Interferon-Gamma-Release assay and absolute CD8 lymphocyte count for acquired immunosuppression monitoring in critically ill patients.

Guided biomarker-personalized immunotherapy is advancing rapidly as a means to rejuvenate immune function in injured patients who are the most immunosuppressed. A recent study introduced a fully automated interferon-γ release assay (IGRA) for monitoring the functionality of T lymphocytes in patients with septic shock. While a significant decrease in IFN-γ release capacity was observed, a significant correlation with CD8 lymphocyte absolute count was also reported, raising the question of whether ex-vivo IFN-γ production would be only a surrogate marker for lymphocyte count or if these two parameters conveyed distinct and complementary information. In a large cohort of more than 353 critically ill patients following various injuries (sepsis, trauma, major surgery), the primary objective of the present study was to simultaneously evaluate the association between ex vivo IFN-γ release and CD8 cell count with regard to adverse outcome. Our findings provide a clear-cut result, as they distinctly demonstrate that IGRA offers higher-quality information than CD8 count in terms of an independent association with the occurrence of an adverse outcome. These results strengthen the case for incorporating IGRA into the array of biomarkers of interest for defining endotypes in sepsis. This holds especially true given that fully automated tests are now readily available and could be used in routine clinical practice.

Immune profiling of critically ill patients with acute kidney injury during the first week after various types of injuries: the REALAKI study.

BACKGROUND: Acute kidney injury (AKI) is common in hospitalized patients and results in significant morbidity and mortality. The objective of the study was to explore the systemic immune response of intensive care unit patients presenting with AKI, especially the association between immune profiles and persistent AKI during the first week after admission following various types of injuries (sepsis, trauma, surgery, and burns). METHODS: REALAKI is an ancillary analysis of the REAnimation Low Immune Status Marker (REALISM) cohort study, in which 359 critically ill patients were enrolled in three different intensive care units. Patients with end-stage renal disease were excluded from the REALAKI study. Clinical samples and data were collected three times after admission: at day 1 or 2 (D1-2), day 3 or 4 (D3-4) and day 5, 6 or 7 (D5-7). Immune profiles were compared between patients presenting with or without AKI. Patients with AKI at both D1-2 and D5-7 were defined as persistent AKI. A multivariable logistic regression model was performed to determine the independent association between AKI and patients' immunological parameters. RESULTS: Three hundred and fifty-nine patients were included in this analysis. Among them, 137 (38%) were trauma patients, 103 (29%) post-surgery patients, 95 (26%) sepsis patients, and 24 (7%) were burn patients. One hundred and thirty-nine (39%) patients presented with AKI at D1-2 and 61 (20%) at D5-7. Overall, 94% presented with persistent AKI at D5-7. Patients with AKI presented with increased pro and anti-inflammatory cytokines and altered innate and adaptive immune responses. The modifications observed in the immune profiles tended to be more pronounced with increasing KDIGO stages. In the logistic regression model, a statistically significant association was observed at D1-2 between AKI and CD10lowCD16low immature neutrophils (OR 3.03 [1.7-5.5]-p < 0.001). At D5-7, increased interleukin-10 (IL-10) levels and reduced ex vivo TNF-α production after LPS stimulation were significantly associated with the presence of AKI (OR 1.38 [1.12-1.71]-p = 0.001 and 0.51 [0.27-0.91]-p = 0.03, respectively). Patients who recovered from AKI between D1-2 and D5-7 compared to patients with persistent AKI at D5-7, tended to correct these alterations. CONCLUSION: Following various types of severe injuries, early AKI is associated with the initial inflammatory response. Presence of AKI at the end of the first week after injury is associated with injury-induced immunosuppression.

Immune Profiling Panel Gene Set Identifies Critically Ill Patients With Low Monocyte Human Leukocyte Antigen-DR Expression: Preliminary Results From the REAnimation Low Immune Status Marker (REALISM) Study.

OBJECTIVES: There is a crucial unmet need for biomarker-guided diagnostic and prognostic enrichment in clinical trials evaluating immune modulating therapies in critically ill patients. Low monocyte expression of human leukocyte antigen-DR (mHLA-DR), considered as a reference surrogate to identify immunosuppressed patients, has been proposed for patient stratification in immunostimulation approaches. However, its widespread use in clinic has been somewhat hampered by technical constraints inherent to flow cytometry technology. The objective of the present study was to evaluate the ability of a prototype multiplex polymerase chain reaction tool (immune profiling panel [IPP]) to identify immunosuppressed ICU patients characterized by a low mHLA-DR expression. DESIGN: Retrospective observational cohort study. SETTING: Adult ICU in a University Hospital, Lyon, France. PATIENTS: Critically ill patients with various etiologies enrolled in the REAnimation Low Immune Status Marker study (NCT02638779). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: mHLA-DR and IPP data were obtained from 1,731 blood samples collected from critically ill patients with various etiologies and healthy volunteers. A partial least square regression model combining the expression levels of IPP markers was trained and used for the identification of samples from patients presenting with evidence of immunosuppression, defined here as mHLADR less than 8,000 antibodies bound per cell (AB/C). The IPP gene set had an area under the receiver operating characteristic curve (AUC) of 0.86 (95% CI 0.83-0.89) for the identification of immunosuppressed patients. In addition, when applied to the 123 patients still in the ICU at days 5-7 after admission, IPP similarly enriched the number of patients with ICU-acquired infections in the immunosuppressed group (26%), in comparison with low mHLA-DR (22%). CONCLUSIONS: This study reports on the potential of the IPP gene set to identify ICU patients presenting with mHLA-DR less than 8,000 AB/C. Upon further optimization and validation, this molecular tool may help in the stratification of patients that could benefit from immunostimulation in the context of personalized medicine.

Fully automated interferon-γ release assay to monitor antigen-independent T cell functionality: A proof of concept study in septic shock.

In sepsis, personalized immunotherapy is being evaluated as a means of restoring immune function in the most severely affected patients. Biomarkers play a crucial role in this process, as there are no clear clinical indicators of immune dysfunction. Functional testing is considered a gold standard for assessing immune function, but this approach faces analytical challenges in terms of clinical implementation. The use of technician-dependent, time-consuming, home-made protocols often leads to poor standardization. This study represents the first beta testing of a fully automated interferon-γ release assay (IGRA) for monitoring the functionality of antigen-independent T lymphocytes. We observed a significant decrease in IFN-γ release capacity, which was associated with typical alterations in immunological cellular parameters (such as low mHLA-DR expression and decreased CD8 T lymphocyte count), in 22 patients with septic shock. Since the test is performed using whole blood and requires no technician intervention, with results available within 4 h, it may offer new possibilities for monitoring patients with immune alterations in routine clinical conditions. Further investigations in larger cohorts of patients are now needed to validate its clinical potential.

Identification of a sub-group of critically ill patients with high risk of intensive care unit-acquired infections and poor clinical course using a transcriptomic score.

BACKGROUND: The development of stratification tools based on the assessment of circulating mRNA of genes involved in the immune response is constrained by the heterogeneity of septic patients. The aim of this study is to develop a transcriptomic score based on a pragmatic combination of immune-related genes detected with a prototype multiplex PCR tool. METHODS: As training cohort, we used the gene expression dataset obtained from 176 critically ill patients enrolled in the REALISM study (NCT02638779) with various etiologies and still hospitalized in intensive care unit (ICU) at day 5-7. Based on the performances of each gene taken independently to identify patients developing ICU-acquired infections (ICU-AI) after day 5-7, we built an unweighted score assuming the independence of each gene. We then determined the performances of this score to identify a subgroup of patients at high risk to develop ICU-AI, and both longer ICU length of stay and mortality of this high-risk group were assessed. Finally, we validated the effectiveness of this score in a retrospective cohort of 257 septic patients. RESULTS: This transcriptomic score (TScore) enabled the identification of a high-risk group of patients (49%) with an increased rate of ICU-AI when compared to the low-risk group (49% vs. 4%, respectively), with longer ICU length of stay (13 days [95% CI 8-30] vs. 7 days [95% CI 6-9], p < 0.001) and higher ICU mortality (15% vs. 2%). High-risk patients exhibited biological features of immune suppression with low monocytic HLA-DR levels, higher immature neutrophils rates and higher IL10 concentrations. Using the TScore, we identified 160 high-risk patients (62%) in the validation cohort, with 30% of ICU-AI (vs. 18% in the low-risk group, p = 0.06), and significantly higher mortality and longer ICU length of stay. CONCLUSIONS: The transcriptomic score provides a useful and reliable companion diagnostic tool to further develop immune modulating drugs in sepsis in the context of personalized medicine.

Monocyte CD169 expression in COVID-19 patients upon intensive care unit admission.

During the second surge of COVID-19 in France (fall 2020), we assessed the expression of monocyte CD169 (i.e., Siglec-1, one of the numerous IFN-stimulated genes) upon admission to intensive care units of 45 patients with RT-PCR-confirmed SARS-CoV2 pulmonary infection. Overall, CD169 expression was strongly induced on circulating monocytes of COVID-19 patients compared with healthy donors and patients with bacterial sepsis. Beyond its contribution at the emergency department, CD169 testing may be also helpful for patients' triage at the ICU to rapidly reinforce suspicion of COVID-19 etiology in patients with acute respiratory failure awaiting for PCR results for definitive diagnosis.

Longitudinal assessment of IFN-I activity and immune profile in critically ill COVID-19 patients with acute respiratory distress syndrome.

BACKGROUND: Since the onset of the pandemic, only few studies focused on longitudinal immune monitoring in critically ill COVID-19 patients with acute respiratory distress syndrome (ARDS) whereas their hospital stay may last for several weeks. Consequently, the question of whether immune parameters may drive or associate with delayed unfavorable outcome in these critically ill patients remains unsolved. METHODS: We present a dynamic description of immuno-inflammatory derangements in 64 critically ill COVID-19 patients including plasma IFNα2 levels and IFN-stimulated genes (ISG) score measurements. RESULTS: ARDS patients presented with persistently decreased lymphocyte count and mHLA-DR expression and increased cytokine levels. Type-I IFN response was initially induced with elevation of IFNα2 levels and ISG score followed by a rapid decrease over time. Survivors and non-survivors presented with apparent common immune responses over the first 3 weeks after ICU admission mixing gradual return to normal values of cellular markers and progressive decrease of cytokines levels including IFNα2. Only plasma TNF-α presented with a slow increase over time and higher values in non-survivors compared with survivors. This paralleled with an extremely high occurrence of secondary infections in COVID-19 patients with ARDS. CONCLUSIONS: Occurrence of ARDS in response to SARS-CoV2 infection appears to be strongly associated with the intensity of immune alterations upon ICU admission of COVID-19 patients. In these critically ill patients, immune profile presents with similarities with the delayed step of immunosuppression described in bacterial sepsis.

DAMPs and RAGE Pathophysiology at the Acute Phase of Brain Injury: An Overview.

Early or primary injury due to brain aggression, such as mechanical trauma, hemorrhage or is-chemia, triggers the release of damage-associated molecular patterns (DAMPs) in the extracellular space. Some DAMPs, such as S100B, participate in the regulation of cell growth and survival but may also trigger cellular damage as their concentration increases in the extracellular space. When DAMPs bind to pattern-recognition receptors, such as the receptor of advanced glycation end-products (RAGE), they lead to non-infectious inflammation that will contribute to necrotic cell clearance but may also worsen brain injury. In this narrative review, we describe the role and ki-netics of DAMPs and RAGE at the acute phase of brain injury. We searched the MEDLINE database for "DAMPs" or "RAGE" or "S100B" and "traumatic brain injury" or "subarachnoid hemorrhage" or "stroke". We selected original articles reporting data on acute brain injury pathophysiology, from which we describe DAMPs release and clearance upon acute brain injury, and the implication of RAGE in the development of brain injury. We will also discuss the clinical strategies that emerge from this overview in terms of biomarkers and therapeutic perspectives.

Multicentric experience with interferon gamma therapy in sepsis induced immunosuppression. A case series.

BACKGROUND: The sepsis-induced immunodepression contributes to impaired clinical outcomes of various stress conditions. This syndrome is well documented and characterized by attenuated function of innate and adaptive immune cells. Several pharmacological interventions aimed to restore the immune response are emerging of which interferon-gamma (IFNγ) is one. It is of paramount relevance to obtain clinical information on optimal timing of the IFNγ-treatment, -tolerance, -effectiveness and outcome before performing a RCT. We describe the effects of IFNγ in a cohort of 18 adult and 2 pediatric sepsis patients. METHODS: In this open-label prospective multi-center case-series, IFNγ treatment was initiated in patients selected on clinical and immunological criteria early (< 4 days) or late (> 7 days) following the onset of sepsis. The data collected in 18 adults and 2 liver transplanted pediatric patients were: clinical scores, monocyte expression of HLA-DR (flow cytometry), lymphocyte immune-phenotyping (flow cytometry), IL-6 and IL-10 plasma levels (ELISA), bacterial cultures, disease severity, and mortality. RESULTS: In 15 out of 18 patients IFNγ treatment was associated with an increase of median HLA-DR expression from 2666 [IQ 1547; 4991] to 12,451 [IQ 4166; 19,707], while the absolute number of lymphocyte subpopulations were not affected, except for the decrease number of NK cells 94.5 [23; 136] to 32.5 [13; 90.8] (0.0625)]. Plasma levels of IL-6 464 [201-770] to 108 (89-140) ng/mL (p = 0.04) and IL-10 from IL-10 from 29 [12-59] to 9 [1-15] pg/mL decreased significantly. Three patients who received IFNγ early after ICU admission (<4 days) died. The other patients had a rapid clinical improvement assessed by the SOFA score and bacterial cultures that were repeatedly positive became negative. The 2 pediatric cases improved rapidly, but 1 died for hemorrhagic complication. CONCLUSION: Guided by clinical and immunological monitoring, adjunctive immunotherapy with IFNγ appears well-tolerated in our cases and improves immune host defense in sepsis induced immuno suppression. Randomized clinical studies to assess its potential clinical benefit are warranted.

Platelet activation and aggregation after aneurysmal subarachnoid hemorrhage.

BACKGROUND: Endovascular techniques have proven beneficial in the treatment of aneurysmal subarachnoid hemorrhage (aSAH), but with high risk of arterial clotting, emboli and dissection. Platelet activation and alterations in hemostasis may contribute to these complications. We investigated platelet activation and aggregation pathways in aSAH patients who underwent endovascular treatment. METHODS: Two blood samples were taken, in the early days after bleeding and during the period at risk of vasospasm. We studied platelet activation through the expression of GpIIbIIIa and P-selectin as well as aggregation rate in the presence of agonists. Platelets from aSAH patients were compared with those from orthopedic postoperative patients (POSTOP). RESULTS: Platelets in aSAH were initially spontaneously activated and remained so over time. aSAH platelets were further activated with rapid aggregation in the presence of agonists, particularly ADP, with behavior comparable to POSTOP platelets. CONCLUSIONS: aSAH platelets showed prolonged increases in activation and aggregation. Therapies targeting the ADP pathway might reduce the risk of clotting and ischemic events in this context among patients requiring multiple endovascular procedures. TRIAL REGISTRATION: Not applicable.

Treatment of cerebral vasospasm following aneurysmal subarachnoid haemorrhage: a systematic review and meta-analysis.

OBJECTIVES: To examine the clinical outcome of aneurysmal subarachnoid haemorrhage (aSAH) patients exposed to cerebral vasospasm (CVS)-targeted treatments in a meta-analysis and to evaluate the efficacy of intra-arterial (IA) approaches in patients with severe/refractory vasospasm. METHODS: Randomised controlled trials, prospective and retrospective observational studies reporting clinical outcomes of aSAH patients exposed to CVS targeted treatments, published between 2006-2016 were searched using PubMed, EMBASE and the Cochrane Library. The main endpoint was the proportion of unfavourable outcomes, defined as a modified Rankin score of 3-6 at last follow-up. RESULTS: Sixty-two studies, including 26 randomised controlled trials, were included (8,976 patients). At last follow-up 2,490 of the 8,976 patients had an unfavourable outcome, including death (random-effect weighted-average, 33.7%; 99% confidence interval [CI], 28.1-39.7%; Q value, 806.0; I 2 = 92.7%). The RR of unfavourable outcome was lower in patients treated with Cilostazol (RR = 0.46; 95% CI, 0.25-0.85; P = 0.001; Q value, 1.5; I 2 = 0); and in refractory CVS patients treated by IA intervention (RR = 0.68; 95% CI, 0.57-0.80; P < 0.0001; number needed to treat with IA intervention, 6.2; 95% CI, 4.3-11.2) when compared with the best available medical treatment. CONCLUSIONS: Endovascular treatment may improve the outcome of patients with severe-refractory vasospasm. Further studies are needed to confirm this result. KEY POINTS: • 33.7% of patients with cerebral Vasospasm following aneurysmal subarachnoid-hemorrhage have an unfavorable outcome. • Refractory vasospasm patients treated using endovascular interventions have lower relative risk of unfavourable outcome. • Subarachnoid haemorrhage patients with severe vasospasm may benefit from endovascular interventions. • The relative risk of unfavourable outcome is lower in patients treated with Cilostazol.

Stent-Retriever Thrombectomy for Acute Anterior Ischemic Stroke with Tandem Occlusion: A Systematic Review and Meta-Analysis.

OBJECTIVES: To assess the efficacy and safety profile of stent-retriever thrombectomy (SRT) in acute anterior ischemic stroke patients with tandem occlusion. MATERIALS AND METHODS: Using the MEDLINE database, we conducted a systematic review and meta-analysis of all studies that included patients with acute ischemic stroke attributable to tandem occlusion who received treatment with SRT between November 2010 and May 2015. RESULTS: The literature search identified 11 previous studies involving a total of 237 subjects out of whom 193 (81.4 %) were treated with acute stent placement for the extracranial internal carotid artery occlusion. Mean initial NIHSS score was 17, and median time from onset to recanalization was 283.5 min. Mean intravenous thrombolysis rate was 63.8 %. In the meta-analysis, the recanalization rate reached 81 % (95 % CI, 73-89). Meta-analysis of clinical outcomes showed a pooled estimate of 44 % (95 % CI, 33-55; 10 studies) for favourable outcome, 13 % (95 % CI, 8-20; 10 studies) for mortality, and 7 % (95 % CI, 2-13; eight studies) for symptomatic intracranial haemorrhage. CONCLUSION: SRT with emergency carotid stenting is associated with acceptable safety and efficacy in acute anterior stroke patients with tandem occlusion compared to natural history. However, the best modality to treat proximal stenosis is based on an individual case basis. KEY POINTS: • Stent retriever thrombectomy of tandem occlusion is efficient and safe. • Emergent carotid stenting during thrombectomy increase symptomatic intracranial haemorrhage without impact mortality. • Thrombectomy of tandem anterior circulation occlusion may be the first therapeutic option.

Ly6Chigh Monocytes Protect against Kidney Damage during Sepsis via a CX3CR1-Dependent Adhesion Mechanism.

Monocytes have a crucial role in both proinflammatory and anti-inflammatory phenomena occurring during sepsis. Monocyte recruitment and activation are orchestrated by the chemokine receptors CX3CR1 and CCR2 and their cognate ligands. However, little is known about the roles of these cells and chemokines during the acute phase of inflammation in sepsis. Using intravital microscopy in a murine model of polymicrobial sepsis, we showed that inflammatory Ly6C(high) monocytes infiltrated kidneys, exhibited altered motility, and adhered strongly to the renal vascular wall in a chemokine receptor CX3CR1-dependent manner. Adoptive transfer of Cx3cr1-proficient monocyte-enriched bone marrow cells into septic Cx3cr1-depleted mice prevented kidney damage and promoted mouse survival. Modulation of CX3CR1 activation in septic mice controlled monocyte adhesion, regulated proinflammatory and anti-inflammatory cytokine expression, and was associated with the extent of kidney lesions such that the number of lesions decreased when CX3CR1 activity increased. Consistent with these results, the pro-adhesive I249 CX3CR1 allele in humans was associated with a lower incidence of AKI in patients with sepsis. These data show that inflammatory monocytes have a protective effect during sepsis via a CX3CR1-dependent adhesion mechanism. This receptor might be a new therapeutic target for kidney injury during sepsis.

Exocytosis acts as a modulator of the ILT4-mediated inhibition of neutrophil functions.

Neutrophils play a major role in inflammatory responses and immune defense against pathogens. Even though expression of inhibitory receptors has been reported on neutrophils, their role remains poorly defined. Here we show that primary human neutrophils expressed immunoglobulin-like transcript 4 (ILT4) inhibitory receptor and that this expression was induced during differentiation of the myelomonoblast PLB-985 cell line into "neutrophil-like" cells. Functional assays indicated that human leukocyte antigen G, the preferred ligand of ILT4, inhibited the phagocytic function of neutrophils. ILT4 engagement also impaired reactive oxygen species production induced through CD32a and both receptors were found colocalized into neutrophil lipid rafts. Moreover, neutrophil degranulation induced through inflammatory stimuli increased ILT4 expression as a result of the rapid translocation of an intracellular pool to the cell surface. Consequently to this ILT4 up-regulation, the human leukocyte antigen G-mediated inhibition of neutrophil phagocytic function was enhanced. Finally, we found that ILT4 up-regulation induced on healthy donor neutrophils following stimulation was impaired in presence of plasma from patients with sepsis. Similarly, ILT4 up-regulation was inhibited in neutrophils from septic patients. Altogether, our results reveal a unique mechanism of regulation of neutrophil functions through ILT4 and its exocytosis that may have implications in inflammatory disorders.