Myeloid-derived suppressor cell function and epigenetic expression evolves over time after surgical sepsis

Sepsis is defined as a life-threatening organ dysfunction induced by a dysregulated host response to infection, and sepsis remains one of the leading causes of preventable deaths in hospitals [1, 2]. The Agency for Healthcare Research and Quality (AHRQ) recently ranked sepsis as the most expensive condition treated in US hospitals, with estimated costs exceeding $24 billion dollars annually [3, 4]. The enormity of the problem has been recognized by the World Health Organization (WHO), which made sepsis a global health priority in 2017 [5].

Due in large part to the improvements in sepsis recognition and in subsequent treatment in intensive care units (ICUs), less than 10% of surgical sepsis patients (who can be adequately resuscitated) now die within the first 14 days of their hospital admission [6]. Although over half of surgical sepsis survivors rapidly recover and are discharged from the ICU within 14 days, nearly 50% of them (or ~ 1/3 of all surgical sepsis patients) develop what has been described as chronic critical illness (CCI). CCI is defined as patients who have prolonged stays in the ICU with unresolved organ dysfunction [7]. CCI can manifest itself as a persistent inflammation, immunosuppression, and catabolism syndrome (PICS) [7‐11], which is associated with dismal long-term outcomes, including poor cognitive and physical function, as well as self-reported quality of life [6, 12]. CCI patients extensively utilize resources as well as accrue significant personal and hospital financial burdens [13].

To date, immune modulation therapy and pharmacotherapeutic agents have proven disappointing in amending septic patient outcomes [14], although some novel agents have demonstrated potential for beneficially moderating septic patients’ immune responses [15, 16]. There are no specific treatments for sepsis survivors who experience CCI, due in part to an inadequate knowledge of its pathobiology [17, 18]. However, we have hypothesized that the persistent low-grade inflammation in PICS patients induces a myelodyscrasia, which includes increased bone marrow and extramedullary proliferation of myeloid-derived suppressor cells (MDSCs) [13, 19].

MDSCs are a heterogeneous group of immature myeloid cells first discovered in cancer patients but found to be important in many disease processes including sepsis [7, 20]. Although MDSCs have several activities, the sine qua non or required ability for a cell to be classified as a MDSC is its ability to suppress lymphocyte proliferation [11, 20]. In sepsis, MDSCs are thought to be beneficial when acutely recruited to inflamed tissues [21], as MDSCs are capable of suppressing acute inflammatory responses and resolving inflammation [22‐24]. However, if this MDSC expansion and infiltration perpetuates, the long-term persistence of MDSCs can induce significant pathophysiology leading to CCI and subsequently PICS [22, 23]. This includes host immunosuppression, an established post-septic pathology that contributes to worsened septic patient outcomes [21, 25]. In murine sepsis models, MDSCs have been found to expand in secondary lymphoid organs within 5 days and to persist for at least 12 weeks with the MDSCs inhibiting T cell proliferation via iNOS and arginase 1 production in part [26‐28]. In human patients, the proportion of the different subsets of MDSCs are noted to expand differently depending on the microbial origin of sepsis [29‐31].

MDSCs are also known to be phenotypically labile cells, capable of changing as well as undergoing terminal differentiation [32, 33]. Thus, MDSCs are a promising cell for immunomodulation therapies [32, 33]. However, the function and characterization of these cells in human sepsis remains undefined. Important to cellular transcriptional/epigenetic modification are microRNAs (miRs). miRNAs are a class of small, non-coding RNAs that regulate gene expression involved in cell development and differentiation. Altered miR expression affects the expansion of immature myeloid cell populations [34]. miRs function at the molecular level and can target proteins that are involved in myeloid lineage differentiation and maturation; thus, they represent a potential MDSC therapeutic target that can be readily manipulated [34].

In murine sepsis, leukocytes that meet the defined cell surface phenotype for MDSCs have a varying functionality depending on the time point from which these cells are isolated after the septic insult [35]. The phenotypic plasticity of these cells over time after human sepsis remains undefined, and a better understanding of MDSC function after the onset of human sepsis is required to successfully apply precision medicine to these patients. While the pathophysiology of sepsis remains highly complex, we examined whether the function of MDSCs evolves over time after sepsis in survivors who develop CCI. We also asked whether changes in the miR expression patterns over time in these sepsis survivors parallel change in MDSC function and phenotype.

Among the 262 sepsis patients included in flow cytometric analysis, the mean age was 59 years, and 121 (46%) were female. Two hundred forty-six (94%) of these patients had at least 1 comorbid condition, and the median number of comorbidities was 2.

Although post-sepsis immunopathology is complex, patients who exhibit CCI have persistent low-grade inflammation and immunosuppression that are known to contribute to their poor outcomes [19, 25]. Studies have shown that MDSCs play an important role in these phenomena [32, 44, 54]. Our research, along with others, has revealed that these cells, at least in regard to cell surface phenotype, are present in the blood of septic patients as soon as 24 h after sepsis onset and are persistent up to 42 days after the host’s initial infection in sepsis survivors with CCI. However, our current study has revealed that myeloid cells 4 days after sepsis, despite having MDSC phenotypic surface markers, are stimulatory toward T cells. This insight is important, as the timing of any immunomodulation after sepsis needs to consider if the host’s circulating immature myeloid cells do in fact require modification. Thus, personalized medicine for sepsis will require we understand the functional status of the host’s immature myeloid cells so that any patients receiving MDSC modification therapy at a specific time point would benefit from the therapy. In addition, since cellular epigenetics can be modified, microRNA expression presents itself as a potential modification therapy for plastic MDSCs in human sepsis [33].

Transcriptomic analysis of the MDSC genome at 14 days post-septic insult revealed a propensity for these cells to upregulate the inflammatory response with simultaneous downregulation of lymphatic cell proliferation. Additional analysis with IPA software revealed significant downregulation of the antigen presentation pathway (Additional file 2: Figure S2). Several studies implicate specific subsets of MDSCs as having unique roles in lymphocyte suppression. The expansion of the granulocytic subset of MDSCs is thought to be prevalent in sepsis [44]. Uhel et al., for example, was able to demonstrate that patients with early expansion of arginase-1 (ARG1)-producing G-MDSCs had strong correlations with increased T cell dysfunction and increased susceptibility to secondary infections in surgical intensive care unit (SICU) [20]. Although G-MDSCs were not the majority of circulating immature myeloid cells in the patients studied for this work, our data indicated that there was a gradual shift to the G-MDSC phenotype as the T lymphocyte proliferation suppressive function of MDSCs increased with time after surgical sepsis. Interestingly, a large portion of the isolated CD33⁺CD11b⁺HLA-DR^low/− did not meet the classic criteria of G- or M-MDSCs as defined in the cancer literature [55]. It is unclear if these CD33⁺CD11b⁺HLA-DR^low/−CD14⁻CD15⁻ cells are the “early MDSC” phenotype described by Bronte [55] or immature myeloid cells still capable of appropriate differentiation. This subpopulation of isolated CD33⁺CD11b⁺HLA-DR^low/− that were non-granulocytic, non-monocytic in nature was not described in our previous work on human MDSCs in sepsis [20]. It should be noted that our current work included all septic patients while our previous publication on human MDSCs included only patients that met the Sepsis-2 criteria for severe sepsis and septic shock. In addition, there has been a dramatic reduction in “in-hospital” mortality due to the implementation of MEWS-SRS early warning systems for sepsis. These factors may have contributed to reduced total numbers of MDSCs and percentages of G-MDSCs observed in our previous publication [20]. Future studies will need to separately analyze these MDSC subtypes and will need to include some more novel cell surface markers. Additionally, the application of single-cell RNAseq and CITEseq techniques to these cells could be very beneficial to our understanding of post-sepsis dyscrasias, especially as myelopoiesis following certain inflammatory events is not only complex, but potentially unique to the disease process.

Many studies aim to target MDSC suppressive by-products, such as arginase-1, inducible nitric oxide synthase (iNOS), nitric oxide, and reactive oxygen species (ROS) [56]. Hübner et al. recently published a study showing that increased serum arginine breakdown in response to MDSC expansion caused dysfunctional T lymphocytes following cardiopulmonary bypass. However, dose-dependent l-arginine supplementation in vitro increased CD4⁺ and CD8⁺ proliferation and enhanced secretion of T cell-related cytokines, such as IFNγ, suggesting that arginine supplementation at specific time points after sepsis may warrant further investigation [57]. Preclinical mouse models have demonstrated that inhibitors of phosphodiesterase-5, sildenafil and tadalafil, downregulate iNOS and ARG1 activities, inhibiting MDSC function and leading to activation of antitumor host immunity and prolonged survival in mice [58‐60]. Another clinical trial utilized tyrosine kinase inhibitors to target MDSCs in patients with renal carcinoma by blocking VEGF and c-kit signaling pathways. Patients receiving the tyrosine kinase inhibitors, sunitinib, had decreased levels of circulating MDSCs, reduced STAT3 activation and ARG1 expression, and displayed elevated activity and proliferation of CD8⁺ cells [61]. Though the septic microenvironment is not as local as certain cancers, similar therapeutic strategies could be of interest.

Though there are several differences between the pathophysiology of cancer and sepsis, it is important to investigate a range of treatment options. In this study, we were able to define unique epigenetic expression patterns in MDSCs isolated at 4 vs 14 days following the host’s initial sepsis onset. Importantly, our work has also illustrated differences between those MDSCs collected from CCI and RAP patients at 14 days following initial sepsis onset. Our work also demonstrated an overlap between the predicted gene targets of the differentially expressed miRs and the differentially expressed genes from septic patients at day 14. Many of the significantly altered miRNAs detected, such as mir-21-5p, mir-181a, miR-106a, and mir-17-5p, are consistent with known regulators of differentiation and maturation of MDSCs in cancer and other autoimmune diseases. miRNA-21-5p expressed in myeloid cells is thought to be a negative regulator of the NF-κB pathway [62], is overexpressed in most tumor types [34, 63], and involved in myeloid progenitor expansion [34, 64, 65]. Our data revealed a significant upregulation of miR-21 at day 4 that shifted toward a significant downregulation by day 14 when MDSCs were potently immunosuppressive against T cell proliferation and capacity to produce cytokines. In septic murine models, in vitro blockade of miR-181 and miR-21 via antagomiR injection introduced a decrease in Gr1⁺CD11b⁺ cells, improved bacterial clearance, and reduced late-sepsis mortality by 74% [66, 67].

It is important to note that several of our miR expression results were not consistent with some of the previous literature (regarding up- vs downregulation), including human cancer results [33]. This illustrates potential heterogeneity in the myelopoietic response to specific diseases as well as the tissue/organ compartment in which the immature myeloid cell resides. It also reinforces the importance of understanding the temporal relation of myelopoiesis in the host after or during a disease process.

Though several of the current treatment options seem promising, our study has revealed the importance of proper timing in MDSC modification so that the targets of these therapies are indeed modifying functionally immunosuppressive MDSCs, rather than more broadly immature myeloid cells, potentially harming the patient. MDSCs may play a dual role during infection and sepsis. During early emergency myelopoiesis, MDSCs expand acutely and provide protection against microbial invasion by producing bactericidal molecules such as ROS and reactive nitrogen species (RNS) that counter the hyperinflammatory response [32]. However, the persistence of these cells can induce deleterious effects in the host.

Our current study was limited in several ways. Our study was restricted in its capacity to investigate a breadth of T lymphocyte produced cytokines, e.g., IL-4 and IL-33 were not in our current analysis. Additionally, we were limited in the ability to isolate MDSC subsets, specifically G-MDSCs, M-MDSCs, and early MDSCs, to investigate the unique suppressive effects of each subset as previous authors have shown. Future studies will need to elucidate the subset-specific mechanisms of MDSC immunosuppression. Currently, the literature only defines early MDSCs by the lack of granulocytic and monocytic specific lineage markers, making it difficult to completely characterize this cell population. However, the use of single-cell RNAseq/CITEseq to examine the transcriptomes of subset-specific human MDSCs after sepsis could help elucidate the properties of this early MDSC cell type. Also of note, there were some limitations of attrition over time. The analyses assumed that those missing data are missing at random and that may or may not be plausible. Lastly, we were unable to administer suggested therapeutics to potentially modify MDSC epigenetics, and thus their immunosuppression capacity.

MDSCs contribute significantly to the host’s immunosuppressive status after human surgical sepsis. This effect is not immediate, but rather evolves over weeks, at which point circulating immature myeloid cells meet the classic criteria for MDSCs as defined in cancer patients. MDSC immunomodulation may well require multi-model therapy to improve septic patient outcomes. This multifaceted approach can potentially include epigenetic modifiers of these cells.