Pathogen-Mediated Inhibition of Anorexia Promotes Host Survival and Transmission

•Feeding status of host regulates pathogen virulence and transmission•Pathogen inhibition of IL-1β prevents anorexia via the gut-brain axis•Pathogen inhibition of anorexia promotes host survival and pathogen transmission•Sickness-induced anorexia creates trade-offs between virulence and transmission Sickness-induced anorexia is a conserved behavior induced during infections. Here, we report that an intestinal pathogen, Salmonella Typhimurium, inhibits anorexia by manipulating the gut-brain axis. Inhibition of inflammasome activation by the S. Typhimurium effector, SlrP, prevented anorexia caused by IL-1β-mediated signaling to the hypothalamus via the vagus nerve. Rather than compromising host defenses, pathogen-mediated inhibition of anorexia increased host survival. SlrP-mediated inhibition of anorexia prevented invasion and systemic infection by wild-type S. Typhimurium, reducing virulence while increasing transmission to new hosts, suggesting that there are trade-offs between transmission and virulence. These results clarify the complex and contextual role of anorexia in host-pathogen interactions and suggest that microbes have evolved mechanisms to modulate sickness-induced behaviors to promote health of their host and their transmission at the expense of virulence. Sickness-induced anorexia is a conserved behavior induced during infections. Here, we report that an intestinal pathogen, Salmonella Typhimurium, inhibits anorexia by manipulating the gut-brain axis. Inhibition of inflammasome activation by the S. Typhimurium effector, SlrP, prevented anorexia caused by IL-1β-mediated signaling to the hypothalamus via the vagus nerve. Rather than compromising host defenses, pathogen-mediated inhibition of anorexia increased host survival. SlrP-mediated inhibition of anorexia prevented invasion and systemic infection by wild-type S. Typhimurium, reducing virulence while increasing transmission to new hosts, suggesting that there are trade-offs between transmission and virulence. These results clarify the complex and contextual role of anorexia in host-pathogen interactions and suggest that microbes have evolved mechanisms to modulate sickness-induced behaviors to promote health of their host and their transmission at the expense of virulence. Infections trigger stereotypical behavioral changes in the host including anorexia, fever, sleep disturbances, social withdrawal, and changes in grooming, collectively referred to as “sickness behaviors” (Dantzer, 2009Dantzer R. Cytokine, sickness behavior, and depression.Immunol. Allergy Clin. North Am. 2009; 29: 247-264Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, Hart, 1988Hart B.L. Biological basis of the behavior of sick animals.Neurosci. Biobehav. Rev. 1988; 12: 123-137Crossref PubMed Scopus (1643) Google Scholar). These behaviors have been proposed to be adaptive strategies to increase the chance of survival from acute illness (Exton, 1997Exton M.S. Infection-induced anorexia: active host defence strategy.Appetite. 1997; 29: 369-383Crossref PubMed Scopus (139) Google Scholar, Hart, 1988Hart B.L. Biological basis of the behavior of sick animals.Neurosci. Biobehav. Rev. 1988; 12: 123-137Crossref PubMed Scopus (1643) Google Scholar, Kyriazakis et al., 1998Kyriazakis I. Tolkamp B.J. Hutchings M.R. Towards a functional explanation for the occurrence of anorexia during parasitic infections.Anim. Behav. 1998; 56: 265-274Crossref PubMed Scopus (183) Google Scholar). Animal studies have suggested that any benefits conferred by anorexia caused by infection are context dependent. In mice systemically infected with Listeria monocytogenes, animals that were force-fed succumbed to infection more rapidly than animals that were allowed to develop the anorexic response (Murray and Murray, 1979Murray M.J. Murray A.B. Anorexia of infection as a mechanism of host defense.Am. J. Clin. Nutr. 1979; 32: 593-596Crossref PubMed Scopus (218) Google Scholar). However, in Drosophila, anorexia was maladaptive for surviving an L. monocytogenes systemic infection (Ayres and Schneider, 2009Ayres J.S. Schneider D.S. The role of anorexia in resistance and tolerance to infections in Drosophila.PLoS Biol. 2009; 7: e1000150Crossref PubMed Scopus (232) Google Scholar). Because many biomedical practices interfere with sickness-induced behaviors, it is important to understand the mechanisms that lead to the induction of these behaviors and the contexts in which they are beneficial or detrimental for the host. Whether sickness-induced anorexia is beneficial for the host is largely dependent on how the fasted state functionally influences host resistance and tolerance defenses (Medzhitov et al., 2012Medzhitov R. Schneider D.S. Soares M.P. Disease tolerance as a defense strategy.Science. 2012; 335: 936-941Crossref PubMed Scopus (1042) Google Scholar, Schneider and Ayres, 2008Schneider D.S. Ayres J.S. Two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases.Nat. Rev. Immunol. 2008; 8: 889-895Crossref PubMed Scopus (0) Google Scholar). Acute starvation and diet restriction studies have primarily focused on how nutrition influences resistance mechanisms (Bedoyan et al., 1992Bedoyan J.K. Patil C.S. Kyriakides T.R. Spence K.D. Effect of Excess Dietary Glucose on Growth and Immune-Response of Manduca-Sexta.J. Insect Physiol. 1992; 38: 525-532Crossref Scopus (13) Google Scholar, Dunn et al., 1994Dunn P.E. Bohnert T.J. Russell V. Regulation of antibacterial protein synthesis following infection and during metamorphosis of Manduca sexta.Ann. N Y Acad. Sci. 1994; 712: 117-130Crossref PubMed Scopus (51) Google Scholar). In contrast to expectations, food restriction had a negative impact on the outcome of infection for the host (Burger et al., 2007Burger J.M. Hwangbo D.S. Corby-Harris V. Promislow D.E. The functional costs and benefits of dietary restriction in Drosophila.Aging Cell. 2007; 6: 63-71Crossref PubMed Scopus (92) Google Scholar, Kristan, 2007Kristan D.M. Chronic calorie restriction increases susceptibility of laboratory mice (Mus musculus) to a primary intestinal parasite infection.Aging Cell. 2007; 6: 817-825Crossref PubMed Scopus (55) Google Scholar, Libert et al., 2008Libert S. Chao Y. Zwiener J. Pletcher S.D. Realized immune response is enhanced in long-lived puc and chico mutants but is unaffected by dietary restriction.Mol. Immunol. 2008; 45: 810-817Crossref PubMed Scopus (83) Google Scholar, Ritz et al., 2008Ritz B.W. Aktan I. Nogusa S. Gardner E.M. Energy restriction impairs natural killer cell function and increases the severity of influenza infection in young adult male C57BL/6 mice.J. Nutr. 2008; 138: 2269-2275Crossref PubMed Scopus (68) Google Scholar, Sun et al., 2001Sun D. Muthukumar A.R. Lawrence R.A. Fernandes G. Effects of calorie restriction on polymicrobial peritonitis induced by cecum ligation and puncture in young C57BL/6 mice.Clin. Diagn. Lab. Immunol. 2001; 8: 1003-1011Crossref PubMed Scopus (88) Google Scholar). However, in a fruit fly model, Drosophila that developed anorexia or were diet restricted during systemic Salmonella infection lived significantly longer despite equivalent pathogen levels compared to infected flies fed a normal diet (Ayres and Schneider, 2009Ayres J.S. Schneider D.S. The role of anorexia in resistance and tolerance to infections in Drosophila.PLoS Biol. 2009; 7: e1000150Crossref PubMed Scopus (232) Google Scholar). More recently, anorexia promoted tolerance in mice infected with L. monocytogenes, indicating that in these contexts, the fasted state promoted tolerance defenses (Wang et al., 2016Wang A. Huen S.C. Luan H.H. Yu S. Zhang C. Gallezot J.D. Booth C.J. Medzhitov R. Opposing Effects of Fasting Metabolism on Tissue Tolerance in Bacterial and Viral Inflammation.Cell. 2016; 166: 1512-1525Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Pathogens and the microbiota are dependent on energy intake of their hosts during infection. Whether anorexia will have a beneficial effect on host outcome will also likely be determined by how microbial virulence is affected under fasted states. The effects of anorexia on pathogen virulence have been most theorized as creating a less hospitable niche, starving pathogens from essential nutrients required for replication (Hart, 1988Hart B.L. Biological basis of the behavior of sick animals.Neurosci. Biobehav. Rev. 1988; 12: 123-137Crossref PubMed Scopus (1643) Google Scholar). Although anorexia may have some positive and undefined effects on inhibiting pathogen growth, it is equally plausible that the fasted state may trigger increased virulence by altering microbial behavior independent of any effects on microbial growth. Microbes vary in their metabolic capacity and foraging strategies to adjust to situations in which nutrients are scarce (Ayres, 2016Ayres J.S. Cooperative Microbial Tolerance Behaviors in Host-Microbiota Mutualism.Cell. 2016; 165: 1323-1331Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Acute starvation in humans is associated with an increased risk of invasive bacterial infections (Page et al., 2013Page A.L. de Rekeneire N. Sayadi S. Aberrane S. Janssens A.C. Rieux C. Djibo A. Manuguerra J.C. Ducou-le-Pointe H. Grais R.F. et al.Infections in children admitted with complicated severe acute malnutrition in Niger.PLoS ONE. 2013; 8: e68699Crossref PubMed Scopus (59) Google Scholar). While this increased risk has been assumed to be dependent on the poor immune status of the patient due to malnutrition (Gordon et al., 2012Gordon J.I. Dewey K.G. Mills D.A. Medzhitov R.M. The human gut microbiota and undernutrition.Sci. Transl. Med. 2012; 4: 137ps12Crossref PubMed Scopus (129) Google Scholar), it is possible that the increased invasiveness and virulence may potentially reflect an adaptive strategy of certain microbes under nutrient-limiting conditions. Thus, the discrepancies shown in previous studies regarding any benefits afforded by sickness-induced anorexia for the host may in part be due to microbe-induced behavioral changes caused by food-restricted conditions that lead to increased virulence of infecting pathogens, the resident microbiota, or both. Salmonella enterica serovar Typhimurium (ST) is a gram-negative bacterium that causes enteric and systemic typhoid-like diseases in diverse animal models and humans. Upon oral infection in mice, ST penetrates the gut epithelial layer, typically by invasion of specialized epithelial cells called M cells (Monack et al., 2004Monack D.M. Mueller A. Falkow S. Persistent bacterial infections: the interface of the pathogen and the host immune system.Nat. Rev. Microbiol. 2004; 2: 747-765Crossref PubMed Scopus (407) Google Scholar). Once in the lamina propria (LP), bacteria are engulfed by innate immune cells, including neutrophils and macrophages, and infection induces the infiltration of T and B cells (Monack et al., 2004Monack D.M. Mueller A. Falkow S. Persistent bacterial infections: the interface of the pathogen and the host immune system.Nat. Rev. Microbiol. 2004; 2: 747-765Crossref PubMed Scopus (407) Google Scholar). The SPI-I type 3 secretion system (T3SS) and associated effectors are important for the gut stage of ST infection (Galán, 1996Galán J.E. Molecular genetic bases of Salmonella entry into host cells.Mol. Microbiol. 1996; 20: 263-271Crossref PubMed Scopus (377) Google Scholar). In adapted salmonellosis such as typhoid fever, with the aid of a second T3SS, SPI-2, and associated effectors (Hensel et al., 1998Hensel M. Shea J.E. Waterman S.R. Mundy R. Nikolaus T. Banks G. Vazquez-Torres A. Gleeson C. Fang F.C. Holden D.W. Genes encoding putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2 are required for bacterial virulence and proliferation in macrophages.Mol. Microbiol. 1998; 30: 163-174Crossref PubMed Scopus (493) Google Scholar), the bacteria disseminate via the lymphatics and bloodstream to systemic organs, including the spleen and liver. The gut phase of salmonellosis in mice induces an anorexic response in the host (Schieber et al., 2015Schieber A.M. Lee Y.M. Chang M.W. Leblanc M. Collins B. Downes M. Evans R.M. Ayres J.S. Disease tolerance mediated by microbiome E. coli involves inflammasome and IGF-1 signaling.Science. 2015; 350: 558-563Crossref PubMed Scopus (136) Google Scholar), and this pathogen can be transmitted to new mice via the fecal-oral route. As ST has evolved mechanisms to manipulate diverse physiological aspects of the mouse, a natural host of ST, it is an excellent pathogen to dissect the relationship among anorexia, host health, pathogen virulence, and transmission. Here, we examined how sickness-induced anorexia affected infection-induced lethality in a transmissible model of infection. We found that the ST effector, Salmonella leucine rich repeat protein (SlrP), negatively regulated virulence and promoted survival of the host by inhibiting the infection-induced anorexic response. We found that SlrP inhibited inflammasome activation and IL-1β maturation in the small intestine (SI), preventing the anorexic feeding program in the hypothalamus that is dependent on the vagus nerve. Failure to inhibit IL-1β and the anorexic response resulted in increased extra-intestinal dissemination and increased virulence of the pathogen but at the expense of pathogen transmission to new hosts. Our study provides mechanistic aspects for how local tissue response to microbes can signal to the brain to induce anorexia and demonstrates that microbes have evolved anti-virulence strategies that inhibit sickness-induced anorexia to promote host survival and pathogen transmission. In ongoing efforts to identify microbial factors that promote host health, we tested the importance of the ST effector, SlrP, in regulating host health during oral infection. SlrP is a member of the novel E3 ubiquitin ligases (NEL) class, a class of ubiquitin ligases that are encoded by some bacteria (Maculins et al., 2016Maculins T. Fiskin E. Bhogaraju S. Dikic I. Bacteria-host relationship: ubiquitin ligases as weapons of invasion.Cell Res. 2016; 26: 499-510Crossref PubMed Scopus (73) Google Scholar, Miao et al., 1999Miao E.A. Scherer C.A. Tsolis R.M. Kingsley R.A. Adams L.G. Bäumler A.J. Miller S.I. Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems.Mol. Microbiol. 1999; 34: 850-864Crossref PubMed Scopus (225) Google Scholar, Tsolis et al., 1999Tsolis R.M. Townsend S.M. Miao E.A. Miller S.I. Ficht T.A. Adams L.G. Bäumler A.J. Identification of a putative Salmonella enterica serotype typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis.Infect. Immun. 1999; 67: 6385-6393Crossref PubMed Google Scholar). The function of NELs and other prokaryotic ubiquitin ligases is traditionally thought to promote virulence during infection (Maculins et al., 2016Maculins T. Fiskin E. Bhogaraju S. Dikic I. Bacteria-host relationship: ubiquitin ligases as weapons of invasion.Cell Res. 2016; 26: 499-510Crossref PubMed Scopus (73) Google Scholar). However, because commensal bacteria and microbes that can colonize the host asymptomatically, including Salmonella species (Boyer and Lemichez, 2004Boyer L. Lemichez E. Targeting of host-cell ubiquitin and ubiquitin-like pathways by bacterial factors.Nat. Rev. Microbiol. 2004; 2: 779-788Crossref PubMed Scopus (22) Google Scholar), also encode mechanisms to interact with the host ubiquitin system, we hypothesized that SlrP may be important for negatively regulating virulence. Specific pathogen-free (SPF) C57BL/6 (B6) mice orally infected with an ST SL1344 strain deficient in slrP (ΔslrP) exhibited faster death kinetics than mice infected with the parental ST SL1344 strain (wild-type, WT) (Figure 1A). ΔslrP-infected animals exhibited a median time to death of 7 days post-infection, while ∼70% of mice infected with the parental strain were alive at day 10 post-infection (Figure 1A). The increased death kinetics of ΔslrP-infected mice were associated with increased weight loss (Figures 1B and 1C). We previously showed that ST infection in B6 mice causes wasting of lean and fat stores (Schieber et al., 2015Schieber A.M. Lee Y.M. Chang M.W. Leblanc M. Collins B. Downes M. Evans R.M. Ayres J.S. Disease tolerance mediated by microbiome E. coli involves inflammasome and IGF-1 signaling.Science. 2015; 350: 558-563Crossref PubMed Scopus (136) Google Scholar). We found that, while ΔslrP-infected mice had slightly higher lean body mass (Figure 1D), they exhibited significantly more wasting of adipose tissue stores compared to WT-infected mice (Figure 1E). These data suggest that SlrP function is necessary for controlling ST virulence and sustaining health of the host during infection in vivo of B6 mice. We performed an in-depth metabolic characterization of infected animals using the Comprehensive Laboratory Animal Monitoring System (CLAMS) and found that mice infected with the ΔslrP strain exhibited differences in the rate of oxygen consumption (VO2, Figure S1A) and CO2 production (VCO2, Figure S1B), resulting in a decreased respiratory exchange ratio (RER) compared to mice infected with WT ST (Figure 2A). In agreement with increased wasting of adipose tissue, lower RER suggests that ΔslrP-infected mice preferentially utilize fat as an energy substrate, resulting in increased lipid oxidation and depletion of fat stores, while WT-infected mice with higher RER use carbohydrates as the preferential fuel source (Schmidt-Nielsen, 1997Schmidt-Nielsen K. Animal physiology: adaptation and environment.Fifth Edition. Cambridge University Press, Cambridge, England; New York, NY, USA1997Google Scholar). We hypothesized that nutrient availability may be compromised in ΔslrP-infected mice due to the development of a more severe anorexic response in the absence of SlrP function. Consistent with our previous findings (Schieber et al., 2015Schieber A.M. Lee Y.M. Chang M.W. Leblanc M. Collins B. Downes M. Evans R.M. Ayres J.S. Disease tolerance mediated by microbiome E. coli involves inflammasome and IGF-1 signaling.Science. 2015; 350: 558-563Crossref PubMed Scopus (136) Google Scholar), we found that mice infected with WT ST exhibited an anorexic response (Figure 2B). However, we found that ΔslrP-infected mice exhibited a more severe anorexic response compared to WT-infected mice that was apparent within the first 24 hr post-infection and became more severe by 24–48 hr post-infection (Figure 2B). Between 0–24 hr post-infection, ΔslrP-infected mice consumed ∼6% less food compared to WT infected animals, and this difference increased to ∼20% less food by 24–48 hr post-infection (Figure 2B). Expression of a WT copy of slrP under the native promoter in the ΔslrP mutant protected B6 mice from infection induced anorexia (Figures S1C and S1D). These data raise the possibility that the more severe anorexic response results from increased pathogen burden, indicating the ΔslrP strain may either grow faster, be cleared less efficiently than the parental strain, or both. However, we found that at both 0–24 and 24–48 hr post-infection—the time frames in which we observe the onset of the more severe anorexic response in ΔslrP-infected mice (Figure 2B)—the pathogen burdens in WT- and ΔslrP-infected mice were comparable in all target tissues of this pathogen (SI, cecum, colon, Peyer’s patches [PP], MLN, liver, and spleen) (Figure 2C). We further found that, at 48 hr post-infection, the percentage of hosts with an extraintestinal dissemination event to be identical between ΔslrP- and WT-infected B6 mice (Figure 2D). Another possibility is that ΔslrP infection induces perturbations in the intestinal microbiota, resulting in a more pathogenic microbiota that leads to increased anorexia. Similar to our findings with SPF mice, germ-free mice monoinfected orally with ΔslrP exhibited a more severe anorexic response compared to germ-free mice that were monoinfected orally with the parental strain (Figure 2E), consuming approximately 50% less food within the first 24 hr post infection. Thus, the more severe anorexic response observed in ΔslrP-infected mice is not due to increased pathogen burdens, differences in pathogen tissue tropism, dissemination, or differential responses of the intestinal microbiota. To determine if the host nutrient status was related to pathogen virulence, we compared infection of food-restricted WT-infected animals to that of WT-infected mice that were fed ad libitum. Food-restricted WT-infected mice exhibited increased wasting that was associated with increased death kinetics compared to WT-infected mice that were fed ad libitum (Figures 2F and 2G). This was specific to infection, as uninfected food-restricted animals exhibited no mortality (Figures S1E and S1F). We next tested whether force-feeding could dampen ΔslrP virulence in B6 mice. ΔslrP-infected mice force-fed a liquefied diet during infection were protected from weight loss and had increased survival comparable to that of WT-infected B6 mice fed ad libitum (Figures 2H and 2I). Taken together, our data suggest that reduced nutrient intake leads to increased virulence of ST oral infection. Since the protection against anorexia mediated by SlrP was neither due to regulation of pathogen burdens nor regulation of the pathogenicity of the intestinal microbiota, we hypothesized that SlrP interacts directly with host physiologies that negatively regulate the sickness-induced anorexic response and, in doing so, regulate ST virulence. Protection from anorexia without a change in pathogen infection levels suggests that SlrP can negatively regulate ST virulence through (1) the inhibition of host processes that promote the anorexic response or (2) through the induction of a disease tolerance mechanism that promotes feeding. To distinguish between these two possibilities, we asked how the absence of SlrP function during ST infection influences the canonical mediators of sickness-induced anorexia. Using the CLAMS monitoring system, we found equivalent activity levels between ΔslrP- and WT-infected mice, indicating that ΔslrP-infected mice are not more anorexic because they are less active during infection (Figure 3A). Although we found no difference in the systemic levels of pathogen between mice infected with either strain at the onset of the more severe anorexic response (0–24 and 24–48 hr), we considered the hypothesis that the absence of SlrP rendered ST more stimulatory of an anorexic response. However, systemic challenge of B6 mice with either ΔslrP or WT ST resulted in an equivalent degree of anorexia (Figure 3B), indicating that ΔslrP is not more stimulatory of an anorexic response when systemic. Consistent with this and our burden analyses (Figure 2C), we found no significant difference in the extent of tissue pathology in ST target tissues liver, spleen, and SI between mice infected with WT or ΔslrP ST (Figures 3C and S2A). Thus, SlrP does not inhibit anorexia by alleviating tissue damage in target organs of this pathogen. SlrP has been shown in in vitro assays to inhibit dendritic cell (DC) migration (McLaughlin et al., 2014McLaughlin L.M. Xu H. Carden S.E. Fisher S. Reyes M. Heilshorn S.C. Monack D.M. A microfluidic-based genetic screen to identify microbial virulence factors that inhibit dendritic cell migration.Integr. Biol. (Camb). 2014; 6: 438-449Crossref PubMed Google Scholar). We found no difference in the levels of DCs or other immune cells associated with ST infection or intestinal inflammation in the LP, MLNs, liver, or spleen between 0–24 and 24–48 hr post-infection, indicating that SlrP does not inhibit anorexia by limiting immune cell infiltration (Figures 3D, 3E, and S2B–S2H). Additionally, levels of TNFα and IL-6, cytokines that can modulate sickness-induced behavioral changes (Dantzer, 2009Dantzer R. Cytokine, sickness behavior, and depression.Immunol. Allergy Clin. North Am. 2009; 29: 247-264Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar), in ST target tissues were not higher in ΔslrP-infected mice at 24 and 48 hr post-infection (Figures 3F, 3G, S2I, and data not shown). Taken together, these data suggest that SlrP does not inhibit the anorexic response by alleviating the primary cause of disease (tissue damage), nor does it inhibit the induction of TNFα, the induction of IL-6, or immune cell infiltration during infection.Figure S2SlrP Modulates Feeding Behavior Independent of Cell Infiltration, Pathology, TNFα, or IL-6, Related to Figure 3Show full caption(A) Histological scoring of spleen, liver, and ileum 72hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 5/group. “ND” indicates none detected – no pathology above limit of detection.(B) Gating strategy for flow cytometric analysis of leukocyte populations in the small intestine LP: T/B lymphocytes (P1), neutrophils (P2), phagocytic macrophages (P3), inflammatory monocytes (P4).(C) Gating strategy for flow cytometric analysis of migratory dendritic cells (P5) in the MLNs.(D) Flow cytometric analysis of percentages of LP and MLN leukocytes 48hr after oral infection of B6 mice with stated strain of S. Typhimurium. Animal numbers shown.(E) Total cellularity (left) and flow cytometric analysis of numbers (top) and percentages (bottom) of LP and MLN leukocytes 24hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 8-9 mice per group.(F) Gating strategy for flow cytometric analysis of leukocyte populations in the spleen and liver: T/B lymphocytes (P1), neutrophils (P2), macrophages (P3), inflammatory monocytes (P4), and dendritic cells (P5).(G) Total cellularity (left) and flow cytometric analysis of numbers (top) and percentages (bottom) of spleen and liver leukocytes 24hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 5 mice per group.(H) Total cellularity (left) and flow cytometric analysis of numbers (top) and percentages (bottom) of spleen and liver leukocytes 48hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 5 mice per group.(I) ELISA measurements of IL-6 in the indicated tissues 24hr after oral infection of B6 mice with WT or ΔslrP S. Typhimurium. n = 4-7 mice per group. ND denotes Not Detected. NS denotes Not Significant.Error bars indicate ± SEM. All comparisons not significant by unpaired Student’s t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Histological scoring of spleen, liver, and ileum 72hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 5/group. “ND” indicates none detected – no pathology above limit of detection. (B) Gating strategy for flow cytometric analysis of leukocyte populations in the small intestine LP: T/B lymphocytes (P1), neutrophils (P2), phagocytic macrophages (P3), inflammatory monocytes (P4). (C) Gating strategy for flow cytometric analysis of migratory dendritic cells (P5) in the MLNs. (D) Flow cytometric analysis of percentages of LP and MLN leukocytes 48hr after oral infection of B6 mice with stated strain of S. Typhimurium. Animal numbers shown. (E) Total cellularity (left) and flow cytometric analysis of numbers (top) and percentages (bottom) of LP and MLN leukocytes 24hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 8-9 mice per group. (F) Gating strategy for flow cytometric analysis of leukocyte populations in the spleen and liver: T/B lymphocytes (P1), neutrophils (P2), macrophages (P3), inflammatory monocytes (P4), and dendritic cells (P5). (G) Total cellularity (left) and flow cytometric analysis of numbers (top) and percentages (bottom) of spleen and liver leukocytes 24hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 5 mice per group. (H) Total cellularity (left) and flow cytometric analysis of numbers (top) and percentages (bottom) of spleen and liver leukocytes 48hr after oral infection of B6 mice with stated strain of S. Typhimurium. n = 5 mice per group. (I) ELISA measurements of IL-6 in the indicated tissues 24hr after oral infection of B6 mice with WT or ΔslrP S. Typhimurium. n = 4-7 mice per group. ND denotes Not Detected. NS denotes Not Significant. Error bars indicate ± SEM. All comparisons not significant by unpaired Student’s t test. Systemic IL-1β is believed to be the predominant mediator of sickness-induced anorexia (Dantzer, 2009Dantzer R. Cytokine, sickness behavior, and depression.Immunol. Allergy Clin. North Am. 2009; 29: 247-264Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). We did not detect any differences in the levels of serum, liver, or spleen IL-1β between ΔslrP- and WT-infected mice within the first 48 hr of infection (Figures 4A and S3A). We did, however, find significantly increased levels of IL-1β in the SI of ΔslrP-infected mice compared to WT-infected mice within the first 48 hr of infection, which correlates with the onset of the more severe anorexic response observed in these mice (Figure 4A). This increased IL-1β was specific to the SI because IL-1β levels of other tissues of the digestive tract including the stomach, cecum, colon, pancreas, PP, and mesenteric lymph node (MLN) were not significantly different between WT and ΔslrP-infected mice (Figure S3B and data not shown). Expression of a WT copy of slrP under the native promoter in the ΔslrP mutant prevented increased levels of IL-1β in the SI of infected mice (Figures S1D and S3C).Figure S3SlrP Modulates Feeding Behavior by Regulating Inflammasome Activation and IL-1β Maturation, Related to Figure 4Show full caption(A) L