Background & Aims The outcome of hepatitis B virus (HBV) infection may be influenced by early interactions between the virus and hepatocyte innate immune responses. To date, the study of such ...interactions during the very early step of infection has not been adequately investigated. Methods We used the HepaRG cell line, as well as primary human hepatocytes to analyze, within 24 h of exposure to HBV, either delivered by a physiologic route or baculovirus vector (Bac-HBV), the early modulation of the expression of selected antiviral/pro-inflammatory cytokines and interferon stimulated genes. Experiments were also performed in the presence or absence of innate receptor agonists to investigate early HBV-induced blockade of innate responses. Results We show that hepatocytes themselves could detect HBV, and express innate genes when exposed to either HBV virions or Bac-HBV. Whereas Bac-HBV triggered a strong antiviral cytokine secretion followed by the clearance of replicative intermediates, a physiologic HBV exposure led to an abortive response. The early inhibition of innate response by HBV was mainly evidenced on Toll-like receptor 3 and RIG-I/MDA5 signaling pathways upon engagement with exogenous agonist, leading to a decreased expression of several pro-inflammatory and antiviral cytokine genes. Finally, we demonstrate that this early inhibition of dsRNA-mediated response is due to factor(s) present in the HBV inoculum, but not being HBsAg or HBeAg themselves, and does not require de novo viral protein synthesis and replication. Conclusions Our data provide strong evidence that HBV viral particles themselves can readily inhibit host innate immune responses upon virion/cell interactions, and may explain, at least partially, the “stealthy” character of HBV.
Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) and current treatments for chronic hepatitis B and HCC are suboptimal. Herein, we identified ...cellular serine/threonine Polo‐like‐kinase 1 (PLK1) as a positive effector of HBV replication. The aim of this study was to demonstrate the proviral role of PLK1 in HBV biosynthesis and validate PLK1 inhibition a potential antiviral strategy. To this end, we employed physiologically relevant HBV infection models of primary human hepatocytes (PHHs) and differentiated HepaRG cells in conjunction with pharmacologic PLK1 inhibitors, small interfering RNA (siRNA)‐mediated knockdown, and overexpression of constitutively active PLK1 (PLK1CA). In addition, a humanized liver Fah−/−/Rag2−/−/Il2rg−/− (FRG) mouse model was used to determine the antiviral effect of PLK1 inhibitor BI‐2536 on HBV infection in vivo. Finally, in vitro PLK1 kinase assays and site‐directed mutagenesis were employed to demonstrate that HBV core protein (HBc) is a PLK1 substrate. We demonstrated that HBV infection activated cellular PLK1 in PHHs and differentiated HepaRG cells. PLK1 inhibition by BI‐2536 or siRNA‐mediated knockdown suppressed HBV DNA biosynthesis, whereas overexpression of PLK1CA increased it, suggesting that the PLK1 effects on viral biosynthesis are specific and that PLK1 is a proviral cellular factor. Significantly, BI‐2536 administration to HBV‐infected humanized liver FRG mice strongly inhibited HBV infection, validating PLK1 as an antiviral target in vivo. The proviral action of PLK1 is associated with the biogenesis of the nucleocapsid, as BI‐2536 leads to its decreased intracellular formation/accumulation. In this respect, our studies identified HBc as a PLK1 substrate in vitro, and mapped PLK1 phosphorylation sites on this protein. Conclusion: PLK1 is a proviral host factor that could be envisaged as a target for combined antiviral and antitumoral strategies against HBV infection and HBV‐mediated carcinogenesis. (Hepatology 2017;66:1750–1765)
Many RNA viruses are found in protozoan parasites. They can be responsible for more serious pathology or treatment failure. For the detection of viral double-stranded RNA (dsRNA), sequence-dependent ...and -independent methods are available, such as quantitative real-time PCR and immunofluorescence, dot blot, ELISA or sequencing. The technique presented here is sequence-independent and is well detailed in the following protocol, taking the example of
RNA virus (LRV) in
) species. To summarise, the protocol is divided into four major steps: RNA extraction from the parasites, RNA purification, enzymatic digestions with DNase I and Nuclease S1, and visualization by gel electrophoresis. This method can be used to detect other viral dsRNA in other parasites. It provides an additional tool, complementary to other techniques previously cited and it is easy and quite fast to achieve.
The oxidative burst generated by the host immune system can restrict intracellular parasite entry and growth. While this burst leads to the induction of antioxidative enzymes, the molecular ...mechanisms and the consequences of this counter-response on the life of intracellular human parasites are largely unknown. The transcription factor NF-E2-related factor (NRF2) could be a key mediator of antioxidant signaling during infection due to the entry of parasites. Here, we showed that NRF2 was strongly upregulated in infection with the human Leishmania protozoan parasites, its activation was dependent on a NADPH oxidase 2 (NOX2) and SRC family of protein tyrosine kinases (SFKs) signaling pathway and it reprogrammed host cell metabolism. In inflammatory leishmaniasis caused by a viral endosymbiont inducing TNF-α in chronic leishmaniasis, NRF2 activation promoted parasite persistence but limited TNF-α production and tissue destruction. These data provided evidence of the dual role of NRF2 in protecting both the invading pathogen from reactive oxygen species and the host from an excess of the TNF-α destructive pro-inflammatory cytokine.
The liver is the largest gland in the human body and functions as an innate immune organ. Liver macrophages called Kupffer cells (KC) constitute the largest group of macrophages in the human body. ...Innate immune responses involving KC represent the first line of defense against pathogens in the liver. Human monocyte-derived macrophages have been used to characterize inflammasome responses that lead to the release of the proinflammatory cytokines IL-1β and IL-18, but it has not yet been determined whether human KC contain functional inflammasomes. We show, to our knowledge for the first time, that KC express genes and proteins that make up several different inflammasome complexes. Moreover, activation of KC in response to the absent in melanoma 2 (AIM2) inflammasome led to the production of IL-1β and IL-18, which activated IL-8 transcription and hepatic NK cell activity, respectively. Other inflammasome responses were also activated in response to selected bacteria and viruses. However, hepatitis B virus inhibited the AIM2 inflammasome by reducing the mRNA stability of IFN regulatory factor 7, which regulated AIM2 transcription. These data demonstrate the production of IL-1β and IL-18 in KC, suggesting that KC contain functional inflammasomes that could be important players in the innate immune response following certain infections of the liver. We think our findings could potentially aid therapeutic approaches against chronic liver diseases that activate the inflammasome.
Co-infection with the hepatitis B virus and hepatitis delta virus (HDV) leads to the most aggressive form of viral hepatitis. Using in vitro infection models, we confirmed that IL-1β, a crucial ...innate immune molecule for pathogen control, was very potent against HBV from different genotypes. Additionally, we demonstrated for the first time a strong and rapid antiviral effect induced by very low doses of IL-1β against HDV. In parallel, using co-culture assays, we demonstrated that monocytes exposed to HBV, and in particular to HBsAg, during differentiation into pro-inflammatory macrophages secreted less IL-1β. Altogether, our data emphasize the importance of developing combined antiviral strategies that would, for instance, reduce the secretion of HBsAg and stimulate the immune system to produce endogenous IL-1β efficient against both HBV and HDV.
Mitochondria regulate steroid hormone synthesis, and in turn sex hormones regulate mitochondrial function for maintaining cellular homeostasis and controlling inflammation. This crosstalk can explain ...sex differences observed in several pathologies such as in metabolic or inflammatory disorders. Nod-like receptor X1 (NLRX1) is a mitochondria-associated innate receptor that could modulate metabolic functions and attenuates inflammatory responses. Here, we showed that in an infectious model with the human protozoan parasite,
, NLRX1 attenuated inflammation in females but not in male mice. Analysis of infected female and male bone marrow derived macrophages showed both sex- and genotype-specific differences in both inflammatory and metabolic profiles with increased type I interferon production, mitochondrial respiration, and glycolytic rate in
-deficient female BMDMs in comparison to wild-type cells, while no differences were observed between males. Transcriptomics of female and male BMDMs revealed an altered steroid hormone signaling in
-deficient cells, and a "masculinization" of
-deficient female BMDMs. Thus, our findings suggest that NLRX1 prevents uncontrolled inflammation and metabolism in females and therefore may contribute to the sex differences observed in infectious and inflammatory diseases.
Leishmania
RNA virus 1 (LRV1) is a double-stranded RNA virus found in some strains of the human protozoan parasite
Leishmania
, the causative agent of leishmaniasis, a neglected tropical disease. ...Interestingly, the presence of LRV1 inside
Leishmania
constitutes an important virulence factor that worsens the leishmaniasis outcome in a type I interferon (IFN)–dependent manner and contributes to treatment failure. Understanding how macrophages respond toward
Leishmania
alone or in combination with LRV1 as well as the role that type I IFNs may play during infection is fundamental to oversee new therapeutic strategies. To dissect the macrophage response toward infection, RNA sequencing was performed on murine wild-type and
Ifnar
-deficient bone marrow–derived macrophages infected with
Leishmania guyanensis
(
Lgy
) devoid or not of LRV1. Additionally, macrophages were treated with poly I:C (mimetic virus) or with type I IFNs. By implementing a weighted gene correlation network analysis, the groups of genes (modules) with similar expression patterns, for example, functionally related, coregulated, or the members of the same functional pathway, were identified. These modules followed patterns dependent on
Leishmania
, LRV1, or
Leishmania
exacerbated by the presence of LRV1. Not only the visualization of how individual genes were embedded to form modules but also how different modules were related to each other were observed. Thus, in the context of the observed hyperinflammatory phenotype associated to the presence of LRV1, it was noted that the biomarkers tumor-necrosis factor α (TNF-α) and the interleukin 6 (IL-6) belonged to different modules and that their regulating specific Src-family kinases were segregated oppositely. In addition, this network approach revealed the strong and sustained effect of LRV1 on the macrophage response and genes that had an early, late, or sustained impact during infection, uncovering the dynamics of the IFN response. Overall, this study contributed to shed light and dissect the intricate macrophage response toward infection by the
Leishmania
-LRV1 duo and revealed the crosstalk between modules made of coregulated genes and provided a new resource that can be further explored to study the impact of
Leishmania
on the macrophage response.
The various symptomatic outcomes of cutaneous leishmaniasis relates to the type and potency of its underlying inflammatory responses. Presence of the cytoplasmic
RNA virus-1 (LRV1) within
, worsens ...lesional inflammation and parasite burden, as the viral dsRNA genome acts as a potent innate immunogen stimulating Toll-Like-Receptor-3 (TLR3). Here we investigated other innate pattern recognition receptors capable of reacting to dsRNA and potentially contributing to LRV1-mediated inflammatory pathology. We included the cytoplasmic dsRNA sensors, namely, the RIG-like receptors (RLRs) and the inflammasome-dependent and -independent Nod-like-receptors (NLRs). Our study found no role for RLRs or inflammasome-dependent NLRs in the pathology of
infection irrespective of its LRV1-status. Further, neither LRV1-bearing
(
+) nor LRV1-negative
(
) activated the inflammasome
. Interestingly, similarly to
,
infection induced the up-regulation of the A20 protein, known to be involved in the evasion of inflammasome activation. Moreover, we observed that
+ promoted the transcription of inflammasome-independent NLRC2 (also called NOD2) and NLRC5. However, only NLRC2 showed some contribution to LRV1-dependent pathology. These data confirmed that the endosomal TLR3 pathway is the dominant route of LRV1-dependent signalling, thus excluding the cytosolic and inflammasome pathways. We postulate that avoidance of the inflammasome pathways is likely an important mechanism of virulence in
infection irrespective of the LRV1-status.
Protozoan parasites contain negatively charged polymers of a few up to several hundreds of phosphate residues. In other organisms, these poly-phosphate (polyP) chains serve as an energy source and ...phosphate reservoir, and have been implicated in adaptation to stress and virulence of pathogenic organisms. In this study, we confirmed first that the polyP polymerase vacuolar transporter chaperone 4 (
) is responsible for polyP synthesis in
parasites. During
culture, polyP is accumulated in logarithmic growth phase and subsequently consumed once stationary phase is reached. However, polyP is not essential since VTC4-deficient (
)
proliferated normally in culture and differentiated into infective metacyclic parasites and into intracellular and axenic amastigotes. In
mouse infections,
knockout showed a delay in lesion formation but ultimately gave rise to strong pathology, although we were unable to restore virulence by complementation to confirm this phenotype. Knockdown of
did not alter the course of
infections in mice, suggesting that polyP was not required for infection, or that very low levels of it suffice for lesion development. At higher temperatures,
promastigotes highly consumed polyP, and both knockdown or deletion of
diminished parasite survival. Thus, although polyP was not essential in the life cycle of the parasite, our data suggests a role for polyP in increasing parasite survival at higher temperatures, a situation faced by the parasite when transmitted to humans.
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