Mesenteric Lymph Nodes Confine Dendritic Cell-Mediated Dissemination of Salmonella enterica Serovar Typhimurium and Limit Systemic Disease in Mice

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1 INFECTION AND IMMUNITY, Aug. 2009, p Vol. 77, No /09/$ doi: /iai Copyright 2009, American Society for Microbiology. All Rights Reserved. Mesenteric Lymph Nodes Confine Dendritic Cell-Mediated Dissemination of Salmonella enterica Serovar Typhimurium and Limit Systemic Disease in Mice Sabrina Voedisch, 1 Christian Koenecke, 1 Sascha David, 2 Heike Herbrand, 1 Reinhold Förster, 1 Mikael Rhen, 3 and Oliver Pabst 1 * Institute of Immunology, Hannover Medical School, Hannover, Germany 1 ; Department of Nephrology, Hannover Medical School, Hannover, Germany 2 ; and Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden 3 Received 9 March 2009/Returned for modification 13 April 2009/Accepted 30 May 2009 In humans with typhoid fever or in mouse strains susceptible to Salmonella enterica serovar Typhimurium (S. Typhimurium) infection, bacteria gain access to extraintestinal tissues, causing severe systemic disease. Here we show that in the gut-draining mesenteric lymph nodes (MLN), the majority of S. Typhimurium-carrying cells show dendritic-cell (DC) morphology and express the DC marker CD11c, indicating that S. Typhimurium bacteria are transported to the MLN by migratory DCs. In vivo FLT-3Linduced expansion of DCs, as well as stimulation of DC migration by Toll-like receptor agonists, results in increased numbers of S. Typhimurium bacteria reaching the MLN. Conversely, genetically impaired DC migration in chemokine receptor CCR7-deficient mice reduces the number of S. Typhimurium bacteria reaching the MLN. This indicates that transport of S. Typhimurium from the intestine into the MLN is limited by the number of migratory DCs carrying S. Typhimurium bacteria. In contrast, modulation of DC migration does not affect the number of S. Typhimurium bacteria reaching systemic tissues, indicating that DC-bound transport of S. Typhimurium does not substantially contribute to systemic S. Typhimurium infection. Surgical removal of the MLN results in increased numbers of S. Typhimurium bacteria reaching systemic sites early after infection, thereby rendering otherwise resistant mice susceptible to fatal systemic disease development. This suggests that the MLN provide a vital barrier shielding systemic compartments from DC-mediated dissemination of S. Typhimurium. Thus, confinement of S. Typhimurium in gutassociated lymphoid tissue and MLN delays massive extraintestinal dissemination and at the same time allows for the establishment of protective adaptive immune responses. Infection with Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever that, following consumption of contaminated food or water, starts with an intestinal phase characterized by colonization of the intestine and transepithelial uptake that provides the pathogen with access to the intestinal mucosa. Disease then progresses to a systemic phase as bacteria spread from the intestine to the spleen and liver. Infection with another Salmonella enterica serovar, S. Typhimurium, in most cases causes locally restricted enteritis in humans without eliciting systemic disease. In contrast, oral infection of susceptible mice with S. Typhimurium, but not S. Typhi, leads to a fatal systemic disease resembling the human disease and is used as a model of human typhoid fever. Notably, susceptible mouse strains that develop fatal systemic disease carry a mutation in the Slc11a1 (formerly natural resistance-associated macrophage protein-1, Nramp-1) gene and include widely used laboratory strains, such as C57BL/6 and BALB/c. In contrast, resistant mouse strains, such as 129Sv, express increased levels of Slc11a1 in infected cells (3, 35), thereby controlling the intracellular replication * Corresponding author. Mailing address: Institute of Immunology, Hannover Medical School, Carl-Neuberg Strasse 1, Hannover, Germany. Phone: Fax: Pabst.Oliver@MH-Hannover.de. Published ahead of print on 8 June of S. Typhimurium and surviving infection. Thus, care needs to be taken when comparing the pathomechanisms in susceptible mouse strains infected with S. Typhimurium and human typhoid fever. Exploiting the M-cell gateway to the gut mucosa is thought to be an important way in which S. Typhimurium overcomes the tight intestinal epithelial barrier (17). M cells continuously sample the gut lumen and transport particulate antigens, including live microbes, across the epithelium to immune cells located in the underlying mucosal tissue. The majority of M cells are located within the follicle-associated epithelia of the gut-associated lymphoid tissue (GALT), such as Peyer s patches (PP), and solitary intestinal lymphoid tissue (SILT) (13). Consistently, in early phases of infection the highest bacterial loads and levels of inflammation are observed at these sites (10). Uptake of S. Typhimurium via PP M cells was shown to cause local damage in the follicle-associated epithelium within 30 min of infection, thus generating gaps in the epithelium that allow rapid bacterial spread to the organs before an immune response can be initiated (17). Apart from M cells associated with lymphoid follicles, a low number of M cells is interspersed throughout the normal epithelium and has been associated with the invasion of S. Typhimurium in mice lacking organized lymphoid tissue in the intestine (15). In addition to the exploitation of these active sampling mechanisms, S. Typhimurium can breach 3170

2 VOL. 77, 2009 MESENTERIC LYMPH NODES LIMIT S. TYPHIMURIUM SPREAD IN MICE 3171 the intestinal barrier through the normal absorptive epithelium (32). Two major virulence determinants of S. Typhimurium are encoded by pathogenicity islands SPI-1 and SPI-2 that translate into two separate type-iii secretion systems (TTSS). The SPI-2-encoded secretion system TTSS-2 mediates the intracellular survival of the pathogens and their persistence in systemic target organs, like the liver and spleen. Consequently, TTSS- 2-deficient S. Typhimurium strains cannot establish persistent infection and mice infected intraperitoneally with these strains will clear the infection (12, 25, 31). In contrast, TTSS-1-defective strains are not reduced in virulence when administered intraperitoneally but are clearly attenuated following oral infection, demonstrating that TTSS-1 is essential for the efficient entry of S. Typhimurium into host tissues (7). Still, TTSS-1-deficient mutants are capable of gaining access to the mucosal tissues, presumably via host-directed sampling mechanisms that act independently of S. Typhimurium-encoded virulence genes. There is evidence for active M-cell-independent bacterial uptake performed by dendritic cells (DC) residing in the lamina propria directly underneath the intestinal epithelium (28). These DC express the chemokine receptor CX 3 CR1 that is essential for the formation of transepithelial extensions by these cells that allow the capture of bacteria directly from the gut lumen (24). Hapfelmeier and colleagues showed that conditional depletion of DC during the phase of transepithelial pathogen uptake strongly reduced the colonization of the lamina propria by TTSS-1-deficient S. Typhimurium (11). In contrast, depleting these DC at later phases of infection, i.e., after epithelial transmigration had occurred, did not influence the systemic spread of the pathogen. Similarly, depletion of DC had no significant influence on the outcome of oral infection with a TTSS-1-sufficient wild-type S. Typhimurium strain (11). Whatever mechanism allows S. Typhimurium to enter host tissues, a central issue in understanding systemic disease development relates to the mechanisms that enable S. Typhimurium to disseminate from the intestine. In tissue, S. Typhimurium infects monocytes/macrophages and neutrophils that show potent antibacterial activity (8, 29, 30) and are essential for host survival. In contrast, S. Typhimurium infection of DC induces their maturation and antigen presentation, thereby initiating adaptive immune responses (for a recent review, see reference 33). Moreover, S. Typhimurium has been observed in B cells, and carriage by any of these cells might allow S. Typhimurium to reach extraintestinal tissues. Indeed, experiments using mice deficient in 2-integrin, a molecule associated with cell migration, showed reduced numbers of S. Typhimurium bacteria in the spleen and liver after oral but not intraperitoneal infection. In particular, cells of the myeloid lineage have been suggested to confer 2-integrin-dependent S. Typhimurium dissemination (36). Thus, at present, multiple mechanisms have been shown to allow for the initial uptake of S. Typhimurium, as well as for the dissemination of the pathogen. However, the actual contributions of the various mechanisms remain enigmatic. In this study, we demonstrate that after oral infection, DC chiefly contribute to S. Typhimurium progression from the intestine to the mesenteric lymph nodes (MLN) but not to hepatosplenic infection. Furthermore, we show that the MLN serve as a vital barrier preventing lethal systemic infection. MATERIALS AND METHODS Ethics statement. All animal experiments have been performed in accordance with institutional guidelines and have been approved by the local committees. Mice. Mice were bred at the central animal facility of Hanover Medical School under specific-pathogen-free conditions or purchased from Charles River (Germany). B6.129P2(C)-Ccr7 tm1rfor /J mice (designated CCR7-deficient mice herein) (6, 26) are characterized by impaired lymphocyte and DC migration. 129S6/SvEvTac-Rag2 tm1fwa mice (designated 129Sv Rag2-deficient mice herein) that fail to generate mature lymphocytes due to impaired V(D)J rearrangement were provided by S. Suerbaum (Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School). In some experiments, C57BL/6 mice received to B16-FL cells (18) by subcutaneous injection. B16-FL melanoma cells were engineered to express Flt3-L, resulting in the in vivo expansion of DC populations. Mobilization of DC was triggered by oral gavage of 10 g of the Toll-like receptor 7/8 (TLR7/8) agonist R848 (Resiquimod) in a volume of 100 l of phosphate-buffered saline (PBS). Mice were orally infected with S. Typhimurium 10 days after B16-FL injection or 2 h after R848 gavage. Mesenteric lymphadenectomy. C57BL/6 and 129Sv mice were anesthetized intraperitoneally. The small intestine and cecum, together with MLN, were exteriorized through a 1-cm-wide incision along the abdomen and kept humid with PBS. Mesenteric lymphadenectomy was performed by microdissection along the length of the superior mesenteric artery to the aortic root. After surgery, the small intestine and cecum were reintroduced into the abdomen, the lesion of the abdominal wall stitched with degradable thread, and the outer skin sealed with wound clips. The animals were infected with S. Typhimurium 6 to 8 weeks after surgery. Cecal ligation and puncture. 129Sv mice were anesthetized using isofluorane; after median laparotomy, a 5-0 silk ligature was placed 10 mm from the cecal tip. The cecum was punctured twice with an 18-gauge needle and gently squeezed to express a 1-mm column of fecal material. The length of 10 mm and the needle size of 18-gauge have been shown to adjust the 50% lethal dose to approximately 24 h. Prewarmed normal saline (1 ml) was injected intraperitoneally. Treatment with fluids was started after 8 h with subcutaneous injection of 1 ml of saline. Twenty-four hours after cecal ligation and puncture, blood samples were taken from severely septic animals. S. Typhimurium infections. All S. Typhimurium strains used in this study were S. Typhimurium strain SL1344 or derivatives of this strain (14). SL1344 aroa and SL1344 aroa sipb were described previously (10). SL1344 aroa causes a self-limiting infection in C57BL/6 mice due to impaired replication in vivo. SL1344 aroa sipb carries an additional mutation inactivating TTSS-1. For histological analysis, a green fluorescent variant of SL1344 aroa expressing green fluorescent protein under the control of the pagc promoter (4) that will only provoke fluorescent signals after successful invasion and location within host cells was used. For infection, S. Typhimurium bacteria were grown to a culture density of bacteria/ml. Bacteria were washed with LB broth containing 3% NaHCO 3 and resuspended to a density of bacteria (attenuated S. Typhimurium) or bacteria (wild-type S. Typhimurium) per 100 l LB/NaHCO 3. Mice were inoculated orally with 100 l of bacterial suspension with a feeding needle or injected intraperitoneally. The numbers of inoculated bacteria and of bacteria present in tissues were determined by plating. In some experiments, animals that were infected with SL1344 aroa sipb were simultaneously infected with an equal number of SL1344 aroa bacteria and CFU corresponding to both strains were determined by differential plating due to their differing antibiotic resistance. No differences were observed in CFU between coinfected mice and mice infected with either SL1344 aroa or SL1344 aroa sipb alone (data not shown). Livers and spleens were dissected and homogenized using an Ultra Turrax. PP and MLN were dissected and minced through a 45- m nylon mesh. Triton X-100 at a final concentration of 0.5% was added to the cell suspensions, which were incubated for 30 s before serial dilutions were plated on LB agar plates. Progression of systemic disease in mice was scored according to three criteria: appearance of the fur, activity, and ability to maintain balance. Each criterion was rated from one (healthy) to three (most severe manifestation), resulting in a total score from three to nine for each animal. Microscopy. For confocal laser-scanning microscopy, spleens and MLN were fixed for 2 or 3 h with 1.5% paraformaldehyde PBS, washed twice with PBS, and embedded in 10% agarose. One-hundred-micrometer-thick sections were obtained by using a Leica VT 1000S vibratome, blocked for 2 h with 5% rat serum in PBS, and stained with antibodies overnight at 4 C with continuous shaking in

3 3172 VOEDISCH ET AL. INFECT. IMMUN. the presence of 0.05% Tween 20. Sections were mounted with Mowiol (Sigma- Aldrich). Slides were analyzed by using an LSM 510 META confocal microscope (Zeiss), and images created using Zeiss LSM5 and Imaris Bitplane software. For liver histology, organs were fixed overnight in 4% paraformaldehyde PBS, dehydrated, and embedded in paraffin. Eight-micrometer-thick sections were obtained with a Leica RM2165 microtome and stained according to the Mallory s Trichrome staining protocol. Slides were analyzed by using a BX61 fluorescence microscope (Olympus), and images created using CellP software. Analysis of sera. Gamma interferon (IFN- ) and tumor necrosis factor alpha (TNF- ) in mouse sera were quantified by cytometric bead assay (mouse Th1/ Th2 cytokine kit; BD Biosciences, Pharmingen) following the manufacturer s instructions. Levels of glutamic-oxaloactetic transaminase (GOT), glutamicpyruvic transaminase (GPT), lactate dehydrogenase (LDH), creatinine, and urea in sera were determined by using the clinical chemistry analyzer AU400 (Olympus). Statistical analysis. Statistical analysis was performed with GraphPadPrism software. All significant values were determined using the unpaired two-tailed t test; error bars represent standard deviations. Statistical differences of the mean values are indicated as follows: *, P value is 0.05; **, P value is 0.01; and ***, P value is RESULTS FIG. 1. High bacterial loads require functional TTSS-1 in PP, spleen, and liver but not in MLN. (A) Mice were orogastrically inoculated with 10 8 wild-type SL1344, 10 9 SL1344 aroa, or10 9 SL1344 aroa sipb ( aroasipb) bacteria. After 2 days, CFU in PP, MLN, spleen, and liver were determined. Each circle represents the result obtained for one infected mouse, shaded boxes indicate values in the 75th percentile, and the median values are marked by horizontal bold lines. Numerals show the number of sterile organs. (B) Mice were infected with green fluorescent SL1344 aroa orally (open bars) or intravenously (filled bars), and S. Typhimurium-carrying cells were classified by expression of CD11c and CD11b using confocal microscopy. Images show examples of types of infected cells in MLN: CD11c CD11b cells (red, upper left), CD11c CD11b cells (blue, upper right), and CD11c CD11b cells (purple, lower left). Projections of cells were generated with Imaris Bitplane software from raw confocal Z-stacks. White scale bars show 10 m. Divergent mechanisms allow for the colonization of gutdraining MLN and systemic compartments. Invasion of epithelial cells in vitro and infection of PP after oral infection in vivo by SPI-1-deficient S. Typhimurium bacteria are reduced 100-fold compared to their levels for SPI-1-sufficient S. Typhimurium, yet SPI-1-deficient S. Typhimurium bacteria largely retain the ability to cause fatal systemic disease in mice (7). This suggests that alternative mechanisms allowing for S. Typhimurium uptake and dissemination might become apparent by comparing strains with retained and inactivated SPI-1-encoded TTSS-1. In this study, we utilized the SPI-1-sufficient wild-type S. Typhimurium strain SL1344, as well as an attenuated derivate lacking the aroa gene (SL1344 aroa), in comparison to a TTSS1-defective variant (SL1344 aroa sipb) to analyze mechanisms of S. Typhimurium dissemination. Two days after oral infection of susceptible C57BL/6 mice with S. Typhimurium SL1344, the highest bacterial load could be observed in PP, whereas approximately 100- and 1,000-fold-lower numbers of bacteria were present in the MLN, liver, and spleen, respectively (Fig. 1A). Comparable bacterial loads were obtained by using a 10-fold-higher inoculum of attenuated, aroa-deficient (SL1344 aroa) S. Typhimurium. This suggests that even though the aroa mutant is less virulent than the parental strain, both strains possess similar invasion properties. In marked contrast, the introduction of an additional mutation in the sipb gene (SL1344 aroa sipb) resulted in roughly 50-fold-reduced numbers of S. Typhimurium in PP and, in the majority of cases, no spread to the spleen and liver was detectable. However, SL1344 aroa sipb largely retained the capacity to colonize the gut-draining MLN, as infection resulted in only fivefold-reduced numbers of S. Typhimurium bacteria in the MLN compared to the numbers in the MLN following infection with SL1344 aroa (Fig. 1A) (these results support previous reports [20, 36]). This indicates that TTSS-1- dependent mechanisms are central for the early colonization of PP, liver, and spleen but are of minor importance for the progression of the infection from the intestine to the gutdraining MLN. The bacterial loads observed in the spleen and liver 2 days after infection with SL1344 and SL1344 aroa varied substantially between individual mice, ranging from sterile to heavily infected organs containing more than 10 4 S. Typhimurium bacteria (Fig. 1A). In contrast, infection with any of the three S. Typhimurium strains, including the TTSS-1-deficient variant, resulted in comparably lower variations in the bacterial burdens observed in PP and MLN. Thus, two independent observations, i.e., the divergent requirements for TTSS-1 and the different interanimal variations, suggest that fundamentally

4 VOL. 77, 2009 MESENTERIC LYMPH NODES LIMIT S. TYPHIMURIUM SPREAD IN MICE 3173 different processes dominate the colonization of the MLN and systemic compartments after oral infection with S. Typhimurium. In MLN, S. Typhimurium bacteria are associated with DC. We next characterized the phenotype of S. Typhimurium-infected cells by immunofluorescent confocal microscopy. To this aim, mice were infected with SL1344 aroa expressing a green fluorescent protein under the control of the pagc promoter (4). Thus, live intracellular bacteria could be selectively identified by green fluorescence. Mice were infected either orally or intravenously and the MLN or spleen, respectively, was analyzed. One-hundred-micrometer-thick sections were stained with antibodies against CD11b and CD11c, and S. Typhimurium-carrying cells classified according to cell morphology and marker expression. Intracellular localization of S. Typhimurium was confirmed by analyzing confocal Z-stacks. Interestingly, the vast majority of all S. Typhimurium-carrying cells could be labeled with CD11b and/or CD11c and only 10% of all S. Typhimurium-bearing cells expressed none of either marker (data not shown). In the spleen and MLN, approximately 50% of all S. Typhimurium-carrying cells coexpressed CD11c and CD11b and showed DC morphology. Twenty-three percent of S. Typhimurium bacteria resided in CD11c CD11b cells, and roughly 27% were observed in CD11c CD11b cells (Fig. 1B). Thus, the majority of S. Typhimuriumcarrying cells displayed characteristics of DC, including typical cell morphology. Moreover, we observed S. Typhimurium in cells coexpressing CD11c and the E integrin chain CD103, which has been suggested as a surrogate marker for lamina propria DC in the MLN (16). These findings can be corroborated by a number of published observations demonstrating that after oral infection, the majority of S. Typhimurium bacteria reside in myeloid cells in the MLN (for a recent review, see reference 33). Moreover, a recent report showed that early TTSS-1-deficient S. Typhimurium bacteria in the intestinal lamina propria were exclusively associated with CD11c-expressing DC (11). Thus, even though DC and other myeloid cells are difficult to distinguish by phenotypic description, these data suggest that a substantial number of live S. Typhimurium bacteria reside within DC. DC carry S. Typhimurium bacteria into MLN. Several observations indicate that DC might contribute decisively to S. Typhimurium dissemination from the intestine to the draining MLN: (i) DC continuously migrate from the intestine to the MLN, thereby transporting commensal bacteria and other antigens (19); (ii) DC migration increases upon microbial stimulation; and (iii) the majority of S. Typhimurium bacteria reside within lamina propria and MLN DC (see above and reference 11). In order to pinpoint the contribution of DC to S. Typhimurium dissemination in vivo, we utilized three independent experimental methods to alter DC numbers and/or their migratory properties. Application of the agonistic TLR7/8 ligand R848 has previously been shown to trigger a massive migration of lamina propria DC into the MLN (40). R848 is a synthetic agonist stimulating TLR7/8 in DC, resulting in the secretion of cytokines and stimulating migration of DC. We thus fed a single dose of R848 2 hours before oral infection with SL1344 aroa. SL1344 aroa is impaired in the production of siderophores and capable of only limited intracellular expansion compared to that of the parental strain (2, 14). Thus, FIG. 2. Increased DC trafficking promotes the dissemination of S. Typhimurium to MLN but not into spleen and liver. Mice received R848 orally 2 hours before infection with SL1344 aroa or were injected with FLT3-L-secreting cells 10 days before infection to expand DC. Bacterial loads were determined 2 days after infection. Each circle represents the result obtained for one infected mouse, shaded boxes indicate values in the 75th percentile, and the median values are marked by horizontal bold lines. Numerals show the number of sterile organs. the numbers of bacteria recovered from various organs more faithfully reflect the rate of dissemination. In line with previously published results, we observed significantly increased numbers of DC in the MLN 16 h after treatment, indicating that DC exited the lamina propria and migrated into the MLN (39; also data not shown). Enhanced migration of DC correlated with significantly increased numbers of S. Typhimurium bacteria in the MLN of R848-fed animals compared to the numbers in the MLN of untreated controls. In contrast, the application of R848 did not influence the numbers of S. Typhimurium bacteria recovered from PP, spleen, and liver (Fig. 2), suggesting that mobilization of intestinal DC selectively enforces the colonization of the MLN. In a second experimental approach, we increased the numbers of DC by subcutaneous implantation of an FLT3-L-secreting tumor. Ten days after implantation, DC numbers in the intestinal lamina propria, PP, MLN, and spleen had increased more than 50-fold (data not shown). Infection of such tumor-bearing mice with SL1344 aroa resulted in increased numbers of S. Typhimurium in the MLN but not in PP, spleen, and liver (Fig. 2). This suggests that increasing the rate of DC migration facilitates the transport of S. Typhimurium from the intestine into the MLN. In contrast, genetic deficiency of chemokine receptor CCR7 impairs migration of DC. Analysis of CCR7-deficient mice revealed that CCR7 is essentially required for DC migration from the intestine to the draining MLN under steady-state and inflammatory conditions (6, 37). Two days after infection, the numbers of S. Typhimurium bacteria in the MLN of CCR7- deficient mice were reduced compared to the numbers in the MLN of wild-type mice, whereas no differences were observed

5 3174 VOEDISCH ET AL. INFECT. IMMUN. FIG. 3. Impaired DC migration in CCR7 / animals correlates with reduced numbers of S. Typhimurium bacteria in MLN but not in liver. Wild-type (filled boxes) or CCR7-deficient (open boxes) mice were infected orally with 10 8 SL1344, 10 9 SL1344 aroa,or10 9 SL1344 aroa sipb ( aroasipb) bacteria as indicated. CFU were determined 2 days after infection. Each circle represents the result obtained for one infected mouse, boxes indicate values in the 75th percentile, and the median values are marked by horizontal bold lines. Numerals show the number of sterile organs. ns, nonsignificant difference. in the colonization of spleen and liver (Fig. 3 and data not shown). Interestingly, lack of CCR7 affected the colonization of the MLN by SL1344 aroa sipb more severely than it did their colonization by SL1344 aroa, indicating that dissemination of TTSS-1-deficient S. Typhimurium might be particularly dependent on DC-mediated transport. Consistently, the survival of CCR7-deficient mice after high-dose infection with SL1344 was not enhanced compared to the survival of wildtype mice (data not shown). In sum, these experiments suggest that DC account for the majority of S. Typhimurium bacteria that are transported from the intestine into the MLN. However, some S. Typhimurium can reach the MLN independent of CCR7 mediation, suggesting that DC-independent dissemination into this site can occur as well. In contrast, DC-bound transport of S. Typhimurium appears to be of minor importance for its reaching systemic sites in wild-type mice. MLN limit systemic spread of S. Typhimurium. DC migration from the intestine to the MLN is confined to lymphatic vessels, and usually DC will not travel beyond the first draining lymph node they enter. We thus speculated that the MLN might act as a vital barrier limiting the lymphogenic dissemination of S. Typhimurium in wild-type mice. To test this hypothesis, MLN were surgically excised from wild-type mice. In such adenectomized mice, MLN afferent and efferent lymphatic vessels will fuse to form a pseudoafferent lymph vessel that directs intestinal lymph back into the circulation without filtration through a lymph node (19, 37). When comparing adenectomized C57BL/6 mice with MLN-sufficient control animals, we observed statistically more bacteria in the livers and a nonsignificant tendency to increased numbers of bacteria in FIG. 4. MLN adenectomy results in strikingly increased numbers of S. Typhimurium bacteria in spleen and liver and renders otherwise resistant 129Sv mice susceptible to fatal bacteremia. (A) MLN-explanted mice (filled boxes) and MLN-sufficient C57BL/6 mice (open boxes) were orogastrically infected with different S. Typhimurium strains as indicated, and CFU determined 2 days postinfection. aroasipb, aroa sipb. (B, C) MLN-explanted (MLNx) and MLNsufficient 129Sv mice (wild type [wt]) and 129Sv Rag-deficient mice were orally infected with nonattenuated SL1344. (B) CFU in MLN, spleen, and liver were determined 7 days after infection. (C) Mice were scored for disease development as described in Materials and Methods. All MLN-sufficient mice (open circles) remained healthy throughout the experiment. In contrast, adenectomized mice (filled circles) developed manifestations of systemic disease. Each circle in panels A and B represents the result obtained for one infected mouse, boxes indicate values in the 75th percentile, and the median values are marked by horizontal bold lines. Numerals indicate the number of sterile organs. ns, nonsignificant difference. Results in panel C are the means and standard deviations for eight mice analyzed in one out of two representative experiments. dpi, days postinfection. the spleens 2 days after infection with each of the three S. Typhimurium strains. In contrast, surgical removal of the MLN did not affect the number of bacteria in PP (Fig. 4A). These results suggest that the MLN limit DC-bound dissemination of S. Typhimurium from the intestine into the periphery. In order to test the protective function of the MLN, we

6 VOL. 77, 2009 MESENTERIC LYMPH NODES LIMIT S. TYPHIMURIUM SPREAD IN MICE 3175 extended our studies to Slc11a1-sufficient 129Sv mice. 129Sv mice survive oral infection with the experimental dose of SL1344 used in this study. Therefore, infection of 129Sv mice allows the study of later time points of infection without using attenuated S. Typhimurium strains. Interestingly, mesenteric adenectomy of 129Sv mice resulted in significantly increased loads of SL1344 in the spleen and liver 7 days after infection (Fig. 4B), suggesting that the absence of the MLN results in an increased bacterial burden at later time points of infection. This effect was not due to generally impaired adaptive immune responses in MLN-deficient mice, as 7 days after infection, Rag2-deficient 129Sv mice, which lack B and T cells, and untreated 129Sv controls harbored similar numbers of S. Typhimurium in MLN, spleen, and liver (Fig. 4B). This indicates that adaptive immune responses do not influence bacterial loads within the first week of infection. We therefore suggest that increased bacterial loads in the livers and spleens of adenectomized mice do not result from impaired adaptive immune control but from S. Typhimurium-loaded DC entering the circulation via pseudoafferent lymph vessels. Notably, mesenteric adenectomy not only increased bacterial loads in the liver and spleen but was accompanied by overt disease, characterized by ruffled fur, lethargy, and rolling behavior (Fig. 4C), that was fatal in four out of eight adenectomized 129Sv mice. In contrast, neither MLN-sufficient wild-type 129Sv nor MLN-sufficient Rag2-deficient 129Sv mice revealed symptoms of disease (Fig. 4C and data not shown), indicating that mesenteric adenectomy results in overt disease development in otherwise resistant mouse strains. Mesenteric adenectomy enhances severe pathology of the liver. In order to understand the pathomechanism in more detail, we compared the progression of disease in C57BL/6 mice with that in 129Sv and adenectomized 129Sv mice. Measurement of transaminases (GOT and GPT) and LDH in the serum was used to monitor liver damage. Creatinine and urea levels in the serum were measured to determine kidney function, and serum levels of proinflammatory cytokines TNF- and IFN- were used as global inflammation markers. Additionally, we analyzed the spleens and the livers of infected mice by histology. Typically, C57BL/6 mice started to develop overt signs of disease 4 days after infection with SL1344 and had to be sacrificed 6 to 7 days postinfection because of severe systemic symptoms of disease. Disease development in C57BL/6 mice correlated with progressively increasing levels of GOT, GPT, and LDH, as well as the cytokines TNF- and IFN-, whereas creatinine and urea levels remained normal (Fig. 5 and data not shown). In S. Typhimurium-infected, MLN-sufficient 129Sv mice, we also observed elevated levels of GOT, LDH, TNF-, and IFN- compared to the levels in noninfected controls (Fig. 5). However, in contrast to the situation in C57BL/6 mice, none of the markers abidingly increased, but instead, they reached low plateau levels only 2 days after infection (Fig. 5). Thus, S. Typhimurium-infected C57BL/6 mice are distinguished by progressively increasing signs of systemic inflammation and liver dysfunction, whereas in 129Sv mice, control of S. Typhimurium expansion correlates with stalled development of liver disease and inflammation. We next extended these measurements to adenectomized 129Sv mice. Mesenteric adenectomy per se did not alter the analyzed serum parameters or cytokine production in uninfected mice (data not shown). Eight days after infection, most adenectomized 129Sv mice showed overt signs of disease, and some of these mice would succumb to infection. The levels of IFN- in adenectomized 129Sv mice were significantly elevated compared to those in MLN-sufficient controls, whereas differences in TNF- levels did not reach statistical significance (Fig. 5). Notably, neither IFN- nor TNF- reached levels typically observed in diseased C57BL/6 mice, indicating that disease development in adenectomized 129Sv mice might be different than in C57/BL6 mice. However, in a fraction of the adenectomized 129Sv mice, the hepatobiliary system was clearly affected, as shown by high serum levels of GOT, GPT, and LDH. GOT levels were significantly higher in adenectomized mice than in MLN-sufficient mice, whereas GPT and LDH showed a nonsignificant trend to increased levels. Histology of liver sections revealed infiltration of granulocytes and macrophages in livers of MLN-sufficient and adenectomized mice 8 days postinfection. However, in agreement with the enhancement of GOT activity in the sera of adenectomized mice in comparison to the levels in MLN-sufficient mice, we observed more extensive signs of tissue damage and fibrosis in livers of the former group (Fig. 6B and C). Similarly, infected adenectomized mice had significantly enlarged spleens compared to the spleens in nonadenectomized mice (Fig. 6D). Thus, mesenteric adenectomy not only resulted in increased bacterial loads in the liver and spleen but also provoked enhanced organ damage, suggesting that lethality of adenectomized 129Sv mice might not be due solely to overwhelming bacteremia and sepsis but might rather result from a more-restricted organ dysfunction, in particular, liver failure. In support of such a scenario, kidney function, measured by serum levels of urea and creatinine, was stable during S. Typhimurium infection (Fig. 5 and data not shown). In contrast, kidney function decreased rapidly in septic mice following cecal ligation and puncture, which is a classical experimental model of bacterial sepsis (Fig. 5). S. Typhimurium loads expand faster in liver and spleen than in MLN. Mesenteric adenectomy did not influence the numbers of S. Typhimurium bacteria in PP, suggesting that the number of S. Typhimurium bacteria initially breaching the intestinal barrier would not be influenced. Instead, in adenectomized mice, S. Typhimurium bacteria residing in intestinal DC, which would be confined to the MLN in MLN-sufficient mice, translocate to the liver and spleen. In order to check if such dislocation affects the overall load of S. Typhimurium in the animals, we carefully determined the expansion rates of S. Typhimurium loads in MLN compared to the rates in the spleens and livers of C57BL/6 and 129Sv mice. Two days after oral infection with SL1344, we observed roughly comparable loads of S. Typhimurium in the MLN, livers, and spleens of C57BL/6 mice compared to the loads in 129Sv mice. However, this situation changed dramatically at later time points. Whereas in C57BL/6 mice, bacterial loads in the MLN increased roughly 10-fold each day, the overall bacterial load in the MLN did not increase substantially over time in 129Sv mice (Fig. 7), indicating that, in 129Sv mice, innate immune control prevents overall expansion of the S. Typhimurium load in this compartment. Strikingly, in the spleen and liver, differences in the course of infection in 129Sv and C57BL/6 mice were less pronounced. The bacterial loads in the spleen and liver in-

7 3176 VOEDISCH ET AL. INFECT. IMMUN. Downloaded from FIG. 5. Mesenteric lymphadenectomy results in increased liver pathology. Levels of liver transaminases and LDH in the sera of infected C57BL/6 or 129Sv mice were determined. Furthermore, cytokines and the kidney-specific serum parameter urea were analyzed. Mice were orogastrically infected with SL1344, and measurements were obtained at several time points (days) as indicated on the x axes. Lymphadenectomized mice (MLNx) were analyzed 8 days after infection. Parameters for mice that underwent cecal ligation and puncture (CLP) were determined 24 h after puncture without S. Typhimurium infection. Each circle represents the result obtained for one infected mouse, shaded boxes indicate values in the 75th percentile, and the median values are marked by horizontal bold lines. In some cases, mean values are given by numbers to allow an easier judgment of the actual measurements. ns, nonsignificant difference; n.d., not determined. creased from day 2 to day 4 of infection in both mouse strains, even though S. Typhimurium loads showed reduced expansion rates in 129Sv mice compared to the rates in C57BL/6 mice (Fig. 7). Thus, the course of infection in 129Sv and C57BL/6 mice did not differ with respect to the rates of invasion and early dissemination, but both strains clearly differed in their ability to control expansion of the S. Typhimurium load at later stages of infection. In particular, the expansion of the S. Typhimurium load in the MLN was almost completely stalled in 129Sv but not C57BL/6 mice. These observations suggest that in 129Sv mice, the MLN, in comparison to the spleen and liver, might be particularly equipped to limit S. Typhimurium expansion. A similar tendency was apparent in C57BL/6 mice during later stages of infection: whereas in the spleens and livers, S. Typhimurium loads showed comparable expansion rates from day 2 to day 5 of infection, in the MLN, expansion clearly slowed down 4 days after infection. DISCUSSION Surviving bacterial infection depends on the timely induction of protective immune responses. During early phases of S. Typhimurium infection, a particularly crucial component of innate defense mechanisms is controlled by the Slc11a1 gene. Mice carrying the wild-type Slc11a1 gene control intracellular bacterial growth within cells of the monocyte/macrophage lineage and are substantially more resistant to S. Typhimurium infection than mice carrying a mutated version of the Slc11a1 gene (3). However, clearance of S. Typhimurium from phagocytes during later stages of infection requires the activation of on October 14, 2018 by guest

8 VOL. 77, 2009 MESENTERIC LYMPH NODES LIMIT S. TYPHIMURIUM SPREAD IN MICE 3177 FIG. 7. S. Typhimurium loads expand more rapidly in spleen and liver than in MLN. C57BL/6 mice (filled boxes) and 129Sv mice (open boxes) were infected with SL1344, and CFU in MLN, spleen, and liver determined at various time points. S. Typhimurium loads expanded rapidly in C57BL/6 mice, whereas expansion was almost completely stalled in MLN of 129Sv mice and was less rapid in spleen and liver. Each circle represents the result obtained for one infected mouse, boxes indicate values in the 75th percentile, and the median values are marked by horizontal bold lines. Numerals indicate the number of sterile organs. ns, nonsignificant difference. CD4 T cells and resistance to S. Typhimurium reinfection depends on Th1-type immunological memory and B-cell-produced antibodies (21, 23). In this study, we demonstrate that the gut-draining MLN form a life-saving firewall protecting the host from rapid extraintestinal pathogen dissemination. Surgical removal of the MLN renders otherwise resistant mice more susceptible to fatal S. Typhimurium infection. Half of all adenectomized 129Sv mice died from S. Typhimurium infection, and all adenectomized mice developed pathology that was absent in MLN-sufficient 129Sv mice. We therefore suggest that confinement of enteropathogenic activity by the MLN is essential for host survival. As shown in this study, mesenteric adenectomy accelerated the colonization of spleen and liver and the bacterial burden in these organs was significantly higher in adenectomized mice than in MLN-sufficient mice. On the other hand, adenectomy did not affect the number of bacteria initially breaching the intestinal barrier and, irrespective of the presence or absence of the MLN, identical numbers of S. Typhimurium bacteria gained access to host tissues. Thus, a FIG. 6. Mesenteric lymphadenectomy results in increased organ pathology. (A, B, C) Liver sections from 129Sv mice stained with Mallory s Trichrome, which indicates collagenolytic necrotic foci (blue). (A) MLN-sufficient noninfected control mouse. (B, C) MLN-sufficient control (B) and MLN-explanted (C) mice were orally infected for 8 days with nonattenuated SL1344. White scale bars show 50 m. (D) Comparison of spleen sizes from the same experiment. Spleen sizes were determined by image analysis and are depicted in arbitrary units. Circles represent spleens of individual mice, horizontal bars the mean measurements, and gray boxes values in the 75th percentile. MLNx, MLN-explanted.

9 3178 VOEDISCH ET AL. INFECT. IMMUN. central question is to understand why infection of the MLN is less detrimental for the host than infection of the spleen and liver. Interestingly, increased numbers of S. Typhimurium bacteria in the livers of adenectomized mice correlated with enhanced liver dysfunction. Thus, one might speculate that the enhanced liver damage in adenectomized mice compared to that in MLN-sufficient mice might trigger enhanced pathology and death. Such detrimental effects might be reinforced by increased expansion rates of S. Typhimurium loads at other sites than the MLN. Whereas bacteria are kept at rather constant numbers from day 2 to day 4 of infection in the MLN of 129Sv mice, S. Typhimurium loads multiplied greatly in the spleens and livers of these animals. Notably, we did not observe any differences in the S. Typhimurium-infected cell types when comparing the spleen and MLN in C57BL/6 mice. At both sites, the majority of infected cells displayed DC morphology and expressed myeloid cell markers. Still, one might speculate that subtle differences in the cells ability to limit intracellular growth or variations in the organ environment might explain overall differences in S. Typhimurium expansion rates. Intracellular replication of S. Typhimurium bacteria eventually will kill the infected cell and result in the release of extracellular bacteria (9). Therefore, bacterial growth in blood-filtrating organs, such as the liver and spleen, might favor systemic sepsis compared to bacterial growth in a lymph node. Consistently, we observed that in heavily diseased C57BL/6 mice that were likely to die within the next hours, the number of bacteria in the blood reached up to bacteria/ml and was accompanied by the production of proinflammatory cytokines. Therefore, preservation of liver function and prevention of bacterial sepsis appear to be the most critical determinants ensuring host survival. The particular function of the MLN in limiting S. Typhimurium dissemination is consistent with the hypothesis that lymphogenic dissemination of S. Typhimurium occurs mostly by DC. The majority of cells that carry S. Typhimurium into the MLN after oral infection expressed DC markers and showed typical DC morphology. Moreover, DC maturation and subsequent migration can be triggered by microbial stimulation, suggesting that DC are prime candidates for transporting S. Typhimurium into the MLN. Consistent with this, in this study, we show that the accumulation of S. Typhimurium in the MLN is reduced in CCR7-deficient mice, whereas experimentally enhanced DC migration correlated with increased numbers of S. Typhimurium reaching the MLN. These observations suggest that the number and availability of migratory DC is a rate-limiting step in the process leading to the colonization of the MLN. This situation is reminiscent of how the intestinal immune system handles commensal bacteria. Commensal bacteria reach the MLN in a strictly CCR7-dependent, DC-bound process and will not disseminate beyond this lymph node. Thus, in healthy mice, the confinement of commensal bacteria within DC in the MLN focuses immune responses on the intestinal immune system and prevents the induction of potentially damaging systemic immune reactions (19). In contrast to inert particles and commensal bacteria, substantial numbers of S. Typhimurium bacteria reached the MLN independent of CCR7-mediated migration processes, indicating that S. Typhimurium can utilize alternative pathways to reach the MLN. In such CCR7-independent dissemination, S. Typhimurium might be transported by other cell types than DC or DC might migrate independent of CCR7. Alternatively, CCR7-independent transport into the MLN might result from extracellular bacteria, e.g., bacteria that escaped from dying infected cells in the PP. These bacteria would be transported by afferent lymph into the MLN. In that case, it is conceivable that the rate of extracellular S. Typhimurium bacterial transport into the MLN would depend on the infection dose and the lytic activity of the S. Typhimurium strain used. In line with such a scenario, we observed that TTSS-1- deficient S. Typhimurium bacteria were more severely compromised than TTSS-1-sufficient strains in CCR7-deficient hosts. Irrespectively, our results indicate that DC-mediated transport takes the lion s share of the lymphogenic spread of S. Typhimurium from the intestine to the MLN. S. Typhimurium-loaded DC migrating to the MLN might originate from various sites, i.e., lymphoid follicles or the normal villous intestinal lamina propria. We have previously reported that inflammation of the intestinal mucosa after S. Typhimurium infection is restricted to PP and SILT (10). Moreover, we readily observed fluorescent S. Typhimurium bacteria in lymphoid follicles but not in normal intestinal villi (10), indicating that invasion of the normal intestinal mucosa would be either rather infrequent or an inconspicuous and short-lived process. We therefore suggest that M cells in lymphoid follicles provide the most frequently used port of entry for pathogenic S. Typhimurium, as well as gut commensal bacteria. Indeed, 100-fold-higher bacterial loads were found to be present in PP than in the spleen and liver 2 days after oral S. Typhimurium infection. Thus, GALT, i.e., PP and SILT, along with the draining MLN, are the first tissues to harbor substantial numbers of S. Typhimurium bacteria early after infection. Consistent with this, the induction of antigen-specific T-cell proliferation is restricted to these organs and does not occur in the liver and spleen (22). However, in the case of TTSS-1-deficient S. Typhimurium bacteria that have severely impaired ability to target M cells, M-cell-independent processes might add substantially to S. Typhimurium uptake (11, 28). In this respect, it is noteworthy that in the initial report describing impaired dissemination of S. Typhimurium in 2- integrin-deficient mice, a TTSS-1-deficient S. Typhimurium strain was used (36). Interestingly, experimental modulation of DC numbers or migratory properties did not influence hepatosplenic colonization, suggesting that DC-mediated transport of S. Typhimurium plays a minor role in systemic disease development. This result is reminiscent of another enteropathogen, Yersinia pseudotuberculosis, which infects the liver and spleen directly from a pool of bacteria replicating in the intestinal lumen, whereas infection of the MLN occurred independent of hepatosplenic infection (1). Consistently, CCR7-deficient mice on a C57BL/6 background succumbed to S. Typhimurium infection with kinetics similar to those for wild-type mice. In contrast to our observations reported here, impaired DC migration was recently suggested to explain reduced colonization of the MLN, as well as the liver and spleen, after oral infection with S. Typhimurium in TLR5-deficient mice. Notably, TLR5 deficiency resulted in increased survival of S. Typhimurium-infected mice (34). Even though at present we cannot fully ex-

10 VOL. 77, 2009 MESENTERIC LYMPH NODES LIMIT S. TYPHIMURIUM SPREAD IN MICE 3179 plain the discrepancy between the observations obtained using TLR5- and CCR7-deficient mice, we suggest that these factors might control distinct properties of DC. In particular, CCR7 is upregulated upon DC maturation, i.e., after barrier breach, and selectively impairs DC migration, whereas TLR5 deficiency might affect other, nonmigration-related aspects of DC function. Thus, DC-mediated transport is very unlikely to directly support transport of S. Typhimurium to the spleen and liver early after infection. This, however, does not rule out that S. Typhimurium bacteria multiplying in the MLN might eventually escape via efferent lymph and thereby enforce systemic infection. In support of such an idea, S. Typhimurium bacteria have been observed in efferent lymph collected from infected calves (27). However, such a process would operate only at later stages of infection and does not explain the early infection of the spleen and liver reported here. Early colonization of the spleen and liver might occur independent of DC at the same time as the colonization of PP and MLN. S. Typhimurium has been observed in the blood within minutes after infection (36, 38), though such a process might depend on the precise bacterial strain and dose analyzed, as suggested by other reports that did not detect S. Typhimurium in the blood at early time points of infection (5, 38). Thus, low numbers of S. Typhimurium bacteria might escape being trapped in the GALT and MLN. Only in particularly sensitive hosts, e.g., Slc11a1-defective C57BL/6 mice, does such lowlevel infection cause fatal disease, whereas in more-resistant hosts, such infection fails to manifest. Notably, the underlying pathomechanisms in adenectomized 129Sv mice and MLNsufficient C57BL/6 mice appear to be different. In particular, only C57BL/6 mice show gradually increasing systemic inflammation, as evidenced by progressively increasing levels of proinflammatory cytokines in their sera. Therefore, infection of adenectomized 129Sv mice provides an interesting model to study the effects of oral S. Typhimurium infection in Slc11a1- sufficient mice. In conclusion, we suggest that in wild-type mice, i.e., Slc11a1-sufficient mice, innate immune control and trapping of S. Typhimurium bacteria in GALT and MLN efficiently forestall systemic disease development. The initial invasion leads to the colonization of GALT, and subsequently, DC-bound transport of S. Typhimurium propagates the infection into the gutdraining MLN. Thus, confinement of S. Typhimurium at these sites delays massive extraintestinal dissemination and at the same time allows for the induction of protective adaptive immune responses. ACKNOWLEDGMENTS S.V. was supported by International Research Training Group 1273 funded by the Deutsche Forschungsgemeinschaft. C.K. is a scholar of the Deutsche Forschungsgemeinschaft (grant KO 3582/1-1). This work was supported by Deutsche Forschungsgemeinschaft grant SFB621- A11 to O.P. We are indebted to Dirk Bumann for providing bacterial strains and Sebastian Suerbaum for providing 129Sv Rag2-deficient mice. We thank Michaela Friedrichsen for excellent technical assistance. REFERENCES 1. Barnes, P. D., M. A. Bergman, J. Mecsas, and R. R. Isberg Yersinia pseudotuberculosis disseminates directly from a replicating bacterial pool in the intestine. J. Exp. Med. 203: Benjamin, W. H., Jr., P. Hall, S. J. Roberts, and D. E. Briles The primary effect of the Ity locus is on the rate of growth of Salmonella typhimurium that are relatively protected from killing. J. Immunol. 144: Blackwell, J. M., T. Goswami, C. A. Evans, D. Sibthorpe, N. Papo, J. K. White, S. Searle, E. N. Miller, C. S. 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