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. Author manuscript; available in PMC: 2018 Aug 15.
Published in final edited form as: J Immunol. 2017 Jul 3;199(4):1362–1371. doi: 10.4049/jimmunol.1601527

Mesenchymal cell-specific MyD88 signaling promotes systemic dissemination of Salmonella Typhimurium via inflammatory monocytes

Donghyun Kim *,†,‡, Sang-Uk Seo *,†,§, Melody Y Zeng *,†, Wan-Uk Kim ‡,, Nobuhiko Kamada ‖, Naohiro Inohara *, Gabriel Núñez *,†
PMCID: PMC5548622  NIHMSID: NIHMS884069  PMID: 28674182

Abstract

Enteric pathogens including Salmonella enteric serovar Typhimurium can breach the epithelial barrier of the host and spread to systemic tissues. In response to infection, the host activates innate immune receptors via the signaling molecule MyD88 that induces protective inflammatory and anti-microbial responses. Most of these innate immune responses have been studied in hematopoietic cells, but the role of MyD88 signaling in other cell types remains poorly understood. Surprisingly, we found that Dermo1-Cre;Myd88fl/fl mice with mesenchymal cell-specific deficiency of MyD88 were less susceptible to orogastric and intraperitoneal S. Typhimurium infection than their Myd88fl/fl littermates. The reduced susceptibility of Dermo1-Cre;Myd88fl/fl mice to infection was associated with lower loads of S. Typhimurium in the liver and spleen. Mutant analyses revealed that S. Typhimurium employs its virulence type III secretion system (T3SS)-2 to promote its growth through MyD88 signaling pathways in mesenchymal cells. Inflammatory monocytes function as a major cell population for systemic dissemination of S. Typhimurium. Mechanistically, mesenchymal cell-specific MyD88 signaling promoted CCL2 production in the liver and spleen and recruitment of inflammatory monocytes to systemic organs in response to S. Typhimurium infection. Consistently, MyD88 signaling in mesenchymal cells enhanced the number of phagocytes including Ly6ChiLy6G− inflammatory monocytes harboring S. Typhimurium in the liver. These results suggest that S. Typhimurium promotes its systemic growth and dissemination through MyD88 signaling pathways in mesenchymal cells.

Keywords: MyD88, Salmonella, Host defense, Mesenchymal cells

Introduction

The enteric pathogen Salmonella enterica, a facultative intracellular pathogen, is a common cause of foodborne gastroenteritis and typhoid fever (1). In humans, gastrointestinal infections are caused by hundreds of S. enterica serovars including the serovar Typhimurium (S. Typhimurium) that serves as a model for studying virulence factors and host defense mechanisms against bacterial pathogens (2). After orogastric infection, S. Typhimurium can engage multiple mechanisms to breach the epithelial barrier and invade into the lamina propria (3). In susceptible mice, S. Typhimurium reaches the mesenteric lymph nodes and spread to systemic organs including the liver and spleen (3). Mammalian hosts deploy an arsenal of defense mechanisms to counter pathogenic microbes including S. Typhimurium. Upon microbial invasion including the oral route, sensing of pathogenic organisms is mediated by several classes of membrane-bound or cytosolic pattern recognition receptors (PRRs). A major class of membrane-bound PRRs is TLRs that recognize a wide array of microbial-associated molecular patterns (MAMPs) (4). In response to S. Typhimurium infection, TLRs are activated by the presence of multiple MAMPs including LPS, lipopeptides and flagellin (5). Ligation of most TLRs by MAMPs leads to recruitment of the adaptor protein MyD88 that mediates intracellular signaling events that culminate in the transcriptional activation of inflammatory and anti-microbial genes (6). In addition, MyD88 serves as a signaling adaptor for IL-1, IL-18, and IL-33 receptors as well as related non-TLR/IL-1R receptors (6).

Pathogens employ virulence factors that enable invasion of host tissues as well as their survival and replication despite recognition by the immune system. Two critical virulence factors of S. Typhimurium are the type III secretion system (T3SS)-1 and -2 encoded by the pathogenicity islands 1 (SPI-1) and 2 (SPI-2), respectively (7–10). The T3SS-1 facilitates invasion of S. Typhimurium into enterocytes and the lamina propria, but is not required for systemic spread after orogastric infection (10). In contrast, the T3SS-2 is activated intracellularly and is critical for bacterial replication inside host cells and the systemic phase of the disease (11, 12). In the streptomycin mouse model that allows efficient gut colonization by S. Typhimurium, pathogen mutants lacking the T3SS-2 can use the T3SS-1 to invade epithelial cells and to elicit early inflammatory responses in the intestine, but are incapable of systemic spread (2, 13, 14). In contrast, S. Typhimurium mutants deficient in the T3SS-1 use an alternative route for transepithelial invasion, replicate in CD11b+CD11c− monocytes/macrophages in the lamina propria where they trigger delayed MyD88-dependent inflammation (14). Although both CD11c+CX3CR1+ DCs and CD11b+CD11c−CX3CR1− macrophages have been implicated in pathogen uptake and/or replication in the lamina propria (3), S. Typhimurium use CD18+ blood phagocytes for extraintestinal dissemination to distant organs by using an effector molecule secreted by T3SS-2 (15, 16). Studies from several laboratories have suggested several routes for S. Typhimurium dissemination from the intestine to mesenteric lymph nodes and to systemic organs including the liver and spleen. A route is dissemination via DCs to mesenteric lymph nodes (MLNs) via lymphatics after initial multiplication in Peyer’s patches (3). However, work from several labs suggest that the DC-MLN route is not essential for systemic dissemination to the liver and spleen (15, 17, 18). Most of the evidence suggests that a hematogenous route via CD18+ phagocytes is the major conduit for S. Typhimurium dissemination from the gut to the liver and spleen (15, 17, 18). However, the mechanisms that S. Typhimurium uses for dissemination to systemic organs such as the liver and spleen during infection remain poorly understood.

Resident mesenchymal cells (RMCs) in adult tissues comprise several populations including interstitial and perivascular fibroblasts, pericytes, and myofibroblasts as well as specialized cells such as hepatic stellate cells (19, 20). In response to microbial stimuli, RMCs can produce multiple pro-inflammatory mediators including cytokines, chemokines, and metabolites of arachidonic acid (21). For example, RMCs produce copious amounts of chemokines, such as CXCL1 and CCL2, that promote the recruitment of neutrophils and Ly6Chi monocytes to the site of infection (21, 22). However, the in vivo role of RMCs in infection by bacterial pathogens including S. Typhimurium remains largely unknown. We provide evidence that mesenchymal cell-specific MyD88 signaling plays an important in the systemic dissemination of S. Typhimurium during infection.

Materials and Methods

Mice

WT, Dermo1-Cre;Myd88fl/fl, Villin-Cre;Myd88fl/fl, and Cd11b-Cre;Myd88fl/fl mice on a C57BL/6 background were bred and kept under specific pathogen-free (SPF) conditions at the University of Michigan Animal Facility. Villin-Cre;Myd88fl/fl and Cd11b-Cre;Myd88fl/fl mice were a gift from Dr. Xiaoxia Li, the Cleveland Clinic (23, 24). Myd88fl/fl mice and Dermo1-Cre mice were purchased from Jackson laboratory and crossed to generate mesenchymal-specific MyD88 deficient Dermo1-Cre;Myd88fl/fl mice. All the experimental procedures were performed in accordance with the protocols approved by the University Committee on Use and Care of Animals (UCUCA) at University of Michigan.

Bacterial strains

Salmonella enterica serovar Typhimurium strain SL1344 and its isogenic mutants ΔSPI1 deficient in the T3SS-1 and ΔssaV deficient in the T3SS-2 were a gift from Denise Monack, Stanford University. The S. Typhimurium strain M525P expressing GFP was a gift from Clare Bryant, University of Cambridge.

S. Typhimurium infections

Groups of age- and sex-matched mice were infected at 7–9 weeks of age. For orogastric infection with S. Typhimurium, mice were pretreated with 20 mg streptomycin by oral gavage and, 24 h later, the mice were orogastrically infected with 5×107 CFU. For systemic infection, mice were injected i.p. with 1×103 CFU of WT or mutant SL1344 strains and with 1×105 CFU of the M525P strain. The mice were euthanized at different times post-infection and samples including organs were collected for analyses. No animals or samples were excluded for analyses in the described mouse experiments.

Analysis of S. Typhimurium loads

Organs and tissues were removed and homogenized in 3–5 ml of PBS. The ceca contents were flushed out with sterile PBS 5 times and suspended in PBS by vortexing for 15 min. Serial dilutions of organ homogenates and cecal contents were plated on MacConkey agar plates supplemented with 50 µg/ml of streptomycin and cultured for 1 day at 37°C to determine CFU. To assess intracellular and extracellular bacteria in the bloodstream, we collected whole blood in heparinized tubes and whole blood and plasma were spread on MacConkey agar plates supplemented with streptomycin. The number of intracellular bacteria was calculated by subtracting extracellular bacterial number in plasma from the corresponding number of colonies in whole blood.

Liver histology and microabscess evaluation

Liver was harvested and fixed in formalin for paraffin embedding. The number of microabscesses in the liver was determined by analysis of more than 3 different fields at 10× magnification per sample in a blinded fashion.

Real-time quantitative PCR (qPCR)

RNAs were isolated using E.Z.N.A Total RNA kit I (Omega Bio-Tek) and the corresponding cDNAs were generated using a high capacity RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer’s instructions. The cDNAs were used for qPCR with gene-specific primer sets and SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instruction using the StepOnePlus Real-Time PCR system (Applied Biosystems). PCR primers (Invitrogen) were as follows: Myd88 (5’-TCCGGCAACTAGAACAGACAGACT-3’; 5’-GCGGCGACACCTTTTCTCAAT-3’), Ccl2 (5’-CCCAATGAGTAGGCTGGAGA-3’; 5’-GCTGAAGACCTTAGGGCAGA-3’), and Gapdh (5’-TGCGACTTCAACAGCAACTC-3’; 5’-GCCTCTCTTGCTCAGTGTCC-3’). The PCR conditions for mRNA quantification were 95°C for 10 min, followed by 40 cycles with denaturation at 95°C for 10 s and annealing and extension at 60°C for 1 min. Cycle threshold (Ct) of respective samples were normalized internally using the average Ct value of Gapdh.

Flow cytometric analysis

Cell surface fluorescence was measured using a FACSCanto II or LSRFortessa (BD Biosciences) instrument and the data analyzed using FlowJo software (TreeStar). Fluorescence-labeled antibodies against CD11b (M1/70), CD11c (N418), CD18 (M18/2), F4/80 (BM8) and Ly6C (HK1.4) were from eBioscience. Antibodies for CD45 30-F11) and Ly6G (1A8) were purchased from BD Pharmingen.

Cell Preparations

Lamina propria cells were isolated as previously described (25). Briefly, the dissected intestinal mucosa was incubated in calcium and magnesium-free HBSS (Gibco) containing 2.5% heat-inactivated FBS and 1 mM DTT (Sigma-Aldrich) to remove mucus. The mucosa was then incubated twice in HBSS containing 1 mM EDTA (Sigma-Aldrich) for 45 min at 37 °C. The tissues were then collected and incubated in HBSS containing 400 U/ml collagenase type 3 and 0.01 mg/ml DNase I (Worthington Biochemical) for 120–180 min at 37 °C. Lamina propria cells were isolated from digested tissue by centrifugation on a Percoll gradient (75% and 40% Percoll). For qPCR analysis, CD45−Thy1+ cells highly enriched in mesenchymal cells (20) were sorted from lamina propria cells using a FACS Aria II instrument (BD Biosciences). For immunoblotting, CD45−Thy1+ cells were isolated by incubating the lamina propria cells first with biotin-labeled antibody for CD45 30-F11) to remove CD45+ cells using the EasySep™ biotin positive selection kit and a magnet (Stemcell Technologies, Inc). The CD45− cell population was then incubated with PE-labeled antibody for Thy1 (53-2.1, BD Pharmingen) and Thy1+ cells selected using the EasySep™ PE positive selection kit and a magnet (Stemcell Technologies, Inc) according to the manufacturer’s instructions. CD3+ T cells were isolated from the lamina propria cells by incubation with PE-labeled antibody against CD3 (145-2C11, BD Pharmingen) and then the EasySep™ PE positive selection kit and a magnet (Stemcell Technologies, Inc). Epithelial cells, BMDMs and BMDCs were prepared as previously described (25–27). Hematopoietic cells in liver were isolated using a similar protocol. Briefly, dissected liver tissues were incubated in HBSS containing 400 U/ml collagenase type 3 and 0.01 mg/ml DNase I (Worthington Biochemical) for 120 min at 37 °C. Hematopoietic cells were obtained by centrifugation of digested tissue on a 40–70% Percoll gradient. Splenocytes and cells from lymph nodes were isolated by grinding the tissue and passing the spleen and lymph node suspensions through a cell strainer.

Immunoblotting

Cells were lysed in RIPA buffer supplemented with complete protease inhibitor cocktail (Roche) and 2 mM DTT (Sigma-Aldrich). Samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were incubated with antibody to MyD88 (ProSci, Inc.). Protein bands detected using the ECL kit (Thermo Scientific). Membranes were stripped using restore stripping buffer (Thermo Scientific) and re-probed for β-actin as a loading control.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism software (GraphPad Software). For comparisons of Ccl2 mRNA amounts between two groups, unpaired, two-tailed unpaired Student t-test was used. Comparisons of pathogen burden, microabscess formation, and cell populations between groups or within each group were performed using the non-parametric Mann–Whitney or Kruskal-Wallis and post Dunn test, respectively. Mouse survival was analyzed using the log-rank Mantel-Cox test. Differences at p < 0.05 were considered to be statistically significant.

Results

Ablation of MyD88 in mesenchymal cells improves survival of Salmonella-infected mice

Mesenchymal cells have been suggested to play a role in the regulation of immune responses against enteric pathogens (21, 22), but experimental evidence in vivo is lacking. To study a role for mesenchymal cells in the innate immune response to S. Typhimurium in the animal, we crossed Myd88fl/fl mice with Dermo1-Cre mice (also called Twist2-Cre) that express the Cre-recombinase selectively in mesenchymal cells during mouse development to generate Dermo1-Cre;Myd88fl/fl mice (28–30). To determine whether the expression of MyD88 was altered in mesenchymal cells, we assessed the RNA and protein expression of MyD88 in lamina propria CD45−Thy1+ cells, that are mesenchymal cells including fibroblasts and myofibroblasts (20, 31), intestinal epithelial cells, bone marrow derived macrophages (BMDMs), dendritic cells (BMDCs) and lamina propria T cells from Dermo1-Cre;Myd88fl/fl and control Myd88fl/fl mice. As expected, the expression of Myd88 mRNA in FACS-sorted lamina propria CD45−Thy1+ cells from Dermo1-Cre;Myd88fl/fl mice was markedly reduced when compared to the same cells from Myd88fl/fl mice (Supplemental Fig. 1A). Consistently, MyD88 protein expression was also greatly reduced in lamina propria CD45−Thy1+ cells from Dermo1-Cre;Myd88fl/fl mice (Left panel in the Supplemental Fig. 1B). In contrast, there was no detectable reduction in the expression of MyD88 in intestinal epithelial cells, BMDMs, BMDCs, and T cells from Dermo1-Cre;Myd88fl/fl when compared to Myd88fl/fl mice (Supplemental Fig. 1B–D). These results indicate that Dermo1-Cre;Myd88fl/fl mice have a deficiency in MyD88 within mesenchymal cells.

To assess an intrinsic role for MyD88 in mesenchymal cells, we first infected Dermo1-Cre;Myd88fl/fl mice and their littermates Myd88fl/fl mice with the virulent S. Typhimurium strain SL1344 using the streptomycin mouse model that allows efficient pathogen colonization via the oral route (8, 9, 13). Surprisingly, Dermo1-Cre;Myd88fl/fl mice exhibited higher survival than Myd88fl/fl mice after orogastric infection (Fig. 1A). Consistent with improved survival, the pathogen loads in the liver and spleen of Dermo1-Cre;Myd88fl/fl mice were lower than in Myd88fl/fl littermates (Fig. 1B). Moreover, histological analyses revealed lower numbers of liver microabscesses in Dermo1-Cre;Myd88fl/fl mice than in Myd88fl/fl mice (Fig. 1C, 1D). In contrast, selective deletion of MyD88 in intestinal epithelial cells (Villin-Cre;Myd88fl/fl) or CD11b-positive cells (Cd11b-Cre;Myd88fl/fl) did not affect mouse survival after orogastric S. Typhimurium infection (data not shown). These results indicate that mesenchymal cell-specific deficiency of MyD88 is associated with reduced dissemination of S. Typhimurium and improved host survival.

FIGURE 1.

FIGURE 1

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Mesenchymal cell-specific MyD88-deficient mice are more resistant to orogastric infection with S. Typhimurium. Myd88fl/fl and Dermo1-Cre;Myd88fl/fl mice were treated with streptomycin and, one day later, the mice were infected orogastrically with 5×107 CFU/mouse of WT S. Typhimurium SL1344 strain. (A) Mouse survival was monitored over time. Data from three independent experiments (n = 14 and 16, respectively). (B) Pathogen loads were assessed in cecal contents, mesenteric lymph nodes (MLNs), liver, and spleen on day 4 after infection. (C) Histological images of liver collected on day 4 after infection and stained with hematoxylin and eosin. Histological images are at 100× magnification and 200× (inset). Arrowheads points to microabscesses. (D) The number of liver microabscesses per microscopic field (lens objective ×10) for each mouse is indicated. Each number represents the average of at least 3 microscopic field per mouse. (B, D) Each dot represents an individual mouse; horizontal lines indicate the mean. The results shown are representative of at least two independent experiments. *p < 0.05 and **p < 0.01 by log-rank Mantel-Cox test (A) and by Mann-Whitney test (B, D). NS, not significant.

MyD88 signaling in mesenchymal cells promotes T3SS-2-mediated Salmonella growth after orogastric infection

S. Typhimurium T3SS-1 and T3SS-2 mediate translocation of effector proteins into host cells to promote pathogen invasion and replication in vivo (8–10, 13). To determine whether the T3SS-1 or T3SS-2 promotes the systemic growth of S. Typhimurium via MyD88 within mesenchymal cells, we infected Dermo1-Cre;Myd88fl/fl and Myd88fl/fl mice with S. Typhimurium strains deficient in T3SS-1 or T3SS-2. In the streptomycin pre-treatment model, the Dermo1-Cre;Myd88fl/fl and Myd88fl/fl littermates had comparable S. Typhimurium T3SS-1 mutant loads in cecal contents, cecal tissue, and mesenteric lymph nodes on day 4 after orogastric infection (Fig. 2A). In contrast, Dermo1-Cre;Myd88fl/fl mice had lower T3SS-1 mutant loads in the liver and spleen than Myd88fl/fl mice (Fig. 2A). Furthermore, Dermo1-Cre;Myd88fl/fl mice exhibited reduced formation of microabscesses in the liver which is consistent with reduced pathogen loads (Fig. 2B, 2C). In addition, both Dermo1-Cre;Myd88fl/fl and Myd88fl/fl littermates had high, but comparable, S. Typhimurium T3SS-2 mutant loads in cecal contents on day 4 after orogastric infection (Fig. 2D). Consistent with previous observations that the T3SS-2 is required for systemic spread (9, 13), both Dermo1-Cre;Myd88fl/fl and Myd88fl/fl mice had low or undetectable S. Typhimurium T3SS-2 mutant loads in the liver and spleen (Fig. 2D). These data indicate that MyD88 signaling within mesenchymal cells regulates T3SS-2-mediated S. Typhimurium systemic dissemination after orogastric infection.

FIGURE 2.

FIGURE 2

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MyD88 signaling in mesenchymal cells promotes T3SS-2-mediated Salmonella growth after orogastric infection. (A–C) Streptomycin-pretreated Myd88fl/fl and Dermo1-Cre;Myd88fl/fl mice were infected orogastrically with 5×107 CFU/mouse of T3SS-1 mutant S. Typhimurium SL1344 strain. (A) Pathogen loads in the cecal contents (g; per gram), cecum tissue, MLNs, liver, and spleen on day 4 after infection. (B) Histological images of liver collected on day 4 after infection and stained with hematoxylin and eosin. Histological images are at 100× magnification and 200× (inset). Arrowheads points to microabscesses. (C) The number of liver microabscesses per microscopic field (lens objective ×10) for each mouse is indicated. Each number represents the average of at least 3 microscopic field per mouse. (D) Streptomycin-pretreated Myd88fl/fl and Dermo1-Cre;Myd88fl/fl mice were infected orogastrically with 5×107 CFU/mouse of T3SS-2 mutant S. Typhimurium SL1344 strain. (A, C, D) Each dot indicates an individual mouse; horizontal lines show the mean. Data shown are representative of at least two independent experiments. *p < 0.05 and **p < 0.01 by Mann-Whitney test. NS, not significant.

MyD88 signaling in mesenchymal cells promotes Salmonella growth after intraperitoneal infection

To determine whether MyD88 can function within mesenchymal cells to control S. Typhimurium infection in the systemic phase of infection, we infected Dermo1-Cre;Myd88fl/fl and Myd88fl/fl mice i.p. with WT S. Typhimurium and assessed mouse survival and pathogen loads in the liver and spleen. We observed increased survival of Dermo1-Cre;Myd88fl/fl mice when compared to Myd88fl/fl littermates (Fig. 3A). Consistently, the pathogen loads in the liver and spleen were lower in Dermo1-Cre;Myd88fl/fl than in Myd88fl/fl mice (Fig. 3B). As expected, the lower pathogen loads in the liver were associated with reduced numbers of microabscesses in the liver of Dermo1-Cre;Myd88fl/fl compared to Myd88fl/fl mice (Fig. 3C, 3D). In accord with results obtained with orogastric infection, after i.p. infection of S. Typhimurium T3SS-1 mutant strain, lower loads of the S. Typhimurium T3SS-1 mutant in the liver and spleen as well as reduced number of liver microabscesses were observed in Dermo1-Cre;Myd88fl/fl mice as compared to Myd88fl/fl littermates (Fig. 3E–3G). In contrast, both Dermo1-Cre;Myd88fl/fl and Myd88fl/fl littermates had significant, but comparable, S. Typhimurium T3SS-2 mutant loads in the liver and spleen on day 3 after i.p. infection (Fig. 3H). These results suggest that T3SS2-mediated S. Typhimurium systemic spread is regulated via MyD88 signaling within mesenchymal cells.

FIGURE 3.

FIGURE 3

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MyD88 signaling in mesenchymal cells promotes T3SS-2-mediated Salmonella growth after intraperitoneal infection. (A–E) Myd88fl/fl and Dermo1-Cre;Myd88fl/fl mice were infected i.p. with 5×103 CFU/mouse of WT S. Typhimurium SL1344 strain. Myd88fl/fl and Dermo1-Cre;Myd88fl/fl mice were infected i.p. with 5×103 CFU/mouse of S. Typhimurium T3SS-1 mutant (E–G) and T3SS-2 mutant (H). (A) Mouse survival was monitored overtime. Data from three independent experiments (n = 11 and 17, respectively). (B, E, H) Pathogen loads were assessed in the liver and spleen on day 3 after infection. (C, F) Histological images of liver collected on day 3 after infection and stained with hematoxylin and eosin. Histological images are at 100× magnification and 200× (inset). Arrowheads points to microabscesses. (D, G) The number of liver microabscesses per microscopic field (lens objective ×10) for each mouse is indicated. Each number represents the average of at least 3 microscopic field per mouse. (B, D, E, G, H) Each dot represents an individual mouse; horizontal lines indicate the mean. The results shown are representative of at least two independent experiments. *p < 0.05, **p < 0.01 and by ****p < 0.0001 was evaluated using the log-rank Mantel-Cox test (A) and by Mann-Whitney test (B, D, E, G, H). Data shown are representative of at least two independent experiments.

Inflammatory monocytes function as a major cell population for systemic dissemination of Salmonella

CD18+ blood leukocytes contribute to extraintestinal dissemination of S. Typhimurium after orogastric infection (15). Although CD18+ leukocytes have been shown to carry S. Typhimurium in the bloodstream, the precise cell population has not been fully characterized. We next determined which blood cell population harbors green fluorescent (GFP)-expressing S. Typhimurium after i. p. infection. Flow cytometric analysis of peripheral blood collected from mice 1 h after i.p. infection revealed that ~ 0.13% of the CD45+ blood cells were positive for GFP-expressing S. Typhimurium when compared to uninfected mice that showed a background staining of ~ 0.046% (Fig. 4A, 4B). The blood cell population that contained more GFP-positive S. Typhimurium were inflammatory monocytes (Ly6ChiLy6G−) followed by neutrophils (Ly6CintLy6G+), while S. Typhimurium were undetectable in F480+Ly6G−Ly6low-neg monocytic cells when compared to uninfected mice (Fig. 4C). CD18 is an integrin that facilitates leukocyte transmigration and is critical for extraintestinal dissemination of S. Typhimurium (15, 16). Notably, CD18 was expressed at higher amounts on the surface of Ly6ChiLy6G− inflammatory monocytes than in Ly6CintLy6G+ neutrophils isolated from the liver and spleen on day 3 after infection with S. Typhimurium (Fig. 5). In addition, when we examined the amount of Salmonella in blood cells and plasma after orogastic or i.p. infection, most S. Typhimurium were associated with blood cells (Supplemental Fig. 2A, 2C), supporting the notion that blood cells play a role as carriers of S. Thyphimurium in systemic dissemination of S. Typhimurium at least during the initial phase of infection. However, there were no difference in the number of blood cells harboring S. Typhimurium between Dermo1-Cre;Myd88fl/fl mice and Myd88fl/fl littermates (Supplemental Fig. 2B, 2D). These results suggest that after infection most S. Typhimurium reside within inflammatory monocytes in the bloodstream independently of MyD88 signaling within mesenchymal cells.

FIGURE 4.

FIGURE 4

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Inflammatory monocytes function as a major cell population for systemic dissemination of Salmonella. Mice were injected i.p. with PBS (n = 10) or 1×106 CFU/mouse of the WT S. Typhimurium M525P strain expressing GFP (n = 9). CD45+ leukocytes (A, B) and phagocyte populations (C) harboring S. Typhimurium in blood were analyzed 1 h after i.p. infection. Each dot indicates an individual animal; horizontal lines represent the mean. **p < 0.01 and ****p < 0.0001 by Mann-Whitney test and #p < 0.05 and ###p < 0.001 by Kruskal-Wallis test. Data shown are representative of two independent experiments.

FIGURE 5.

FIGURE 5

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Inflammatory monocytes express high amounts of surface CD18. Mice were infected i.p. with 1×103 CFU/mouse of T3SS-1 mutant S. Typhimurium SL1344 strain. The expression of CD18 on CD45+CD11b− cells (CD11b−), CD45+CD11b+Ly6CintLy6G+ cells (PMN; neutrophils) and CD45+CD11b+Ly6ChiLy6G− cells (Mono; inflammatory monocytes) from liver tissue (A, B) and spleen (C, D) were analyzed by flow cytometry on day 3 after infection. The X-axis of flow plot was normalized to the same scale to facilitate comparison (A, C). Each dot indicates an individual animal; horizontal lines represent the mean (B, D). **p < 0.01 by Mann-Whitney test.

Mesenchymal cell-specific MyD88-deficient mice exhibit impaired CCL2 production and recruitment of inflammatory monocytes during Salmonella infection

To understand the mechanism by which MyD88 signaling within mesenchymal cells regulates the systemic phase of S. Typhimurium infection, we first assessed the number of phagocyte populations in the liver of Dermo1-Cre;Myd88fl/fl mice and Myd88fl/fl littermates after orogastric infection with WT S. Typhimurium. Within the diverse CD45+CD11b+ phagocyte population, the percent and total number of Ly6ChighLy6G− cells that contain inflammatory monocytes, but not LyCintLy6G+ neutrophils, were reduced in the liver of Dermo1-Cre;Myd88fl/fl mice when compared to Myd88fl/fl littermates (Fig. 6A–C). Consistently, the expression of Ccl2 mRNA, a chemokine that regulates the recruitment of blood monocytes to sites of inflammation, was lower in the liver of Dermo1-Cre;Myd88fl/fl mice than in Myd88fl/fl littermates (Fig. 6D). However, there was no difference in Ccl2 mRNA expression and inflammatory monocyte numbers in MLNs between Dermo1-Cre;Myd88fl/fl and Myd88fl/fl littermates (Supplemental Fig. 3). Comparable results were obtained when Dermo1-Cre;Myd88fl/fl mice and Myd88fl/fl littermates were infected i.p. with the WT S. Typhimurium. Lower amounts of Ccl2 mRNA were detected in the liver of Dermo1-Cre;Myd88fl/fl mice than in Myd88fl/fl littermates after i.p. infection (Fig. 6E). Reduced numbers of Ly6ChighLy6G− cells, but not LyCintLy6G+ cells, were also found in the liver of Dermo1-Cre;Myd88fl/fl mice, compared to Myd88fl/fl littermates after i.p. infection with WT S. Typhimurium (Fig. 6F–H). Furthermore, when the mice were infected orogastrically or i.p. with S. Typhimurium T3SS-1 mutant, the Ccl2 mRNA expression and phagocyte population in the liver and spleen were consistent with data obtained from infection with WT S. Typhimurium (Supplemental Fig. 4). These results indicate that MyD88 signaling in mesenchymal cells regulates CCL2 expression in the liver and spleen and the recruitment of inflammatory monocytes to systemic organs upon S. Typhimurium infection.

FIGURE 6.

FIGURE 6

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Mesenchymal cell-specific MyD88-deficiency is associated with reduced CCL2 production and recruitment of inflammatory monocytes. (A–D) Streptomycin-pretreated Myd88fl/fl and Dermo1-Cre;Myd88fl/fl mice were infected orogastrically with 5×107 CFU/mouse of WT S. Typhimurium SL1344 strain. (A–C) Representative flow cytometric analysis (A), percentage (B) and total numbers (C) of inflammatory monocytes and neutrophils in liver tissue on day 4 after infection. (D) Amounts of Ccl2 mRNA normalized to Gapdh expression in the liver on indicated days after infection. Data are shown as the mean ± s.d. (E–H) Streptomycin-pretreated Myd88fl/fl and Dermo1-Cre;Myd88fl/fl mice were infected i.p. with 5×103 CFU/mouse of S. Typhimurium SL1344 WT strain. (E) Amounts of Ccl2 mRNA normalized to Gapdh expression in the liver on day 3 after infection. Data are shown as the mean ± s.d. (F–H) Representative flow cytometric analysis (F), percentage (G) and total numbers (H) of inflammatory monocytes and neutrophils in liver tissue on day 3 after infection. Each dot indicates an individual animal; horizontal lines represent the mean. Results are representative of at least two independent experiments. *p < 0.05, ***p < 0.001, and ****p < 0.0001 by unpaired t- test (A, E) or by Mann-Whitney test (C, D, G, H). NS, not significant.

MyD88 signaling in mesenchymal cells enhances the number of phagocytes harboring Salmonella in the liver

We next assessed whether MyD88 acts in mesenchymal cells to regulate the number of phagocytes harboring S. Typhimurium. To determine this, we infected Dermo1-Cre;Myd88fl/fl mice and Myd88fl/fl littermates with the GFP+-S. Typhimurium strain i.p. and analyzed the phagocyte population in the liver harboring the pathogen by flow cytometry using a gating strategy depicted in Fig. 7A. A higher percentage of CD45+ hematopoietic cells harboring GFP-S. Typhimurium was detected in the liver of Myd88fl/fl mice than in Dermo1-Cre;Myd88fl/fl littermates (Fig. 7B). After gating on GFP+CD45+ cells, flow cytometric analysis revealed that inflammatory monocytes (Ly6ChiLy6G−) were the phagocyte population that harbored more GFP+-S. Typhimurium in Myd88fl/fl mice (Fig. 7C). Furthermore, the number of Ly6ChiLy6G− cells harboring S. Typhimurium was reduced in Dermo1-Cre;Myd88fl/fl mice as compared to Myd88fl/fl mice (Fig. 7C). Consistent with the analyses in blood, S. Typhimurium was also found to reside within neutrophils (Ly6CintLy6G+) and other CD11b+ and CD11b− cell populations in the liver of Myd88fl/fl mice after i.p. infection, although at lower numbers than in inflammatory monocytes (Fig. 7C). Importantly, the number of inflammatory monocytes, neutrophils and some, but not all, CD11b+ and CD11b− populations harboring S. Typhimurium were reduced in Dermo1-Cre;Myd88fl/fl mice (Fig. 7C). These results suggest that MyD88 signaling in mesenchymal cells enhances the number of Ly6ChiLy6G− inflammatory monocytes, but also that of other phagocyte populations, harboring S. Typhimurium.

FIGURE 7.

FIGURE 7

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MyD88 signaling in mesenchymal cells enhances the number of phagocytes harboring S. Typhimurium in the liver. Myd88fl/fl and Dermo1-Cre;Myd88fl/fl mice were inoculated i.p. with the S. Typhimurium M525P strain expressing GFP or injected i.p. with PBS (sham). (A) Representative flow cytometry plots showing gating scheme for analysis of S. Typhimurium-GFP. (B) CD45+ leukocytes harboring S. Typhimurium in the liver tissue on day 3 after infection were analyzed by flow cytometry. Each dot indicates an individual animal; horizontal lines represent the mean. (C) Enumeration of seven phagocyte populations: inflammatory monocytes, neutrophils (PMN), macrophages (MΦ), dendritic cells (DCs) or other phagocyte cells. Data are expressed as the mean ± s.e.m. *p < 0.05 by Mann-Whitney test for comparison between Myd88fl/fl and Dermo1-Cre;Myd88fl/fl mice. #p < 0.05, ##p < 0.01 and ###p < 0.001 by Kruskal-Wallis test for comparisons to inflammatory monocytes in Myd88fl/fl mice. Data shown are all values from two independent experiments.

Discussion

The MyD88 adaptor mediates signaling via innate immune receptors including those of the TLR/IL-1R family that are involved in the detection of pathogens and/or induction of protective immune responses (6). In accord with this notion, mice with whole-body deletion of MyD88 are impaired in the clearance of bacterial pathogens including S. Typhimurium and are more susceptible to infection (13, 32, 33). Surprisingly, we provide evidence that mutant mice with mesenchymal cell-specific deficiency of MyD88 exhibit reduced pathogen loads in tissues and are less susceptible to infection with S. Typhimurium. Thus, although MyD88 signaling generally activate host defense mechanisms that promote pathogen eradication, our results indicate that S. Typhimurium can use MyD88 signaling within mesenchymal cells to promote its own dissemination into host tissues. Mutant analyses indicate that S. Typhimurium employs virulence factor T3SS-2 to promote its growth through MyD88 signaling pathways in mesenchymal cells. The notion that pathogens including S. Typhimurium benefit from inflammatory pathways to promote their own expansion particularly at mucosal sites is not new (34). For example, S. Typhimurium-induced inflammation triggers the generation of nitrate and neutrophil-derived oxidation products that boost the growth of the pathogen in the inflamed gut (35, 36). Collectively, these results suggest that S. Typhimurium hijacks host signaling pathways that are involved in protection against the pathogen to promote its own expansion in the gut and in the systemic phase of the infection.

Analyses of Dermo1-Cre;Myd88fl/fl and control Myd88fl/fl mice revealed that intrinsic deficiency of MyD88 in mesenchymal cells was associated with impaired recruitment of Ly6ChighLy6G− inflammatory monocytes to the spleen and liver after orogastric or i.p. infection with S. Typhimurium. The latter finding could be explained, at least in part, by reduced amounts of CCL2, the chemokine that mobilizes bone marrow monocytes to the blood circulation and is critical for the recruitment of Ly6ChighLy6G− monocytes to the sites of infection (22, 37, 38). Both MyD88fl/fl and Dermo1-Cre;MyD88fl/fl mice had comparable Ccl2 mRNA expression and numbers of inflammatory monocytes in the MLNs. A likely interpretation of the results is that the DC-MLN route that appears to play a minor role in systemic spread (15, 17, 18) is not regulated via MyD88 signaling in mesenchymal cells. The results are more compatible with the notion that MyD88 signaling in mesenchymal cells regulates dissemination of S. Typhimurium via the hematogenous route involving recruitment of inflammatory monocytes harboring the pathogen to the liver and the spleen. Mesenchymal cells including fibroblasts produce copious amounts of CCL2 in response to bacterial infection (21, 22). Thus, it is possible that CCL2 production induced by S. Typhimurium is mediated, at least in part, via MyD88-dependent pathways such as TLR2 and TLR4 in fibroblasts and/or other mesenchymal cells. In a non-excluding and alternative possibility, cytokines such as IL-1β or IL-18 that are produced in response to S. Typhimurium infection in vivo could stimulate mesenchymal cells to produce CCL2 via MyD88. The observation that most S. Typhimurium reside within Ly6ChighLy6G− inflammatory monocytes is consistent with previous studies that identified CD18+ phagocytes as the main carriers of the pathogen in the bloodstream (15). Notably, CD18 is required for extraintestinal dissemination of S. Typhimurium after orogastric infection (15, 16). Although the role of CD18 in the systemic spread of S. Typhimurium remains unclear, it is likely that the requirement of this integrin reflects its function in mediating extravasation of leukocytes from blood to tissues (39). Intriguingly, CD18 was found to be expressed on the surface of both Ly6ChighLy6G− inflammatory monocytes and Ly6CintLy6G+ neutrophils, although the former cells express higher amounts of surface CD18. In addition to S. Typhimurium, CD18+ phagocytes cells are also required for efficient translocation of Yersinia enterocolitica from the intestine to the spleen (40). Thus, the higher amounts of surface CD18 could contribute to the enhanced ability of Ly6ChighLy6G− inflammatory monocytes to harbor and disseminate certain pathogens from the bloodstream to host tissues. In addition, S. Typhimurium manipulates the maturation of the Salmonella-containing vacuole using its T3SS-2 (41, 42). Thus, reduced anti-microbial activity of Ly6ChighLy6G− inflammatory monocytes compared to neutrophils could also play a role in the increased ability of monocytes to transport live S. Typhimurium.

Collectively, our studies suggest that S. Typhimurium employs its virulence T3SS-2 to promote its systemic growth and dissemination through MyD88 signaling pathways in mesenchymal cells. Although MyD88 signaling pathways are better known for the induction of protective responses against the pathogen, our results suggest that they can also be exploited by S. Typhimurium to promote its own expansion in vivo. In response to infection, inflammatory monocytes are also recruited by the host to induce protective immune responses (22, 43). These results indicate that the same signaling pathways induced via MyD88 can promote the clearance, but also, the dissemination of the invading pathogen.

Supplementary Material

1

Acknowledgments

The authors are grateful to Xiaoxia Li for mutant mice, Denise Monack and Clare Bryant for bacterial strains and Lisa Haynes for animal husbandry.

This work was supported by grants R01AI063331 and R01DK091191 (G.N.) from the National Institutes of Health, a grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2015R1A3A2032927) (W.-U.K.) and a grant from NRF funded by the Korea government (MSIP) (2016R1C1B2008089) (S.-U.Seo).

Abbreviations used in this article

BMDCs

bone marrow derived dendritic cells

BMDMs

bone marrow derived macrophages

RMCs

resident mesenchymal cells

S. Typhimurium

Salmonella enterica serovar Typhimurium

T3SS

type III secretion system

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