Immunomodulatory proteins from hookworms reduce cardiac inflammation and modulate regulatory responses in a mouse model of chronic Trypanosoma cruzi infection

Abstract

Introduction: Hookworms are parasitic helminths that secrete a variety of proteins that induce anti-inflammatory immune responses, stimulating increased CD4 + Foxp3+ regulatory T cells and IL-10 production. Hookworm-derived recombinant proteins AIP-1 and AIP-2 have been shown to reduce inflammation in mouse models of inflammatory bowel disease and inflammatory airway disease by inducing CD4+Foxp3+ cells and IL-10 production. In contrast, chronic infection with the protozoal parasite Trypanosoma cruzi, the causative agent of Chagas disease, leads to chronic inflammation in tissues. Persistence of the parasites in tissues drives chronic low-grade inflammation, with increased infiltration of inflammatory cells into the heart, accompanied by increased production of inflammatory cytokines. There are no current antiparasitic drugs that effectively reduce or prevent chronic myocarditis caused by the onset of Chagas disease, thus new therapies are urgently needed. Therefore, the impact of AIP-1 and AIP-2 on myocarditis was investigated in a mouse model of chronic T. cruzi infection.

Methods: Female BALB/c mice infected with bioluminescent T. cruzi H1 strain trypomastigotes for 70 days were treated once daily for 7 days with 1mg/kg AIP-1 or AIP-2 protein by intraperitoneal injection. Control mice were left untreated or treated once daily for 14 days with 25mg/kg aspirin in drinking water. At 84 days of infection, splenocytes, cardiac tissue and serum were collected for evaluation.

Results: Treatment with both AIP-1 and AIP-2 proteins significantly reduced cardiac cellular infiltration, and reduced cardiac levels of IFNγ, IL-6 and IL-2. AIP-2 treatment reduced cardiac expression of COX-2. Further, while incubation with AIP-1 and AIP-2 proteins did not induce a significant upregulation of an immunoregulatory phenotype in dendritic cells (DC), there was a modest upregulation of CD11c +CD11b+MHCII+SIRPα+ expression, suggesting a regulatory phenotype. Ex-vivo stimulation of splenocytes from the treatment groups with AIP-1 loaded DC induced reduced levels of cytotoxic and pro-inflammatory T cells, stimulation with AIP-2 loaded DC specifically induced enhanced levels of CD4+CD25+Foxp3+ regulatory T cells among treatment groups.

Discussion: All in vivo and in vitro results demonstrate that hookworm-derived AIP-1 and AIP-2 proteins reduce T. cruzi induced cardiac inflammation, possibly through multiple anti-inflammatory mechanisms.

Keywords: Trypanosoma cruzi; anti-inflammatory; hookworm; immunomodulatory; myocarditis.

Figure 1

Study timeline and treatments. Bft stands for Blood form trypomastigotes. N/A, Not Applicable. Image created with Biorender.

Figure 2

Cardiac cellular infiltrate in heart tissues stained with Hematoxylin and Eosin (H&E). Representative images of left ventricular sections from (A) Uninfected (B) Infected Untreated (C) AIP-1 (D) AIP-2 (E) aspirin treated mice. Bar = 100µm (F) Graph of cellular infiltration in cardiac tissues. Data from individual mice are shown. n=10. Statistical Analysis = Mann-Whitney, p<0.05. Each treatment group is compared to the Infected Untreated group and error bars are defined by Mean with SD. ***p ≤ 0.001; ****p ≤ 0.0001.

Figure 3

Cardiac fibrosis in heart tissues stained with Picrosirius Red. Representative images of left ventricular sections from (A) Uninfected (B) Infected Untreated (C) AIP-1 (D) AIP-2 (E) aspirin treated mice. Bar = 100µm (F) Graph of the percentage of fibrosis in cardiac tissues. Data from individual mice are shown. n=10 Statistical Analysis = Mann-Whitney, p<0.05. Each treatment group is compared to the Infected Untreated group and error bars are defined by Mean with SD. **p ≤ 0.01; ***p ≤ 0.001.

Figure 4

Pro- and anti-inflammatory cytokines measured in cardiac tissue lysate. The concentration of (A) IFNγ, (B) IL-2, (C) IL-4, (D) IL-6, (E) IL-10, and (F) TNFα were measured by Luminex. Data from individual mice are shown. n=10. Statistical Analysis = Mann-Whitney, p<0.05. Each treatment group is compared to the Infected Untreated group and error bars are defined by Mean with SD. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Figure 5

Pro- and anti-inflammatory cytokines measured in serum. The concentration of (A) IFNγ, (B) IL-2, (C) IL-4, (D) IL-6, (E) IL-10, and (F) TNFα were measured by Luminex. Data from individual mice are shown. n=10. Statistical Analysis = Mann-Whitney, p<0.05. Each treatment group is compared to the Infected Untreated group and error bars are defined by Mean with SD. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Figure 6

Relative quantification of gene expression levels in cardiac tissues. Expression of (A) Arg1, (B) Mmp9, (C) Nos2, (D) Cox2, (E) NFκB, and (F) STAT-1 mRNA levels were measured from cardiac tissues by RT-PCR. Data from individual mice are shown. n=10. Statistical Analysis = Mann-Whitney, p<0.05. Each treatment group is compared to the Infected Untreated group and error bars are defined by Mean with SD. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001.

Figure 7

Parasite burdens. Parasite burdens were measured at the end of the study in (A) blood and (B) cardiac tissue by quantitative PCR (qPCR). Data from individual mice are shown. n=10. Statistical Analysis, Mann-Whitney; p<0.05. Error bars are defined by Mean with SD.

Figure 8

Secreted cytokines from in vitro restimulated splenocytes. Splenocytes from uninfected (A), Infected untreated (B), AIP-1 treated (C), AIP-2 treated (D), and aspirin treated (E) mice were harvested, and co-cultured with DC incubated with AIP-1, AIP-2 proteins or T. cruzi lysate. 48-hour culture supernatants were then evaluated for pro and anti-inflammatory cytokines (IFN-γ, IL-4, IL-10, IL-17a TNFα,) by Luminex analysis. Statistical analyses was performed using ANOVA with Tukey’s multiple comparison test comparing the mean of each column to the mean of every other column *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Error bars are defined by Mean with SD.

Figure 9

Immunophenotype of T cells before and after co-culturing splenocytes with DC. Splenocytes harvested from uninfected-untreated, infected-untreated, AIP-1 treated, AIP-2 treated, or aspirin-treated mice were co-cultured with DC loaded with AIP-1 protein, AIP-2 protein, T. cruzi lysate, or no protein (control DC). Flow cytometry analysis was performed on pre-co-culture, unstimulated splenocytes (baseline) and DC-stimulated splenocytes after 13 days of co-culture. The overall gating strategy for flow cytometry analysis is shown in (A). Splenocytes were analyzed for proliferation (CD8+KI67+), cytotoxicity (CD8+GranzymeB+), inflammation (CD8+TNFα+) and regulatory phenotypes (CD4+CD25+Foxp3+). Comparisons were made between all groups among uninfected untreated control mice (B), infected untreated mice (C), infected AIP-1 treated mice (D), infected AIP-2 treated mice (E) and infected aspirin treated mice (F). Statistical analyses was performed using Kruskal-Wallis with Dunn’s multiple comparisons tests comparing the mean rank of each column to the mean rank of every other column. Error bars are defined by mean with SD. * p<0.05.