Human neutrophil-like cells demonstrate antimicrobial responses to the chronic cyst form of Toxoplasma gondii

Abstract

The protozoan parasite Toxoplasma gondii infects approximately 2.5 billion people worldwide. Infection induces a rapid dissemination of parasites throughout the body followed by the formation of lifelong cysts within neurons of the host brain. Both stages require a dynamic immune response comprised of both innate and adaptive cells. Neutrophils are a primary responding cell to acute infection and have been observed in the brain during murine chronic infection. Previous studies investigating human neutrophils found that invasion by Toxoplasma tachyzoites inhibits apoptosis of neutrophils, prolonging their survival under inflammatory conditions. Here, we demonstrate the differentiation of two distinct subsets following exposure of human neutrophil-like-cells (HNLC) to Toxoplasma cysts. In vitro stimulation and imaging studies show cyst-specific induction of cytokines and cyst clearance by HNLCs. Further testing demonstrates that aged HNLCs perform less phagocytosis of cysts compared to non-aged HNLCs. In conclusion, this study identifies a novel response of HNLCs to Toxoplasma cysts and may indicate a role for neutrophils in the clearance of cysts during human infection with Toxoplasma.

Keywords: Toxoplasma gondii; chronic cysts; human; immune response; neutrophils.

Figure 1.. Two different types of human neutrophil-like cells (HNLCs) are present following exposure to T. gondii.

HNLCs were exposed to either T. gondii tachyzoites or chronic-stage cysts for 4 hours. Coverslips were stained with immunofluorescent antibodies, and cells were imaged at 40x magnification. A) Immunofluorescence staining of HNLCs following addition of tachyzoites and cysts (blue = DAPI, green = CD15, red = T. gondii, scale bar = 25μm, scale bar of zoomed tachyzoite image = 10 μm). B) Total number of CD15+ cells and percent of CD15+ cells from 7 randomized regions of interest (ROIs) per sample (n=3/group). C) Percent of parasite positive cells from 15 randomized ROIs in a total of 6 “Tachyzoite” wells.

Figure 2.. Cyst-induced responses of human neutrophils.

HNLCs were stimulated with media alone, LPS (or PMA for NET production), T. gondii Me49 tachyzoites, tachyzoite antigen, T. gondii Me49 cysts, or cyst antigen. Following stimulation, cell supernatants were tested for cytokine and chemokine production via LEGENDplex assay and NET production via PicoGreen assay. A) Quantification of cytokine production by HNLCs via LEGENDplex assay. B-C) Quantification of extracellular DNA from re-stimulated brain mononuclear cells (BMNCs) (B) and HNLCs (C). For all significance measures, unpaired Student T-test was performed and p-values are as follows: * = p<0.05, ** = p<0.01.

Figure 3.. HNLCs attack and phagocytose cyst material.

A) Live time-lapse imaging after exposure of differentiated HNLCs to T. gondii cysts for 3 hours (40x magnification). Figure shows time-lapse images of brightfield cells overlayed with fluorescent cysts (green) at 5min, 60min, 120min, and 180min post-cyst exposure. Scale bar = 15μm. Arrows indicate HNLC migration towards cysts. B-D) HNLCs were exposed to cysts for 60 minutes with or without inhibition of phagocytosis using Cytochalasin D. Representative flow cytometry plots (B) of un-exposed cells, normal cells, and inhibited cells following 60min cyst exposure. Flow plots were analyzed after gating on live cells, and numbers on flow plots represent the average percentage of expression (n=4). Numbers in parentheses of upper right quadrants equal average percentages of expression of all CD11b+ cells. Quantification of CD11b+FITC+ (showing cell uptake of cyst material) (C) and CD11b+FITC− (cells without cyst material) (D) frequencies for each exposure condition. Statistical significance was determined by One-Way ANOVA (n=4 per group), **** = p<.0001.

Figure 4.. Characterization of HNLCs demonstrates age-dependent differences in response to cyst exposure.

Differentiated HNLCs were either exposed to cysts for 60min following completion of differentiation or were exposed to cysts after 2 weeks of aging following complete differentiation. A) Representative flow cytometry plots of CXCR4 and CD15 expression by unexposed Non-Aged (left) and Aged (right) cells. B) Representative flow cytometry plots (left) and quantification (right) of CD11b+FITC+ (showing cell uptake of cyst material) and CD11b+FITC− (cells without cyst material) frequencies in Non-aged and Aged cells after cyst exposure. Flow plots were analyzed after gating on live cells, and numbers on flow plots represent the average percentage of expression (n=4). C) Quantification of CXCR4 and CD15 frequencies in Non-aged and Aged CD11b+FITC+ (left) and CD11b+FITC− (right) populations after cyst exposure. For all quantification, statistical significance was determined by One-Way ANOVA (n=4 per group), * = p<0.05, ** = p<0.01, *** = p<0.001 and **** = p<0.0001.