Protozoan Parasite Babesia microti Subverts Adaptive Immunity and Enhances Lyme Disease Severity

Abstract

Lyme disease is the most prominent tick-borne disease in the United States. Co-infections with the tick-transmitted pathogens Babesia microti and Borrelia burgdorferi sensu stricto are becoming a serious health problem. B. burgdorferi is an extracellular spirochete that causes Lyme disease while B. microti is a protozoan that infects erythrocytes and causes babesiosis. Testing of donated blood for Babesia species is not currently mandatory due to unavailability of an FDA approved test. Transmission of this protozoan by blood transfusion often results in high morbidity and mortality in recipients. Infection of C3H/HeJ mice with B. burgdorferi and B. microti individually results in inflammatory Lyme disease and display of human babesiosis-like symptoms, respectively. Here we use this mouse model to provide a detailed investigation of the reciprocal influence of the two pathogens on each other during co-infection. We show that B. burgdorferi infection attenuates parasitemia in mice while B. microti subverts the splenic immune response, such that a marked decrease in splenic B and T cells, reduction in antibody levels and diminished functional humoral immunity, as determined by spirochete opsonophagocytosis, are observed in co-infected mice compared to only B. burgdorferi infected mice. Furthermore, immunosuppression by B. microti in co-infected mice showed an association with enhanced Lyme disease manifestations. This study demonstrates the effect of only simultaneous infection by B. burgdorferi and B. microti on each pathogen, immune response and on disease manifestations with respect to infection by the spirochete and the parasite. In our future studies, we will examine the overall effects of sequential infection by these pathogens on host immune responses and disease outcomes.

Keywords: Babesia microti; Borrelia burgdorferi; Lyme disease; adaptive immune response; babesiosis; co-infection; tick-borne co-infection.

FIGURE 1

Effect of B. burgdorferi strain (N40) co-infection on B. microti (Bm) growth. (A) Percent of RBCs infected with B. microti in C3H mice infected either with B. microti alone, or with B. microti and N40, was determined over 21 days p.i. Each data point represents average parasitemia ± s.d. (n = 5). Both groups of mice exhibited peak parasitemia at 13th day p.i. with significantly high peak parasitemia in Bm-infected mice (^*p < 0.05, df = 8, F = 1.72). (B) Hemoglobin levels were measured for 21 days p.i. N40-infected mice were used as controls. (C) Examination of blood smears showed lower erythrocytes density and higher parasitemia in Bm-infected mice (left) than co-infected mice (right). Bar represents 10 μm and arrows indicate parasitized RBCs.

FIGURE 2

Outline of animal experiments to determine host response and disease manifestations after infection with B. burgdorferi N40 strain and/or B. microti (Bm). (A) Experimental plan and samples analyses at the acute phase (11th day p.i.) of infection of C3H/HeJ female mice. (B) Evaluation of the impact of infection on the host and pathogens when the parasitemia becomes undetectable microscopically (21st day p.i.).

FIGURE 3

Babesia microti (Bm) infection caused splenic enlargement in C3H mice at the acute phase of infection and differentially increase splenocytes compared to N40-infected mice. (A) Overall experimental plan and samples analyses after infection of C3H/HeJ female mice. (B) Splenic weights were determined on day 11 p.i. Spleens of Bm infected and co-infected mice were significantly heavier than spleens from N40 infected mice, which showed moderate increase in weight compared to naïve mice. (C) Gating scheme used for FACS analysis of splenic CD45+ cells is shown. (D) Quantitative analyses of splenic B cells, T cells, CD4+ and CD8a+ cells, and macrophages in each group of mice. Each cell type is represented as percentage of total CD45+ splenic leukocytes (mean ± s.d., n = 5). Two-tailed unpaired student t-test for unequal variance between the paired groups was used to determine significance (NS, not significant, ^*p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^****p < 0.0001).

FIGURE 4

Effect of B. microti (Bm) and N40 co-infection on brain and joint colonization by B. burgdorferi, and their effect on joint inflammation during the acute phase of infection. (A) High numbers of spirochetes were observed in joints and brains of B. burgdorferi infected mice, while the N40 burden was significantly higher in joints of co-infected mice as determined by a two-tailed unpaired student t-tests for unequal variance between the paired groups (^∗∗p < 0.01, df = 8, F = 3.60). (B) B. burgdorferi infection caused only mild joints inflammation during the acute phase of disease as indicated by change in synovial space (arrow 1), synovial hyperplasia and erosion of cartilage (arrow 2), and lymphocytic infiltration (arrow 3) while respective markers show higher inflammation in co-infected mice and no inflammation in Bm-infected mice. Bar represents 100 μm. (C) Images of the head region (left dorsal, right ventral) of N40-infected and co-infected mice captured by live imaging using IVIS-200 after i.p. injection of D-luciferin substrate showed an increase in bioluminescence, particularly in the frontal region of brain of co-infected mice. (D) Mice were deeply anesthetized, perfused with PBS and fixative before euthanasia. Brain sections were labeled with anti-B. burgdorferi antibodies conjugated to FITC and nuclei of host cells stained with DAPI. Endothelial cells were labeled using anti-CD31 antibodies tagged with PE (red) and are marked in the figure by arrowheads. Green spirochetes were detected in brain sections from N40 infected and co-infected mice (arrows) when the sections were examined using Nikon Ti2 microscope. B. microti infected mice used as a negative control did not show any spirochetes. Bar represents 100 μm.

FIGURE 5

Babesia microti (Bm) infection caused pronounced splenomegaly affecting splenic architecture in C3H mice at day 21 p.i. (A,B) A significant but moderate increase in spleen size is observed in N40 infected mice (2) compared to naïve mice (1) while very pronounced splenomegaly is observed in Bm infected (3) mice (^∗∗∗p < 0.001, df = 3, F = NA) and co-infected (4) mice (^∗∗∗p < 0.001, df = 5, F = NA). (C,D) H&E stained spleen sections displayed normal architecture with a clear demarcation between the white and red pulp (arrows 1 and 3, respectively) and marginal zone (arrow 2), in uninfected (C), and N40-infected (D) mice. (E,F) Demarcation between red and white pulp was indistinguishable in spleens of co-infected (E), and B. microti infected (F) mice. Bar in microscopic images represents 100 μm.

FIGURE 6

Flow cytometry analysis of splenic leukocytes from infected mice at day 21 p.i. Percentage of each cell type in each mouse is calculated using total CD45+ cells and data presented as mean ± s.d. Increase in F4/80+ macrophage percentage remained significantly higher only in co-infected mice (n = 10) compared to the naïve, uninfected mice (n = 5). Significant but only moderate increase in T and B cells was observed in Bm infected mice (n = 10). Increase in CD19+B cells, total CD3+ T cells, and CD8a+ cells in N40 infected (n = 9) and Bm infected mice was observed at this stage while significant reduction in co-infected mice occurred compared to respective cells in mice infected with each pathogen individually. Increases in CD4+ T cells relative to naïve mice in Bm-infected and N40 infected mice were higher than in co-infected mice. Each bar represents the mean ± s.d. (NS, not significant, ^*p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^****p < 0.0001).

FIGURE 7

Determination of the specific antibody response in N40 infected and co-infected mice at day 21 p.i. ELISA analysis using N40 protein extract probed with pooled plasma of either N40 infected or co-infected mice indicated a significant reduction (^****p < 0.0001, df = 8, F = 1.55) in the specific antibodies against B. burgdorferi in co-infected mice. No reactivity was observed in the negative control (No Ag) or using naïve mice plasma.

FIGURE 8

Increased colonization of organs by N40 and exacerbation of Lyme disease in mice by co-infection with B. microti (Bm). (A) Representative real-time images using IVIS-200 displaying higher spirochetal burden in co-infected than N40-infected mice at 2 weeks, and (B) 3 weeks of infection. (C) Burden of N40 in tissues determined using duplex qPCR assay with horizontal lines representing the mean N40 recA copy number. Joint and brain of co-infected mice showed significantly (^∗∗∗p < 0.001) higher spirochete burden as compared to mice infected with N40 alone while B. burgdorferi burden in hearts was not significantly different (NS). (D) More severe arthritis manifested by change in synovial space (arrow 1), synovial hyperplasia and erosion of cartilage (arrow 2), and higher lymphocytic infiltration (arrow 3) were observed in co-infected mice as compared to the N40-infected mice. (E) Opsonophagocytosis of N40 by mouse J774.1 macrophages was observed after 2 h of co-incubation of plasma from N40-infected mice with spirochetes such that macrophages showed significant phagocytosis detected as red, internalized bacteria (arrowheads) and some green extracellular spirochetes (arrows). The macrophages are marked blue. (F) Although phagocytosis occurred after opsonization of N40 with plasma from co-infected mice, it showed significantly lower internalized spirochetes compared to those using plasma from N40-infected mice (E). (G) Incubation of B. burgdorferi using plasma from B. microti infected mice, and (H) uninfected, naïve mice showed no phagocytosis of the spirochetes after 2 h of co-incubation of B. burgdorferi with macrophages. Bar represents 100 μm in Figures 7E–H.

FIGURE 9

Borrelia burgdorferi N40 strain infection with or without B. microti co-infection resulted in colonization of the forebrain at day 21 p.i., as shown in multiple sections by immunostaining. (A) Deeply anesthetized mice were perfused with PBS and fixative before euthanasia. Brain sections were labeled with anti-B. burgdorferi antibodies conjugated to FITC (white arrows) and endothelial cells using anti-CD31 antibodies tagged with PE (red), marked by arrowheads. The nuclei of the host cells are stained blue by DAPI. Aggregates of green spirochetes (arrows) were detected in brain sections from N40 infected and co-infected mice when examined by Nikon Eclipse Ti A1 scanning confocal microscope. Control B. microti infected mice brain did not show any spirochetes. (B) Additional sections of brains of mice infection with N40 alone or co-infected with B. microti also showed presence of the spirochetes when the sections were examined using Nikon Ti2 microscope illuminated using a Lumencor Spectra X light engine and images captured with a Hamamatsu ORCA Flash4.0 V3 sCMOS camera and Nikon NIS Elements software. Arrows mark green spirochetes in the image while arrowheads depict red endothelial cells. Bar represents 100 μm.