ABSTRACT
Chagas disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a public health problem affecting 6 to 8 million people, mainly in Latin America. The role of microRNAs in the pathogenesis of Chagas disease has not been well described. Here, we investigate the role of microRNA-155 (miR-155), a proinflammatory host innate immune regulator responsible for T helper type 1 and type 17 (Th1 and Th17) development and macrophage responses during T. cruzi infection. For this, we compared the survival and parasite growth and distribution in miR-155−/− and wild-type (WT) C57BL/6 mice. The lack of miR-155 caused robust parasite infection and diminished survival of infected mice, while WT mice were resistant to infection. Immunological analysis of infected mice indicated that, in the absence of miR-155, there was decreased interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. In addition, we found that there was a significant reduction of CD8-positive (CD8+) T cells, natural killer (NK) cells, and NK-T cells and increased accumulation of neutrophils and inflammatory monocytes in miR-155−/− mice. Collectively, these data indicate that miR-155 is an important immune regulatory molecule critical for the control of T. cruzi infection.
INTRODUCTION
Trypanosoma cruzi, a causative agent of Chagas disease, is endemic in areas of Central and South America, with up to 25 million people at risk for infection. Chronically infected individuals transmigrate to other regions, making this a worldwide problem with 6 to 8 million people infected; approximately 10,000 of those die yearly from chronic sequela of the disease (1). This infection is primarily transmitted by the feeding of hematophagous triatomine insects but can also be acquired through ingestion of parasite-contaminated meat and beverages, via maternal-fetal transmission, and through blood and tissue donation (2). While the acute and chronic stages of the disease can be mostly asymptomatic, an estimated 20 to 30% of those infected develop chronic manifestations of the disease, which include heart failure and mega-organ syndromes (3). Chagas-induced cardiomyopathy is the leading cause of heart failure in Latin America (4). Chagas disease is treatable with either benznidazole or nifurtimox in the acute and indeterminate stages of the disease (5), which can prevent progression to the chronic phase. However, these medications are often not well tolerated, leading to noncompliance and incomplete therapy. Newer, more effective chemotherapeutic agents are needed for Chagas disease. Additionally, there are no vaccines available for this infection. Understanding the immune response to T. cruzi is important for the design of effective vaccines and uncovering potential drug targets.
The immune response in Chagas disease is complex, and various immune cells play different roles in the establishment and control of infection (6, 7). Differential host immune responses are thought to contribute to susceptibility or resistance of infection (8, 9). These include small, noncoding RNAs, termed microRNAs (miRNAs), which are important in immune regulation through modulation of gene expression at both transcriptional and posttranscriptional levels (10). More than 2,000 microRNAs have been described, including a multitude that have been linked to human disease (11). miRNA-155 (miR-155), a particularly well-characterized miRNA, has been shown to be a chief regulator of hematopoietic cells, including monocytes, macrophages, T cells, and B cells, affecting the expression of proinflammatory and anti-inflammatory cytokines (12–14). Additionally, miR-155 binds with the heat shock protein 40 genes Dnaja2 and Dnajb1 and leads to T helper type 1 and type 17 (Th1 and Th17) cell subset development and promotion of antigen presentation by dendritic cells (15, 16). miR-155 drives the inflammatory response of macrophages by targeting suppressor of cytokine signaling 1 (SOCS1), a STAT inhibitory factor, which inhibits signal transducer and activator of transcription 1 (STAT-1) and negatively regulates interferon gamma (IFN-γ). miR-155 is an important immune regulator in visceral leishmaniasis (17), Toxoplasma and tuberculosis (12, 18). miR-155 induces both Th1 and Th2 immune responses and helps to control the infection by Leishmania donovani (17). Likewise, miR-155 has a dual role during Mycobacterium tuberculosis infection by maintaining the survival of M. tuberculosis-infected macrophages and providing an adaptive immune response by promoting the survival and function of M. tuberculosis-specific T cells (12).
Th1 and Th2 responses occur throughout the course of T. cruzi infection. Initial production of interleukin 12 (IL-12) stimulates the synthesis of proinflammatory soluble mediators such as IFN-γ, TNF-α, and nitrogen oxide (NO) in macrophages (19, 20). Increased production of NO and additional proinflammatory cytokines IL-1, IL-6, and IL-18, which activate inflammatory cells, is essential for intracellular killing of parasites (19, 21). The expansion of Th2 cells and production of IL-10 are promoted by IL-4 and control the effects of excessive immune activation (19). In addition, parasite-specific antibodies, largely IgG, as well as CD8-positive (CD8+) lymphocytes, contribute to the reduction of overall parasitic load (19, 22). Despite all these immunological events, T. cruzi causes persistent chronic infection in 20 to 30% of the infected individuals. This could be due to various reasons, including the inhibitory effect on mononuclear phagocytic system, escape from phagolysosomal trapping inside the cells and migration to the cytoplasm for replication, different morphological life cycle forms in the infected individuals, presence of mucin-like sialylated molecules for host complement inhibition, and inhibition of IL-2 gene transcription in T cells (23). Utilizing comparative infection of wild-type and mutant mice devoid of miR-155, we sought to understand the role of miR-155 expression on the immune response and outcome of T. cruzi infection. We found the absence of miR-155 greatly enhances the susceptibility to parasite infection by downregulating an effective Th1 response.
RESULTS
miR-155 is essential for the resistance of C57BL/6 mice from T. cruzi infection.miR-155 has recently been known to play a role in immune modulation against some experimental parasitic diseases (17, 18, 24). We hypothesized that the lack of miR-155 would potentiate the infection of T. cruzi in mice. To test this, we infected WT and miR-155−/− mice with high-dose (105) T. cruzi tissue-cultured trypomastigotes intraperitoneally. All of the miR-155−/− infected mice died after 40 days of infection, whereas the infected WT mice showed no signs of clinical infection (Fig. 1A). To confirm the T. cruzi infection in WT and miR-155−/− mice, we extracted total DNA from the spleen tissue of T. cruzi-infected WT and miR-155−/− mice and ran PCR analysis using T. cruzi-specific TcZ1 and TcZ2 primers. The primer pair TcZ1 and TcZ2 amplifies a 188-bp sequence that is present within a 195-bp repetitive element of the nuclear DNA microsatellite region of T. cruzi (25, 26). The amplified 188 bp of T. cruzi-specific nuclear DNA region was clearly visible in both WT and miR-155−/− mice (Fig. 1B). We also microscopically examined blood for the presence of parasites in both T. cruzi-infected WT and miR-155−/− mice. After 40 days of postinfection, the parasitemia was high in miR-155−/−-infected mice in comparison to WT mice, in which it was undetectable (Fig. 1C). The examination of heart tissue sections from WT and miR-155−/− mice revealed high numbers of amastigotes in the tissue of miR-155−/− mice (Fig. 1D), whereas we found no parasites in the hearts of infected WT mice. These data clearly demonstrate that in the absence of miR-155, normally resistant mice are highly susceptible to invasive parasite infection that is 100% lethal.
(A) Kaplan-Meier survival curve comparing the survival of WT and miR-155−/− mice infected with 105 tissue-cultured trypomastigotes intraperitoneally (n = 8 in each group). The T. cruzi–infected miR-155−/− mice died after 40 days of infection, whereas WT mice did not develop disease. (B) Detection of T. cruzi in the spleens of infected WT and miR-155−/− mice (n = 3 in each group as shown). Results are shown as a T. cruzi-specific PCR amplification of genomic DNA using TcZ1 and TcZ2 primers (188 bp). The template for control lane (Ctrl) is the genomic DNA isolated from Brazil strain of T. cruzi. Tissue from noninfected control animals was negative for parasite DNA (not shown). Mkr, DNA size ladder. (C) Parasitemia of infected mice on day 40 postinfection. Blood was collected from euthanized mice, and blood smears were examined from Giemsa-stained slides by use of ×100 magnification under oil immersion. A total of 200 fields were counted for parasites from each of the mice (n = 5) from each group (WT and miR-155−/−), and the average value was determined. ND, not detected. (D) Histological examination of heart tissue of T. cruzi-infected mice. WT and miR-155−/− mice were infected with T. cruzi, and the hearts were collected, fixed, and examined by light microscopy after staining with hematoxylin and eosin. Fifty fields from sections of each heart (n = 5) were examined. The images were taken using Leica DMi1 with LAS v4.12 software. This figure shows representative duplicate photomicrographs of hearts of T. cruzi-infected WT and miR-155−/− mice. The black circles for miR-155−/− mice represent the amastigote pseudocysts inside the cells of heart tissue.
Lack of miR-155 results in decreased Th1 cytokine production.miR-155 is a critical mediator to provide T-cell-dependent immune responses and cytokine production (27), factors that are critical for the control of intracellular pathogens. To further study the immune effects of the lack of miR-155 in the context of T. cruzi infection, we examined the production of cytokines in the blood of WT and miR-155−/− mice infected with T. cruzi. The production of IFN-γ and TNF-α was significantly reduced in miR-155−/− mice compared to WT controls (Fig. 2). Likewise, the production of IL-13 was dramatically decreased in miR-155−/− mice. Since IL-13 and IL-4 regulate the M2 macrophage to enhance parasite clearance through Th2 activation (28), the significant reduction of IL-13 in miR-155−/− mice likely led to a decreased Th2 response during T. cruzi infection that led to robust infection and tissue pathology. Taken together, these findings suggest that the robust infection in miR-155−/− mice is due to the lower production of protective cytokines.
Evaluation of Th1 cytokines in the serum of WT and miR-155−/− mice. Serum levels of IFN-γ, TNF-α, IL-13, and IL-4 were measured from T. cruzi-infected WT and miR-155−/− mice. Significant decreases in IFN-γ, TNF-α, and IL-13 were observed in miR-155−/− mice. Data are shown from one representative experiment of three separate experiments and presented as a mean ± standard error of the mean (SEM). ***, P < 0.001; *, P < 0.05; ND, not detected.
miR-155 deficiency results in significant decreases of CD8+ T cells, NK cells, and NK-T cells in the spleen of T. cruzi-infected mice.Natural killer (NK) cells, NK-T cells, and CD8+ T cells are involved in parasite clearance in vivo by direct killing of extracellular trypomastigotes through the perforin-independent mechanism and the production of the robust amount of IFN-γ (7, 29–33). Thus, to better understand the effect of miR-155 on these immune cells during T. cruzi infection, the splenic cell populations of CD8+ T cells, NK cells, and NK-T cells were comparatively analyzed from parasite-infected miR-155−/− and WT mice. We found significant decreases in the number of all the indicated cell populations in miR-155−/−-infected mice (Fig. 3), suggesting that miR-155 is important for the maintenance of cytotoxic cell populations of NK and NK-T cells that control the T. cruzi infection. In our study, the subsequent parasitic dissemination in miR-155−/− mice could be due to the decreased number of CD8+ T cells and NK cells that were not able to initiate the trypanocidal effector mechanisms.
Depletion of CD8+ T cells, NK cells, and NK-T cells in miR-155−/− mice. Spleens were collected from each mouse of WT and miR-155−/− groups. The splenocytes were labeled with cell-specific fluorescent-labeled antibodies and analyzed by flow cytometry. Results were calculated and expressed as a mean ± SD of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05.
miR-155 deficiency leads to accumulation of neutrophils and inflammatory monocytes in the spleens of T. cruzi-infected mice.It is known that the neutrophils and inflammatory monocytes (Ly6Chi CCR2+) are primarily recruited in the spleen and can lead to tissue damage and disease pathogenesis (34, 35). To demonstrate the influence of miR-155 on inflammatory monocytes and neutrophils as an innate immunity mechanism during T. cruzi infection, splenic cells were isolated from WT and miR-155−/− mice and stained with CD11b. Infection with T. cruzi elicited the accumulation of both inflammatory monocytes (CD11b+/Ly6Chi) and neutrophils (CD11b+/LY6G) in miR-155−/− mice (Fig. 4).
Accumulation of splenic neutrophils and inflammatory monocytes in miR-155−/− mice. Flow cytometric analysis of splenic neutrophils and inflammatory monocytes of parasite-infected WT and miR-155−/− mice. Data are expressed as a percentage of LY6Chi and LY6Cmed LY6G+ cells of the total CD11b gated cells (inflammatory monocytes are CD11b+ Ly6Chi cells and neutrophils are CD11b+ LY6Cmed LY6G+). Data are presented as a mean ±SD of four to five mice per experiment. The asterisks indicate the statistical significance of the difference between WT and miR-155−/− mice. ***, P < 0.001; **, P < 0.01; *, P < 0.05.
DISCUSSION
In this study, we show that the lack of miR-155 leads to pronounced susceptibility of mice to T. cruzi infection by mediating the downregulation of Th1 cytokines and CD8+, NK, and NK-T cells necessary for host control of parasite infection. Although microRNAs are important regulators of the immune system, the role of host microRNAs in T. cruzi infection has not been well studied. However, there are several studies that show the important role of microRNAs during infection by intracellular parasites. A recent study demonstrated that miR-155 is important in resolving Leishmania infection by inducing T-cell proliferation and IFN-γ production through the negative regulation of SOCS1 and SHIP1. Our data of lower expression of IFN-γ, TNF-α, and IL-13 in miR-155−/− mice are in agreement with the previous report in which miR-155 exhibits an important host factor to control the infections (17). Previous studies showed that several host microRNAs, including miR-155, are deregulated in Chagas disease during the course of infection. Moreover, the expression of miR-155 in cardiac tissue is higher in the acute phase of Chagas disease (36, 37).
The expression of several cytokines in Chagas disease determines the protection or progression of the disease (38, 39). The higher expression of IFN-γ activates macrophages in Chagas disease, which promotes control of infection. However, the expression of IL-4 and IL-10 favors the growth of intracellular parasites leading to chronic infection (40). In our study, the decrease of protective cytokines (IFN-γ and TNF-α) and the exacerbation of infection in miR-155−/− mice indicate that miR-155 is vital for control of acute infection. Moreover, our results support previous studies showing that IFN-γ and TNF-α are important in controlling the infection.
It is well-known that miR-155 regulates the activation of NK and NK-T cells (41, 42), which are important for increased extracellular parasite cytotoxicity, and provides protection from T. cruzi infection (43). Our data indicate that both NK and NK-T cells were reduced as a result of the absence of miR-155 expression, and we surmise that this leads to the lack of control of parasite infection in the miR-155−/− mice. Additionally, the depletion of CD8+ T cells in miR-155−/− mice is presumed to have contributed to the overwhelming infection we observed. This conclusion is supported in past studies that have shown that CD8+ T cells provide immune protection through multiple mechanisms, including the production of IFN-γ, and that the depletion of CD8+ T cells produces uncontrollable parasite infection in mice (33, 44). Our findings indicate that the loss of miR-155 −/− expression leads to the depletion of key immune cell subsets that are important for controlling T. cruzi infection.
Neutrophils and inflammatory monocytes are generally the first-line cell types to encounter microbial pathogens in the initial stages of infection; however, the role of monocytes in control of parasitic infection varies depending on the parasite involved. A recent study showed that inflammatory monocytes are essential to control Toxoplasma gondii infection in mice (45). However, another study showed that inflammatory monocytes facilitate the growth of the parasites in the spleen in visceral leishmaniasis (46). T. cruzi induces neutrophil recruitment to the site of infection, where parasites are phagocytized and eliminated by phagolysosome-mediated lysis through the production of myeloperoxidase and reactive oxygen species (47). We found a significant increase in splenic neutrophils (Ly6cmed Ly6G+ cells) in infected miR-155−/− mice, which may be due to higher parasitic load in the spleens in these animals rather than the direct effect of miR-155 knockout on neutrophil phenotypes. We also observed increased inflammatory cells in the heart tissue of these animals correlating with enhanced parasite infiltration, which may reflect a global increase in neutrophilic response in the miR-155−/− mice. Aberrant miR-155 expression has recently been shown to adversely affect neutrophil migration (48), secretion of proinflammatory cytokines (49), and production of neutrophil extracellular traps (50), suggesting that appropriate expression of miR-155 is important for many aspects of normal neutrophil function. In the complete absence of miR-155 expression in our model, neutrophil accumulation may be related to multiple levels of dysfunction, contributing to the inability to effectively control parasites. We also found elevated levels of splenic inflammatory monocytes (Ly6CHi cells) in infected miR-155−/− mice. Recruitment of these cells has been linked to the susceptibility of Leishmania (46) and may be acting similarly. However, it is unknown whether they may also be dysfunctional secondary to miR-155 deficiency.
In conclusion, our results indicate that the absence of miR-155 expression leads to enhanced susceptibility of C57BL/6 mice to T. cruzi infection. The inability to control parasite infection is related to the downregulation of Th1 cytokines, a decrease in CD8+, NK, and NK-T cells, and possibly the dysfunctionality of inflammatory monocytes and granulocytes. It is clear that miR-155 is a critical polyfunctional master-regulator of immune function that is critical for host control of a large number of microbial pathogens.
MATERIALS AND METHODS
Chemicals and reagents.Chemicals were purchased from Sigma. All antibodies used for flow cytometry were purchased from BioLegend (San Diego, CA, USA). Cell culture media Dulbecco modified Eagle medium (DMEM) and RPMI 1640, phosphate-buffered saline, fetal bovine serum, and penicillin/streptomycin were purchased from Life Technologies (Gibco, Grand Island, NY, USA).
Cell cultures and parasites.Rat heart myoblasts (H9C2) were cultured at 37°C in DMEM supplemented with 10% fetal bovine serum, 100 μg/ml of streptomycin, and 100 μg/ml penicillin. Epimastigotes of the Brazil strain of T. cruzi were cultured at 26°C in liver digested-neutralized tryptone (LDNT) medium supplemented with 20 mg/liter hemin (Sigma), 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. The virulence of parasite stocks was maintained by regular passage into AJ mice (purchased from The Jackson Laboratory, Bar Harbor, ME, USA) by serial passage. Blood-stage trypomastigotes were inoculated into monolayers of H9C2 cells, and the tissue culture trypomastigotes were grown to infect mice in this study.
Mice and experimental infection.Wild-type (WT) and mi-R155−/− C57BL/6 mice were purchased from The Jackson Laboratory (miR-155 stock number 07745, BL/6 stock number 000664; Bar Harbor, ME, USA). The Ethical Committee for Animal Research of Ohio State University approved the animal protocols. The animals were maintained according to the rules and regulations of the University Laboratory Animal Resources (ULAR). All experiments were performed in 6- to 8-week-old mice. Mice were infected with 105 trypomastigotes of T. cruzi in 100 μl of saline solution by intraperitoneal inoculation. Clinical parameters of infected mice were closely monitored daily to determine the overall survival. All mice were harvested at 40 days postinfection when the miR-155−/− mice were terminally ill.
Measurement of parasitic load.The blood was collected by cardiac puncture at the time of harvest after 40 days of T. cruzi infection, and blood smears were prepared from each group of the mice (n = 5) of WT and miR-155−/−, and slides were stained by Giemsa. The parasitic load was calculated by counting the number of parasites in 200 high-power immersion fields, and the parasitic numbers were enumerated by calculating the average of total count divided by total fields analyzed.
Flow cytometry.Splenic cells from WT and miR-155−/− mice were used to evaluate the immune cell types. Cell suspensions were prepared according to the previously described protocol (17). In brief, spleens from all mice were removed and minced to make a single-cell suspension after lysis of red blood cells using ACK lysis buffer. For immunostaining, the cells were stained with CD11b, Ly6C, and Ly6G. Cells were acquired through a fluorescence-activated cell sorter (BD Bioscience, San Jose, CA, USA), and the data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).
Histology.Mice were euthanized at 40 days postinfection, and hearts were removed and fixed in 10% buffered formalin. Tissue sections (5 μM) were stained with hematoxylin and eosin and examined under a light microscope for parasite clumps. Images were taken using a Leica DMi1 with the Leica Application Suite (LAS) v4.12 software.
Cytokine assay.Blood was collected from T. cruzi-infected WT and miR-155−/− mice at the time of harvest after 40 days of infection. Cytokines IFN-γ, TNF-α, IL-4, and IL-13 were analyzed by enzyme-linked immunosorbent assay (ELISA) as previously described (17). The capture and detection antibodies were purchased from BioLegend (San Diego, CA, USA). The recombinant mouse cytokine standards were purchased from BD Bioscience (San Jose, CA, USA). The cytokine concentrations were measured by using a SpectraMax microplate reader and Softmax Pro Software (Molecular Devices LLC, Sunnyvale, CA, USA).
Total DNA preparation and PCR amplification.Total genomic DNA was extracted from spleen tissue of WT and miR-155−/− mice after 40 days of infection using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instruction. PCR amplifications were performed using Ranger DNA polymerase (Bioline, UK) with each 20-μl reaction mixture. The primers used for the PCR amplification were TcZ1 (5′-CGAGCTCTTGCCCACACGGGTGCT-3′) and TcZ2 (5′-CCTCCAAGCAGCGGATAGTTCAGG-3′). The amplification was conducted in a thermocycler with the following conditions: initial denaturation at 95°C for 3 min; 35 cycles at 95°C for 30 s, 52°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min.
Statistical analysis.Statistical significance was calculated using a Student's t test to determine statistical significance of differences among the groups. All results are expressed as mean ± standard deviation (SD). All statistical analyses were performed in GraphPad Prism software. A P value of <0.05 was considered significant and is indicated with an asterisk.
ACKNOWLEDGMENTS
B.K.J., S.V., G.R.S., and G.V. performed experiments and analyzed data. B.K.J. and S.V. wrote and edited the manuscript. A.R.S. designed experiments and analyzed data. B.S.M. designed experiments, analyzed data, and edited the manuscript.
The work in this paper was supported by grants from the NIH (R21AI131227 to B.S.M.). Work in the Satoskar lab is supported by grants from the National Institutes of Health.
We have no conflicts of interest to declare.
FOOTNOTES
- Received 17 December 2019.
- Returned for modification 27 January 2020.
- Accepted 13 April 2020.
- Accepted manuscript posted online 20 April 2020.
- Copyright © 2020 American Society for Microbiology.
