Importance of the CCR5-CCL5 axis for mucosal Trypanosoma cruzi protection and B cell activation

Abstract

Trypanosoma cruzi is an intracellular parasite and the causative agent of Chagas disease. Previous work has shown that the chemokine receptor CCR5 plays a role in systemic T. cruzi protection. We evaluated the importance of CCR5 and CCL5 for mucosal protection against natural oral and conjunctival T. cruzi challenges. T. cruzi-immune CCR5(-/-) and wild-type C57BL/6 mice were generated by repeated infectious challenges with T. cruzi. CCR5(-/-) and wild-type mice developed equivalent levels of cellular, humoral, and protective mucosal responses. However, CCR5(-/-)-immune mice produced increased levels of CCL5 in protected gastric tissues, suggesting compensatory signaling through additional receptors. Neutralization of CCL5 in CCR5(-/-)-immune mice resulted in decreased mucosal inflammatory responses, reduced T. cruzi-specific Ab-secreting cells, and significantly less mucosal T. cruzi protection, confirming an important role for CCL5 in optimal immune control of T. cruzi replication at the point of initial mucosal invasion. To investigate further the mechanism responsible for mucosal protection mediated by CCL5-CCR5 signaling, we evaluated the effects of CCL5 on B cells. CCL5 enhanced proliferation and IgM secretion in highly purified B cells triggered by suboptimal doses of LPS. In addition, neutralization of endogenous CCL5 inhibited B cell proliferation and IgM secretion during stimulation of highly purified B cells, indicating that B cell production of CCL5 has important autocrine effects. These findings demonstrate direct effects of CCL5 on B cells, with significant implications for the development of mucosal adjuvants, and further suggest that CCL5 may be important as a general B cell coactivator.

Figure 1

CCR5^−/− T. cruzi-immune mice develop similar levels of T. cruzi-specific serum IgG and fecal extract IgA as compared with wild-type mice. CCR5^−/− and C57BL/6J wild-type mice were infected orally (Fig. 1A, C) or conjunctivally (Fig. 1B, D) with T. cruzi and challenged 8 weeks later via the same route. Four weeks later, serum (Fig. 1A, B) and fecal pellets (Fig. 1C, D) were collected from individual T. cruzi-immune (n=7–8/group) and naïve (n=3/group) mice, and T. cruzi-specific IgG (Fig. 1A, B) and IgA (Fig. 1C, D), respectively, studied via ELISA. No statistically significant differences were detected between T. cruzi-immune CCR5^−/− and wild-type mice [Mann Whitney U-Test]. Immune = T. cruzi-immune mice; WT B6 = C57BL/6J mice

Figure 2

CCR5^−/− mice have similar T. cruzi-specific IgG and IFN-γ ELISPOT responses as compared to wild-type mice in the spleen. CCR5^−/− and wild-type C57BL/6 mice were orally or conjunctivally challenged several times with T. cruzi to generate T. cruzi-immune mice. Eleven- to thirteen-days post T. cruzi rechallenge, spleen cells from orally (Fig. 2A, C) and conjunctivally (Fig, 2B, D) challenged mice were isolated and evaluated for T. cruzi trans-sialidase (TS) specific IgG (Fig. 2A, B) and T. cruzi-specific IFN-γ (Fig. 2C, D) ELISPOT responses. 1×10⁶ spleen cells (SC) were added per well to assess T. cruzi-specific IgG ASC responses (Fig. 2A, B). In order to detect T. cruzi-specific IFN-γ responses, SC (5×10⁵) were pulsed with T. cruzi (Tc) lysate (10 μg/mL), trans-sialidase (TS; 10 μg/mL), or three individual H-2K^b CD8 epitopes (pep ASP2, VNHRFTLV; pep TSSA, ANYNFTLV; and pep 77.2, VDYNFTIV) at 2.5 μg/mL overnight at 37°C. No significant differences were detected between T. cruzi-immune CCR5^−/− and wild-type mice [Mann-Whitney U Test]. TS = trans-sialidase, ASC = antibody secreting cell, SFC = spot-forming cell, SC = spleen cells. Immune = T. cruzi-immune mice. WT = C57BL/6 mice. n = 7–8 mice/group.

Figure 3

CCR5 is not required for mucosal protection against T. cruzi. CCR5^−/− and wild-type C57BL/6 mice were orally or conjunctivally challenged several times with T. cruzi to generate T. cruzi-immune mice. Eleven- to thirteen-days after T. cruzi rechallenge, mice were sacrificed, gastric DNA (Fig. 3A) and nasal cavity DNA (Fig. 3B) were isolated and the number of T. cruzi molecular equivalents were quantitated using a T. cruzi-specific qPCR assay. Spleen cells and draining lymph node cells were isolated and assessed for parasite outgrowth using a standard parasite limiting dilution assay (Fig. 3C-F). *p < 0.05, **p < 0.01, ***p < 0.005; [Mann-Whitney U Test]. Control = primary infected mice, Immune= T. cruzi-immune mice, SC = spleen cells, ME = T. cruzi molecular equivalents. ND = not detectable. n = 5–8/group.

Figure 4

CCL5 neutralization in CCR5^−/− T. cruzi-immune mice leads to decreased gastric mucosal T. cruzi protection. CCR5^−/− and wild-type C57BL/6 mice were orally challenged several times with T. cruzi to generate T. cruzi-immune mice. Immune and naïve mice were then orally rechallenged with T. cruzi. In Figure 4A, T. cruzi-immune mice (n=3/group), along with naïve controls, were sacrificed on day 3 and gastric RNA isolated. Data represent fold changes (± standard error) in CCL5 gene expression compared with naïve CCR5^−/− or wild-type mice (calculated using 2^−ΔΔCt method with gapdh as a housekeeping gene). Negative controls without added reverse transcriptase were included to confirm the removal of gastric DNA (data not shown). *p < 0.05 [t test]. In Figure 4B, T. cruzi-immune CCR5^−/− mice were treated with 250 μg of mouse neutralizing α-CCL5 or isotype control IgG1κ (Sigma) mAb I.P. every other day starting 4 days prior to oral T. cruzi challenge and continuing through day 10 after rechallenge. Total gastric DNA was isolated 12 days after rechallenge and assessed for the number of T. cruzi genomes (molecular equivalents; ME) using T. cruzi-specific real-time PCR. Lines represent median values. n=8–9/group. These results represent two independent experiments with the results pooled together. *p < 0.05 [Mann-Whitney U test].

Figure 5

CCL5 neutralization in CCR5^−/− T. cruzi-immune mice leads to decreased T. cruzi-specific IgG and IgA antibody secreting cells in the spleen. CCR5^−/− mice were challenged multiple times orally with T. cruzi. Six weeks after the last challenge, mice were treated with 250 μg of neutralizing anti-CCL5 or isotype control IgG1κ mAb I.P. and rechallenged orally with T. cruzi as described in Figure 4. Twelve days after rechallenge, treated CCR5^−/− mice (n=5/group), as well as naïve, age-matched CCR5^−/− control mice (n=3), were sacrificed to assess T. cruzi-specific IgG (Fig. 6A) and IgA (Fig. 6B) antibody secreting cell (ASC) responses in the spleen. *p < 0.05, **p < 0.01 [Mann-Whitney U test]. These data represent 1 of 2 independent experiments with similar results. Immune = T. cruzi-immune mice; IgG1κ = IgG1κ isotype control antibody treated; α-CCL5 = anti-CCL5 neutralizing antibody treated. n=5/group.

Figure 6

CCL5 neutralization in CCR5^−/− T. cruzi-immune mice leads to decreased gastric inflammatory chemokines in the gastric mucosa. CCR5^−/− mice were orally challenged multiple times with T. cruzi. Six weeks after the last challenge mice were treated with 250 μg neutralizing anti-CCL5 or isotype control IgG1κ mAb I.P. and rechallenged orally with T. cruzi as described in figure 4. Total gastric RNA was isolated 12 days post-challenge. After genomic DNA removal and cleanup, qRT-PCR was used to measure chemokine gene expression. Data represents fold changes (± standard error) in gene expression as compared to age-matched naïve CCR5^−/− mice (n=3) (calculated using 2^−ΔΔCt method with gapdh as a housekeeping gene). Negative controls without added reverse transcriptase were included to confirm the removal of gastric DNA. Immune = T. cruzi-immune, IgG1κ = IgG1κ isotype-control antibody treated. α-CCL5 = anti-CCL5 neutralizing antibody treated. *p < 0.05, **p < 0.01 [Mann-Whitney U Test]. n=5/group.

Figure 7

The direct effects of CCL5 on B cells. Highly purified B cells (> 98% CD19⁺) were isolated from naïve C57BL/6 mice and cultured with or without LPS (0.5 μg/mL) ± recombinant mouse CCL5 (400 pg/mL) and proliferative responses were measured via [³H]-thymidine incorporation (Fig. 7A) or CFSE dilution (Fig. 7B). Culture supernatants were taken 7 days after stimulation and assessed for total IgM secretion via ELISA (Fig. 7C). Culture supernatants were taken at days 2, 3, 5 and 7 after stimulation with or without LPS (10 μg/mL) or anti-CD3/CD28, and CCL5 protein production was measured via ELISA (Fig. 7D). In a separate experiment, highly purified B cells were stimulated with or without LPS (10 μg/mL) ± anti-CCL5 mAb or IgG1κ isotype control antibody and proliferative responses measured via [³H]-thymidine incorporation on days 2 and 3 post stimulation (Fig. 7E). Culture supernatants were taken 7 days after stimulation, total IgM secretion was measured via ELISA and the percent suppression was calculated ((LPS_alone-LPS_{antibody treated})/LPS_alone) x 100 = % suppression (Fig. 7F). Data represent two to three independent experiments with multiple triplicates of pooled samples with similar results obtained in each experiment.