Effects of Mycobacterium vaccae NCTC 11659 and Lipopolysaccharide Challenge on Polarization of Murine BV-2 Microglial Cells

Previous studies have shown that the in vivo administration of soil-derived bacteria with anti-inflammatory and immunoregulatory properties, such as Mycobacterium vaccae NCTC 11659, can prevent a stress-induced shift toward an inflammatory M1 microglial immunophenotype and microglial priming in the central nervous system (CNS). It remains unclear whether M. vaccae NCTC 11659 can act directly on microglia to mediate these effects. This study was designed to determine the effects of M. vaccae NCTC 11659 on the polarization of naïve BV-2 cells, a murine microglial cell line, and BV-2 cells subsequently challenged with lipopolysaccharide (LPS). Briefly, murine BV-2 cells were exposed to 100 µg/mL whole-cell, heat-killed M. vaccae NCTC 11659 or sterile borate-buffered saline (BBS) vehicle, followed, 24 h later, by exposure to 0.250 µg/mL LPS (Escherichia coli 0111: B4; n = 3) in cell culture media vehicle (CMV) or a CMV control condition. Twenty-four hours after the LPS or CMV challenge, cells were harvested to isolate total RNA. An analysis using the NanoString platform revealed that, by itself, M. vaccae NCTC 11659 had an “adjuvant-like” effect, while exposure to LPS increased the expression of mRNAs encoding proinflammatory cytokines, chemokine ligands, the C3 component of complement, and components of inflammasome signaling such as Nlrp3. Among LPS-challenged cells, M. vaccae NCTC 11659 had limited effects on differential gene expression using a threshold of 1.5-fold change. A subset of genes was assessed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR), including Arg1, Ccl2, Il1b, Il6, Nlrp3, and Tnf. Based on the analysis using real-time RT-PCR, M. vaccae NCTC 11659 by itself again induced “adjuvant-like” effects, increasing the expression of Il1b, Il6, and Tnf while decreasing the expression of Arg1. LPS by itself increased the expression of Ccl2, Il1b, Il6, Nlrp3, and Tnf while decreasing the expression of Arg1. Among LPS-challenged cells, M. vaccae NCTC 11659 enhanced LPS-induced increases in the expression of Nlrp3 and Tnf, consistent with microglial priming. In contrast, among LPS-challenged cells, although M. vaccae NCTC 11659 did not fully prevent the effects of LPS relative to vehicle-treated control conditions, it increased Arg1 mRNA expression, suggesting that M. vaccae NCTC 11659 induces an atypical microglial phenotype. Thus, M. vaccae NCTC 11659 acutely (within 48 h) induced immune-activating and microglial-priming effects when applied directly to murine BV-2 microglial cells, in contrast to its long-term anti-inflammatory and immunoregulatory effects observed on the CNS when whole-cell, heat-killed preparations of M. vaccae NCTC 11659 were given peripherally in vivo.


Introduction
Stress-related psychiatric disorders, including anxiety disorders, mood disorders, and trauma-and stressor-related disorders, such as posttraumatic stress disorder (PTSD), are a significant burden worldwide [1,2].Psychosocial stress and chronic low-grade inflammation are considered risk factors for the development of stress-related psychiatric disorders [3,4].The "Old Friends" hypothesis posits that increasing rates of inflammation, inflammatory diseases, and stress-related psychiatric disorders in industrialized nations are due, at least in part, to reduced exposure to immunoregulatory microorganisms (i.e., microorganisms that promote a balanced expression of regulatory and effector T cells) [5].Consequently, one strategy to reduce the risk of these conditions would be to use immunoregulatory approaches to mitigate stress-induced inflammatory responses [6,7].
One of the "Old Friends" is M. vaccae NCTC 11659, a soil-derived bacterium with antiinflammatory and immunoregulatory properties that, when given via the subcutaneous or oral route, can prevent inflammatory conditions, such as allergic airway inflammation in a murine model of allergic asthma [8][9][10], or prevent outcomes of stressor exposures that are driven by inappropriate inflammation [6].Both the effects of M. vaccae NCTC 11659 on allergic airway inflammation [10] and its effects on negative outcomes of stressor exposure (e.g., stress-induced colitis and stress-induced exaggeration of anxiety-like or fear-like behaviors [11]) are dependent on regulatory T cells (Treg) in the periphery.
In addition to these effects of M. vaccae NCTC 11659 on peripheral immune signaling when given via the subcutaneous or oral route, it also influences innate immune cells within the central nervous system (CNS), i.e., microglia.Immunization with M. vaccae NCTC 11659 via the subcutaneous route can, (1) by itself, decrease hippocampal Nfkbia and Nlrp3 mRNA expression and (2) prevent stress-induced microglial priming, as assessed by LPS-induced increases in Il1b and Nfkbia mRNA expression in freshly isolated hippocampal microglia [12][13][14][15].However, the direct effects of M. vaccae NCTC 11659 on microglia, as might occur following the translocation of the live bacillus across the blood-cerebrospinal fluid barrier, for example, after the intravenous administration of a heat-killed suspension of Mycobacterium, as recently conducted using Mycobacterium indicus pranii (Mycobacterium w) in critically ill COVID-19 patients [16,17] or as recently conducted using the intravenous administration of the Bacillus Calmette-Guérin (BCG) vaccine in macaques [18], have not been studied.
Therefore, we set out to determine the effects of M. vaccae NCTC 11659, relative to vehicle control conditions, applied directly to murine BV-2 microglial cells when followed, 24 h later, by an immune challenge with LPS or vehicle control conditions.For a detailed description of the history of the development of M. vaccae NCTC 11659 used here, see [19].
For a description of the alternative designations and different preparations and production processes of M. vaccae NCTC 11659 used in clinical trials or preclinical studies, see [20].

Experiment 1: LPS Dose-Response
Lipopolysaccharide (LPS) is an endotoxin derived from the outer membrane of Gramnegative bacteria, and it is known to induce a robust immune response.In BV-2 cells, LPS increases Il1b mRNA expression [21] and induces the release of functional IL-1β, a proinflammatory cytokine [22].In contrast, LPS decreases Il10 mRNA and interleukin 10 (IL-10) protein expression, an anti-inflammatory cytokine, in murine BV-2 cells [23].Finally, LPS enhances NF-κB signaling in BV-2 cells [23], and studies using siRNA knockdown of Nfkb1, which encodes NFκB1 p50, suggest that Nfkb1 plays a key role in the proinflammatory response to LPS and M1 polarization [24].Therefore, we conducted dose-response studies to evaluate the effects of LPS on Il1b, Il10, and Nfkb1 mRNA expression in murine BV-2 cells.LPS induced dose-dependent increases in Il1b and Nfkb1 (Figure 1A,C).In contrast, as expected, LPS induced a decrease in Il10 mRNA expression (Figure 1B).Taken together, these data demonstrate that, under the conditions used here, LPS dose-dependently induced the expected proinflammatory response and shift toward the M1 immunophenotype of BV-2 cells.We selected the 250 ng/mL LPS concentration for further experiments since it induced a greater-than-2-fold change in all genes studied and induced the maximum response in Nfkb1 mRNA expression.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 3 of 20 description of the history of the development of M. vaccae NCTC 11659 used here, see [19].For a description of the alternative designations and different preparations and production processes of M. vaccae NCTC 11659 used in clinical trials or preclinical studies, see [20].

Experiment 1: LPS Dose-Response
Lipopolysaccharide (LPS) is an endotoxin derived from the outer membrane of Gram-negative bacteria, and it is known to induce a robust immune response.In BV-2 cells, LPS increases Il1b mRNA expression [21] and induces the release of functional IL-1β, a proinflammatory cytokine [22].In contrast, LPS decreases Il10 mRNA and interleukin 10 (IL-10) protein expression, an anti-inflammatory cytokine, in murine BV-2 cells [23].Finally, LPS enhances NF-κB signaling in BV-2 cells [23], and studies using siRNA knockdown of Nfkb1, which encodes NFκB1 p50, suggest that Nfkb1 plays a key role in the proinflammatory response to LPS and M1 polarization [24].Therefore, we conducted doseresponse studies to evaluate the effects of LPS on Il1b, Il10, and Nfkb1 mRNA expression in murine BV-2 cells.LPS induced dose-dependent increases in Il1b and Nfkb1 (Figure 1A,C).In contrast, as expected, LPS induced a decrease in Il10 mRNA expression (Figure 1B).Taken together, these data demonstrate that, under the conditions used here, LPS dose-dependently induced the expected proinflammatory response and shift toward the M1 immunophenotype of BV-2 cells.We selected the 250 ng/mL LPS concentration for further experiments since it induced a greater-than-2-fold change in all genes studied and induced the maximum response in Nfkb1 mRNA expression.
Figure 1.Effects of lipopolysaccharide (LPS) on Il1b, Il10, and Nfkb1 mRNA expression in murine BV-2 cells.Murine BV-2 microglial cells were treated with 0, 1, 10, 100, 250, or 1000 ng/mL LPS for 24 h.Cells were harvested, and cDNA was prepared from total RNA.Gene expression was measured using real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and is represented relative to the highest Ct value for each gene using the 2 −∆∆Ct method, with Actb, which encodes β-actin, as a reference.Data represent mean quantification values (Ct) + SEM, i.e., relative expression of (A) Il1b, (B) Il10, and (C) Nfkb1 mRNA.Data were analyzed using a one-way ANOVA followed by Fisher s LSD test using a single pooled error value, if appropriate, at a two-tailed alpha level of 0.05.* p < 0.05, Fisher s LSD test compared to cell media vehicle (CMV) control group.For all experimental conditions, there were 8 technical replicates (n = 8) from n = 1 experiment.

Experiment 2: Principal Coordinate Analysis of All Samples Used for the NanoString Platform
The analysis of all samples used for the NanoString platform using a principal coordinate analysis (PCoA) revealed the close clustering of samples within each treatment group (Figure 1).The PC1 axis accounted for 95.48% of the variation, predominantly representing the variation among groups due to treatment with LPS.The PC2 axis accounted for 3.36% of the variation, predominantly representing the variation among groups due to treatment with M. vaccae NCTC 11659.Together, PC1 and PC2 accounted for 98.84% of Figure 1.Effects of lipopolysaccharide (LPS) on Il1b, Il10, and Nfkb1 mRNA expression in murine BV-2 cells.Murine BV-2 microglial cells were treated with 0, 1, 10, 100, 250, or 1000 ng/mL LPS for 24 h.Cells were harvested, and cDNA was prepared from total RNA.Gene expression was measured using real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and is represented relative to the highest Ct value for each gene using the 2 −∆∆Ct method, with Actb, which encodes β-actin, as a reference.Data represent mean quantification values (Ct) + SEM, i.e., relative expression of (A) Il1b, (B) Il10, and (C) Nfkb1 mRNA.Data were analyzed using a one-way ANOVA followed by Fisher's LSD test using a single pooled error value, if appropriate, at a two-tailed alpha level of 0.05.* p < 0.05, Fisher's LSD test compared to cell media vehicle (CMV) control group.For all experimental conditions, there were 8 technical replicates (n = 8) from n = 1 experiment.

Experiment 2: Principal Coordinate Analysis of All Samples Used for the NanoString Platform
The analysis of all samples used for the NanoString platform using a principal coordinate analysis (PCoA) revealed the close clustering of samples within each treatment group (Figure 1).The PC1 axis accounted for 95.48% of the variation, predominantly representing the variation among groups due to treatment with LPS.The PC2 axis accounted for 3.36% of the variation, predominantly representing the variation among groups due to treatment with M. vaccae NCTC 11659.Together, PC1 and PC2 accounted for 98.84% of the total variation.An analysis using PERMANOVA revealed significant differences among the groups (F (4,7) = 2.69, p < 0.001; Figure 2).the total variation.An analysis using PERMANOVA revealed significant differences among the groups (F(4,7) = 2.69, p < 0.001; Figure 2).

Figure 2.
The principal coordinate analysis (PCoA) plot provides a visual representation of the pattern of proximity among all samples in the study based on an analysis of 248 endogenous inflammation-related genes using the nCounter Mouse Inflammation v2 Panel in the NanoString nCounter platform.The figure was generated using the vegan, ecodist, and ggplot2 packages in RStudio.The sample sizes of all experimental conditions were n = 3 technical replicates.PC1 explains 95.48% of the variability, and PC2 explains 3.36%.Abbreviations: BBS, borate-buffered saline; CMV, cell media vehicle; LPS, lipopolysaccharide; M. vaccae NCTC 11659, Mycobacterium vaccae NCTC 11659; PC1, principal coordinate 1; PC2, principal coordinate 2.

Effects of M. vaccae NCTC 11659 on mRNA Expression in BV-2 Microglial Cells Based on Analysis Using the NanoString Platform
Studies using M. vaccae NCTC 11659 in vivo have shown that, in the absence of subsequent stress exposure or LPS challenge, M. vaccae NCTC 11659 has an adjuvant-like effect, e.g., increasing bronchopulmonary Il1b, Il6, and Tnf mRNA expression 12 h to 3 days following intratracheal administration [25] and resulting in a persistent increase in the secretion of IL-6 from mesenteric lymph node cells stimulated with an anti-CD3 antibody in vitro [11] or a persistent increase in hippocampal Il6 mRNA expression [20].Likewise, in vitro studies have revealed that the treatment of human THP-1-derived-macrophages with M. vaccae NCTC 11659 increases the expression of genes involved in proinflammatory signaling, including Il1b, Ccl2, and numerous other canonical inflammatory markers [19] (Holbrook et al., 2023).In the present study, 48 h following the exposure of BV-2 cells to 100 µg/mL M. vaccae NCTC 11659, 5 of the 248 endogenous genes in the CodeSet (i.e., 2%) were differentially expressed (p < 0.05 and fold change either less than -1.5 or greater than 1.5) within the CMV groups between M. vaccae NCTC 11659 conditions (i.e., 100 µg/mL M. vaccae NCTC 11659/CMV vs. BBS/CMV) (Figure 3A).Of these five differentially expressed genes, three were upregulated (C3, Ccl2, and Tnf) and two were downregulated (Arg1 and Smad7, which encodes Smad7.Smad7 inhibits transforming growth factor beta Figure 2. The principal coordinate analysis (PCoA) plot provides a visual representation of the pattern of proximity among all samples in the study based on an analysis of 248 endogenous inflammationrelated genes using the nCounter Mouse Inflammation v2 Panel in the NanoString nCounter platform.The figure was generated using the vegan, ecodist, and ggplot2 packages in RStudio.The sample sizes of all experimental conditions were n = 3 technical replicates.PC1 explains 95.48% of the variability, and PC2 explains 3.36%.Abbreviations: BBS, borate-buffered saline; CMV, cell media vehicle; LPS, lipopolysaccharide; M. vaccae NCTC 11659, Mycobacterium vaccae NCTC 11659; PC1, principal coordinate 1; PC2, principal coordinate 2.

Effects of M. vaccae NCTC 11659 on mRNA Expression in BV-2 Microglial Cells Based on Analysis Using the NanoString Platform
Studies using M. vaccae NCTC 11659 in vivo have shown that, in the absence of subsequent stress exposure or LPS challenge, M. vaccae NCTC 11659 has an adjuvant-like effect, e.g., increasing bronchopulmonary Il1b, Il6, and Tnf mRNA expression 12 h to 3 days following intratracheal administration [25] and resulting in a persistent increase in the secretion of IL-6 from mesenteric lymph node cells stimulated with an anti-CD3 antibody in vitro [11] or a persistent increase in hippocampal Il6 mRNA expression [20].Likewise, in vitro studies have revealed that the treatment of human THP-1-derived-macrophages with M. vaccae NCTC 11659 increases the expression of genes involved in proinflammatory signaling, including Il1b, Ccl2, and numerous other canonical inflammatory markers [19] (Holbrook et al., 2023).In the present study, 48 h following the exposure of BV-2 cells to 100 µg/mL M. vaccae NCTC 11659, 5 of the 248 endogenous genes in the CodeSet (i.e., 2%) were differentially expressed (p < 0.05 and fold change either less than -1.5 or greater than 1.5) within the CMV groups between M. vaccae NCTC 11659 conditions (i.e., 100 µg/mL M. vaccae NCTC 11659/CMV vs. BBS/CMV) (Figure 3A).Of these five differentially expressed genes, three were upregulated (C3, Ccl2, and Tnf ) and two were downregulated (Arg1 and Smad7, which encodes Smad7.Smad7 inhibits transforming growth factor beta (TGFβ) signaling by preventing the formation of Smad2/Smad4 complexes that initiate TGFβ signaling [26]) (for a list of differentially expressed genes, see Table S1; for a list of genes that had p-values less than 0.05, see Table S2).Note that a large number of genes (47 genes with fold changes <1.5 but > 0; 61 genes with fold changes >-1.5 but <0) had significantly different expression levels between groups based on p-values but did not meet the criterion for an absolute value that exceeded the Log2 fold change.
(TGFβ) signaling by preventing the formation of Smad2/Smad4 complexes that initiate TGFβ signaling [26]) (for a list of differentially expressed genes, see Table S1; for a list of genes that had p-values less than 0.05, see Table S2).Note that a large number of genes (47 genes with fold changes <1.5 but > 0; 61 genes with fold changes >-1.5 but < 0) had significantly different expression levels between groups based on p-values but did not meet the criterion for an absolute value that exceeded the Log2 fold change.The x-axis represents the log ratio of the fold change, and the y-axis represents the negative log of the p-value, which was derived from a moderated t-test.We used the limma R library [27] in order to calculate fold changes and p-values in ROSALIND ® .Each dot in each of the figure panels represents a different gene.(A) The pale cornflower-blue dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the M. vaccae NCTC 11659/CMV-treated group relative to the BBS/CMV-treated group.The melon dots (upper right of the volcano plot) represent genes in the M. vaccae NCTC 11659/CMV-treated group that were expressed at higher levels relative to the BBS/CMV-treated group.(B) The pale-cornflower-blue dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the BBS/LPS-treated group relative to the BBS/CMV-treated group.The sea-blue dots (upper right of the volcano plot) represent genes in the BBS/LPS-treated group that were expressed at higher levels relative to the BBS/CMV-treated group.(C) The sea-blue dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the M. vaccae NCTC 11659/LPS-treated group relative to the BBS/LPS-treated group.The metallic-red dots (upper right of the volcano plot) represent genes in the M. vaccae NCTC 11659/LPS-treated group that were expressed at higher levels relative to the BBS/LPS-treated group.(D) The melon dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the M. vaccae NCTC 11659/LPS-treated group relative to the M. vaccae NCTC 11659/CMV-treated group.The metallic-red dots (upper right of the volcano plot) represent genes in the M. vaccae NCTC 11659/LPS-treated group that were expressed at higher levels relative to the M. vaccae NCTC 11659/CMV-treated group.Black dots in panels (A-D) represent genes that were not found to be differentially expressed between the groups.The black dashed vertical lines represent Log2 fold changes that exceed |0.6|, i.e., genes with a fold change of at least 1.5.The black dashed horizontal line in panels (A-D) indicates a p-value of 0.05, expressed as the -log10 p-value, with values derived from a moderated t-test.Volcano plots were generated using RStudio with ggplot2 and ggrepel packages.See Table S3 for a list of definitions of gene symbols.

Effects of LPS on mRNA Expression in Murine BV-2 Microglial Cells Based on Analysis Using the NanoString Platform
Previous studies have demonstrated that 250 ng/mL LPS (Escherichia coli 0111:B4) potently induces inflammation in BV-2 microglial cells and human THP-1-derived macrophages [19,21,28].Twenty-four hours following the exposure of BV-2 cells to 250 ng/mL LPS, relative to CMV control conditions, 71 of the 248 endogenous inflammationrelated genes in the CodeSet (i.e., 28.6%) were differentially expressed, as assessed by a moderated t-test (i.e., satisfied the criteria of p < 0.05 and fold change either less than -1.5 or greater than 1.5) (i.e., BBS/250 ng/mL LPS vs. BBS/CMV) (Figure 3B).Of these 71 differentially expressed genes, 36 were upregulated and 35 were downregulated (for a list of differentially expressed genes, see Table S4, and for a list of genes that had p-values less than 0.05, see Table S5).Note that a large number of genes had significantly different expression levels between groups based on p-values but did not meet the criterion for an absolute value that exceeded the Log2 fold change.

Differential Gene Expression between M. vaccae NCTC 11659/LPS as Compared to BBS/LPS Based on Analysis Using the NanoString Platform
Previous studies have shown that 100 µg/0.1 mL M. vaccae NCTC 11659, given by s.c.injection once a week for three weeks, can attenuate the stress-induced exaggeration of inflammatory hippocampal gene expression [20].In THP-1-derived macrophages, pretreatment with 300 µg/mL M. vaccae NCTC 11659 24 h prior to LPS exposure, relative to the BBS/LPS condition, increases the expression of a number of genes involved in anti-inflammatory signaling, including Il10, Il10rb, Tgfb2, Tgfbr1, and Mrc1, and decreases the expression of genes encoding proinflammatory cytokines, such as Il12b [19].In the present study, among cells exposed to LPS-challenge conditions, exposure to 100 µg/mL M. vaccae NCTC 11659 relative to the BBS control condition (i.e., 100 µg/mL M. vaccae NCTC 11659/250 ng/mL LPS vs. BBS/250 ng/mL LPS) led to the differential expression of 3 of the 248 genes in the CodeSet (i.e., 1.2%) (p < 0.05 and fold change either less than -1.5 or greater than 1.5) when assessed 48 h following exposure to M. vaccae NCTC 11659 (Figure 3C).These three differentially expressed genes (in order of descending absolute fold change) were Ccl7 (upregulated), Csf3 (upregulated), and Cysltr1 (downregulated; for a list of differentially expressed genes, see Table S6; for a list of genes that had p-values less than 0.05, see Table S7).Note that a large number of genes had significantly different expression levels between groups based on p-values but did not meet the criteria for an absolute value that exceeded the Log2 fold change.

Differential Gene Expression between M. vaccae NCTC 11659/LPS as Compared to M. vaccae NCTC 11659/CMV Based on Analysis Using the NanoString Platform
The NanoString panel that was used includes genes (such as Il1b, Il6, Tnf, Nos2, C-C chemokine ligand Ccl2, Tlr2, Tgfb, Il10, Il4, etc.) that can provide insight into whether or not the murine BV-2 microglial cells are polarized toward a proinflammatory phenotype or polarized toward an anti-inflammatory phenotype [29,30].Among M. vaccae NCTC 11659treated cells, 24 h of exposure to 250 ng/mL LPS, relative to the CMV control condition (i.e., 100 µg/mL M. vaccae NCTC 11659/250 ng/mL LPS vs. 100 µg/mL M. vaccae NCTC 11659/CMV), led to the differential expression of 72 of the 248 genes in the CodeSet (i.e., 29.03%) (p < 0.05 and fold change either less than -1.5 or greater than 1.5) (Figure 3D) (for a list of differentially expressed genes, see Table S8; for a list of genes that had p-values less than 0.05, see Table S9).Note that a large number of genes had significantly different expression levels between groups based on p-values but did not meet the criterion for an absolute value that exceeded the Log2 fold change.Despite preexposure to M. vaccae NCTC 11659, LPS challenge, relative to the CMV control condition, increased the markers of M1 polarization, including Il1b, Il6, Tnf, Nos2, and C-C chemokine ligand Ccl2, while it decreased Arg1, a marker of M2 polarization, suggesting that M. vaccae NCTC 11659 was not sufficient to prevent an LPS-induced shift toward an M1 microglia phenotype.

Validation of Effects of M. vaccae NCTC 11659 and LPS on Arg1 Gene Expression in BV-2 Microglial Cells Using Real-Time RT-PCR
The analysis using the NanoString platform indicated the decreased expression of Arg1 in murine BV-2 microglial cells following exposure to either M. vaccae NCTC 11659 or LPS (Figure 3A,B).To validate these Arg1 gene expression results, Arg1 gene expression was analyzed using real-time RT-PCR, using Actb as a housekeeping gene.We analyzed Actb for differential expression across treatment groups and found no differences in expression (Figure S2).The analysis of Arg1 expression revealed an interaction between M. vaccae NCTC 11659 and LPS (F (1,6) = 40.70,p < 0.001; Figure 4A, Tables S10 and S11).The post hoc analysis revealed a decrease in Arg1 expression in the M. vaccae NCTC 11659/CMV group relative to the BBS/CMV control condition (p < 0.05), as well as decreased Arg1 expression in the BBS/250 ng/mL LPS group relative to the BBS/CMV vehicle condition (p < 0.0001).
Although not detected based on the analysis using the NanoString platform, among the LPS-challenged groups, exposure to M. vaccae NCTC 11659 increased Arg1 expression (p < 0.001).Among M. vaccae NCTC 11659-exposed cells, LPS induced a significant, but attenuated, decrease in Arg1 expression (p < 0.001) (Figure 4A).It is important to note that, although M. vaccae NCTC 11659 attenuated the LPS-induced decrease in Arg1 mRNA expression, it did not prevent it.
absolute value that exceeded the Log2 fold change.Despite preexposure to M. vaccae NCTC 11659, LPS challenge, relative to the CMV control condition, increased the markers of M1 polarization, including Il1b, Il6, Tnf, Nos2, and C-C chemokine ligand Ccl2, while it decreased Arg1, a marker of M2 polarization, suggesting that M. vaccae NCTC 11659 was not sufficient to prevent an LPS-induced shift toward an M1 microglia phenotype.

Validation of Effects of M. vaccae NCTC 11659 and LPS on Arg1 Gene Expression in BV-2 Microglial Cells Using Real-Time RT-PCR
The analysis using the NanoString platform indicated the decreased expression of Arg1 in murine BV-2 microglial cells following exposure to either M. vaccae NCTC 11659 or LPS (Figure 3A,B).To validate these Arg1 gene expression results, Arg1 gene expression was analyzed using real-time RT-PCR, using Actb as a housekeeping gene.We analyzed Actb for differential expression across treatment groups and found no differences in expression (Figure S2).The analysis of Arg1 expression revealed an interaction between M. vaccae NCTC 11659 and LPS (F(1,6) = 40.70,p < 0.001; Figure 4A, Tables S10 and S11).The post hoc analysis revealed a decrease in Arg1 expression in the M. vaccae NCTC 11659/CMV group relative to the BBS/CMV control condition (p < 0.05), as well as decreased Arg1 expression in the BBS/250 ng/mL LPS group relative to the BBS/CMV vehicle condition (p < 0.0001).Although not detected based on the analysis using the NanoString platform, among the LPS-challenged groups, exposure to M. vaccae NCTC 11659 increased Arg1 expression (p < 0.001).Among M. vaccae NCTC 11659-exposed cells, LPS induced a significant, but attenuated, decrease in Arg1 expression (p < 0.05) (Figure 4A).It is important to note that, although M. vaccae NCTC 11659 attenuated the LPS-induced decrease in Arg1 mRNA expression, it did not prevent it., and (F) Nlrp3 was measured using real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and is represented relative to the highest Ct value for each gene using the 2 −∆∆Ct method.Data in panels A-F represent means + SEM.Data were analyzed using a two-factor ANOVA followed by Fisher's LSD test using a pooled error value, if appropriate, and a two-tailed alpha level of 0.05.* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.For all experimental conditions, there were 3 technical replicates (n = 3) from n = 1 experiment.Abbreviations: BBS, boratebuffered saline; CMV, cell media vehicle; LPS, lipopolysaccharide; NCTC, National Collection of Type Cultures.For definitions of gene symbols, see Table S3.For sample sizes, see Table S10.

Validation of Effects of M. vaccae NCTC 11659 and LPS on Genes Encoding Proinflammatory
Cytokines and Chemokine Ligands (i.e., Il1b, Il6, Tnf, and Ccl2 Gene Expression) in BV-2 Microglial Cells Using Real-Time RT-PCR The analysis using the NanoString platform revealed that exposure to M. vaccae NCTC 11659 alone increased the expression of Tnf and Ccl2, while LPS alone or in the presence of M. vaccae NCTC 11659 increased the expression of Il1b, Il6, Tnf, and Ccl2 in BV-2 microglial cells (Figure 3A,B,D).To validate these results from the NanoString analysis, Il1b, Il6, Tnf, and Ccl2 gene expression levels were analyzed using real-time RT-PCR, using Actb as a housekeeping gene.

Validation of Effects of M. vaccae NCTC 11659 and LPS on Nlrp3 mRNA Expression in BV-2 Microglial Cells Using Real-Time RT-PCR
Our prior work suggests that Nlrp3 inflammasome priming plays a pivotal role in stress-induced neuroinflammatory and microglial priming [31].Conversely, immunization with whole-cell, heat-killed M. vaccae NCTC 11659 by itself decreases hippocampal Nlrp3 mRNA expression and attenuates stress-induced hippocampal microglial priming when assessed one week later [13].The analysis using the NanoString platform demonstrated an increase in the expression of Nlrp3 mRNA following exposure to LPS among BV-2 microglial cells previously exposed to M. vaccae NCTC 11659 (Figure 3D).To validate these results, Nlrp3 mRNA expression was analyzed using real-time RT-PCR, using Actb as a housekeeping gene.The analysis of Nlrp3 mRNA expression revealed main effects for both M. vaccae NCTC 11659 (F (1,6) = 11.63,p < 0.05; Figure 4F and Table S10) and LPS (F (1,6) = 78.61,p < 0.0001; Figure 4F and Table S10).The post hoc analysis revealed that Nlrp3 mRNA expression was greater in the BBS/250 ng/mL LPS group relative to the BBS/CMV vehicle condition (p < 0.01; Figure 4F).Among M. vaccae NCTC 11659-treated groups, LPS increased Nlrp3 mRNA expression (i.e., M. vaccae NCTC 11659/LPS group relative to the M. vaccae NCTC 11659/BBS control condition (p < 0.001; Figure 4F)).Likewise, greater Nlrp3 mRNA expression was observed in the M. vaccae NCTC 11659/250 ng/mL LPS group relative to the BBS/250 ng/mL LPS vehicle condition (p < 0.01) (Figure 4F and Table S10), suggesting that M. vaccae NCTC 11659 promoted neuroinflammatory and microglial priming.For access to all data for all studies reported here, see File S1.

Discussion
Here, we report that the exposure of murine BV-2 microglial cells to M. vaccae NCTC 11659 by itself induced an "adjuvant-like" effect, increasing the expression of canonical proinflammatory genes, such as Il1b, Il6, and Tnf.As expected, the exposure of murine BV-2 microglial cells to LPS induced a strong polarization of BV-2 microglia toward an inflammatory phenotype relative to the CMV vehicle control condition, including increases in the expression of genes encoding canonical proinflammatory cytokines, chemokine ligands, and the Nlrp3 inflammasome.In most cases, pretreatment with M. vaccae NCTC 11659 was found to enhance the effects of LPS, suggesting that M. vaccae NCTC 11659 exaggerated the polarization of BV-2 cells, inducing neuroinflammatory and microglial priming responses.However, treatment with M. vaccae NCTC 11659 protected against the LPS-induced reduction in Arg1, suggesting that M. vaccae NCTC 11659 attenuated the LPS-induced shift toward an inflammatory metabolic phenotype [29,30,[32][33][34][35]. Together, these data suggest that exposure to M. vaccae NCTC 11659 induces an atypical microglial phenotype, characterized by enhanced markers of neuroinflammation and microglial priming, while at the same time attenuating the LPS-induced decrease in Arg1, suggesting an attenuation of the LPS-induced inflammatory metabolic phenotype.

M. vaccae NCTC 11659 by Itself Polarized Murine BV-2 Microglial Cells toward a Proinflammatory Phenotype
Treatment with M. vaccae NCTC 11659 alone induced an "adjuvant-like" effect in BV-2 microglial cells, which, based on real-time RT-PCR, was evidenced by the increase in the gene expression of Il1b, Il6, and Tnf.This was expected as, when animals are immunized with M. vaccae NCTC 11659 without subsequent exposure to a stressor, M. vaccae NCTC 11659 induces small fold-change increases in markers of inflammation.For example, the intratracheal administration of M. vaccae NCTC 11659 in s.c.M. vaccae NCTC 11659immunized mice (days -28 and -14) induces increases in bronchopulmonary Il1b, Il6, and Tnf mRNA expression 12 h to 3 days later [25].Similarly, the s.c.administration of M. vaccae NCTC 11659 in unstressed, single-housed control mice increases the secretion of IL-6 from freshly isolated mesenteric lymph node cells stimulated with an anti-CD3 antibody, assessed three weeks following immunization [11].Finally, the s.c.administration of M. vaccae NCTC 11659 increases hippocampal Il6 mRNA expression in unstressed control rats, assessed one week following immunization [20].Consistent with these significant, but small fold-change, increases in proinflammatory gene expression, 47 genes in the CodeSet were significantly increased, as assessed at p < 0.05, but did not exceed the threshold of 1.5-fold change conventionally required for differential expression.

LPS Strongly Polarizes Murine BV-2 Microglial Cells toward a Proinflammatory Phenotype
The exposure of murine BV-2 microglial cells to LPS, as expected, induced a strong polarization toward a proinflammatory phenotype when assessed 24 h later, with the differential expression of 28.6% of the genes analyzed relative to the CMV control condition, including increases in the expression of genes encoding canonical proinflammatory cytokines such as IL-1β, IL-6, and Tnf, and chemokine ligands such as Ccl2.The induction of inflammatory mRNA expression by LPS in murine BV-2 cells is consistent with previous studies [23,[36][37][38].Conversely, LPS decreased the expression of Arg1 and Mrc1 (Cd206, encoding mannose receptor C-Type 1), likewise consistent with the polarization of murine BV-2 microglial cells toward a more inflammatory phenotype [29,35].

M. vaccae NCTC 11659 Enhanced the Polarizing Effects of LPS on Inflammatory mRNA Expression in Murine BV-2 Microglial Cells, Consistent with Neuroinflammatory and Microglial Priming
Among cells exposed to LPS, exposure to M. vaccae NCTC 11659 primed murine BV-2 microglial cells to respond to LPS with exaggerated increases in the expression of canonical inflammatory markers, i.e., Tnf and Nlrp3, which plays a pivotal role in stress-induced neuroinflammatory and microglial priming [31].These effects were confirmed using realtime RT-PCR.The overall effects of M. vaccae NCTC 11659 in enhancing inflammatory responses induced by LPS in microglia are distinct from the effects of M. vaccae NCTC 11659 on peripheral immune cells.Previous studies have shown that human THP-1-derived macrophages respond to exposure to M. vaccae NCTC 11659 with attenuated LPS-induced decreases in Il10:Il12b, Il10:Il23a, Tgfb1:Il12b, and Tgfb1:Il23a mRNA expression ratios [19].
Previous studies conducted in vivo demonstrate that the peripheral administration (i.e., s.c.) of M. vaccae NCTC 11659 attenuates stressor-induced inflammation, neuroinflammation, and microglial priming while promoting stress resilience [6,[11][12][13]20].The current study implies that the ability of M. vaccae NCTC 11659 to attenuate stressor-induced neuroinflammation and microglial priming is not due to direct exposure to the residential immune cells in the CNS but rather through the peripheral immune system, secondarily impacting microglial cells.Another possibility is that the anti-inflammatory effects of M. vaccae NCTC 11659 emerge over a longer time frame.Studies conducted using immunization with M. vaccae NCTC 11659 tend to evaluate impacts on stress-induced inflammatory responses at least one week following immunization, whereas the studies conducted here evaluated responses to LPS one day following treatment with M. vaccae NCTC 11659 [6,[11][12][13]20].Indeed, increases in the serum concentrations of anti-inflammatory cytokines, such as IL-4, IL-10, and TGFβ, in mice peak four to six weeks following infection with a strain of M. vaccae isolated from bovine submaxillary lymph nodes [39], suggesting a delayed and persistent anti-inflammatory response to M. vaccae in vivo.

M. vaccae NCTC 11659 Attenuated LPS-Induced Reduction in Arg1 Gene Expression
The dramatic LPS-induced reduction in Arg1 was blunted by previous exposure to M. vaccae NCTC 11659.These data suggest that the direct exposure of BV-2 microglia to M. vaccae NCTC 11659 induces a complex microglial phenotype characterized by increased neuroinflammation and microglial priming, in association with the attenuation of LPSinduced decreases in Arg1.Since Arg1 is characteristic of microglia involved in wound healing, the phagocytosis of debris, inflammation inhibition, homeostasis restoration, and extracellular matrix protection [35], this finding raises the question of whether M. vaccae NCTC 11659 might, at the same time, (1) induce microglial priming and (2) promote a metabolic phenotype consistent with recovery from tissue damage.Future studies should evaluate the time course of gene expression in the current model to determine whether M. vaccae NCTC 11659 accelerates the transition to an anti-inflammatory phenotype following the initial immune activation.

Comparisons of the Effects of M. vaccae NCTC 11659 on the Immunophenotype of Microglia Following Administration In Vivo versus In Vitro
Previous studies have shown that when M. vaccae NCTC 11659 is given in vivo by subcutaneous injection, it can prevent microglial priming in rats when assessed one week later [12][13][14].Interestingly, when given in vivo in rats, M. vaccae NCTC 11659 decreases hippocampal Nfkbia [12,13] and Nlrp3 [13] mRNA expression, consistent with the finding that M. vaccae NCTC 11659 can prevent stress-induced hippocampal neuroinflammation and the priming of hippocampal microglia [12][13][14]20] when assessed one week after subcutaneous administration.In contrast, M. vaccae NCTC 11659 appears to have starkly different effects when applied directly to BV-2 cells, a hippocampal microglial cell line, when assessed 48 h following exposure.Specifically, under LPS-challenge conditions, the direct preexposure of BV-2 cells to M. vaccae NCTC 11659 enhanced LPS-induced increases in Nlrp3 mRNA expression, suggesting that either (1) the ability of the in vivo administration of M. vaccae NCTC 11659 to decrease Nlrp3 mRNA expression is not due to the direct effects of the bacterium on microglial cells or (2) the ability of M. vaccae NCTC 11659 to decrease Nlrp3 mRNA expression in the CNS occurs over a longer time course.One exception to these differences in the effects of M. vaccae NCTC 11659 when administered in vivo versus in vitro is the effect on Arg1 mRNA expression, a marker of an M2-like immunophenotype.M. vaccae NCTC 11659 increases Arg1 mRNA expression when administered either in vivo [12] or, specifically under LPS-challenge conditions, in vitro, suggesting that this is a highly conserved effect of M. vaccae NCTC 11659 on microglial cells.Since Arg1 expression in microglia is characteristic of an M2 anti-inflammatory phenotype [40,41], this may represent a conserved bias toward an atypical M2-like microglia phenotype in response to M. vaccae NCTC 11659 both in vivo and in vitro.This may be beneficial in some conditions, such as neurodegenerative diseases [40,42,43] and psychiatric disorders [30].

Limitations
One limitation of this report is that this study was conducted in vitro.It will be important to determine whether whole-cell, heat-killed M. vaccae NCTC 11659, when administered in vivo by either s.c.injection, intradermal injection, or other routes (e.g., oral, intranasal, intragastric, intratracheal, and intravenous routes), accesses the CNS, as observed with M. tuberculosis [44].Alternatively, M. vaccae NCTC 11659 may be phagocytosed by circulating monocytes or dendritic cells, which can enter the circulation and translocate across the blood-cerebrospinal fluid barrier or blood-brain barrier [44][45][46], in which case, it may have very different effects on microglial phenotypes.In addition, it will be important to fully characterize the time course of the effects of M. vaccae NCTC 11659 on inflammatory responses to a subsequent immune challenge beyond the 48 h time point studied here.Although we have characterized the immunophenotype of BV-2 cells in response to M. vaccae NCTC 11659 and LPS, the effects of treatment on the external morphology of the cells are also an important concern and should be addressed in future studies.Another point to consider is that the BV-2 cell line is derived from female mice.It will be important to see whether primary microglial cells, including microglial cells extracted from male mice, have a similar response to M. vaccae NCTC 11659.

Clinical Implications
The data from the current study suggest that the ability of immunization with M. vaccae NCTC 11659 or its component parts in vivo to attenuate LPS-induced inflammatory responses in microglia, as observed previously [12,13], appears to be due to the regulation of the peripheral immune system rather than due to direct actions on microglial cells.However, future studies of murine BV-2 microglia with extended time courses are required to determine whether the anti-inflammatory effects of M. vaccae NCTC 11659 are evident at time points when the anti-inflammatory effects of M. vaccae NCTC 11659 are observed in vivo, typically one to two weeks following treatment.In addition, future studies using primary cultures of microglia are needed to confirm that BV-2 cells and freshly isolated microglia respond to M. vaccae NCTC 11659 in a similar way.Nevertheless, the ability of M. vaccae NCTC 11659 to increase Arg1 mRNA expression, as observed now both in vivo [12] and in vitro, may lead to a less aggressive M1-like inflammatory environment, which may be beneficial in some conditions, including neurodegenerative diseases [40,42,43] and psychiatric disorders [30].

Conclusions and Future Directions
Although M. vaccae NCTC 11659 has been studied primarily in the context of the "Old Friends" hypothesis, the current study highlights how whole-cell, heat-killed M. vaccae NCTC 11659 acutely (i.e., within 48 h) enhanced LPS-induced inflammation in murine BV-2 cells, suggestive of enhanced neuroinflammation and microglial priming.These data indicate that the ability of M. vaccae NCTC 11659, when administered in vivo, to prevent stress-induced microglial priming in association with stress resilience effects in rodent models either is due to M. vaccae NCTC 11659's effects on the peripheral immune system, which indirectly attenuates stress-induced neuroinflammation and microglial priming, or evolves over a longer time course, or is due to a combination of these factors.In support of the hypothesis that the strong anti-inflammatory and immunoregulatory effects of M. vaccae NCTC 11659 develop over a longer time course, early responses to the administration of M. vaccae NCTC 11659 in vivo are dominated by proinflammatory responses (i.e., increased Il1b, Il6, and Tnf mRNA expression 12-72 h following administration) [25], whereas the anti-inflammatory and immunoregulatory effects of M. vaccae NCTC 11659 are typically observed later, i.e., one week after administration [11][12][13][14]20].Future studies are needed to determine whether M. vaccae NCTC 11659 or its component parts can access the CNS compartment, either as a single bacillus or in a "Trojan horse"-like manner, as has been observed with M. tuberculosis.

Murine BV-2 Microglial Cells
BV-2 microglial cells are an immortalized murine cell line that has been infected with a v-raf/v-myc oncogene-carrying retrovirus (J2) [47].We selected murine BV-2 microglial cells to evaluate the effects of M. vaccae NCTC 11659 on naïve and LPS-stimulated immune responses, as this cell line reproduces the effects of LPS on primary microglia with high fidelity while reducing the need for continuous cell preparation and animal experimentation [48].Cells were obtained from frozen stocks maintained in Gibco TM Recovery™ Cell Culture Freezing Medium (Cat.No. 12648-010, Thermo Fisher Scientific, Waltham, MA, USA).Briefly, for freezing cells, cells were cultured to be approximately 70-80% confluent, viable, and healthy before freezing.Fresh medium was added 5 h before freezing.For a 100 mm culture plate, 1.5-2 mL of trypsin plus 5 mL of medium was added to neutralize the trypsin.Cells were centrifuged at 1500 r.p.m. for 5 min, and the supernatant was discarded.Cells were resuspended in Gibco TM Recovery™ Cell Culture Freezing Medium and gently mixed to maintain a homogeneous cell suspension.Aliquots of 1 mL of cell suspension were dispensed into sterile cryogenic storage vials.Cryovials containing the cells were placed in an isopropanol chamber and stored at −80 • C overnight.The next day, vials containing frozen cells were transferred to liquid nitrogen.
Cells were received on passage four.Because they were semi-suspended, BV-2 cells were shaken to be lifted during passage.Cells were not used for more than 20 passages.For the experiments described here, BV-2 cells were cultured in Gibco TM Advanced DMEM/F12 (Cat.No. 12634010; Thermo Fisher Scientific) medium supplemented with Hyclone 10% fetal bovine serum (FBS; Cat.No. F9423, Sigma-Aldrich, Saint Louis, MO, USA) and Gibco TM 1% penicillin/streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin; Cat.No. 15140122, Thermo Fisher Scientific) under standard culture conditions (37 • C in a humidified 5% CO 2 incubator).The cells were plated on sterile 24-well tissue culture plates (Cat.No. 10062-896; VWR North American, Radnor, PA, USA).Each well was plated with 200,000 BV-2 cells in 0.5 mL of medium.To harvest BV-2 cells, the cells were washed with PBS prior to the addition of Gibco TM Trypsin-EDTA (0.25%) (Cat.No. 25200056, Thermo Fisher Scientific); the cells were then centrifuged (500 r.p.m. for 5 min at 21-23 • C) and resuspended in DMEM/F12 medium.

M. vaccae NCTC 11659
The material used here was provided as a 10 mg/mL stock suspension in sterile borate-buffered saline; strain National Collection of Type Cultures (NCTC) 11659, batch C079-ENG#1, provided by BioElpida (Lyon, France).For the heat-killing of M. vaccae NCTC 11659, the culture was centrifuged at 3000× g at 4 • C for ten minutes to pellet the cells, the growth medium was removed, and cells were weighed and resuspended in sterile borate-buffered saline (BBS) to a concentration of 10 mg/mL.Cells were transferred to a sealed sterile glass container and autoclaved at 121 • C for 15 min.The sterile heat-killed bacterial stock was stored at 4 • C, and M. vaccae NCTC 11659 was further diluted to a final in-well concentration of 96.99 µg/mL for the treatment of BV-2 cells.Stocks were swirled each time before they were pipetted into the wells to ensure that the suspension was distributed equally.After pipetting M. vaccae NCTC 11659 or vehicle into the wells, the plates were gently swirled to evenly distribute the treatments.For quality control, the M. vaccae NCTC 11659 material was tested to confirm its immunoregulatory effects (e.g., increased Il10:Il12a and Il10:Il12b mRNA expression) in murine bone-marrow-derived dendritic cells.

LPS
This study used lipopolysaccharide (LPS; Escherichia coli O111:B4; Cat.No. L2630, Sigma-Aldrich) as an immune challenge, while the remaining wells were challenged with a cell culture media vehicle (CMV) control condition.It is well documented that LPS (Escherichia coli O111:B4) induces a proinflammatory response, identified by canonical proinflammatory markers, in BV-2 microglial cells [23,36,37].

Experimental Timeline
The experimental timeline is illustrated in Figure 5.The experiment was conducted once, and the statistical analyses were performed on technical replicates.On Day 0, we treated six wells (200,000 BV-2 cells in 0.5 mL of medium) with a whole-cell, heat-killed preparation of M. vaccae NCTC 11659 (15 µL of 3.33 mg/mL M. vaccae NCTC 11659 in a final volume of 515 µL per well, resulting in a final concentration of 96.99 µg/mL, hereafter referred to as 100 µg/mL, of M. vaccae NCTC 11659) and six wells with 15 µL of vehicle (sterile BBS).Twenty-four hours later, on Day 1, we challenged three of the M. vaccae NCTC 11659-treated wells and three of the BBS-treated wells with 10 µL of 13.25 µg/mL LPS stock, resulting in each well having a concentration of 252.4 ng/mL LPS, hereafter referred to as 250 ng/mL (LPS; Escherichia coli O111:B4; Cat.No. L2630, Sigma-Aldrich), while the remaining wells were challenged with a cell culture media vehicle (CMV) control condition.On Day 2, the collection of BV-2 cells and the purification of total RNA were carried out, as described in Section 4.5.

RNA Isolation
Twenty-four hours after treating the cells with 250 ng/mL LPS (E. coli O111:B4) or CMV, total RNA was extracted from BV-2 cells using the QIAGEN RNeasy Mini kit (Cat.No. 74104, QIAGEN, Hilden, Germany).For each well, RNA extraction yielded >30 µg of RNA.Following the determination of RNA concentrations in each sample using a NanoDrop One machine (Cat.No. ND-ONE-W, Thermo Fisher Scientific), the RNA samples were diluted using nuclease-free water to 5 ng/µL.RNA samples were frozen at -80 °C be-

RNA Isolation
Twenty-four hours after treating the cells with 250 ng/mL LPS (E. coli O111:B4) or CMV, total RNA was extracted from BV-2 cells using the QIAGEN RNeasy Mini kit (Cat.No. 74104, QIAGEN, Hilden, Germany).For each well, RNA extraction yielded >30 µg of RNA.Following the determination of RNA concentrations in each sample using a NanoDrop One machine (Cat.No. ND-ONE-W, Thermo Fisher Scientific), the RNA samples were diluted using nuclease-free water to 5 ng/µL.RNA samples were frozen at -80 • C before submitting samples to the Veterans Health Administration, Rocky Mountain Regional Veterans Affairs Medical Center (RMRVAMC) Core Equipment facility.

NanoString nCounter Gene Expression
An analysis of NanoString nCounter gene expression was conducted according to vendor instructions.Briefly, 25 ng of total RNA per sample was processed with the NanoString nCounter system (NanoString, Seattle, WA, USA) using the nCounter Mouse Inflammation v2 Panel (Cat.No. XT-CSO-MIN2-12; NanoString).The NanoString nCounter ® Mouse Inflammation v2 Panel is a multiplex gene expression analysis platform that enables the analysis of 254 genes representing a range of inflammation-related pathways.The CodeSet includes 248 murine inflammation-related genes and 6 genes designated as housekeeping genes.A number of published studies have used the nCounter Mouse Inflammation v2 Panel ; to the best of our knowledge, however, this is the first study to use this platform to study murine BV-2 cells.
The 25 ng samples of RNA were mixed with the Reporter Codeset and Capture Probeset and then incubated at 65 • C for at least 16 h to allow adequate hybridization.Hybridization buffer was added to bring the samples to a volume of 30 µL, which was then loaded into the cartridge and run on the nCounter Sprint profiler.The data were normalized through RUV-III [77].The normalized data were then exported into ROSALIND ® (San Diego, CA, USA), a cloud-based genomics and gene expression analysis platform, and were analyzed using OnRamp software (version 3.35.12.0,OnRamp Bioinformatics, Inc., San Diego, CA, USA).

Real-Time RT-PCR and Primers
Real-time RT-PCR was used to analyze gene expression in two separate studies: Experiment 1, which evaluated the dose-dependent effects of LPS, and Experiment 2, which evaluated the effects of M. vaccae NCTC 11659, LPS, and their interaction.The methods used to conduct real-time RT-PCR for Experiment 1 and Experiment 2 were identical.
Aliquots of the stock samples of extracted mRNA used for the analysis using NanoString in Experiment 2 were also used for cDNA synthesis in order to perform real-time RT-PCR.For Experiments 1 and 2, approximately 500 ng of total RNA was used for the preparation of cDNA, which was conducted using Invitrogen SuperScript TM II Reverse Transcriptase (Cat.No. 18064014, Thermo Fisher Scientific) according to manufacturer instructions.Approximately 50 ng per well of cDNA was used as a template material for conducting real-time RT-PCR.Real-time RT-PCR was carried out in duplicate using a CFX96 Touch Real-Time PCR Detection System (Cat.No. 1845097, Bio-Rad, Hercules, CA, USA), together with QuantiTect SYBR Green Master Mix (Cat.No. 204145, QIAGEN, Hilden, Germany).For both Experiments 1 and 2, gene expression was normalized using Actb, which encodes beta-actin.Real-time RT-PCR data were represented as a fold increase relative to the lowest amount of mRNA expressed on the plate for each gene using the 2 -∆∆Ct method.
Primer sequences (Table 1) other than Arg1 were designed using the PrimerQuest TM Tool (Integrated DNA Technologies (IDT) website; https://www.idtdna.com/pages,accessed on 8 August 2019, Coralville, IA, USA).Primer sequences were designed to span exon/exon boundaries in order to exclude the amplification of genomic DNA.The murine Arg1 primer sequence (Table 1) was obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/, accessed on 28 December 2023, Cambridge, MA, USA).Sequence speci-ficity was tested using the Basic Local Alignment Search Tool at NCBI [77].Primers were obtained from IDT. Primer specificity was verified by melt curve analysis.

Statistical Analysis
Statistical approaches to the analysis of NanoString data and real-time RT-PCR data are outlined in detail below.

NanoString Analysis
The analysis of reference genes using the NanoString platform revealed that treatment conditions altered the expression of reference genes in the nCounter Mouse Inflammation v2 Panel, resulting in inadequate normalization (Figure S1).Therefore, we conducted the normalization of NanoString data in the statistical software program R using Removing Unwanted Variation-III (RUV-III), which selects stably expressed endogenous genes to use as housekeeping genes post hoc [78].
RUV-III-normalized data were imported into ROSALIND ® .Normalized data were analyzed using ROSALIND ® (https://rosalind.bio/,accessed on 1 October, 2023), with a HyperScale architecture developed by ROSALIND ® , Inc.The limma R library [27] was used to calculate fold changes and p-values in ROSALIND ® using a moderated t-test.The moderated t-test is often used for the analysis of studies with small sample sizes and can be interpreted in a similar manner to Student's t-test, but with the noteworthy difference that the standard errors are moderated using empirical Bayes methods [79,80].The statistical software program R was used to make principal coordinate analysis (PCoA) plots using the ggplot2, vegan, and ecodist packages.The statistical software program R was used to make volcano plots using the ggplot2 and ggrepel packages.Scatterplots were made using the ggplot2 and ggrepel R packages using the Log base 2 fold changes and p-values obtained from ROSALIND ® .

Real-Time RT-PCR Analysis
Relative gene expression was calculated using the 2 −∆∆Ct method [81], and relative gene expression data were analyzed using a two-way ANOVA followed by Fisher's LSD test, if appropriate, at a two-tailed alpha level of 0.05 using a single pooled error term for Fisher's LSD test [82].

Figure 3 .
Figure 3. Figure illustrating volcano plots representing differentially expressed genes between pairs of treatment groups.The x-axis represents the log ratio of the fold change, and the y-axis represents the negative log of the p-value, which was derived from a moderated t-test.We used the limma R library [27] (Ritchie et al., 2015) in order to calculate fold changes and p-values in ROSALIND ® .Each dot in each of the figure panels represents a different gene.(A) The pale cornflower-blue dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the M. vaccae NCTC 11659/CMV-treated group relative to the BBS/CMV-treated group.The melon dots (upper right of the volcano plot) represent genes in the M. vaccae NCTC 11659/CMV-treated group that were expressed at higher levels relative to the BBS/CMV-treated group.(B) The pale-cornflower-blue dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the BBS/LPS-treated group relative to the BBS/CMV-treated group.The sea-blue dots (upper right of the volcano plot) represent genes in the BBS/LPS-treated group that were expressed at higher levels relative to the BBS/CMV-treated group.(C) The sea-blue dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the M. vaccae NCTC 11659/LPS-treated group relative to the BBS/LPS-treated group.The metallic-red dots (upper right

Figure 3 .
Figure3. Figure illustrating volcano plots representing differentially expressed genes between pairs of treatment groups.The x-axis represents the log ratio of the fold change, and the y-axis represents the negative log of the p-value, which was derived from a moderated t-test.We used the limma R library[27] in order to calculate fold changes and p-values in ROSALIND ® .Each dot in each of the figure panels represents a different gene.(A) The pale cornflower-blue dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the M. vaccae NCTC 11659/CMV-treated group relative to the BBS/CMV-treated group.The melon dots (upper right of the volcano plot) represent genes in the M. vaccae NCTC 11659/CMV-treated group that were expressed at higher levels relative to the BBS/CMV-treated group.(B) The pale-cornflower-blue dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the BBS/LPS-treated group relative to the BBS/CMV-treated group.The sea-blue dots (upper right of the volcano plot) represent genes in the BBS/LPS-treated group that were expressed at higher levels relative to the BBS/CMV-treated group.(C) The sea-blue dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the M. vaccae NCTC 11659/LPS-treated group relative to the BBS/LPS-treated group.The metallic-red dots (upper right of the volcano plot) represent genes in the M. vaccae NCTC 11659/LPS-treated group that were expressed at higher levels relative to the BBS/LPS-treated group.(D) The melon dots (upper-left portion of the volcano plot) represent genes that were expressed at lower levels in the M. vaccae NCTC 11659/LPS-treated group relative to the M. vaccae NCTC 11659/CMV-treated group.The metallic-red dots (upper right of the volcano plot) represent genes in the M. vaccae NCTC 11659/LPS-treated group that were expressed at higher levels relative to the M. vaccae NCTC 11659/CMV-treated group.Black dots in panels (A-D) represent genes that were not found to be differentially expressed between the groups.The black dashed vertical lines represent Log2 fold changes that exceed |0.6|, i.e., genes with a fold change of at least 1.5.The black dashed horizontal line in panels (A-D) indicates a p-value of 0.05, expressed as the -log10 p-value, with values derived from a moderated t-test.Volcano plots were generated using RStudio with ggplot2 and ggrepel packages.See TableS3for a list of definitions of gene symbols.

Table 1 .
Primers used for real-time RT-PCR.Primers are listed from 5 ′ to 3 ′ .