Upregulation of Neuroinflammation-Associated Genes in the Brain of SARS-CoV-2-Infected Mice

Neurological manifestations are a significant complication of coronavirus disease 2019 (COVID-19), but the underlying mechanisms are yet to be understood. Recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced neuroinvasion and encephalitis were observed in K18-hACE2 mice, leading to mortality. Our goal in this study was to gain insights into the molecular pathogenesis of neurological manifestations in this mouse model. To analyze differentially expressed genes (DEGs) in the brains of mice following SARS-CoV-2 infection, we performed NanoString gene expression analysis using three individual animal samples at 1, 3, and 6 days post-infection. We identified the DEGs by comparing them to animals that were not infected with the virus. We found that genes upregulated at day 6 post-infection were mainly associated with Toll-like receptor (TLR) signaling, RIG-I-like receptor (RLR) signaling, and cell death pathways. However, downregulated genes were associated with neurodegeneration and synaptic signaling pathways. In correlation with gene expression profiles, a multiplexed immunoassay showed the upregulation of multiple cytokines and chemokines involved in inflammation and cell death in SARS-CoV-2-infected brains. Furthermore, the pathway analysis of DEGs indicated a possible link between TLR2-mediated signaling pathways and neuroinflammation, as well as pyroptosis and necroptosis in the brain. In conclusion, our work demonstrates neuroinflammation-associated gene expression profiles, which can provide key insight into the severe disease observed in COVID-19 patients.


Introduction
Since the first outbreak in China in 2019, coronavirus disease 2019 (COVID-19) has spread rapidly and globally, with a mortality rate of 2% resulting in an ongoing pandemic.The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency increases the urgency of establishing a comprehensive understanding of the molecular pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).Typical clinical presentations of COVID-19 can be characterized by mild or asymptomatic conditions; some patients experience more severe disease and develop systemic inflammation, tissue damage, acute respiratory distress syndrome, thromboembolic complications, cardiac injury, and/or cytokine storm [1,2].Furthermore, SARS-CoV-2 infection is also associated with a wide variety of neurological manifestations, such as headache, loss of taste and smell, ataxia, meningitis, cognitive dysfunction, memory loss, seizures, and impaired consciousness, as well as long-term neurological problems in more than 30% of adults [3,4].However, the mechanism by which SARS-CoV-2 infection causes neurological diseases remains unclear.
We previously showed that the intranasal infection of SARS-CoV-2 in K18-hACE2 mice resulted in brain encephalitis characterized by the secretion of cytokines and chemokines, leukocyte infiltration, hemorrhage, and neuronal cell death [5].SARS-CoV-2 infects cells within the nasal turbinate, eye, and olfactory bulb, suggesting SARS-CoV-2's entry into the brain by this route after intranasal infection.In addition, histopathological analyses revealed that neuroinflammation potentially resulted in the severe disease observed in SARS-CoV-2-infected K18-hACE2 mice.In a follow-up study, we demonstrated that the neuronal cultures obtained from K18-hACE2 mice are permissive to SARS-CoV-2 infection and support productive virus replication.Furthermore, SARS-CoV-2 infection upregulated the expression of genes involved in antiviral immunity and inflammation in the brain [6].
To further investigate the molecular pathogenesis of neurological manifestations in K18-hACE2 mice following SARS-CoV-2 infection, we profiled expression changes of approximately 786 selected genes covering major neuroinflammation pathways using the NanoString nCounter technology.Furthermore, we identified several significantly dysregulated and functionally interesting genes associated with neuroinflammation during SARS-CoV-2 infection.This study shed a new light on the molecular pathways and genes involved in neuroinflammation and cell death, which can help to develop targeted treatments and interventions for COVID-19 patients with neurological complications.

SARS-CoV-2 Infection of K18-hACE2 Mice Modulates Host Gene Expression in Brain Tissue
We previously established SARS-CoV-2 pathogenesis in a K18-hACE2 mouse model and demonstrated that intranasal inoculation with SARS-CoV-2 resulted in neuroinvasion and neurotropism [5,6].To investigate the characteristics of SARS-CoV-2-induced immune response in the brain, K18-hACE2 mice were intranasally inoculated with SARS-CoV-2 (USA-WA1/2020), and brain tissues were collected on days 1, 3 and 6 post-infection.No significant weight loss was observed at day 1 and day 3 post-infection.As previously reported, infected animals started showing clinical symptoms of disease such as weight loss as well as neurological symptoms starting day 4 after the infection [5,6].By 6 days, all mice infected with the virus lost between 10 and 15% body weight.We euthanized three individual animals at 1, 3, and 6 days post-infection.We euthanized mock control group animals on day 6.We identified the DEGs by comparing virus-infected animals with the mock control group animals.First, we confirmed the time-dependent increase in SARS-CoV-2 genome copies in the infected brains of K18-hACE2 mice (Figure 1A).Next, RNA was extracted from the brain tissues and analyzed using the NanoString nCounter technology to uncover gene expression patterns.We selected differentially expressed genes (DEGs) using the following criteria: fold change (FC) > 5 and <−2 were considered as the range for upregulated and downregulated genes, respectively.Figure 1B presents the number of DEGs at days 1, 3, and 6 post-SARS-CoV-2 infection.In total, 104 genes were significantly upregulated, and 89 genes were significantly downregulated at 6 days postinfection (D6), whereas there were no upregulated genes and only six downregulated genes at 1 day post-infection (D1).To show gene dysregulation, we generated a Venn diagram, and it showed 16 genes, including Cxcl10 (encoding IP-10), Ccl2 (encoding MCP-1), Zbp1, and Irf7, commonly dysregulated in response to SARS-CoV-2 infection (Figure 1C).Of note, most DEGs were detected only at D6, which correlated with high viral RNA in the brain and body weight loss.
(encoding RANTES), Cxcl9 (encoding MIG), and Ccl3 (encoding MIP-1α) showed an increase of more than 100-fold at D6.Among the downregulated genes, Eomes, Fos, and Dxl1 were commonly downregulated in SARS-CoV-2-infected mice, and Grm2, BDNF, and Gria4 were significantly downregulated at D3 and D6 (Table 1).To examine which signaling pathways are involved in the expression of significant DEGs, we performed heatmap analysis (Figure 2).Notably, genes including Ddx58 (encoding RIG-I), Ifih1 (encoding MDA5), and Tlr2, involved in Toll-like receptor (TLR) and RIG-I-like receptor (RLR) signaling as well as genes associated with the cytosolic DNA sensing pathway were found to be highly upregulated (Figure 2A,B).In addition to the genes associated with pattern recognition receptor (PRR) signaling, genes closely linked with nuclear factor kappa B (NF-κB) and tumor necrosis factor (TNF) signaling pathways were also found to be upregulated at D6 (Figure 2D,E).Furthermore, in terms of the NF-κB signaling pathway, transcriptional factors involved in the canonical pathway, Cd40 (encoding p50) and Nfkb2 (encoding p100), and transcriptional factors involved in the non-canonical pathway, Nfkbia (encoding IκBα) and Relb, were activated [7].We previously reported the activation of the inflammatory response in the brain [6].Here, we examine the genes responsible for cytokine and chemokine production.Numerous genes encoding the CCL family, such as Ccl2, Ccl3, and Ccl5; the CXCL family, such as Cxcl9 and Cxcl10; the TNF family, such as tnfsf10 and tnfrsf1b; and the interleukin (IL) family, such as Il1a, and Il1b were significantly upregulated in the brain after SARS-CoV-2 infection (Figure 2C).Notably, the expression of Cxcl10, Tnf, Il1a, and Il1b, the final products of the PRR signaling pathway, increased by 20-40-fold compared to mock groups.
sensing pathway were found to be highly upregulated (Figure 2A,B).In addition to the genes associated with pattern recognition receptor (PRR) signaling, genes closely linked with nuclear factor kappa B (NF-κB) and tumor necrosis factor (TNF) signaling pathways were also found to be upregulated at D6 (Figure 2D,E).Furthermore, in terms of the NF-κB signaling pathway, transcriptional factors involved in the canonical pathway, Cd40 (encoding p50) and Nfkb2 (encoding p100), and transcriptional factors involved in the noncanonical pathway, Nfkbia (encoding IκBα) and Relb, were activated [7].We previously reported the activation of the inflammatory response in the brain [6].Here, we examine the genes responsible for cytokine and chemokine production.Numerous genes encoding the CCL family, such as Ccl2, Ccl3, and Ccl5; the CXCL family, such as Cxcl9 and Cxcl10; the TNF family, such as tnfsf10 and tnfrsf1b; and the interleukin (IL) family, such as Il1a, and Il1b were significantly upregulated in the brain after SARS-CoV-2 infection (Figure 2C).Notably, the expression of Cxcl10, Tnf, Il1a, and Il1b, the final products of the PRR signaling pathway, increased by 20-40-fold compared to mock groups.Downregulated genes were also analyzed.Surprisingly, several genes that play major roles in synapse signaling and neuronal functions, such as the retrograde endocannabinoid, long-term potentiation, and neurotrophin signaling pathways, were downregulated by SARS-CoV-2 (Figure 3).In detail, synapse signaling pathways can be divided into five categories, as follows: gamma-aminobutyric acidergic, cholinergic, dopaminergic, serotonergic, and glutamatergic synapse signaling [8].Among these synapse signaling pathways, most of the downregulated genes contribute to glutamatergic synapse signaling, such as Gria4, Grin2b, Grm2, and Grm3 (Figure 3B).We also evaluated the genes associated with tight junction proteins to see if SARS-CoV-2 infection impaired tissue integrity and enhanced brain-blood barrier permeability.We detected four genes, Jam2, Prkaca, Dig1, and Mapk10, that were involved in tight junction and cellular adhesion mechanisms and were downregulated (Figure 3G).
(KEGG pathway: mmu04668), and (F) cell death (KEGG pathway: mmu04210 and mmu04217).Red indicates the genes were upregulated in response to SARS-CoV-2 infection.Downregulated genes were also analyzed.Surprisingly, several genes that play major roles in synapse signaling and neuronal functions, such as the retrograde endocannabinoid, long-term potentiation, and neurotrophin signaling pathways, were downregulated by SARS-CoV-2 (Figure 3).In detail, synapse signaling pathways can be divided into five categories, as follows: gamma-aminobutyric acidergic, cholinergic, dopaminergic, serotonergic, and glutamatergic synapse signaling [8].Among these synapse signaling pathways, most of the downregulated genes contribute to glutamatergic synapse signaling, such as Gria4, Grin2b, Grm2, and Grm3 (Figure 3B).We also evaluated the genes associated with tight junction proteins to see if SARS-CoV-2 infection impaired tissue integrity and enhanced brain-blood barrier permeability.We detected four genes, Jam2, Prkaca, Dig1, and Mapk10, that were involved in tight junction and cellular adhesion mechanisms and were downregulated (Figure 3G).To validate gene expression profiling, we performed a Luminex assay on brain homogenates and measured the protein level of cytokines and chemokines.We used three To validate gene expression profiling, we performed a Luminex assay on brain homogenates and measured the protein level of cytokines and chemokines.We used three individual animals at 1, 3, and 6 days post-infection.We also used three mock control group animals.Correlating with NanoString data, the pro-inflammatory cytokines IL-1α, IL-1β, TNF-α, and IFN-γ were significantly increased compared to the mock control at D3 and D6 (Figure 4).
individual animals at 1, 3, and 6 days post-infection.We also used three mock control group animals.Correlating with NanoString data, the pro-inflammatory cytokines IL-1α, IL-1β, TNF-α, and IFN-γ were significantly increased compared to the mock control at D3 and D6 (Figure 4).

SARS-CoV-2 Infection Causes Neuro-Inflammatory Response Following Antiviral Response Activation
To characterize the molecular function and biological process of DEGs, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.GO analysis showed that diverse immune responses, such as defense response to virus, immune system processes, and innate immune response were activated at D3 and persisted through to D6 (Table 2).Along with the immune system, responses like the chemokine-mediated signaling pathway, the killing of cells of other organisms, and neutrophil chemotaxis slightly increased at D3.However, multiple biological pathways involving the inflammatory response, innate immune response, and cellular response to lipopolysaccharides were strongly increased at D6.In terms of molecular function, protein binding, chemokine activity, and cytokine activity were detected at both D3 and D6.

SARS-CoV-2 Infection Causes Neuro-Inflammatory Response Following Antiviral Response Activation
To characterize the molecular function and biological process of DEGs, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.GO analysis showed that diverse immune responses, such as defense response to virus, immune system processes, and innate immune response were activated at D3 and persisted through to D6 (Table 2).Along with the immune system, responses like the chemokine-mediated signaling pathway, the killing of cells of other organisms, and neutrophil chemotaxis slightly increased at D3.However, multiple biological pathways involving the inflammatory response, innate immune response, and cellular response to lipopolysaccharides were strongly increased at D6.In terms of molecular function, protein binding, chemokine activity, and cytokine activity were detected at both D3 and D6.Correlating with GO analysis, the KEGG pathways showed the sequential activation of innate immune and inflammatory responses, followed by cytokine and chemokine production and signaling pathways (Table 3).At D3, PRR signaling pathways like cytosolic DNA sensing, RIG-I-like receptor signaling, and Toll-like receptor signaling dominated the pro-inflammatory cytokine production-related signaling pathways, like TNF signaling, and several virus pathways, including coronavirus disease-COVID-19.At day 6, the pro-inflammatory cytokine production pathways, such as cytokine-cytokine receptor interaction and TNF signaling, were ranked high, followed by virus pathways such as coronavirus disease-COVID-19.Overall, the number of genes involved in the detected KEGG pathways increased on D6 to about 20-25 compared to D3. Next, ingenuity pathway analysis (IPA) was performed to delineate the SARS-CoV-2mediated activation of neuro-inflammatory pathways in depth.Figure 5A shows graphical summaries of the brains of SARS-CoV-2-infected mice.IRF3 and IRF7 were elevated at both D3 and D6.In addition, IFNG, IL1A, and IL1B showed substantial crosstalk between TLR signaling pathway mediators such as TLR3, TLR7, and MYD88 as well as key molecules of innate immune responses such as DDX58, MAVS, IRF3, and IRF7.
Correlating with heatmaps and KEGG pathway analyses, comparative analyses for canonical pathways showed that Toll-like receptor signaling, the role of pattern recognition receptors in recognition bacteria and viruses, the role of RIG-I-like receptors in antiviral innate immunity, and activation of IRF by cytosolic pattern recognition receptors were activated in response to SARS-CoV-2 infection in the brain, in order of significance (Figure 5B).Specifically, several pathways related to cytokine and chemokine production pathways, such as the role of hypercytokinemia/hyperchemokinemia in the pathogenesis of influenza, NF-kB signaling, and the TNFR signaling pathway, were detectable at D6, in sequence.We also found that the synaptogenesis signaling pathway and synaptic long term potentiation were significantly downregulated.However, neurological complications such as the coronavirus pathogenesis pathway and neuroinflammation signaling pathway were increased.In addition to neuronal function alterations, the quantity of neurons, cells, and progenitor cells as well as the development of neurons were decreased in response to SARS-CoV-2 infection.In contrast, apoptosis, the apoptosis of neurons, and neuronal cell death were elevated.Furthermore, individual analyses for disease and disorders highlighted that neurological diseases are associated with SARS-CoV-2 infection (Figure 5C).Nervous system development and function, neurological disease, inflammatory disease, and infectious disease were significantly disrupted, as were antimicrobial response and inflammatory responses at D3.However, inflammatory responses and inflammatory disease were increased at D6, suggesting that inflammation in the brain tissue accelerated as SARS-CoV-2 infection progressed.To explore the mechanisms behind SARS-CoV-2-induced neuroinflammation, we generated network maps with a significantly increased z-score.At D6, TLR1/2 and TLR2/6 heterodimers and their downstream mediators MYD88 and TRAF6 were highly upregulated, and these are linked with NF-kB-and IRF-dependent cytokine production pathways (Figure 6A).Together with the PRR signaling pathway, we identified several genes at D6 related to cell death pathways such as pyroptosis, necroptosis, and apoptosis (Figure 2F).As shown by our previous report, cell death pathways such as pyroptosis and necroptosis are more activated than apoptosis in response to SARS-CoV-2 infection.These pathways are related to upstream inflammation triggered by IL-1β secretion (Figures 4 and 6B) [6].

Discussion
Multiple research groups have suggested the possibility of neurotropism and neurological pathology caused by SARS-CoV-2 [2,[9][10][11][12].Additionally, we and others have reported that the inoculation of SARS-CoV-2 in K18-hACE2 mice resulted in neuroinvasion and neurological diseases [5,13].In the present study, we explored the molecular underpinnings of COVID-19 neurological complications using SARS-CoV-2-infected mice.First, we found that genes linked to TLR signaling and cell death pathways were upregulated, while genes associated with neurodegeneration and synaptic signaling were downregulated.We also identified the potential role of TLR2 in SARS-CoV-2-induced neuroinflammation and its potential link to other neurodegenerative diseases.Furthermore, the study's insights into the imbalance in synapse signaling and synaptic dysfunction provide crucial information for investigating potential long-term neurological consequences in COVID-19 survivors.
Our comprehensive gene expression analysis indicates the specific induction of RLR

Discussion
Multiple research groups have suggested the possibility of neurotropism and neurological pathology caused by SARS-CoV-2 [2,[9][10][11][12].Additionally, we and others have reported that the inoculation of SARS-CoV-2 in K18-hACE2 mice resulted in neuroinvasion and neurological diseases [5,13].In the present study, we explored the molecular underpinnings of COVID-19 neurological complications using SARS-CoV-2-infected mice.First, we found that genes linked to TLR signaling and cell death pathways were upregulated, while genes associated with neurodegeneration and synaptic signaling were downregulated.We also identified the potential role of TLR2 in SARS-CoV-2-induced neuroinflammation and its potential link to other neurodegenerative diseases.Furthermore, the study's insights into the imbalance in synapse signaling and synaptic dysfunction provide crucial information for investigating potential long-term neurological consequences in COVID-19 survivors.
Our comprehensive gene expression analysis indicates the specific induction of RLR and TLR signaling pathways, as shown by heatmaps (Figure 2A,B).In addition, the key mediators involved in NF-κB, TNF, and JAK/STAT signaling were also increased by SARS-CoV-2 infection, consistent with previous publications [14][15][16].In addition to RLR and NLR, the essential role of TLRs was highlighted.Accumulating evidence suggests the involvement of TLRs in neurodegenerative diseases.TLRs are typically expressed in neurons, astrocytes, and microglia.In detail, microglia express all TLRs, whereas resting astrocytes express low levels of TLR2, 4, 5, and 9 and activate astrocytes expressing TLR2 [17][18][19][20].During SARS-CoV-2 infection, the TLR2/MyD88 signaling pathway can detect envelope and spike proteins of SARS-CoV-2, and these are linked with NF-κB-mediated inflammatory responses [16,21,22].In our study, genes involved in TLR2/MyD88 signaling pathways were activated by SARS-CoV-2 infection, and pro-inflammatory cytokines like IL-1α, IL-1β, TNF-α, and IFN-γ were increased in the brain (Figures 4 and 6A).Considering our data, TLR2 may affect neuroinflammation and neuronal cell death.Moreover, it is well known that TLR2 accelerates the pathology of neurological disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and neuroinflammation.Relevant to this information, the blockage of TLR2-MyD88 interaction prevented neuroinflammation and attenuated AD's pathology [23,24].Therefore, further studies are needed to examine whether TLR2 directly induces neuroinflammation and cell death at cellular levels.
It has been reported that SARS-CoV-2 infection activates various forms of cell death, including pyroptosis, apoptosis, and necroptosis [25][26][27][28].We previously reported that SARS-CoV-2 infection activated necroptosis via ZBP1, RIPK1/3, and MLKL, with possible extrinsic apoptosis or pyroptosis activation, as shown by the increase in the expression of caspases [6].Since pyroptosis is closely linked with inflammation by the cleavage of cytokine precursors and the activation of microglia or astrocytes, it could possibly contribute to neuroinflammation [29][30][31].Necroptosis is distinguished from pyroptosis as RIPK1 and RIPK3 mediate them; however, both induce cell lysis and release damage-associated molecular patterns (DAMPs) to cause inflammation [32].In CNS, both RIPK1 and RIPK3 are implicated in playing a role in neurological diseases such as multiple sclerosis, amyotrophic lateral sclerosis (ALS), AD, and PD by promoting neuroinflammation, cytokine production by microglia, and astrocyte and neuronal cell death [33][34][35][36].Our IPA data demonstrate the activation of pyroptosis and necroptosis by SARS-CoV-2 as well as the crosstalk between these cell death pathways and IL-1α and IL-1β production (Figures 4 and 6B).Additionally, as previously mentioned, there is a four-fold increase in caspase expression, including Casp1, Casp7, and Casp8, in comparison to the mock control (Figure 2F).ZBP1 expression is implicated in inflammatory cell death.Since SARS-CoV-2 infection is closely associated with both cell death and inflammatory responses, more research is needed to understand how the virus triggers various cell death pathways and triggers inflammatory responses.
It should also be noted that genes involved in synaptogenesis signaling and longterm synaptic potentiation were differentially expressed, as detected by comparative IPA analysis for canonical pathways (Figure 5B).Importantly, we observed that the genes involved in neuroactive ligand-receptor interaction and synapse signaling pathways were significantly downregulated (Figure 3A,B).Neurotransmitters that can be divided into excitatory and inhibitory neurotransmitters are used in synaptic signaling pathways.Functionally, GABA, glycine, and serotonin are the main inhibitory neurotransmitters, while glutamate, aspartate, dopamine, epinephrine, and norepinephrine are considered excitatory neurotransmitters [8].The balance of excitatory and inhibitory synapse signaling must be tightly regulated and maintained for synapse plasticity and the efficient functioning of signal transduction.Otherwise, an imbalance in synapse signaling might result in neurological complications such as AD, PD, and ALS [37].According to our data, most DEGs related to synapse signaling are involved in excitatory neurotransmitters, especially glutamatergic excitatory synapse signaling, which mediates four of the major glutamic receptors: N-methyl-D-aspartate receptors that consist of the Grin family, AMPA receptors that consist of the Gria family, Kaniate receptors that consist of the Grik family, and metabotropic glutamate receptors that consist of the Grm family [38].In particular, several receptors belonging to the Gria, Grin, and Grm families were significantly suppressed in SARS-CoV-2-infected mouse brains, suggesting that SARS-CoV-2 induces an imbalance in synapse signaling and synaptic dysfunction.Together with alterations in synapse signaling, several genes involved in retrograde endocannabinoid signaling, long-term potentiation, and neurotrophin signaling were slightly downregulated (Figure 3D-F).One of the limitations of this study is that we did not compare SARS-CoV-2-induced transcriptional changes with other stimuli such as LPS.Another limitation includes the use of a mouse model of severe COVID-19.Additionally, there is need for further clinical evidence to establish a direct link between these molecular changes and human neurological complications In conclusion, our study has expanded the role of neuroinflammation-related genes during SARS-CoV-2 infection in neurological complications.In SARS-CoV-2-infected brains, the gene expression patterns were significantly disrupted, resulting in the robust activation of the immune response and inflammation.It is possible that the activation of the TLR signaling pathway, especially TLR2, may contribute to inflammation via NF-κB and TNF signaling pathways and programmed cell death, such as pyroptosis, in the brain.As novel variants of SARS-CoV-2 emerge and threaten public health, studies of the influence of SARS-CoV-2 variants are needed and can provide important insight into SARS-CoV-2 neuropathogenesis.Also, it would be interesting to test whether TLR2-targeting drugs can mitigate SARS-CoV-2-induced neuroinflammation and have any potential in therapeutic application.

SARS-CoV-2 Infection in Mice
This study was performed following the National Institutes of Health and the Institutional Animal Care and Use Committee (IACUC) guidelines.The protocol was approved by the Georgia State University IACUC (protocol number A20044).To infect mice with SARS-CoV-2, we performed all the animal experiments in the Animal Biosafety Level 3 laboratory (ABSL3).Mice that met the human endpoint criteria were euthanized to limit suffering.As previously described, SARS-CoV-2 (USA-WA1/2020) was isolated from an oropharyngeal swab from a patient in Washington, USA (BEI NR-52281) [39].Virus titration was performed using VeroE6 cells [5,39].Hemizygous K18-hACE2 mice were obtained from the Jackson Laboratory (Bar Harbor, ME) [5].Six-week-old K18-hACE2 mice were transferred to the ABSL-3 facility and acclimated to the local surroundings before initiating experiments.Nine animals were intranasally inoculated with 10 5 PFU of SARS-CoV-2 (USA-WA1/2020), whereas three animals (mock control group) were inoculated with equivalent amounts of PBS [5].Approximately similar numbers of male and female mice were used.During experiments, mice were checked for body weight, appetite, activity, and neurological signs every day following SARS-CoV-2 infection.On days 1, 3, and 6 after inoculation, the mice were euthanized to collect brain tissues.We euthanized three individual animals at 1, 3, and 6 days post-infection.We euthanized mock control group animals on day 6.We identified the DEGs by comparing virus-infected animals with the mock control group animals.The mock and SARS-CoV-2-infected mice were anesthetized using isoflurane and perfused with PBS.Later, RNA was collected from the brain.

RNA Extraction and Evaluation
Frozen brain tissues harvested from mock (n = 3) and infected animals (n = 9) were weighed and lysed in RLT buffer (Qiagen), and RNA was extracted using an RNeasy MiniKit (Qiagen, Hilden, Germany) using the manufacturer's instructions [40].To measure the purity and quantity of total RNA, a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) were used.The following criteria were maintained: wavelength absorbance ratio A260/280 ~2.0 and A260/230 ~2.0.The percentage of RNA fragments ≥300 and nucleotides DV300 was ≥50%, and the integrity RIN was >4. by using MILLIPLEX Analyst software 5.1.Data are presented as the mean ± standard deviation (SD).Statistical comparisons between different treatments were performed using Mann-Whitney U tests or Student's t-tests, and the results were considered statistically significant at p < 0.05.

Figure 2 .
Figure 2. Upregulated genes in the brains of SARS-CoV-2-infected mice.Heatmaps showing the fold change in expression levels of specific genes involved in the following signaling pathways: (A) Tolllike receptor (TLR) signaling pathway (KEGG pathway: mmu04620), (B) RIG-I-like receptor (RLR)

Figure 4 .
Figure 4. Cytokine and chemokine levels are differentially expressed in the brains of SARS-CoV-2infected mice.To measure cytokine secretion levels, brain homogenates from mock-(Ctl) and SARS-CoV-2-inoculated mice were collected at days 3 (D3) and 6 (D6) after infection.Cytokine secretion levels in brain homogenates were examined via MILLIPLEX using the manufacturer's instructions.Five-parameter logistics of the spline curve fitting method were used as the standard curve, and data were calculated using MILLIPLEX Analyst software 5.1.Each point represents the mean ± SD.Statistical analysis: * p < 0.05, ** p < 0.01, *** p < 0.001, compared to mock control groups.

Figure 4 .
Figure 4. Cytokine and chemokine levels are differentially expressed in the brains of SARS-CoV-2-infected mice.To measure cytokine secretion levels, brain homogenates from mock-(Ctl) and SARS-CoV-2-inoculated mice were collected at days 3 (D3) and 6 (D6) after infection.Cytokine secretion levels in brain homogenates were examined via MILLIPLEX using the manufacturer's instructions.Five-parameter logistics of the spline curve fitting method were used as the standard curve, and data were calculated using MILLIPLEX Analyst software 5.1.Each point represents the mean ± SD.Statistical analysis: * p < 0.05, ** p < 0.01, *** p < 0.001, compared to mock control groups.

Pathogens 2024 ,
13, x FOR PEER REVIEW 9 of 16 necroptosis are more activated than apoptosis in response to SARS-CoV-2 infection.These pathways are related to upstream inflammation triggered by IL-1β secretion (Figures 4 and 6B) [6].

Figure 5 .Figure 5 .
Figure 5. Top canonical pathways activated in the SARS-CoV-2-infected brain.(A) Graphical abstracts were generated for the overall changes in gene expression patterns using ingenuity pathway analysis (IPA) at days 3 (D3) and 6 (D6) after infection.(B) Comparative canonical pathways and (C) individual analyses for disease and disorders in response to SARS-CoV-2 infection were generated using IPA tools.The orange line indicates the default p value significance threshold of 0.05.

Figure 6 .
Figure 6.Identification of top canonical pathways activated by SARS-CoV-2-infected brains.Network maps show the pathways regulated by SARS-CoV-2 infection.At 6 days post-infection, the gene expression related to (A) PRR signaling merged with the neuroinflammation signaling pathway, while (B) pyroptosis and necroptosis are represented using the ingenuity pathway analysis (IPA) tool.

Figure 6 .
Figure 6.Identification of top canonical pathways activated by SARS-CoV-2-infected brains.Network maps show the pathways regulated by SARS-CoV-2 infection.At 6 days post-infection, the gene expression related to (A) PRR signaling merged with the neuroinflammation signaling pathway, while (B) pyroptosis and necroptosis are represented using the ingenuity pathway analysis (IPA) tool.

Table 1 .
List of top up-and downregulated genes in the brains of SARS-CoV-2-infected K18-hACE2 mice at 1, 3, and 6 days post-infection.FC = fold change.

Table 1 .
List of top up-and downregulated genes in the brains of SARS-CoV-2-infected K18-hACE2 mice at 1, 3, and 6 days post-infection.FC = fold change.