity (Fig. 2B). The upregulation of DEGs involved in the immune response was most conspicuous at 6 dpi (Fig. 2C). Particularly, genes of proinflammatory cytokines (Il1b, Il6, and Tnf) and complement components (C1qa, C3, and C4b) in the inflammatory response were upregulated at 6 dpi (Fig. 2D). In addition, genes in the IFN signaling pathway (Oas1a, Oas2, Stat1, Stat2, Irf1, and Irf7) were upregulated at 6 dpi ( Fig. 2E). Although interferon-stimulated genes (ISGs) were not expected to be differentially expressed in response to the viral infection, ISG expression can be induced by an IFNAR-independent pathway, such as the downstream of MAVS signaling in myeloid dendritic cells (38). Collectively, RNA-seq data from the brains of ZIKV-infected mice showed distinct immune and inflammatory signatures at 6 dpi.
ZIKV infection results in infiltration and proinflammatory activation of macrophages, but not microglia in the Ifnar1 2/2 mouse brain. To identify immune cells in the brain that are responsible for the proinflammatory responses to ZIKV infection, we isolated the mouse brain after ZIKV infection. The brain homogenates were used to isolate immune cells in the brain, including microglia and macrophages, by 30 and 70% Percoll gradient centrifugation, followed by flow cytometry analysis of cell surface markers such as CD11b and CD45, as illustrated in Fig. 3A. The isolate consisted of two populations, namely, macrophages (CD11b 1 and CD45 High ) and microglia (CD11b 1 and CD45 Low ). While the number of microglia in the brain did not change, that of macrophages increased ;4-fold in response to ZIKV infection, possibly indicating macrophage brain infiltration ( Fig. 3C and D). This pattern of alteration by ZIKV infection was different from that obtained by SARS-CoV-2 infection in our previous study, wherein microglia were significantly depopulated (39). Next, we analyzed the IL-1b, IL-6, and TNF-a responses in each of the two populations ( Fig. 4A). Interestingly, only the numbers of IL-1b-positive macrophages, but not those of microglia, dramatically increased ( Fig. 4B and C). These results suggest a role for macrophages in IL-1b-mediated inflammation in the brains of ZIKV-infected mice.
We then investigated whether IL-1b expression in macrophages was mediated by direct ZIKV infection. To address this, we used an anti-flavivirus envelope protein antibody (4G2) to stain ZIKV-infected (10 4 PFU) cells along with IL-1b in the two isolated populations (Fig. 5A). As previous studies have demonstrated that microglia and macrophages are susceptible to ZIKV infection (19,(40)(41)(42), we observed that they were infected with ZIKV ( Fig. 5B and C). While ZIKV infection of microglia in Ifnar1 2/2 mice did not lead to microglial activation ( Fig. 4B and 5D), infection of macrophages resulted in the induction of IL-1b ( Fig. 4C and 5E). Notably, when we measured IL-1b 1 populations of ZIKV 1 versus ZIKVfor both microglia and macrophages, IL-1b 1 population of ZIKV 1 macrophages but not ZIKV 1 microglia, was significantly increased by ZIKV infection, indicating that ZIKV-infected macrophages are the source of IL-1b in the brain ( Fig. 5F and G). These findings demonstrate that IL-1b induction in the brains of ZIKV-infected Ifnar1 2/2 mice was likely due to ZIKV-infected macrophages, but not microglia.
ZIKV infection of THP-1 cells induces IL-1b secretion and inflammatory cell death. Next, we used human monocyte THP-1 cells to confirm our in vivo observations. Given the key role of NLRP3 inflammasome in innate immune responses by activating caspase-1 to promote IL-1b secretion and pyroptosis (43), we examined whether ZIKV infection stimulates IL-1b secretion through NLRP3 inflammasome activation in THP-1 cells. When THP-1 cells were infected with ZIKV at a multiplicity of infection (MOI) of 0.1, the viral RNA and envelope protein levels were increased in a time-dependent manner, as shown by RT-qPCR and Western blotting, respectively ( Fig. 6A and B). ELISA revealed that, compared to the mock infection, ZIKV infection significantly augmented IL-1b secretion over time (Fig. 6C). In addition to the increase in IL-1b secretion, cell death was promoted by ZIKV infection in THP-1 cells, possibly due to pyroptosis (Fig. 6D). Consequently, we determined whether caspase-1 and GSDMD cleavage resulting from activation of the NLRP3 inflammasome were induced by ZIKV infection. Caspase-1 and Gasdermin D (GSDMD) cleavage was observed from 1 dpi, followed by IL-1b maturation and secretion (Fig. 6E). Thus, ZIKV infection of THP-1 cells activates the NLRP3 inflammasome and consequently induces IL-1b maturation and secretion.
The increase in C3 levels by ZIKV infection is mediated by IL-1b signaling. Previous studies have revealed that IL-1b and other proinflammatory cytokines induce the transcription factor CCAAT/enhancer binding protein b (C/EBP-b); this transcription factor directly activates the promoter of C3, which plays a crucial role in the activation of the complement system and contributes to innate immune responses (28,44,45). To validate the role of C/EBP-b in IL-1b-mediated induction of C3 expression by ZIKV infection in THP-1 cells, we infected cells with ZIKV at 5 MOI and analyzed the activation of P38, Erk1/ 2, and C/EBP-b using Western blotting. Indeed, ZIKV infection induced C/EBP-b expression and its activation through phosphorylation, which was conducted by activated p38 but not Erk1/2, resulted in the elevation of C3 expression (Fig. 7A). C3 induction was suppressed by C/EBP-b knockout in ZIKV-infected THP-1 cells (Fig. 7B). Diacerein, an inhibitor of IL-1b production (46), and an IL-1R antagonist effectively reduced the ZIKV-mediated C3 induction in a dose-dependent manner, thereby suggesting the involvement of IL-1b in the induction of C3 gene expression by ZIKV infection (Fig. 7C and D). The reduction of C3 secretion by Diacerein and the IL-1R antagonist was confirmed by ELISA ( Fig. 7E and F). Therefore, we demonstrated that ZIKV infection in THP-1 cells promotes C/EBP-b expression through IL-1b induction, eventually resulting in increased C3 levels. In the brains of Ifnar1 2/2 mice, the induction and activation of C/EBP-b by ZIKV infection were determined at 6 dpi using RT-qPCR and Western blot analysis ( Fig. 8A and B). The increased expression and activation of C/EBP-b led to the induction of C3 mRNA and protein expression as determined by RT-qPCR and ELISA, respectively ( Fig. 8C and D). We also detected C3 induction in mouse sera after ZIKV infection, which indicated systemic C3 induction (Fig. 8E). To evaluate the functional consequences of C3 induction and complement activation by ZIKV infection, we determined the C5a levels in the brain. As expected, the C5a levels in the brains of ZIKV-infected mice significantly increased at 6 dpi (Fig. 8F). Taken together, our findings indicate that the induction of C3 and complement activation by ZIKV infection is mediated by the IL-1b signaling pathway.

DISCUSSION
To date, the neuropathogenesis of ZIKV infection remains unclear. Understanding the neuropathogenesis of ZIKV infection would help to understand ZIKV-associated neurological diseases. ZIKV infects human fetal brain microglia and macrophages (20). This induces high levels of proinflammatory cytokines which are implicated in ZIKVderived neuroinflammation, including IL-1b that is mediated by the NLRP3 inflammasome (47,48). C3 is induced in most neurodegenerative diseases and is upregulated by proinflammatory cytokines such as IL-1b (25)(26)(27)(28). Furthermore, IL-1b and C3 expression is increased in different viral infections of the CNS (29,49). However, whether IL-1b and C3 expression in the brain is affected by ZIKV infection remains to be investigated. Here, we used adult Ifnar1 2/2 mice as a ZIKV infection mouse model to examine proinflammatory responses in the CNS upon ZIKV infection. We demonstrated that ZIKV infection of the brain in this animal model augments IL-1b expression in infiltrating macrophages and elicits IL-1b-mediated inflammation, thereby leading to the destructive consequences of neuroinflammation.
Neurons are targets in the CNS for ZIKV, JEV, and WNV infection (50)(51)(52). They are likely the first responders to immune responses against neurotropic viral infections, producing type I IFNs and expressing MHC class I molecules (53,54). Upon initiation of an inflammatory response in the CNS, proinflammatory cytokines are critical for recruitment of monocytes to the CNS (49). When we infected the Ifnar1 2/2 mice with ZIKV, increased proinflammatory cytokine levels and macrophage infiltration were observed in the brain at 6 dpi ( Fig. 1C and D and Fig. 3B and D). These results imply that ZIKV infection can elicit neuronal inflammation and, consequently, monocyte infiltration in this animal model.
Microglia and macrophages have been found to be of the same origin (55) and have similar functions, such as production of inflammatory mediators (56). However, they play different roles in the brain. Microglia may protect the injured brain, whereas macrophages concurrently damage the brain (57). In this study, although both microglia and macrophages were infected with ZIKV at 6 dpi ( Fig. 5B and E), macrophages showed proinflammatory activation with upregulated IL-1b expression, but microglia were not observed in the brains of infected Ifnar1 2/2 mice (Fig. 4B and C and Fig. 5E and F). Proinflammatory activated macrophages can accelerate the number of circulating immune cells and increase their infiltration into the brain, in addition to playing critical roles in pathophysiological processes. Further studies are warranted to determine why ZIKV-infected microglia were not activated in the brains of Ifnar1 2/2 mice.
In ZIKV-GBS, particularly the acute inflammatory demyelinating polyneuropathy variant, cytokine-mediated inflammation and macrophage activation may lead to peripheral nerve injury, and complement activation may be associated with demyelinating neuropathies (8,58). Given that IL-1 signaling is considered as the upper hierarchical cytokine signaling cascade in the CNS (30), ZIKV infection in the brain of Ifnar1 2/2 mice induces IL-1b expression in infiltrating macrophages (Fig. 3D, Fig. 4C, and Fig. 5F) and elicits IL-1b-mediated inflammation and macrophage activation, which can be associated with the destructive consequences of neuroinflammation, such as peripheral neuropathy. Our findings also suggest that ZIKV-induced IL-1b secretion may lead to the expansion of encephalitogenic T cells (59) and pyroptosis in CNS peripheral myeloid and lymphoid cells; these cells can mediate neuroinflammation in multiple CNS diseases (60).
Complement is thought to have a protective effect, but exaggerated or insufficient activation of the complement system can cause neuropathies and contribute to neurodegeneration and neuroinflammation (25,61). ZIKV infection upregulated C3 expression through IL-1b-mediated signaling ( Fig. 7 and 8), possibly disrupting the balance of the complement system, which can mediate myelin phagocytosis by macrophages (61). The anaphylatoxins (C3a, C4a, and C5a) produced during complement activation play a major role in the pathogenesis of inflammatory disorders, including ischemia/ reperfusion injury, and are involved in various neurodegenerative disorders (62). An increase in C5a in the cerebrospinal fluid was also detected during the exacerbation of were infected with ZIKV at 5 MOI. C3 and C/EBP-b proteins in cell lysates were quantitatively analyzed by Western blotting. Actin served as the loading control. (C to F) THP-1 cells were infected with ZIKV at 5 MOI, followed by treatment with an IL-1R antagonist as indicated (C) or 100 ng/mL (E), or with Diacerein as indicated (D) or 50 mM (F). C3 levels in cell lysates were assessed by Western blot (C and D). Actin served as the loading control. Secreted C3 levels in supernatants were measured by ELISA (E and F). Statistically significant differences between the groups were determined using Student t test (E and F). ***, P , 0.001; ****, P , 0.0001. Bars indicate means 6 the SEM.
In summary, ZIKV infection of the brain induced the expression of proinflammatory cytokines, including IL-1b, IL-6, IFN-g , and TNF-a, in Ifnar1 2/2 mice. RNA-seq analysis revealed that the expression of genes involved in immune responses to viral infection and C/EBP-b (LAP) protein levels in lysates of the brain homogenates were assessed by Western blotting. Actin served as the loading control. (C) C3 mRNA levels in the brain homogenates were determined by RT-qPCR. (D) C3 protein levels in lysates of the brain homogenates were assessed by ELISA. (E) C3 protein levels in mouse sera were measured by ELISA. (F) C5a protein levels in lysates of the brain homogenates were quantitatively analyzed by ELISA. Statistically significant differences between the groups were determined using Student t test (A, C, D, and F) and one-way analysis of variance (ANOVA; E). ***, P , 0.001; ****, P , 0.0001. Bars indicate means 6 the SEM. and inflammatory responses in the brains of these mice was significantly upregulated by ZIKV infection. Notably, infiltration and proinflammatory activation of macrophages, but not microglia, were observed at 6 dpi in the ZIKV-infected Ifnar1 2/2 mice. ZIKV infection of THP-1 cells induced pyroptosis, increased IL-1b release, and consequently increased C3 expression. Increases in C3 and C5a levels in the brains of infected mice were verified. Overall, our data suggest that ZIKV infection in the brains of Ifnar1 2/2 mice augments IL-1b expression in infiltrating macrophages and elicits IL-1b-mediated inflammation through macrophage activation, which can be associated with the destructive consequences of neuroinflammation.
Mononuclear cell isolation. Mononuclear cells isolation in the mouse brain was performed following the protocols as previously described (39,68,69). Mock-or ZIKV-infected mice were anesthetized with isoflurane, followed by perfusion with 10 mL of cold 1Â DPBS (Gibco) into the left ventricle to remove blood from the tissues. The brains were transferred to a six-well plate containing cold Hanks' balanced salt solution (HBSS; Gibco), and the plates were kept on ice. The generation of brain cell suspensions by a 70-mm-poresize cell strainer (SPL; Gyeonggi-do, South Korea) was prepared in 10 mL per brain of digestion cocktail containing 0.5 mg/mL DNase I (Roche, Basel, Switzerland) and 1 mg/mL collagenase A (Roche) in HBSS. The suspension was incubated at room temperature for 30 min, followed by centrifugation for 7 min at 300 Â g and 18°C. The cell pellet was resuspended in 30% Percoll (Sigma-Aldrich) in HBSS and then slowly layered over 70% Percoll in HBSS in a 15 mL-conical tube. Approximately 2 mL of the interphase volume was collected into a new tube after gradient centrifugation for 40 min at 200 Â g at 18°C. Isolated mononuclear cells were washed three more times in a volume of 500 mL of HBSS containing 0.01 M HEPES (Gibco), using a microcentrifuge for 7 min at 600 Â g at 4°C.
RT-qPCR. Quantitative RT-PCR (QuantStudio 3; Applied Biosystems, Foster City, CA) was performed using one-step Prime script III RT-qPCR mix (TaKaRa Bio, Shiga, Japan). The viral RNA of ZIKV NS3 was detected by customized probe-based qPCR assay (Integrated DNA Technologies, Coralville, IA). The Il1b, Il6, Tnf, Ifng, Cebpb, and C3 genes were also detected using individual customized probes (Integrated DNA Technologies). The sequences of the qPCR probes and primers used in this study are listed in Table  S1 in the supplemental material.
RNA-seq and analysis. The sequencing library was prepared using the TruSeq Stranded mRNA Sample Prep kit and sequenced on NovaSeq 6000 (Illumina, San Diego, CA), yielding more than 6G bases of sequence for each sample. Adaptor sequences were removed from the sequenced reads using Cutadapt (version 3.1) (70) and aligned to the hybrid reference genomes of humans (GRCh38.p13_ENS100) and ZIKV with the STAR aligner (version 2.7.6a) (71). Aligned reads were quantified at the gene level by HTSeq (version 0.13.5) (72) with "intersection-nonempty" mode. Genes with lower than five counts for the total count per gene were removed for further analysis. Differentially expressed genes were analyzed with DESeq2 (version 1.30.1) (73) using absolute (log 2 -fold change) of .1 and an adjusted P value (Benjamini-Hochberg) of ,0.01 as the cutoff. Multidimensional scaling analysis was performed with the Clustermap function in the Python Seaborn package (version 0.11.1) using genes with a mean fragments per kilobase of transcript per million (FPKM) of .1 among the samples and transformed to log 2 (FPKM 1 1). Overrepresentation analysis of the DEGs enriched in GO Biological Process 2018 was performed with EnrichR (74) and an adjusted P value (Benjamini-Hochberg) of ,0.05.
Statistical analysis. All experiments were performed at least three times. All data were analyzed using Prism 8.0 software (GraphPad, San Diego, CA). A P value of ,0.05 was considered statistically significant. Specific analysis methods are described in the figure legends.

SUPPLEMENTAL MATERIAL
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