Mécanismes de subversion de l'immunité innée par le virus de l'Hépatite C (VHC)

L'hépatite C pose un problème de santé publique majeur, dans la mesure où le risque de développer une infection chronique est relativement élevé (40 à 60%) et où la résistance au traitement de choix - l’interféron alpha pégylé et la ribavirine - touche près de la moitié des patients. Cette persistence virale repose avant tout sur de puissantes stratégies d’évasion du système immunitaire inné de l’hôte par le virus. Dans ce projet, nous nous sommes intéressés à la caractérisation de la réponse antivirale dans des hépatocytes primaires humains normaux et chroniquement infectés avec le VHC, un domaine encore largement inconnu dû à la difficulté d’obtenir ce type de matériel primaire. Nous avons étudié la fonctionnalité de deux voies majeures de détection des pathogènes viraux suite à l’exposition d’hépatocytes primaires humains à de l’ARNdb intracellulaire, via le récepteur et adaptateur RIG-I/MDA5-CARDIF, et extracellulaire via TLR3-TRIF, mimant ainsi les étapes précoces de la détection d’un virus par la cellule hôte. Nous avons établi par RT-PCR quantitatif et analyse transcriptomique par microarray, que ces deux voies de stimulation sont fonctionnelles dans des hépatocytes primaires normaux et que leur activation entraîne à la fois l’expression de gènes antiviraux communs (ISG56, ISG15, CXCL10, …) mais aussi spécifiques avec les gènes IL28A, IL28B et IL29 qui sont une signature de l’activation de la voie de détection de l’ARNdb intracellulaire. La protéine virale NS3/4A joue un rôle majeur à la fois dans le clivage de la polyprotéine virale initiale, mais aussi en interférant avec les cascades de signalisation engagées suite à la détection par la cellule hôte de l’ARN du VHC. Plus particulièrement, nous avons démontré que l’expression ectopique de NS3/4A dans des hépatocytes primaires humains normaux entraîne une diminution significative de l’induction des gènes antiviraux dûe au clivage de CARDIF au cours de l’activation de la voie de signalisation médiée par RIG-I. Nous avons également démontré que l’expression de la NS3/4A entraîne des modifications de l’expression de gènes-clé impliqués dans la régulation de l’apoptose et du programme de mort cellulaire, en particulier lorsque la voie TLR3 est induite. L’ensemble de ces effets sont abolis en présence de BILN2061, inhibiteur spécifique de NS3/4A. Malgré les stratégies de subversion de l’immunité innée par le VHC, nous avons démontré l’induction significative de plusieurs ISGs et chemokines dans des hepatocytes primaires provenant de patients chroniquement infectés avec le VHC, sans toutefois détecter d’interférons de type I, III ou certains gènes antiviraux précoces comme CCL5. Ces observations, concomitantes avec une diminution de l’expression de CARDIF et une correlation inverse entre les niveaux d’ARNm des ISGs et l’ARN viral révèlent une réponse antivirale partielle dûe à des mécanismes interférents sous-jacents. Cette réponse antivirale détectable mais inefficace est à mettre en lien avec l’échec du traitement classique PEG-IFN-ribavirine chez la moitié des patients traités, mais aussi en lien avec l’inflammation chronique et les dommages hépatiques qui mènent ultimement au développement d’une fibrose puis d’une cirrhose chez une grande proportion de patients chroniquement infectés.Hepatitis C infection is a worldwide health problem since the risk to develop a persistent infection is relatively elevated (40 to 60%) and nearly half of the infected patients do not respond to the classical anti-HCV therapy based on a combination of PEG-IFNα and ribavirin. Viral persistence is based on powerful evasion strategies of the host’s innate immune system. In our study, we characterized antiviral response in primary human normal and chronically HCV-infected hepatocytes, a cutting-edge in our field due to the difficulty to isolate this particular cell type. In order to better define the antiviral response in freshly isolated human primary hepatocytes, we stimulated these cells with extracellular and intracellular dsRNA to trigger TLR3/TRIF and RIG-I-MDA5/CARDIF-mediated antiviral signaling pathways. By using qRT-PCR and microarray analysis, we report that both detection pathways are functional in normal human hepatocytes, their activation leading to the expression of both common (IFIT1, OASL, ISG15 and CXCL10) and specific genes (IL28A, IL28B and IL29), these last ones being a signature of the intracellular dsRNA-mediated pathway. HCV NS3/4A plays a key role in the viral polyprotein processing and upon viral RNA detection by interfering with the host’s antiviral signalling cascades. We report that major antiviral genes induction following activation of RIG-I mediated pathway are severely impaired in ectopically NS3/4A expressing normal hepatocytes due to CARDIF cleavage, but can be restored by specific NS3/4A inhibitor BILN2061. Our microarray analysis also revealed a role for NS3/4A following TRL3-mediated pathway activation on regulation of apoptosis and programmed cell death, which could be linked to strategies for the virus to persist in its host. Despite HCV strategies to circumvent the host’s immune defense system, we observed significant upregulation of ISGs and chemokines in liver biopsies and corresponding isolated hepatocytes from chronically HCV-infected patients. However, no type I and III interferon, neither key-antiviral genes (e.g., CCL5) were detected, underlying an ongoing –but inefficient- antiviral response unable to eradicate the virus. Moreover, we obtained significant inverse correlations between ISGs mRNAs and viral RNA in addition to CARDIF decrease, clearly unravelling efficient viral interfering strategies in a context of chronic HCV infection. This sustained -albeit incomplete- hepatic innate immune response is certainly associated to the failure of the classical IFN-based therapy in half of the infected patients and to the chronic inflammation causing liver damages and eventually leading to hepatocarcinoma which is often observed at late stage of the disease

Requirement of NOX2 and Reactive Oxygen Species for Efficient RIG-I-Mediated Antiviral Response through Regulation of MAVS Expression

The innate immune response is essential to the host defense against viruses, through restriction of virus replication and coordination of the adaptive immune response. Induction of antiviral genes is a tightly regulated process initiated mainly through sensing of invading virus nucleic acids in the cytoplasm by RIG-I like helicases, RIG-I or Mda5, which transmit the signal through a common mitochondria-associated adaptor, MAVS. Although major breakthroughs have recently been made, much remains unknown about the mechanisms that translate virus recognition into antiviral genes expression. Beside the reputed detrimental role, reactive oxygen species (ROS) act as modulators of cellular signaling and gene regulation. NADPH oxidase (NOX) enzymes are a main source of deliberate cellular ROS production. Here, we found that NOX2 and ROS are required for the host cell to trigger an efficient RIG-I-mediated IRF-3 activation and downstream antiviral IFNβ and IFIT1 gene expression. Additionally, we provide evidence that NOX2 is critical for the expression of the central mitochondria-associated adaptor MAVS. Taken together these data reveal a new facet to the regulation of the innate host defense against viruses through the identification of an unrecognized role of NOX2 and ROS

Intact dendritic cell pathogen-recognition receptor functions associate with chronic hepatitis C treatment-induced viral clearance.

Although studies have addressed the exhaustion of the host's immune response to HCV and its role in treatment, there is little information about the possible contribution of innate immunity to treatment-induced clearance. We hypothesized that because intact myeloid dendritic cell (MDC) pathogen sensing functions are associated with improved HCV-specific CD8+ T cell functionality in some chronically infected patients, it might enhance HCV clearance rate under standard interferon therapy. To investigate this hypothesis, TLR-induced MDC activation and HCV-specific CD8+ T cell response quality were monitored longitudinally at the single-cell level using polychromatic flow cytometry in chronically infected patients undergoing interferon therapy. We correlated the immunological, biochemical and virological data with response to treatment. We demonstrate that the clinical efficacy of interferon-induced viral clearance is influenced by the extent to which HCV inhibits MDC functions before treatment, rather than solely on a breakdown of the extrinsic T cell immunosuppressive environment. Thus, viral inhibition of MDC functions before treatment emerges as a co-determining factor in the clinical efficacy of interferon therapy during chronic HCV infection

MDC TLR functionality is associated with the likelihood of achieving SVR following pegIFN and ribavirin treatment.

<p>(A) Heat maps of week 0 (pre-treatment) FACS measured IL-12 and TNFα (columns) protein expression profiles (CP) for lineage⁻CD16⁻CD45⁺CD11c⁺MHC-II^br MDCs activated with TLR agonists from viremics starting therapy and followed longitudinally (n = 20) as a log₂ fold-change in MFI expression relative to TLR stimulated MDCs from a reference group of aviremic controls that cleared HCV after IFN therapy (n = 12) (yellow, higher than; blue, lower than; white, no change versus protein expression in aviremics). (B-D) Individual HCV plasma viral RNA loads (PVL, B), ALT (C) or AST (D) levels were determined before, during and after antiviral therapy. Red lines represent SVR; black lines NR. Vertical gray lines indicate period of antiviral treatment. (E) Relative levels of IRG mRNAs in PBMCS of NRs and SVRs at 4 and 12 weeks of treatment, normalized to pre-treatment levels, as determined by qPCR. Error bars represent mean ± SEM. (F) Correlation between MDC inhibition (sum of IL-12 and TNF-α TRIF-dependent TLR MFI fold-change, log₂) prior to treatment initiation and the end-of-treatment changes of PVL from pre-treatment (n = 20).</p

Clinical data of the 34 patients clustered according to MDC functionality suffering from chronic HCV and undergoing pegIFN treatment.

<p>In parentheses are %; NA, not applicable.</p>1<p>yr ± SD.</p>2<p>log₁₀ IU x ml⁻¹ ± SD.</p

Differences in functional phenotype of the HCV-specific CD8 responses during therapy between SVRs and NRs.

<p>(A) Pre- and on-treatment FACS density plots in response to a 5.5 hour incubation with 2 µgml⁻¹ of HCV NS5 peptide pools showing the frequencies of memory CD8⁺ T cells displaying the depicted combinations of CD107a mobilizing and intracellular production of IFNγ, IL-2, and TNF-α for patient P70. Gating was done on viable memory CD14⁻CD19⁻CD3⁺CD4⁻CD8⁺ cells that were not CD27⁺CD45RO⁻. Background activity against CD28/CD49d costimulation alone has been subtracted. (B) Flow cytometric analysis of polyfunctionality within total genotype 1 HCV-specific CD8 memory T-cells is shown at pre-treatment prior to start of therapy. The bar chart shows each of the 15 possible response profiles on the x-axis as the percentage of the total cytokine response on the y-axis. The filled bar represent the interquartile range and the line the median. (C) Summary of functional profile in SVR (red outline) and NR during treatment. The distinct cellular subsets shown in panel B were grouped by number of functions, so each section of the pie charts represent the mean proportion of HCV-specific CD8⁺ T cells grouped by the number of functions expressed independently of any particular function and matching the color code used in panel B. Statistically significant differences at pre-treatment between SVRs an NRs (*P<0.05, by Mann Whitney test) are indicated by the asterix. (D) The frequency of total HCV-specific CD8 memory T-cell responses at pre-treatment (week 0) and during treatment is shown in each individual as a percentage of their total memory CD8 T-cell population. (E) pegIFN-induced breakdown of the immunosuppressive environment is similar in nonresponders versus responders. Relative levels of immune suppressive genes in PBMCs of NRs (black) and SVRs (red), normalized to pretreatment levels, as determined by qPCR. Error bars represent mean ± SEM (Bonferoni's One-way Anova post-test, **P<0.001). (F) Relationship between the proportion of CD107a mobilization on HCV-specific CD8⁺ T cells at 12 weeks after treatment initiation and the 12-week treatment changes of PVL from pre-treatment (n = 10, analyzed using Pearson correlation). Each symbol corresponds to one subject: SVRs are in red, NRs are in black.</p

Characteristics of the twenty HCV chronic patients longitudinally followed during treatment course.

a<p>log₁₀ IU/ml.</p>b<p>By trypan blue count; visits during and following treatment.</p>c<p>Frequencies are total cytokine-producing (IFN-γ, IL-2, TNFα) and degranulating (CD107a⁺) cells out of the CD8⁺ memory subset at pre-treatment. All positive responses to HCV pools were summed to determine the total antigen-specific response within the memory peripheral blood T cell populations. Due to HCV peptide reagent availability, only genotype 1 patients could be stimulated. ND: not determined.</p>d<p>Number of HCV proteins detected by CD8⁺ T cells.</p

Lower rate of SVR in HCV chronic patients with impaired MDC TLR functionality.

<p>PBMCs were cultured in the presence of brefeldin A and TLR agonists for 6(A) Representative FACS plots of IL-6⁺ gated lineage⁻CD16⁻CD45⁺CD11c⁺MHC-II^br MDCs positive for TNF-α or IL-12 before treatment are shown following LPS or 3M-002 stimulation as stratified by CP cluster. Numbers on the left side of and above the bracketed lines indicate the geometric MFI and the percentages of cytokine–expressing MDCs in the designated area, respectively. (B) TNF-α and IL-12 expression (mean ± SD) as a log₂ geometric MFI fold-induction above unstimulated control. Statistical comparisons between CP groups and aviremic control group were calculated by the Dunnett one-way ANOVA post test (***P<0.001). (C) MDC cytokine profile-stratified analysis of SVR and NR rates indicates that the difference between CP-N and CP-D patients irrespective of HCV genotypes is significant (n = 34 subjects; *P = 0.0048 by Fisher's exact two-tail test).</p

Poly(I:C) and Lipopolysaccharide Innate Sensing Functions of Circulating Human Myeloid Dendritic Cells Are Affected In Vivo in Hepatitis C Virus-Infected Patients▿

The role of peripheral dendritic cells (DCs) in hepatitis C virus (HCV) infection is unclear. To determine if persistent infection exerts an inhibitory pressure on HCV-specific innate responses, we analyzed DC function in blood through quantification of cell-associated HCV RNA levels in conjunction with multiparametric flow cytometry analysis of pathogen recognition receptor-induced cytokine expression. Independently of the serum viral load, fluorescence-activated cell sorter-purified total DCs had a wide range of cell-associated HCV genomic RNA copy numbers (mean log10, 5.0 per 106 cells; range, 4.3 to 5.8). Here we report that for viremic patients with high viral loads in their total DCs, the myeloid DC (MDC) subset displayed impaired expression of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) but normal IL-6 or chemokine CCL3 expression in response to poly(I:C) and lipopolysaccharide (LPS). IL-6-expressing cells from this subgroup of viremic patients demonstrated a significant increase (sixfold more) in TNF-α− IL-12− cell frequency compared to healthy donors (mean, 38.8% versus 6.5%; P < 0.0001), indicating a functional defect in a subpopulation of cytokine-producing MDCs (∼6% of MDCs). Attenuation of poly(I:C) and LPS innate sensing was HCV RNA density dependent and did not correlate with viremia or deficits in circulating MDC frequencies in HCV-infected patients. Monocytes from these patients were functionally intact, responding normally on a per-cell basis following stimulation, independent of cell-associated HCV RNA levels. Taken together, these data indicate that detection of HCV genomic RNA in DCs and loss of function in the danger signal responsiveness of a small proportion of DCs in vivo are interrelated rather than independent phenomena