CE15 - Immunologie, Infectiologie et Inflammation

Exploring the dynamics of X-linked gene expression in female B cells: implication in humoral immunity to influenza virus – XiToll-BFlu

Understanding the X-linked genetic mechanisms responsible for the strongest humoral immune response in females

The response initiated by TLR7 activation in innate immune cells and B cells is an essential line of defense against RNA viruses. We propose that ICX escape from TLR7 plays a major role in innate and adaptive immunity against certain pathogens such as RNA viruses, as well as in protective immunity in vaccination against influenza virus.

Analysis of the impact of the escape from ICX of the Tlr7 gene using genetic models on the humoral immune response directed against viral nanoparticles or the influenza virus.

There is a sex bias in humoral immunity in humans and mice. In adults of childbearing age, basal immunoglobulin concentration, B cell count and antibody responses are higher in females compared to males. This difference is also observed in the immune response against RNA viruses (influenza viruses). In mice, it has been suggested that a sex-related difference in Tlr7 expression in B cells may be implicated in explaining the greater antibody production in females. Escape of the TLR7 gene to inactivation of the X chromosome (ICX) has been proposed as a mechanism contributing to this sex bias. Indeed, we have recently shown that TLR7 escapes ICX in the immune cells of women and men with Klinefelter syndrome (Souyris et al., Sci.Immunol. 2018). Bi-allelic B cells were enriched within IgG-producing plasma cell lineage cells in activation and differentiation models using TLR7-specific ligands, suggesting a causal link between TLR7 bi-allelism and enhanced B cell responsiveness. In this project, a consortium formed by the groups of J.C. Guéry (CPTP, Toulouse), specialist in the study of sex-related factors in immunity, J. Chaumeil (Institut Cochin, Paris), specialist in the mechanisms of 'ICX, and the epigenetic and nuclear organization of genes in immune cells, and M. Ducatez (ENVT, Toulouse), specialist in influenza surveillance and pathogenesis, will address this hypothesis. Our objectives are to develop and validate new genetic tools in mice, not only to monitor immune cells presenting a bi-allelic expression of Tlr7 during the humoral response against RNA nanoparticles and RNA viruses (virus of H1N1 flu and COVID-19 SARS-CoV2); but also, to impose mono-allelism of Tlr7, or to inhibit cellular mosaicism linked to random inactivation and escape to XCI of the Tlr7 gene in females in order to demonstrate a direct causal link between the Bi-allelic expression of Tlr7 in B cells and the generation of protective immunity against influenza or SARS-CoV2 vaccination. We will study the molecular mechanism of Tlr7 XCI escape and how it can be modulated. Overall, our project will provide unprecedented insight into the nature of the mechanisms responsible for female predominance in TLR7-induced humoral immunity, and could open avenues for the development of new vaccine strategies against RNA viruses.

This project is essentially based on animal experimentation with the development and characterization of original genetic models making it possible to trace the expression of candidate genes on the X chromosomes of females; but also to evaluate the selective gene invalidation on the inactive X. Classic methods of cellular immunology are used, as well as more recent methods such as high-dimensional flow cytometry and NGS analyzes on sorted populations (RNA-seq) and single cells (sc-RNA-seq). RNA FISH and DNA FISH techniques are also used to assess the 3-D conformation of certain loci with respect to the territories of active and inactive X chromosomes. Finally, we use immunization models with viral nanoparticles to study the humoral response in vivo in mice in the different genetic models that we are developing. Finally, models of influenza virus infections will also be in place in order to study the impact of the escape from ICX of certain genes on the protective humoral response in the context of a natural infection.

The results of the project are currently confidential. They are the subject of articles in preparation.

The analysis of the impact of ICX escape of the Tlr7 gene on antiviral immunity is a breakthrough project, using cutting-edge technologies. It could open up new fields of investigation and could lead to the development of sex-specific biomarkers to guide the diagnosis and management of certain infectious and autoimmune diseases in women. We believe that to achieve effective treatment for all individuals in the age of precision medicine, men and women will need to be treated differently, in order to be effectively and equitably protected.

Publications
1. Youness A, Miquel CH, and Guéry JC. Escape from X chromosome inactivation and the female predominance in autoimmune diseases. Int J Mol Sci. 2021 23;22(3):1114. doi: 10.3390/ijms22031114. PMID: 33498655
2. Cenac C, Ducatez M, Guéry JC. Hydroxychloroquine inhibits proteolytic processing of endogenous TLR7 protein in human primary plasmacytoid dendritic cells. Eur J Immunol 2022 Jan;52(1):54-61. doi: 10.1002/eji.202149361. pub 2021 Oct 21. PMID: 34580855

Communications
International
1. JC guéry, “X chromosome inactivation escape: a driving force in SLE pathogenesis?”; June, 2nd 2021 Webinar EULAR Virtual Congress 2021
2. JC Guéry, “X chromosome inactivation escape in autoimmune pathogenesis”; American College of Rheumatology Convergence Meeting, November 5 -10, 2021.
3. JC Guéry, “Mechanisms underlying sex differences in innate & adaptive immunity to ssRNA viruses”, 14 Congress of the European Society of Gynecology, 11-13 Nov 2021, Venezia, Italy
4. JC Guéry, « Deconstructing the sex bias in allergy and autoimmunity: from sex hormones and beyond”; BSI Congress 2021, Nov 28-Dec 1, Edinburgh, UK

France
1. JC Guéry, “TLR7 escapes X chromosome inactivation in human immune cells: connecting two X chromosomes with increased risk of lupus”; Webinar October 12th, 2020 ; Institut Imagine, Necker, Paris.
2. JC Guéry, « X chromosome inactivation plasticity in female immune cells: a driving force in immunity”; March, 29th 2021; Webinar CRCT, Toulouse
3. JC Guéry, “Biais de sexe dans les maladies auto-immunes » ; 21 Octobre 2021; BMS– Auto-ImmunoSEM 2021 Pavillon Wagram, Paris17ème
4. JC Guéry, “Biais de sexe dans la pandémie COVID : stéroïdes sexuels et chromosomes X”; 11éme Congrès du GEMVi, Paris 19-20 novembre 2021.

The nature and the strength of the immune response differ between women and men, resulting in sex-based differences in the prevalence, manifestations and outcome of autoimmune and infectious diseases. While women are able to mount more vigorous immune responses to infections, they also suffer more from autoimmunity and subsequent inflammation-induced tissue damage. A growing body of data shows that not only sex hormones but also sex-chromosome associated loci regulate biological pathways common to autoimmune and infectious diseases. This is particularly clear for TLR7, a single-stranded RNA receptor encoded by an X-linked gene. The response initiated by TLR7-mediated sensing of ssRNA by innate immune cells and B cells is an essential line of defense against RNA viruses. However, TLR7 can also respond to endogenous ssRNA containing ligands, potentially leading to autoimmunity or inflammation if not properly controlled. The difference in humoral immunity is one of the best conserved sex differences in immunology. Generally, among adults of reproductive ages, females have greater antibody responses than males, higher basal immunoglobulin (Ig) levels, and higher B cell numbers. Sex-based differences in the immune response to RNA viruses (i.e. influenza virus) are also well documented. Among adults, females produce higher neutralizing antibody titers compared with males following seasonal influenza vaccination. In mice it has been suggested that sex-based differences in expression of Tlr7 in B cells may contribute to greater antibody production in females than males. Gene dosage effects due to escape from X-chromosome inactivation (XCI) could possibly contribute to this sex differences. Indeed, in female mammals, one of the two X chromosomes is randomly inactivated to equalize the dosage of X-linked gene expression between sexes. XCI is established at an early stage of female embryogenesis and results in cellular mosaicism, where about one-half of the cells in a tissue express the maternal X chromosome and the other half the paternal one. However, in certain tissues or individuals, 15-23% of X-linked human genes escape XCI and are thus expressed from both the active (Xa) and inactive (Xi) X chromosomes. Indeed, we recently demonstrated that TLR7 escapes XCI in immune cells from women and Klinefelter syndrome males (Souyris et al., Sci. Immunol. 2018). Strikingly, B cells expressing TLR7 biallelically were more responsive than monoallelic cells at specific checkpoints of B cell differentiation that involve signaling through TLR7.
We propose a major role for the XCI-escape of TLR7 in generating plasticity and diversity within the female immune cell compartment, with a likely impact on innate and adaptive immunity against selected pathogens or self-components containing ssRNA. A consortium formed by the group of JC Guéry (CPTP, Toulouse), specialist in the study of sex differences in immunity, J. Chaumeil (Cochin Institute, Paris), specialist in XCI, and in epigenetic & nuclear organization in immune cells, and M. Ducatez (ENVT, Toulouse), specialist in influenza surveillance and pathogenesis will address this hypothesis in this project. We will develop and validate new genetic tools in mice not only to track immune cells with bi-allelic expression of Tlr7 during the course of B cell responses to ssRNA nanoparticules and to inactivated RNA viruses (Influenza, SARS-Cov2); but also, to enforce Tlr7 mono-allelism in order to provide a direct causal link between Tlr7 bi-allelic expression in B cells and the generation of protective humoral immunity to influenza and COVID-19 vaccination in female. We will investigate the molecular mechanism of XCI-escape of Tlr7 and how this can be modulated. Altogether, our project will provide unprecedented knowledges on the nature of the mechanism underlying the female predominance in TLR7-driven B cell immunity, and could open avenues for the development of novel vaccine strategies.

Project coordination

Jean-Charles GUÉRY (CENTRE DE PHYSIOPATHOLOGIE DE TOULOUSE PURPAN)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Inserm - Inst Cochin Institut Cochin
CPTP CENTRE DE PHYSIOPATHOLOGIE DE TOULOUSE PURPAN
IHAP Interactions hôtes-agents pathogènes

Help of the ANR 580,226 euros
Beginning and duration of the scientific project: February 2021 - 42 Months

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