Stat3 and C/EBPß synergize to induce miR-21 and miR-181b expression during sepsis.

Myeloid-derived suppressor cells (MDSCs) increase late sepsis immunosuppression and mortality in mice. We reported that microRNA (miR) 21 and miR-181b expression in Gr1+CD11b+ myeloid progenitors increase septic MDSCs in mice by arresting macrophage and dendritic cell differentiation. Here, we report how sepsis regulates miR-21 and miR-181b transcription. In vivo and in vitro binding studies have shown that C/EBPα transcription factor, which promotes normal myeloid cell differentiation, binds both miRNA promoters in Gr1+CD11b+ cells from sham mice. In contrast, in sepsis Gr1+CD11b+ MDSCs miR-21 and miR-181b promoters bind both transcription factors Stat3 and C/EBPß, which co-imunoprecipitate as a single complex. Mechanistically, transcription factor Rb phosphorylation supports Stat3 and C/EBPß accumulation at both miRNA promoters, and C/EBPß or Stat3 depletion by siRNA in sepsis Gr1+CD11b+ MDSCs inhibits miR-21 and miR-181b expression. To further support this molecular path for MDSC accumulation, we found that Stat3 and C/EBP binding at miR-21 or miR-181b promoter was induced by IL-6, using a luciferase reporter gene transfection into naive Gr1+CD11b+ cells. Identifying how sepsis MDSCs are generated may inform new treatments to reverse sepsis immunosuppression.

MicroRNA 21 (miR-21) and miR-181b couple with NFI-A to generate myeloid-derived suppressor cells and promote immunosuppression in late sepsis.

The sepsis initial hyperinflammatory reaction, if not treated early, shifts to a protracted state of immunosuppression that alters both innate and adaptive immunity and is associated with elevated mortality. Myeloid-derived suppressor cells (MDSCs) are myeloid progenitors and precursors that fail to differentiate into mature innate-immunity cells and are known for their potent immunosuppressive activities. We previously reported that murine MDSCs expand dramatically in the bone marrow during late sepsis, induced by cecal ligation and puncture, and demonstrated that they contribute to late-sepsis immunosuppression. However, the molecular mechanism responsible for generating these immature Gr1(+) CD11b(+) myeloid cells during sepsis remains unknown. We show here that sepsis generates a microRNA (miRNA) signature that expands MDSCs. We found that miRNA 21 (miR-21) and miR-181b expression is upregulated in early sepsis and sustained in late sepsis. Importantly, we found that simultaneous in vivo blockade of both miRNAs via antagomiR (a chemically modified miRNA inhibitor) injection after sepsis initiation decreased the bone marrow Gr1(+) CD11b(+) myeloid progenitors, improved bacterial clearance, and reduced late-sepsis mortality by 74%. Gr1(+) CD11b(+) cells isolated from mice injected with antagomiRs were able to differentiate ex vivo into macrophages and dendritic cells and produced smaller amounts of the immunosuppressive interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß) after stimulation with bacterial lipopolysaccharide, suggesting that immature myeloid cells regained their maturation potential and have lost their immunosuppressive activity. In addition, we found that the protein level of transcription factor NFI-A, which plays a role in myeloid cell differentiation, was increased during sepsis and that antagomiR injection reduced its expression. Moreover, knockdown of NFI-A in the Gr1(+) CD11b(+) cells isolated from late-septic mice increased their maturation potential and reduced their production of the immunosuppressive mediators, similar to antagomiR injection. These data support the hypothesis that sepsis reprograms myeloid cells and thus alters the innate immunity cell repertoire to promote immunosuppression, and they demonstrate that this process can be reversed by targeting miR-21 and miR-181b to improve late-sepsis survival.

NFI-A disrupts myeloid cell differentiation and maturation in septic mice.

Mounting evidence supports that sepsis-associated immunosuppression increases mortality. As potential contributors to poor sepsis outcomes, myeloid-derived suppressor cells, which are Gr1(+) CD11b(+) innate-immune cell progenitors unable to differentiate and possess suppressive activities, expand dramatically in septic mice by a process requiring increased microRNA-21 and microRNA-181b expression. The inhibition of these microRNAs in vivo in septic mice restores Gr1(+) CD11b(+) cell differentiation and maturation and improves survival. Here, we show that during sepsis-induced generation of myeloid-derived suppressor cells, transcription factor nuclear factor 1 A type represses cyclin-dependent kinase inhibitor p21 to arrest differentiation of Gr1(+) CD11b(+) cells. Our findings include the following: 1) Gr1(+) CD11b(+) myeloid cells from late septic mice genetically lacking nuclear factor 1 A type cannot suppress CD4(+) T cell proliferation and activation; 2) the reconstitution of nuclear factor 1 A type in microRNA-21 and microRNA-181b-depleted Gr1(+) CD11b(+) myeloid-derived suppressor cells inhibits cyclin-dependent kinase inhibitor p21 and restores the immune-suppressor phenotype; 3) ex vivo nuclear factor 1 A type knockdown in Gr1(+) CD11b(+) myeloid-derived suppressor cells from late septic mice restores cyclin-dependent kinase inhibitor p21 expression and promotes monocyte and dendritic cell differentiation; and 4) ectopic nuclear factor 1 A type expression in normal Gr1(+) CD11b(+) cells generates an immunosuppressive phenotype. We suggest that therapeutically targeting nuclear factor 1 A type during late sepsis might improve survival.

Processing Body Formation Limits Proinflammatory Cytokine Synthesis in Endotoxin-Tolerant Monocytes and Murine Septic Macrophages.

An anti-inflammatory phenotype with pronounced immunosuppression develops during sepsis, during which time neutrophils and monocytes/macrophages limit their Toll-like receptor 4 responses to bacterial lipopolysaccharide (LPS/endotoxin). We previously reported that during this endotoxin-tolerant state, distinct signaling pathways differentially repress transcription and translation of proinflammatory cytokines such as TNFα and IL-6. Sustained endotoxin tolerance contributes to sepsis mortality. While transcription repression requires chromatin modifications, a translational repressor complex of Argonaute 2 (Ago2) and RNA-binding motif protein 4 (RBM4), which bind the 3'-UTR of TNFα and IL-6 mRNA, limits protein synthesis. Here, we show that Dcp1 supports the assembly of the Ago2 and RBM4 repressor complex into cytoplasmic processing bodies (p-bodies) in endotoxin-tolerant THP-1 human monocytes following stimulation with LPS, resulting in translational repression and limiting protein synthesis. Importantly, this translocation process is reversed by Dcp1 knockdown, which restores TNFα and IL-6 protein levels. We also find this translational repression mechanism in primary macrophages of septic mice. Because p-body formation is a critical step in mRNA translation repression, we conclude that Dcp1 is a major component of the translational repression machinery of endotoxin tolerance and may contribute to sepsis outcome.