Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction.

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

Citrulline enteral administration markedly reduces immunosuppressive extrafollicular plasma cell differentiation in a preclinical model of sepsis.

The sustained immunosuppression associated with severe sepsis favors an increased susceptibility to secondary infections and remains incompletely understood. Plasmablast and plasma cell subsets, whose primary function is to secrete antibodies, have emerged as important suppressive populations that expand during sepsis. In particular, sepsis supports CD39hi plasmablast metabolic reprogramming associated with adenosine-mediated suppressive activity. Arginine deficiency has been linked to an increased risk of secondary infections in sepsis. Overcoming arginine shortage by citrulline administration efficiently improves sepsis-induced immunosuppression and secondary infections in the cecal ligation and puncture murine model. Here, we aimed to determine the impact of citrulline administration on B cell suppressive responses in sepsis. We demonstrate that restoring arginine bioavailability through citrulline administration markedly reduces the dominant extrafollicular B cell response, decreasing the immunosuppressive LAG3+ and CD39+ plasma cell populations, and restoring splenic follicles. At the molecular level, the IRF4/MYC-mediated B cell reprogramming required for extrafollicular plasma cell differentiation is shunted in the splenic B cells of mice fed with citrulline. Our study reveals a prominent impact of nutrition on B cell responses and plasma cell differentiation and further supports the development of citrulline-based clinical studies to prevent sepsis-associated immune dysfunction.

Enteral citrulline supplementation versus placebo on SOFA score on day 7 in mechanically ventilated critically ill patients: the IMMUNOCITRE randomized clinical trial.

BACKGROUND: Restoring plasma arginine levels through enteral administration of L-citrulline in critically ill patients may improve outcomes. We aimed to evaluate whether enteral L-citrulline administration reduced organ dysfunction based on the Sequential Organ Failure Assessment (SOFA) score and affected selected immune parameters in mechanically ventilated medical intensive care unit (ICU) patients. METHODS: A randomized, double-blind, multicenter clinical trial of enteral administration of L-citrulline versus placebo for critically ill adult patients under invasive mechanical ventilation without sepsis or septic shock was conducted in four ICUs in France between September 2016 and February 2019. Patients were randomly assigned to receive enteral L-citrulline (5 g) every 12 h for 5 days or isonitrogenous, isocaloric placebo. The primary outcome was the SOFA score on day 7. Secondary outcomes included SOFA score improvement (defined as a decrease in total SOFA score by 2 points or more between day 1 and day 7), secondary infection acquisition, ICU length of stay, plasma amino acid levels, and immune biomarkers on day 3 and day 7 (HLA-DR expression on monocytes and interleukin-6). RESULTS: Of 120 randomized patients (mean age, 60 ± 17 years; 44 [36.7%] women; ICU stay 10 days [IQR, 7-16]; incidence of secondary infections 25 patients (20.8%)), 60 were allocated to L-citrulline and 60 were allocated to placebo. Overall, there was no significant difference in organ dysfunction as assessed by the SOFA score on day 7 after enrollment (4 [IQR, 2-6] in the L-citrulline group vs. 4 [IQR, 2-7] in the placebo group; Mann‒Whitney U test, p = 0.9). Plasma arginine was significantly increased on day 3 in the treatment group, while immune parameters remained unaffected. CONCLUSION: Among mechanically ventilated ICU patients without sepsis or septic shock, enteral L-citrulline administration did not result in a significant difference in SOFA score on day 7 compared to placebo. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02864017 (date of registration: 11 August 2016).

SARS-CoV-2-Induced ARDS Associates with MDSC Expansion, Lymphocyte Dysfunction, and Arginine Shortage.

PURPOSE: The SARS-CoV-2 infection can lead to a severe acute respiratory distress syndrome (ARDS) with prolonged mechanical ventilation and high mortality rate. Interestingly, COVID-19-associated ARDS share biological and clinical features with sepsis-associated immunosuppression since lymphopenia and acquired infections associated with late mortality are frequently encountered. Mechanisms responsible for COVID-19-associated lymphopenia need to be explored since they could be responsible for delayed virus clearance and increased mortality rate among intensive care unit (ICU) patients. METHODS: A series of 26 clinically annotated COVID-19 patients were analyzed by thorough phenotypic and functional investigations at days 0, 4, and 7 after ICU admission. RESULTS: We revealed that, in the absence of any difference in demographic parameters nor medical history between the two groups, ARDS patients presented with an increased number of myeloid-derived suppressor cells (MDSC) and a decreased number of CD8pos effector memory cell compared to patients hospitalized for COVID-19 moderate pneumonia. Interestingly, COVID-19-related MDSC expansion was directly correlated to lymphopenia and enhanced arginase activity. Lastly, T cell proliferative capacity in vitro was significantly reduced among COVID-19 patients and could be restored through arginine supplementation. CONCLUSIONS: The present study reports a critical role for MDSC in COVID-19-associated ARDS. Our findings open the possibility of arginine supplementation as an adjuvant therapy for these ICU patients, aiming to reduce immunosuppression and help virus clearance, thereby decreasing the duration of mechanical ventilation, nosocomial infection acquisition, and mortality.

Venoarterial extracorporeal membrane oxygenation induces early immune alterations.

BACKGROUND: Venoarterial extracorporeal membrane oxygenation (VA-ECMO) provides heart mechanical support in critically ill patients with cardiogenic shock. Despite important progresses in the management of patients under VA-ECMO, acquired infections remain extremely frequent and increase mortality rate. Since immune dysfunctions have been described in both critically ill patients and after surgery with cardiopulmonary bypass, VA-ECMO initiation may be responsible for immune alterations that may expose patients to nosocomial infections (NI). Therefore, in this prospective study, we aimed to study immune alterations induced within the first days by VA-ECMO initiation. METHODS: We studied immune alterations induced by VA-ECMO initiation using cytometry analysis to characterize immune cell changes and enzyme-linked immunosorbent assay (ELISA) to explore plasma cytokine levels. To analyze specific changes induced by VA-ECMO initiation, nine patients under VA-ECMO (VA-ECMO patients) were compared to nine patients with cardiogenic shock (control patients). RESULTS: Baseline immune parameters were similar between the two groups. VA-ECMO was associated with a significant increase in circulating immature neutrophils with a significant decrease in C5a receptor expression. Furthermore, we found that VA-ECMO initiation was followed by lymphocyte dysfunction along with myeloid-derived suppressor cells (MDSC) expansion. ELISA analysis revealed that VA-ECMO initiation was followed by an increase in pro-inflammatory cytokines such as IL-6, IL-8 and TNF-α along with IL-10, a highly immunosuppressive cytokine. CONCLUSION: VA-ECMO is associated with early immune changes that may be responsible for innate and adaptive immune alterations that could confer an increased risk of infection.

Impaired efferocytosis and neutrophil extracellular trap clearance by macrophages in ARDS.

Exaggerated release of neutrophil extracellular traps (NETs) along with decreased NET clearance and inability to remove apoptotic cells (efferocytosis) may contribute to sustained inflammation in acute respiratory distress syndrome (ARDS). Recent studies in experimental models of ARDS have revealed the crosstalk between AMP-activated protein kinase (AMPK) and high-mobility group box 1 (HMGB1), which may contribute to effectiveness of efferocytosis, thereby reducing inflammation and ARDS severity.We investigated neutrophil and NET clearance by macrophages from control and ARDS patients and examined how bronchoalveolar lavage (BAL) fluid from control and ARDS patients could affect NET formation and efferocytosis. Metformin (an AMPK activator) and neutralising antibody against HMGB1 were applied to improve efferocytosis and NET clearance.Neutrophils from ARDS patients showed significantly reduced apoptosis. Conversely, NET formation was significantly enhanced in ARDS patients. Exposure of neutrophils to ARDS BAL fluid promoted NET production, while control BAL fluid had no effect. Macrophage engulfment of NETs and apoptotic neutrophils was diminished in ARDS patients. Notably, activation of AMPK in macrophages or neutralisation of HMGB1 in BAL fluid improved efferocytosis and NET clearance.In conclusion, restoration of AMPK activity with metformin or specific neutralisation of HMGB1 in BAL fluid represent promising therapeutic strategies to decrease sustained lung inflammation during ARDS.

Early Expansion of Circulating Granulocytic Myeloid-derived Suppressor Cells Predicts Development of Nosocomial Infections in Patients with Sepsis.

RATIONALE: Sepsis induces a sustained immune dysfunction responsible for poor outcome and nosocomial infections. Myeloid-derived suppressor cells (MDSCs) described in cancer and inflammatory processes may be involved in sepsis-induced immune suppression, but their clinical impact remains poorly defined. OBJECTIVES: To clarify phenotype, suppressive activity, origin, and clinical impact of MDSCs in patients with sepsis. METHODS: Peripheral blood transcriptomic analysis was performed on 29 patients with sepsis and 15 healthy donors. A second cohort of 94 consecutive patients with sepsis, 11 severity-matched intensive care patients, and 67 healthy donors was prospectively enrolled for flow cytometry and functional experiments. MEASUREMENTS AND MAIN RESULTS: Genes involved in MDSC suppressive functions, including S100A12, S100A9, MMP8, and ARG1, were up-regulated in the peripheral blood of patients with sepsis. CD14posHLA-DRlow/neg monocytic (M)-MDSCs were expanded in intensive care unit patients with and without sepsis and CD14negCD15pos low-density granulocytes/granulocytic (G)-MDSCs were more specifically expanded in patients with sepsis (P < 0.001). Plasma levels of MDSC mediators S100A8/A9, S100A12, and arginase 1 were significantly increased. In vitro, CD14pos- and CD15pos-cell depletion increased T-cell proliferation in patients with sepsis. G-MDSCs, made of immature and mature granulocytes expressing high levels of degranulation markers, were specifically responsible for arginase 1 activity. High initial levels of G-MDSCs, arginase 1, and S100A12 but not M-MDSCs were associated with subsequent occurrence of nosocomial infections. CONCLUSIONS: M-MDSCs and G-MDSCs strongly contribute to T-cell dysfunction in patients with sepsis. More specifically, G-MDSCs producing arginase 1 are associated with a higher incidence of nosocomial infections and seem to be major actors of sepsis-induced immune suppression.

Human resistin promotes neutrophil proinflammatory activation and neutrophil extracellular trap formation and increases severity of acute lung injury.

Although resistin was recently found to modulate insulin resistance in preclinical models of type II diabetes and obesity, recent studies also suggested that resistin has proinflammatory properties. We examined whether the human-specific variant of resistin affects neutrophil activation and the severity of LPS-induced acute lung injury. Because human and mouse resistin have distinct patterns of tissue distribution, experiments were performed using humanized resistin mice that exclusively express human resistin (hRTN(+/-)(/-)) but are deficient in mouse resistin. Enhanced production of TNF-α or MIP-2 was found in LPS-treated hRtn(+/-/-) neutrophils compared with control Rtn(-/-/-) neutrophils. Expression of human resistin inhibited the activation of AMP-activated protein kinase, a major sensor and regulator of cellular bioenergetics that also is implicated in inhibiting inflammatory activity of neutrophils and macrophages. In addition to the ability of resistin to sensitize neutrophils to LPS stimulation, human resistin enhanced neutrophil extracellular trap formation. In LPS-induced acute lung injury, humanized resistin mice demonstrated enhanced production of proinflammatory cytokines, more severe pulmonary edema, increased neutrophil extracellular trap formation, and elevated concentration of the alarmins HMGB1 and histone 3 in the lungs. Our results suggest that human resistin may play an important contributory role in enhancing TLR4-induced inflammatory responses, and it may be a target for future therapies aimed at reducing the severity of acute lung injury and other inflammatory situations in which neutrophils play a major role.

Neutrophil function and bactericidal activity against Staphylococcus aureus after cardiac surgery with cardiopulmonary bypass.

Staphylococcus aureus is the main bacterial pathogen encountered in mediastinitis after cardiac surgical procedures; it remains a devastating complication with a high mortality rate. As neutrophils have a primordial role in the defense against staphylococcus infection and cardiopulmonary bypass (CPB) is known to induce immunosuppression, the aim of this study was to investigate CPB impact on neutrophil functions. Patients without known immunosuppression scheduled for cardiac surgery with CPB were included. Bone marrow and blood samples were harvested before, during, and after surgery. Neutrophil phenotypic maturation and functions (migration, adhesion, neutrophil extracellular trap [NET] release, reactive oxygen species (ROS) production, phagocytosis, and bacteria killing) were investigated. Two types of Staphylococcus aureus strains (one from asymptomatic nasal carriage and another from mediastinitis infected tissues) were used to assess in vitro bacterial direct impact on neutrophils. We found that CPB induced a systemic inflammation with an increase in circulating mature neutrophils after surgery. Bone marrow sample analysis did not reveal any modification of neutrophil maturation during CPB. Neutrophil lifespan was significantly increased and functions such as NET release and ROS production were enhanced after CPB whereas bacteria killing and phagocytosis were not impacted. Results were similar with the two different isolates of Staphylococcus aureus. These data suggest that CPB induces a recruitment of mature neutrophils via a demargination process rather than impacting their maturation in the bone marrow. In addition, neutrophils are fully efficient after CPB and do not contribute to postoperative immunosuppression.

MONOCYTIC MYELOID-DERIVED SUPPRESSOR CELL EXPANSION AFTER CARDIAC SURGERY WITH CARDIOPULMONARY BYPASS INDUCES LYMPHOCYTE DYSFUNCTION.

ABSTRACT: Cardiac surgery with cardiopulmonary bypass (CPB) is associated with an immune paresis that predisposes to the development of postoperative infections and sepsis. Among factors responsible for CPB-induced immunosuppression, circulating myeloid-derived suppressor cells (MDSCs) have been found to induce early lymphocyte apoptosis and lymphocyte proliferation inhibition. However, the mechanisms involved are not fully understood. In this study, we found that the main lymphocyte subsets decreased significantly 24 h after cardiac surgery with CBP. As expected, cardiac surgery with CPB induced a monocytic MDSC expansion associated with an increased T-cell apoptosis and decreased proliferation capacity. Noteworthy, granulocytic MDSCs remain stable. Myeloid-derived suppressor cell depletion restored the ability of T-cell to proliferate ex vivo . After CPB, indoleamine 2,3-dioxygenase activity and IL-10 plasma level were increased such as programmed death-ligand 1 monocytic expression, whereas plasma level of arginine significantly decreased. Neither the inhibition of indoleamine 2,3-dioxygenase activity nor the use of anti-programmed death-ligand 1 or anti-IL-10 blocking antibody restored the ability of T-cell to proliferate ex vivo . Only arginine supplementation restored partially the ability of T-cell to proliferate.

AMPK activates Parkin independent autophagy and improves post sepsis immune defense against secondary bacterial lung infections.

Metabolic and bioenergetic plasticity of immune cells is essential for optimal responses to bacterial infections. AMPK and Parkin ubiquitin ligase are known to regulate mitochondrial quality control mitophagy that prevents unwanted inflammatory responses. However, it is not known if this evolutionarily conserved mechanism has been coopted by the host immune defense to eradicate bacterial pathogens and influence post-sepsis immunosuppression. Parkin, AMPK levels, and the effects of AMPK activators were investigated in human leukocytes from sepsis survivors as well as wild type and Park2-/- murine macrophages. In vivo, the impact of AMPK and Parkin was determined in mice subjected to polymicrobial intra-abdominal sepsis and secondary lung bacterial infections. Mice were treated with metformin during established immunosuppression. We showed that bacteria and mitochondria share mechanisms of autophagic killing/clearance triggered by sentinel events that involve depolarization of mitochondria and recruitment of Parkin in macrophages. Parkin-deficient mice/macrophages fail to form phagolysosomes and kill bacteria. This impairment of host defense is seen in the context of sepsis-induced immunosuppression with decreased levels of Parkin. AMPK activators, including metformin, stimulate Parkin-independent autophagy and bacterial killing in leukocytes from post-shock patients and in lungs of sepsis-immunosuppressed mice. Our results support a dual role of Parkin and AMPK in the clearance of dysfunctional mitochondria and killing of pathogenic bacteria, and explain the immunosuppressive phenotype associated Parkin and AMPK deficiency. AMPK activation appeared to be a crucial therapeutic target for the macrophage immunosuppressive phenotype and to reduce severity of secondary bacterial lung infections and respiratory failure.

Comparative immune profiling of acute respiratory distress syndrome patients with or without SARS-CoV-2 infection.

Acute respiratory distress syndrome (ARDS) is the main complication of coronavirus disease 2019 (COVID-19), requiring admission to the intensive care unit (ICU). Despite extensive immune profiling of COVID-19 patients, to what extent COVID-19-associated ARDS differs from other causes of ARDS remains unknown. To address this question, here, we build 3 cohorts of patients categorized in COVID-19-ARDS+, COVID-19+ARDS+, and COVID-19+ARDS-, and compare, by high-dimensional mass cytometry, their immune landscape. A cell signature associating S100A9/calprotectin-producing CD169+ monocytes, plasmablasts, and Th1 cells is found in COVID-19+ARDS+, unlike COVID-19-ARDS+ patients. Moreover, this signature is essentially shared with COVID-19+ARDS- patients, suggesting that severe COVID-19 patients, whether or not they experience ARDS, display similar immune profiles. We show an increase in CD14+HLA-DRlow and CD14lowCD16+ monocytes correlating to the occurrence of adverse events during the ICU stay. We demonstrate that COVID-19-associated ARDS displays a specific immune profile and may benefit from personalized therapy in addition to standard ARDS management.

Short-term survival of acute respiratory distress syndrome patients due to influenza virus infection alone: a cohort study.

BACKGROUND: Influenza virus (IV)-related pathophysiology suggests that the prognosis of acute respiratory distress syndrome (ARDS) due to IV could be different from the prognosis of ARDS due to other causes. However, the impact of IV infection alone on the prognosis of ARDS patients compared to that of patients with other causes of ARDS has been poorly assessed. METHODS: We compared the 28-day survival from the diagnosis of ARDS with an arterial oxygen tension/inspiratory oxygen fraction ratio ≤150 mmHg between patients with and without IV infection alone. Data were collected prospectively and analysed retrospectively. We first performed survival analysis on the whole population; second, patients with IV infection alone were compared with matched pairs using propensity score matching. RESULTS: The cohort admitted from October 2009 to March 2020 consisted of 572 patients, including 73 patients (13%) with IV alone. On the first 3 days of mechanical ventilation, nonpulmonary Sequential Organ Failure Assessment scores were significantly lower in patients with IV infection than in the other patients. After the adjusted analysis, IV infection alone remained independently associated with lower mortality at day 28 (hazard ratio 0.51, 95% CI 0.26-0.99, p=0.047). Mortality at day 28 was significantly lower in patients with IV infection alone than in other patients when propensity score matching was used (20% versus 38%, p=0.02). CONCLUSIONS: Our results suggest that patients with ARDS following IV infection alone have a significantly better prognosis at day 28 and less severe nonpulmonary organ dysfunction than do those with ARDS from causes other than IV infection alone.

Frontline Science: HMGB1 induces neutrophil dysfunction in experimental sepsis and in patients who survive septic shock.

Sepsis is accompanied by the initial activation of proinflammatory pathways and long-lasting immunosuppression that appears to contribute to late-occurring mortality. Although high-mobility group box 1 (HMGB1) is involved in many aspects of inflammation, its role in sepsis-induced immune suppression remains unclear. In this study, we examined HMGB1's contribution to neutrophil NADPH oxidase activity dysfunction and associated neutrophil-dependent bacterial clearance in mice subjected to sepsis and in patients who survive septic shock. Using a murine model of polymicrobial septic peritonitis, we demonstrated that treatment with anti-HMGB1 Ab significantly diminished sepsis-induced dysfunction of neutrophil NADPH oxidase activity. In a subsequent set of experiments, we found that blocking HMGB1 preserved the ability of neutrophils from patients recovering from septic shock to activate NADPH oxidase. Taken together, our data suggest that HMGB1 accumulation in the late phase of sepsis plays a specific role in the development of postsepsis immunosuppression and specifically affects neutrophil-dependent antibacterial defense mechanisms. Thus, blocking HMGB1 may be a promising therapeutic intervention to diminish the adverse effects of sepsis-induced immunosuppression.

Immune Dysfunction After Cardiac Surgery with Cardiopulmonary Bypass: Beneficial Effects of Maintaining Mechanical Ventilation.

UNLABELLED: Cardiac surgery with cardiopulmonary bypass (CPB) induces postoperative immunosuppression and impaired pulmonary function. Maintaining mechanical ventilation (MV) during CPB improves pulmonary function and diminishes postoperative systemic inflammation. However, there are no data about the influence of maintaining MV during CPB on postoperative immune dysfunction. METHODS: Fifty patients were prospectively divided into two groups: without MV during bypass (n = 25) and dead space MV with positive end-expiratory pressure (n = 25). PaO2 (arterial oxygen tension)/FIO2 (inspired oxygen fraction) ratio, CXCL10 (C-X-C motif chemokine 10), CCL2 (chemokine ligand 2), tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), human leukocyte antigen-DR antigen (HLA-DR), monocytic myeloid-derived suppressor cells (Mo-MDSCs, CD14HLA-DR monocytes), and blood cell count were collected before and after surgery. RESULTS: Cardiopulmonary bypass induced a marked immunosuppression with a significant increase in plasmatic levels of TNF-α and IL-10 and a significant decrease in HLA-DR monocytic expression. The postoperative proportion of Mo-MDSCs was subsequently significantly increased. Maintaining MV during CPB significantly improved PaO2/FIO2 ratio and decreased postoperative plasmatic levels of TNF-α and IL-10 compared with patients without MV during CPB. Furthermore, nonventilated patients had a lower lymphocyte count after surgery compared with patients with MV during CPB. CONCLUSION: Our study suggests that maintaining MV during CPB for cardiac surgery decreases postoperative immune dysfunction and could be an interesting strategy to diminish the occurrence of postoperative nosocomial infection without hampering the surgical procedure. However, these findings have to be confirmed in a clinical trial using the incidence of nosocomial infection as an endpoint.

Neutrophils trigger a NF-κB dependent polarization of tumor-supportive stromal cells in germinal center B-cell lymphomas.

Both tumor-associated neutrophils (TAN) and cancer-associated fibroblasts (CAFs) display specific phenotypic and functional features and contribute to tumor cell niche. However, their bidirectional crosstalk has been poorly studied, in particular in the context of hematological malignancies. Follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL) are two germinal center-derived lymphomas where various cell components of infiltrating microenvironment, including TAN and CAFs, have been demonstrated to favor directly and indirectly malignant B-cell survival, growth, and drug resistance. We show here that, besides a direct and contact-dependent supportive effect of neutrophils on DLBCL B-cell survival, mediated through the BAFF/APRIL pathway, neutrophils and stromal cells cooperate to sustain FL B-cell growth. This cooperation relies on an overexpression of IL-8 by lymphoma-infiltrating stromal cells that could thereafter efficiently promote neutrophil survival and prime them to neutrophil extracellular trap. Conversely, neutrophils are able to activate stromal cells in a NF-κB-dependent manner, inducing their commitment towards an inflammatory lymphoid stroma phenotype associated with an increased capacity to trigger malignant B-cell survival, and to recruit additional monocytes and neutrophils through the release of CCL2 and IL-8, respectively. Altogether, a better understanding of the lymphoma-supporting effects of neutrophils could be helpful to design new anti-tumor therapeutic strategies.