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Protein undernutrition reduces the efficacy of praziquantel in a murine model of Schistosoma mansoni infection [1]

['Joseph Bertin Kadji Fassi', 'Laboratory Of Animal Physiology', 'Department Of Animal Biology', 'Physiology', 'Faculty Of Science', 'University Of Yaoundé I', 'Yaoundé', 'Centre For Schistosomiasis', 'Parasitology', 'Hermine Boukeng Jatsa']

Date: 2022-08 

Thirty-day-old mice were fed with a low-protein diet, and 40 days later, they were individually infected with fifty Schistosoma mansoni cercariae. A 28-day-treatment with praziquantel at 100 mg/kg for five consecutive days followed by distilled water begins on the 36 th day post-infection. Mice were sacrificed on the 64 th day post-infection. We determined the parasitological burden, liver and intestine histomorphometry, liver injury, and immunomodulation parameters. Praziquantel treatment of infected mice fed with a standard diet (IN-PZQ) resulted in a significant reduction of worm and egg burdens and a normalization of iron and calcium levels. The therapy also improved schistosomiasis-induced hepatopathy and oxidative stress. The anti-inflammatory and immunomodulatory activities of praziquantel were also significant in these mice. When infected mice receiving the low-protein diet were treated with praziquantel (ILP-PZQ), the body weight loss and hepatomegaly were not alleviated, and the worm and liver egg burdens were significantly higher than those of IN-PZQ mice (P < 0.001). The treatment did not reduce the increased activities of ALT and γ-GGT, the high malondialdehyde concentration, and the liver granuloma volume. The iron and calcium levels were not ameliorated and differed from those of IN-PZQ mice (P < 0.001 and P < 0.05). Moreover, in these mice, praziquantel treatment did not reverse the high level of IL-5 and the low mRNA expression of CCL3/MIP-1α and CXCL-10/IP-10 induced by S. mansoni infection.

Almost 90% of people requiring schistosomiasis preventive chemotherapy in 2018 lived in sub-Saharan Africa. Besides, 205.3 million children under five years suffer and die of undernutrition in low- and middle-income countries. The physiopathology of schistosomiasis mansoni involves liver damage, oxidative stress, and perturbation of the immune response. These disturbances are intensified by undernutrition. Praziquantel is used to treat schistosomiasis, but its efficacy on the comorbidity of S. mansoni infection and undernutrition has not been investigated. We conducted this study to assess the effectiveness of praziquantel on S. mansoni infection in mice fed with a low-protein diet. We recorded growth retardation, hepatomegaly, and high worm and egg burdens in mice fed with a low-protein diet and treated with PZQ. Moreover, the treatment did not reverse the liver function injury, oxidative stress, high iron level, and low calcium level. The proinflammatory cytokine IL-5 was still high, and the gene expression of some macrophage-associated chemokines was reduced. Therefore, this study demonstrated that in a murine model of a low-protein diet, the efficacy of praziquantel on S. mansoni infection was reduced. It also underlines the importance of targeting protein deficiency and malnutrition in populations living in schistosomiasis endemic areas for efficient disease control.

The relationship between protein malnutrition of the host and S. mansoni infection is a very complex mechanism, not entirely determined since each can increase the other. Protein malnutrition is a factor that can alter the host-parasite environment system, aggravating the course of schistosomiasis by breaking the equilibrium in the relationship among the components of this system [ 16 – 20 ]. Clinical and experimental studies have demonstrated the interference of malnutrition in the outcome of schistosomiasis or vice versa. Assis et al. [ 17 , 21 ] showed that S. mansoni infection negatively impacts schoolchildren’s growth at low or moderate levels. The association between inadequate dietary intake and heavy S. mansoni infection increased the risk of stunting children. Other authors indicated that the association between undernutrition and S. mansoni infection leads to growth retardation, intensifies liver injuries, and decreases the humoral immune response in mice [ 19 , 22 , 23 ]. Moreover, feeding mice dams with a restricted-protein diet lead firstly to neonatal malnutrition of offspring during lactation and secondly to an increased egg output and liver damage in S. mansoni-infected pups [ 20 ]. More specifically, morphometric studies revealed that undernutrition of the host impairs the somatic development, the reproductive system, and the tegumental structure of male adult S. mansoni worms recovered from undernourished infected mice [ 18 ]. In clinical studies, praziquantel treatment improved the anthropometric indexes, nutritional status, and haemoglobin level of S. mansoni or S. japonicum-infected children and adolescents [ 17 , 24 ]. However, the literature lacks data on the effect of praziquantel on the comorbidity of S. mansoni infection and undernutrition. This study aimed to assess the efficacy of praziquantel on S. mansoni infection in mice fed with a low-protein diet.

Undernutrition and schistosomiasis are public health problems and often share the same geographical areas. Approximately 700 million persons, primarily children, are at risk of schistosomiasis in 78 endemic countries. It is estimated that more than 229 million people worldwide, with almost 90% of them living in sub-Saharan Africa, required preventive chemotherapy in 2018. [ 1 ]. Estimates by the World Health Organization show that 462 million adults are underweight, 205.3 million children under five years suffer from undernutrition, and about 45% of deaths are linked to undernutrition and mainly occur in low- and middle-income countries [ 2 ]. Schistosoma mansoni infection induces liver damage through the granulomatous inflammatory formation around eggs trapped in the sinusoidal periportal spaces and the generation of reactive oxygen species (ROS) [ 3 – 5 ]. Undernourished people are generally more susceptible to infections and increased morbidity and mortality [ 6 – 10 ]. Protein malnutrition induces structural changes in the lymphoid organs and impairs the innate and adaptative immune response. It is thus recognized as the cause of frequent immunodeficiency [ 11 – 15 ].

Blood collected in dry tubes was centrifuged at 3500 rpm for 15 min, and the serum obtained was stored at -70°C for biochemical analysis. Therefore, we determined the total protein level using the Biuret method [ 34 ] and the albumin level using the BIOLABO kit according to the method described by Doumas et al. [ 35 ]. In addition, we measured the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) according to the Reitman and Frankel method by using Bioclin kits [ 36 ]. Furthermore, we estimated the activities of alkaline phosphatase (ALP) and Gamma-glutamyltransferase (GGT) as described by Burtis et al. [ 37 ] and Szasz et al. [ 38 ], respectively. We also determined the total bilirubin, glucose, iron, calcium, total cholesterol, and triglycerides according to the protocol described in the BIOLABO kits by Burtis et al. [ 37 ]. In addition, the protocol to assay HDL cholesterol was described by Badimon et al. [ 39 ]. As a result, LDL cholesterol level was calculated as follows:

S. mansoni ova were counted in the feces the day before the sacrifice (63 rd day post-infection). Feces were individually collected from each infected mouse, weighed, and homogenized in 10% buffered formaldehyde. Two aliquots of 100 μL each were counted on a light microscope to determine the number of eggs. After sacrifice, the mice’s liver and intestine were removed, rinsed with PBS, weighed, and digested separately in 4% KOH solution at 37°C for 6h. Tissue suspensions were centrifuged at 1500 rpm for 5 min, and the supernatant was removed [ 33 ]. Using a light microscope, the number of eggs was determined in two aliquots of 100 μL each using a light microscope. Results were expressed as the mean number of eggs per gram of feces or tissue for the liver and intestine.

The diet did not induce any variation of the mRNA expression of cytokines and chemokines in healthy mice (HN), except for the gene expression of FGF-1 and CCL3, which are low in HLP mice. S. mansoni infection significantly decreased the mRNA expression of TGF-β1 by 81.71% and 79.68%, and FGF-1 by 79.25% and 64.45% in the standard diet feed (IN) and low-protein diet-feed mice (ILP), respectively, as compared to their healthy controls. We also recorded significant reductions of mRNA expression of chemokines CCL2/MCP-1, CCL3/MIP-1α, and CXCL-10/IP-10. In addition, we noted a decrease of IFN-γ mRNA expression and an increase of FoxP3 IL-10 and IL-13 mRNA expression in the liver of infected mice compared to healthy mice, although it was not statistically significant. Oral administration of praziquantel to infected mice completely reversed the diminished TGF-β1 gene expression. It was 3.70-fold and 2.85-fold higher in infected mice receiving the standard diet (IN-PZQ) or the low-protein diet (ILP-PZQ), respectively, than in their infected untreated controls. Praziquantel treatment did not restore the mRNA expression of FGF-1 and MCP-1 in infected mice, whatever their diet. Regarding the gene expression of chemokines, significant increases of the mRNA expression of CXCL-10/IP-10 and CCL3/MIP-1α were 40.59-fold and 5.96-fold, respectively, higher in IN-PZQ mice than those of IN mice. However, their levels did not change in infected mice fed with a low-protein diet and treated with praziquantel (ILP-PZQ) ( Fig 9 ). Finally, PZQ treatment restored the gene expression of TGF-β1 but failed to do it for CXCL-10/IP-10 and CCL3/MIP-1α in infected mice fed with a low-protein diet.

IL-17A and IL-10 were not detected in the sera of healthy mice receiving a standard or a low-protein diet. However, S. mansoni infected mice secreted significant levels of IL-17A and IL-10, particularly in infected mice receiving the standard diet. IL-17A and IL-10 were 75.40% and 69.95%, respectively, lower in the ILP group than in the IN group. The administration of praziquantel to infected mice significantly reduced IL-17A levels by 71.45% and 94.08% and IL-10 levels by 82.27% and 94.71% for IN-PZQ and ILP-PZQ groups, respectively, as compared to their infected untreated controls. The level of TGF-β1 was not modified by S. mansoni infection, whatever the type of diet administered to mice. However, it increased significantly after praziquantel treatment, to 1.81-fold and 3.05-fold in IN-PZQ and ILP-PZQ groups, respectively, compared to their healthy controls ( Fig 8 ). PZQ treatment restores the cytokines Th1, Th2, Th17, and Treg levels of infected mice receiving the standard or the low-protein diet, except for the IL-5 level of infected mice receiving the low-protein diet.

In both standard and low-protein diet-feed infected mice, treatment with praziquantel restores the SOD and CAT activities. We recorded 49.35% and 63.96% increase of SOD activity and 33.29% and 57.22% increases of catalase activity in IN-PZQ and ILP-PZQ groups of mice, respectively, when compared to their infected-untreated controls groups. However, praziquantel treatment did not reverse the reduced concentration of GSH in IN-PZQ mice. On the contrary, GSH concentration significantly increased by 47.34% in ILP-PZQ mice compared to ILP mice (P < 0.001). Furthermore, PZQ treatment did not restore the nitrite levels, whatever the type of diet, and it was significantly low in ILP-PZQ mice compared to ILP mice (P < 0.05), as well as to IN-PZQ mice (P < 0.05). PZQ treatment improved the MDA concentration and SOD and CAT activities of IN-PZQ and ILP-PZQ mice but did not bring the MDA concentration of ILP-PZQ mice near the normal range.

Administration of a low-protein diet to healthy mice (HLP) resulted in a significant reduction of 45.29% of the red blood cell count (P < 0.001) as compared to healthy mice receiving a standard diet (HN). S. mansoni infection significantly reduced the red blood cell count, hematocrit, and lymphocytes. At the same time, we recorded an increase in white blood cell count and eosinophil percentage in IN mice compared to HN mice. In infected-untreated mice receiving the low-protein diet (ILP), we noted a decrease in lymphocyte percentage (P < 0.001) but an increase of white blood cell counts (P < 0.001) as compared to healthy mice of the HLP group. Only one mouse expressed eosinophilia in this group. Treatment with praziquantel restored the hematocrit, the total leukocytes, lymphocytes, and eosinophils percentage in mice receiving the standard or the low-protein diet compared to their controls. The red blood cell concentration of all the infected mice receiving praziquantel remained significantly lower than that of IN mice (P < 0.01 for IN-PZQ and P < 0.001 for ILP-PZQ) ( Table 4 ).

As shown in Fig 5 , the low-protein diet significantly decreased the total proteins, albumin, glucose, and calcium concentrations in HLP mice compared to HN mice. When infected, mice submitted to the standard or the low-protein diet (IN or ILP group) showed significant decreases in total proteins, albumin, glucose, and calcium levels and a significant increase in iron concentration compared to their respective controls (HN and HLP groups). Administration of PZQ to S. mansoni-infected mice receiving the standard diet did not reestablish the expected concentration of proteins, albumin, and glucose. Still, it significantly restored the iron and calcium concentrations (P < 0.001). Compared to ILP mice, we recorded significant increases of total proteins level by 35.54% and glucose concentration by 52.94% in ILP-PZQ mice. However, these increases were not enough to get these concentrations close to normal ones since the total proteins, albumin, and glucose levels were still significantly lower in ILP-PZQ mice than in HN mice (P < 0.05 and P < 0.001). Albumin, iron and calcium concentrations of ILP-PZQ mice did not improve after PZQ treatment. The iron level of ILP-PZQ mice significantly remained higher than that of IN-PZQ mice (P < 0.001). At the same time, their calcium concentration was low compared to that of the IN-PZQ mice (P < 0.05). PZQ treatment ameliorated total proteins and glucose levels in infected mice receiving the low-protein diet but failed to normalize them compared to healthy mice receiving the standard diet. Moreover, PZQ treatment did not improve albumin, iron and calcium levels in these mice.

Administration of a low-protein diet to healthy mice (HLP) induced a significant reduction of aspartate aminotransferase (AST) activity by 47.71% as compared to healthy mice receiving a standard diet (HN). S. mansoni infection induced a significant increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) activities, and total bilirubin concentration in mice receiving a standard diet (IN) or a low-protein diet (ILP). Oral administration of praziquantel to infected mice receiving a standard diet resulted in a significant reduction of ALT (12.53%), ALP (42.11%), and GGT (55.19%) activities, as well as total bilirubin concentration (46.12%) as compared to those of their untreated controls. In infected mice receiving the low-protein diet and treated with praziquantel (ILP-PZQ), only ALP activity and total bilirubin level were reduced by 37.48% and 40.14%, respectively, when compared to those of the untreated controls. Activities of AST, ALT and GGT remained higher than those of ILP controls mice, and the differences were statistically significant for AST (P < 0.05) and GGT activities (P < 0.001) ( Fig 4 ). PZQ treatment reduced ALP activity and total bilirubin concentration and failed to improve AST, ALT, and GGT in S. mansoni-infected mice receiving the low-protein diet.

S. mansoni infection significantly increased mice’s liver, spleen, and intestine weights receiving either the standard diet (IN) or the low-protein diet (ILP). However, the IN mice were more affected by the infection than the ILP mice, as denoted by the significant difference in hepatosplenomegaly (P < 0.001) and intestine enlargement (P < 0.01) between the two groups. Oral administration of PZQ to normally nourished and infected mice (IN-PZQ group) resulted in a significant reduction (P < 0.001) of the liver (32.83%), spleen (60.64%), and intestine (32.07%) weights as compared to those of infected-untreated mice. PZQ treatment then reestablished the liver, intestine and spleen weights of IN-PZQ mice as they were close to the normal range of HN naïve mice. On the contrary, PZQ treatment of infected mice receiving the low-protein diet (ILP-PZQ) did not significantly reduce the liver and intestine weight indexes that are still similar to those of ILP mice. Only the spleen weight of ILP-PZQ mice significantly decreased by 43.01% as compared to their healthy controls (P < 0.01) ( Fig 2 ). Then, PZQ treatment did not improve hepatomegaly and intestine enlargement induced by the infection in mice receiving the low-protein diet.

As shown in Fig 1 , healthy mice receiving either a standard diet or a low-protein diet gained weight during the experimentation. On the contrary, infected mice (IN and ILP groups) significantly lost weight compared to their respective healthy controls HN and HLP (P < 0.001) from the tenth week of experimentation (4 weeks p.i) to the end (10 weeks p.i). Oral administration of praziquantel to infected mice receiving the standard diet induced a normalization of body weight as the body weight variation increased in the IN-PZQ group compared to IN group (P < 0.01). On the contrary, the body weight of infected mice receiving the low-protein diet and treated with PZQ (ILP-PZQ group) was not improved after the treatment. The body weight variation of mice belonging to the ILP-PZQ group wasn’t statistically different from that of the ILP group at the end of the experimentation. The Lee index, an indicator of the nutritional status, did not vary after S. mansoni infection or after PZQ treatment of mice receiving the standard diet. However, this index was reduced in S. mansoni infected mice fed with a low-protein diet than in controls (P < 0.05). PZQ treatment did not significantly improve the body weight reduction in mice fed a low-protein diet.

Discussion

Protein malnutrition and schistosomiasis are health problems that affect millions of people. A low-protein diet increases the risk of illness and death [7,8]. Protein malnutrition can impair immune function and affect hematopoiesis, biochemical and histological parameters [7,10,46–50]. Consequently, a low-protein diet increases susceptibility to infections and induces an imbalance between food intake and the need to ensure the most favourable growth [47,51–53]. As revealed by the Lee index, the nutritional state of healthy mice receiving the low-protein diet was normal in the current study. Since the bromatological characteristics of the low-protein diet and the standard one revealed that the total metabolizable energy was similar, it can be understandable that the low-protein diet used to feed healthy mice has supplied food intake without altering energy expenditure, body fat, lean mass, and body weight [54,55]. While infected with S. mansoni, mice receiving either the standard or the low-protein diet significantly lost weight, probably due to anaemia and hypoglycemia. Indeed, the current study clearly showed a reduction in red blood cell count, hematocrit and glucose concentration in infected mice as S. mansoni adult worms use haemoglobin and glucose for their nutrition, energy supply, and egg-laying [56]. As determined by anthropometric indexes, growth retardation has been associated with chronic S. mansoni infection in children [17]. The Lee index also provides information on the health and growth of individuals and is often cited as a reliable indicator of nutritional status [57,58]. In this study, the Lee index of infected mice receiving the low-protein diet was significantly reduced, demonstrating the negative impact of undernutrition and schistosomiasis on growth [21,59]. Moreover, the total proteins, albumin, and glucose concentrations of mice fed with the low-protein diet significantly decreased. Hypoproteinemia and hypoalbuminemia could be the consequence of reducing protein supply in the diet, leading to a decrease of protein biosynthesis by the host. The combined effects of protein and glucose deficiencies and S. mansoni infection have undoubtedly impaired the nutritional status of the mice and, therefore, their growth. Contrary to that of infected mice fed a low-protein diet, oral administration of PZQ improves the body weight of infected mice receiving the standard diet. The low body weight of the infected mice fed with the low-protein diet and treated with PZQ could be linked to the persistence of anaemia and protein and glucose deficiencies after PZQ treatment. The red blood cell counts, and the concentrations of total proteins, albumin, and glucose of these mice remained low compared to those of healthy mice. An adverse synergistic action of malnutrition and S. mansoni infection on host glycemia and hemoglobinemia have been previously described [23,24].

In the current study, the worm recovery rates of infected-untreated mice were similar in both diet groups, indicating the capacity of worms to accomplish their life cycle in a final host fed with a low-protein diet [18,60]. However, some authors have mentioned that male adult S. mansoni isolated from mice fed with a low-protein diet present thinning of the tegument, large tubercles on the dorsal region, vacuolated areas on the subtegumental region, and small testes [18]. On the other hand, Wolowczuk et al. [61] pointed out that even if a low-protein diet may induce morphological changes in schistosomes and produce dwarf worms, these changes would not be enough to impair their reproductive capacity; dwarf worms would still be able to mate and lay eggs. This correlates with our study, where egg loads in feces and liver were comparable in both diet groups. S. mansoni-infected mice also exhibited hepatosplenomegaly and intestine enlargement, undoubtedly due to egg deposition in the hepatic and intestine parenchyma. Enlargement of the spleen could result from passive congestion of blood flow and reticuloendothelial hyperplasia [60,62]. Oral administration of PZQ to infected mice receiving the standard diet (IN-PZQ group) resulted in a significant reduction of hepatomegaly, splenomegaly, and intestine enlargement. This could be the consequence of the considerable decrease of egg burden in the liver and intestine after PZQ treatment, as recorded in this study and by some authors [30,63–65]. The reduction of hepatic egg load of infected mice fed with the low-protein diet and treated with PZQ (ILP-PZQ) was insufficient to induce a reduction of hepatomegaly. Indeed, despite PZQ treatments, the worm and hepatic egg burdens of infected mice receiving the low-protein diet were higher than those of infected mice receiving the standard diet. These findings justify the persistence of anemia and low glucose levels in these mice and indicate that a low-protein diet could reduce PZQ efficacy. This is probably linked to intestinal malabsorption of PZQ as França et al. [13] have demonstrated that in undernourished mice, intestinal villi lose their brush border and become small and irregular, thus impairing intestinal function. Ibrahim et al. [12] pointed out that intestinal inflammation resulting from enteric pathogens disrupts intestinal barrier function. Undernutrition coupled with granulomatous intestinal inflammation, as recorded in infected mice fed with a low-protein diet, could thus explain the malabsorption of PZQ. The poor efficacy of PZQ in protein-undernourished and infected mice could also be understandable by exploring the PZQ mechanism of action. PZQ acts on schistosome motility by disrupting calcium ions homeostasis in the worm. It alters the worm’s membrane permeability, causing an uncontrolled and rapid calcium ion influx that leads to sustained muscle contraction and paralysis [66–68]. It has been demonstrated that PZQ-induced disruption of schistosome tegument and muscular contraction depends on calcium concentration in the worm’s environment [67,69–72]. In the current study, calcium levels remained low in infected mice receiving the low-protein diet and treated with PZQ. The extracellular calcium could therefore be insufficient to sustain schistosome muscle contractions which leads to their paralysis and death. This might also explain the important worm and egg burdens of infected mice receiving the low-protein diet after PZQ treatment.

Embolization of schistosome eggs in the liver induces impairment of hepatic metabolism. Following da Silva et al. [73], who demonstrated lipid metabolism alterations in hepatosplenic schistosomiasis, the present study showed significant decreases in total cholesterol, cholesterol LDL, and triglyceride in infected untreated mice receiving the standard or the low-protein diet. Because S. mansoni worms cannot synthesize cholesterol required for their growth and egg production, they absorb it from the host’s bloodstream, hence dropping total cholesterol, cholesterol LDL, and triglycerides [74]. Hepatocellular injury leading to the modification of transaminases (ALT and AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and total bilirubin (BIL) levels is also common in S. mansoni infection [30,45,63,65]. The presence of S. mansoni eggs in the liver parenchyma initiates an inflammatory process that gives rise to the formation of granulomas. These granulomatous lesions injure hepatocytes, which lose their membrane integrity and release transaminases in the bloodstream, thus increasing their activity [60,75]. The present investigation also showed increased PAL, GGT, and BIL concentration, implying an impairment of hepato-biliary function [73]. Since the liver is the site of iron metabolism, hepatocyte injury could increase iron concentration as recorded in S. mansoni-infected mice during this study and in patients with chronic liver diseases [76]. Administration of PZQ to infected mice receiving the standard diet or the low-protein diet ameliorates the lipid profile, ALP activity, and BIL concentration. ALT and GGT activities and iron levels were improved only in infected mice receiving the standard diet. These results demonstrated that the liver is recovering from the injury induced by S. mansoni infection. The concomitant reduction of worm burden and egg load in the liver after PZQ treatment has contributed to reducing the hepatocellular damage and probably initiated liver regeneration. Oliva-Vilarnau et al. [77] have demonstrated that calcium is a critical component of hepatic growth factors signalling during liver regeneration after injury. Intracellular calcium has been associated with mitogens epithelial growth factor (EGF) and hepatocyte growth factor (HGF) in hepatocytes. As recorded in this study, the normalization of the calcium concentration of infected mice receiving the standard diet after PZQ treatment could be essential for their liver regeneration. Remarkably, in S. mansoni infected mice receiving the low-protein diet, the ALT and GGT activities and iron and calcium concentrations did not recover after PZQ treatment. This impaired recovery is probably due to the high liver egg burden and hypocalcemia in these mice, indicating a diminished efficiency of PZQ treatment in this low-protein setting again.

The infection induced liver oxidative stress in the current study, marked by malondialdehyde overload and depletion of antioxidants and nitrites. During schistosomiasis, the granuloma-inflammatory cells generate reactive oxygen species (ROS) such as superoxide and hydroxyl radicals involved in the production of lipid peroxides. This leads to an increased concentration of MDA in the liver. Because of its implication in the generation of peroxynitrite, the oxidization of nitric oxide (NO) to nitrites, and consequently the concentration of nitrites, diminish. Therefore, the host will immediately use antioxidants to counteract the harmful action of ROS overload. The consequence will be the reduction of hepatic SOD, CAT, and GSH levels [4,30,45,63–65,78,79]. Administration of PZQ to S. mansoni-infected mice fed with a standard or a low-protein diet reduced lipid peroxidation (MDA) and improved enzymatic, non-enzymatic antioxidants levels (SOD, CAT, and GSH). However, despite PZQ treatment to infected mice fed with the low-protein diet (ILP-PZQ), MDA concentration was still higher, and nitrites were lower than those of infected mice fed with the standard diet treated with PZQ (IN-PZQ). It implies that despite the treatment, lipoperoxidation due to ROS and peroxynitrite production occurs, probably because of the high liver egg burden of the ILP-PZQ group of mice. Indeed, the action of PZQ on oxidative stress is indirect through its schistosomicidal effect on adult worms. By limiting the recruitment of inflammatory cells, the source of ROS, in the vicinity of schistosome eggs, PZQ indirectly reduces ROS production [64,80,81].

The generation of oxidative stress is intimately linked to the granulomatous inflammatory cells’ activity. Therefore, reducing ROS production would reduce inflammatory response or vice versa. We assessed the effects of PZQ treatment on the inflammatory status of S. mansoni-infected mice by performing histomorphometry of the liver and intestine and determining the immunological status of the mice. Histopathological examination of the liver and the mall intestine sections of infected mice fed with the standard or the low-protein diet and treated with PZQ (IN-PZQ and ILP-PZQ, respectively) revealed fewer and smaller granulomas than in infected untreated mice. These observations were confirmed by the reducing number of liver and intestine granulomas. The hepatic granulomas volume also decreased in the IN-PZQ group of mice but not in the ILP-PZQ group. Other authors obtained similar results on S. mansoni-infected mice receiving a standard diet. They correlated this anti-inflammatory activity of PZQ to its schistosomicidal effect resulting in the reduction of eggs laying in the liver and intestine [30, 45, 82–84]. The decrease in granulomas volume after PZQ treatment of infected mice could also be consistent with the reduction of fibrosis. This is materialized by the decline of collagen types I and III or their biomarker hydroxyproline [64, 82, 84]. The non-reduction of the hepatic granulomas volume of infected mice receiving the low-protein diet and treated with PZQ could be explained by the continuous recruitment of inflammatory cells due to the critical egg burden in their liver. It has been demonstrated that schistosomiasis and undernutrition comorbidity led to increased egg production and liver damage marked by high density and large areas of exudative granulomas [19, 20].

Malnutrition considerably impairs the immune system by causing atrophy of primary lymphoid organs, compromising complement components and phagocyte function, and decreasing the biological function of lymphocytes, macrophages, and Kupffer cells [12,13]. In the current study, protein deficiency did not induce significant Th1, Th2, Th17, and Treg levels. In contrast, several authors have reported reduced proliferation and effector function of lymphocytes T, a low level of blood lymphocytes T, and a shift of a Th1 towards a Th2 cytokine response in children with severe malnutrition and fasted mice or mice fed with a low-protein diet [13–15,85–88]. The lack of variation in cytokine levels in our study could be due to the moderate low-protein diet used to feed mice (14.60% of protein), as compared to the very low-protein diet (2% of protein) used by others [89]. The involvement of T lymphocytes, especially CD4+ T cells, in the immune response against schistosomiasis is essential [90]. During the migratory phase of schistosomula, from 3 to 5 weeks post-infection, the dominant immune response is Th1. When parasites mature, mate and begin to lay eggs at 5 to 6 weeks post-infection, female worms release fertilized eggs that stimulate a Th2 immune response via their soluble egg antigens. The Th2 response reaches a peak at approximately 8 weeks post-infection and is down-modulated with progression to chronic infection [91–94]. In the current study, the immune response at 9 weeks of S. mansoni infection shifted from a Th1 to a Th2 response in infected mice receiving the standard or the low-protein diet. Indeed, the serum levels of TNF-α and IFN-γ, as well as the liver mRNA expression of CCL2/MCP-1, FGF, CCL3/MIP 1-α, CXCL-10/IP-10, and IFN-γ decreased significantly. Concurrently, the serum levels of IL-4, IL-5, IL-13, and the liver mRNA expression of FoxP3 increased. During the early acute stage of S. mansoni infection, a Th1 response is initiated and is characterized by the increased concentration of inflammatory cytokines and chemokines [95]. The elevated expression of eosinophil-, neutrophil- and macrophage- associated chemokines such as CCL2/ MCP-1, CCL3/MIP 1-α, and CXCL-10/IP-10 is concomitant to the migration of eosinophils and neutrophils from the circulation to the site of the granulomatous inflammation, and the recruitment and activation of hepatic stellate cells (HSCs). [96–98]. HSCs can also be activated during the chronic stage of schistosomiasis, and in turn, they can produce chemokines like CCL2, CCL3, and CXCL-10 following liver injury [99]. The reduction of the mRNA expression of CCL2, CCL3, FGF, CXCL-10, and IFN-γ at the 9th-week post-infection reflects a downmodulation of the Th1 response by reducing the recruitment of inflammatory cells and HSCs to the granulomatous site. This indicates a maximal granuloma growth, as revealed by the highest IL-2 production [100]. The increased serum level of IL-17A during this period is also correlated to the granulomatous lesions associated with severe liver pathology [95,101]. Hepatic granuloma formation and fibrosis are upregulated by Th2 and Th17 cells, mainly secreting IL-4 and IL-17A, respectively [102–104], and downregulated by Th1 and Treg cells [105,106]. Regulatory T cells secrete IL-10 and TGF-β that suppress the activation of dendritic cells, mediate Th1 and Th2 responses and inhibit granuloma development and fibrosis during S. mansoni infection to promote host survival [90,95,107,108]. During this study, increasing the serum level of IL-10 and its mRNA expression in infected mice demonstrates its regulatory activity by inhibiting the production of cytokines Th1 like TNF-α [107]. IL-10 can also play its regulatory role in periportal fibrosis by blocking the activation of quiescent HSCs [109,110]. The reduction of the mRNA expression of TGF-β1 in S. mansoni-infected mice after 9 weeks could also reflect the modulation of liver fibrosis. It has been reported that high levels of TGF-β1 are associated with liver fibrosis and pulmonary arterial hypertension in S. mansoni infection [111,112]. The normalization of Th1, Th2, Th17, and Treg cytokine levels (IL-2, IL-5, IL-13, IL-17A, and TGF-β1) and the mRNA expression of some chemokines and Treg cytokines (CCL3, CXCL-10 and TGF-β1) after PZQ treatment is the consequence of drug-induced clearance of S. mansoni worms. This reduces the number of eggs trapped in the liver, and subsequently the recruitment and migration of inflammatory cells around the eggs. Indeed, in the current study, PZQ treatment re-establish normal levels of total leukocytes and eosinophils that were significantly increased and of lymphocytes that was decreased by S. mansoni infection. Leukocytosis, eosinophilia and lymphocytopenia are common during granulomatous inflammatory diseases as schistosomiasis. Blood total leukocytes and eosinophils increase to fight the infection and lymphocytes migrate at the sites of inflammation, being attracted by chemokines and cytokines. This migratory process leads to a decrease of their blood count [113,114]. The capacity of PZQ to normalize the total leukocyte, eosinophil and lymphocyte blood counts therefore demonstrates its ability to limit the immunogenic action of S. mansoni eggs and to alleviate the infection. Because of the persistent high worm and egg burdens in the group of infected mice receiving the low-protein diet and treated with PZQ, the levels of IL-5 and the mRNA expression of CCL3 and CXCL-10 were not ameliorated by the treatment. FoxP3 was barely detectable in non-infected mice, but expressed in the liver of infected untreated mice and of infected mice treated with PZQ. This is consistent with its down-regulatory role on Th1 and Th2 cytokines production and on the fibrogranulomatous inflammation via the inhibition of the profibrogenic activity of IL-4 and IL-13 [115,116].

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[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0010249

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