ABSTRACT
Infections with Trichuris trichiura are among the most common causes of intestinal parasitism in children worldwide, and the diagnosis is based on microscopic egg identification in the chronic phase of the infection. During parasitism, the adult worm of the trichurid nematode maintains its anterior region inserted in the intestinal mucosa, which causes serious damage and which may open access for gut microorganisms through the intestinal tissue. The immune-regulatory processes taking place during the evolution of the chronic infection are still not completely understood. By use of the Swiss Webster outbred mouse model, mice were infected with 200 eggs, and tolerance to the establishment of a chronic Trichuris muris infection was induced by the administration of a short pulse of dexamethasone during nematode early larval development. The infected mice presented weight loss, anemia, an imbalance of the microbiota, and intense immunological cell infiltration in the large intestine. It was found that mice have a mixed Th1/Th2/Th17 response, with differences being found among the different anatomical locations. After 45 days of infection, the parasitism induced changes in the microbiota composition and bacterial invasion of the large intestine epithelium. In addition, we describe that the excretory-secretory products from the nematode have anti-inflammatory effects on mouse macrophages cultured in vitro, suggesting that T. muris may modulate the immune response at the site of insertion of the worm inside mouse tissue. The data presented in this study suggest that the host immune state at 45 days postinfection with T. muris during the chronic phase of infection is the result of factors derived from the worm as well as alterations to the microbiota and bacterial invasion. Taken together, these results provide new information about the parasite-host-microbiota relationship and open new treatment possibilities.
INTRODUCTION
Infections with soil-transmitted helminths affect more than a quarter of the world’s population, causing morbidity and disability, especially in people living in regions with poor access to water and public health (1). According to a 2013 estimate, whipworms of the species Trichuris trichiura infect 477 million individuals worldwide, with the highest prevalence being in children (2). Trichuriasis, as well as other helminthiases, is present at a high incidence, especially in people with nutritional deficiency (3, 4). Other species of the genus Trichuris with a global distribution are parasites of other mammals, including animals of economic and veterinary interest (5). All trichurid nematodes present a unique strategy for their parasitism, forming an intratissue niche inside the cells of the mammalian large intestine, causing alterations and disruption of the tissue cover (6). In humans, the colitis induced by Trichuris is being studied as a possible major environmental driver of inflammatory bowel disease (7). The specific host-parasite relationship developed for trichurid nematodes was studied before (8–11), but the immune-regulatory processes involved in maintenance of the chronic infection remain unknown.
Trichuris muris has been widely used in experimental mouse models to study host-parasite interactions. In both mice and humans, Trichuris eggs hatch in the small intestine, and the larvae then migrate to the large intestine, invading the intestinal mucosa (8). The nematode grows and moves inside the epithelial layer, with the anterior end remaining inserted in the host tissue and the posterior region remaining free in the lumen, causing damage to the intestinal epithelium (12). Once they are fixed, Trichuris worms cause inflammation at the site of intracellular attachment and induce a colitis-like pathology (13).
A distinctive morphological characteristic of the Trichuris genus is the presence of a bacillary band in the anterior region of the body, which is formed by bacillary glands, stichocytes, and cuticular inflations and which is hypothesized to play a role in parasite invasion and maintenance in the host tissue (14, 15). The parasite’s anterior end excretes and secretes products (excretory-secretory [ES] products) that can be collected from the media of in vitro cultures and that have been described to be factors governing host-parasite interactions (16). Considering the particular niche of Trichuris, which grows inside the intestinal epithelium, it seems reasonable to expect that the ES products from this nematode may have an important role in parasitism. Previous works identified several proteins and microRNAs secreted by T. muris (17), and proteins isolated from ES products were also tested as a vaccine, with promising results (18); however, the effects on the immune response have not been fully elucidated.
It was shown that T. muris infection affects the composition of the gut microbiota, decreasing the diversity of bacterial communities and changing the abundance of some particular genera (19, 20). On the other hand, contact between commensal bacteria and T. muris eggs is required for hatching of the eggs (21). During a Trichuris infection, depletion of the microbiota with antibiotic treatment leads to the establishment of a chronic infection, indicating that intestinal bacteria are associated with the worm establishment inside the intestine (21, 22). However, the influence of the intestinal microbiota in the regulation of the host immune response and in the pathophysiological evolution of the infection remains to be established.
The pathophysiology of trichuriasis is complex, and its development depends on host genetic characteristics, nematode mechanical and biochemical actions, ES product activity, and changes to the microbiota, with all these different factors having effects on the host immune response. Two major immune responses were characterized in laboratory mice, and these depended on the inbred mouse strain: a Th2-type immune response, which leads to parasite expulsion, and a Th1-type response, which is associated with the establishment of a chronic infection (8). Although the Th2-type response is associated with host protection and Th1 responses are associated with susceptibility to infection, the immunological balance in a chronic Trichuris infection was only barely explored (18, 23). An outbred mouse strain was used as an experimental model in this study, and tolerance to the establishment of a chronic infection was induced by the administration of a short pulse of dexamethasone during early larval development of the nematode in an attempt to mimic the conditions of populations in which the individuals have a nonuniform genetic background and an immune profile state that allows infection.
The present study characterizes the alterations promoted by T. muris in the epithelial layers of the large intestine as well as the local and systemic immune responses at a specific time point in the initial chronic phase of infection, which was at 45 days postinfection. It was found that mice have a mixed Th1/Th2/Th17 response, with differences being seen among the different anatomical locations. It was shown that chronic infection induces changes in the microbiota composition and bacterial invasion of the large intestine submucosa. This study also found that ES products have anti-inflammatory effects on mouse macrophages, suggesting that T. muris may modulate the immune response at the site of insertion in mouse tissue. It is proposed here that the host’s immune status observed during chronic T. muris infection is the result of factors from the nematode as well as changes in the microbiota composition and bacterial invasion. These novel data can contribute to the development of new guidelines for treatments with anthelmintics and antibiotics as well as the administration of prebiotics and/or probiotics for modulation of the microbiota.
RESULTS
Chronic T. muris infection causes a reduction in body weight and alterations in abdominal organs.Initially, the impact of chronic T. muris infection was observed in mice when the animals’ health status was evaluated after 45 days of infection. The infected mice started to eliminate eggs on the 35th day after infection, and at that time, remarkable hair loss was observed in the infected animals. The infection reduced the mouse body weight, with the average weight being 36.53 ± 3.22 g for the control mice and 33.17 ± 5.29 g for the infected mice. During necropsy, it was possible to identify macroscopic alterations in the abdominal cavity, such as greater viscera adhesion and cecal hypertrophy and rigidity. An average of 82.5 ± 7.14 nematodes was recovered from each infected animal.
T. muris infection promotes rupture and disorganization of the intestinal epithelium, accompanied by immune cell infiltration.Histopathological analysis allowed identification of the normal aspect of the three layers of the large intestine in the control animals and structural alterations in the tissues of the chronically infected mice, with the anterior region of T. muris being found to be inserted in the mucosa of the host intestinal epithelium of the infected mice (Fig. 1A). In the control mice, the mucosa muscular layer was very thin and almost imperceptible. However, in the infected mice, it was possible to visualize that layer very clearly (Fig. 1A, right). Morphometric analyses showed a significant thickening of the mucosa (466 ± 143 μm), submucosa (327 ± 127 μm), and muscularis (169 ± 69 μm) layers in the infected mice compared with the thickness of the mucosa (239 ± 89 μm), submucosa (83 ± 19 μm), and muscularis (71 ± 23 μm) layers of the control animals (Fig. 1B).
Histological section of large intestine (cecum) stained with hematoxylin and eosin, showing the morphology and morphometry of the large intestine layers. (A) (Left) Section from control animals showing the integrity of the mucosa, submucosa, and muscularis layers. (Right) Section from infected mice showing the nematode T. muris inserted in the mucosa and submucosa with infiltration. Bars, 100 μm. (Insets) PAS staining showing goblet cells in control and infected mucosal tissue. Bars, 50 μm. (B) Graph representing the morphometry results for the mucosa, submucosa, and muscularis of control and infected mouse large intestine. Data show the mean ± SEM. (C) Graph showing goblet cell and enterocyte quantification in the mucosa. Data show the mean ± SEM. Significant differences in the results between the groups for the results shown in panels B (n = 5) and C (n = 12) were determined by the Mann-Whitney test. *, P ≤ 0.05. Abbreviations: Mu, mucosa; Mm, muscularis mucosa; Sm, submucosa; M, muscularis; bv, blood vessel; N, nematode; gc, goblet cells; e, enterocytes.
Morphological characterization of the cells found in the intestinal epithelium showed that the thickness alteration observed in chronically infected mice was promoted by changes in the volume and number of each infected mouse’s own tissue cells and by inflammatory cell infiltration. In the mucosal layer of the infected mice, an increase in the number and volume of goblet cells compared with the number and volume of goblet cells in the controls was observed (Fig. 1A, insets for control and infected mice, and Fig. 1C). The histopathological images enabled the identification of a polymorphonuclear cell infiltrate in the intestinal sections of the infected mice (Fig. 2A), contributing to the increase in the height of the mucosal and submucosal layers. Quantification of the stained cells in the submucosa showed that the amounts of neutrophils, eosinophils, macrophages, and lymphocytes were significantly higher in the infected mice than in the control animals (Fig. 2B).
Histological section of large intestine (cecum) stained with Giemsa, showing the cell morphology present in the mucosa and submucosa. (A) (Left) Control tissue without cellular infiltrate. The mucosa and submucosa have rare cells inside. (Right) In infected tissue, it is possible to identify and quantify the cells present in the mucosa and submucosa forming a cellular infiltrate. Bars, 20 μm. (B) Graph showing the results of identification and quantification of the cells present in the submucosa of control and infected animals. Data show the mean ± SEM. Significant difference between the groups were determined by the Mann-Whitney test. *, P ≤ 0.05 (n = 5). Abbreviations: Mu, mucosa; Sm, submucosa; E, eosinophil; N, neutrophil; L, lymphocyte; Mϕ, macrophage.
Hematological and biochemical alterations promoted by chronic T. muris infection.Subsequently, the hematological parameters for control and infected mice were analyzed. The infected mouse group showed a significant increase in the leucocyte (white blood cell [WBC]) numbers, together with a decrease in the glucose concentrations in the blood, compared with the findings for the control group (Table 1). In T. muris-infected mice, significant decreases in the number of red blood cells (RBCs) and hematocrit (Hct) were also detected, and these decreases were accompanied by an increase in the mean corpuscular hemoglobin concentration (MCHC) compared with that in the noninfected mice (Table 1). Significant differences in the lipid parameters measured in the serum of infected and control mice were not observed (Table 1), indicating that the nematode infection does not change lipid metabolism or absorption.
Hematological and biochemical parameters of control mice and mice infected by T. muris
Chronic T. muris infection induces an increase in the numbers of CD11b+ cells in PPs and MLNs.It was further analyzed whether the chronic nematode infection can induce alterations in Peyer’s patches (PPs), mesenteric lymph nodes (MLNs), and spleen (SPL). Infected animals presented an increase in the mass of MLNs and SPL (Fig. 3A), whereas a significant increase in the number of cells was observed only in MLNs (Fig. 3B). These results indicate that T. muris infection induces hypertrophy and hyperplasia of MLNs and hypertrophy of the SPL. No differences in the weights of or total cell number in PPs were identified between the control and the infected mice (Fig. 3A and B).
Differences in the mass and cellularity in immune organs from control and infected mice. (A) Mass of the mesenteric lymph nodes (MLNs), Peyer`s patches (PPs), and spleen (SPL) isolated from control and T. muris-infected mice. (B) Number of total cells in MLNs, PPs, and SPL isolated from control and infected mice obtained by flow cytometry analysis. Significant differences between the groups were determined by the Mann-Whitney test. *, P ≤ 0.05 (n = 8).
The MLN and PP phenotyping revealed no statistically significant differences in the percentage of CD3+ lymphocytes (Fig. 4), but an increase in the percentage of CD11b+ cells in infected mice compared with that in control mice was found, indicating that the chronic infection increased the percentage of natural killer, monocytes, macrophages, and/or neutrophils in these organs (Fig. 5A and B), and more details were exhibited by flow cytometry (Fig. 6).
Flow cytometry analysis (dot plot [left] and histograms [right]) of CD3+ cells (fluorescence [FL2] height) from the Peyer’s patches of control mice (A), Peyer’s patches of infected mice (B), mesenteric lymph nodes of control mice (C), and mesenteric lymph nodes of infected mice (D). The graphs are representative of those from 8 independent experiments. SSC, side scatter; FSC, forward scatter.
Differences in CD11b+ cell percentages in mesenteric lymph nodes (MLNs) and Peyer`s patches (PPs) from control and infected mice. (A) Percentage of CD11b+ cells in MLNs isolated from control and infected mice. (B) Percentage of CD11b+ cells in PPs isolated from control and infected mice. Significant differences between the groups were determined by the Mann-Whitney test. *, P ≤ 0.05 (n = 8).
Flow cytometry analysis (dot plot [left] and histograms [right]) of CD11b+ cells (fluorescence [FL1] height) from Peyer’s patches of control mice (A), Peyer’s patches of infected mice (B), mesenteric lymph nodes of control mice (C), and mesenteric lymph node of infected mice (D). The graphs are representative of those from 8 independent experiments.
T. muris-infected mice exhibited higher levels of Th1, Th2, and Th17 cytokines in mesenteric lymph nodes and serum but not in large intestine tissue than control mice.To better characterize the immune response in a chronic T. muris infection, the levels of Th1, Th2, and Th17 cytokines were measured in three different anatomical locations of the control and the infected mice. Both the MLNs and serum of infected mice presented a significant increase in the levels of the Th1-type cytokines gamma interferon (IFN-γ), interleukin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNF-α) and the Th2-type cytokines IL-10 and IL-4 as well as IL-17 compared with those seen in the MLNs and serum of the control animals (Fig. 7A and B). The level of IL-2 was augmented in MLNs and serum, but the increase was statistically significant only in MLNs (Fig. 7A and B). However, an increase in IFN-γ levels was observed only in homogenates of the large intestines of the infected mice and not in those of the noninfected mice, with no differences in the levels of the other cytokines tested being seen (Fig. 7C).
Cytokine secretion in mesenteric lymph nodes, serum, and large intestine of control and T. muris-infected mice. After isolation and homogenization, the amounts of cytokines in mesenteric the lymph nodes (A), serum (B), and large intestine fragment (C) were measured using an ELISA kit. Significant differences between the groups were determined by the Mann-Whitney test. *, P ≤ 0.05 (n = 8).
To check the activation state of the immune cells in chronically infected mice, leukocytes were isolated from the mouse peritoneal cavity. The number of total cells isolated from the infected mice was significantly higher than the number isolated from the uninfected mice (Fig. 8A). The isolated intraperitoneal immune cells were then allowed to adhere in a culture dish, and the majority of these were peritoneal macrophages. The lipopolysaccharide (LPS) susceptibilities of the cells isolated from both groups of animals were tested. As observed in Fig. 8B, the intraperitoneal macrophages isolated from infected mice released significantly more nitrite (NO2−) in the extracellular medium when they were stimulated with 1, 10, or 100 μg/ml LPS than cells obtained from the control animals.
Measurement of the total number of peritoneal cells and NO2− production by macrophages of mice infected or not with T. muris. (A) Peritoneal cells from control and infected mice were quantified using a Neubauer chamber. (B) Cultures of peritoneal macrophages from mice infected or not with T. muris were maintained under culture conditions for 3 to 5 days and stimulated with LPS (1, 10, or 100 μg/ml) for 24 h. The supernatant was collected and analyzed for the NO2− concentration using the Greiss method. The nitrite concentration was related to the amount of protein in each well, obtained by the Bradford assay after cell lysis. Data show the mean ± SEM from four independent experiments with four mice per group in each experiment. Significant differences between the groups were determined by the Mann-Whitney test. *, P < 0.05.
Excretory-secretory products derived from T. muris have anti-inflammatory properties.The bacillary band of T. muris is located in the anterior part of the body, specifically, in the region inserted into host tissue during infection. Because this particularity promotes the direct contact of the parasite bacillary glands with the host tissue cells and the inflammatory infiltrate, it was wondered whether the ES products derived from these glands could have anti-inflammatory properties and maybe explain the low cytokine levels detected in the large intestine. To evaluate the effect of ES products from T. muris, RAW 264.7 macrophages were preincubated with ES products (10 μg/ml) and subsequently treated with LPS. After that, the amounts of nitric oxide (NO) and two proinflammatory cytokines, IL-1β and TNF-α, released into the culture supernatants were measured. As it is possible to see in Fig. 9A, pretreatment with the ES products from T. muris was able to inhibit NO2− production by macrophages treated with LPS (5 and 10 ng/ml). Accordingly, the release of IL-1β and TNF-α, triggered by LPS (100 ng/ml) plus ATP (3 mM), was also inhibited by the pretreatment with the ES products (Fig. 9B and C).
Effect of ES products treatment on NO2−, IL-1β, and TNF-α production in RAW 264.7 macrophages stimulated with LPS. (A) RAW 264.7 macrophages were treated with ES products (10 μg/ml) for 24 h and then stimulated with LPS (5 or 10 ng/ml) for more 24 h. The amount of NO2− in the supernatant was measured using the Greiss method, and the concentration was related to the amount of proteins. (B and C) RAW 264.7 macrophages were treated with ES products (10 μg/ml) for 24 h and then stimulated with LPS (100 ng/ml) for 6 h and ATP (3 mM) for an additional 2 h. The IL-1β (B) or TNF-α (C) concentration in the supernatant was measured using the appropriate ELISA kit, and the concentration was related to the amount of proteins. Data show the mean ± SEM from four independent experiments. Significant differences between the groups were determined by the Mann-Whitney test. *, P < 0.05 (n = 5).
To rule out the possibility that the inhibition of NO and cytokine release observed after pretreatment with the T. muris ES product was due to any cytotoxic effect, a viability assay was performed with RAW 264.7 macrophages maintained in culture together with ES products (10 μg/ml) for 24 and 48 h. As expected, the presence of ES products in the culture medium did not have an impact on macrophage viability (Fig. 10).
Effect of T. muris ES products on the viability of RAW 264.7 cells. Cells were exposed to ES products (10 μg/ml) for 24 h (A) and 48 h (B), and then cell viability was measured using the MTT assay. Untreated macrophages (culture medium) were considered 100% viable, and lysed macrophages were used as a positive toxicity control. The percentage of macrophages in culture medium plus T. muris ES products (10 μg/ml) was expressed in relation to the value for the control (100%). Data show the mean ± SEM from three independent experiments.
T. muris infection modifies the gut microbiota and promotes bacterial invasion of the intestinal epithelium.As it was previously shown that T. muris infection affects the gut microbiota (19, 20), it was decided to study the bacterial composition in the intestines of mice chronically infected with this nematode. Analysis of fecal cultures of infected and noninfected mice showed a significant increase in the quantity of Escherichia coli and members of the Proteus genus in infected animals compared with that in control animals (Table 2). In addition, T. muris-infected mice exhibited large amounts of Bacteroides in their feces, but this genus could not be detected in the feces of the noninfected animals (Table 2). An increase in total anaerobes was observed in T. muris-infected mice compared with uninfected mice; however, the difference was not significant.
Analysis of bacteria in feces by biochemical tests
When the identification of the bacterial species from infected and noninfected mouse feces was performed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry, similar but broad results were obtained for the two groups of mice. The bacterial species Enterococcus faecalis, Enterococcus faecium, and Enterococcus gallinarum were identified only in the infected animals. It was not possible to detect members of this bacterial genus in the uninfected animals. The presence of the bacterial species E. coli and Proteus mirabilis in both the infected and noninfected groups was also confirmed by MALDI-TOF mass spectrometry (Table 3).
Identification of bacterial species in feces using MALDI-TOF mass spectrometry
Further histological examination of the intestinal mucosa showed that in samples from the control group of mice, bacteria were located only in crypts of Lieberkühn, restricted on the epithelium surface, as expected (Fig. 11A). However, when infected tissues were observed, it was possible to identify bacteria not only in the crypts of Lieberkühn but also inside the submucosal layer (Fig. 11A, bottom). Using Giemsa stain, the presence of cocci and bacilli was detected in the large intestine submucosa obtained from the infected animals (Fig. 11B and C).
Histological sections of large intestine (cecum) stained with Giemsa for analyses of bacterial invasion in the submucosa layer. (A) (Top) Control tissue with bacteria in the intestine lumen and inside the crypt of Lieberkühn (arrowhead). (Inset) Detail of the control tissue showing bacilli in the crypt of Lieberkühn (arrowhead). (Bottom) Infected tissue with bacteria inside the crypt of Lieberkühn (arrowhead) and invasive bacteria (arrow) in the submucosa layer. (Inset) Detail of the infected tissue showing bacilli in the crypt of Lieberkühn (arrowhead). Bars, 50 μm. Inset bars, 10 μm. (B) Submucosa with invasive coccus-shaped bacteria, lymphocytes, and macrophages. Bar, 10 μm. (C) A large number of the invasive bacilliform bacteria in the submucosa, neutrophils, and eosinophils were also identified. Bar, 10 μm. The data in panels B and C are representative of those from 5 experiments. Abbreviations: c, coccus-shaped bacteria; b, bacilliform bacteria; Mu, mucosa; Sm, submucosa; bv, blood vessel; M, muscularis.
Two different experiments using fluorescence microscopy were performed to obtain evidence of the presence of bacteria. In the first one, intestinal slices from the control and infected mice were stained with DAPI (4′,6-diamidino-2-phenylindole), and it was possible to observe a large number of bacilli near the anterior nematode region (Fig. 12A and B), suggesting that the epithelial damage caused by the nematode promotes bacterial invasion. The invasive process was confirmed by fluorescent in situ hybridization (FISH) experiments, where it was observed that, in the cecum of mice from the control group, the bacteria (green) were present only in the mucosa layer and were localized in the crypts of Lieberkühn (Fig. 12C and D), whereas in samples from infected mice, the bacteria were identified inside the submucosa (Fig. 12E and F).
Fluorescence microscopy, using DAPI and fluorescence in situ hybridization (FISH), of sections of control and infected tissues showing bacterial invasion. (A and B) DAPI experiments showing the anterior region of the nematode inserted in the intestinal tissue and bacilliform bacteria (arrows) close to the nematode cuticle. In panel B, it is possible to identify the bacteria (arrows) and the bacillary band of the T. muris nematode. (C and D) FISH experiments showing the three layers of the uninfected cecum tissue and the bacteria (green) present only in the mucosa. (E and F) FISH experiments showing infected tissue invaded by bacteria (green) in the submucosa (arrows). (D and F) Merged figures showing bacteria (green) and host tissue (DAPI). Bars, 10 μm (A and B) and 50 μm (C to F). The images are representative of those from 5 experiments. Abbreviations: N, nematode; c, cuticle; Bb, bacillary band; Mu, mucosa; Sm, submucosa; M, muscularis.
DISCUSSION
Whipworms show an unusual biological niche, living for long periods inside the host’s intestinal epithelium, often without causing an excessive pathology. The intimate contact of the nematode with host tissues and microbiota requires specific adaptations, such as the secretion of immunomodulatory factors by the parasite. While most of the pathological studies have examined the acute immune response to T. muris and the balance between resistance and susceptibility to infection (8, 12, 24–26), only recently a growing number of works have analyzed the animals’ immunopathological processes during chronic infection (18–20, 27–30). Normally, outbred mouse strains show similar proportions of susceptible individuals and individuals resistant to infection. Outbred strains display a genetic variability that is closer to conditions in nature than that seen for inbred strains. Both humans and wild-type animals are susceptible to infection, with individual immunosuppressive conditions favoring susceptibility and the establishment of a chronic infection (31–33). It was decided to use an outbred mouse strain with induced tolerance to infection because it was considered that such a model must approximate the situation in the human colon during chronic trichuriasis (34).
A significant thickening was detected in the three layers of the large intestine in mice chronically infected with T. muris. Hyperplasia and hypertrophy of goblet cells were also observed in the infected animals compared with the findings for the control group. These results are in agreement with the data shown by others (35), obtained using an experimental model of C57BL/6 mice infected with Nippostrongylus brasiliensis. In this model, the infected animals also showed intestinal goblet cell hyperplasia (35), and recently, the epithelial hyperplasia observed in C57BL/6 mice chronically infected with T. muris was associated with cancer development (20). On the other hand, other groups did not find differences in the numbers of goblet cells or alterations in the thickening of the large intestine layers in C57BL/6 mice infected by T. muris at 35 days compared with the findings for control mice (36). The dissimilar findings may reside in the distinct genetic backgrounds of the mouse strains or infection times, because the analysis in this study was conducted on day 45, while the study of Ramanan and coworkers (36) was conducted on day 35.
Protein malnutrition is a clinical condition reported in infections caused by soil-transmitted helminths, including trichuriasis (37). In this work, no differences in the quantities of enterocytes between the control and the infected mice were observed; however, histopathological studies showed that malnourished children with persistent diarrhea present a positive relationship between the degree of malnutrition and the decrease in the size of enterocytes and their nuclei (38). The impact of trichuriasis on enterocyte biology is an important issue in human and veterinary health that remains to be revealed.
The parasitism lesions developed by T. muris in this experimental model promoted an infiltrate in the submucosa with increased number of neutrophils, eosinophils, macrophages, and lymphocytes. Eosinophilic infiltration was also described in biopsy specimens from patients with trichuriasis in South Korea (39). A similar mucosal cell infiltrate was registered in the C57BL/6 mouse model of chronic T. muris infection (20), and a large influx of macrophages into the large intestine of infected BALB/c and AKR mice was also reported (24).
It was shown that the chronic phase of T. muris infection produces alterations in hematological and biochemical parameters. One remarkable alteration in hematological values was the significant increase in the numbers of the leukocytes in infected mice, which probably indicates an immunological response to the tissue damage and bacterial invasion (see below). A significant reduction in the number of red blood cells as well as in the hematocrit percentage was observed, as was a slight, but not significant, decrease in the iron content in the blood of the infected animals, which may indicate iron deficiency anemia. Similar results were obtained in humans infected with T. trichiura (40, 41), and a study showed that C57BL/6 mice infected with a low dose of T. muris eggs did not have anemia (28), suggesting that the parasitic load directly impacts the hematocrit. These results show that outbred animals, such as Swiss Webster have a heterogeneous response more similar to that seen in human infections than that seen in infections in isogenic mice.
The weight loss observed in the infected animals in the present study may have been related to the glucose deficiency in the blood. Previous in vitro experiments showed that the T. muris bacillary band, the nematode region inserted into the host tissue during parasitism, accumulates and absorbs glucose (15). In addition, our results showed that the infection promotes bacterial invasion and a significant increase in the numbers of anaerobes and E. coli bacteria in the intestine. It seems possible that alterations in the microbiota also modify carbohydrate metabolism, as was described before in the case of pigs infected with T. suis (9).
A significant hypertrophy of the MLNs and SPL of mice chronically infected with T. muris was observed; however, hyperplasia was detected only in the MLNs, the lymph nodes draining the large intestine. Greater numbers of macrophages were observed in the mucosa of resistant mouse strains than in the mucosa of susceptible strains (24), suggesting a key role for these cells in infection. Phenotypic characterization showed a higher proportion of CD11b+ cells in the MLNs and PPs, indicating that monocytes, neutrophils, natural killer cells, granulocytes, and/or macrophages migrate to regions close to the parasite location as a consequence of the infection. Increased numbers of CD11b+ cells in MLNs were also described before in a C57BL/6 mouse model of T. muris infection (26). PPs and MLNs are the two main sites where immune responses against intestinal pathogens can be initiated; however, data about T. muris infection are still limited at present.
The type of immune response generated against T. muris during the first days of infection determines the balance between susceptibility and resistance to infection. A Th2-type response is associated with parasite expulsion, whereas a Th1-type response is associated with the establishment of a chronic infection (12). However, characterization of the immune response using murine models with 45 days of infection is rare, despite the fact this condition could be closer to the situation in humans with long periods of parasitism. In the study described here, the amounts of responses of the Th1, Th2, and Th17 types were increased in the MLNs, PPs, and serum of infected mice compared with those of the uninfected animals, compatible with a mixed immune response. In the large intestine, only the levels of IFN-γ in infected mice were significantly higher than those in the control mice, with no differences in the amounts of the other cytokines measured being seen. The depletion of IFN-γ in susceptible AKR mice resulted in expulsion of the parasite (23), suggesting an important role of this Th1 cytokine in the maintenance of a chronic infection. The treatment of normally resistant BALB/K mice with an anti-IL-4 receptor antibody resulted in a chronic T. muris infection (23), showing that IL-4 participates in parasite expulsion. Under the conditions used in this study, the levels of IL-4 remained low in the large intestine and IFN-γ was the only Th1 cytokine present at significantly larger amounts in the large intestine of infected mice than in that of the control mice, maintaining an environment where the parasite could be detected in the host. The levels of IFN-γ in cultures of MLNs from AKR mice chronically infected with T. muris were higher than those in uninfected mice (27) and were also higher than the levels produced by the same cell type isolated from resistant BALB/c mice (26), showing that the level of this Th1-type cytokine is elevated during the development of infection, in agreement with our results. CD4+ T cells from the colons of T. muris-infected C57BL/6 mice displayed an increase in IFN-γ production when they were isolated and stimulated with phorbol myristate acetate and ionomycin in the culture compared with that for cells from noninfected mice (18, 36). Also using C57BL/6 mice, Chenery and coworkers showed that a chronic infection with T. muris causes IFN-γ-expressing T-cell accumulation in the bone marrow, demonstrating a Th1 cell-driven response to infection (28). However, up to now there have been no records of studies covering the several cytokine classes that were measured in this work at 45 days of chronic T. muris infection in three different tissues. In schistosomiasis and filariasis, it was observed in individuals with a chronic pathology that the parasite causes a damaging immunopathology, with high levels of Th1-, Th2-, and Th17-type cytokines being found in serum (42). Reinfection with Fasciola hepatica potentiates a mixed Th1/Th2/Th17/regulatory T cell response in Wistar rats (43). A mixed Th1/Th2 cytokine response was also observed in mice treated with Taenia solium metacestode antigens (44). Intestinal gene expression in pigs infected with T. suis showed an increased expression of genes for the Th1-type cytokines IL-1α and IL-6, as well as genes involved in the release of the Th2-type cytokine IL-13 (45). Besides, it seems logical to think that the immune state of the whole individual after 45 days of infection is a mix of not only the immune response elicited by the nematode parasite antigens but also the immune response induced by the bacterial invasion resulting from rupture of the intestinal epithelial barrier as well as the inflammation that occurs as a consequent of the tissue damage itself.
Surprisingly, the amounts of cytokines in the large intestine showed no difference between infected and control mice (with the exception of IFN-γ), despite the fact that a massive infiltrate with large amounts of macrophages in the submucosa was observed. This fact leads us to wonder about the macrophage activation state in the infected mice. It was found that the number of intraperitoneal cells, mainly monocytes and neutrophils, recruited during infection was significantly higher in the infected group than in the control group, suggesting peritonitis. In addition, the differentiated macrophages isolated from infected mice were more responsive to LPS treatment than those isolated from uninfected mice, indicating that these cells are in an activated state.
It has been known for a long time that helminths secret products that can interact with the host, and immunomodulatory activity has been found in the ES products of several species of parasitic worms (16). Recently, two different groups characterized the ES products of T. muris and identified more than 100 proteins and 20 microRNAs (17, 46). Little is known about the effects of ES products from T. muris on immune cells. D’Elia and Else showed that the ES products from T. muris stimulate the secretion of IL-10 and IL-6 from bone marrow-derived macrophages and dendritic cells (47). It was shown here, for the first time, that the ES products of this nematode inhibit the inflammatory response of macrophages stimulated with bacterial LPS. This could be the explanation for the low levels of cytokines found in the large intestine, where the worm is in close contact with the host and the ES products can have potent immunomodulatory effects.
The intestinal microbiota contributes to nutrient digestion and absorption and also modulates the individual immune response. Trichuriasis can promote an imbalance in these functions, since the parasite can affect the microbiota composition. Swine infected with T. suis present changes in their microbiota (22), and this could be related to the decrease in carbohydrate metabolism and amino acid biosynthesis observed in parasitized animals (9). It was previously shown that T. muris infection decreases the diversity of the microbiota, increasing the abundance of members of the Lactobacillus genus (19), and that the alterations in the microbiota composition can be reversed after clearance of the infection (20). In agreement with these findings, in the present study, it was shown that T. muris infection promotes a significant increase in total anaerobes, with a dramatic increase in the numbers of Escherichia coli bacteria and Bacteroides spp. MALDI-TOF mass spectrometry confirmed the results of the biochemical tests for E. coli and Proteus and identified three different species of Enterococcus. A similar increase in the abundance of Bacteroides spp. was also described in C57BL/6 mice chronically infected with T. muris (19, 20) and in pigs infected with T. suis (9). In a report describing findings different from those described above, Ramanan and coworkers reported that chronic T. muris infection inhibits Bacteroides vulgatus colonization and promotes the establishment of a protective microbiota enriched in the Clostridiales (36). In the same work, the authors described no differences in corporal weight or morphological alterations between the intestines of infected C57BL/6 mice and those of uninfected mice (36). A finding that may explain the discrepancies in the results obtained by these different research groups comes from a recent study that compared the immune response and the microbiota changes induced by chronic infection with T. muris in C57BL/6 mice maintained under controlled laboratory conditions or in an outdoor environment (48). The mice placed in an outdoor environment that was similar to their natural habitat harbored larger worm biomasses than the laboratory mice and also showed decreased Th2-type and increased Th1-type immune responses, with higher levels of inflammatory cytokines being seen in the intestine and MLNs (48). These interesting results indicate that the interactions between the host and the nematode are strongly affected by the genetic background and the environment, the latter of which also affects the diet. Changes in diet can affect the microbiota; in addition, changes in the microbiota can shape the immune response. As a consequence of all these interactions, the immune state during a helminth infection is the result of the cross talk of all these factors in each individual host. In humans, the data related to microbiota changes induced by trichuriasis also conflict. The composition of the microbiota in children from Ecuador infected with T. trichiura did not show significant differences from that in uninfected children (49). On the other hand, an indigenous population in Malaysia infected with helminths showed a reduction in the alpha diversity of their microbial communities after treatment with albendazole, indicating that the parasite probably increases the diversity of the microbiota (50). Once more, it is expected that differences in geographic location, diet, genetic background, age, and gender may lead to different results among studies.
Because the adult worm promotes tissue rupture, forming syncytial tunnels (6), several authors hypothesized that these disruptions in the mucosa may allow the entrance of cecal bacteria (6, 29, 51). In this work, invading bacteria were identified in the large intestine (cecum) submucosa of T. muris-infected mice. These lesions on the intestinal tissues serve as a means of access for the lumen bacteria and may promote aggravation of the pathology. The presence of bacteria inside the host tissues could be responsible for the mixed Th1/Th2/Th17 profile observed in infected mice. It was hypothesized that macrophages in the mucosa and submucosa in close contact with bacteria maintain a slow production of proinflammatory cytokines due to the secretion of products from the nematode. This seems to be an evolutionary strategy to avoid a deleterious inflammatory response that could be harmful to the parasite, as well as to the host. It is important to note that no bacteria were identified in the muscularis layer of the large intestine or in the bloodstream, suggesting that immune cells in the submucosa prevent the spread of these microorganism by the host body. More experiments should be carried out to elucidate the association of these invasive bacteria with the nematode infection process and with the host’s inflammatory state.
The evolution of the mammalian immune system happened in intimate association with the establishment of helminth parasitism in metazoans (52). Complex mechanisms in the relationship among the host immune response, the gut microbiome, and parasite survival strategies are involved. These three players participate in the host health state, and an understanding of this cross talk is fundamental to understand the clinical evolution of this infection and think about new therapeutic strategies. This study contributes, for the first time, new data from a model with an outbred mouse strain. Alterations in the intestinal tissue, changes in the microbiota composition, and a mixed immune response, probably resulting from invasion by intestinal bacteria, together with the effects of the worm and ES products, were described (Fig. 13).
Illustration showing the large intestine (mucosa, submucosa, and muscularis) infected with T. muris (nematodes), excretory-secretory (ES) product release (blue arrows), the bacterial invasion process (red arrows), and the immunological response (interleukins). The infographic is a summary of the results of this study showing the tissue damage caused by the nematodes. The damage promotes bacterial invasion into the tissue and modulation of the immunological response as a consequence of ES product secretion. It is possible to identify an increase in the amounts of interleukins in Peyer’s patches and mesenteric lymph nodes; however, this was not observed for the interleukins dosed directly in the large intestine (tissue).
MATERIALS AND METHODS
Ethics statement.The parasite life cycle was maintained in the Otto Wucherer Helminth Biology Laboratory, and all the animal protocols were approved by the Ethics Committee for Animal Experimentation of the Health Sciences Center of the Federal University of Rio de Janeiro under protocol IBCCF 149, according to Brazilian federal law (Law 11.794/2008, regulated by Decree 6.899/2009), based on the Guide for the Care and Use of Laboratory Animals (53), prepared by the National Research Council of the U.S. National Academy of Sciences, and the Australian Code of Practice for Care and Use of Animals for Scientific Purposes (54). All the animals received humane care in compliance with the above-mentioned guides, used by the Ethics Committee to approve the protocol.
Experimental infections and collection of excretory-secretory products.Eggs from the Trichuris muris Edinburgh strain, provided by Francisco Bolas (Universidad Complutense de Madrid), were washed twice in dechlorinated water and incubated at 28 ± 2°C for 45 days. After this period, the percentage of embryonated eggs was estimated by light microscopy. Two hundred embryonated eggs in 0.3 ml of dechlorinated water were administered orally to 20 laboratory-bred male Swiss Webster mice aged 4 to 6 weeks, and the control group was administered 0.3 ml of dechlorinated water. Infected and control mice were immunomodulated with 50 μl of dexamethasone sodium phosphate (8 mg/ml) and dexamethasone acetate (10 mg/ml) (Duo-Decacron; Aché Laboratory) at 7, 9, and 11 days postinfection. The immunomodulation has an influence only in the first days of infection (drug half-life, 2 h) and was performed in order to establish a parasitic load in the chronically infected mice. After 45 days of infection, the mice were euthanized (in a CO2 chamber) and necropsied, and the different tissues were recovered. The health conditions of the experimental mice were evaluated as described previously (55).
For the collection of T. muris excretory-secretory (ES) products, adult worms were gently removed from the large intestine tissue (cecum) with the aid of a round brush (no. 4) and immediately transferred into and incubated in 24-well plates with serum-free RPMI 1640 medium containing 2 mM l-glutamine (Gibco) along with 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich) at 37°C in 5% CO2. After 24 h, the supernatant was collected and maintained overnight in an endotoxin removal column, according to the manufacturer’s specifications (Thermo Scientific).
Gut histology: morphometric and morphological experiments.Large intestine fragments taken from the cecum region were fixed in 8% formaldehyde at pH 7.4 for 24 h and transferred to 4% formalin. The tissue was dehydrated in a graded ethanol series (30% to absolute), subjected to diafanization with xylene (Merck), and embedded in paraffin (Sigma-Aldrich). Tissue sections (5 μm) were obtained and stained with hematoxylin-eosin (Sigma-Aldrich), Giemsa (Merck), and periodic acid-Schiff (PAS).
Morphometric and morphological analyses were performed using the software Bel View (version 6.2.3.0; Bel Engineering, Monza, Italy), and images were obtained using a Nikon Eclipse 80i microscope equipped with a Nikon DS-Ri1 digital camera. The three cecum layers in five different areas in five animals from each group (the control and the infected groups) were measured at random. For morphological experiments in the submucosa, the cells in three areas of 50 μm2 randomly selected from five animals from each group were quantified and identified. For enterocyte and goblet cell quantification, three crypts of Lieberkühn were randomly selected from four animals from each group and analyzed.
Hematological and biochemical analyses.During the necropsy, cardiac puncture was performed for the collection of blood (0.8 to 1.0 ml) from the infected and the control mice. The blood samples were stored in vials containing EDTA as an anticoagulant for hematological analysis and in microtubes without anticoagulant for biochemical analysis. The samples intended for biochemical analysis were centrifuged at 500 × g for 5 min to obtain serum for the experiments. Altogether, the total leukocyte (WBC) count (in 106 cells per microliter), red blood cell (RBC) count (in 106 cells per microliter), hematocrit (in percent), hemoglobin (HGB) concentration (in grams per deciliter), mean corpuscular volume (MCV; in femtoliters), mean corpuscular hemoglobin (MCH) amount (in picograms), mean corpuscular hemoglobin concentration (MCHC; in grams per deciliter), platelet count (in 106 cells per microliter), iron concentration (in milligrams per deciliter), glucose concentration (in milligrams per deciliter), C-reactive protein concentration (in milligrams per deciliter), total protein concentration (in grams per deciliter), total cholesterol fractions (low-density lipoprotein, high-density lipoprotein, and very-low-density lipoprotein; in milligrams per deciliter), and triglyceride concentration (in milligrams per deciliter) were determined. The samples were evaluated in the Clinical Analyses of Laboratory of Animals platform (RPT12C platform; Fiocruz) and in the Laboratory of Lipids platform (LabLip platform; UERJ), which are certified by the Analytical Quality Control Program of the National Program of Quality Control (PNCQ) of the Brazilian Society of Clinical Analyses (SBAC). For the hematological and biochemical analyses, the samples were analyzed using a Poch 100iV Sysmex system (Kobe, Japan) and J & J Vitros 250 clinical system (Johnson & Johnson, USA), respectively.
Phenotypic analysis.The lymph nodes (LNs), Peyer’s patches (PPs), and spleen (SPL) were removed macroscopically, weighed, and homogenized in RPMI 1640 medium (Gibco) using a cell strainer (BD). Cells were counted using a hemocytometer chamber and maintained om RPMI 1640 plus 10% fetal bovine serum (FBS; Gibco), 100 U/ml of penicillin, and 100 μg/ml of streptomycin until use.
Cells obtained from PPs, SPL, and MLNs were washed twice with phosphate-buffered saline (PBS), blocked for 45 min with 10% horse serum, washed again, and incubated for 1 h with fluorescein isothiocyanate-conjugated antibody against Mac-1 (CD11b; eBioscience, USA) or CD3 (eBioscience, USA). The cells were washed twice in PBS, and at least 10,000 events were obtained for each sample using a FACScan flow cytometer (Becton, Dickinson, USA).
The flow cytometry data were analyzed using the WinMDI program (Multiple Document Interface Flow Cytometry Application, version 2.8; The Scripps Research Institute, La Jolla, CA, USA). The percentage of cells positive for each antigen was applied to the total number of cells in each sample from each mouse to estimate the absolute number of cells of each cell type. The region of living cells was determined using the parameters forward scatter versus side scatter.
Identification of bacteria in intestinal tissue.The samples stained with Giemsa (Merck) and marked with 300 μM DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) were used for the identification of invasive bacteria. For the fluorescence experiment, the tissue sections were permeabilized with Triton X-100 (Sigma-Aldrich), washed in PBS, stained in 300 μM DAPI for 1 min, washed in PBS again, and mounted with 0.5% N-propyl gallate (Sigma-Aldrich). Ten histological sections from five animals in each group were analyzed using light microscopy in the bright-field and epifluorescence (358- and 461-nm excitation and emission, respectively) modes. The images were obtained using a Nikon Eclipse 80i microscope equipped with a Nikon DS-Ri1 digital camera.
For fluorescence in situ hybridization (FISH), the cecum fragments were collected and fixed in 10% buffered formalin for 48 h. Subsequently, this tissue was incubated at 4°C in 10% and 30% sucrose for 24 h, posteriorly included in OCT gel (Tissue-Tek), and frozen in liquid nitrogen. Thin sections (5 μm) were recovered on slides prepared with polylysine (Sigma/Aldrich) on a cryostat (Leica CM1850). FISH was performed on each slide using the conditions and buffers described previously (56), using 30% formamide in the hybridization buffer and probes for eubacteria. After this procedure, the slides were marked with DAPI at a concentration of 0.1 μg/ml for 10 min, and the slide was coated with N-propyl gallate (Sigma/Aldrich) and observed with a Zeiss microscope equipped with an AxioImager AxioCam RMC microscope (Zeiss, Germany).
Macrophage experiments.Peritoneal macrophages were obtained by lavage of the peritoneal cavity with 10 ml of Dulbecco’s modified Eagle medium (DMEM; Gibco, USA). After isolation from the peritoneal cavity, the monocytes-macrophages pass through an adhesion process and need to be maintained in culture for at least 3 days for stabilization (57). The number of total viable cells of mice, infected with T. muris or not, was determined using trypan blue (Sigma-Aldrich) in a Neubauer chamber. The numbers of peritoneal macrophages and cells of the RAW 264.7 macrophage cell line were counted (2 × 105 cells/well), and the cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml of streptomycin, and 2 mM glutamine in 24-well plates.
The production of nitric oxide (NO) was measured by determination of the nitrite (NO2−) concentration in the supernatant using the Griess colorimetric method (58). In brief, peritoneal macrophages from control or infected mice were stimulated with 1 μg/ml of LPS for 24 h, and the amount of NO2− in the supernatants was measured. The RAW 264.7 cell line was treated with ES products (10 μg/ml) for 24 h and was stimulated with LPS (10 ng/ml) for an additional 24 h, and then the NO2− concentration in the supernatants was measured. The absorbance at 550 nm was measured by spectrophotometry, and the values were quantified against a sodium nitrite standard curve. After the experiment, to avoid any variation in cell number, the cells in each well were lysed (the lysis buffer consisted of 0.1% Triton X-100 and a cOmplete protease inhibitor cocktail set [Sigma-Aldrich]), and the protein concentration was measured by the Bradford assay, based on the use of Coomassie brilliant blue G-250, using albumin as a standard (59). The nitrite concentration was related to the amount of proteins as the number of micromolar NO2−/number of micrograms of protein.
Measurement of cytokine concentrations in immune organs, serum, and cell culture supernatants.Blood samples were obtained by cardiac puncture, allowed to clot at room temperature, and centrifuged at 500 × g for 10 min, and serum samples were collected and immediately frozen at −80°C until use. In addition, lymph nodes (LNs), PPs, SPL, and large intestine fragments were also collected and homogenized for cytokine analysis. Each 1 mg of LNs was homogenized in 100 μl PBS, and for large intestine fragments, we used the same weight (1 mg) with 50 μl PBS. The organs and tissues were immersed in ice-cold PBS supplemented with a complete protease inhibitor cocktail set (Sigma-Aldrich), washed, macerated, and passed through a 70-μm-mesh-size cell strainer (BD). After homogenization, the samples were centrifuged at 4°C and 500 × g for 15 min, and the supernatant was removed and immediately frozen at −80°C until use. The quantity of IL-1β in serum, LNs, and large intestine fragment supernatant was measured by use of an enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer’s instructions (Peprotech, USA). Cytokine (IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17A, and IL-10) levels in serum, LNs, and large intestine fragment supernatant were determined using a commercial kit (cytometric bead array [CBA] mouse Th1/Th2/Th17; BD Pharmingen), according to the manufacturer’s instructions. The supernatants were incubated with cytokine-specific antibody-coated microspheres and phycoerythrin (PE)-conjugated detection antibody at room temperature for 2 h with protection from the light. A standard curve was prepared by incubating the microspheres at different concentrations (0 to 5,000 pg/ml). After incubation, 500 μl of wash solution was added to each tube and the tubes were centrifuged at 200 × g for 5 min. The supernatant was discarded, and the pellet was resuspended in 300 μl of wash solution. Data were acquired on a FACSCanto II flow cytometer (BD Bioscience, USA) and analyzed using FCAP Array software (BD Bioscience). The cytokine detection limits were as follows: IL-2, 0.1 pg/ml; IL-4, 0.03 pg/ml; IL-6, 1.4 pg/ml; IFN-γ, 0.5 pg/ml; TNF-α, 0.9 pg/ml; IL-17A, 0.8 pg/ml; and IL-10, 16.8 pg/ml. The effects of ES products on IL-1β production were measured using RAW 264.7 macrophages incubated or not with ES products (10 μg/ml) for 24 h and then treated with 100 ng/ml LPS for 6 h. After this period, ATP (3 mM) was added and the mixture was incubated under the same conditions described above for an additional 2 h. The supernatants were collected, and the quantities of IL-1β and TNF-α were measured by use of an ELISA kit, according to the manufacturer’s instructions, with a limit of detection of 10 pg/ml (Peprotech, USA).
Cell viability assays.Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) assay. RAW 264.7 macrophages (2 × 106 cells/ml) were allowed to adhere to 96-well tissue culture plates for 24 h at 37°C in an atmosphere of 5% CO2. Macrophages were incubated with T. muris ES products (10 μg/ml) in the culture medium for 24 and 48 h. MTT (2.5 mg/ml) was added to the culture medium, and the culture was incubated for 3 h at 37°C. The resulting formazan crystals were solubilized by adding 10% dimethyl sulfoxide (DMSO) after removal of the supernatants. The absorbance of the culture at 570 nm was read using a microplate reader (Bio-Tek, Winooski, VT, USA). Untreated macrophages were lysed by the addition of 0.1% Triton X-100 as a positive toxicity control, and macrophages without any treatment were considered 100% viable.
Fecal cultures.To analyze the effects of the T. muris infection on the gut microbiota, about 0.09 g of feces, equivalent to approximately four fecal pellets, was collected immediately before the necropsy of five control mice and five infected mice. The fecal samples were maintained separately for each host. The animal stool was used for the cultivation of aerobic and anaerobic bacteria. For identification by biochemical tests, fresh stool samples from each animal were placed in tubes and diluted with 3 ml of PBS (for investigation of Gram-negative bacilli and enterococci) or 3 ml of thioglycolate broth (for investigation of total anaerobes and Bacteroides spp.). The samples were agitated on a mechanical shaker, and serial 10-fold dilutions were prepared in PBS or thioglycolate broth. Ten microliters of each dilution was plated in triplicate on the surface of the culture medium. For cultivation of the bacteria, Enterococcosel agar, blood agar, MacConkey agar, and brucella agar (all from Difco, USA) and bacteroides bile-esculin agar (Oxoid, UK) were used. The culture media were incubated at 37°C either under aerobic conditions or under anaerobic conditions using an anaerobic gas-generating kit in an anaerobic jar (Oxoid, UK) for 24 to 48 h. Following incubation, the number of CFU per 1 ml of fecal samples was calculated from the number of CFU per milliliter of the appropriate dilution obtained. Bacterial identification was carried out by biochemical tests. The identification of Enterobacteriaceae from the colonies grown on the MacConkey medium was performed by biochemical tests. The strains were seeded in the following media: double sugar-urea medium (DAU), sulfate-indole-agar motility medium (SIM; Kasvi) plus filter paper tape containing the Kovac reagent, and Simmons citrate medium (Kasvi). After sowing, the media were incubated in an oven at 37°C, and after 24 h, the result was read according to the variation in the color of the medium and turbidity indicating mobility or not.
The bacterial colonies identified by the biochemical tests, as explained above, were isolated on Trypticase soy agar (Oxoid, UK) for analyses using matrix assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry equipment (Microflex LT; Bruker). For bacterial species identification, MALDI Biotyper (version 3.1) software and FlexControl (version 3.4) software were used.
Statistical analyses.Differences among experimental groups in all the experiments were evaluated by the nonparametric Mann-Whitney test, with a P value of ≤0.05 being considered significant. All data were analyzed using GraphPad Prism (version 5) software and GraphPad InStat software (GraphPad, USA).
ACKNOWLEDGMENTS
We are grateful to the staff of the Fiocruz RPT12C platform and the Laboratory of Lipids platform (UERJ; LabLip platform) for hematological and biochemical analyses, Francisco Bolas-Fernandez from the Universidad Complutense de Madrid for the donation of Trichuris muris eggs, Pedro Ernesto Lopes Leão of the Microbiology Microscopy Center, Padrón-Lins-UFRJ, for support with the FISH experiments, and Sergio Fracalanzza and Larissa Alvarenga Batista Botelho from LIMM-UFRJ for mass spectrometry analyses. We thank Arian Carvalho de Alcantara for creation of the illustrations.
This work was supported by individual grants from the Brazilian agencies FAPERJ-JCNE (Productive Fellowship Program, grant 202.660/2018), CNPq, and CAPES for a Ph.D. scholarship and postdoctoral fellowship support.
FOOTNOTES
- Received 15 August 2019.
- Returned for modification 9 September 2019.
- Accepted 6 December 2019.
- Accepted manuscript posted online 16 December 2019.
- Copyright © 2020 American Society for Microbiology.
