key: cord-0312943-5w0flb5n
authors: Balderrama-Gutierrez, Gabriela; Milovic, Ana; Cook, Vanessa J.; Islam, M. Nurul; Zhang, Youwen; Kiaris, Hippokratis; Belisle, John T.; Mortazavi, Ali; Barbour, Alan G.
title: The infection-tolerant mammalian reservoir of Lyme disease and other zoonoses broadly counters the inflammatory effects of endotoxin
date: 2020-12-14
journal: bioRxiv
DOI: 10.1101/2020.12.13.422519
sha: 9092f791f072b6cbb5ebd64a16451091523e72c8
doc_id: 312943
cord_uid: 5w0flb5n

Animals that are competent natural reservoirs of zoonotic diseases commonly suffer little morbidity from the pathogens they persistently harbor. The mechanisms of this infection tolerance and the trade-off costs are poorly understood. We used exposure to a single dose of lipopolysaccharide (LPS) endotoxin as an experimental model of inflammation to compare the responses of the cricentine rodent Peromyscus leucopus, the white-footed deermouse, to that of Mus musculus, the standard laboratory model for pathogenesis studies. Four hours after injection with either LPS or saline, blood and spleen and liver tissues were collected postmortem and subjected to RNA-seq, untargeted metabolomics, and specific RT-qPCR. This was followed by analysis of differential expression at the gene, pathway, and empirical network levels. The deermice showed the same signs of sickness as the mice with LPS exposure, and in addition demonstrated comparable increases in levels of corticosterone and expression of interleukin (IL)-6, tumor necrosis factor, IL-1b, and acute phase reactants, including C-reactive protein. But whereas the M. musculus response to LPS was best-characterized by network analysis as cytokine-associated, the P. leucopus response was dominated by pathway terms associated with neutrophil activity. Dichotomies between the species in expression profiles of arginase 1 and nitric oxide synthase 2, as well as the ratios of IL-10 to IL-12, were consistent with a type M1 polarized macrophage in the mice and a type M2 or alternatively-activated response in the deermice. Analysis of metabolites in the plasma and RNA in the tissues revealed differences between the two species in tryptophan metabolism during response to LPS. Two up-regulated genes in particular signified the difference between the species: Slpi (secretory leukocyte proteinase inhibitor) and Ibsp (integrin-binding protein sialoprotein). The latter was previously unrecognized in the context of inflammation or infection. Key RNA-seq findings in P. leucopus were replicated in a second LPS experiment with older animals, in a systemic bacterial infection model, and with cultivated fibroblasts. Taken together, the results indicate that the deermouse possesses several adaptive traits to moderate effects of inflammation and oxidative stress ensuing from infection. This seems to be at the cost of infection persistence and that is to the benefit of the pathogen.

controls (mean of 7 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] ) (p = 0.008). But there was not an elevation in nine P. leucopus after LPS compared with six controls: 7 (3-11) vs 7 (3-12) (p = 0.9).

Metabolomics of plasma. We carried out untargeted metabolomics by LC/MS of the plasma of the LPS-treated and control P. leucopus and M. musculus. Of the 8125 molecular features (MF) identified in the M. musculus plasma samples, 123 (1.5%) differed between LPS-treated and control animals with a FDR < 0.05 and an absolute fold change of > 2.0. Of the 7714 MF identified in the P. leucopus plasma samples, 215 (2.8%) correspondingly differed between treated and control animals. Subsequent analysis focused on pathway enrichment, which allowed cross-species comparison of differences in the identifiable metabolites between treated and untreated animals. For the M. musculus and the P. leucopus samples the number of identified pathways with at least one KEGGdefineable compound were 76 and 73, respectively, of which 73 were in common (Table S2 ). Figure  2A plots pathway enrichment in deermice against that in mice for each of the pathways in common. In both species there was significant enrichment of the steroid hormone biosynthesis pathway in animals 4 h after LPS injection. This finding was consistent with the results of the immunoassays of corticosterone in the plasma of both species. This similarity in response held for most of the other pathways in which enrichment in a pathway could be determined. But there were exceptions, and we highlight three of these.

Of the nine putative constituents asigned to the KEGG vitamin B6 metabolism pathway, eight were significant hits in P. leucopus plasma and five in M. musculus samples. The three additional metabolites detected in deermice were pyridoxine (C00314), pyridoxamine (C00534), and 2-oxo-3hydroxy-4-phosphobutanoate (C06054). Pyridoxal (C00847) and pyridoxal 5'-phosphate (C00250) were enriched in both species of LPS-treated animals. The latter two vitamers were reported to have anti-inflammatory effects in an LPS model by reducing expression of IL-1b and suppressing NF-kB activation in macrophages and, moreover, protected mice from lethal endotoxic shock [55] . Among patients with sepsis, higher concentrations in vitamin B6 metabolite biomarkers in the blood were associated with greater likelihood of survival in two clinical research studies [56, 57] .

There was overall enrichment of the complex tryptophan metabolism in LPS-treated animals of both species, but the magnitude was greatest among the P. leucopus, which had 30 significant hits out of a possible 41 compounds in the tryptophan metabolism pathway (Figure 2A ; Table S2 ). Abundances of specific tryptophan metabolites were determined from peak areas. Tryptophan itself was at a significantly lower abundance in plasma of LPS-treated P. leucopus compared untreated animals but at a marginally higher level in M. musuculus after LPS treatment compared to controls ( Figure 2B and Table S3 ). Two metabolites of the indole 2,3-dioxygenase (Ido1) pathway, kynurenine and anthranilic acid, were similar in concentration between the two species under both conditions. In the melatonin pathway the concentration of serotonin was significantly lower in treated deermice than in untreated counterparts.

The third pathway, biotin metabolism, demonstrated the greatest magnitude difference between species (Figure 2A) , with only biotin (KEGG C00120) itself identified in mice, and three additional metabolites enriched in the plasma of LPS-treated deermice: biotinyl-5'-AMP (C05921), biocytin (C05552), and L-lysine (C00047). Biotin metabolism was reported as one of the pathways, along with vitamin B6 metabolism and tryptophan metabolism, associated with early Lyme disease in a study of urine metabolites of humans [58] . But since biotin is not synthesized by mammals, this difference between the groups may be attributable to differences in food intake during four hours of the experiment.

Differentially expressed genes in each species. We performed RNA-seq for 3 tissues (blood, spleen, and liver) of each species 4 h after injection of LPS or saline alone to explore the species-and tissue-specific responses. After calculating gene expression levels, we performed differential gene expression analysis using 24,295 and 35,805 annotated transcripts for P. leucopus or M. musculus, respectively, as reference sets in separate analyses for each species (Tables S4-9 ). The volcano plots of Figure 3 and Figure S2 show hundreds of genes that were either up-or down-regulated in the blood and two organs of each species by the criteria of ≥4 fold change and FDR of <0.05. With the exception of the spleens of P. leucopus, there were more up-regulated than down-regulated genes in each of the comparisons. The ratio of up-to down-regulated genes ranged from ~1x for mouse blood to ~5x for P. leucopus blood. The highest total numbers of DEGs were in the livers of each species: 1553 for P. leucopus and 3250 for M. musculus.

Examples of up-regulated DEGs shared between the two species in their responses to LPS were IL-10 (Il10) and granulocyte colony-stimulating factor (Csf3) in all tissues, IL-6 (Il6), granulocyte-macrophage colony-stimulating factor (Csf2), and pentraxin 3 (Ptx3) in the spleen and liver, and superoxide dismutase 2 (Sod2) in the spleen. A notable difference in the responses of P. leucopus and M. musculus was consistent with the finding of the nitric oxide assays of the plasma of these animals. Inducible nitric oxide synthase or nitric oxide synthase 2 (Nos2) transcript levels were a mean of 493x higher in the blood of LPS-treated mice than in the controls (FDR p = 10 -78 ). But in the blood of deermice Nos2 expression was barely detectable (TPM of 0.08 vs. 4.8 in mice), and expression of Nos2 was indistinguishable between the two conditions (FDR p = 0.34). In contrast, arginase 1 (Arg1), which by its action reduces the amount of arginine available for Nos2 to produce nitric oxide, was 21x higher in expression in the blood of LPS-treated P. leucopus than in controls (FDR p = 10 -47 ), while in M. musculus blood Arg1 expression 4 h after LPS injection was 6x lower than baseline expression (FDR p = 0.04). This reciprocal expression profile for Nos2 and Arg1 between the two species was also observed in the spleen. The products of Nos2 and Arg1 are informative biomarkers for categorizing polarized macrophage responses [59] . The disposition of P. leucopus to M2 polarization, characterized by high Arg1 and low Nos2 expression in activated macrophages, is consistent with the ability of deermice to moderate inflammation.

Two other genes whose expression profiles served to distinguish the two species in their responses to LPS were integrin-binding sialoprotein (Ibsp) and secretory leukocyte peptidase inhibitor (Slpi). These were first and fourth ranked of the up-regulated DEGs in the blood for P. leucopus (Table S4 ), but they ranked numbers 6232 and 33,541, respectively, among measured transcripts in the blood for M. musculus (Table S7 ). The fold differences between Ibsp and Slpi in the LPS-treated deermice and control animals were 2903x (FDR p = 10 -174 ) and 1280x (FDR p = 10 -57 ) higher, respectively. In contrast, Ibsp was lowly expressed in mice under both conditions (TPM of 0.001 and FDR p = 1.0), and Slpi in expression was decreased by 3x in the treated animals (FDR p = 0.04).

Sex-specific responses to LPS in P. leucopus. For the DEGs highlighted to this point the sex of the animal was not discernibly a major co-variable. To identify sex-specific responses the DEG analysis was applied to the RNA-seq of the blood and spleen of P. leucopus treated with LPS. The comparison groups were the 6 females and 6 males, as well as blood for the 4 females and 4 males that were controls. There was little overlap between the 71 sex-specific DEGs in LPS blood, the 56 in LPS spleen, and the 39 in control blood (Table S10) . But the few DEGs that were in common between at least two of the groups were notable. In all groups, including the controls, a non-coding RNA (XR_003736827.1) was expressed at 500-1000x higher levels in females than in males in all tissues. This ncRNA was revealed as the Xist or "inactive X specific transcript", which functions to inactivate genes on the second X chromosome of females and is orthologous to NR_001570.2 of M. musculus. D1Pas1, an autosomal DEAD-box RNA helicase, which is expressed in the testis in mice [60] , was markedly lower in expression in both treated and control females. Two genes that were more highly expressed by~1000-fold in the blood of females that received LPS over both their male counterparts and control females were the X-linked lysine-specific demethylase 6A (Kdm6a; XM_028854130.1) and adiponectin receptor 2 (Adipor2; XR_003735732.1). Kdm6a has been implicated in risk of autoimmunity and reported to regulate multiple immune response genes [61] . Adiponectin, an adipokine, has an anti-inflammatory effect [62] and expression of its receptor is reportedly affected by macrophage polarization phenotype [63] . Adipor2 was also highly expressed in the spleen of LPS-treated females.

Functional processes distinguishing and shared between species. To further define similarities and difference between the responses of the two species to LPS we identified gene groups categorized by GO terms as P. leucopus-specific, M. musculus-specific, or shared, meaning that within each pairing the enrichment groups that were up-or down-regulated. For this analysis 14,685 one-to-one orthologs were used for the comparisons. Figure 4 summarizes this analysis for blood, spleen, and liver. The genes associated with each GO term for which there was significant enrichment are listed in Table S11 .

In the blood both species showed up-regulation of some components of cytokine-mediated signaling and cellular responses to cytokines, as well as neutrophil activities. For cytokine-associated genes these included aconitate decarboxylase 1 (Acod1), chemokine C-X-C motif ligand 10 (Cxcl10), Cxcl11, Il1a, Il10, interleukin 1 receptor (Il1r), interleukin 1 receptor antagonist (Il1rn), superoxide dismutase 2 (Sod2), and TNF. Neutrophil-associated genes up-regulated in the blood of both species, including CD14 antigen (Cd14), Cd177, formyl peptide receptor 1 (Fpr1), Fpr2, lipocalin 2 (Lcn2), matrix metallopeptidase 8 (Mmp8), Ptx3, and toll-like receptor 2 (Tlr2). The Peromyscus-specific upregulated profile in the blood featured an expanded set of genes constituting GO terms for neutrophil activities, including arachidonate 5-lipoxygenase (Alox5), Cd33, C-X-C motif receptor 2 (Cxcr2), leucine-rich alpha-2 glycoprotein (Lrg), Mmp9, resistin (Retn), and Slpi. The blood samples of M. musculus uniquely featured GO terms related to the following: (1) blood coagulation and fibrinolysis, including apolipoprotein H (Apoh), coagulation factor II (F2), coagulation factor XII (F12), histidine-rich glycoprotein (Hrg), plasminogen (Plg), protein C (Proc), and thrombomodulin (Thbd), and (2) type 1 interferon signalling and cellular responses, including bone marrow stromal cell antigen 2 (Bst2), signal transducer and activator of transcription 2 (Stat2), and XIAP-associated factor 1 (Xaf1).

There were 297 DEGs for the spleen that were shared between Peromyscus and Mus, a further indication that the deermice were responding in many ways similarly to mice in their early responses to this dose of LPS (Table S11) . But there were distinguishing features as well. Upregulated GO terms for the P. leucopus spleen were tryptophan metabolic and catabolic processes, specifically indoleamine 2,3-dioxygenase 1 (Ido1) and Ido2. For M. musculus uniquely-associated GO terms that were up-regulated in the spleen dealt with production and differentiation of myeloid cells, macrophages, and osteoclasts, and included the interleukins IL-12b (Il12b) and interleukin 17 members IL-17A (Il17a), Il17f, and IL-23 (Il23a). Down-regulated genes populating GO terms related to assembly of skeletal and cardiac myofibrils included cardiac muscle actin 1 (Actc1), myomesin 2 (Myom2), and Myom3 in M. musculus.

In the liver samples four up-regulated GO terms that distinguished the deermice from the mice were phosphatidylserine metabolic process and phosphatidylserine acyl-chain remodeling, which included phospholipase A2, group IIA (Pla2g2a), Pla2g5, and Pla1a, regulation of IL-5 and IL-13 secretion, both of which included the transcription factor GATA binding protein 3 (Gata3), [64] , and tumor necrosis factor receptor superfamily, member 21 (Tnfrsf21), which reportedly is a determinant of influenza A virus susceptibility in mice [65] . Two distinguishing GO terms associated with down-regulated genes in the livers of LPS-treated P. leucopus were response to retinoic acid and cellular response to retinoic acid, both sets of which included wingless-type MMTV integration site family, member 11 (Wnt11), Wnt5A, and frizzled class receptor 10 (Fzd10), which mediates Wnt signaling in neuron development [66] .

A distinguishing GO term for a set of up-regulated genes in the liver of M. musculus was positive regulation of I-kB kinase/NF-kB signaling, which included NLR family, apoptosis inhibitory protein 5 or Naip5 (Birc), Ccl19, inhibitor of kappaB kinase epsilon (Ikbke), Il12b, Il1a, LPS-induced TN factor (Litaf), myeloid differentiation primary response gene 88 (Myd88), receptor tyrosinekinase orphan receptor 1 (Ror1), Tlr2, and tumor necrosis factor (ligand) superfamily, member 10 (Tnfsf10). Distinguishing GO terms for a set of down-regulated genes in the liver of the LPS-treated mice regard the androgen receptor signaling pathway and include the transcription factor forkhead box H1 (Foxh1), hairy/enhancer-of-split related with YRPW motif-like (Heyl), which is associated with repression of transforming growth factor beta (Tgfb) signaling [67] , and Nodal (Nodal), which bind various TGFb receptors during development [68] .

In summary, this layering of established GO terms over the DEG analysis provided consolidation and further evidence that P. leucopus and M. musculus have much in common in how they respond in the blood, spleen, and liver in the first few hours after exposure to LPS. But there were also several functional processes in which the species differed. One feature of the P. leucopus response that particularly stood out in this analysis was the activation of neutrophils and other phagocytes. Included in the lists of specific genes under all the neutrophil-associated GO terms was Slpi, which was first identified as an inhibitor of serine proteases such as neutrophil elastase [69] . In M. musculus the notable distinguishing GO terms concerned blood coagulation and fibrinolysis, processes that are important in the pathophysiology of sepsis [70] .

Gene network patterns across species. To further delineate the gene expression networks without a prior categorization, we performed WGCNA to empirically identify groups of genes (modules) that distinguished specific species and/or treatments in responses to LPS. Gene network analysis allowed identification of patterns across the three tissues. Since blood and the two organs inherently differed in their expression profiles across the genome, and one tissue could overshadow the patterns in other tissues, we made a matrix in which each column represented a sample and each row represented a gene per tissue. Each gene had a suffix to identify the tissue of origin. Twentyfour modules were identified, and GO term enrichment was assessed for unique genes for each module. Figure 5 summarizes the results for four modules: darkorange2, darkseagreen3, brown4, and lightblue. The associated GO terms and constituent genes of the four modules are listed in Table  S12 .

The darkorange module comprised genes that were up-regulated in LPS-treated P. leucopus and not in either control P. leucopus or LPS-treated or control M. musculus. The blood made the largest contribution with 1472 genes to this module, followed by liver and spleen with 933 and 776, respectively, each. The highest three ranked GO terms related to neutrophils: neutrophil mediated immunity (accession 0002446), neutrophil activation involved in immune response (0002283), and neutrophil degranulation (0043312). The three GO terms share several neutrophil-associated genes that were up-regulated, including those for lysosomal cysteine proteases cathepsin S (Ctss) and cathepsin B (Ctsb), and lysosomal membrane protein 2 (Lamp2). Slpi was also among the contributing genes for each of the three neutrophil GO terms.

The lightblue module comprised genes that were up-regulated in LPS-treated M. musculus but not in either control mice or in LPS-treated or control P. leucopus. In contrast to the darkorange module, the liver made the largest contribution to the lightblue module with 1170 genes, followed by spleen with 566 genes and blood with 33 genes. The three highest ranked GO terms for this module were cytokine-associated: cytokine-mediated signaling pathway (0019221), cellular response to cytokine stimulus (0071345), and response to cytokine (0034097). While the darkorange module also included two cytokine-associated GO terms at a lower rank than the three neutrophilassociated GO terms, only a minority of genes (41 of 170) in the combined list overlapped between the two species for the cytokine-mediated signaling pathway GO term. Selected up-regulated genes unique to LPS-treated M. musculus under this GO term included the following: cytokines Il1a, Il12, Il17, Il22, Il27, Il33, and Ifng; the chemokines Ccl5 (RANTES), Cxcl9, and Cxcl13; macrophage markers Cd86 and Cd80; and transcription factors Jak3 and Stat4. Selected DEGs associated with LPS-treated P. leucopus for this GO term included the following: Alox5, annexin 1 (Anxa1), Casp3, leptin (Lep), Sod2, Tgfb1, and tissue inhibitor of metalloproteinases (Timp1); the cytokines Il1b, Il10, Il19, and Interferon 1 beta (Ifnb1); and the transcription factors Gata3, Irf8, Stat1, and Stat3.

The two other modules highlighted in Figure 5 and Table S12 are the darkseagreen module, which distinguished control P. leucopus from the other 3 groups, and the brown module, which featured GO terms that were down-regulated in comparison to controls in both sets of LPS-treated animals. For the dark sea green module the spleen was the major contributor with 4791 genes followed by liver with 3189 genes and blood with 73 genes. The five GO terms contained from 241 to 1001 genes. The two with most genes in common were plasma membrane bounded cell projection assembly (0120031) and cilium assembly (0060271). For the brown module the liver RNA-seq with a contribution of 876 genes far out-numbered the contributions of spleen with 6 genes or blood with 3 genes. Genes common to the top three GO terms in this module were all kinases: tyrosine-protein kinase ABL (Abl1), tyrosine-protein kinase CSK (Csk), mitogen-activated protein kinase 3 or ERK1 (Mapk3), and megakaryocyte-associated tyrosine kinase (Matk). Mapk3 is a determinant of macrophage polarization [71] .

Selected genes in blood samples. Results from untargeted investigations led us to examine coding sequences that represented processes that either typified one or the other of the species' distinguishing responses to LPS or were shared for one or more of the three tissues. Some these genes were identified in the individual DEG analyses summarized in Figure 3 . These included several genes, such as for IL-6, Nos2, and Sod2, commonly up-regulated after exposure to LPS or during sepsis in mice and humans. Other genes were chosen for the well-characterized roles in the pathways and other functional processes, including regulatory, that were highlighted for us by the GO term analysis and module-based analysis summarized in Figure 4 and Figure 5 , respectively. A few were selected because in P. leucopus they were either DEGs that were novel in this context or remarkable for the scale of the changes in expression in this species. An example of the former was the integrin-binding sialoprotein and its Ibsp gene. An example of the latter was the secretory leukocyte peptidase inhibitor and its Slpi gene.

For this cross-species differential expression analysis there was a single reference set that contained the pairs of orthologous protein coding sequences of the mRNAs for both P. leucopus and M. musculus. Figure 6A shows results for 9 genes of blood samples from 8 M. musculus controls (MC), 12 M. musculus LPS-treated (ML), 8 P. leucopus controls (PC), and 12 P. leucopus LPS-treated (PL). A preliminary analysis did not reveal significant differences between females and males of either species for this, as well as for the other two tissues, so the results are not stratified by sex. The deermice and mice were similar in the direction of the changes in expression for Il1rn, Lcn2, and Sod2, but the fold changes were greater in P. leucopus for Lcn2 and Sod2 than in M. musculus (Table  S13) . Four other genes were observed to decrease in expression in the blood of mice 4 h after LPS and increase, by several orders of magnitude in some cases, in the deermice: Alox5, which transforms essential fatty acids into leukotrienes, Slpi, which is commonly associated with neutrophil GO terms, transforming growth factor beta (Tgfb), which can be activated when Slpi cleaves an inhibitor [72] , and Ibsp, which is associated with known of the GO terms featured in Figure 4 and has been little noted with regard to inflammation. We confirmed the markedly higher expression of Slpi in P. leucopus blood after LPS by RT-qPCR ( Table 1) .

The most remarkable difference between the species was in their comparative expression at baseline and after LPS of Nos2 and Arg1, a phenomenon we had also noted in the previous RNAseq analysis. With the exception of one animal, we detected little expression of Arg1 in the blood of mice either in controls or LPS-treated animals. In contrast, Nos2 expression was many fold higher in M. musculus after LPS than in controls. The opposite occurred in P. leucopus: expression of Nos2 was undetectable in controls and marginally increased in some treated animals. At the same time Arg1 was already a high level of expression in control animals and was on average ~10-fold higher in those that received LPS. The Nos2 findings in mice and deermice were consistent with our measurements of nitric oxide in the plasma. The RNA-seq results of Nos2 and Arg1 were confirmed by species-specific RT-qPCR assays of the same samples but with different cDNA libraries from those for RNA-seq (Table 1) .

Selected genes in spleen samples. Figure 6B shows box-whisker plots of 9 selected genes for RNA samples from the spleens of the 40 animals. This study confirmed the low expression of Arg1 before and after LPS in M. musculus and the high baseline expression of this gene in P. leucopus and a further ~10-fold elevation in LPS-treated animals ( Table S14 ). Genes that increased in the spleen in both mice and deermice with LPS exposure, albeit with different degrees of fold-change, were Il6, Il10, Il1b, Csf2, Csf3, CCl2, Cxcl11, and Tnf. These were indications that P. leucopus was affected by LPS and in several relevant pathways was responding similarly to M. musculus.

There were also increases in both species with LPS of expression of pentraxin 3 (Ptx3), which is a pattern-recognition molecule involved in innate immune responses, and indoleamine 2,3dioxygenase 1 (Ido1), which is the first and rate-limiting enzyme of tryptophan catabolism through the kynurenine pathway [73] . But the baseline levels in P. leucopus were as high as those of LPStreated M. musculus and were even higher in the deermice treated with LPS. In the case of Ptx3, this was more than a hundred-fold. Expression of interleukin-12 (IL-12) was approximately the same in normalized reads in control P. leucopus and M. musculus. It declined in P. leucopus treated with LPS but not in similarly exposed M. musculus. The relationship between IL-10 and IL-12 expression in the spleen in individual animals is plotted in Figure S3 of Supplementary Materials.

Diversity of responses within study populations. To investigate whether the outbred deermouse population was more heterogeneous or diverse in responses than the inbred BALB/c strain mice, we carried out a pairwise analysis of coefficients of determination (R 2 ) within each of the two species (12 animals each) as well as for all 24 animals that received LPS. When self pairings were excluded, this represented 66 pairs each for P. leucopus and M. musculus and 144 pairs of each of LPS-treated P. leucopus paired with each of the LPS-treated M. musculus. To control for the wide differences in expression noted between species for some mRNAs, the 17 chosen genes were ones for which the inter-species fold differences were within an order of magnitude and represented those that were more highly expressed in mice as well as those more highly expressed in deermice. The median fold difference between P. leucopus and M. musculus for these genes was 1.2. We used spleen because of longer reads for this sample and similar numbers of up-and down-regulated DEGs in both species ( Figure 3 ). The genes were the following: Alox5, Ccl2, Csf2, Csf3, Cxcl11, Gapdh, Hmox1, Il1b, Il1rn, Il6, Lcn2, Mt-Co1, Nfe2l2, Slpi, Sod2, Tgfb1, and Tnf. Figure S4 shows the distributions of R 2 values of the sets of pairs. As expected correlations were lower between mixed species pairs than intra-species pairs (upper panel); the mean and median R 2 were 0.699 and 0.701. The seven highest intra-species pairwise R 2 values (0.987-0.994) were observed among the M. musculus, and the mean and median of the 66 R 2 values were marginally higher at 0.940 and 0.954 for the mice than the 0.937 and 0.948 for the deermice. But, overall, P. peromyscus animals in this experiment were not notably more diverse in their responses for this set of genes than were the sample of inbred mice under the same experimental conditions (p = 0.68; Mann-Whitney U p = 0.19). This was evidence that the DEGs, enrichment GO terms, and modules that distinguished between the two species were likely not accounted for by greater variances or a divergent sub-group among the deermice.

Selected genes in liver samples. Figure 6C comprises analogous plots of expression of 9 selected genes in the livers of LPS-treated and untreated animals (Table S15 ). In both species albumin (Alb) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh), a commonly used "housekeeping" gene control for transcription assays, declined about the same extent 4 h after the LPS injection. Sod2 was also elevated in the both sets of treated animals, but the fold-change was greater in the deermice. In contrast to Sod2, Tlr2 increased more than a hundred-fold in LPS-treated M. musculus but only marginally from its comparatively higher baseline level in P. leucopus.

Other evidence that P. leucopus was responding similarly to the endotoxin as mice was the comparable rise in both sets of treated animals in what are considered "acute phase reactants" for evaluating infection and inflammation: C-reactive protein (Crp), haptoglobin (Hp), and a serum amyloid A (Saa) [74] . In liver tissue Arg1 expression was comparatively high in both species among the controls, but, as in the blood and spleen, the levels were elevated in P. leucopus and declined in M. musculus. Nos2 expression was marginally higher in the livers of LPS-treated deermice than in their blood samples, but this was still many fold lower than observed in the treated mice.

Pairwise analysis of selected DEGs in both species. Paired relationships between a sub-set of these genes in each of the 3 tissues are depicted in Figure 7 . The responses of the two species are largely indistinguishable for Crp, Hp, Alb, and Saa3 among the liver proteins. Both species also displayed correlated responses between Tnf and Il1b and between Il1rn and Il1b, another indication that P. leucopus like M. musculus "recognizes" the LPS. Up to a certain point events after exposure to LPS in both species appear similar. What distinguished between species was the higher ratio of Il10 to Tnf expression in P. leucopus under both conditions, but in particular among the LPS-treated animals. Other pairs of biomarkers that uniquely characterized the responses of P. leucopus to endotoxin were Alox5 and Sod2, Ido1 and Ptx3, and, most starkly, Arg1 and Nos2. The profile of high Nos2-low Arg1 expression in response to LPS typifies a type M1 macrophage response in the mouse, while the low Nos2-high Arg1 profile we observed in P. leucopus is more characteristic of a type M2 or alternatively-activated macrophage response [59] . Further evidence of a dichotomy between species that corresponded to polarized macrophage categories was the Il10 to Il12 ratio in the spleen [75] . In control M. musculus the mean ratio (95% CI) was 0.036 (0.026-0.046). It was tenfold higher in LPS-treated mice: 0.361 (0.251-0.471). At baseline the Il10 to Il12 ratio was a hundred fold higher in control deermice--3.47 (2.32-4.63)--compared to what was observed in mice. Among the P. leucopus treated with LPS the ratio was even higher at 191 (123-259) than the corresponding group of M. musculus.

We found similar profiles in blood and spleen samples of older animals in an experiment that was carried out before the deermouse-mouse comparison but under identical experimental conditions, i.e. 10 µg per gm dose or buffer alone as control and samples taken after 4 h. The 16 animals (12 females) had mean and median ages of 79 and 81 weeks, respectively, with a range of 54 to 94 weeks (Table S1 ). Nine animals received LPS and 7 buffer alone. Figure S5 comprises boxplots of log-transformed normalized unique reads for pairs of genes in either the blood or spleen of these animals. We observed again in this second experiment low baseline and after treatment expression of Nos2 in the blood but high baseline expression of Arg1 with a further increase in the LPS-treated (Table S16 ). Both Slpi and Ibsp increased more than a hundred-fold in expression in the blood after LPS. In the spleen Il1b, Tnf, Il6, Il10, Ccl2, Cxcl11, Csf2, and Csf3 all were increased in the LPS treated animals, as were two genes that notably more up-regulated in the younger sample of P. leucopus than in comparably-aged M. musculus: Ido1 and Ptx3.

Systemic bacterial infection. Five P. leucopus were infected with the relapsing fever agent Borrelia hermsii on day 0. Another 3 animals received buffer alone. Infection of the blood was directly confirmed by microscopy on day 4. The animals were euthanized on day 5, just before the appearance of neutralizing antibody was anticipated [76] . The infected animals had enlarged spleens, as well as large numbers of bacteria in the spleens, as assessed by quantitative PCR (Table  S17) . We used previously collected PE100 reads for the blood of these animals [37] for first measuring unique reads mapping to mRNA sequences of selected genes. As we observed in the blood of LPStreated deermice, Nos2 expression was not or barely detectable in either the controls and infected animals. Arg1 expression was high at baseline and was further elevated at 5 days into infection. Two genes of P. leucopus that clearly distinguished this species from M. musculus in its short-term response to LPS were Slpi and Ibsp; both of these were about a hundred-fold more highly expressed in infected animals than in controls.

RNA-seq was carried out with the same reference set and settings as for the samples in comparative response study. Given the smaller sample size of the infection study, we focused on more highly expressed genes. Of the 46,154 transcripts in the full reference set, 1773 had mean TPM values of ≥ 50 in at least one of the two groups in each experiment. This latter set was used as the basis for determining and then comparing fold changes between the LPS condition with a duration in hours and the systemic bacterial infection condition with a duration in days (Table S18 ). Figure 8 summarizes the pair-wise fold-changes for the individual genes. More than two-thirds of the DEGs in the infection experiment were also represented among the DEGs in the LPS experiment among the P. leucopus. There was a high correlation in the direction and magnitude of the fold-changes between two experiments. We noted again the substantial up-regulation of Slpi and Ibsp in the shorter term LPS experiment and the longer term infection experiment. Other coding sequences upregulated under both conditions were Acod1, Alox5, Arg1, Cxcl2, Il1rn, Il1b, Lcn2, and Mmp8. A discordant DEG was Dusp (dual specificity phosphatase; also known as mitogen-activated protein kinase phosphatase or "Mkp-1"), which was up-regulated after LPS but down-regulated in the blood of infected animals. Dusp has been implicated in regulating lipid metabolism during sepsis [77] .

LPS treatment of P. leucopus fibroblasts. Low-passage cultures of fibroblasts isolated from ear tissue from five LL stock deermice were split into pairs, and then one member of each pair was treated with LPS for 4 h and the other member with saline alone. Of the 46,141 transcripts in the reference set, 18,462 had a mean TPM of > 1 in either the control or LPS group, and these were used the DEG analysis (Table S19) . For protein coding sequences the 10 highest TPMs in order among control samples were ferritin heavy chain (Fth), Slpi, secreted protein acidic and cysteine rich (Sparc), eukaryotic translation elongation factor 1 alpha (Eef1a1), ferritin light chain (Ftl), vimentin (Vim), serpin family H member 1 (Serpinh1), ribosomal protein lateral stalk subunit P1 (Rplp1), collagen type I alpha 1 chain (Col1a1), and Gapdh. There were 324 genes that were up-regulated by criterion of fold-change ≥ 4 and FDR < 0.05, and 17 genes that were down-regulated, with an additional 80 down-regulated by criterion of fold-change ≥ 2 and FDR < 0.5 ( Figure 9 ). Among those displaying the marked increases in expression between the control and LPS conditions were two subunits of nuclear factor kappa B (Nfkb), two forms of Saa, Nos2, Csf3, Sod2, and phospholipase A2 group IIA (Pla2g2a), which is associated with inflammation during system bacterial infection [78] . Slpi transcripts were not only surprisingly abundant in the fibroblast cultures under usual cultivation conditions, but by RNA-seq and pairwise comparisons expression further increased by 1.5 (1.2-1.9) fold in those pair members exposed to LPS (p = 0.02). By the same type of pair analysis with the coding sequence of mRNA as the reference, expression of Nos2 increased 792 (124-5073) fold in the LPS treated fibroblasts (p = 10 -5 ). This was evidence that the absent or scant expression of inducible nitric oxide synthase we observed in both control and LPS-treated P. leucopus animals was not attributable to a genotypic incapacity to transcribe the Nos2 gene.

Conjunctivitis. After identification of a variety of DEGs under conditions of LPS treatment of whole animals and isolated cells or of systemic infection, we returned to the disease sign we observed in P. leucopus but not in M. musculus, namely, conjunctivitis severe enough to cause eyelid closure within 2-4 hours of the LPS injection. Six of 12 animals receiving the LPS overtly manifested conjunctivitis during the experiment. Table 2 lists DEGs in the blood or spleen from RNA-seq with absolute fold-changes between animals with conjunctivitis and those without of >2 and FDR values of ≤0.05. Of note in the blood of deermice with conjunctivitis were 21x higher expression of Cxcl13, 12x higher expression of Ccl6, but 46x lower expression of lysozyme (with FDR of < 10-4 ), an antimicrobial enzyme and constituent of tears that coat and protect the conjunctiva [79] . In spleen samples the conjunctivitis animals were distinguished by 6-7 fold higher expression (with FDR <0.005) of two forms of carbonic anhydrase, an enzyme of relevance for the eye disorder glaucoma, because of its role in producing aqueous humor in the eye [80] .

The study yielded abundant information about a small mammal that is emerging as an informative model organism, not only for the study of pathogenesis and immunology of infectious diseases but also for the fields of aging, behavior, ecology, and reproductive biology [33] . The question driving this investigation was how do deermice largely avoid morbidity and mortality when infected with pathogens that otherwise are disabling if not fatal for humans? "Sickness" has various definitions [81] , including one operationally based on behavior for animal studies [82] . P. leucopus and M. musculus became equivalently sick by the behavioral criteria of reduced activity, mutual huddling, and lower food intake. Those P. leucopus that had conjunctivitis, which rendered them functionally blind, arguably were even sicker than the M. musculus. Nevertheless, under conditions that frequently led to death or the moribund state in various strains of M. musculus in several studies, P. leucopus receiving the same or even much higher doses of LPS pulled through and recovered.

To address this central question, we mainly depended on RNA-seq of whole blood, spleen, and liver. The study did not specifically examine differences in mRNA isoforms or small non-coding RNAs. These results were complemented by analysis of a limited set of coding sequences, specific RT-qPCR assays, direct detection of the compounds in the blood, and metabolomics. The most extensive and best documented experiment was on the response among several young adult animals of both sexes to a single dose of LPS at approximately the LD50 for mice. But we obtained similar results with P. leucopus of considerably older age under the same conditions and with animals that had systemic bacterial infection of 5 days' duration. While we noted some differences between sexes of P. leucopus in the experiment, for the most part females and males responded the same to LPS, at least in the short term. The experiment with P. leucopus fibroblast cells in culture exposed to LPS showed that the some of the findings, such as high expression of Slpi, are reproduced in vitro and, thus, could be exploited by transgenic and silencing technologies.

Integration of the metabolomics results for the plasma with the transcriptomics results for the three tissues was limited. The sources of some of the metabolites in the blood would likely have been organs, e.g. the adrenal glands, or gut microbiota not subjected to RNA-seq. Nevertheless, the metabolomics confirmed that P. leucopus responded similarly to M. musculus in many respects at the same dose of LPS. Consequently, the overall milder outcome of LPS challenge in the deermice is probably not attributable to something as pleiotropic as deficiency of the receptor for LPS or the proximate signal transduction step.

One of the striking differences between the two species in their responses to LPS was overall predominance in the GO term and de novo module analyses of neutrophil-associated genes for P. leucopus and cytokine-associated genes for M. musculus (Figures 4 and 5) . The blood was the greatest contributor to the set of genes that distinguished LPS-treated P. leucopus from treated and untreated M. musculus and from control deermice, while the liver provide the majority of genes that distinguished LPS-treated M. musculus from deermice and from control mice. The comparatively heightened neutrophil transcriptional activity in P. leucopus paralleled the appearance of conjunctivitis with purulent exudates in the eyes of deermice but not mice. While increased activity of neutrophils could lead to tissue damage from elastase and other proteases and by reactive oxygen species, this may be ameliorated in the deermice by such factors as the leukocyte protease inhibitor Slpi and Sod2, a defense against oxygen radicals.

Slpi is a non-glycosylated 12 kDa cationic, cysteine-rich protein, which was known to be increased in expression in response to LPS and lipoteichoic acids and under the stimulus of TNF and IL-1b (reviewed in [83] ). It inhibits neutrophil proteases like cathepsins and elastase, but also prevents degradation of the NFkB inhibitory proteins IkBa and IkBb through its anti-protease activity [84] . Slpi knockout or deficient animlas had impaired wound healing and increased inflammation [85] , elevated Nos2 activity in macrophages [86] , and increased susceptibility to LPSinduced shock [87] .

While LPS-treated P. leucopus increased expression of TLR signaling adapter MyD88 and downstream inflammatory cytokines, such as IL-6, TNF, and IL-1b, to approximately the same degree as in M. musculus, this was countered by greater expression of cytokines associated with M2 polarization, such as IL-10 and TGFb, as well as other anti-inflammatory mediators, like Anax1, in the context of lesser expression of IL-12 and IFNg. The GO term cytokine-mediated signaling pathway served to distinguish both LPS-treated P. leucopus and LPS-treated M. musculus from their untreated counterparts, but the sets of constituent genes that populated the GO term for each species overlapped for only a minority ( Figure 5 ).

Another notable difference was the contrasting expression of arginase 1 and inducible nitric oxide synthase, or Nos2, in the two species. This can be mapped as well onto macrophage polarization, where type M1 macrophages in a simplified scheme feature high Nos2 and low Arg1 and most varieties of type M2, or alternatively activated macrophages, are characterized by low Nos2 and high Arg1 [59] . The differences between species in their IL-10 to IL-12 ratios before and after LPS [75] was also consistent with a greater disposition of P. leucopus to a type M2 response ( Figure S3 ). Further categorization into the M2 sub-types recognized in mice was inconclusive at this point. Nos2 gene knock-out demonstrate reduced susceptiblity to the toxicity of LPS but at the cost of greater susceptibility to Listeria monocytogenes or to Leishmania major infections [48, 88] . While experimental infections with these particular intracellular pathogens have not been reported for P. leucopus, this species does not seem to be particularly at risk of morbidity or mortality from the obligate intracellular bacterial pathogen Anaplasma granulocytophila or the Powassan encephalitis virus [89] , that exploit P. leucopus as a competent reservoir [1] .

Integrin-binding sialoprotein (Ibsp) or bone sialoprotein 2 (BSP2) is a special and revelatory case in this study. There were marked increases in the expression of this gene in P. leucopus but not in M. musculus upon exposure to LPS. The Ibsp protein has predominantly been associated with bone and tooth morphogenesis with no reported association with inflammation or innate immunity. Consequently, it would not be expected to be included in many of the innate immunity and inflammation pathways and GO terms that distinguished LPS-treated from untreated animals of either or both species. But there is justification for viewing Ibsp as P. leucopus' functional substitute of osteopontin (Spp1), another small integrin-binding glycoprotein that is heavily modified posttranslationally [90] . Both BSP2 and osteopontin (BSP1) bind to alpha v beta 3 integrins, and their genes are within a hundred kilobases of each other on chromosome 4 of humans and chromosome 22 of P. leucopus [36] . Osteopontin is a biomarker of sepsis in humans [91] and reportedly acts through Stat1 degradation to inhibit Nos2 transcription [92] .

Could observed differences between the two species in these experiments be attributable in part to differences in their microbiomes? The gut metagenomes were determined from preexperiment fecal pellets from the animals in this study, and the results have been reported [38] . In general, the deermice and mice had similar distributions and frequencies of bacteria at the taxonomic level of family and in the representation and proportions of different biosynthetic, metabolic, catabolic, and regulatory functions. But there were also substantive differences in the gut microbiota between the species that plausibly could account for some of the distinguishing responses to LPS. The first was the abundant presence in the intestine in P. leucopus, but not in M. musculus, of a protozoan, identified as a new species of the parabasalid genus Tritrichomonas. The presence of Tritrichomonas muris in some populations of the same inbred strains of M. musculus altered their cellular immune responses to other microorganisms and antigens [93] . Further distinguishing the gut microbiota was the greater abundance and diversity in P. leucopus of Lactobacillus spp., the primary niche for which was the forestomach [38] . In two studies of the use of Lactobacillus spp. as probiotics, feeding of mice with L. paracasei or L. plantarum resulted in less inflammation in comparision to controls when challenged with an influenza virus or Klebsiella pnemonia, respectively [94, 95] . In the present study a tryptophan metabolite that distinguished the plasma samples of the two species was indolepropionic acid (Table S3) , which at baseline was threefold higher in concentration in deermice than in mice but was nine-fold lower concentration in LPStreated P. leucopus. It was only marginally lower between the corresponding sets of M. musculus. This compound is the metabolic product of certain intestinal microbes and reportedly has antiinflammatory and anti-oxidant properties [96, 97] .

In summary, P. leucopus differed in many respects from M. musculus in these experiments in its response to LPS. Under conditions in which about half the mice would be expected to die within 48 h, the deermice survived the acute insult and recovered, a phenomenon that modeled the tolerance of these animals to infection. In their outcomes after LPS exposure the naïve P. leucopus resembled M. musculus that had become accustomed to LPS by prior exposure to a low dose of LPS [98] . A characteristic profile of these pre-treated mice is comparatively lesser expression of proinflammatory mediators and greater expression of genes of phagoctyes [99] , similarly to what we observed in naïve P. leucopus. The phenotype of Slpi knockout mice resembles that of animals that have been tolerized to LPS in their suppression by administered IFNg [100] , a cytokine that is an alternative to LPS as an inducer of Nos2 expression in macrophages [101] .

Whether this points to a single pathway or even a single gene remains to be determined. But we doubt that the phenomenon of infection tolerance can be reduced to a simple explanation. The evidence rather is of multiple adaptive traits representing different aspects of innate immunity, metabolism, oxidative stress management, and perhaps the microbiome that serve to sustain P. leucopus populations amidst the varied infectious agents they face. This is also to the benefit of these agents in this trade-off, because it renders P. leucopus a competent vertebrate reservoir for them and one of impact for human populations in endemic areas.

Animals. Adult outbred P. leucopus of the LL stock were obtained from the Peromyscus Genetic Stock Center (PGSC) of the University of South Carolina [102] . The LL stock colony was founded with 38 animals captured near Linville, NC between 1982 and 1985 and has been closed since 1985. Sib-matings are avoided, and complete pedigree records are kept. Animals of the LL stock closed colony had mitochondria of the same genome sequence with little or no heteroplasmy [37] . Adult BALB/cAnNCrl (BALB/c) and C.B.17 strain Severe Combined Immundeficiency (SCID) M. musculus were purchased from Charles River. All animals in the LPS experiments spent at least two weeks at the UCI facilities before the experiment.

Animals were maintained in the AAALAC-accredited U.C. Irvine vivarium with 2-5 animals per cage according to sex and on 12 hours light-12 hours dark lighting schedule, temperature of 21-23° C, humidity of 30-70%, water ad libitum, and a diet of 8604 Teklad Rodent (Harlan Laboratories). For prior to injections animals were lightly anesthetized with 2.5% isoflurane in presence of 2 liters/min oxygen. The rodents were euthanized by carbon dioxide overdose and intracardiac exsanguination at the termination of the experiment or if they were moribund, unable to eat or drink, or otherwise distressed. Dissection was carried out immediately. Instruments were cleaned first and then sterilized between dissections.

The study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. University of California Irvine protocol AUP-18-020 was approved by the Institutional Animal Care and Use Committee (IACUC). The protocol for the comparative study of P. leucopus and M. musculus for responses to LPS after 4 h was in addition approved by the Animal Care and Use Review Office of the United States Army Medical Research and Materiel Command. P. leucopus studied at the PGSC were under IACUCapproved protocol 2349-101211-041917 of the University of South Carolina.

Tables S1 and S21 of Supplementary Materials respectively provide information on each of the animals in the LPS experiments and corresponding National Center for Biotechnology Information (NCBI) (http://ncbi.nlm.nih.gov) BioProject and BioSample identifying numbers and descriptions for these samples. The gut metagenomes from feces collected from the 20 P. leucopus and 20 M. musculus animals 1-2 days before the comparative experiment have been described [38] and are available from MG-RAST database (https://www.mg-rast.org) under accession numbers mgm4832531.3-mgm4832578.

LPS susceptibility and dose responses. Thirty adult P. leucopus, divided into five groups of three females and three males, were each injected intraperitoneally (i.p.) on day 0 with a 50 µl volume of Escherichia coli O111:B4 LPS purified by ion exchange and with <1% protein and <1% RNA (Sigma Aldrich; catalog L3024), which was diluted in sterile, endotoxin-free 0.9% saline (Sigma Aldrich) to achieve the following doses in µg/gm (mg/kg) body weight: 10, 50, 100, 200, and 300. The doses were administered in randomized order over the period 1400-1700 h of a single day. Animals were returned to their cages with ad libitum food and water and then monitored every 12 h for the following signs: reduced activity by criterion of huddling with little or movement for > 5 min, ruffled fur, hyperpnea or rapid respiration rate, and conjunctivitis by the criterion of closed eyes with crusting observable on the eyelids. The primary endpoint was death during the period between observations or the moribund state (immobility, rapid respiration, and not able to feed or drink) at the scheduled monitoring time or when notified in the interim by vivarium attendants.

LPS effects at 4 h. Animals were anesthetized with isoflurane and injected i.p. with a single dose of E. coli O111:B4 LPS at a concentration of 10 µg/gm body weight in a 50 µl volume as described above. The control group was anesthetized and then injected with the 0.9% saline alone. The experiment started at 0800 h with 10 min intervals between animals and with alteration of LPS and control injections. At 4.0 hr after their injection the animals were euthanized. After opening the chest, exsanguination was performed by cardiac puncture and blood was transferred to a heparin sulfate coated tubes (Becton-Dickinson Microtainer). Anticoagulated blood was centrifuged to pellet blood cells for 3 min at 4600 x g at 4° C. Plasma and blood pellet was kept separately at -80° C until further analysis. Liver and spleen were extracted, flash-frozen in liquid nitrogen, and stored at -80 °C until RNA extraction.

Experimental infection. Infection of a group of adult P. leucopus LL stock with the relapsing fever agent Borrelia hermsii strain MTW of genomic group II was described by Barbour et al. [37] . Peromyscus species are natural hosts of genomic group II strains of B. hermsii [103] . In brief, animals were anesthetized and then injected on day 0 with 10 3 bacteria by each of i.p. and subcutaneous routes in 50 µl volumes of PBS and diluted plasma from infected SCID mice, as described [104] . On day 4 a drop of tail vein blood was mixed with PBS an examined as a wet mount by phase microscopy to confirm infection. On day 5 animals were euthanized with carbon dioxide and terminal exsanguination. Whole blood was dispensed into heparin-coated tubes, and the spleens were removed by dissection, weighed, and then flash frozen in liquid nitrogen. Confirmation of infection and quantitation of bacterial burdens in the spleens was carried by quantitative PCR, as described [105] .

Fibroblast cell culture and LPS treatment. Fresh ear punches were collected from five LL stock P. leucopus animals (2 females and 3 males) during routine marking procedures at the time of weaning at ~3 weeks of age. The PGSC identification numbers (and mating pair for each animal) were 22608 (H-1075), 22609 (H-1075), 22610 (H-1121), 22611 (H-1121), and 22614 (H-1127). After the ear punch tissue was bathed in 70% ethanol for 2 min, it was placed in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone FetalClone II; Thermo Scientific), minced, and then treated with 5 mg/ml collagenase type I (Millipore) for 1 h. After undigested debris was removed, the disassociated cells were cultivated in the same medium at 37° C and in 5% CO2. Cells were passed when adherent layers reached 45-90% confluency and for no more than 7 passages. For the experiment individual cultures were split into pairs and they were incubated at initial concentrations of 3 x 10 5 cells per well for 24 h. LPS or saline alone was added medium for final LPS concentration of 1 µg/ml and then the incubation was continued for 4 h. After disassociation of the fibroblast layer with trypsin and then addition of RNAlater (Thermo Scientific), the cells were harvested and stored at a concentration of ~10 6 /ml in -80 ℃ until RNA extraction.

Analyses of blood. Nitric oxide was analyzed from frozen plasma samples using Nitric Oxide Assay Kit (Invitrogen) according to manufacturer's instructions and nitrite concertation was calculated based on standard curve measuring optical density at 540 nm on a plate reader (BioTek Synergy2). Corticosterone was measured colorimetrically in plasma using DetectX Corticosterone Enzyme Immunoassay Kit K014-H1 (Arbor Assays) based on corticosterone-peroxidase conjugate. After incubation, the reaction is read at 450nm on a BioTek Synergy2 plate reader.

Metabolomics. Untargeted detection and analysis of metabolites in plasma of P. leucopus and M. musculus were carried out essentially as described [106] . In brief, 40 µl volumes of plasma, which had been stored frozen at -80° C, were extracted with 120 µl methanol containing as internal standards phenylalanine d5 (175 ng/mL), 1-methyl tryptophan (37.5 ng/mL), and arachnidonoyl amide (30 ng/mL). After precipitated proteins were removed by centrifugation, the supernatant was dried under vacuum and then suspended in 50% methanol. Aliquots of 10 µl were subjected to high pressure liquid chromatography (HPLC) and quadrupole (Q) time-of-flight (TOF) mass spectrometry (MS) with periodic inclusion of pooled samples for quality control. Metabolites were separated on an Agilent Technologies Poroshell C8 column (100 x 2.1 mm, 2.7 µm) with a gradient of acetonitrile and water both containing 0.1% formic acid with an Agilent 1260 HPLC pump under conditions previously described [106] . The eluent was introduced into an Agilent Technologies 6520 Q-TOF-MS instrument equipped with an electrospray ionization source. The parameters for the analysis were a capillary voltage of 4000 V, fragmenter voltage of 120 V, gas at 310° C, gas flow of 10 L/min, and nebulizer pressure of 45 psig. Data were acquired in the positive ion mode at a scan range of 75-1700 for the mass-to-charge ratio (m/z) and at a rate of 1.67 spectra per second. The raw data files were converted to the XML-based mzML format with Proteowizard [107] . The files were then processed for peak picking, grouping, and retention time correction with the XCMS suite of software [108] . The centWave algorithm was applied to detect chromatographic peaks with these parameter settings: ≤ 30 ppm for m/z deviation in consecutive peaks, signal to noise ratio of 10, a prefilter of 3 scans with peak intensity of ≥ 750, and 10 to 45 s for peak width. Molecular features (MF), defined by m/z and retention time, were grouped across samples using a bandwidth of 15 and overlapping m/z slice of 0.02. Retention time correction was performed with the retcor algorithm of XCMS. Finally, peak area was normalized by median fold normalization method as previously described [109] . Pathway enrichment analysis was performed with Mummichog network analysis software v. 2 [110] , which predicts functional activity from spectral feature tables without a priori identification of metabolites. This was implemented on-line at Metaboanalyst (http://www.metaboanalyst.ca). The parameter settings were mass accuracy of 20 ppm, positive ion mode, and cut-off p value of 0.05 for the Fisher's exact test of observed and expected hits of a given pathway. Pathway enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/) terms was determined separately for the P. leucopus and M. musculus sets of LPS-treated and control animals. A cross-species comparison of enrichments for pathways in common was plotted. Identifiable metabolites constituting the pathways that distinguished between LPS-treated and control animals were listed for each species. As a measure of abundance, the peak areas of selected metabolites were extracted from raw data set using Skyline software [111] .

RNA extractions. RNA from blood was extracted using NucleoSpin RNA Blood Mini kit (Macherey-Nagel). Lysis buffer and proteinase K were added to the frozen pellet and shaken while thawing. RNA from liver and spleen was extracted from 30 mg of frozen tissues, mechanically homogenized and further lysed in RLT buffer with 2-mercaptoethanol on TissueLyser (Qiagen) with 3 mm beads then extracted according to the protocol using RNeasy Mini Kit (Qiagen).

Extraction of total RNA from blood of P. leucopus infected with B. burgdorferi was described by Long et al. [36] . For extraction of total RNA from freshly obtained and chilled anticoagulated blood was mixed with an equal volume of Buffer EL of the QIAamp RNA Blood Mini Kit (Qiagen) and then processed with the kit and Qiagen QIAshredder spin columns according to the manufacturer's protocol. For extraction of RNA from frozen liver the Qiagen TissueLyser instrument with 3 mm stainless steel beads and the RNeasy Mini Kit (Qiagen) was used. Nucleic acid concentrations were determined with a Qubit fluorometer (ThermoFisher Scientific). The quality of the extracted RNA assessed by Agilent 2100 BioAnalyser with the Nano RNA chip. The RNA was stored in RNase-free distilled water at -80° C.

Reverse transcriptase quantitative PCR. RT-qPCR assays were developed and implemented for measurement of transcripts of genes for the following proteins of P. leucopus: nitric oxide synthase 2 (Nos2), arginase 1 (Arg1), secretory leukocyte peptidase inhibitor (Slpi), and glyceraldehyde 3-phosphate dehydrogenase (Gapdh). cDNA was synthesized from extracted RNA using iScript Reverse Transcription kit and iScript Supermix (BioRad) for qPCR, according to the manufacturer's instructions. The reaction mix was incubated in a thermal cycler for 5 min at Standards were the corresponding PCR products cloned into the E. coli plasmid vector pUC57 and stored frozen at -80° C in single-use aliquots after plasmid purification, as described.

RNA-seq. Library preparation with the Illumina TruSeq mRNA stranded kit was carried out as described [36] . The libraries were normalized and then multiplexed to achieve 12 samples per flow cell on an Illumina HiSeq 4000 instrument and 100 or 150 cycles of paired-end read chemistry at the U.C. Irvine Genomic High Throughput Facility. The quality of sequencing reads was analyzed using FastQC (Babraham Bioinformatics). The reads were trimmed of low-quality reads (Phred score of <15) and adapter sequences, and corrected for poor-quality bases using Trimmomatic [112] . Paired-end reads in fastq files were quantified using kallisto v. 0. 46.1 ([113] using the GENCODE annotation v. 21 (https://www.gencodegenes.org) for mouse and GCF_004664715.1_Pero_o.1 _rna from NCBI for P. leucopus [36] . The mean (95% confidence interval) coefficients of determination (R 2 ) between paired replicates of 4 RNA extracts (blood of 4 LPS-treated P. leucopus), but with independent cDNA libraries and sequencing lanes was 0.994 (0.990-0.997) for 8055 genes with mean transcripts-per-million (TPM) >1, while the mean R 2 for the 24 discordant pairs for these samples was 0.975 (0.973-0.977). Sequencing reads were deposited with Sequence Read Archive (Table 3) .

Differential expression. We used edgeR v. 3.28.1 [114] for differential gene expression (DEG) analysis. Genes were called differentially expressed if their absolute fold-change between conditions was > 4.0 and the false-discovery rate (FDR) was <0.05. To compare fold-changes across species, we merged the output tables from the DEG analyses and retained 14,685 orthologous genes that were synonymously annotated between both species, out of total 24,295 annotated genes for P. leucopus and 35,805 for M. musculus (Tables S4-S9 of Supplementary Materials). To screen for DEGs that varied in magnitude by species, we required an absolute fold change of > 5.0 (log2 = 2.5) in one species and < 5.0 in the other. For DEGs designated as "shared" between the species, the absolute fold change was > 5.0 in both. Enrichment analysis was done for each one of the gene groups, separated by up or done regulation. Enrichment of Gene Ontology (GO; http://geneontology.org) terms for biological processes was computed using EnrichR (https://amp.pharm.mssm.edu/Enrichr) [115] and plotted using ggplot (v. 3.3.2) of the R package [116] . The GO terms were sorted by ascending p value.

RNA-seq of a limited set of protein coding sequences (CDS) of both species in the same DEG analysis was carried out using CLC Genomics Workbench v. 20 (Qiagen). Paired-end reads were mapped with a length fraction of 0.4, similarity fraction of 0.9, and penalties of 3 for mismatch, insertion, or deletion to the CDS of sets of corresponding orthologous mRNAs of P. leucopus and M. musculus. Accession numbers are given in Table S20 . Counted reads were those that were speciesspecific. For example, for Arg1 the coding sequences for both P. leucopus and M. musculus were in the reference set but for P. leucopus only the unique reads for Arg1 for that species were tabulated. The cross-hybridization of reads, e.g. number of M. musculus reads mapping to P. leucopus Arg1, was no more than 5% of that of the homologously mapped gene, e.g. M. musculus Arg1, and usually <1%. Expression values were unique reads normalized for total reads across all the samples without adjustment for reference sequence length. These were log10-transformed.

Weighted correlation network analysis. We used WGCNA [117] to identify densely interconnected genes (modules) for the 6 datasets across species and tissues by building a matrix of gene expression with genes TPM >1 in one or more individual. We selected a power ( ) of 13 for a soft threshold for the weighted network and specified a minimum of 100 genes per module. To merge modules with similar gene expression profiles, we carried out a dynamic tree cut, with an eigengene dissimilarity threshold of 0.2 that generated the final eigengene profiles, where eigengene is the first principal component of module expression matrix [118] . The inferred modules were distinguished by assigned hexadecimal color code (https://www.color-hex.com/color-names.html), e.g. darkorange2. Modules, associated GO terms, and constituent genes that are not presented in this paper have been deposited with Dryad (http://datadryad.org) under the dataset name "Peromyscus_WGCNA_supplement" (https://doi.org/7280/D1B38G).

Statistics. Means are presented with 95% confidence intervals. Parametric (t test) and nonparametric (Mann-Whitney) tests of significance were 2-tailed. Unless otherwise stated, the t test p value is given. Adjustment of p values for multiple testing was by the Benjamini-Hochberg method [119] , as implemented in edgeR, CLC Genomics Workbench (see above), or False Discovery Rate Online Calculator (https://tools.carbocation.com/FDR). For categorical data an exact Likelihood Ratio Test was performed with StatExact v. 6 (Cytel Statistical Software). Other methods are given in software programs or suites cited above. Table S11 . Figure 5 . Four selected eigengene modules by network analysis of differential responses of P. leucopus or M. musculus to LPS. The different modules are distinguished by color of the hexadecimal scheme: darkorange2 for up-regulated in LPS-treated P. leucopus, darkseagreen4 for comparatively higher expression in untreated P. leucopus than other three groups, brown4 for down-regulated in both species after LPS treatment, and lightblue for up-regulated in LPStreated M. musculus. The top 5 GO terms by adjusted p value are shown for each module. The DEGs constituting each of the GO term sets from this analysis are listed in Table S12 along with p values and odds ratios. The other 20 modules from this analysis are available at the Dryad dataset repository (https://doi.org/7280/D1B38G). Table S13 -S15. Figure 8 . Comparison of DEGs of RNA-seq of P. leucopus treated with LPS and P. leucopus systemically infected with bacterial agent Borrelia hermsii. There were 12 LPS-treated animals with 8 controls, and 5 infected animals with 3 controls (Tables S17 and S18). Panel A. Venn diagram of numbers of DEGs in each experiment and the overlap between them. Panel B. The scatter plot is of log2-transformed fold-changes between study and control conditions for infection experiment (y-axis) versus short-term LPS experiment (x-axis). Selected genes are indicated by label adjacent to a red symbol for the datapoint. (Table S19 ) are given on x-axis and false discovery rate p values are given on y-axis. For conciseness the upper limit for the -log10 values for this graph was 13.5. The exact or approximate locations of selected differentially-expressed genes are shown. 

Infection resistance and tolerance in Peromyscus spp., natural reservoirs of microbes that are virulent for humans

Tickborne diseases -confronting a growing threat

The Mammals of North America

Comparing the relative potential of rodents as reservoirs of the Lyme disease spirochete (Borrelia burgdorferi)

Antibody response in whitefooted mice (Peromyscus leucopus) experimentally infected with the Lyme disease spirochete (Borrelia burgdorferi)

Duration of Borrelia burgdorferi infectivity in white-footed mice for the tick vector Ixodes scapularis under laboratory and field conditions in Ontario

Antigenic variation in vector-borne pathogens

Longitudinal study of infection with Borrelia burgdorferi in a population of Peromyscus leucopus at a Lyme disease-enzootic site in Maryland

Borrelia burgdorferi infection in a natural population of Peromyscus leucopus mice: a longitudinal study in an area where Lyme Borreliosis is highly endemic

The Lyme disease pathogen has no effect on the survival of its rodent reservoir host

Experimental infection of the white-footed mouse with Borrelia burgdorferi

Experimental Borrelia burgdorferi infection in Peromyscus leucopus

Borrelia burgdorferi has minimal impact on the Lyme disease reservoir host Peromyscus leucopus. Vector Borne Zoonotic Dis

Disentangling Genetic Variation for Resistance and Tolerance to Infectious Diseases in Animals

Two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases

Tolerance of infections

Heritable variation in host tolerance and resistance inferred from a wild host-parasite system

Costs and benefits of plant resistance to herbivory

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MD: National Institute of Allergy and Infectious Diseases

An immunological marker of tolerance to infection in wild rodents

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Experimental infection model for Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus)

SARS-CoV-2 infection, neuropathogenesis and transmission among deer mice: Implications for reverse zoonosis to New World rodents

North American deer mice are susceptible to SARS-CoV-2

Viruses in bats and potential spillover to animals and humans. Current opinion in virology

Going to Bat(s) for Studies of Disease Tolerance

Vertebrate defense against parasites: Interactions between avoidance, resistance, and tolerance

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Deer mice as laboratory animals

Peromyscus mice as a model for studying natural variation

Peromyscus as a model of human disease

Muroid rodent phylogenetics: 900-species tree reveals increasing diversification rates

The genome of Peromyscus leucopus, natural host for Lyme disease and other emerging infections

Genomes, expression profiles, and diversity of mitochondria of the White-footed Deermouse Peromyscus leucopus, reservoir of Lyme disease and other zoonoses

Lactobacilli and other gastrointestinal microbiota of Peromyscus leucopus, reservoir host for agents of Lyme disease and other zoonoses in North America

Animal models of borreliosis

Lipopolysaccharide (LPS)). In: Delves PJ, editor. Encyclopedia of Immunology

Tolerance to bacterial pyrogens: I. Factors influencing its development. The Journal of experimental medicine

Gene-specific control of inflammation by TLRinduced chromatin modifications

Inhibition of lipopolysaccharide-induced signal transduction in endotoxin-tolerized mouse macrophages: dysregulation of cytokine, chemokine, and toll-like receptor 2 and 4 gene expression

Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene

MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction

The susceptibility of mice to bacterial endotoxins

Species differences in Kupffer cells and endotoxin sensitivity

Altered immune responses in mice lacking inducible nitric oxide synthase

Lipopolysaccharide-induced lethality and cytokine production in aged mice

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The wild-derived inbred mouse strain SPRET/Ei is resistant to LPS and defective in IFN-beta production

Inhibition of autophagy with 3-methyladenine is protective in a lethal model of murine endotoxemia and polymicrobial sepsis

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

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Vitamin B6 prevents IL-1β protein production by inhibiting NLRP3 inflammasome activation

Metabolomics in pneumonia and sepsis: an analysis of the GenIMS cohort study

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Identification of urine metabolites as biomarkers of early Lyme disease

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Mouse D1Pas1, a DEAD-box RNA helicase, is required for the completion of first meiotic prophase in male germ cells

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Peromyscus Genetic Stock Center Coumbia, SC: University of South Carolina

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A crossplatform toolkit for mass spectrometry and proteomics

XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification

Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards

Predicting network activity from high throughput metabolomics

Skyline: an open source document editor for creating and analyzing targeted proteomics experiments

Trimmomatic: a flexible trimmer for Illumina sequence data

Near-optimal probabilistic RNA-seq quantification

edgeR: a Bioconductor package for differential expression analysis of digital gene expression data

Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool

R: A language and environment for statistical computing Vienna, Austria: R Foundation for Statistical Computing

WGCNA: an R package for weighted correlation network analysis

Large-scale gene network analysis reveals the significance of extracellular matrix pathway and homeobox genes in acute myeloid leukemia: an introduction to the Pigengene package and its applications

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Dose response of P. leucopus to LPS. Groups of 6 adult animals received different intraperitoneal (i.p.) doses on a mg per gm body weight basis of Escherichia coli LPS at time 0 and then monitored for physical signs of sickness (conjunctivitis, inactivity, and hyperpnea) and survival over the succeeding 7 days (168 h). Panel B. Experimental design of comparative study of short-term effects of LPS on P. leucopus and Mus musculus

Phosphatidyl serine acyl-chain remodeling Regulation of interleukin-5 secretion Regulation of Interleukin-13 secretion Phosphatidyl serine metabolic process

Cytokine-mediated signaling pathway Cytokine-mediated signaling Regulation of infalmmatory response Positive regulation of T-cell proliferation Pos. regulation of I-KappaB kinase

We thank Khalil Bassam, Angelique Cortez, and Hanjuan Shao for their assistance in these studies. 

Cancer Center Support Grant (P30CA-062203) at the University of California, Irvine and NIH shared instrumentation grants 1S10RR025496-01, 1S10OD010794-01, and 1S10OD021718-01. The PGSC was supported by NSF grant DBI1755670.