Transcriptomic and innate immune responses to Yersinia pestis in the lymph node during bubonic plague

Abstract

A delayed inflammatory response is a prominent feature of infection with Yersinia pestis, the agent of bubonic and pneumonic plague. Using a rat model of bubonic plague, we examined lymph node histopathology, transcriptome, and extracellular cytokine levels to broadly characterize the kinetics and extent of the host response to Y. pestis and how it is influenced by the Yersinia virulence plasmid (pYV). Remarkably, dissemination and multiplication of wild-type Y. pestis during the bubonic stage of disease did not induce any detectable gene expression or cytokine response by host lymph node cells in the developing bubo. Only after systemic spread had led to terminal septicemic plague was a transcriptomic response detected, which included upregulation of several cytokine, chemokine, and other immune response genes. Although an initial intracellular phase of Y. pestis infection has been postulated, a Th1-type cytokine response associated with classical activation of macrophages was not observed during the bubonic stage of disease. However, elevated levels of interleukin-17 (IL-17) were present in infected lymph nodes. In the absence of pYV, sustained recruitment to the lymph node of polymorphonuclear leukocytes (PMN, or neutrophils), the major IL-17 effector cells, correlated with clearance of infection. Thus, the ability to counteract a PMN response in the lymph node appears to be a major in vivo function of the Y. pestis virulence plasmid.

FIG. 1.

Progression of histopathology in the draining lymph nodes of rats after intradermal injection of WT or pYV⁻ Y. pestis. Left and middle columns, lymph node sections stained for PMNs (brown) and Y. pestis (red) by immunohistochemistry (IHC); right column, lymph node sections stained by hematoxylin and eosin (H&E). (A) Uninfected lymph node showing normal B cell follicle (F) and T cell-rich paracortex (PC) architecture. (B and C) Y. pestis WT-infected lymph nodes dissected at 36 h (bubonic stage) (B) or 60 h (septicemic stage) (C) after infection. (D and E) Y. pestis pYV⁻-infected lymph nodes dissected at 36 h (D) or 60 h (E) after infection. In the H&E sections, yellow arrowheads indicate areas of neutrophil infiltration, green arrowheads indicate fields of extracellular bacteria, the black arrowhead indicates hemorrhage, and red arrowheads indicate eosinophils. Scale bars, 0.5 mm (left column), 50 μm (middle column, A3, and D3), or 10 μm (B3, C3, and E3).

FIG. 2.

Changes in immune cell populations in rat lymph nodes during infection with WT or pYV⁻ Y. pestis. (A to E) Percentages of T cells, B cells, macrophages, and neutrophils in lymph node cell suspensions prepared from uninfected rats (PBS controls) or from rats at 36 or 60 h after intradermal injection of 10⁸ pYV⁻ or 10³ WT Y. pestis organisms. Samples (n = 5 for each group) were labeled with antibodies against specific immune cell markers and analyzed by fluorescence-activated cell sorting (FACS). The means and standard deviations (SD) are indicated. *, P < 0.05; **, P < 0.001 (compared to PBS controls, as determined by one-way ANOVA with Dunnett's posttest). P values for significant differences between infected groups as determined by Tukey's posttest are also shown. (F) The percentage of neutrophils correlated with the bacterial load in the lymph nodes of rats infected with pYV⁻ Y. pestis (gray circles) (P = 0.0015; r² = 0.7346 by Pearson correlation analysis) but not in rats infected with WT Y. pestis (black circles) (P = 0.9943; r² = 0.000001). B, samples from rats in the bubonic stage of disease at 36 h; S, samples from rats in the septicemic stage of disease at 60 h.

FIG. 3.

Differential white blood cell (WBC) counts of peripheral blood collected from uninfected rats (PBS controls) or from rats at 36 and 60 h after infection with WT or pYV⁻ Y. pestis. Bars indicate the means and SD of the percentage of neutrophils (left axis), and black circles indicate the means and SD of the total number of WBC (right axis) in the blood samples (n = 5 rats for the WT 60-h group; n = 3 for all other groups). *, P < 0.05; **, P < 0.001 (percentage of neutrophils compared to PBS controls by one-way ANOVA with Dunnett's posttest). There was no significant difference in total WBC among the different groups.

FIG. 4.

Bacterial loads in inguinal lymph node samples used for microarray analysis. The dashed line indicates the sensitivity level of the Q-PCR method used.

FIG. 5.

(A) Principal-component analysis (PCA) representation of replicate rat gene expression profiles in the draining lymph node at 36 and 60 h after intradermal injection of sterile PBS, 10³ WT Y. pestis organisms, or 10⁸ pYV⁻ Y. pestis organisms. WT-infected samples were separated into two groups based on whether or not bacteria had disseminated from the lymph node to the blood: bubonic (sterile spleen) and septicemic (viable bacteria in the spleen). (B) Molecular and cellular function classification of genes whose expression was altered by infection with WT or pYV⁻ Y. pestis. The functions shown are the top three functional groups from the WT-infected samples. (C) Venn diagrams representing the numbers of rat genes upregulated or downregulated ≥2-fold in the lymph nodes of infected rats compared to uninfected control rats.

FIG. 6.

Common and differential transcriptional response patterns of immune response genes in the lymph nodes of rats during infection with WT or pYV⁻ Y. pestis. Complete expression data and descriptions of the genes shown can be found in Tables S1 to S6 in the supplemental material.

FIG. 7.

Extracellular cytokine and chemokine levels in lymph node fluid collected from uninfected (PBS control) rats and from rats infected with WT or pYV⁻ Y. pestis. B, samples from rats with bubonic plague; S, samples from rats with septicemic plague. *, P < 0.05 compared to PBS controls; **, P < 0.05 compared to all other samples (PBS control, pYV⁻-infected, and WT-infected bubonic samples) (as determined by one-way ANOVA with Dunnett's and Tukey's posttests).