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.

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.

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.

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.

CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice.

CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.

IL-25 and type 2 innate lymphoid cells induce pulmonary fibrosis.

Disease conditions associated with pulmonary fibrosis are progressive and have a poor long-term prognosis with irreversible changes in airway architecture leading to marked morbidity and mortalities. Using murine models we demonstrate a role for interleukin (IL)-25 in the generation of pulmonary fibrosis. Mechanistically, we identify IL-13 release from type 2 innate lymphoid cells (ILC2) as sufficient to drive collagen deposition in the lungs of challenged mice and suggest this as a potential mechanism through which IL-25 is acting. Additionally, we demonstrate that in human idiopathic pulmonary fibrosis there is increased pulmonary expression of IL-25 and also observe a population ILC2 in the lungs of idiopathic pulmonary fibrosis patients. Collectively, we present an innate mechanism for the generation of pulmonary fibrosis, via IL-25 and ILC2, that occurs independently of T-cell-mediated antigen-specific immune responses. These results suggest the potential of therapeutically targeting IL-25 and ILC2 for the treatment of human fibrotic diseases.

Acanthamoeba interactions with the blood-brain barrier under dynamic fluid flow.

Acanthamoeba granulomatous encephalitis (AGE), caused by Acanthamoeba castellanii, is a fatal infection of immunocompromised individuals. The pathogenesis of blood-brain barrier (BBB) breach remains unknown. Using a novel in vitro BBB infection model under flow conditions, demonstrates that increases in flow rates lead to decreased binding of A. castellanii to host cells. This is a distinct departure from previous findings under static conditions. However, similarly to static conditions binding of A. castellanii to host cells is host mannose dependent. Disruption of the host cell monolayer was independent of amoeba binding, but dependent on secreted serine proteases. For the first time we report the binding dynamics of A. castellanii under physiological conditions, showing that BBB disruption is not directly linked to binding, instead it is reliant on secreted proteases. Our results offer a platform on which therapies designed at modulating physiological parameters can improve the outcome of infection with A. castellanii.

Parasite limiting macrophages promote IL-17 secretion in naive bovine CD4⁺ T-cells during Neospora caninum infection.

Neospora caninum infects bovine hosts giving rise to pro-inflammatory immune responses that can result in foetal death or spontaneous abortion, this appears to be mediated by the actions of IFN-γ on cell activation and migration/trafficking. Yet successful vaccination or natural immunity is also strongly correlated with IFN-γ production. We utilised in vitro infection of bovine macrophages to prime naive T-cell responses. Naive T-cells in contact with infected macrophages produce both IFN-γ and IL-17 in a pattern that is dependent on whether the priming macrophage was protected or non-protected. Our results may explain the apparent dual role of IFN-γ during infection if a second major pro-inflammatory cytokine, IL-17, is produced simultaneously.

Toxoplasma gondii peroxiredoxin promotes altered macrophage function, caspase-1-dependent IL-1ß secretion enhances parasite replication.

Alternatively activated macrophages (AAM) are a key feature Th2 immunity and have been associated with a variety of roles during helminth infection. The role this cell subset plays in protozoan infection remain relatively unexplored, herein we describe the effects of a redox enzyme (rTgPrx) derived from Toxoplasma gondii on murine macrophage phenotype in vitro. RTgPrx has been previously associated with the maintainance of parasite oxidative balance. Here our experiments show that rTgPrx promotes AAM as indicated by high arginase-1 (arg-1), YM1 and FIZZ expression via both signal transducer and activator of transcription (STAT)6-dependent and -independent mechanisms. Additionally rTgPrx treatment reduced caspase-1 activity and IL-1ß secretion, while simultaneously increasing IL-10 release. Furthermore the in vitro replication of T. gondii (RH strain) was enhanced when macrophages were treated with rTgPrx. This is in contrast with the previously described effects of a Plasmodium berghei ANKA 2-cys-peroxiredoxin that promotes pro-inflammatory cytokine production. These results highlight the role of T. gondii derived redox enzymes as important immune modulators and potentially indicate a role for AAM in modulating immunopathology and promoting parasite replication during T. gondii infection.

Possible role for Toll-like receptors in interaction of Fasciola hepatica excretory/secretory products with bovine macrophages.

Alternative activation of macrophages (Mphi) during helminth infection is a characteristic feature of the host immune response. Alternatively activated macrophages (AAMphi) are distinguished from others by high arginase 1 (Arg-1) activity, low nitric oxide (NO), and high interleukin 10 (IL-10) production. In murine models, these cells have been shown to possess anti-inflammatory properties. They have also been implicated in exacerbating a subsequent infection with a secondary pathogen. In this study we used cattle experimentally infected with Fasciola hepatica to monitor the kinetics of IL-4 and IL-10 over the course of infection. Using naïve Mphi in vitro, we examined the effects of exposure to F. hepatica excretory/secretory products (FhepES) alone or in combination with IL-4. Our results suggest that FhepES may work in combination with IL-4 to produce AAMphi. The effects of FhepES on the subsequent responses to lipopolysaccharide (LPS) and purified protein derivative from Mycobacterium bovis (PPD-B), which are bovine Toll-like receptor 4 (TLR4) and TLR2 antagonists, respectively, were also examined. We found that Mphi stimulated with FhepES together with LPS or PPD-B have reduced NO or gamma interferon production, respectively. The ability of FhepES to produce AAMphi was found to be heat labile and partially dependent on glycan residues. A possible role for TLR recognition is discussed.