The CXCL12/CXCR4 signaling pathway : a new host factor involved in human papillomavirus-induced carcinogenesis

Les papillomavirus humains (HPV), dont on dénombre plus de 300 types différents, infectent spécifiquement les épithéliums. Ces infections sont communes et généralement asymptomatiques. Cependant, lorsqu’elles persistent, elles peuvent donner lieu à des lésions bénignes, telles que les verrues, ou cancéreuses, telles que le cancer du col de l’utérus. Les facteurs de l’hôte impliqués dans la persistance et la pathogénie des infections par les HPV restent largement méconnus. Les premières évidences du rôle de l’axe de signalisation CXCL12/CXCR4 dans la pathogénie virale proviennent d’observations faites dans le contexte d’un déficit immunitaire rare, le syndrome WHIM. En effet, ce syndrome est dû à des dysfonctions de l’axe CXCL12/CXCR4 − causées par des mutations de CXCR4 conduisant à un gain de fonction de l’axe CXCL12/CXCR4 − et est caractérisé par une susceptibilité sélective des patients à des infections sévères, persistantes et parfois malignes par les HPV. Au vu de cette susceptibilité, l’objectif de ma thèse a été d’approfondir cet éventuel lien causal entre les dysfonctions de l’axe CXCL12/CXCR4 et la pathogenèse associée aux infections par les HPV et de caractériser les mécanismes moléculaires en jeu.Afin de répondre à cette problématique, je me suis intéressée dans la première partie de mes travaux de thèse aux conséquences des dysfonctions de l’axe CXCL12/CXCR4 − à travers le gain de fonction de CXCR4 associé au syndrome WHIM − sur le cycle biologique d’HPV18 étudié dans des cultures organotypiques épithéliales tridimensionnelles. Ces travaux nous ont permis de mettre en évidence que les dysfonctions de CXCR4 limitaient la production virale au profit de la mise en place d’un processus de transformation cellulaire. Les mécanismes en jeu impliquent une augmentation de la prolifération cellulaire et un changement du profil d’expression des protéines virales en faveur des oncoprotéines et au détriment de celles impliquées dans la réplication virale.Dans la seconde partie de mes travaux, je me suis attachée à déterminer les effets du blocage de l’axe CXCL12/CXCR4 dans un modèle murin de néoplasie épithéliale induite par HPV16 (souris K14-HPV16). Le traitement de ces souris par l’AMD3100, un antagoniste sélectif de CXCR4, induit une tendance à la normalisation se manifestant par une diminution significative de l’hyperplasie induite par HPV16. Cet effet est associé à une réduction de l’hyperprolifération des kératinocytes et de l’infiltrat de cellules immunitaires dans le derme.En conclusion, ce travail de thèse identifie l’axe CXCL12/CXCR4 comme un facteur de l’hôte impliqué dans la carcinogenèse induite par les HPV, et révèle le bénéfice de stratégies thérapeutiques basées sur le blocage de cet axe.Human papillomaviruses (HPVs), which encompass almost 300 different types identified so far, specifically infect epitheliums. Most of the time, HPVs are associated with asymptomatic infections suggesting an efficient control by the host immune system. However, when these infections persist, HPVs can cause cutaneous warts but also mucosal lesions that can progress to dysplasia and cancer (e.g. cervical cancers). The host factors involved in HPV persistence and derived-pathogenesis remain quite obscure. The first evidence for a role of the CXCL12/CXCR4 signaling axis in HPV pathogenesis came from observations made in the context of a rare immunodeficiency disorder, the WHIM syndrome. This syndrome is caused by dysfunctions of the axis formed by the chemokine CXCL12 and its receptor CXCR4 – caused by inherited heterozygous mutations in CXCR4 leading to a gain-of-function of the CXCL12/CXCR4 axis – and featured by a high susceptibility to severe, persistent and sometimes malignant HPV infections. In light of this susceptibility, the aim of my thesis was to characterise the molecular mechanisms involved and to find out whether it extend to a more general interplay between the CXCL12/CXCR4 axis and HPV biological cycle and pathogenesis.In the first part of my work, I investigated the consequences of CXCL12/CXCR4 dysfunctions – through the CXCR4 gain-of-function – on the HPV18 life cycle in three-dimensional organotypic epithelial cultures. We found that CXCR4 dysfunctions limited the viral replication at the benefit of cell transformation. The mechanisms included an increased in cell proliferation and a change in viral protein expression profile in favour of oncoproteins and at the expense of proteins involved in viral replication.In the second part of my work, I determined the impact of the CXCL12/CXCR4 blockade on a murin model of HPV16-induced neoplasia (K14-HPV16 mice). Treatment of these mice by AMD3100, a selective antagonist of CXCR4, results in a normalisation of HPV-induced lesions manifested by a significant decrease of skin hyperplasia. This effect is associated with a reduction in keratinocyte hyperproliferation and immune cell infiltration in dermis.To conclude, this thesis work identifies the CXCL12/CXCR4 axis as a new host factor involved in human papillomavirus-induced carcinogenesis, and reveals the benefit of therapeutic strategies based on the blockade of this axis

In vitro coordination of Fe-protoheme with amyloid β is non-specific and exhibits multiple equilibria

International audienc

Altered chemotactic response to CXCL12 in patients carrying GATA2 ă mutations

International audienceGATA2 deficiency formerly described as MonoMAC syndrome; dendritic ă cells, monocytes, B cells, and natural killer cell deficiency; familial ă myelodysplastic syndrome/acute myeloid leukemia; or Emberger syndrome ă encompasses a range of hematologic and nonhematologic anomalies, mainly ă characterized by monocytopenia, B lymphopenia, natural killer cell ă cytopenia, neutropenia, immunodeficiency, and a high risk of developing ă acute myeloid leukemia. Herein, we present 7 patients with GATA2 ă deficiency recruited into the French Severe Chronic Neutropenia ă Registry, which enrolls patients with all kinds of congenital ă neutropenia. We performed extended immunophenotyping of their whole ă blood lymphocyte populations, together with the analysis of their ă chemotactic responses. Lymphopenia was recorded for B and CD4(+) T cells ă in 6 patients. Although only 3 patients displayed natural killer cell ă cytopenia, the CD56(bright) natural killer subpopulation was nearly ă absent in all 7 patients. Natural killer cells from 6 patients showed ă decreased CXCL12/CXCR4-dependent chemotaxis, whereas other lymphocytes, ă and most significantly B lymphocytes, displayed enhanced CXCL12induced ă chemotaxis compared with healthy volunteers. Surface expression of CXCR4 ă was significantly diminished in the patients' natural killer cells, ă although the total expression of the receptor was found to be equivalent ă to that of natural killer cells from healthy individual controls. ă Together, these data reveal that GATA2 deficiency is associated with ă impaired membrane expression and chemotactic dysfunctions of CXCR4. ă These dysfunctions may contribute to the physiopathology of this ă deficiency by affecting the normal distribution of lymphocytes and thus ă potentially affecting the susceptibility of patients to associated ă infections

AMD3100 treatment reduces the size of the tumors induced by injection of HPV18-positive keratinocytes expressing CXCR41013 in nude mice.

<p>Tumor growth curve of the average tumor volume (mm3 ± SEM) in each group (n = 6 for AMD3100-treated mice and n = 7 for PBS-control mice) as a function of time. Representative results from one of two independent experiments are shown. **p < 0.01 (two-way ANOVA).</p

CXCR4¹⁰¹³ expression in raft cultures promotes keratinocyte proliferation and reduces apoptosis.

<p>(A) Detection of Ki-67 expression by immunohistochemical staining in proliferating HPV18-positive CXCR4^wt and CXCR4¹⁰¹³ raft culture sections. (B) Apoptotic cells (green) in HPV18-positive CXCR4^wt and CXCR4¹⁰¹³ rafts identified by TUNEL assay. Nuclei (blue) were counterstained with DAPI. Images are representative of three independent experiments. Scale bars = 100 μm. (C) Quantification of Ki-67-positive and apoptotic cells from the histological analyses. Results are expressed as the percentage of positive cells out of the total number of epithelial cells. Values are means ± SEM. ***p < 0.001. (D) Western blots (left) and densitometric analyses (right) showing relative levels of p53 in HPV18-positive CXCR4^wt and CXCR4¹⁰¹³ rafts. p53 protein levels were normalized to GAPDH protein levels and arbitrarily set at 1 for CXCR4^wt rafts. Values are means ± SEM. ***p < 0.001.</p

Abortive HPV18 infection in rafts cultures expressing CXCR4¹⁰¹³.

<p>Images of HPV18-positive CXCR4^wt and CXCR4¹⁰¹³ raft culture sections depicting HPV DNA by in situ hybridization (A) and HPV18-E2 (B), HPV18-E4 (C), and HPV18-L1 (D) by immunohistochemical staining. Quantifications of HPV DNA-, E2-, E4- and L1-positive cells from histological analyses are expressed as the percentage of positive cells out of the total number of epithelial cells. Values are means ± SEM. **p < 0.01, ***p < 0.001. Images are representative of three independent experiments. Scale bars = 100 μm, inset scale bar = 10 μm.</p

Expression of HPV oncoproteins and their surrogate markers is enhanced in CXCR4¹⁰¹³ rafts.

<p>(A) Western blots (left) and densitometric analyses (right) showing relative levels of HPV18-E6 and HPV18-E7 expression in HPV18-positive CXCR4^wt and CXCR4¹⁰¹³ rafts. E6 and E7 protein levels were normalized to GAPDH protein levels and arbitrarily set at 1 for CXCR4^wt rafts (staining controls are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006039#ppat.1006039.s005" target="_blank">S5 Fig</a>). Values are means ± SEM. **p < 0.01. HPV18-positive CXCR4^wt and CXCR4¹⁰¹³ raft sections stained for p16 expression by immunohistochemistry (B) and for MCM2 expression by immunofluorescence (C). Detection of MCM2 expression by western blot (D). MCM2 protein levels were normalized to GAPDH protein levels and arbitrarily set at 1 for CXCR4^wt rafts. Means ± SEM are shown. ***p < 0.001. Images are representative of three independent experiments. Scale bars = 100 μm.</p

Inhibition of the ATM DNA damage pathway in CXCR4¹⁰¹³-expressing rafts.

<p>Western blots and densitometric analyses showing relative levels of involucrin and p53 (A), HPV18-E6 (B), pATM, ATM, pCHK2 and CHK2 (C) in HPV18-positive CXCR4^wt and CXCR4¹⁰¹³ NIKS cells differentiated in high calcium media (Ca) for indicated times. Levels of target proteins were normalized to GAPDH protein levels and arbitrarily set at 1 for undifferentiated (Ca 0 h) CXCR4^wt NIKS cells. Values are means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.</p

Blocking CXCR4¹⁰¹³ activity by the selective antagonist AMD3100 restores the productive HPV life cycle.

<p>(A) Sections of HPV18-positive CXCR4^wt and CXCR4¹⁰¹³ raft cultures treated (AMD3100-treated) or not (untreated) with AMD3100 and stained with hematoxylin and eosin. (B) Representative HPV18-positive CXCR4¹⁰¹³ raft cultures sections treated or not with AMD3100 and stained for HPV18-E4 (upper panel) or HPV18-L1 (lower panel). Images are representative of three independent experiments. Scale bars = 100 μm, inset scale bar = 10 μm. Quantifications of E4- and L1-positive cells in untreated and AMD3100-treated (treated) rafts from histological analyses are expressed as the percentage of positive cells out of the total number of epithelial cells. Values are means ± SEM. (C-D) Western blots (left) and densitometric analyses (right) showing relative levels of HPV18-E6 and HPV18-E7 proteins in HPV18-positive CXCR4¹⁰¹³ (C) and CXCR4^wt (D) raft cultures treated or not with AMD3100. E6 and E7 protein levels were normalized to GAPDH and arbitrarily set at 1 for untreated raft cultures. Values are means ± SEM. **p < 0.01 and ***p < 0.001.</p