Infection des cellules dendritiques périphériques du sang par le virus de l'hépatite C

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Inhibition de la réception des signaux de danger via les TLR TRIF-dépendants dans les cellules dendritiques myéloïdes infectées avec le virus de l'hépatice C in vivo : mécanisme d'évasion de l'immunité innée dans l'infection chronique

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

The mitochondrial protease HtrA2 restricts the NLRP3 and AIM2 inflammasomes.

Activation of the inflammasome pathway is crucial for effective intracellular host defense. The mitochondrial network plays an important role in inflammasome regulation but the mechanisms linking mitochondrial homeostasis to attenuation of inflammasome activation are not fully understood. Here, we report that the Parkinson\u27s disease-associated mitochondrial serine protease HtrA2 restricts the activation of ASC-dependent NLRP3 and AIM2 inflammasomes, in a protease activity-dependent manner. Consistently, disruption of the protease activity of HtrA2 results in exacerbated NLRP3 and AIM2 inflammasome responses in macrophages ex vivo and systemically in vivo. Mechanistically, we show that the HtrA2 protease activity regulates autophagy and controls the magnitude and duration of inflammasome signaling by preventing prolonged accumulation of the inflammasome adaptor ASC. Our findings identify HtrA2 as a non-redundant mitochondrial quality control effector that keeps NLRP3 and AIM2 inflammasomes in check

Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors

<p>Abstract</p> <p>Background</p> <p>The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established.</p> <p>Methods</p> <p>Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells.</p> <p>Results</p> <p>We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect.</p> <p>Conclusion</p> <p>MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.</p

Comment le virus de l’hépatite C détourne la réponse immunitaire adaptative orchestrée par les cellules dendritiques

La fonction des cellules dendritiques (CD) est d’amorcer et d’orienter la réponse adaptative grâce à leur capacité à reconnaître, à l’aide des récepteurs de type PRR (pattern recognition receptors), un nombre restreint de structures moléculaires propres aux micro-organismes et communes à de nombreux agents pathogènes. La stratégie de ces sentinelles n’est pas de reconnaître toute la gamme des millions de motifs antigéniques existants mais un petit nombre d’entre eux et d’intégrer cette information afin d’aider le système adaptatif à faire la distinction entre peptides du soi et peptides dangereux. Nous verrons dans cette revue que le virus de l’hépatite C interrompt la signalisation en aval de certains PRR, ce qui lui permet d’échapper à la détection par l’immunité innée et d’inhiber la capacité des CD à stimuler une réponse adaptative antivirale

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

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

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