Both hemolytic anemia and malaria parasite-specific factors increase susceptibility to Nontyphoidal Salmonella enterica serovar typhimurium infection in mice

Abstract

Severe pediatric malaria is an important risk factor for developing disseminated infections with nontyphoidal Salmonella serotypes (NTS). While recent animal studies on this subject are lacking, early work suggests that an increased risk for developing systemic NTS infection during malaria is caused by hemolytic anemia, which leads to reduced macrophage microbicidal activity. Here we established a model for oral Salmonella enterica serotype Typhimurium challenge in mice infected with Plasmodium yoelii nigeriensis. Initial characterization of this model showed that 5 days after coinoculation, P. yoelii nigeriensis infection increased the recovery of S. Typhimurium from liver and spleen by approximately 1,000-fold. The increased bacterial burden could be only partially recapitulated by antibody-mediated hemolysis, which increased the recovery of S. Typhimurium from liver and spleen by 10-fold. These data suggested that both hemolysis and P. yoelii nigeriensis-specific factors contributed to the increased susceptibility to S. Typhimurium. The mechanism by which hemolysis impaired resistance to S. Typhimurium was further investigated. In vitro, S. Typhimurium was recovered 24 h after infection of hemophagocytic macrophages in 2-fold-higher numbers than after infection of mock-treated macrophages, making it unlikely that reduced macrophage microbicidal activity was solely responsible for hemolysis-induced immunosuppression during malaria. Infection with P. yoelii nigeriensis, but not antibody-mediated hemolysis, reduced serum levels of interleukin-12p70 (IL-12p70) in response to S. Typhimurium challenge. Collectively, studies establishing a mouse model for this coinfection suggest that multiple distinct malaria-induced immune defects contribute to increased susceptibility to S. Typhimurium.

FIG. 1.

Strategy for determination of species of murine malaria parasites. Boxes I, II, III, and IV represent variable regions of merozoite surface protein 1 (MSP-1), while the flanking regions are conserved among the different murine Plasmodium species (7). Brackets indicate the primer pairs (primers 1 to 10) used to verify species and subspecies.

FIG. 2.

Colonization of mice during infection with individual pathogens and during coinfection. (A) Parasitemia levels in mice infected with P. yoelii nigeriensis alone (squares) and during coinfection with S. Typhimurium (circles). Data represent means ± standard deviations (SD) for five mice. (B to D) Organ colonization by S. Typhimurium 5 days after i.g. infection of untreated mice, mice injected i.p. with naïve cells (RBC), or mice injected with red blood cells containing P. yoelii nigeriensis (Pyn). Numbers of CFU from the spleen (B), liver (C), and PP (D) are represented as the means ± standard errors of results from groups of 9 to 17 mice. Statistical significance was analyzed by using a Kruskal-Wallis nonparametric ANOVA followed by Dunn's multiple-comparisons test. P values for each pairwise comparison are shown. ns, not significant.

FIG. 3.

Effect of S. Typhimurium infection on pathology in spleen and liver. (A) Mice were injected with naïve RBC or P. yoelii nigeriensis-infected RBC (Pyn) and subsequently gavaged with S. Typhimurium (S.Tm) or LB. On day 5 postinfection, the spleen weight was calculated as a percentage of the body weight. (B) Histopathological lesions in HE-stained sections of liver and spleen after infection with P. yoelii nigeriensis (magnification, ×200) or S. Typhimurium (magnification, ×200) or coinfection with P. yoelii nigeriensis and S. Typhimurium (magnification, ×400). (Left) Arrowheads point to splenic macrophages filled with hemozoin pigment. Thick arrows indicate neutrophil infiltrates in the red pulp (RP) after S. Typhimurium infection. (Right) Thin arrows show the adherence of monocytes on vascular endothelial cells. Asterisks indicate microabscess formation (arrow). Images are representative of groups of 5 mice. WP, white pulp.

FIG. 4.

P. yoelii nigeriensis infection and antibody-mediated hemolysis elicit similar degrees of anemia. Mice were inoculated i.p. with 5 × 10⁶ RBC containing P. yoelii nigeriensis (+Pyn) or with an equivalent amount of uninfected blood (−Pyn) (uninfected controls). To induce noninfectious hemolytic anemia, mice were injected i.p. with rabbit anti-mouse RBC IgG (α-RBC) or nonspecific rabbit IgG (ns) (controls). Erythrocyte counts were determined at day 5 after infection or treatment. Data shown are means ± SD for 5 mice per treatment group. Differences between treatment groups and their respective controls were analyzed by a Student's t test. Data shown are from a representative experiment that was performed twice. P values are shown above each data set.

FIG. 5.

Hemolytic anemia increases systemic S. Typhimurium colonization. Shown are data for organ colonization by S. Typhimurium 5 days after i.g. inoculation with bacteria and i.p. inoculation with either PBS, nonspecific IgG, or anti-erythrocyte IgG. Log CFU from the spleen (A), liver (B), and PP (C) are represented as means ± standard errors of logarithmically transformed data from 6 mice. Statistical significance was analyzed by using ANOVA followed by a Tukey's HSD test. P values for each pairwise comparison are shown. ns, not significant.

FIG. 6.

Effect of RBC uptake on intracellular replication of S. Typhimurium. Results are represented as means ± standard deviations from three independent experiments. Opsonized or nonopsonized sheep RBC were incubated with J774 macrophages at a ratio of 20:1 for 2 h at 37°C. Control macrophages were not treated with RBC. Macrophages were then infected with S. Typhimurium (MOI of 5) and lysed 1 and 24 h after infection to enumerate CFU. Statistical significance was determined by ANOVA followed by a Tukey's HSD test. ns, not significant.

FIG. 7.

Malaria parasite infection, but not hemolytic anemia, blunts the IL-12 response to S. Typhimurium infection. IL-12p70 in serum was assayed by enzyme-linked immunosorbent assay (ELISA) at 5 days after infection. (A) Mice were singly infected with S. Typhimurium (Stm) or coinfected with P. yoelii nigeriensis. (B) Mice were mock treated with PBS (untreated controls) or inoculated with nonspecific IgG or with anti-RBC IgG to induce anemia. Results are the means ± standard deviations for 3 to 6 mice per treatment group. Statistical significance was analyzed by ANOVA followed by a Tukey's HSD test. P values for each pairwise comparison are indicated. ns, not significant.