Myeloid-derived suppressor cells (MDSCs) are a heterogeneous Gr1⁺CD11b⁺population of immature cells containing granulocytic and monocytic progenitors, which expand under nearly all inflammatory conditions and are potent repressors of T-cell responses. Studies of MDSCs during inflammatory responses, including sepsis, suggest they can protect or injure. Here, we investigated MDSCs during early and late sepsis. To do this, we used our published murine model of cecal ligation and puncture (CLP)-induced polymicrobial sepsis, which transitions from an early proinflammatory phase to a late anti-inflammatory and immunosuppressive phase. We confirmed that Gr1⁺CD11b⁺MDSCs gradually increase after CLP, reaching ∼88% of the bone marrow myeloid series in late sepsis. Adoptive transfer of early (day 3) MDSCs from septic mice into naive mice after CLP increased proinflammatory cytokine production, decreased peritoneal bacterial growth, and increased early mortality. Conversely, transfer of late (day 12) MDSCs from septic mice had the opposite effects. Early and late MDSCs studiedex vivoalso differed in their inflammatory phenotypes. Early MDSCs expressed nitric oxide and proinflammatory cytokines, whereas late MDSCs expressed arginase activity and anti-inflammatory interleukin 10 (IL-10) and transforming growth factor β (TGF-β). Late MDSCs had more immature CD31⁺myeloid progenitors and, when treatedex vivowith granulocyte-macrophage colony-stimulating factor (GM-CSF), generated fewer macrophages and dendritic cells than early MDSCs. We conclude that as the sepsis inflammatory process progresses, the heterogeneous MDSCs shift to a more immature state and from being proinflammatory to anti-inflammatory.

Following the TLR-dependent initiation phase of acute systemic proinflammatory responses such as sepsis, an adaptive phase represses or activates a specific pattern of gene expression until the inflammation resolves. Here, we used the THP-1 sepsis cell model of bacterial LPS/endotoxin tolerance to show that TLR4-induced miR-146a supports the feed-forward adaptive processes that silence transcription and disrupt translation of acute proinflammatory genes. First, we found that miR-146a regulates a pathway that promotes the binding of transcription repressor RelB to the TNF-α promoter, a step known to precede histone and DNA modifications, which generate facultative heterochromatin to silence acute proinflammatory genes. However, once RelB binding occurred, miR-146a inhibition could not reverse compacted chromatin, and endotoxin tolerance persisted. Second, we observed that miR-146a regulates a pathway that supports assembly of the translation repressor complex of TNF-α by preventing the interaction of the RNA-binding protein effector Ago2 and RBM4. We also determined that once endotoxin tolerance is established, and specific genes have been reprogrammed, transcription and translation disruption can be reversed only by simultaneously depleting RelB and inhibiting miR-146a. Thus, miR-146a induction supports the TLR4-dependent shift from initiation to gene-specific repression at two levels. Our results also imply that therapies designed to reverse endotoxin tolerance as potential therapies for sepsis should be directed at the transcription and translation pathways of reprogramming.

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 β (TGF-β) 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.

RUNX1 overlapping RNA (RUNXOR) is a long non-coding RNA and plays a pivotal role in the differentiation of myeloid cells via targeting runt-related transcription factor 1 (RUNX1). We and others have previously reported that myeloid-derived suppressor cells (MDSCs) expand and inhibit host immune responses during chronic viral infections; however, the mechanisms responsible for MDSC differentiation and suppressive functions, in particular the role of RUNXOR–RUNX1, remain unclear. Here, we demonstrated that RUNXOR and RUNX1 expressions are significantly upregulated and associated with elevated levels of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), signal transducer and activator of transcription 3 (STAT3), and reactive oxygen species (ROS) in MDSCs during chronic hepatitis C virus (HCV) infection. Mechanistically, we discovered that HCV-associated exosomes (HCV-Exo) can induce the expressions of RUNXOR and RUNX1, which in turn regulates miR-124 expression via STAT3 signaling, thereby promoting MDSC differentiation and suppressive functions. Importantly, overexpression of RUNXOR in healthy CD33⁺ myeloid cells promoted differentiation and suppressive functions of MDSCs. Conversely, silencing RUNXOR or RUNX1 expression in HCV-derived CD33⁺ myeloid cells significantly inhibited their differentiation and expressions of suppressive molecules and improved the function of co-cultured autologous CD4 T cells. Taken together, these results indicate that the RUNXOR–RUNX1–STAT3–miR124 axis enhances the differentiation and suppressive functions of MDSCs and could be a potential target for immunomodulation in conjunction with antiviral therapy during chronic HCV infection.

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/EBPβ, which co‐imunoprecipitate as a single complex. Mechanistically, transcription factor Rb phosphorylation supports Stat3 and C/EBPβ accumulation at both miRNA promoters, and C/EBPβ 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.