Interplays of liver fibrosis-associated microRNAs: Molecular mechanisms and implications in diagnosis and therapy.

microRNAs (miRNAs) are a class of non-coding functional small RNA composed of 21-23 nucleotides, having multiple associations with liver fibrosis. Fibrosis-associated miRNAs are roughly classified into pro-fibrosis or anti-fibrosis types. The former is capable of activating hepatic stellate cells (HSCs) by modulating pro-fibrotic signaling pathways, mainly including TGF-ß/SMAD, WNT/ß-catenin, and Hedgehog; the latter is responsible for maintenance of the quiescent phenotype of normal HSCs, phenotypic reversion of activated HSCs (aHSCs), inhibition of HSCs proliferation and suppression of the extracellular matrix-associated gene expression. Moreover, several miRNAs are involved in regulation of liver fibrosis via alternative mechanisms, such as interacting between hepatocytes and other liver cells via exosomes and increasing autophagy of aHSCs. Thus, understanding the role of these miRNAs may provide new avenues for the development of novel interventions against hepatic fibrosis.

microRNAs in parasite-induced liver fibrosis: from mechanisms to diagnostics and therapeutics.

Chronic parasite infections in the liver pose a global threat to human and animal health, often occurring with liver fibrosis that leads to cirrhosis, liver failure, and even cancer. Hepatic fibrogenesis is a complex yet reversible process of tissue repair and is associated with various factors, including immune cells, microenvironment, gut microbiome, and interactions of the different liver cells. As a profibrogenic or antifibrogenic driver, microRNAs (miRNAs) are closely involved in parasite-induced hepatic fibrosis. This article updates the current understanding of the roles of miRNAs in hepatic fibrogenesis by parasite infections and discusses the strategies using miRNAs as candidates for diagnostics and therapeutics.

Identification and expression of a transforming growth factor beta (TGF-ß) homologue in the tropical liver fluke Fasciola gigantica.

Liver flukes, Fasciola spp., are veterinary and medically important parasites infecting numerous species of economically important animals in addition to humans on a global scale. The components of transforming growth factor beta (TGF-ß) signalling are widely distributed throughout the animal kingdom and are considerably conserved. Through shared common signal transduction mechanisms, crosstalk of TGF-ß signalling between a host and the parasite during infection is possible. Herein, we have identified and undertaken the molecular characterisation of a putative TGF-ß homologue from the tropical liver fluke F. gigantica (FgTLM). A FgTLM cDNA was 3557 bp in length, it encoded for 620 amino acid polypeptide which consisted of 494 amino acids of prodomain and 126 amino acids comprising the mature protein. FgTLM displayed characteristic structures of mammalian TGF-ß ligands that were unique to the inhibin-ß chain, monomer of activin. A phylogenetic analysis revealed the high degree of conservation with TGF-ß molecules from trematode species. Interestingly, the sequence of amino acid in the active domain of FgTLM was completely identical to FhTLM from F. hepatica. FgTLM expressed throughout the lifecycle of F. gigantica but was highly expressed in developmental active stages. The dynamics of expression of FgTLM during the developmental stages of F. gigantica was comparable to the pattern of TGF-ß expression in F. hepatica. Our findings demonstrated that FgTLM exhibits a high level of similarity to FhTLM in the context of both amino acid sequence and the life stage expression patterns. These similarities underline the possibility that the FgTLM molecule might have the same properties and functions as FhTLM in biological processes of the immature parasites and host immune evasion. Consequently, the specific biological functions of FgTLM on either parasite or relevant hosts need to be defined experimentally.

Vivaxin genes encode highly immunogenic, non-variant antigens on the Trypanosoma vivax cell-surface.

Trypanosoma vivax is a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal livestock disease across sub-Saharan Africa. Previously, we identified diverse T. vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum shows that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now call Vivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-ß8) that encodes one particularly immunogenic vivaxin protein and which is highly expressed during infections but displays minimal polymorphism across the parasite population. Vaccination of mice with VIVß8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals but, ultimately, it does not prevent disease. Although VIVß8 is localized across the cell body and flagellar membrane, live immunostaining indicates that VIVß8 is largely inaccessible to antibody in vivo. However, our phylogenetic analysis shows that vivaxin includes other antigens shown recently to induce immunity against T. vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of the T. vivax cell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence host-parasite interactions.

Cleaved CD95L perturbs in vitro macrophages responses to Toxoplasma gondii.

Toxoplasma gondii infects approximately 1-2 billion people, and manipulation of the macrophage response is critical to host and parasite survival. A cleaved (cl)-CD95L form can promote cellular migration and we have previously shown that cl-CD95L aggravates inflammation and pathology in systemic lupus erythematosus (SLE). Findings have shown that CD95L is upregulated during human infection, therefore we examined the effect of cl-CD95L on the macrophage response to T. gondii. . We find that cl-CD95L promotes parasite replication in macrophages, associated with increased arginase-1 levels, mediated by signal transducer and activator of transcription (STAT)6. Inhibition of both arginase-1 and STAT6 reversed the effects of cl-CD95L. Phospho-kinase array showed that cl-CD95L alters Janus Kinases (JAK)/STAT, mammalian target of rapamycin (mTOR), and Src kinase signals. By triggering changes in JAK/STAT cl-CD95L may limit anti-parasite effectors.

Trichinella spiralis cystatin, TsCstN, modulates STAT4/IL-12 to specifically suppress IFN-γ production.

We have previously identified a cystatin, TsCstN, derived from the L1 stage of Trichinella spiralis and have shown that this protein is internalised in macrophages. Here we sought to address if this macrophage-TsCstN interaction could alter downstream T-cell priming. Using LPS-primed macrophages to stimulate T-cells in a co-culture system with or without TsCstN we assessed the resultant T-cell outcomes. IFN-γ, both protein and mRNA, but not IL-17A was negatively regulated by inclusion of TsCstN during macrophage priming. We identified a cell-cell contact independent change in the levels of IL-12 that led to altered phosphorylated STAT4 levels and translocation. TsCstN also negatively regulated the autonomous response in the myotubule cell line, C2C12. This work identifies a potential pathyway for L1 larvae to evade protective Th1 based immune responses and establish muscle-stage T. spiralis infection.

A host-independent role for Fasciola hepatica transforming growth factor-like molecule in parasite development.

The trematode parasite Fasciola hepatica causes chronic infection in hosts, enabled by an immunosuppressed environment. Both host and parasite factors are known to contribute to this suggesting that avoidance of immunopathology is beneficial to both parties. We have previously characterised a parasite transforming growth factor (TGF)-like molecule, FhTLM, that interacts with host macrophages to prevent antibody-dependent cell cytotoxicity (ADCC). FhTLM is one of many described helminth TGF homologues and multiple helminths are now known to utilise host immune responses as developmental cues. To test whether, or how, F. hepatica uses FhTLM to manipulate host immunity, we initially examined its effects on the CD4 T-cell phenotype. Despite inducing IL-10, there was no induction of FoxP3 within the CD4 T-cell compartment. In addition to inducing IL-10, a wide range of chemokines were elicited from both CD4 T-cells and macrophages. However, no growth or survival advantage was conferred on F. hepatica in our co-culture system when CD4 T-cells, macrophages, or eosinophils were tested. Finally, using RNA interference we were able to verify a host-independent role for FhTLM in parasite growth. Despite the similarities of FhTLM with other described helminth TGF homologues, here we demonstrate species-specific divergence.

Stem cell-derived enteroid cultures as a tool for dissecting host-parasite interactions in the small intestinal epithelium.

Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant and species-specific models. Increasingly, 3D stem cell-derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review, we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments.

Evasion of Host Immunity During Fasciola hepatica Infection.

Fasciola hepatica, the common liver fluke, causes infection of livestock throughout temperate regions of the globe. This helminth parasite has an indirect lifecycle, relying on the presence of the mud snail to complete its transition from egg to definitive host (Beesley et al., Transbound Emerg Dis 65:199-216, 2017). Within the definitive host, the parasite excysts in the intestine forming a newly excysted juvenile (NEJ) and migrates via the peritoneal cavity to the liver. Disease resulting from infection can be acute or chronic depending on the host and the number of parasites present. Sheep may succumb to a fatal acute infection if the challenge of metacercariae is great enough. However, in cattle chronic disease is the most likely outcome with parasites surviving for long periods of time. Annual losses are estimated to be in the region of US$ 2000 million to the agricultural industry (Beesley et al., Transbound Emerg Dis 65:199-216, 2017). Management of the disease depends heavily on chemotherapy with triclabendazole being the drug of choice, consistent use for over 20 years has resulted in drug-resistant strains emerging worldwide (Beesley et al., Int J Parasitol 47:11-20, 2017). A more sustainable approach to control would be through vaccination and indeed a lead candidate has been identified, cathepsin L1. Despite these promising results the parasite continues to confound our own and host efforts to generate long-lasting and effective immunity. In this brief review we focus our attention on those mechanisms that the parasite utilises to circumvent the innate based defense mechanisms within the host.

A novel cystatin derived from Trichinella spiralis suppresses macrophage-mediated inflammatory responses.

Trichinella spiralis can modulate host immune responses to retain a suitable environment for its long-term survival. Incidentally, the parasite elicits regulatory effects through immunomodulatory molecule release, which can suppress host inflammation and may be used for the treatment of unrelated inflammatory diseases in someday. Here we identified and characterized a novel T. spiralis cystatin (TsCstN), which inhibits inflammation mediated by LPS-treated macrophages.Proteins contained in the excretory-secretory (ES) product of muscle-stage T. spiralis (ES-L1) were fractionated, and each was treated with mouse bone marrow-derived macrophages (mBMDMs) before LPS stimulation. The fractions that exhibited high immunomodulatory property by decreasing pro-inflammatory cytokines or increasing anti-inflammatory cytokines were identified by mass spectrometry. Incidentally, the conserved hypothetical protein (Tsp_04814) was selected for further characterization as it presented the most significant MS score. An annotation of Tsp_04814 using protein structural homology comparison suggested that it has high structural similarity to human cystatin E/M (TM score 0.690). The recombinant T. spiralis novel cystatin (rTsCstN) was expressed in Escherichia coli at a molecular weight of approximately 13 kDa. Mouse anti-rTsCstN polyclonal antibody (pAb) could detect native TsCstN in crude worm antigens (CWA) and ES-L1 and be predominantly localized in the stichosome and subcuticular cells. rTsCstN inhibited cysteine proteases in vitro, especially cathepsin L, at an optimal pH of 6. Besides, rTsCstN could be internalized into mBMDMs, which were mostly distributed in the cytoplasm and lysosome both before and after LPS stimulation. To evaluate the rTsCstN immunomodulatory properties on mBMDMs, rTsCstN was incubated with mBMDM before LPS stimulation; this demonstrated that rTsCstN suppressed pro-inflammatory cytokine production and MHC class II expression.T. spiralis L1-derived TsCstN was characterized as a novel cysteine protease inhibitor. The protein elicits an anti-inflammatory property by suppressing pro-inflammatory cytokines and interfering with the antigen presentation process through depletion of MHC class II expression.

An analysis of the immune compartment within bovine adipose tissue.

Adipose tissue (AT) has wide functions as an active endocrine organ acting as a site of nutrient storage and thermogenesis. Recently it has been identified as having a key role in murine and human immunity and inflammation. Type 1 or type 2 immune responses and their respective cytokines have been linked to white or brown AT, respectively. Most dramatic is the involvement of type-2 innate lymphoid cells (ILC2s) in stimulating eosinophil recruitment via interleukin (IL)-13 which in turn stimulates alternative macrophage activation via IL-4/IL-13. Recruited leukocytes are capable of influencing the cellular composition and function of adipose tissue and present a route to combat human obesity, however these processes are poorly understood in ruminants. Here we have characterised the resident leukocytes populations within bovine mesenteric AT (MAT) and subcutaneous AT (SAT), compared with the corresponding mesenteric lymph node (MLN). Concurring with related studies, we find bovine AT has its own resident leukocyte populations where eosinophils and neutrophils dominate. Importantly the proportion of eosinophils or neutrophils corresponded to the adipocyte size found in both depots. Further exploration of this area may have important implications on the food production industry or could be applied to improve the course of pathogenesis during disease.

Prevalence of vertically transmitted Neospora caninum amongst beef cattle in Phayao, Thailand.

Neospora caninum, the causative agent of neosporosis, is recognised as a significant trigger of abortion and productivity losses in cattle worldwide. Current information regarding to the prevalence of N. caninum in Thailand is limited due to the limitations of detection methods and the difficulty of recovering of viable parasite. Vertical transmission is the main route of N. caninum infection in cattle. Therefore, detection of N. caninum DNA in placental tissue could be a possible means of laboratory diagnosis of neosporosis in live animals, particularly in the context of transplacental transmission. The aim of this study was to investigate the prevalence of transplacentally transmitted N. caninum infection in female beef cattle in the northern Thai province of Phayao by detection of N. caninum DNA in bovine placenta by PCR. A total of 96 bovine placentas were collected from 7 districts of Phayao. Our result indicated that overall PCR prevalence of N. caninum in cattle in this area was 36.5% varying from 16.7-50.0% between districts. The districts with the highest prevalence of infection were Muang (50.0%) and Mae Chai (44.7%). The proportion of N. caninum infection was quite high suggesting that newborn calves were at risk of congenital infection. This study provides a current snapshot of the status of bovine neosporosis in Phayao which could lead to the development of effective strategies for prevention and control this disease.

IL-17 and IL-22 elicited by a DNA vaccine encoding ROP13 associated with protection against Toxoplasma gondii in BALB/c mice.

Toxoplasma gondii, an intracellular parasitic protozoan, is capable of infecting man and all warm-blooded animals. Cell-mediated immunity is vital in mounting protective responses against T. gondii infection. Recent studies have shown that T-helper (Th) 17 responses may play a key role in parasite control. In this current study, we constructed a DNA vaccine encoding T. gondii ROP13 in a pcDNA vector. Groups of BALB/c mice were immunized intramuscularly with pcROP13 or controls and challenged with the RH strain of T. gondii. The results showed that immunization with pcROP13 could elicit an antibody response against T. gondii. The expression of the canonical Th17 cytokines, interleukin (IL)-17 and IL-22, were significantly increased after immunization with pcROP13 compared with control groups ( p < 0.05). Furthermore, vaccination resulted in a significant decrease in parasite load ( p < 0.05). The induction of Th17 related cytokines, using a ROP13 DNA vaccine, against T. gondii should be considered as a potential vaccine approach for the control of toxoplasmosis.

Fasciola hepatica, TGF-ß and host mimicry: the enemy within.

Helminths parasites undergo developmental changes and migration within their definitive host, in addition to establishing chronic infection. Essential to this is the evasion of host immune responses; the canonical Th2 response is effective at removing parasites resident in the intestine. Conversely, helminths also promote the development of antigen-specific anergy and regulation. This often limits pathology but allows parasite survival, parasite effectors mediating this are the subject of intense study. They may be useful as future vaccine targets or xenogenic therapeutics. Fasciola hepatica possesses a family of TGF-like molecules of which one member, FhTLM, is capable of promoting intrinsic and extrinsic effects. Here we review the extrinsic effects of FhTLM on the host macrophage and its consequences for protective immunity. This review also discusses the specificities of FhTLM in light a very recent description of a nematode TGF-ß mimic and the effects of endogenous TGF-ß.

Bovine Neonatal Monocytes Display Phenotypic Differences Compared With Adults After Challenge With the Infectious Abortifacient Agent Neospora caninum.

The neonatal period represents a window of susceptibility for ruminants given the abundance of infectious challenges in their environment. Maternal transfer of immunity does not occur in utero but post-parturition, however this does not compensate for potential deficits in the cellular compartment. Here we present a cellular and transcriptomic study to investigate if there is an age-related difference in the monocyte response in cattle during intra-cellular protozoan infection. We utilized Neospora caninum, an obligate intracellular protozoan parasite that causes abortion and negative economic impacts in cattle worldwide, to study these responses. We found neonatal animals had a significant greater percentage of CD14+ monocytes with higher CD80 cell surface expression. Adult monocytes harbored more parasites compared to neonatal monocytes; additionally greater secretion of IL-1ß was observed in neonates. Microarray analysis revealed neonates have 535 genes significantly upregulated compared to adult with 23 upregulated genes. Biological pathways involved in immune response were evaluated and both age groups showed changes in the upregulation of tyrosine phosphorylation of STAT protein and JAK-STAT cascade pathways. However, the extent to which these pathways were upregulated in neonates was much greater. Our findings suggest that neonates are more resistant to cellular invasion with protozoan parasites and that the magnitude of the responses is related to significant changes in the JAK-STAT network.

The Chronic Stages of Bovine Fasciola hepatica Are Dominated by CD4 T-Cell Exhaustion.

Fasciola hepatica infection of ruminants leads to non-resolving chronic infection, as patency develops, there is switching to a TGF-ß and IL-10 led response. Here, we explore the responses of CD4 T-cells within the major draining lymph nodes. We found minimal expression of Foxp3 within CD4 cells but elevated levels within the γδ (WC1+) population. There is a strong T-cell-intrinsic exhaustion phenotype within the hepatic lymph node (HLN) characterized by a lack of antigen-specific proliferation and cytokine secretion. CD4 T-cells recovered from the HLN had high levels of PD-1 expression and low levels of IL-2 secretion. Exogenous IL-2 partially rescued this defect; when combined with neutralization of IL-10 and TGF-ß, full restoration of proliferation, and cytokine production was achieved. Moreover, there is a clear uncoupling of the mechanisms that facilitate this regulation with parasite-specific proliferation and cytokine secretion being governed by independent means. These data would suggest that there is a CD4 T-cell-intrinsic regulation in place early in chronic infection, potentially leading to failure in resistance to reinfection.

A rapid IL-17 response to Cryptosporidium parvum in the bovine intestine.

Cryptosporidium parvum causes diarrhoea, due to villi damage, in livestock and humans globally. Immunity develops after repeated infections but initial infections can be severe, highlighting the importance of early infection dynamics. We have modelled early C. parvum infection in bovine jejunum biopsies. IL-17A accumulated over time peaking at 9h post-infection, with no effect of infection on IL-1ß; antibiotics positively influenced IL-17A as higher levels were found in cultures with antibiotics. Infection of primary fibroblasts resulted in lower plaque formation when fibroblasts were primed with IL-17A. Our results indicate a role for IL-17A in reducing C. parvum-dependent host cell damage.

CD95 and the MRL-lpr Mouse Model.

CD95 (Fas-ligand) is a key mediator of cell death in multiple setting, thus its loss within the MRL-lpr (Faslpr) homozygote mice results in spontaneous autoimmunity. This is characterized by the development of arthritis and immune complex glomerulonephrosis making this strain a useful model for studying systemic lupus erythematosus. Herein we describe a method to exploit the heterozygote offspring of this strain in a model to study the effects of a CD95L blocking peptide on lupus-like disease in vivo.

A Trematode Parasite Derived Growth Factor Binds and Exerts Influences on Host Immune Functions via Host Cytokine Receptor Complexes.

The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allowing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-ß RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-ß RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory receptor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is dependent on TGF-ß RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody-dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages-again dependent on TGF-ß RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for dampened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced effector response targeting juvenile parasites which we demonstrate extends to an abrogation of the ADCC response. Thus suggesting that FhTLM is a stage specific evasion molecule that utilises host cytokine receptors. These findings are the first to clearly demonstrate the interaction of a helminth cytokine with a host receptor complex resulting in immune modifications that facilitate the non-protective chronic immune response which is characteristic of F. hepatica infection.

Mapping B-cell responses to Salmonella enterica serovars Typhimurium and Enteritidis in chickens for the discrimination of infected from vaccinated animals.

Serological surveillance and vaccination are important strategies for controlling infectious diseases of food production animals. However, the compatibility of these strategies is limited by a lack of assays capable of differentiating infected from vaccinated animals (DIVA tests) for established killed or attenuated vaccines. Here, we used next generation phage-display (NGPD) and a 2-proportion Z score analysis to identify peptides that were preferentially bound by IgY from chickens infected with Salmonella Typhimurium or S. Enteritidis compared to IgY from vaccinates, for both an attenuated and an inactivated commercial vaccine. Peptides that were highly enriched against IgY from at least 4 out of 10 infected chickens were selected: 18 and 12 peptides for the killed and attenuated vaccines, respectively. The ten most discriminatory peptides for each vaccine were identified in an ELISA using a training set of IgY samples. These peptides were then used in multi-peptide assays that, when analysing a wider set of samples from infected and vaccinated animals, diagnosed infection with 100% sensitivity and specificity. The data describes a method for the development of DIVA assays for conventional attenuated and killed vaccines.