Estelle Dortu

The varicella-zoster virus ORF47 kinase interferes with host innate immune response by inhibiting the activation of IRF3.

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-κB, is a key regulator of IFN-β expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-β and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-β and ISG15.

Human papillomavirus entry into NK cells requires CD16 expression and triggers cytotoxic activity and cytokine secretion

Human papillomavirus (HPV) infections account for more than 50% of infection‐linked cancers in women worldwide. The immune system controls, at least partially, viral infection and around 90% of HPV‐infected women clear the virus within two years. However, it remains unclear which immune cells are implicated in this process and no study has evaluated the direct interaction between HPVs and NK cells, a key player in host resistance to viruses and tumors. We demonstrated an NK‐cell infiltration in HPV‐associated preneoplastic cervical lesions. Since HPVs cannot grow in vitro, virus‐like particles (VLPs) were used as a model for studying the NK‐cell response against the virus. Interestingly, NK cells displayed higher cytotoxic activity and cytokine production (TNF‐α and IFN‐γ) in the presence of HPV‐VLPs. Using flow cytometry and microscopy, we observed that NK‐cell stimulation was linked to rapid VLP entry into these cells by macropinocytosis. Using CD16+ and CD16− NK‐cell lines and a CD16‐blocking antibody, we demonstrated that CD16 is necessary for HPV–VLP internalization, as well as for degranulation and cytokine production. Thus, we show for the first time that NK cells interact with HPVs and can participate in the immune response against HPV‐induced lesions.

Novel Association between Vasoactive Intestinal Peptide and CRTH2 Receptor in Recruiting Eosinophils A POSSIBLE BIOCHEMICAL MECHANISM FOR ALLERGIC EOSINOPHILIC INFLAMMATION OF THE AIRWAYS

Background: Ligand receptor ligation regulates immune-inflammatory cell chemotaxis. Results: Vasoactive intestinal peptide and prostaglandin D2 share the CRTH2 receptor in inducing eosinophil chemotaxis. Conclusion: There is a strong association between VIP and CRTH2 in eosinophil chemotaxis. Significance: This is the first evidence that may indicate that CRTH2 could modulate the neuroimmunoregulatory axis in allergic eosinophil inflammation. We explored the relation between vasoactive intestinal peptide (VIP), CRTH2, and eosinophil recruitment. It is shown that CRTH2 expression by eosinophils from allergic rhinitis (AR) patients and eosinophil cell line (Eol-1 cells) was up-regulated by VIP treatment. This was functional and resulted in exaggerated migratory response of cells against PGD2. Nasal challenge of AR patients resulted in a significant increase of VIP contents in nasal secretion (ELISA), and the immunohistochemical studies of allergic nasal tissues showed significant expression of VIP in association with intense eosinophil recruitment. Biochemical assays showed that VIP-induced eosinophil chemotaxis from AR patients and Eol-1 cells was mediated through the CRTH2 receptor. Cell migration against VIP was sensitive to protein kinase C (PKC) and protein kinase A (PKA) inhibition but not to tyrosine kinase or p38 MAPK inhibition or calcium chelation. Western blot demonstrated a novel CRTH2-mediated cytosol-to-membrane translocation of PKC-ϵ, PKC-δ, and PKA-α, -γ, and -IIαreg in Eol-1 cells upon stimulation with VIP. Confocal images and FACS demonstrated a strong association and co-localization between VIP peptide and CRTH2 molecules. Further, VIP induced PGD2 secretion from eosinophils. Our results demonstrate the first evidence of association between VIP and CRTH2 in recruiting eosinophils.

Human papillomavirus oncoproteins induce a reorganization of epithelial-associated γδ T cells promoting tumor formation

Significance Of all tumor-infiltrating leukocytes, T cells bearing γδ T-cell receptors have been associated with the most favorable prognosis. However, we show here, in a mouse model of carcinogenesis induced by human papillomavirus (HPV) oncoproteins, that γδ T cells promoted the development of HPV-induced lesions. Indeed, HPV-oncoprotein expression induced an infiltration of γδ T cells producing IL-17A, a proangiogenic cytokine, and decreased density of antitumor Vγ5+ γδ T subsets. Supporting the clinical relevance of our observations, IL-17A+ γδ T cells were detected in human cervical cancer, where HPV oncoproteins are highly expressed, but not in less advanced cervical lesions. These results support the notion that viral oncoproteins can induce a switch from antitumoral to protumoral γδ T subsets in solid tumors. It has been shown that γδ T cells protect against the formation of squamous cell carcinoma (SCC) in several models. However, the role of γδ T cells in human papillomavirus (HPV)-associated uterine cervical SCC, the third-leading cause of death by cancer in women, is unknown. Here, we investigated the impact of γδ T cells in a transgenic mouse model of carcinogenesis induced by HPV16 oncoproteins. Surprisingly, γδ T cells promoted the development of HPV16 oncoprotein-induced lesions. HPV16 oncoproteins induced a decrease in epidermal Skint1 expression and the associated antitumor Vγ5+ γδ T cells, which were replaced by γδ T-cell subsets (mainly Vγ6+ γδlowCCR2+CCR6−) actively producing IL-17A. Consistent with a proangiogenic role, γδ T cells promoted the formation of blood vessels in the dermis underlying the HPV-induced lesions. In human cervical biopsies, IL-17A+ γδ T cells could only be observed at the cancer stage (SCC), where HPV oncoproteins are highly expressed, supporting the clinical relevance of our observations in mice. Overall, our results suggest that HPV16 oncoproteins induce a reorganization of the local epithelial-associated γδ T-cell subpopulations, thereby promoting angiogenesis and cancer development.

γδ T cells could promote cancer progression of HPV-induced lesions via pro-angiogenic mechanisms

High-risk human papillomavirus infection is the etiological agent of cervical cancer, the third cause of cancer-associated death in women worldwide. Gamma delta T cells (γδ T cells) represent a small population of T cells expressing a T cell receptor (TCR) composed of gamma and delta chains. Their role in the context of HPV-induced lesions was not investigated yet, but we previously showed an infiltration of γδ T cells in this cancer, suggesting a relationship between HPV-induced lesions and γδ T cells. The goal of this project is to study the role of γδ T cells in the immune response against HPV-induced tumours. In order to study the role of γδ T cells in HPV-induced lesions, we have established a mouse model by crossing transgenic mice expressing HPV16 oncogenic genes, which develop spontaneous skin lesions, with γδ T cell-deficient mice. Surprisingly, depletion of γδ T cells significantly delays development of HPVinduced lesions. In parallel, we observed by immunohistochemistry an increase of leukocyte infiltration in HPV-induced lesions in absence of γδ T cells. Then, we evaluated by flow cytometry the proportions of immune cell populations present in the mouse skin and we found a larger proportion of CD4+ T cells in HPV and HPV γδ T cell-deficient mice compared to normal mice. Since γδ T cells could induce angiogenesis when infiltrating tumors, we measured blood vessels density in mice skin sections and we observed a significantly increase of blood vessels density in HPV mice compared to HPV γδ T cells-deficient mice. Our results suggest that γδ T cells could promote cancer progression in the context of HPV-induced lesions. We will further characterise these cells to understand in which cellular and molecular mechanisms they are involved.

Characterization of hepatitis C virus-induced nasal mucosa remodelling

Previous studies have suggested that a panel of antibodies, including CD34, factor XIIIa, CD10 and ST-3, can help to distinguish DFSP from DF. However, the reliability of these antibodies is not absolute. For this reason, continued efforts have been made to characterize the immunoreactivity of alternative markers. In this study we observed a constant and strong expression of cathepsin K in DFs, but its complete absence in DFSPs. This finding suggests that cathepsin K is potentially a novel and sensitive positive marker of DF versus DFSP. The sensitivity and specificity of cathepsin K are comparable or even superior to that of currently available markers of DF. The inclusion of this novel marker in the immunohistochemical panel to differentiate between DF and DFSP may assist in making a correct diagnosis, especially when CD34 proves inadequate. However, care must be taken in making a final diagnosis with these markers alone; the other potential diagnoses that might arise in a small biopsy specimen showing spindle cells must also be considered.