AUTHOR=Ding Lin , Sheriff Sulaiman , Sontz Ricky A. , Merchant Juanita L. 

TITLE=Schlafen4+-MDSC in Helicobacter-induced gastric metaplasia reveals role for GTPases

JOURNAL=Frontiers in Immunology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1139391

DOI=10.3389/fimmu.2023.1139391

ISSN=1664-3224

ABSTRACT=<sec><title>Introduction</title><p>MDSCs express SCHLAFEN 4 (SLFN4) in <italic>Helicobacter</italic>-infected stomachs coincident with spasmolytic polypeptide-expressing metaplasia (SPEM), a precursor of gastric cancer. We aimed to characterize SLFN4<sup>+</sup> cell identity and the role of Slfn4 in these cells.</p></sec><sec><title>Methods</title><p>Single-cell RNA sequencing was performed on immune cells sorted from PBMCs and stomachs prepared from uninfected and 6-month <italic>H. felis</italic>-infected mice. Knockdown of Slfn4 by siRNA or PDE5/6 inhibition by sildenafil were performed in vitro. Intracellular ATP/GTP levels and GTPase activity of immunoprecipitated <italic>Slfn4</italic> complexes were measured using the GTPase-Glo assay kit. The intracellular level of ROS was quantified by the DCF-DA fluorescent staining, and apoptosis was determined by cleaved Caspase-3 and Annexin V expression. <italic>Gli1CreERT2 x Slfn4</italic><sup>fl/fl</sup> mice were generated and infected with <italic>H. felis</italic>. Sildenafil was administered twice over 2 weeks by gavaging <italic>H. felis</italic> infected mice ~4 months after inoculation once SPEM had developed.</p></sec><sec><title>Results</title><p><italic>Slfn4</italic> was highly induced in both monocytic and granulocytic MDSCs from infected stomachs. Both <italic>Slfn4</italic><sup>+</sup>-MDSC populations exhibited strong transcriptional signatures for type-I interferon responsive GTPases and exhibited T cell suppressor function. SLFN4-containing protein complexes immunoprecipitated from myeloid cell cultures treated with IFNa exhibited GTPase activity. Knocking down Slfn4 or PDE5/6 inhibition with sildenafil blocked IFNa induction of GTP, SLFN4 and NOS2. Moreover, IFNa induction of <italic>Slfn</italic><sup>+</sup>-MDSC function was inhibited by inducing their reactive oxygen species (ROS) production and apoptosis through protein kinase G activation. Accordingly, in vivo disruption of Slfn4 in <italic>Gli1CreERT2 x Slfn4</italic><sup>fl/fl</sup> mice or pharmacologic inhibition by sildenafil after Helicobacter infection also suppressed SLFN4 and NOS2, reversed T cell suppression and mitigated SPEM development.</p></sec><sec><title>Conclusion</title><p>Taken together, SLFN4 regulates the activity of the GTPase pathway in MDSCs and precludes these cells from succumbing to the massive ROS generation when they acquire MDSC function.</p></sec>