Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved September 15, 2014 (received for review May 30, 2014)
Significance
A minority of infected hosts is thought to be responsible for the majority of pathogen transmission events. Surprisingly little is known about what distinguishes superspreader hosts from the rest of the infected population. Using a mouse model of Salmonella infection, we show that levels of Salmonella are equivalent between antibiotic-treated superspreader and nonsuperspreader hosts; however, superspreader hosts are uniquely able to tolerate antibiotic treatment, unlike nonsuperspreader hosts. We find that nonsuperspreaders have a hyperinflammatory response to antibiotic treatment, resulting in increased inflammatory myeloid cells that contribute to the morbidity observed. Superspreaders display neither an increased frequency of inflammatory myeloid cells nor morbidity upon antibiotic treatment. Our data describe tolerance mechanisms unique to superspreader hosts that enable sustained pathogen transmission.
Abstract
Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>108 cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.
A growing body of work has demonstrated that a minority of infected hosts is responsible for the majority of new infections within the population. Woolhouse et al. first formulated the 80/20 rule of host–pathogen interactions, wherein 20% of the infected hosts (“superspreaders”) are responsible for 80% of the infections (1). For example, analysis of cattle herds infected with Escherichia coli O157:H7 has demonstrated that high-shedding individuals (8–20% of the infected herd) are responsible for the majority of the pathogen transmission to uninfected members of the herd (2⇓⇓–5). The identification of these superspreaders is of key importance for disease treatment and clearance (1, 6⇓–8). However, comparatively little is known about the host immune response that contributes to the superspreader state.
An infected host can fight pathogenic infection by two distinct processes—resistance and tolerance. Resistance encompasses a diverse set of mechanisms used by the host to control pathogen invasion and replication. Tolerance, conversely, employs different mechanisms that help the host organism tolerate the damage caused by both the pathogenic infection and the resulting immune response, thereby maintaining host health (9⇓–11). Although very little is known about the full spectrum of tolerance mechanisms, the few studies in animals suggest that, because pathogens and immunopathology can potentially affect almost any physiological process, tolerance is not restricted to a single protective pathway (9, 12, 13). Unlike resistance mechanisms, tolerance strategies do not have direct negative consequences for the pathogen and therefore should place no selective pressures upon the pathogen (12, 14, 15). For these reasons, tolerance mechanisms have been hypothesized to play a role in the maintenance of the asymptomatic superspreader state (11, 12, 15). However, an experimental link between tolerance and transmission has not been demonstrated.
Upon oral infection with Salmonella enterica serovar Typhimurium, in our mouse model of Salmonella transmission, 30% of infected hosts shed the pathogen at high levels (>108 Salmonella per gram of feces). These superspreader hosts are able to efficiently infect naive cagemates (16) and possess a distinct immune phenotype compared with the majority of the infected hosts [which shed the pathogen at lower levels and are nonsuperspreaders (17)]. Importantly, both superspreader and nonsuperspreader hosts carry identical pathogen burdens across all tissues except the intestinal tract. The host microbiota plays an important role in protecting the host from acute Salmonella infection (18, 19) and in the establishment of the superspreader state (16). Frequent subtherapeutic antibiotic use is common among livestock animals, and the resulting disruption of host gut flora or dysbiosis has long-lasting effects on the health of the host (20). Here, we demonstrate that superspreader hosts are uniquely able to tolerate antibiotic treatment and importantly, this tolerance is not maintained in nonsuperspreader hosts.
Results
Superspreaders Are Tolerant of Oral Antibiotic Treatment.
We have previously shown that administration of a single dose of 5 mg of streptomycin to nonsuperspreader hosts results in the increase of intestinal Salmonella burden to superspreader levels overnight (>108 cfu/g) (16, 17). We observed, however, that, unlike naturally occurring superspreaders, these antibiotic-induced superspreaders displayed signs of morbidity and even death. It has been shown that streptomycin alone can induce gastrointestinal inflammation (21). Thus, we treated superspreaders with antibiotics to control for potential effects of the antibiotic on the microbiota and host immune response. After streptomycin treatment, both superspreaders and nonsuperspreaders had identical levels of fecal Salmonella (Fig. 1A). Surprisingly, superspreader hosts treated with antibiotics did not lose weight, remaining at 98% (±2.5%) of their initial weight. In contrast, nonsuperspreader hosts treated with antibiotics lost an average of 15% (±9.7%) of their body weight (Fig. 1B). Indeed, nonsuperspreaders lost significantly more weight compared with superspreaders at 5 d after antibiotic treatment (Fig. 1C).
Fig. 1.
Superspreaders are tolerant to oral antibiotic treatment. Mice were infected for 30 d, and their transmission status was identified. (A–F) Superspreaders (SS; black circles) and nonsuperspreaders (non-SS; red squares) were treated with 5 mg of streptomycin (A–D) or neomycin (E and F) via oral gavage. Weight and fecal Salmonella burden were recorded for the indicated period. Data shown is representative of a minimum of two experiments with a total of 10–15 mice per condition. (A and B) Each data point represents an individual mouse. (C) Mice from two independent experiments were pooled with a total of 10–15 mice per condition, and weight data are shown from 5 d after streptomycin treatment. (D) Salmonella bacterial burden of indicated organs (left y axis) and whole blood (right y axis) in streptomycin-treated superspreaders and nonsuperspreaders. (E and F) Fecal Salmonella burden was quantified in neomycin treated superspreader and nonsuperspreader hosts (E), and weight data were recorded at indicated times (F). (G) Mice were infected for 4 d (shedding status cannot be confirmed at this time point) and treated with 5 mg of streptomycin (red squares) or PBS (black squares), and weight was recorded for the time period indicated. *P < 0.05; **P < 0.005; ****P < 0.0001. ns, not significant.
To determine the cause of this weight loss, we first investigated the pathogen and commensal bacterial burden in these antibiotic-induced superspreaders. We measured the levels of Salmonella in systemic tissues, such as the spleen and mesenteric lymph nodes, and determined that the bacterial burden did not differ significantly between antibiotic-treated superspreader and nonsuperspreader hosts (Fig. 1D). Commensal outgrowth, as measured by anaerobic growth on brain and heart infusion blood agar, did not differ significantly between the groups. Finally, neither Salmonella Typhimurium (Fig. 1D) nor commensal bacteria was detected in the blood, confirming that weight loss was not due to bacterial sepsis mediated by Salmonella or culturable commensal outgrowth.
Because the Salmonella Typhimurium strain we use is resistant to streptomycin, it was possible that the weight loss observed in streptomycin-treated nonsuperspreaders was induced by the overnight increase (100- to 100,000-fold) in Salmonella numbers in the gastrointestinal tract. To control for this increase, we treated superspreader and nonsuperspreader hosts orally with neomycin, an antibiotic to which the Salmonella Typhimurium strain is sensitive. Upon treatment with a single dose (5 mg) of neomycin, the fecal Salmonella levels in nonsuperspreaders still rose to superspreader levels (Fig. 1E). Two weeks after oral antibiotic treatment, nonsuperspreaders lost significantly more weight (up to 12 ± 10% of their initial body weight; Fig. 1F). These results indicate that superspreaders are protected from antibiotic-induced gastrointestinal disruptions.
Salmonella-Infected Hosts Are Not Tolerant to Antibiotic Treatment During the First Week of Infection.
Superspreaders have a distinct immune phenotype compared with hosts that are not colonized with high levels of Salmonella in the intestinal tract (17). The superspreader immune phenotype consists of an active, prolonged innate immune response that is characterized by higher levels of neutrophils in intestinal and systemic sites and a suppressed systemic adaptive CD4 T-cell response. In contrast, nonsuperspreaders have lower levels of gastrointestinal and systemic inflammation, but an active, robust CD4 T-cell response (17). Our previous studies demonstrated that the immune state of superspreaders is fully established by 2 wk after infection. This finding raises the question: Is the tolerance to antibiotic treatment gained by superspreaders? To test this possibility, we treated hosts with an oral antibiotic before the adaptive immune response was initiated. Because we had shown previously that the adaptive CD4 T-cell response is not activated until 7–9 d after infection (17), the treatment of hosts with an antibiotic 4 d after Salmonella infection would eliminate the potential contribution of Salmonella-specific CD4 T-cell responses. Mice treated with streptomycin at day 4 after infection lost significantly more weight than PBS-treated cagemates (Fig. 1G), suggesting that a Salmonella-specific T-cell response is not required for the weight loss observed after antibiotic treatment. Together, these results indicate that the superspreader-specific tolerance to antibiotic treatment develops later during infection.
Antibiotic-Treated Nonsuperspreaders Have Increased Acute-Phase Response.
Having established that antibiotic-driven weight loss could occur before the onset of Salmonella-specific adaptive T-cell responses, we next characterized the inflammatory response in these hosts. We examined cytokine levels in the serum and intestinal wash of antibiotic-treated superspreaders and nonsuperspreaders. There was no significant difference in the average serum cytokine level between the two groups. However, there was a wide variation in the levels of proinflammatory cytokines in antibiotic-treated nonsuperspreaders (Fig. 2 A and B), which was influenced by the two hosts with the largest weight loss (>20% of their initial body weight). Indeed, the only mice that had detectable levels of serum IL-1β and TNF-α were moribund (Fig. 2A). This pattern was also seen in intestinal washes, with the most moribund mice having the highest levels of proinflammatory cytokines, especially IL-1β, TNF-α, and IL-6 (Fig. 2B). Together, these data suggest that a severe inflammatory response may underlie the morbidity observed in antibiotic-treated nonsuperspreaders.
Fig. 2.
Antibiotic-mediated weight loss is correlated with activation of the acute-phase response. Mice were infected for 30 d, and their transmission status was identified. Superspreaders (black circles) and nonsuperspreaders (red squares) were treated with 5 mg of antibiotic via oral gavage. Superspreaders and nonsuperspreaders were treated with streptomycin and sacrificed 6 d after antibiotic treatment. Data shown are representative of two experiments with a total of 8–10 mice in each condition. (A and B) Serum (A) and intestinal cytokine (B) levels were measured by ELISA. Each point represents an individual mouse, and data are representative of two experiments with 10–15 mice per group. Nonsuperspreader hosts that lost >20% of their total body weight are represented by filled squares (Inset). (C) Fecal samples from streptomycin-treated superspreaders and nonsuperspreaders were processed, and host-derived proteins were identified by using mass spectrometry. Data show normalized spectral counts from the 17 identified proteins in the acute-phase response pathway depicted in a heat map where intensity was normalized within each protein and warmer colors represent increased spectral counts as depicted in the key. Host weight was normalized from red to blue with warmer colors depicting increased host weight. All proteins were correlated to host weight loss using Spearman’s correlation. *Significant correlations. (D) Host weight data from C is quantified, and significance is calculated by using the two-tailed Mann–Whitney test. **P < 0.005.
To confirm that morbidity was linked to an inflammatory response, we used a nontargeted proteomics approach to analyze host-secreted proteins in the feces. We found a large number of proteins belonging to the acute-phase response. Of the 17 identified acute-phase proteins, 10 were significantly positively correlated to weight loss (Fig. 2C). These results indicate that the morbidity observed in antibiotic-treated nonsuperspreader hosts (Fig. 2D) is linked to activation of the acute-phase response.
Antibiotic-Treated Nonsuperspreaders Have Increased Splenic Inflammatory Myeloid Cells.
Next, we investigated the inflammatory response by analyzing the frequency of Ly6C+ CD11b+ inflammatory myeloid cells in the mesenteric lymph nodes and spleen. These cells encompass both inflammatory monocytes and neutrophils (Fig. S1A), cell types that have been shown to secrete IL-1β and TNF-α (22, 23). We found that antibiotic-treated nonsuperspreaders had significantly more inflammatory myeloid cells in the spleen compared with antibiotic-treated superspreaders 6 d after treatment (Fig. 3 A and B). Upon analysis of the subsets of inflammatory myeloid cells, we found that the spleens from all of the antibiotic-treated nonsuperspreader mice contained significantly more inflammatory monocytes than antibiotic-treated superspreaders (Fig. S1B). A similar trend was seen for splenic neutrophil frequencies (Fig. S1C), with the difference in neutrophil frequencies between antibiotic-treated superspreader and nonsuperspreaders being significant after removing the sickest mice (mice that lost >20% of their initial body weight; Fig. S1C).
Fig. 3.
Antibiotic-treated nonsuperspreaders have increased inflammatory myeloid cells in the spleen, but not the gut. Mice were infected for 30 d, and their shedding status was identified. Superspreaders (black) and nonsuperspreaders (red) were treated with 5 mg of antibiotic via oral gavage. (A and B) Splenic cells were collected from streptomycin-treated superspreaders and nonsuperspreaders 6 d after antibiotic treatment. (A) Representative FACS plot shows frequency of CD11b+ Ly6C+ inflammatory myeloid cells gated on nonlymphocytes. (B) Total frequency of myeloid inflammatory cells expressed as a percentage of nonlymphocytes. (C and D) Ceca were fixed, and hematoxylin/eosin-stained sections were scored for histopathological changes. A representative figure of a cecal section from each group is displayed in C, and histopathological scores are represented in D. (E) Bars represent colonic neutrophils measured as a frequency of CD11b+ cells 6 d after streptomycin treatment. *P < 0.05.
This increase in inflammatory cells is not limited to systemic sites. Indeed, naturally occurring superspreaders and antibiotic-induced superspreaders have cecal and colonic inflammatory cell infiltrates. In contrast to the spleen, the levels of cecal and colonic inflammation were the same in both groups of mice, as revealed by histopathological analysis (Fig. 3 C and D). Additionally, the frequency of colonic neutrophils in antibiotic-treated superspreaders and nonsuperspreaders was identical and did not vary with morbidity (Fig. 3E), further confirming that superspreaders and nonsuperspreaders have similar levels of intestinal and colonic inflammatory cells after antibiotic treatment. Together, our results indicate that the increased morbidity in nonsuperspreader mice is linked to increased inflammatory myeloid cells in the spleen, but not the gut.
CD4 T Cells Contribute to Antibiotic-Induced Morbidity but Not Superspreader Tolerance.
Although antibiotic-mediated morbidity can occur before the development of Salmonella-specific adaptive immune responses (Fig. 1G), we wondered whether CD4 T cells contribute to morbidity in antibiotic-treated nonsuperspreaders. Neutralization of CD4 T cells in mice that had been infected with Salmonella for 30 d was sufficient to rescue antibiotic-treated nonsuperspreaders from significant weight loss (Fig. 4A and Fig. S2). There was a concurrent decrease in the numbers of splenic CD11b+Ly6C+ inflammatory myeloid cells and the constituent groups of inflammatory monocyte and neutrophils in CD4 T-cell–depleted mice (Fig. 4B). However, bacterial burdens in the spleen were significantly increased in CD4-neutralized mice compared with controls (Figs. 1D and 4C). Importantly, superspreader hosts treated with CD4 T-cell–neutralizing antibody did not lose weight and remained asymptomatic after antibiotic treatment, indicating that CD4 T cells are not required for maintenance of superspreader tolerance (Fig. 4A).
Fig. 4.
CD4 T cells contribute to antibiotic-induced morbidity but not superspreader tolerance. Mice were infected for 30 d, and their shedding status was identified. Superspreaders (black circles) and nonsuperspreaders (red squares) were treated with 5 mg of streptomycin via oral gavage. Mice were treated with CD4-neutralizing antibodies or the isotype control (n = 4 or 5 in each group) 1 d before antibiotic treatment and at 2 and 4 d after antibiotic treatment. (A) Each point represents an individual mouse, and the weight data were collected daily. (B) Splenocytes were collected and representative cell populations were enumerated. In the isotype group, a single mouse was excluded due to morbidity. (C) Splenic Salmonella burden was quantified. *P < 0.05; **P < 0.005.
Neutralizing IL-1β and TNF-α Alleviates Antibiotic-Treated Nonsuperspreader Morbidity.
Based on the links between the levels of inflammatory myeloid cells, proinflammatory cytokines, fecal acute-phase proteins, and morbidity in antibiotic-treated nonsuperspreader mice, we hypothesized that suppression of the acute-phase cytokines might restore tolerance in antibiotic-treated nonsuperspreaders. To test this hypothesis, we neutralized IL-1β and TNF-α—the two acute-phase cytokines detected in moribund antibiotic-treated nonsuperspreader hosts. Nonsuperspreaders treated with neomycin or streptomycin were injected with neutralizing antibodies against IL-1β and TNF-α or with the isotype control antibody. The mice that received the IL-1β– and TNF-α–neutralizing antibodies lost significantly less weight compared with antibiotic-treated nonsuperspreaders injected with the isotype control antibodies (Fig. 5 A and B). As expected, the neutralizing antibodies did not affect the weights of antibiotic-treated superspreader hosts (Fig. 5A). Importantly, the frequencies of inflammatory myeloid cells and neutrophils in antibiotic-treated nonsuperspreader hosts were significantly reduced (Fig. 5 C and D). This decrease in frequency was mirrored by significant decreases in the numbers of total inflammatory myeloid cells, inflammatory monocytes, and neutrophils in the spleens of acute-phase cytokine-neutralized hosts (Fig. 5E). Thus, neutralization of acute-phase cytokines was sufficient to suppress the expansion of the inflammatory myeloid cell population and bring them to levels comparable to those observed in superspreader hosts. Systemic and fecal Salmonella levels were not affected by treatment with neutralizing antibodies (Fig. 5 F and G), confirming that neutralization of acute-phase cytokines resulted in increased tolerance of the bacterial burden.
Fig. 5.
Neutralization of acute-phase cytokines ablates weight loss in antibiotic-treated nonsuperspreaders. Mice were infected for 30 d, and their transmission status was identified. Superspreaders (SS; black circles) and nonsuperspreaders (Non-SS; red squares) were treated with 5 mg of neomycin (A and C–F) or streptomycin (B and G) via oral gavage. Data shown are representative of two experiments with a total of 8–10 mice in each condition. Along with the antibiotic, mice were injected intraperitoneally with neutralizing antibodies against IL-1β and TNF-α (filled shapes) and the isotype control (open shapes). (A and B) Each point represents an individual mouse, and the weight data were measured at 12 d after neomycin treatment (A) and 6 d after streptomycin treatment (B). Splenocytes were collected from mice in A, and inflammatory myeloid cells (C) and neutrophils (D) were quantified as a frequency of nonlymphocytes. Cell populations were enumerated in E. Bacterial burden of selected tissues was quantified at 12 d after neomycin treatment (F) and 6 d after streptomycin treatment (G) *P < 0.05; **P < 0.005. ns, not significant.
Discussion
Disease tolerance to pathogen infection has been characterized in genetically distinct mouse and Drosophila strains (14, 24, 25), as well as in a mouse model of viral and bacterial coinfection (26). Our results demonstrate that tolerance can also be induced in a subset of individuals infected with the same pathogen in a genetically similar host population. Unlike resistance, tolerance has been proposed to not have negative consequences for the pathogen (9⇓–11, 15). However, recent theoretical work has predicted that the use of therapeutic mechanisms that alleviate symptoms of illness in infected hosts but do not alter pathogen burden can have negative epidemiological consequences for host populations (13). We have identified a disease-associated tolerance mechanism that is specific to superspreaders and enables the host to tolerate inflammation mediated by oral antibiotics, thereby facilitating continued pathogen transmission. Nonsuperspreaders, however, display morbidity upon antibiotic treatment. This morbidity is correlated with the activation of the acute-phase inflammatory response and the increase in systemic inflammatory myeloid cells. Neutralization of the acute-phase response cytokines in these nonsuperspreader hosts reduces morbidity while leaving pathogen burden unchanged, thereby enabling continued pathogen transmission. Our results further underline the importance of identification and treatment of highly infectious superspreader hosts.
We have previously shown that superspreader hosts have more systemic inflammation compared with nonsuperspreader hosts, yet appear perfectly healthy. However, upon antibiotic treatment, superspreader hosts have a relatively reduced inflammatory response compared with nonsuperspreaders. Our data suggest that the previously described superspreader-specific immune phenotype allows superspreaders to tolerate antibiotic insult and resulting inflammation. For example, superspreaders have dampened responses to some cytokines (IL-6 in particular) and lower basal phosphorylated STAT proteins (17). Thus, superspreader hosts may be better equipped to tolerate higher levels of proinflammatory cytokines, and we speculate that these tolerance mechanisms protect the host from morbidity associated with antibiotic-driven dysbiosis. One hypothesis we tested was whether superspreaders had higher levels of IL-1 receptor agonist, thereby inhibiting the effect of IL-1β via competitive binding to the IL-1 receptor (27). However, we found that superspreaders did not have higher levels of IL-1 receptor agonist in serum or intestinal wash (Fig. S3), indicating an alternate mechanism(s) of tolerance of acute-phase cytokines. Intriguingly, acute-phase cytokine neutralization in antibiotic-treated superspreaders did not result in suppression of the neutrophils or inflammatory monocytes, suggesting that proliferation of these cell types may be differentially regulated in superspreaders. Although inflammatory monocytes (28) are observed during chronic Salmonella infection, little is known about their functionality in the context of transmission. We wonder whether induction and regulation of inflammatory monocytes and neutrophils may differ based on the transmission status of the infected host.
We observed that moribund antibiotic-treated nonsuperspreaders had lower frequencies of neutrophils in the spleen, potentially indicating a belated attempt to control antibiotic-driven inflammation. For example, we show that at 7 d after streptomycin treatment, surviving nonsuperspreader hosts have lower levels of splenic neutrophils compared with 3 d after streptomycin treatment (Fig. S4A). In contrast, there was a sustained increase in colonic neutrophils in all of the antibiotic-treated nonsuperspreaders (Fig. S4B). We have shown previously that an influx of systemic neutrophils is sufficient to suppress Tbet+ CD4 T cells. Consistent with our previous findings, we saw lower frequencies of TH1 cells in antibiotic-treated nonsuperspreaders (Fig. S5 A and B). This suppression is inadequate for protection, because depletion of CD4 T cells is required to rescue antibiotic-treated nonsuperspreaders from weight loss. The mechanism that mediates this rescue remains unclear. We observed fewer inflammatory monocytes and neutrophils in CD4-depleted mice, providing an independent line of evidence that these cells are linked to the morbidity observed in antibiotic-treated nonsuperspreaders. However, unlike the neutralization of acute-phase cytokines, we observed increased splenic bacterial loads in CD4-depleted mice. Although naturally occurring superspreaders have a blunted CD4 T-cell response, we show here that CD4-depleted superspreaders continue to be tolerant of antibiotics, suggesting that CD4 T cells are not required for tolerance.
The mice used in our study are siblings and cagemates, potentially indicating that intracage microbiota variation does not impact development of disease-associated tolerance in superspreaders. A month-long infection with Salmonella Tyhpimurium may result in differential modulation of the gut microbiota in superspreaders and nonsuperspreaders, resulting in a superspreader-specific microbiota that is protective against antibiotic treatment. However, because superspreader and nonsuperspreader hosts were housed in the same cage before antibiotic treatment, our results suggest that disease-associated tolerance develops independent of cage effects. To confirm that the protective nature of the microbiota is not transferrable, we cohoused superspreaders and nonsuperspreaders after antibiotic treatment; however, this cohousing did not prevent morbidity in the nonsuperspreader hosts (Fig. S6). It is possible that the components of the microbiota that mediate tolerance are not transferrable by cohousing and instead require a more direct method of transfer. Because uninfected mice treated with neomycin or streptomycin do not display morbidity and have only transient inflammation (21), presence of a pathogen is still required. This finding raises the question of whether Salmonella in superspreaders mediates tolerance. Although we have previously published the finding that the Salmonella cultured from superspreaders does not have heritable differences in virulence compared with nonsuperspreaders (16), there could be nonheritable differences in Salmonella that mediate tolerance within superspreaders. These transcriptional differences could be driven by interactions with a superspreader-specific microbiota or differential localization within the superspreader gut.
Some host-adapted pathogens are capable of establishing longstanding carrier states in specific hosts, which can lead to pathogen shedding for extended periods of time. However, the long-term effects on the host remain to be determined. It is possible that the tolerance to antibiotic-induced inflammation observed in superspreader hosts could have a deleterious effect in other cases of immune challenge, such as a secondary infection or potential induction of autoimmunity. Here we show that superspreader tolerance is acquired sometime between 4 d and 4 wk after infection. The determination of the host immune and microbiota effects mediating this tolerance will likely lead to new modalities of treatment of proinflammatory diseases, such as inflammatory bowel disease and ulcerative colitis.
Materials and Methods
Infections.
The 129 × 1/SvJ (strain 000691) or 129S1/SvImJ (strain 002448) mice were purchased from Jackson Laboratories and infected at 6–8 wk of age. Mice were infected with Salmonella Typhimurium strain SL1344 grown shaking overnight in Luria broth supplemented with streptomycin at a final concentration of 100 μg/mL. Bacteria were spun down, washed, and resuspended in PBS at a concentration of 1010 cfu/mL and 108 cfu in a volume of 10 μL fed to mice via pipet tip. Food was removed, and mice were starved 16 h before infection. Mice were placed into a fresh cage after infection and antibiotic treatment. Superspreader and nonsuperspreader status was confirmed by plating fecal pellets on antibiotic-selective plates, and shedding status was determined as described (17). All animal experiments were conducted in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines and approved by the Stanford University Institutional Animal Care and Use Committee.
Antibiotic Administration.
Mice were oral gavaged with 5 mg of the appropriate antibiotic suspended in 100 μL of distilled water at either 30 or 4 d after infection. After antibiotic treatment, mice were moved to fresh cages and weighed daily by using the Dune electrical balance (Braintree; DCT201). Mice were weighed at the same time of day to control for circadian-related variations in feeding behavior. Typically after antibiotic treatment, mice from the same group were housed together, with the exception of the cohousing experiment in which three nonsuperspreaders and two superspreaders mice were placed in a cage together.
Single Cell Isolation.
Single cell solutions were obtained as described (17). Briefly, splenocytes were obtained by grinding spleens between two glass slides, mesenteric lymph nodes were dissociated by using a pestle grinder, and all solutions were passed through a 70-μm strainer. Colonic tissue was thoroughly cleaned, cut into small pieces, and digested in a RPMI medium containing 10% (vol/vol) fetal calf serum, 0.5% Hepes, 0.1% β-Mercaptomethanol, and 1 mg/mL each of Collagenase A and Trypsin Inhibitor (C9891; T6522; Sigma) and DNase I (Roche; 1010415900).
Fluorescence-Activated Cell Staining.
Cells were fixed with paraformaldehyde (Electron Microscopy Sciences; 15710) at a final concentration of 1.6% for 10 min at room temperature. Cells were permeabilized with methanol, washed twice with FACS buffer (0.5% BSA, 0.02% sodium azide, and PBS, with a pH of 7.2), and stained with the granulocyte or T-cell panel of markers. The granulocyte panel included CD11b (PE-CF594), Ly6G (FITC), Ly6C (PE), MHC-II (APC), F4/80 (APCCy7), CD11c (PerCPCy5.5), and CD3, B220 (PacBlue). The T-cell panel included CD4 (PE-CF594), Tbet (PE), FoxP3 (PacBlue), CD11b (APCCy7), CD44 (APC), Gr1 (PECy5.5), and CD19 (FITC). Cell numbers were obtained by using CountBright beads (Invitrogen; 11570066).
Cytokine Neutralization.
Neutralizing antibodies against TNF-α (clone TN3-19.12; BE0244), anti–IL-1β (clone B122; BE0246), and isotype control (hamster polyclonal IgG; BE0091) were all obtained from Bio-X-Cell. Mice were injected intraperitoneally with 5 μg of each antibody or 10 μg of the isotype control. Injections were performed daily for streptomycin experiments or every second day for neomycin experiments for a total of six or seven injections per experiment.
For CD4 neutralization experiments, mice were injected with 150 μg of CD4-neutralizing antibody (clone GK1.5; BE00031) or isotype control (Rat IgG2b; BE0090) intraperitoneally 1 d before antibiotic treatment and again at days 2 and 4 after antibiotic treatment.
Cytokine Analysis.
TNF-α (88-7324), IFN-γ (88-7314), IL-10 (88-7804), and IL-6 (88-7064) ELISA kits were purchased from eBiosciences. IL-1β (DY401) and IL-1 receptor agonist (DY408) kits were purchased from R&D Systems. Serum was obtained via cardiac puncture, and intestinal wash was obtained by flushing 1 mL of phospho-buffered saline containing protease inhibitor (Roche; 05892953001) through the length of the intestine over a 70-μm cell strainer.
Histology.
Tissues evaluated histologically were fixed in 10% buffered neutral formalin and routinely processed for paraffin embedding and stained with hematoxylin and eosin. A semiquantitative scoring system of 0 to +6, (0 = no significant lesion, +1 = minimal, +2 = mild, +3 = moderate, +4 = marked, +5 = severe, and +6 = massive) was used to evaluate the severity of pathological changes in the ceca of infected mice including inflammation, edema, ulceration, and necrosis. The distribution (focal, multifocal, regionally extensive, or diffuse) and location (i.e., limited to lamina propria, lamina propria + submucosa, or transmural, through all layers of the cecal wall) of pathology was also noted in addition to assigning severity scores.
Proteomics.
Protein was extracted from fecal pellets, digested with trypsin, and analyzed by liquid chromatography tandem mass spectrometry as described (29). Two fractions from the C4 chromatography (40% and 60% acetonitrile) were analyzed, and no technical replicates were collected. Data were filtered to a 1% peptide false discovery rate (FDR) and 5% protein FDR by using in-house software (30). Acute-phase response proteins were identified through the use of IPA (Ingenuity Systems), and Spearmann correlations were calculated in Prism 6.
Statistics.
Significance was calculated by using the Mann–Whitney U two-tailed test unless otherwise mentioned.
Acknowledgments
We thank Dr. David Schneider for several thought-provoking conversations on tolerance and Katherine Ng for her insight and help with experiments. S.G. was supported by National Institutes of Health Grant R01 A1095396. This work was supported by a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Diseases award (to D.M.M.).
Footnotes
- ↵1To whom correspondence should be addressed. Email: dmonack{at}stanford.edu.
Author contributions: S.G. and D.M.M. designed research; S.G. performed research; J.S.L. and J.E.E. contributed new reagents/analytic tools; S.G., J.S.L., and D.M.B. analyzed data; and S.G. and D.M.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1409968111/-/DCSupplemental.
References
Disease-associated tolerance in transmitters
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