Liver Granulomas Research Articles

The IL-33/ST2 pathway is not essential to Th2 stimulation but is key for modulation and survival during chronic infection with Schistosoma mansoni in mice

Morbidity during chronic schistosomiasis has been associated with the induction and modulation of type-2 granulomatous inflammatory response induced by antigens secreted by the eggs, which become trapped in capillary venules of the host tissues, especially in the liver and intestines. IL-33, an alarmin released after cell damage, binds to its ST2 (suppressor of tumorigenicity 2) receptor, expressed in an variety of immune cells, including ILC2 and macrophages, and stimulates the early production of IL-5 and IL-13, which leads to eosinophil infiltration and activation of a Th2 response. However, the role of IL-33/ST2 activation on Schistosoma-induced granuloma formation and modulation is mostly unknown. In the current work, we comparatively evaluated the immune response and granuloma formation in wild-type BALB/c (WT) and BALB/c mice genetically deficient in the IL-33 receptor (ST2−/−) experimentally infected with Schistosoma mansoni. Mice were infected with 25 or 50 S. mansoni cercariae and followed for up to 14 weeks to assess mortality. Mice from each experimental group were comparatively evaluated for parasite burden, liver immune response, and granuloma appearance during acute and chronic schistosomiasis. Our data showed that the number of circulating worms and eggs retained in the liver and eliminated in the feces was similar in WT and ST2−/− infected mice, but infected ST2−/− mice presented an enhanced rate of mortality. Interestingly, the production of type-2 cytokines by soluble egg antigens (SEA)-stimulated spleen cells, the serum concentrations of IL-5 and Immunoglobulin (Ig)-E, and the level of parasite-reactive IgG1 were similar in infected mice of both experimental groups. The concentrations of IL-4, IL-5, IL-13, and IFN-γ in liver homogenate of infected mice also did not differ between the strains at acute schistosomiasis, but there was a significant increase in IL-17 levels in ST2−/− infected mice at this phase. On the other hand, IL-4, IL-13, IL-10, IL-17, and IFN-γ concentrations were reduced and the ratios of IL-4/IFN-γ and IL-17/IFN-γ were higher in liver homogenate of chronically infected ST2−/− mice, suggesting unbalanced Th2 and Th17 responses. Moreover, liver granulomas of ST2−/− mice were larger and disorganized, showing an intense cellular infiltrate, rich in eosinophils and neutrophils. Our results suggest that the absence of the IL-33/ST2 pathway is not essential for the Schistosoma-induced Th2 response, but is necessary to prevent host mortality by modulating granuloma-mediated pathology.

Analysis of the Efficiency of Different Antituberculous Drugs and Approaches to Treat BCG-Induced Granulomatosis in Mice and Abundance and Localization of Mycobacterium tuberculosis in the Liver.

In 3 months after infection with Mycobacterium tuberculosis (MBT) from BCG vaccine, male BALB/с mice received intraperitoneal injections of isonicotinic acid hydrazide, dextrazide, or liposome-encapsulated dextrazide, or inhalation of liposome-encapsulated dextrazide 2 times a week for 6 months. In 6 months, no MBT were detected in macrophages outside granulomas in treated mice. Macrophages containing MBT can incorporate into granulomas and leave them after suppression of MBT persistence. Liposome-encapsulated dextrazide showed the maximum therapeutic efficiency: the total MBT level in granuloma macrophages and volume density of destruction foci in the liver parenchyma decreased by 5.1 and 5.3 times, respectively, in comparison with the corresponding parameters in mice treated with isonicotinic acid hydrazide. Inhalations of liposome-encapsulated dextrazide prevented the destructive processes in liver granulomas due to macrophage migration from granulomas, which reduced granuloma sizes and destructive potential of granuloma lysosomes and therefore improved their diffusion-dependent trophics.

Association Between Carbamazepine Toxicity, Liver Bile Duct Injury, Granuloma and Inflammatory Cells Infiltration in Female Mice

The liver is an important organ in the body that can be affected by many drugs and toxins. The hepatotoxinscan cause oxidant stress that lead to activation of inflammatory cells and cause liver damage. Drug inducedbile duct injuries are related to drug toxicity, multiple drugs have been known to cause the development ofliver granulomas. Carbamazepine (CBZ) among other antiepileptic drugs is believed to cause hepatic injury.In this study we investigated the effect of (CBZ) 20mg/kg/day on female mice liver after 14 and 30 daysof treatment. The histological findings showed that (CBZ) can cause histological alterations in the livercomponents such as bile duct proliferation, biliary hypertrophy, ductopenia, inflammatory cells infiltrationand granulomas.

Regulation of Liver Fibrosis during Murine Schistosomiasis Mansoni

Background: The main pathology of Schistosoma mansoni (S. mansoni) infection is induced by a granulomatous tissue reaction against the parasite eggs. Unfortunately, no therapy has been proven to prevent the progression of hepatic fibrosis associated with granulomatous hypersensitivity to parasite eggs. Accumulating evidence has demonstrated a critical pathogenic role for both interleukin 17 (IL-17) and interleukin 13 (IL-13) in organ fibrosis. Objective: The present study investigated the role of IL-17 and IL-13 in the pathogenesis of liver fibrosis during S. mansoni infection. Material and Methods: Thirty female C57BL/6 mice were divided into three groups, normal, infected, and anti- mouse IL-17 treated groups. The infected and anti-mouse IL-17 groups were infected with 40±5 cercariae of S. mansoni per mouse. Neutralizing rat anti-mouse IL-17 mAb or an isotype-matched rat IgG mAb was first administered intraperitoneally 3 weeks after S. mansoni infection (62.5μg per mouse) then at the same dose every third day until 2 days before killing. Serum IL-17, IL-13, and proinflammatory cytokines levels were determined by ELISA. Liver granulomas were measured by an ocular micrometer. Results: Serum level of IL-17 was significantly higher in infected mice compared with non-infected animals. Reducing IL-17 activity using anti-IL-17 monoclonal antibodies improves liver functions and reduces the size of the liver granulomas. Meanwhile, Th-2 profibrogenic cytokine IL-13 was also decreased in infected/anti-IL-17 mAB- treated mice. IL-17-induced proinflammatory mediators (IL-1Land TNF-that involved in liver fibrosis were markedly reduced in anti-IL-17 mAB-treated mice. Conclusion: IL-17 and IL-13 contribute to granulomatous inflammatory and fibrosing reactions in murine schistosomiasis.

S2457 Histoplasma Hepatitis Presenting as Mirizzi Syndrome

INTRODUCTION: Hepatitis secondary to Histoplasmosis is a pathology that is complex and difficult to diagnose. We report a case that presented as Mirizzi syndrome with respiratory dysfunction that proved to be disseminated histoplasmosis with hepatic involvement. CASE DESCRIPTION/METHODS: A 50 y/o female on adalimumab for rheumatoid arthritis presented with right-upper quadrant pain, a 5-day history of jaundice, fever and shortness of breath. Liver chemistries were elevated with alkaline phosphatase of 733 IU/L, total bilirubin of 7 mg/dl, AST 612 IU/L and ALT 202 IU/L. Abdominal ultrasonography revealed gallstones and mildly dilated common bile duct. A chest CT showed diffuse bilateral groundglass opacities. Based on the complex presentation and timing (mid March, 2020) the working diagnoses were gallstone disease with potential SARS-CoV2 infection. Endoscopic Retrograde Cholangiopancreatography exposed Mirizzi syndrome and a long-stent was placed into the bile duct. COVID-19 testing was negative. Liver enzymes showed minimal improvement and respiratory symptoms continued to worsen. Other laboratory tests as well as a detailed toxin intake (including alcohol) were negative. Due to the lack of improvement the patient underwent bronchoscopy and liver biopsy. The former showed buddying yeast in the lungs and the latter liver granulomas with PAS positive yeast consistent with Histoplasma. Liver biopsy cultures were positive for Histoplasma capsulatum. The patient was placed on Itraconazole and discharged home upon improvement. Eight weeks post discharge her liver enzymes have almost completely normalized and symptoms have resolved. DISCUSSION: Disseminated histoplasmosis affecting the liver is a relatively rare and often lethal complication of this infection. The confirmation of extra pulmonary disease is critical as the length of therapy extends from several weeks to one year. Histoplasma hepatitis has been reported in individuals undergoing immunosuppression, such as our patient, and a consistently reported characteristic has been a high AST to ALT ratio which can lead to an initial suspicion of alcoholic hepatitis. This should be kept in mind if the provided history is not compatible with toxin-related (alcohol) liver disease. Our patient also had a clinical presentation compatible with Mirizzi syndrome further obscuring the diagnosis. In this regard, alternative diagnoses should be thought if there are no dramatic improvements of liver chemistries following recanalization of the bile duct.

Regulation of Liver Fibrosis during Murine Schistosomiasis Mansoni

Background: The main pathology of Schistosoma mansoni (S. mansoni) infection is induced by a granulomatous tissue reaction against the parasite eggs. Unfortunately, no therapy has been proven to prevent the progression of hepatic fibrosis associated with granulomatous hypersensitivity to parasite eggs. Accumulating evidence has demonstrated a critical pathogenic role for both interleukin 17 (IL-17) and interleukin 13 (IL-13) in organ fibrosis. Objective: The present study investigated the role of IL-17 and IL-13 in the pathogenesis of liver fibrosis during S. mansoni infection. Material and Methods: Thirty female C57BL/6 mice were divided into three groups, normal, infected, and anti- mouse IL-17 treated groups. The infected and anti-mouse IL-17 groups were infected with 40±5 cercariae of S. mansoni per mouse. Neutralizing rat anti-mouse IL-17 mAb or an isotype-matched rat IgG mAb was first administered intraperitoneally 3 weeks after S. mansoni infection (62.5μg per mouse) then at the same dose every third day until 2 days before killing. Serum IL-17, IL-13, and proinflammatory cytokines levels were determined by ELISA. Liver granulomas were measured by an ocular micrometer. Results: Serum level of IL-17 was significantly higher in infected mice compared with non-infected animals. Reducing IL-17 activity using anti-IL-17 monoclonal antibodies improves liver functions and reduces the size of the liver granulomas. Meanwhile, Th-2 profibrogenic cytokine IL-13 was also decreased in infected/anti-IL-17 mAB- treated mice. IL-17-induced proinflammatory mediators (IL-1Land TNF-that involved in liver fibrosis were markedly reduced in anti-IL-17 mAB-treated mice. Conclusion: IL-17 and IL-13 contribute to granulomatous inflammatory and fibrosing reactions in murine schistosomiasis.

In vivo assessment of the antischistosomal activity of curcumin loaded nanoparticles versus praziquantel in the treatment of Schistosoma mansoni

Schistosomiasis is a serious parasitic infection affecting millions worldwide. This study aimed to explore the anti-schistosomal activity of curcumin and curcumin loaded gold-nanoparticles (Cur-GNPs) with or without praziquantel (PZQ). We used six groups of the C57BL/6 mice in which five groups were infected with Schistosoma Mansoni (S. mansoni) cercariae and exhibited, separately, to different treatment regimens of curcumin, curcumin loaded nanoparticle, and PZQ, in addition to one untreated group which acts as a control. Mice were sacrificed at the 8th week where both worms and eggs were counted in the hepatic and porto-mesenteric vessels in the liver and intestine, respectively, in addition to a histopathological examination of the liver granuloma. Curcumin caused a significant reduction in the worms and egg count (45.45%) at the 3rd week. A significant schistosomicidal effect of PZQ was found in all groups. Cur-GNPs combined with PZQ 97.4% reduction of worm burden in the 3rd week and the highest reduction in the intestinal and hepatic egg content, as well, besides 70.1% reduction of the granuloma size. The results suggested the curcumin in combination with PZQ as a strong schistosomicidal regimen against S. mansoni as it alters the hematological, biochemical, and immunological changes induced.

Investigating Immunization With Nucleotide Enzymes of Schistosoma mansoni: Nucleoside Diphosphate Kinase and Adenylosuccinate Lyase as New Antigenic Targets Against Schistosomiasis.

Schistosomiasis, caused by Schistosoma mansoni trematode worm, affects more than 1.5 million people in Brazil. The current treatment consists in the administration of Praziquantel, the only medicine used for treatment for more than 40 years. Some of the limitations of this drug consist in its inactivity against schistosomula and parasite eggs, the appearance of resistant strains and non-prevention against reinfection. Thus, the objective of this study was to evaluate the effect of immunization with recombinant functional enzymes of the purine salvage pathway of S. mansoni, Nucleoside Diphosphate Kinase (NDPK) and Adenylosuccinate Lyase (ADSL), to evaluate the host immune response, as well as the parasite load after vaccination. For this, Balb/c mice were divided into 5 groups: control (uninfected and untreated), non-immunized/infected, NDPK infected, ADSL infected, and NDPK + ADSL infected. Immunized groups received three enzyme dosages, with a 15-day interval between each dose, and after 15 days of the last application the animals were infected with 80 cercariae of S. mansoni. On the 47th day after the infection, fecal eggs were counted and, on the 48th day after the infection, the evaluation of leukocyte response, parasite load, antibody production, cytokines quantification, and histopathological analysis were performed. The results showed that immunizations with NDPK, ADSL or NDPK + ADSL promoted a discreet reduction in eosinophil counts in lavage of peritoneal cavity. All immunized animals showed increased production and secretion of IgG1, IgG2a, and IgE antibodies. Increased production of IL-4 was observed in the group immunized with the combination of both enzymes (NDPK + ADSL). In addition, in all immunized groups there were reductions in egg counts in the liver and intestine, such as reductions in liver granulomas. Thus, we suggest that immunizations with these enzymes could contribute to the reduction of schistosomiasis transmission, besides being important in immunopathogenesis control of the disease.

The immunomodulatory activity of Chenopodium ambrosioides reduces the parasite burden and hepatic granulomatous inflammation in Schistosoma mansoni-infection

Ethnopharmacological relevanceFolk medicine reports have described the use of Chenopodium ambrosioides as an anti-inflammatory, analgesic, and anthelmintic herb. These effects, including its activity against intestinal worms, are already scientifically observed. However, the immunological mechanisms of this species in the treatment of Schistosoma mansoni infection are unknown.Aim of the study: To evaluate the immunological and anti-Schistosoma mansoni effects of a crude Chenopodium ambrosioides hydro-alcoholic extract (HCE). Materials and methodsFor the in vitro analysis, cercariae and adult worms were exposed to different concentrations (0 to 10,000 μg/mL) of the HCE. For the in vivo evaluation, Swiss mice were infected with 50 cercariae of S. mansoni and separated into groups according to treatment as follows: a negative control (without treatment), a positive control (treated with Praziquantel®), HCE1 Group (treated with HCE during the cutaneous phase), HCE2 Group (treated with HCE during the lung phase), HCE3 Group (treated with HCE during the young worm phase), and HCE4 Group (treated with HCE during the adult worm phase). The animals treated with HCE received daily doses of 50 mg/kg, by gavage, for seven days, corresponding to the different developmental stages of S. mansoni. For comparison, a clean control group (uninfected and untreated) was also included. All animals were euthanized 60 days post-infection to allow the following assessments to be performed: a complete blood cells count, counts of eggs in the feces and liver, the quantification of cytokines and IgE levels, histopathological evaluations of the livers, and the analysis of inflammatory mediators. ResultsHCE treatment increased the mortality of cercariae and adult worms in vitro. The HCE treatment in vivo reduced the eggs in feces and liver. The number and area of liver granulomas, independent of the phase of treatment, were also reduced. The treatment with HCE reduced the percentage of circulating eosinophils, IgE, IFN-γ, TNF-α, and IL-4. In contrast, the treatment with the HCE, dependent on the phase, increased IL-10 levels and the number of peritoneal and bone marrow cells, mainly of T lymphocytes, B lymphocytes, and macrophages. This effect could be due to secondary compounds presents in this extract, such as kaempferol, quercetin and derivatives. ConclusionsThis study demonstrates that Chenopodium ambrosioides has antiparasitic and immunomodulatory activity against the different phases of schistosomiasis, reducing the granulomatous inflammatory profile caused by the infection and, consequently, improving the disease prognosis.

Cell therapy as a new approach on hepatic fibrosis of murine model of Schistosoma mansoni-infection.

Schistosomiasis is an acute and chronic disease of the genus Schistosoma triggered by blood flukes. Schistosomiasis is a disease occurring in, or endemic to, tropical and subtropical regions. A new concept was implemented to deal with schistosomiasis from natural plant sources. Curcumin's common name is Turmeric. Curcumin has proven to be main active component in Curcuma longa L. and has a wide range of anti-phrastic effects. Previous studies have shown the role of bone marrow mesenchymal stem cells (BMSCs) therapy in hepatic fibrosis recovery. The current study was, therefore, intended to examine therapeutic role of BMSCs and Turmeric in murine schistosomiasis mansoni. Mice were divided into five groups: a negative control group (non-infected non-treated), a positive control group (infected non-treated), a BMSCs treated group; Turmeric treated group, and untreated group. BMSCs derived from male mice were injected intraperitoneally into female mice receiving S. mansoni cercariae through the subcutaneous route. Liver histopathology and immuno-histochemical examinations were evaluated. BMSCs intraperitoneal injection resulted in a significant reduction of liver collagen, granuloma size, and significant increase of OV-6 expression in the Schistosomiasis-treated mice group. There was overall improvement in pathological changes of the liver. Unfortunately, group IV showed a mild improvement in the granuloma size and fibrosis compared to corresponding BMSCs treatment group, although with vacuolated liver cells. BMSCs have a regenerative potential in liver tissue histopathology by decreasing liver fibrosis and granulomas. Turmeric, by contrast, could not be used as an anti-fibrotic, according to the findings.

A Novel In Vitro Mouse Model to Study Mycobacterium tuberculosis Dissemination Across Brain Vessels: A Combination Granuloma and Blood‐Brain Barrier Mouse Model

In vitro culture models of the blood-brain barrier (BBB) provide a useful platform to test the mechanisms of cellular infiltration and pathogen dissemination into the central nervous system (CNS). We present an in vitro mouse model of the BBB to test Mycobacterium tuberculosis (Mtb) dissemination across brain endothelial cells. One-third of the global population is infected with Mtb, and in 1%-2% of cases bacteria invade the CNS through a largely unknown process. The "Trojan horse" theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB-granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infections, isolation of primary astroglial and endothelial cells, and assembly of the in vitro BBB model is presented. These techniques can be used to analyze the interaction of adaptive and innate immune system cells with brain endothelial cells, cellular transmigration, BBB morphological and functional changes, and methods of bacterial dissemination. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of primary mouse brain astrocytes and endothelial cells Basic Protocol 2: Isolation of primary mouse bone marrow-derived dendritic cells Support Protocol 1: Validation of dendritic cell purity by flow cytometry Basic Protocol 3: Isolation of primary mouse peripheral blood mononuclear cells Support Protocol 2: Isolation of primary mouse spleen cells Support Protocol 3: Purification and validation of CD4+ T cells from PBMCs and spleen cells Basic Protocol 4: Isolation of liver granuloma supernatant and determination of organ load Support Protocol 4: In vivo and in vitro infection with mycobacteria Basic Protocol 5: Assembly of the BBB co-culture model Basic Protocol 6: Assembly of the combined in vitro granuloma and BBB model.

An Insight into the Potential Parasitological Effect ofSchistosoma mansoniAntigens in Infected Mice: Prophylactic Role of Cercarial Antigen

Schistosomiasis is a multifactorial disease that includes environmental, behavioral, parasitic, vector, and host factors. This study aimed to assess the protective effect of single and polyvalent antigens from cercarial antigen preparations (CAPs), soluble worm antigen preparations (SWAPs), and soluble egg antigens (SEAs) which were used as candidate vaccines in an experimental model of Schistosoma mansoni-infected mice. The efficiency of the antigens was tested by determining their effects on fecal egg count, egg viability analysis, and tissue egg counts. Histological and morphometric analyses of granulomas in liver and intestine tissues were performed. In the present study, all immunized groups showed a significant reduction in the average fecal egg count and tissue egg load compared with infected mice. The most substantial reduction in fecal egg count was observed in the combined vaccinated group (23.23 ± 3.2). The group vaccinated with CAP before infection showed the highest reduction in tissue egg load (liver and intestine: 85.22 and 91.70%, respectively). Immunized animals showed a highly significant reduction in the numbers of hepatic granulomas compared with the infected non-immunized group. In conclusion, combining these different antigens (CAP, SWAP, and SEA) augments the protective immunity compared with other immunized groups.

Schistosoma mansoni FES Tyrosine Kinase Involvement in the Mammalian Schistosomiasis Outcome and Miracidia Infection Capability in Biomphalaria glabrata.

Schistosomiasis is a neglected tropical disease (NTD) caused by helminthes from the Schistosoma genus. This NTD can cause systemic symptoms induced by the deposition of parasite eggs in the host liver, promoting severe complications. Functional studies to increase knowledge about parasite biology are required for the identification of new drug targets, because the treatment is solely based on praziquantel administration, a drug in which the mechanism of action is still unknown. Protein kinases are important for cellular adaptation and maintenance of many organisms homeostasis and, thus, are considered good drug targets for many pathologies. Accordingly, those proteins are also important for Schistosoma mansoni, as the parasite relies on specific environmental signals to develop into its different stages. However, the specific roles of protein kinases in S. mansoni biology are not well understood. This work aims at investigating the tyrosine-protein kinase FES (Feline Sarcoma) functions in the maintenance of S. mansoni life cycle, especially in the establishment of mammalian and invertebrate hosts’ infection. In this regard, the verification of Smfes expression among S. mansoni stages showed that Smfes is more expressed in infective free-living stages: miracidia and cercariae. Schistosomula exposed to SmFES-dsRNA in vitro presented a reduction in movement and size and increased mortality. Mice infected with Smfes-knocked-down schistosomula exhibited a striking reduction in the area of liver granuloma and an increased rate of immature eggs in the intestine. Female adult worms recovered from mice presented a reduced size and changes in the ovary and vitellarium; and males exhibited damage in the gynecophoral canal. Subsequently, miracidia hatched from eggs exposed to SmFES-dsRNA presented changes in its capability to infect and to sense the snail mucus. In addition, the SmFES RNAi effect was stable from miracidia to cercariae. The establishment of infection with those cercariae reproduced the same alterations observed for the knocked-down schistosomula infection. Our findings show that SmFES tyrosine kinase (1) is important in schistosomula development and survival; (2) has a role in adult worms pairing and, consequently, female maturation; (3) might be essential for egg antigen expression, thus responsible for inducing granuloma formation and immunomodulation; and (4) is essential for miracidia infection capability. In addition, this is the first time that a gene is kept knocked down during three different S. mansoni life stages and that a tyrosine kinase is implicated in the parasite reproduction and infection establishment in the mammalian host. Accordingly, SmFES should be explored as an alternative to support schistosomiasis treatment and morbidity control.

Bevacizumab as a potential anti-angiogenic therapy in schistosomiasis: A double-edged, but adjustable weapon.

Investigating the anti-angiogenic effect of bevacizumab on chronic schistosomiasis mansoniin atrial to hinder the Schistosome-induced angiogenesis and porto-systemic shunting complications. The immunohistochemical expression of CD34, VEGF-R1, PCNA and α-SMA (angiogenesis markers) was analysed in the lung, liver and gastrointestinal junctions of chronic Smansoniinfected mice after intraperitoneal injection of bevacizumab. The effect of prolongedadministration of bevacizumab with praziquantel was also assessed through parasitic load, protective index, granuloma and fibrous tissue evaluation. A regression in thevascular activity and microvascular density wasobserved in the infected mice after receivingbevacizumab. They had a significantly less VEGF-R1, PCNA, CD-34 and α-SMA expression in comparison to the infected untreatedmice. The least tissue egg count was reported in mice received bevacizumab for 6weeks (Mean=27120). However, they had persistent liver granulomas, and massively amalgamated fibrosis. Interestingly, the least faecal egg and tissue worms counts (Mean=112, 13.4), and thehighest protection index (39.26) were reported in mice received bevacizumab for 3weeks, with marked granuloma, and fibrous tissue resolution. Bevacizumabhas a promising protective effect against the Schistosoma-induced angiogenesis. As an adjuvant to praziquantel, it is important to adjust the appropriateduration of administration that achieves the best schistosomicidal effect without impeding granuloma and fibrous tissue resolution.

Inhibition of the NAD salvage pathway in schistosomes impairs metabolism, reproduction, and parasite survival.

NAD, a key co-enzyme required for cell metabolism, is synthesized via two pathways in most organisms. Since schistosomes apparently lack enzymes required for de novo NAD biosynthesis, we evaluated whether these parasites, which infect >200 million people worldwide, maintain NAD homeostasis via the NAD salvage biosynthetic pathway. We found that intracellular NAD levels decline in schistosomes treated with drugs that block production of nicotinamide or nicotinamide mononucleotide–known NAD precursors in the non-deamidating salvage pathway. Moreover, in vitro inhibition of the NAD salvage pathway in schistosomes impaired egg production, disrupted the outer membranes of both immature and mature parasites and caused loss of mobility and death. Inhibiting the NAD salvage pathway in schistosome-infected mice significantly decreased NAD levels in adult parasites, which correlated with reduced egg production, fewer liver granulomas and parasite death. Thus, schistosomes, unlike their mammalian hosts, appear limited to one metabolic pathway to maintain NAD-dependent metabolic processes.

Xiaochaihu decorction relieves liver fibrosis caused by Schistosoma japonicum infection via the HSP47/TGF-\u03b2 pathway

BackgroundHepatic fibrosis caused by chronic infection with Schistosoma japonica remains a serious public health problem in the world. Symptoms include inflammation, liver granuloma and fibrosis, whilst treatment options are still limited. This study aims to investigate whether and how traditional Chinese medicine Xiaochaihu decoction (XCH) could mitigate liver fibrosis caused by S. japonicum infection.MethodsBALB/c mice were infected with S. japonicum cercariae and treated with XCH for 16 weeks. Liver pathological changes were assessed by H&E and Masson staining. NIH3T3 and Raw264.7 cells were treated with S. japonicum egg antigens with or without XCH treatment. Quantitative real-time PCR, western blot, immunfluorescence and ELISA were performed to determine the changes of levels of fibrogenic markers.ResultsXCH protected mouse liver from injuries and fibrosis caused by S. japonicum infection and considerably reduced egg burden in a dose-dependent manner. Infection with S. japonicum caused elevation of serum ALT, AST, ALP, HA and PIIINP levels and reduction of ALB and GLOB levels, which was markedly suppressed by XCH. The upregulation of TGF-β1, Hsp47, α-SMA, Col1A1 and Col3A1 in S. japonicum-infected mouse liver was also significantly inhibited by XCH. Schistosoma japonicum egg antigens promoted the expression of Hsp47, TGF-β1, Timp-1, α-SMA, Col1A1 and Col3A1 in NIH3T3 cells, and TGF-β1, CTGF, IL-13, IL-17 and IL-6 in Raw264.7 cells, which was inhibited by XCH, LY2157299 and shRNA-Hsp47.ConclusionsThese results demonstrated that the hepatic protective effects of Xiaochaihu decoction were mediated by HSP47/TGF-β axis.

Relevance of inducible nitric oxide synthase for immune control of Mycobacterium avium subspecies paratuberculosis infection in mice

ABSTRACT Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease (JD), an incurable chronic intestinal bowel disease in ruminants. JD occurs worldwide and causes enormous economic burden in dairy industry. Research on JD pathobiology is hampered by its complexity which cannot completely be mimicked by small animal models. As a model the mouse allows dissecting some pathogenicity features of MAP. However, for unknown reasons MAP exhibits reduced growth in granulomas of infected mice compared to other Mycobacterium avium subspecies. Here, we characterized immune reactions of MAP-infected C57BL/6 mice. After infection, mice appeared fully immunocompetent. A strong antigen-specific T cell response was elicited indicated by IFNγ production of splenic T cells re-stimulated with MAP antigens. Function of splenic dendritic cells and proliferation of adoptively transferred antigen-specific CD4+ T cells was unaltered. Isolated splenic myeloid cells from infected mice revealed that MAP resides in CD11b+ macrophages. Importantly, sorted CD11b+CD11c− cells expressed high level of type 2 nitric oxide synthase (NOS2) but only low levels of pro- and anti-inflammatory cytokines. Correspondingly, MAP-infected MAC2 expressing myeloid cells in spleen and liver granuloma displayed strong expression of NOS2. In livers of infected Nos2−/− mice higher bacterial loads, more granuloma and larger areas of tissue damage were observed 5 weeks post infection compared to wild type mice. In vitro, MAP was sensitive to NO released by a NO-donor. Thus, a strong T cell response and concomitant NOS2/NO activity appears to control MAP infection, but allows development of chronicity and pathogen persistence. A similar mechanism might explain persistence of MAP in ruminants.

Wnt5A Signaling Restrains Leishmania donovani infection in a mouse model

Abstract Objective In view of the emergence of drug resistant strains of L. donovani and persistence of Kala-azar, it is important to understand how host derived factors promote immune resistance to L. donovani infection. Accordingly, considering the role of Wnt5A in maintenance of immune homeostasis, our objective is to evaluate the potential of host Wnt5A signaling in restraining L. donovani infection independent of drug sensitivity or resistance, utilizing a mouse model. Methods Effect of Wnt5A depletion on L. donovani infection was deciphered using Wnt5A heterozygous mice with reference to wild type cohorts, where mice were infected with L. donovani promastigotes through iv route. Effect of augmented Wnt5A signaling on L. donovani infection was investigated by iv injection of recombinant Wnt5A protein (rWnt5A) prior to infection. Infection load in liver and spleen was enumerated by Leishman Donovan Unit (LDU) and evaluated by histology. Plasma cytokine levels were checked by ELISA. Results Wnt5A heterozygous mice display substantially more disorganized splenic germinal centers and liver granulomas and about 50–60% more LDU upon L. donovani infection as compared to their wild type partners. rWnt5A treatment causes 40–80% reduction in infection load, which correlates with decrease or abolition of splenic germinal center disorganization and liver granulomas, and increase in pro-inflammatory cytokine to anti-inflammatory cytokine ratio. Conclusion Our results indicate that (i) Wnt5A signaling protects the host from L. donovani infection independent of drug sensitivity or resistance and (ii) rWnt5A has potential as a prophylactic agent for prevention of L. donovani infection and incidence of Kala-azar.

Macrophage replacement in Mycobacterium tuberculosis-induced murine lung granulomas

Abstract Monocytes/macrophages are the dominant cell type in mycobacterial granulomas. Macrophages both provide a survival niche for the bacteria and play a central role in bacterial control. Most of them are recruited from the blood with a Ly6C+ phenotype and they differentiate into F4/80+ macrophages in the lung lesions. It is well demonstrated that monocytes have access to pre-formed granulomatous lesions, but the rate of cell recruitment and the fate of the recruited cells are not well understood. Previously, testing BCG-induced liver granulomas using a transplantation model, we detected continuous cell replacement in the preformed lesions. Here, using intravital staining and cytofluorimetric detection of CD45+ blood cells in combination with IHC, we show that CD11b+ cells can access the lung parenchyma and Mtb-induced lesions over the course of 24 hours in Mtb H37Rv infected animals. We measured the level of monocyte/macrophage infiltration in both acute and chronic infection in C57BL/6 and C3HeB/FeJ mice. Although monocytes have a short lifespan in the blood, using intravenous cell transfer of tdTomato-expressing sentinel cells we show that they can live longer than a week in the lung lesions of Mtb-infected animals. Interestingly, a population of adoptively transferred CD11b+tdTomato+ cells can be detected for more than 10 days in the blood vessels in the infected lung. Gene expression profiling of recently or earlier recruited (CD11b+F4/80− or CD11b+F4/80+) cells sorted from the lung shows differential inflammatory gene expression between these populations. The continuous cell replacement that is required to maintain granulomatous lesions offers a novel possibility to manipulate granuloma pathology for a therapeutic advantage.

Chronic whipworm infection exacerbates Schistosoma mansoni egg-induced hepatopathology in non-human primates

BackgroundSchistosomiasis continues to inflict significant morbidity and mortality in the tropical and subtropical regions of the world. The disease endemicity overlaps with the transmission of other parasitic diseases. Despite the ubiquity of polyparasitism in tropical regions, particularly in rural communities, little is known about the impact of multiple helminth infections on disease progression. In this pilot study, we describe the influence of chronic Trichuris trichiura infection on Schistosoma mansoni egg-induced hepatopathology in infected baboons.MethodsBaboons with or without underlying whipworm infection were challenged with S. mansoni cercariae to establish schistosomiasis. Adult S. mansoni worms were recovered by perfusion and enumerated, hepatic granulomas were quantified via light microscopy, and transcriptional profiling of tissues were completed using RNA sequencing technologies.ResultsCo-infection with both S. mansoni and T. trichiura resulted in higher female schistosome worm burden and significantly larger liver granuloma sizes. Systems biology analyses of peripheral blood mononuclear cells (PBMC) revealed pathways associated with increased liver damage in co-infected baboons.ConclusionsUnderlying chronic whipworm infection intensified schistosome egg-induced liver pathology in infected baboons. RNA-Seq analysis provided insight into pathways associated with increased liver damage, corroborating histological findings.