Microglia influence host defense, disease, and repair following murine coronavirus infection of the central nervous system

Abstract The present study examines functional contributions of microglia in host defense, demyelination, and remyelination following infection of susceptible mice with a neurotropic coronavirus. Treatment with PLX5622, an inhibitor of colony stimulating factor 1 receptor (CSF1R) that efficiently depletes microglia, prior to infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality compared with control mice that correlated with impaired control of viral replication. Single cell RNA sequencing (scRNASeq) of CD45+ cells isolated from the CNS revealed that PLX5622 treatment resulted in muted CD4+ T cell activation profile that was associated with decreased expression of transcripts encoding MHC class II and CD86 in macrophages but not dendritic cells. Evaluation of spinal cord demyelination revealed a marked increase in white matter damage in PLX5622‐treated mice that corresponded with elevated expression of transcripts encoding disease‐associated proteins Osteopontin (Spp1), Apolipoprotein E (Apoe), and Triggering receptor expressed on myeloid cells 2 (Trem2) that were enriched within macrophages. In addition, PLX5622 treatment dampened expression of Cystatin F (Cst7), Insulin growth factor 1 (Igf1), and lipoprotein lipase (Lpl) within macrophage populations which have been implicated in promoting repair of damaged nerve tissue and this was associated with impaired remyelination. Collectively, these findings argue that microglia tailor the CNS microenvironment to enhance control of coronavirus replication as well as dampen the severity of demyelination and influence repair.

tor 1 receptor (CSF1R) that efficiently depletes microglia, prior to infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality compared with control mice that correlated with impaired control of viral replication. Single cell RNA sequencing (scRNASeq) of CD45+ cells isolated from the CNS revealed that PLX5622 treatment resulted in muted CD4+ T cell activation profile that was associated with decreased expression of transcripts encoding MHC class II and CD86 in macrophages but not dendritic cells. Evaluation of spinal cord demyelination revealed a marked increase in white matter damage in PLX5622-treated mice that corresponded with elevated expression of transcripts encoding disease-associated proteins Osteopontin (Spp1), Apolipoprotein E (Apoe), and Triggering receptor expressed on myeloid cells 2 (Trem2) that were enriched within macrophages. In addition, PLX5622 treatment dampened expression of Cystatin F (Cst7), Insulin growth factor 1 (Igf1), and lipoprotein lipase (Lpl) within macrophage populations which have been implicated in promoting repair of damaged nerve tissue and this was associated with impaired remyelination. Collectively, these findings argue that microglia tailor the CNS microenvironment to enhance control of coronavirus replication as well as dampen the severity of demyelination and influence repair. replicates within glial cells with relative sparing of neurons (Bergmann, Lane, & Stohlman, 2006;Templeton & Perlman, 2007;Weiss & Leibowitz, 2011). Expression of type I interferon (IFN-I) is critical in helping control viral replication as mice lacking IFN-I receptor exhibit increased mortality associated with enhanced viral replication (Ireland, Stohlman, Hinton, Atkinson, & Bergmann, 2008). In addition, localized expression of T cell chemotactic chemokines including CCL5, CXCL9, and CXCL10 within the CNS contribute to host defense by attracting virus-specific CD4+ and CD8+ T cells into the CNS that further control viral replication through secretion of interferon-γ (IFN-γ) and cytolytic activity (Bergmann et al., 2004;Glass et al., 2004;Glass & Lane, 2003a;Glass & Lane, 2003b;Liu et al., 2000;Liu, Armstrong, Hamilton, & Lane, 2001;Marten, Stohlman, & Bergmann, 2001;Parra et al., 1999).

Main points
Antibody-secreting cells (ASCs) are also capable of responding to CXCL9 and CXCL10 and aid in host defense (Phares, Marques, Stohlman, Hinton, & Bergmann, 2011;. Nonetheless, sterile immunity is not achieved and the majority of animals that survive the acute stage of disease develop immune-mediated demyelination in which both virus-specific T cells and macrophages amplify the severity of white matter damage associated with hind-limb paralysis (Bergmann et al., 2006;Hosking & Lane, 2009;Templeton & Perlman, 2007).

While the functional roles of T cells and B cells in both host
defense and disease in JHMV-infected mice have been extensively studied, there is increasing interest in better understanding how resident cells of the CNS contribute to these events. Microglia are considered the resident immune cells of the CNS and aid in a diverse array of functions including maintaining CNS homeostasis as well as contributing to various disease-associated conditions (Hammond, Robinton, & Stevens, 2018;Salter & Stevens, 2017;Tejera & Heneka, 2019;Wolf, Boddeke, & Kettenmann, 2017). Moreover, microglia are immunologically competent and capable of rapidly responding to infection and/or damage via specific expression of surface receptors culminating in morphologic changes accompanied by secretion of proinflammatory cytokines/chemokines that function in amplifying neuroinflammation. Recently, the functional role of microglia in contributing to host defense in response to CNS infection with neurotropic viruses has been examined. These studies have been greatly aided by findings demonstrating that mice lacking colony stimulating factor 1 receptor (CSF1R−/−) lack microglia emphasizing the importance of this signaling pathway in microglia development (Ginhoux et al., 2010). Subsequent studies by Green and colleagues (Elmore et al., 2014) showed that blocking CSF1R signaling in adult mice through administration of CSF1R antagonists is also important in survival of microglia in adult mice. Recent studies have employed treatment of mice with PLX5622, a brain penetrant and selective antagonist of the CSF1R that results in a dramatic reduction in microglia, to better understand functional roles of these cells in preclinical models of neurodegenerative disease (Acharya et al., 2016;Dagher et al., 2015;Elmore et al., 2014;Spangenberg et al., 2019). In addition, PLX5622-mediated targeting of microglia results in increased susceptibility to West Nile virus (WNV) Seitz, Clarke, & Tyler, 2018), Japanese encephalitis virus (JEV) (Seitz et al., 2018), Theiler's murine encephalomyelitis virus (TMEV) (Sanchez et al., 2019a;Waltl et al., 2018), and JHMV (Wheeler, Sariol, Meyerholz, & Perlman, 2018) arguing for a protective role for microglia against acute viral-induced encephalitis.
The current study was undertaken to evaluate how microglia tailor the immunological landscape in response to JHMV infection within the brain and spinal cord at different stages of infection with regard to pathways associated with both host defense and neuropathology.
We believe microglia will be critical in aiding in host defense through regulating a number of different pathways including antigen presentation and T cell activation as well as augmenting demyelination. To address this, we used a comprehensive set of analytical approaches including single cell RNA sequencing (scRNAseq), flow cytometry, and histopathological techniques to assess disease outcome in JHMVinfected mice treated with PLX5622 at defined times postinfection.
Our findings emphasize an important role for microglia in aiding in host defense in response to JHMV infection of the CNS as well as influencing both the severity of spinal cord demyelination and remyelination in a model of murine coronavirus-induced neurologic disease.

| Mice and viral infection
Five-week-old C57BL/6 male mice were purchased from The Jackson Laboratory. Mice were infected intracranially (i.c.) with 250 plaque forming units (PFU) of JHMV strain J2.2v-1 in 30 μl of sterile Hanks balanced sterile solution (HBSS) and animals were euthanized at days 3, 7, 12, and 21 postinfection (p.i.). Clinical disease in JHMV-infected mice was evaluated using a previously described scale . To determine viral titers within brains, experimental animals were sacrificed at defined times p.i., brains isolated, homogenized and plaque assay were performed on the DBT astrocytoma cell line as described previously (Hirano, Murakami, Fujiwara, & Matsumoto, 1978

| Cell isolation and flow cytometry
Flow cytometry was performed to identify inflammatory cells entering the CNS using established protocols (Blanc, Rosen, & Lane, 2014;Chen et al., 2014). In brief, single cell suspensions were generated from tissue samples by grinding with frosted microscope slides.
Immune cells were enriched via a two-step Percoll cushion (90 and 63%) and cells were collected at the interface of the two Percoll layers. Before staining with fluorescent antibodies, isolated cells were incubated with anti-CD16/32 Fc block (BD Biosciences, San Jose, CA) at a 1:200 dilution. Immunophenotyping was performed using commercially available antibodies specific for the following cell surface markers: CD4, CD8, CD11b (BD Biosciences, San Jose, CA), and CD45 (eBioscience, San Diego, CA). The following flow cytometric gating strategies were employed for inflammatory cells isolated from the CNS: macrophages (CD45 hi CD11b+) and microglia (CD45 lo CD11b+). APC-conjugated rat anti-mouse CD4 and a PE-conjugated tetramer specific for the CD4 immunodominant epitope present within the JHMV matrix (M) glycoprotein spanning amino acids 133-147 (M133-147 tetramer) to determine total and virus-specific CD4 + cells, respectively (Chen et al., 2014;Marro, Grist, & Lane, 2016a); APC-conjugated rat anti-mouse CD8a and a PEconjugated tetramer specific for the CD8 immunodominant epitope present in the spike (S) glycoprotein spanning amino acids 510-518 (S510-518) to identify total and virus-specific CD8 + cells, respectively (Chen et al., 2014;Marro et al., 2016a). Data were collected using a BD LSR Fortessa X-20 flow cytometer and analyzed with FlowJo software (Tree Star Inc.).

| scRNASeq
Immune cells were isolated as described above from brain (day 7 p.i.) and spinal cord (day 14 p.i.) and stained with DAPI and APC Database. Expression levels and distribution of population-specific immune cell markers were then analyzed to further refine the identified clusters and expose any subpopulations that should be separated as independent clusters. Once the clusters were established and identified, plots were generated using Seurat, ggpubr, and fgsea R packages.

| Histology
Mice were euthanized at defined times points according to IACUCapproved guidelines and the length of spinal cord extending from thoracic vertebrate 6-10 was cryoprotected in 30% sucrose, cut into 1-mm transverse blocks and processed to preserve the craniocaudal orientation and subsequently embedded in O.C.T. (VWR, Radnor, PA).
Eight micron (μm)-thick coronal sections were cut and sections were stained with hematoxylin/eosin (H&E) in combination with luxol fast blue (LFB) and between 4 and 8 sections/mouse analyzed. Areas of total white matter and demyelinated white matter were determined with ImageJ Software and demyelination was scored as a percentage of total demyelination from spinal cord sections analyzed (Blanc et al., 2015;Blanc, et al., 2014;Dickey, Worne, Glover, Lane, & O'Connell, 2016;Marro, Grist, & Lane, 2016b).

| Electron microscopy and g-ratio analysis
For electron microscopy (EM) analysis of spinal cords, mice were sacrificed and underwent cardiac perfusion with 0.1 M cacodylate buffer containing 2% paraformaldehyde/2% glutaraldehyde. Serial ultrathin sections of spinal cords embedded in Epon epoxy resin were stained with uranyl acetate-lead citrate and analyzed as previously described (Liu, Keirstead, & Lane, 2001). Images at ×1200 magnification were analyzed for g-ratio using ImageJ software. In adult animals there is a relationship between axon circumference and myelin sheath thickness (number of lamellae) expressed by the g-ratio (axon diameter/total fiber diameter); in remyelination this relationship changes such that myelin sheaths are abnormally thin for the axons they surround (Smith, Bostock, & Hall, 1982). An abnormally thin myelin sheath, relative to axonal diameter, was used as the criterion for oligodendrocyte remyelination. Absence of a myelin sheath was used as the criterion for demyelination. For most axons, two measurements were conducted with a minimum of 400 axons analyzed per experimental group. In all cases, slides were blinded and read independently by two investigators.

| Statistical analysis
GraphPad Prism was used to perform statistical analyses. Data for each experiment is presented as mean ± SEM. For flow cytometry analysis unpaired Student's t test was used to determine significance and a p value of <.05 was considered statistically significant. Wilcoxon test was used for analyzing gene expression in scRNASeq clusters and the resulting p values were corrected for multiple comparisons by Holm-Sidak method and a p value of <.05 was considered statistically significant.

| PLX5622 treatment increases susceptibility to JHMV-induced neurologic disease
To evaluate the contribution of microglia to disease progression in JHMV-infected mice, the CSF1R inhibitor PLX5622 was administered as previous studies have reported this pharmacologic approach effectively depletes >90% of microglia (Acharya et al., 2016;Najafi et al., 2018). Mice were treated with PLX5622 (1,200 mg/kg) 7 days prior to infection and continued on the drug for the duration of the experiment. Treatment with PLX5622 resulted in an overall increase in mortality with 25% of PLX5622-treated mice surviving to day 21 p.i. whereas 75% of control-chow treated mice survived to this time ( Figure 1a). The increase in mortality in PLX5622-treated mice correlated with increased viral titers within the brains and spinal cords at days 3, 7, and 12 p.i. compared with control animals; however, by day 21 p.i. viral titers were not detected (ND) in experimental groups F I G U R E 1 PLX5622 treatment increases susceptibility to JHMV-induced neurologic disease. Mice were fed either PLX5622 or control chow for 7 days prior to i.c. infection with JHMV (250 PFU) and subsequently remained specific chow for the duration of the experiment. PLX5622 treatment led to (a) increased mortality compared to control mice that was associated with an (b) impaired ability to control viral replication within the brains and spinal cords at days 3, 7, and 12 p.i. compared with control mice. Representative flow cytometric data from JHMV-infected mice treated with either PLX5622 or control chow and gating on microglia (CD45 lo CD11b + cells) or macrophages (CD45 hi CD11b + cells) in (c) brains at day 7 p.i., and (d) spinal cords at day 14 p.i. PLX5622-treatment resulted in reduced numbers of microglia in brains and spinal cords compared with control mice. Data are derived from a minimum of three independent experiments with a minimum of 3 mice/time points. Data in B, C, and D are presented as average ± SEM. ND, not detected; *p < .05, ****p < .0001 ( Figure 1b). We confirmed efficient microglia (CD45 lo CD11b+) depletion in PLX5622-treated mice within the brain at day 7 p.i. (Figure 1c) and spinal cord at day 14 p.i. (Figure 1d) using flow cytometry.
PLX5622 treatment did not affect numbers of macrophages (CD45 hi CD11b+) within brains and spinal cords of experimental mice (Figure 1c,d). These findings support earlier work indicating that PLX5622-targeting of microglia impacts efficient immune-mediated control of viral replication following infection with neurotropic viruses Sanchez et al., 2019b;Seitz et al., 2018;Waltl et al., 2018;Wheeler et al., 2018).

| PLX5622 treatment and immune cell infiltration into the brains of JHMV-infected mice during acute disease
Our findings reveal that PLX5622 treatment of mice increases susceptibility to JHMV-induced neurologic disease associated with impaired ability to control viral replication. In order to better understand the effects of PLX5622 treatment on influencing the immune cell composition of the CNS we used 10× Genomics scRNAseq technology.
Experimental mice were fed either control chow or chow containing PLX5622 for 7 days prior to infection and remained on chow until sacrificed at either day 7 p.i. or 14 p.i., at which point, live CD45+ cells were sorted from the brains or spinal cords, respectively. The respective tissues were found appropriate to study host defense and disease pathogenesis. The immune response to JHMV infection peaks around day 7 p.i. in the brain and ensuing spinal cord demyelination is present at day 14 p.i. We aggregated data from 4,806 cells taken from control-treated (n = 6) and 3,868 cells from PLX5622-treated (n = 6) mice brain tissue at day 7 p.i. and performed unsupervised clustering analysis based on similarity of gene expression signatures using Seurat single cell genomics R package (Ekiz et al., 2019) (Table 1). This approach revealed 16 distinct cell clusters representative of both lymphoid and myeloid linages at day 7 p.i. (Figure 2a). To better understand the overlapping expression of marker genes and identification of cell clusters, we employed a recently described algorithm that compares the gene expression signatures of cell clusters with publicly available ImmGen database (Ekiz et al., 2019). As previously described (Ekiz et al., 2019), this algorithm calculates an aggregate identity score for each scRNAseq cell cluster as a measure of molecular similarity to the ImmGen subsets. Through combinations of these two approaches, we identified three CD8+ T cell subsets [naïve, effector (Eff.), and memory (Mem.)], two macrophage subsets (Mac 1 and Mac 2), four dendritic cell (DC) subsets (plasmacytoid, NADPH [Nox2], XCR1 [Xcr1], CCL22 [Ccl22]), and single subsets of CD4+ T cells, regulatory T cells (Treg), natural killer (NK) cells, B cells, microglia, neutrophils (neuts), and monocytes at day 7 p.i. (Figure 2a). In order to verify the algorithm-assisted identification of cell clusters, we examined expression of known cellular markers in our data set; expression of these markers corresponded with the respective identities of the distinct clusters (Figure 2b,c). Our initial preliminary analyses focusing on samples separately are in agreement with the results of this aggregated approach.
We next analyzed differences in CD45+ cells between PLX5622-treated and control mice at day 7 p.i. following JHMV infection. When data from the cellular genotypes were plotted side-byside, treatment-dependent dynamics within the tissues started to emerge (Figure 3a,b). Importantly, we were able to show that

| PLX5622-treatment alters infiltration and activation phenotype of T cells during acute disease
Control of JHMV replication within the CNS is associated with infiltration of activated virus-specific CD4+ and CD8+ T cells (Marten et al., 2001;Pearce, Hobbs, McGraw, & Buchmeier, 1994;Williamson & Stohlman, 1990). At day 7 p.i., PLX5622 treatment did not significantly alter CD4+ T cell infiltration yet there was an increase in CD8+ T cells (p < .05) compared with control mice as determined by flow cytometric analysis ( Figure S1a). There were no differences in virus-specific CD4+ and CD8+ T cells specific for Overview of experimental conditions showing treatment, sacrifice time points, tissue collected, and total number of CD45+ cells isolated as well as reads/cell following scRNAseq analysis immunodominant epitopes present within the Matrix (M) ( Figure S1b) and Spike (S) glycoproteins ( Figure S1c) as determined by tetramer staining (Blanc, et al., 2014;Marro et al., 2016b).
Evaluation of defined factors associated with T cell activation at day 7 p.i. revealed reduced expression of the Th1-associated transcription factor Tbet (Tbx21) (p < .01) and this was associated with reduced (p < .05) expression of Tnf transcripts, but not Ifng transcripts, in PLX5622-treated mice compared to control mice ( Figure 4a). We also determined reduced expression of activation markers CD69 (Cd69) and CD44 (Cd44, p < .05) in CD4+ T cells from the brains of PLX5622-treated mice compared to control mice at day 7 p.i. (Figure 4a). In addition, the CD4+ T cells subset from PLX5622-treated mice also expressed reduced transcripts for Il2ra We next performed scRNAseq on CD45+ cells enriched from the spinal cords of JHMV-infected mice treated with either PLX5622 or control at day 14 p.i. Using the same approach as described above using aggregated data from 2,725 cells taken from control-treated (n = 6) and 4,891 cells from PLX5622-treated (n = 6) mice (Table 1) CNS viral infection has been identified Sanchez et al., 2019a;Seitz et al., 2018;Waltl et al., 2018;Wheeler et al., 2018).
Perlman and colleagues  have shown that microglia are required for optimal host defense in response to JHMV infection of the CNS. Targeted depletion of microglia through administration of PLX5622 revealed a role in limiting mortality that was associated with impaired control of JHMV replication. The increase in susceptibility to disease did not appear to be due to altered expression of IFN-I but more likely a reflection of impaired antigen-presentation due to muted MHC Class II expression by macrophages infiltrating the CNS of PLX5622-treated mice and this likely resulted in dampened T cell responses . We believe the increase in expression of IFN-I response genes in PLX5622-treated mice most likely reflects the overall increase in viral titers within the brains. Similarly, microglia depletion led to increased mortality in mice infected with WNV associated with diminished activation of APCs and limited reactivation of virus-specific T cells that led to reduced viral clearance Seitz et al., 2018). These findings clearly implicate microglia in enhancing optimal host responses following CNS viral infection, in part, by influencing antigen-presentation that affects virusspecific T cell responses.
We undertook the present study to better understand how microglia contribute to host defense as well as demyelination and repair following JHMV infection of the CNS using sophisticated molecular, cellular, and histologic approaches. Employing PLX5622 to deplete microglia, we found increased mortality associated with  (Figure 3d,e). These findings may reflect the increase in viral titers within the CNS of PLX5622-treated mice at these times but also argue that microglia are not solely responsible for production of IFN-I.
We detected altered T cell responses within the CNS following PLX5622 treatment as determined by both flow cytometry and scRNASeq. There were increased numbers of total CD8+ T cells (p < .05) as well as virus-specific CD8+ T cells within the brains of JHMV-infected mice treated with PLX5622 compared to controls at day 7 p.i. We also found a trend towards increased total CD4+ T cells and virus-specific CD4+ T cells in PLX5622-treated mice compared to controls although these differences were not significant. In terms of T cell activation, we detected differential responses in T cell subsets at day 7 p.i. In CD4+ T cells, there was a reduction in transcripts associated with Th1-polarized activation for example, T-bet (Tbx21) in PLX5622-treated mice compared to controls although there were no differences in Ifng transcripts in CD4+ T cells in experimental groups.
PLX5622-treatment also resulted in reduced expression of CD4+ T cell surface activation markers including CD44, CD69, and components of IL-2 receptor in PLX5622-treated mice compared with controls whereas there was an overall increased activation phenotype associated with  (Diamond & Farzan, 2013). It is important to note that both populations of cells were present at a low frequency and there were no dramatic differences between experimental groups, although CD40+ macrophages were present at a much higher frequency compared to Ifit + macrophages. Furthermore, there was a small population of Galectin+ microglia in spinal cords in control mice yet not in PLX5622-treated mice and previous studies argue for a role for certain isoforms of Galectin in potentially contributing to demyelination in patients with multiple sclerosis (MS) (de Jong et al., 2018) while other isoforms are considered important in driving oligodendrocyte differentiation associated with remyelination (Thomas & Pasquini, 2018). Our findings that spinal cord demyelination was significantly increased in PLX5622-treated mice supports an emerging role for microglia in restricting the severity of white matter and this is consistent with a recent study from our group indicating that microglia influence the severity of demyelination in JHMVinfected mice (Brown et al., 2019). While we are currently exploring the molecular and cellular mechanisms by which microglia may modulate the CNS microenvironment in mice persistently infected with JHMV, evidence presented in the current study indicates that PLX5622 treatment resulted in increased expression of transcripts encoding for Osteopontin, APOE, and TREM2 all of which have been implicated in contributing to demyelination (Chabas et al., 2001;Krasemann et al., 2017;Ulrich & Holtzman, 2016). Interestingly, expression of all three transcripts were enriched within macrophage populations suggesting a specific effect by which microglia may suppress expression and limit myelin damage.
Emerging studies have pointed to a protective role for microglia in limiting neuropathology and promoting repair (Baaklini, Rawji, Duncan, Ho, & Plemel, 2019;Lee, Hamanaka, Lo, & Arai, 2019;Lloyd & Miron, 2019). In support of this concept are recent studies from Miron and colleagues (Lloyd & Miron, 2019) showing an important role for microglia in enhancing remyelination in a toxin-model of demyelination that is aided by microglial death and subsequent microglial repopulation; here, in this study, the absence of microglia prevents their further death and repopulation and is associated with increased white matter damage. Although mechanisms by which microglia may support remyelination have not been completely defined, it is though that these cells aid in clearance of myelin debris and/or secrete growth factors/cytokines that influence maturation of oligodendrocyte progenitor cells (OPCs) into mature myelin-producing oligodendrocytes. What is also clear is that microglia are heterogenous in terms of transcriptome and protein expression which is likely regulated during disease and this would influence the role of these cells in enhancing or muting disease progression and potential repair.
In support of a protective role for microglia in restricting neuropathology and promoting repair is our data demonstrating that PLX5622 treatment of JHMV-infected mice results in an increase in white matter damage associated with impaired remyelination (Figure 7a,b,d, and e). In addition, scRNASeq also shows reduced expression of genes encoding proteins previously associated with remyelination including Cystatin F (Durose et al., 2019;Ma et al., 2011;Shimizu et al., 2017), Insulin growth factor 1 (IGF1) (Hlavica et al., 2017;Wlodarczyk et al., 2017;Ye et al., 2002) and Lipoprotein lipase (Bruce et al., 2018) within macrophages isolated from the spinal cords of PLX5622-treated mice (Figure 7f). These findings further support the notion that microglia may either directly or indirectly influence remyelination within the spinal cord by contributing to controlling expression of genes encoding proteins that regulate OPC maturation. We are currently pursuing the functional contributions of Cystatin F, IGF1, and Lipoprotein lipase in contributing to remyelination in JHMV-infected mice.
We would caution that although demyelination was worsened and remyelination was impaired when microglia are depleted, this may

CONFLICT OF INTEREST
The authors declare no competing financial interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.