Profiling the dysregulated immune response in sepsis: overcoming challenges to achieve the goal of precision medicine.

Sepsis is characterised by a dysregulated host immune response to infection. Despite recognition of its significance, immune status monitoring is not implemented in clinical practice due in part to the current absence of direct therapeutic implications. Technological advances in immunological profiling could enhance our understanding of immune dysregulation and facilitate integration into clinical practice. In this Review, we provide an overview of the current state of immune profiling in sepsis, including its use, current challenges, and opportunities for progress. We highlight the important role of immunological biomarkers in facilitating predictive enrichment in current and future treatment scenarios. We propose that multiple immune and non-immune-related parameters, including clinical and microbiological data, be integrated into diagnostic and predictive combitypes, with the aid of machine learning and artificial intelligence techniques. These combitypes could form the basis of workable algorithms to guide clinical decisions that make precision medicine in sepsis a reality and improve patient outcomes.

Association of Asymmetric and Symmetric Dimethylarginine with Inflammation in the Population-Based Study of Health in Pomerania.

The amino acids arginine (Arg), asymmetric (ADMA) and symmetric dimethylarginine (SDMA) are related to nitric oxide (NO) metabolism and potential markers of two different disease entities: cardiovascular disease such as atherosclerosis and systemic inflammation in critically ill patients with sepsis. Although very different in their pathophysiological genesis, both entities involve the functional integrity of blood vessels. In this context, large population-based data associating NO metabolites with proinflammatory markers, e.g., white blood cell count (WBC), high-sensitivity C-reactive protein (hsCRP), and fibrinogen, or cytokines are sparse. We investigated the association of Arg, ADMA and SDMA with WBC, hsCRP, and fibrinogen in 3556 participants of the Study of Health in Pomerania (SHIP)-TREND study. Furthermore, in a subcohort of 456 subjects, 31 inflammatory markers and cytokines were analyzed. We identified Arg and SDMA to be positively associated with hsCRP (ß coefficient 0.010, standard error (SE) 0.002 and 0.298, 0.137, respectively) as well as fibrinogen (ß 5.23 × 10-3, SE 4.75 × 10-4 and 0.083, 0.031, respectively). ADMA was not associated with WBC, hsCRP, or fibrinogen. Furthermore, in the subcohort, Arg was inversely related to a proliferation-inducing ligand (APRIL). SDMA was positively associated with osteocalcin, tumor necrosis factor receptor 1 and 2, and soluble cluster of differentiation 30. Our findings provide new insights into the involvement of Arg, ADMA, and SDMA in subclinical inflammation in the general population.

Kinetics of human leukocyte antigen receptor HLA-DR+ monocytes and T lymphocytes during remission induction therapy in ANCA-associated vasculitis.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is characterized by small vessel inflammation and the presence of autoantibodies against cytoplasmic proteases, most often proteinase-3 and myeloperoxidase. Peripheral blood monocytes are an important source of local macrophage accumulation within parenchymal organs, as evidenced by their presence in early lesions in ANCA-associated glomerulonephritis. Major histocompatibility complex (MHC) II cell surface receptor human leukocyte antigen receptor (HLA-DR) allows antigen presentation to T cells and is crucial for the initiation of an immune response. We herein report HLA-DR abundance in AAV and the kinetics of HLA-DR+ monocytes and T lymphocytes during remission induction therapy in AAV. Life-threatening AAV with pulmonary hemorrhage and renal involvement was associated with the presence of HLA-DR in a considerable population of peripheral blood monocytes and T lymphocytes, and relapsing disease manifested despite persistent B cell depletion after remission induction with rituximab. Moreover, remission induction in AAV with steroids, plasma exchange and intravenous cyclophosphamide, and improvement of clinical symptoms were associated with a decrease in HLA-DR+ differing between monocytes and T lymphocytes. Particularly, persistent suppression of HLA-DR+ monocytes was observed during remission induction, while an initial decrease in HLA-DR+ T lymphocytes was followed by recovery of this population during the further course. Detailed insights into HLA-DR kinetics could pave the way towards an increased understanding of immunopathology and identify patients that could mostly benefit from distinct remission induction regimens.

Erythrocytes increase endogenous sphingosine 1-phosphate levels as an adaptive response to SARS-CoV-2 infection.

Low plasma levels of the signaling lipid metabolite sphingosine 1-phosphate (S1P) are associated with disrupted endothelial cell (EC) barriers, lymphopenia and reduced responsivity to hypoxia. Total S1P levels were also reduced in 23 critically ill patients with coronavirus disease 2019 (COVID-19), and the two main S1P carriers, serum albumin (SA) and high-density lipoprotein (HDL) were dramatically low. Surprisingly, we observed a carrier-changing shift from SA to HDL, which probably prevented an even further drop in S1P levels. Furthermore, intracellular S1P levels in red blood cells (RBCs) were significantly increased in COVID-19 patients compared with healthy controls due to up-regulation of S1P producing sphingosine kinase 1 and down-regulation of S1P degrading lyase expression. Cell culture experiments supported increased sphingosine kinase activity and unchanged S1P release from RBC stores of COVID-19 patients. These observations suggest adaptive mechanisms for maintenance of the vasculature and immunity as well as prevention of tissue hypoxia in COVID-19 patients.

Case Report: Interferon- γ Rescues Monocytic Human Leukocyte Antigen Receptor (mHLA-DR) Function in a COVID-19 Patient With ARDS and Superinfection With Multiple MDR 4MRGN Bacterial Strains.

Background: CD14+ monocytes present antigens to adaptive immune cells via monocytic human leukocyte antigen receptor (mHLA-DR), which is described as an immunological synapse. Reduced levels of mHLA-DR can display an acquired immune defect, which is often found in sepsis and predisposes for secondary infections and fatal outcomes. Monocytic HLA-DR expression is reliably induced by interferon- γ (IFNγ) therapy. Case Report: We report a case of multidrug-resistant superinfected COVID-19 acute respiratory distress syndrome (ARDS) on extracorporeal membrane oxygenation (ECMO) support. The resistance profiles of the detected Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Citrobacter freundii isolates were equipped with resistance to all four antibiotic classes including carbapenems (4MRGN) and Cefiderocol in the case of K. pneumoniae. A causal therapeutic antibiotic strategy was not available. Therefore, we measured the immune status of the patient aiming to identify a potential acquired immune deficiency. Monocyte HLA-DR expression identified by FACS analysis revealed an expression level of 34% positive monocytes and suggested severe immunosuppression. We indicated IFNγ therapy, which resulted in a rapid increase in mHLA-DR expression (96%), rapid resolution of invasive bloodstream infection, and discharge from the hospital on day 70. Discussion: Superinfection is a dangerous complication of COVID-19 pneumonia, and sepsis-induced immunosuppression is a risk factor for it. Immunosuppression is expressed by a disturbed antigen presentation of monocytes to cells of the adaptive immune system. The case presented here is remarkable as no validated antibiotic regimen existed against the detected bacterial pathogens causing bloodstream infection and severe pneumonia in a patient suffering from COVID-19 ARDS. Possible restoration of the patient's own immunity by IFNγ was a plausible option to boost the patient's immune system, eliminate the identified 4MRGNs, and allow for lung recovery. This led to the conclusion that immune status monitoring is useful in complicated COVID-19-ARDS and that concomitant IFNγ therapy may support antibiotic strategies. Conclusion: After a compromised immune system has been detected by suppressed mHLA-DR levels, the immune system can be safely reactivated by IFNγ.

Dynamics of Vascular Protective and Immune Supportive Sphingosine-1-Phosphate During Cardiac Surgery.

Introduction: Sphingosine-1-phosphate (S1P) is a signaling lipid and crucial in vascular protection and immune response. S1P mediated processes involve regulation of the endothelial barrier, blood pressure and S1P is the only known inducer of lymphocyte migration. Low levels of circulatory S1P correlate with severe systemic inflammatory syndromes such as sepsis and shock states, which are associated with endothelial barrier breakdown and immunosuppression. We investigated whether S1P levels are affected by sterile inflammation induced by cardiac surgery. Materials and Methods: In this prospective observational study we included 46 cardiac surgery patients, with cardiopulmonary bypass (CPB, n=31) and without CPB (off-pump, n=15). Serum-S1P, S1P-sources and carriers, von-Willebrand factor (vWF), C-reactive protein (CRP), procalcitonin (PCT) and interleukin-6 (IL-6) were measured at baseline, post-surgery and at day 1 (POD 1) and day 4 (POD 4) after surgical stimulus. Results: Median S1P levels at baseline were 0.77 nmol/mL (IQR 0.61-0.99) and dropped significantly post-surgery. S1P was lowest post-surgery with median levels of 0.37 nmol/mL (IQR 0.31-0.47) after CPB and 0.46 nmol/mL (IQR 0.36-0.51) after off-pump procedures (P<0.001). The decrease of S1P was independent of surgical technique and observed in all individuals. In patients, in which S1P levels did not recover to preoperative baseline ICU stay was longer and postoperative inflammation was more severe. S1P levels are associated with its sources and carriers and vWF, as a more specific endothelial injury marker, in different phases of the postoperative course. Determination of S1P levels during surgery suggested that also the anticoagulative effect of heparin might influence systemic S1P. Discussion: In summary, serum-S1P levels are disrupted by major cardiac surgery. Low S1P levels post-surgery may play a role as a new marker for severity of cardiac surgery induced inflammation. Due to well-known protective effects of S1P, low S1P levels may further contribute to the observed prolonged ICU stay and worse clinical status. Moreover, we cannot exclude a potential inhibitory effect on circulating S1P levels by heparin anticoagulation during surgery, which would be a new pro-inflammatory pleiotropic effect of high dose heparin in patients undergoing cardiac surgery.

Urinary Levels of SARS-CoV-2 Nucleocapsid Protein Associate With Risk of AKI and COVID-19 Severity: A Single-Center Observational Study.

Background: Acute kidney injury (AKI) is very common in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) disease 2019 (COVID-19) and considered as a risk factor for COVID-19 severity. SARS-CoV-2 renal tropism has been observed in COVID-19 patients, suggesting that direct viral injury of the kidneys may contribute to AKI. We examined 20 adult cases with confirmed SARS-CoV-2 infection requiring ICU supportive care in a single-center prospective observational study and investigated whether urinary markers for viral infection (SARS-CoV-2 N) and shedded cellular membrane proteins (ACE2, TMPRSS2) allow identification of patients at risk for AKI and outcome of COVID-19. Objectives: The objective of the study was to evaluate whether urinary markers for viral infection (SARS-CoV-2 N) and shedded cellular membrane proteins (ACE2, TMPRSS2) allow identification of patients at risk for AKI and outcome of COVID-19. Results: Urinary SARS-CoV-2 N measured at ICU admission identified patients at risk for AKI in COVID-19 (HR 5.9, 95% CI 1.4-26, p = 0.0095). In addition, the combination of urinary SARS-CoV-2 N and plasma albumin measurements further improved the association with AKI (HR 11.4, 95% CI 2.7-48, p = 0.0016). Finally, combining urinary SARS-CoV-2 N and plasma albumin measurements associated with the length of ICU supportive care (HR 3.3, 95% CI 1.1-9.9, p = 0.0273) and premature death (HR 7.6, 95% CI 1.3-44, p = 0.0240). In contrast, urinary ACE2 and TMPRSS2 did not correlate with AKI in COVID-19. Conclusions: In conclusion, urinary SARS-CoV-2 N levels associate with risk for AKI and correlate with COVID-19 severity.

Case Report: Interferon-γ Restores Monocytic Human Leukocyte Antigen Receptor (mHLA-DR) in Severe COVID-19 With Acquired Immunosuppression Syndrome.

Background: The major histocompatibility complex (MHC) class II characterized by monocytes CD14+ expression of human leukocyte antigen receptors (HLA-DR), is essential for the synapse between innate and adaptive immune response in infectious disease. Its reduced expression is associated with a high risk of secondary infections in septic patients and can be safely corrected by Interferon-y (IFNy) injection. Coronavirus disease (COVID-19) induces an alteration of Interferon (IFN) genes expression potentially responsible for the observed low HLA-DR expression in circulating monocytes (mHLA-DR). Methods: We report a case of one-time INFy injection (100 mcg s.c.) in a superinfected 61-year-old man with COVID-19-associated acute respiratory distress syndrome (ARDS), with monitoring of mHLA-DR expression and clinical tolerance. Observations: Low mHLA-DR pretreatment expression (26.7%) was observed. IFNy therapy leading to a rapid increase in mHLA-DR expression (83.1%). Conclusions: Severe ARDS in a COVID-19 patient has a deep reduction in mHLA-DR expression concomitantly with secondary infections. The unique IFNy injection was safe and led to a sharp increase in the expression of mHLA-DR. Based on immune and infection monitoring, more cases of severe COVID-19 patients with low mHLA-DR should be treated by IFNy to test the clinical effectiveness.

Reference ranges for sphingosine-1-phosphate in neonates.

OBJECTIVES: Sphingosine-1-phosphate (S1P) is a signalling lipid involved in embryonic development, physiological homeostasis, and pathogenic processes in multiple organ systems. Disturbance of S1P homeostasis has been associated with various human diseases in which the immune response and vascular integrity are severely compromised. Up-to-date, no study has analyzed S1P levels in neonates. The objective of this study was to determine S1P serum concentrations in neonates and establish S1P reference ranges. METHODS: S1P levels in the umbilical cord blood of 460 term and preterm neonates were compared to a previously described cohort of healthy adult blood donors. S1P levels were further correlated with demographic characteristics, cellular sources of S1P, and inflammatory markers. RESULTS: The median S1P serum level in neonates was 1.70 µmol/L (IQR 1.41-1.97 µmol/L) and significantly higher than normal values reported in adults. S1P levels correlated positively with the number of red blood cells (p<0.001) and negatively with neutrophil precursors (p=0.028). CONCLUSIONS: Elevated S1P levels in neonates compared to adults possibly result from higher S1P content in its cellular sources due to the essential role of S1P during embryogenesis. Generated S1P ranges may be used as reference ranges for future studies in neonates.

SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization.

Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to farmed mink has been observed in Europe and the US. In the infected animals, viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink are mostly compatible with efficient entry into human cells and its inhibition by soluble angiotensin-converting enzyme 2 (ACE2). In contrast, mutation Y453F reduces neutralization by an antibody with emergency use authorization for coronavirus disease 2019 (COVID-19) therapy and sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink.

Camostat Mesylate May Reduce Severity of Coronavirus Disease 2019 Sepsis: A First Observation.

Severe acute respiratory syndrome coronavirus 2 cell entry depends on angiotensin-converting enzyme 2 and transmembrane serine protease 2 and is blocked in cell culture by camostat mesylate, a clinically proven protease inhibitor. Whether camostat mesylate is able to lower disease burden in coronavirus disease 2019 sepsis is currently unknown. DESIGN: Retrospective observational case series. SETTING: Patient treated in ICU of University hospital Göttingen, Germany. PATIENTS: Eleven critical ill coronavirus disease 2019 patients with organ failure were treated in ICU. INTERVENTIONS: Compassionate use of camostat mesylate (six patients, camostat group) or hydroxychloroquine (five patients, hydroxychloroquine group). MEASUREMENTS AND MAIN RESULTS: Clinical courses were assessed by Sepsis-related Organ Failure Assessment score at days 1, 3, and 8. Further, viral load, oxygenation, and inflammatory markers were determined. Sepsis-related Organ Failure Assessment score was comparable between camostat and hydroxychloroquine groups upon ICU admission. During observation, the Sepsis-related Organ Failure Assessment score decreased in the camostat group but remained elevated in the hydroxychloroquine group. The decline in disease severity in camostat mesylate treated patients was paralleled by a decline in inflammatory markers and improvement of oxygenation. CONCLUSIONS: The severity of coronavirus disease 2019 decreased upon camostat mesylate treatment within a period of 8 days and a similar effect was not observed in patients receiving hydroxychloroquine. Camostat mesylate thus warrants further evaluation within randomized clinical trials.

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.

Current gaps in sepsis immunology: new opportunities for translational research.

Increasing evidence supports a central role of the immune system in sepsis, but the current view of how sepsis affects immunity, and vice versa, is still rudimentary. The European Group on Immunology of Sepsis has identified major gaps that should be addressed with high priority, such as understanding how immunological alterations predispose to sepsis, key aspects of the immunopathological events during sepsis, and the long-term consequences of sepsis on patient's immunity. We discuss major unmet topics in those three categories, including the role of key immune cells, the cause of lymphopenia, organ-specific immunology, the dynamics of sepsis-associated immunological alterations, the role of the microbiome, the standardisation of immunological tests, the development of better animal models, and the opportunities offered by immunotherapy. Addressing these gaps should help us to better understand sepsis physiopathology, offering translational opportunities to improve its prevention, diagnosis, and care.

Analyses of sphingosine-1-phosphate in the context of transfusion: how much is in stored blood products and in patient blood?

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bloodborne lipid that regulates vascular tone and endothelial permeability. S1P concentrations are reduced in critically ill patients. As hematopoietic cells produce S1P, this study intends to investigate S1P concentrations in blood products during storage and in patient plasma after blood transfusion. STUDY DESIGN AND METHODS: S1P concentrations were measured in 83 red blood cell (RBC) units and 73 platelet concentrates (PCs) before and after storage. In addition, 26 critically ill patients who received one or two RBC units were recruited to measure S1P plasma levels before and three times within 24 hours after transfusion. RESULTS: The highest S1P concentrations were found in fresh PCs. S1P concentrations in PCs are reduced by 60% when stored at room temperature for 4 days, whereas in RBCs S1P concentrations remained stable when stored at 4°C within 35 days. S1P concentrations in PCs and RBCc were 2.5 to 6 times higher compared to patient plasma. Plasma S1P levels in critically ill patients, however, transiently decreased after transfusion of RBCs and recover to pretransfusion values within the following 24 hours. CONCLUSION: S1P concentrations in blood products are significantly higher compared to human plasma S1P levels, even though plasma S1P levels decreased after RBC transfusion in critically ill patients and reached pretransfusion values within 24 hours.

Loss of sphingosine 1-phosphate (S1P) in septic shock is predominantly caused by decreased levels of high-density lipoproteins (HDL).

BACKGROUND: Sphingosine 1-phosphate (S1P) is a signaling lipid essential in regulating processes involved in sepsis pathophysiology, including endothelial permeability and vascular tone. Serum S1P is progressively reduced in sepsis patients with increasing severity. S1P function depends on binding to its carriers: serum albumin (SA) and high-density lipoproteins (HDL). The aim of this single-center prospective observational study was to determine the contribution of SA- and HDL-associated S1P (SA-S1P and HDL-S1P) to sepsis-induced S1P depletion in plasma with regard to identify future strategies to supplement vasoprotective S1P. METHODS: Sequential precipitation of lipoproteins was performed with plasma samples obtained from 100 ICU patients: surgical trauma (n = 20), sepsis (n = 63), and septic shock (n = 17) together with healthy controls (n = 7). Resultant fractions with HDL and SA were analyzed by liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-MS/MS) for their S1P content. RESULTS: Plasma S1P levels significantly decreased with sepsis severity and showed a strong negative correlation with increased organ failure, quantified by the Sequential Organ Failure Assessment (SOFA) score (rho - 0.59, P < 0.001). In controls, total plasma S1P levels were 208 µg/L (187-216 µg/L). In trauma patients, we observed an early loss of SA-S1P (- 70%) with a concurrent increase of HDL-S1P (+ 20%), resulting in unaltered total plasma S1P with 210 µg/L (143-257 µg/L). The decrease of plasma S1P levels with increasing SOFA score in sepsis patients with 180.2 µg/L (123.3-253.0 µg/L) and in septic shock patients with 99.5 µg/L (80.2-127.2 µg/L) was mainly dependent on equivalent reductions of HDL and not SA as carrier protein. Thus, HDL-S1P contributed most to total plasma S1P in patients and progressively dropped with increasing SOFA score. CONCLUSIONS: Reduced plasma S1P was associated with sepsis-induced organ failure. A constant plasma S1P level during the acute phase after surgery was maintained with increased HDL-S1P and decreased SA-S1P, suggesting the redistribution of plasma S1P from SA to HDL. The decrease of plasma S1P levels in patients with increasing sepsis severity was mainly caused by decreasing HDL and HDL-S1P. Therefore, strategies to reconstitute HDL-S1P rather than SA-S1P should be considered for sepsis patients.

Symmetrical (SDMA) and asymmetrical dimethylarginine (ADMA) in sepsis: high plasma levels as combined risk markers for sepsis survival.

BACKGROUND: Nitric oxide (NO) regulates processes involved in sepsis progression, including vascular and immune function. NO is generated by nitric oxide synthases (NOS) from L-arginine. Cellular L-arginine uptake is inhibited by symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA) is a competitive inhibitor of NOS. Increased inhibitor blood concentrations lead to reduce NO bioavailability. The aim of this study was to determine whether plasma concentrations of SDMA and ADMA are markers for sepsis survival. METHOD: This prospective, single center study involved 120 ICU patients with sepsis. Plasma SDMA and ADMA were measured on admission (day 1), day 3 and day 7 by mass spectrometry together with other laboratory markers. The sequential organ failure assessment (SOFA) score was used to evaluate sepsis severity. Survival was documented until day 28. Groups were compared using the Mann-Whitney U test, chi-squared test or non-parametric analysis of variance (ANOVA). Mortality was assessed using Kaplan-Meier curves and compared using the log-rank test. Specific risk groups were identified using a decision tree algorithm. RESULTS: Median plasma SDMA and ADMA levels were significantly higher in non-survivors than in survivors of sepsis: SDMA 1.14 vs. 0.82 µmol/L (P = 0.002) and ADMA 0.93 vs. 0.73 µmol/L (P = 0.016). ANOVA showed that increased plasma SDMA and ADMA concentrations were significantly associated with SOFA scores. The 28-day mortality was compared by chi-square test: for SDMA the mortality was 12% in the lower, 25% in the intermediate and 43% in the 75th percentile (P = 0.018); for ADMA the mortality was 18-20% in the lower and intermediate but 48% in the 75th percentile (P = 0.006). The highest mortality (61%) was found in patients with plasma SDMA > 1.34 together with ADMA levels > 0.97 µmol/L. CONCLUSIONS: Increased plasma concentrations of SDMA and ADMA are associated with sepsis severity. Therefore, our findings suggest reduced NO bioavailability in non-survivors of sepsis. One may use individual SDMA and ADMA levels to identify patients at risk. In view of the pathophysiological role of NO we conclude that the vascular system and immune response are most severely affected when SDMA and ADMA levels are high.

Human leucocyte antigen (HLA-DR) gene expression is reduced in sepsis and correlates with impaired TNFα response: A diagnostic tool for immunosuppression?

BACKGROUND: Sepsis is defined as a dysregulated immune response to infection. Impaired immune response in sepsis, often described as endotoxin tolerance, is characterized by unresponsiveness of monocytes on lipopolysaccharide (LPS) stimulation to release tumor necrosis factor α (TNFα). Furthermore, decreased monocyte surface protein expression of human leucocyte antigen DR (HLA-DR) is a marker for changes of the innate immune response during sepsis. Quantitative polymerase chain reaction (qPCR) and flow-cytometry (FACS) have been used to measure protein or gene expression of HLA-DR. We aimed to determine whether changes in mRNA expression of HLA-DR are associated with impaired TNFα response in human sepsis. METHODS: Surface protein together with mRNA expression of HLA-DR were measured by FACS and qPCR in a cohort of 9 sepsis patients and compared to 10 pre-operative control patients in a prospective study. In addition, 20 patients with post-surgical inflammation, 20 patients with sepsis or septic shock were included and TNFα was determined following ex vivo stimulation of whole blood with 500 pg/mL LPS. Total RNA was prepared from whole blood and subjected to qPCR analysis for expression analysis of HLA-DR alpha (HLA-DRA) to correlate TNFα response with HLA-DRA expression. RESULTS: Patients with sepsis presented higher numbers of monocytes in peripheral blood (P<0.001) but decreased surface protein and mRNA HLA-DR levels when compared to controls. In all patients mRNA expression of HLA-DRA was decreased by approximately 70% compared to controls (P<0.01) and was lowest in patients with sepsis or septic shock (P<0.01). TNFα response to LPS was decreased in all patients (median 319 pg/mL versus controls 1256 pg/mL; P<0.01) and lowest in patients with sepsis or septic shock (median 128 pg/mL; P<0.01). HLA-DRA correlated positively with TNFα response in all study participants (r +0.60, P<0.001) and within patients (r +0.67, P<0.001). The TNFα:HLA-DRA ratio correlated negatively with severity and the Sequential Organ Failure Assessment (SOFA) score (Spearman's rho -0.59, P<0.001). CONCLUSION: In this study, HLA-DRA expression was associated with a functional assay of the innate immune response. Future interventional studies aimed at the immune response during sepsis could make use of these methods for optimizing target groups based on biological plausibility and intervention effectiveness.