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Elucidation of the function of DDX3X in innate immunity

Final Report Summary - DDX3X FUNCTION (Elucidation of the function of DDX3X in innate immunity)

- Summary of the project objectives:
Objective 1 comprised to generate mice carrying an allele that allows for conditional deletion of DDX3X in specific tissues by Cre/loxP-mediated recombination. The purpose of the first objective is to obtain mice that carry the conditionally deleted DDX3X allele in their germline and to obtain mice with a deletion of DDX3X in various tissues necessary for further analyses of DDX3X function.
Objective 2 comprised to analyse the function of DDX3X on regulating IFN-I responses. To assess the in vivo role of DDX3X in the IFN-I pathway, we aimed to analyse DDX3X whole-body knock-out mice or tissue-specific DDX3X knock-out mice. We proposed to analyse various cell types lacking functional DDX3X protein in response to different pattern recognition receptor (PRR) stimuli. If DDX3X indeed plays a role in the TBK1/IKK-ε – IRF3 pathway, we would expect lower IFN-I levels in infected MEFs, macrophages, or dendritic cells lacking DDX3X protein. Moreover, mice lacking functional DDX3X should be more susceptible to viral infections than wild type mice. In summary, through work proposed in objective 2 we aimed at showing a crucial role for DDX3X in the response to viral infection in vivo, via its role in the IFN-I pathway.
Objective 3 aimed at characterising the role of DDX3X in acute lung injury. Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury (ALI), has been the most prevalent cause of death in individuals infected with SARS virus, or different influenza viruses. Previously, TLR4 has been shown to be a susceptibility gene for acute lung injury. It was suggested that DDX3X acts downstream of TLR4, therefore, suggesting the possibility of an involvement of DDX3X in ALI. The work suggested in objective 3 aimed at shedding light on mechanisms leading to acute lung injury, which is important for discovering novel drug targets to treat ALI.

- Description of the work performed and the main results achieved so far:
Objective 1:
Mouse embryonic stem cells were successfully targeted to obtain an allele with DDX3X exon2 (located on the X-chromosome) flanked by loxP sites (DDX3Xfl). The β-actin-Cre mouse line was used to delete DDX3X exon2 in order to obtain the DDX3X deficient allele (DDX3X-). DDX3X+/- female mice are viable, however, DDX3X-/y males seem to be early embryonic lethal (before d10.5). In order to study DDX3X deleted tissues we crossed conditional DDX3X mice to vav-icre mice to delete DDX3X in hematopoietic cells. Deletion efficiency in spleen and various hematopoietic cell types is close to 100%, as assessed by q-RT-PCR and Western Blotting.
DDX3X deficient MEFs were generated by tamoxifen-induced deletion of DDX3X in DDX3Xfl/y cre-ERT2+/- mice. Furthermore, DDX3Xfl/fl cre-ERT2+/- MEFs were generated to avoid a potential compensatory effect by DDX3Y from the Y-chromosome. Moreover, DDX3Xfl/fl cre-ERT2+/- mice were utilised to derive bone marrow macrophages, myeloid dendritic cells and plasmacytoid dendritic cells in vitro.
Objective 2:
Experiments were initially performed with DDX3X deficient MEFs that were obtained from DDX3Xfl/y cre-ERT2+/- embryos. Moreover, DDX3X deficient myeloid dendritic cells, plasmacytoid dendritic cells, and macrophages that were differentiated in vitro from bone marrow cells of DDX3Xfl/y vav-icre+/- mice. Stimulation of various pattern recognition receptors as well as stimulation of inflammasomes was performed in multiple ways and induction of cytokines was assayed. One consistent result seems to be a strong reduction of IFNβ response in DDX3X deficient MEFs, mDCs, and macrophages upon pdA:dT stimulation indicating a role for DDX3X in the detection pathway of cytosolic DNA. Furthermore, VSV-infection of DDX3X deficient MEFs consistently resulted in impaired IFN-I and TNFα response.
Moreover, we observed reduced sensitivity of the inflammasome response in bone marrow derived macrophages in response to various stimuli.
Whole animal in vivo infections with Listeria monocytogenes , VSV, and H1N1 are ongoing and we already got preliminary data indicating that DDX3Xfl/y vav-icre+/- mice exhibit highly increased susceptibility to L. monocytogenes infection.
There are several ways to model acute lung injury in mice: induction through acid aspiration or induction through inactivated or live virus infections. Virus infection experiments are set up and ongoing, however, no preliminary data can be reported yet. Our focus was to obtain clean results in the context of DDX3X and its role in IFN and inflammasome responses, therefore, objectives 1 and 2 were prioritized during the past funding period.