Long Non-Coding RNA Hotairm1 Promotes S100A9 Support of MDSC Expansion during Sepsis.

Myeloid-derived suppressor cells (MDSCs) expand during mouse and human sepsis, but the mechanism responsible for this is unclear. We previously reported that nuclear transport of S100A9 protein programs Gr1+CD11b+ myeloid precursors into MDSCs in septic mice. Here, we show that long non-coding RNA Hotairm1 converts MDSCs from an activator to a repressor state. Mechanistically, increased Hotairm1 expression in MDSCs in mice converted S100A9 from a secreted proinflammatory mediator to an immune repressor by binding to and shuttling it from cytosol to nucleus during late sepsis. High Hotairm1 levels were detected in exosomes shed from MDSCs from late septic mice. These exosomes inhibited lipopolysaccharide-stimulated secretion of S100A9 from early sepsis Gr1+CD11b+ cells. Importantly, Hotairm1 knockdown in late sepsis Gr1+CD11b+ MDSCs prevented S100A9 cytosol to nuclear transfer and decreased repression of proimmune T cells. Notably, ectopic expression of Hotairm1 in early sepsis Gr1+CD11b+ cells shuttled S100A9 to the nucleus and promoted the MDSC repressor phenotype. In support of translating the mechanistic concept to human sepsis, we found that Hotairm1 binds S100A9 protein in CD33+CD11b+HLA-DR- MDSCs during established sepsis. Together, these data support that Hotairm1 is a plausible molecular target for treating late sepsis immune suppression in humans and its immune repressor mechanism may be cell autonomous.

HuR promotes miRNA-mediated upregulation of NFI-A protein expression in MDSCs during murine sepsis.

Myeloid-derived suppressor cells (MDSCs) contribute to high mortality rates during sepsis, but how sepsis induces MDSCs is unclear. Previously we reported that microRNA (miR)-21 and miR-181b reprogram MDSCs in septic mice by increasing levels of DNA binding transcription factor, nuclear factor 1 (NFI-A). Here, we provide evidence that miR-21 and miR-181b stabilize NFI-A mRNA and increase NFI-A protein levels by recruiting RNA-binding proteins HuR and Ago1 to its 3' untranslated region (3'UTR). We also find that the NFI-A GU-rich element (GRE)-binding protein CUGBP1 counters miR-21 and miR-181b dependent NFI-A mRNA stabilization and decreases protein production by replacing 3'UTR bound Ago1 with Ago2. We confirmed the miR-21 and miR-181b dependent reprogramming pathway in MDSCs transfected with a luciferase reporter construct containing an NFI-A 3'UTR fragment with point mutations in the miRNA binding sites. These results suggest that targeting NFI-A in MDSCs during sepsis may enhance resistance to uncontrolled infection.

S100A9 maintains myeloid-derived suppressor cells in chronic sepsis by inducing miR-21 and miR-181b.

Myeloid-derived suppressor cells (MDSC) expand during sepsis, suppress both innate and adaptive immunity, and promote chronic immunosuppression, which characterizes the late/chronic phase of sepsis. We previously reported that the transcription factors Stat3 and C/EBPß synergize to induces the expression of microRNA (miR)-21 and miR-181b to promote MDSC expansion in a mouse model of polymicrobial sepsis that progresses from an early/acute proinflammatory phase to a late/chronic immunosuppressive stage. We also showed that Gr1+CD11b+ cells, the precursors of MDSCs, from mice genetically deficient in the inflammatory protein S100A9 lack miR-21 or miR-181b in late sepsis, and are not immunosuppressive. In the present study, we show that S100A9 induces miR-21 and miR-181b during the late sepsis phase. We find that S100A9 associates with and stabilizes the Stat3-C/EBPß protein complex that activates the miRNA promoters. Reconstituting Gr1+CD11b+ cells from S100A9 knockout mice with late sepsis with S100A9 protein restores the Stat3-C/EBPß protein complex and miRNA expressions, and switches the Gr1+CD11b+ cells into the immunosuppressive, MDSC phenotype. Importantly, we find that this process requires IL-10 mediated signaling, which induces S100A9 translocation from the cytosol to the nucleus. These results demonstrate that S100A9 promotes MDSC expansion and immunosuppression in late/chronic sepsis by inducing the expression of miR-21 and miR-181b.

IL-10 induces an immune repressor pathway in sepsis by promoting S100A9 nuclear localization and MDSC development.

The myeloid-related protein S100A9 reprograms Gr1+CD11b+ myeloid precursors into myeloid-derived suppressor cells (MDSCs) during murine sepsis. Here, we show that the immunosuppressive cytokine IL-10 supports S100A9 expression and its nuclear localization in MDSCs to function as immune repressors. To support this new concept, we showed that antibody mediated IL-10 blockade in wild-type mice after sepsis induction inhibited MDSC expansion during late sepsis, and that ectopic expression of S100A9 in Gr1+CD11b+ cells from S100A9 knockout mice switched them into the MDSC phenotype only in the presence of IL-10. Knockdown of S100A9 in MDSCs from wild-type mice with late sepsis confirmed our findings in the S100A9 knockout mice. We also found that while both IL-6 and IL-10 can activate S100A9 expression in naive Gr1+CD11b+ cells, only IL-10 can induce S100A9 nuclear localization. These results support that IL-10 drives the molecular path that generates MDSCs and enhances immunosuppression during late sepsis, and inform that targeting this immune repressor path may improve sepsis survival in mice.

Nfia deletion in myeloid cells blocks expansion of myeloid-derived suppressor cells during sepsis.

Sepsis-induced immunosuppression increases the risk of chronic infection and reduces survival. Myeloid-derived suppressor cells (MDSCs) expand in the bone marrow and spleen during murine polymicrobial sepsis, contributing to immunosuppression. A better understanding of molecular controls of MDSC production is needed to identify treatment targets. We previously reported that miR-21 and miR-181b couple with transcription factor NFI-A to induce MDSCs during murine sepsis. Here, we expand upon these observations by showing that conditional deletion of the Nfia gene in the myeloid lineage precludes MDSC development. NFI-A-deficient Gr1+CD11b+ myeloid cells are not immunosuppressive and differentiate normally into macrophages and dendritic cells. In contrast, ectopically expressed NFI-A prevents differentiation of these immature Gr1+CD11b+ cells, while converting them into MDSCs. In addition, NFI-A-deficient Gr1+CD11b+ cells decreased, and cells transfected with NFI-A increase expression of miR-21 and miR181b. Our results support a myeloid cell loop in which NFI-A and miR-21 and miR-181b sustain Gr1+CD11b+ MDSC-dependent immunosuppression during sepsis.

Intracellular S100A9 Promotes Myeloid-Derived Suppressor Cells during Late Sepsis.

Myeloid precursor cell reprogramming into a myeloid-derived suppressor cell (MDSC) contributes to high mortality rates in mouse and human sepsis. S100A9 mRNA and intracellular protein levels increase during early sepsis and remain elevated in Gr1+CD11b+ MDSCs after pro-inflammatory sepsis transitions to the later chronic anti-inflammatory and immunosuppressive phenotype. The purpose of this study was to determine whether intracellular S100A9 protein might sustain Gr1+CD11b+ MDSC repressor cell reprogramming during sepsis. We used a chronic model of sepsis in mice to show that S100A9 release from MDSCs and circulating phagocytes decreases after early sepsis and that targeting the S100a9 gene improves survival. Surprisingly, we find that intracellular S100A9 protein translocates from the cytosol to nucleus in Gr1+CD11b+ MDSCs during late sepsis and promotes expression of miR-21 and miR-181b immune repressor mediators. We further provide support of this immunosuppression pathway in human sepsis. This study may inform a new therapeutic target for improving sepsis outcome.

Expression of C/EBPß in myeloid progenitors during sepsis promotes immunosuppression.

Sepsis-induced myeloid-derived suppressor cells (MDSCs) contribute to immunosuppression associated with sepsis. We reported that the CCAAT enhancer-binding protein C/EBPß activates microRNA (miR)-21 and miR-181b expressions, which induce transcription factor NFI-A to support the generation and expansion of MDSCs in the bone marrow and spleens of septic mice. Here, using a conditional knockout mouse model lacking C/EBPß in the myeloid lineage, we find that without C/EBPß, myeloid progenitor cells could not express miR-21 or miR-181b, and ectopic expression of C/EBPß in the C/EBPß-deficient myeloid progenitors activated the expression of the two miRNAs. Moreover, C/EBPß-reconstituted myeloid cells expressed IL-10 and reduced T cell proliferation and function, similar to control MDSCs that express C/EBPß. Exogenous expression of miR-21 and miR-181b in the C/EBPß-deficient myeloid progenitors from septic mice produced similar results. Notably, NFI-A-dependent transactivation of NF-kB MDSC generating pathway was reversed in the C/EBPß-deficient myeloid progenitors from septic mice. Together, these results support that decreasing C/EBPß expression prevents MDSC generation and decreases immunosuppression in septic mice, providing a target for sepsis treatment.