key: cord-0920031-wyggm522 authors: Coudereau, Rémy; Waeckel, Louis; Cour, Martin; Rimmele, Thomas; Pescarmona, Rémi; Fabri, Astrid; Jallades, Laurent; Yonis, Hodane; Gossez, Morgane; Lukaszewicz, Anne‐Claire; Argaud, Laurent; Venet, Fabienne; Monneret, Guillaume title: Emergence of immunosuppressive LOX‐1+ PMN‐MDSC in septic shock and severe COVID‐19 patients with acute respiratory distress syndrome date: 2021-04-28 journal: J Leukoc Biol DOI: 10.1002/jlb.4covbcr0321-129r sha: 15c66b5bc5e15857f3f73a8135e4d2363f031df4 doc_id: 920031 cord_uid: wyggm522 Myeloid‐derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with immunosuppressive properties. In cancer patients, the expression of lectin‐type oxidized LDL receptor 1 (LOX‐1) on granulocytic MDSC identifies a subset of MDSC that retains the most potent immunosuppressive properties. The main objective of the present work was to explore the presence of LOX‐1+ MDSC in bacterial and viral sepsis. To this end, whole blood LOX‐1+ cells were phenotypically, morphologically, and functionally characterized. They were monitored in 39 coronavirus disease‐19 (COVID‐19, viral sepsis) and 48 septic shock (bacterial sepsis) patients longitudinally sampled five times over a 3 wk period in intensive care units (ICUs). The phenotype, morphology, and immunosuppressive functions of LOX‐1+ cells demonstrated that they were polymorphonuclear MDSC. In patients, we observed the significant emergence of LOX‐1+ MDSC in both groups. The peak of LOX‐1+ MDSC was 1 wk delayed with respect to ICU admission. In COVID‐19, their elevation was more pronounced in patients with acute respiratory distress syndrome. The persistence of these cells may contribute to long lasting immunosuppression leaving the patient unable to efficiently resolve infections. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with immunosuppressive properties as they are potent repressors of T-cell responses. 1 Two main subtypes of MDSC are currently described. 2 More recently, MDSC have been reported to be involved in other clinical contexts including acute and chronic inflammation. 4 The salient feature of MDSC in these conditions is their ability to inhibit T cell function and thus to contribute to the occurrence of subsequent immunosuppression. As an illustrative example, sepsis, defined as a life-threatening organ dysfunction caused by a dysregulated inflammatory host response to infection, 5 is characterized by a delayed step of immunosuppression. 6 Recent studies have demonstrated the association between elevated circulating MDSC (both PMN-and M-MDSC) and increased and/or higher risk of nosocomial infections during sepsis. [7] [8] [9] Interestingly, the most severe cases of coronavirus disease-19 (COVID-19, caused by SARS-CoV-2) are viral sepsis by virtue of international definition (i.e., infection and one organ failure that is acute respiratory failure). Once COVID-19 patients develop acute respiratory distress syndrome (ARDS), they similarly present with features of immunosuppression: altered type-I IFN signaling, 10 decreased monocyte HLA-DR (mHLA-DR), 11 lymphopenia, 12 and altered lymphocyte functions. 13 A couple of recent articles reported on the early emergence of PMN-MDSC especially in the most severe COVID-19 patients. 14, 15 Although there is a growing interest in monitoring MDSC in clinical practice, the lack of a specific phenotypic marker usable in whole blood protocol renders their assessment difficult on a routine basis due to the necessity to perform cell purification before staining. This is especially true for PMN-MDSC, which are investigated among PBMCs after isolation by density gradient centrifugation. 3 In 2016, Condamine et al. reported that the expression of lectin-type oxidized LDL receptor 1 (LOX-1) identified a subset of MDSC that retained the most potent immunosuppressive properties. 16 Importantly, LOX-1 staining can be performed in whole blood. 16 Since this pioneering description, LOX-1 expression in MDSC have been confirmed in few human cancer studies. 17, 18 In this context, the question of the emergence of LOX-1+ PMN-MDSC in bacterial and viral sepsis remained unsolved. The main objective of the present work was thus to highlight the presence of these cells in two etiologies of sepsis, that is, bacterial and viral and to verify that these cells identified in whole blood possessed all the characteristics of MDSC. Fresh peripheral blood was collected using EDTA anticoagulant tubes. A total of 100 μl of whole blood was labelled with antibodies: Fresh PBMCs were isolated from whole blood by Ficoll density gra- Freshly purified T cells were stimulated with anti-CD2/3/28 coatedbeads (T cell Activation/Expansion kit, 2 beads per T cell, Biolegend, Bergisch Gladbach, Germany) and cultured alone or cocultured with heterologous CD45dim LOX1+ or CD45+ LOX1-PMN at a 1:1 ratio for 72 h at 37 • C and 5% CO 2 . T cell functionality was assessed by measuring IFN-γ production in culture supernatant (ELLA, Proteinsimple, San Jose, CA, USA), according to the manufacturer's instructions. Results regarding expression of CD45 dim LOX1 + cells in patients are presented as individual values and Tukey boxplots. For clinical parameters, continuous data and biologic parameters are presented as medians and interquartile ranges (IQR) whereas categoric data are presented as numbers of cases and percentages among total population (median and IQR). Results regarding IFN-γ production of T cells in coculture are presented as means ± SD. Nonparametric Mann-Whitney, Fisher, and χ 2 tests were used to assess differences between HV and patients or differences between viral and bacterial sepsis, and the nonparametric Wilcoxon paired test was used to assess variations between patients themselves at different time points. P-values lower than 0.05 were considered statistically significant. As a preliminary step, we first developed a whole blood protocol to assess LOX-1 expression on circulating neutrophils. As regularly performed in clinical flow cytometry, we used CD45 expression to draw a leukogate in order to exclude debris. Then, neutrophils were identified based on CD15 positivity and lack of CRTH2 (in order to exclude eosinophils). As depicted in Figure 1 , we were able to identify a subset of neutrophils expressing LOX-1. These cells presented with diminished CD45 expression (Fig. 1A) and an immature phenotype as they did not express CD16 nor CD10 (Fig. 1A) . We next performed LOX-1 staining after Ficoll purification on cells from both Ficoll ring and pellet. As expected, LOX-1+ CD15+ neutrophils were found in the Ficoll ring with PBMCs and were absent from the pellet (Supporting Information Fig. S1 ). We confirmed that these LOX-1+ cells presented with low CD10 and CD16 expressions. In addition, after staining with May-Grünwald Giemsa, sorted CD45 dim CD15+ LOX-1+ population appeared as neutrophil precursors presenting with immature morphology (i.e., poorly segmented reniform nucleus). In contrast, CD45+ LOX-1 negative cells (found in the pellet) appeared as mature neutrophils exhibiting a well-segmented nucleus (Supporting Information Fig. S1 ). In order to further qualify LOX-1+ neutrophils as MDSC, we next investigated their suppressive functions. 2 Due to the limited biologic resources (i.e., small amount of available blood from severely ill ICU patients), we assessed inhibition of IFN-γ production by T cells that require fewer cells than proliferation assay. To this end, Sepsis, which by international definition is defined as an organ dysfunction due to a dysregulated host response to an infection, 19 is characterized by the occurrence of delayed immunosuppression that associates with poor outcome and nosocomial infections. 6 ,20 In this condition, MDSC emergence has been described and is reported to contribute to mechanisms sustaining this state of immunosuppression. 7, 21 Although mainly due to bacterial and fungal etiologies, viral origin is also a cause for sepsis. COVID-19 is an illustrative example of viral sepsis because patients infected by SARS-CoV-2 may develop (in worst cases) ARDS (that is a major pulmonary dysfunction). Interestingly, COVID-19 patients also present with marked immunosuppression characterized by severe lymphopenia, decreased mHLA-DR expression, or increased IL-10 concentrations. 11, [22] [23] [24] In addition to lymphopenia, T cell altered functionality, and decreased mHLA-DR, the current results illustrate that LOX-1+ MDSC occurrence constitutes another similarity between sepsis and cancer. 25 Considering their potent immunosuppressive properties (T cell inhibition, arginine depletion, reactive oxygen species synthesis, production of PGE2, and anti-inflammatory cytokines), it is reasonable to speculate on their participation in septic patients worsening due to immunosuppression. In line, their elevation is a marker of severity associated with occurrence of ARDS. Another striking result of the present study is the similar rise of these immunosuppressive cells in bacterial and viral sepsis and the delayed maximal appearance of LOX-1+ MDSC because they peaked approximately 1 wk after ICU admission in both etiologies of sepsis. This agrees with previous results showing that decreased mHLA-DR, observed in septic shock and COVID-19 patients, is associated with unfavorable outcomes in a delayed manner, that is, from day 4 after septic shock. 6, 20 Over days persistence of low mHLA-DR predicted nosocomial infections occurrence as well as mortality, 8, 26, 27 whereas inaugural values did not. Here, at ICU discharge, patients still presented elevated LOX-1+ MDSC values. Importantly, despite constant improvements in the management of patients, the long-term mortality in sepsis has remained dramatically high. 28 Increased mortality may last few years after sepsis. 29 The first cause of rehospitalization and death after sepsis are infectious diseases evoking a role for a long-term persistence of immunosuppression. This phenomenon is hypothesized to be due to persistent inflammation, immunosuppression, and catabolism syndrome, and/or chronic critical illness, defined as Nonparametric Mann-Whitney test was used to assess differences between HV and patients. The nonparametric Wilcoxon paired test was used to assess variations between patients themselves at day 0/2 and day 3/5; *P < 0.05, **P < 0.01 more than 14 d spent in ICU associated with organ dysfunction and immunosuppression. In both cases, chronic low-grade inflammation perpetuates long-term immunosuppression that in turn favors additional infectious events. In that respect, proinflammatory compounds such as alarmins (S100A9/S100A8, HMGB-1) are believed to induce MDSC proliferation/differentiation during late bacterial sepsis by promoting immune repressor functions in bone marrow. 9,30-33 Most importantly, S100A8 (also known as calgranulin) has been demonstrated to be an important early maker of severity in COVID-19 patients. 34 We can thus speculate that this nonresolving inflammation observed in septic shock and COVID-19 patients, which is also a feature of various cancers may represent a common mechanism leading to the induction of MDSC known to lower immune surveillance. 25 Nevertheless, our study has limitations. 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Hospices Civils de Lyon, Immunology Department: Fabienne Additional supporting information may be found online in the Supporting Information section at the end of the article.