Alexandre Pachot

Increased percentage of CD4+CD25+ regulatory T cells during septic shock is due to the decrease of CD4+CD25- lymphocytes.

Objective:The elevation of the percentage of regulatory CD4+CD25+ T lymphocytes (Treg) has been recently described during septic shock. The objective of the present study was to investigate whether this increased percentage was due to Treg proliferation. Design:Observational study. Setting:Adult intensive care units in a university hospital. Subjects:Patients with septic shock (n = 54) and healthy individuals (n = 30). Interventions:None. Measurements and Main Results:In patients, we first confirmed the increased percentage of Treg among CD4+ lymphocytes in comparison with healthy individuals. Surprisingly, regarding absolute counting, we demonstrated that both T CD4+ lineages (CD25+ and CD25−) were diminished immediately after the onset of shock. Then, whereas Treg returned rapidly to healthy donors values, CD4+CD25− T lymphocytes remained dramatically reduced. Finally, Foxp3 (Treg-related gene) messenger RNA quantification enabled us to definitively rule out a lack of proliferation since it was not increased during shock. Conclusion:The increased percentage of Treg after shock is due not to their proliferation but to a decrease in CD4+CD25− T lymphocyte number. We hypothesize that it might be due to a resistance of Treg to apoptosis processes occurring during septic shock.

IL-17RA and IL-17RC Receptors Are Essential for IL-17A-Induced ELR+ CXC Chemokine Expression in Synoviocytes and Are Overexpressed in Rheumatoid Blood

IL-17A is a cytokine secreted by the newly described Th17 cells implicated in rheumatoid arthritis (RA). Less is known about its receptors in synoviocytes. IL-17RA and IL-17RC were found to be overexpressed in RA peripheral whole blood and their expression was detected locally in RA synovium. In vitro, IL-17A synergized with TNF-α to induce IL-6, IL-8, CCL-20, and matrix metalloproteinase-3. Using microarrays, a specific up-regulation of Glu-Leu-Arg+ CXC chemokines was observed in IL-17A-treated synoviocytes. Using both posttranslational inhibitions by silencing interfering RNA and extracellular blockade by specific inhibitors, we showed that both IL-17RA and IL-17RC are implicated in IL-17A-induced IL-6 secretion, whereas in the presence of TNF-α, the inhibition of both receptors was needed to down-regulate IL-17A-induced IL-6 and CCL-20 secretion. Thus, IL-17A-induced IL-6, IL-8, and CCL20 secretion was dependent on both IL-17RA and IL-17RC, which are overexpressed in RA patients. IL-17A-induced pathogenic effects may be modulated by IL-17RA and/or IL-17RC antagonism.

Decreased monocyte human leukocyte antigen-DR expression after severe burn injury: Correlation with severity and secondary septic shock.

Objective:Severe thermal injury causes immune dysfunctions involving both pro- and anti-inflammatory mechanisms. It subsequently leads to a state of immune deficiency that shares some similarities with sepsis-induced immunosuppression. A hallmark of the latter is established by decreased monocyte human leukocyte antigen-DR (mHLA-DR) measurements. The main objective of the current study was to characterize the appearance and the duration of low mHLA-DR expression after severe burn as well as to determine its correlation with mortality and septic complications. Design:Observational study. Setting:Burn unit (intensive care unit) in a university hospital. Patients:Severe burn patients (total burn surface area >30%, n = 14) and healthy individuals (n = 29). Interventions:None. Measurements and Main Results:Patients were immunologically monitored during 15 days. We quantified mHLA-DR expression with a standardized flow cytometry protocol. Every patient presented with decreased mHLA-DR expression at days 2–3 after burn. Then, from days 4–6, this expression increased in patients who would survive whereas it remained low in nonsurvivors. As early as days 7–10 after burn, patients who were going to develop secondary septic shock exhibited significantly lower mHLA-DR expression in comparison with patients recovering without severe septic complications. Using quantitative reverse transcriptase-polymerase chain reaction, at days 4–6, we found that the RNA level of the nonpolymorphic HLA-DRA chain and the transcription factor class II transactivator were also significantly decreased compared with healthy controls; however, plasma cytokines (interleukin-6, tumor necrosis factor-α, and interleukin-10) did not provide any significant prognostic information. Conclusions:Severe burn injury induced a marked reduction in mHLA-DR expression at both protein and messenger RNA levels. Its persistent decrease was associated with mortality and the development of septic complications.

Human CD4+CD25+ Regulatory T Lymphocytes Inhibit Lipopolysaccharide-Induced Monocyte Survival through a Fas/Fas Ligand-Dependent Mechanism

Although it is known that septic shock induces immunosuppression, the mechanism for this phenomenon is not well understood. Monocytes play a central role in septic shock pathophysiology, which is also characterized by an increased proportion of natural regulatory T (Treg) cells. We therefore investigated whether Treg could be involved in the decreased monocyte expression of CD14 and HLA-DR observed during septic shock. We demonstrated that human Treg inhibit LPS-induced retention of monocyte CD14. Because loss of CD14 is a hallmark of monocyte apoptosis, this suggests that Treg inhibit monocyte survival. This effect was largely mediated through the release of a soluble mediator that was not identical with either IL-10 or IL-4. The Fas/FasL pathway participated in the effect as it was blocked by anti-FasL Abs and reproduced by Fas agonist and recombinant soluble FasL. Furthermore, expression of FasL was much higher on Treg than on their CD25− counterparts. Collectively, these results indicate that Treg act on monocytes by inhibiting their LPS-induced survival through a proapoptotic mechanism involving the Fas/FasL pathway. This may be an important mechanism for septic shock-induced immunosuppression and may offer new perspectives for the treatment of this deadly disease.

Decreased Expression of the Fractalkine Receptor CX3CR1 on Circulating Monocytes as New Feature of Sepsis-Induced Immunosuppression

Although it is known that septic shock rapidly induces immune dysfunctions, which contribute to the impaired clearance of microorganisms observed in patients, the mechanisms for this phenomenon remain incompletely understood. We recently observed, in a microarray study, an altered circulating leukocyte CX3CR1 mRNA expression associated with patients’ mortality. As monocytes play a central role in septic shock pathophysiology and express high levels of CX3CR1, we therefore further investigated the alteration of CX3CR1 expression and of its ligand fractalkine (CX3CL1) on those cells in this clinical condition. We observed that CX3CR1 expression (both mRNA and protein) was severely down-regulated in monocytes and consequently associated with a lack of functionality upon fractalkine challenge. Importantly, nonsurvivors presented with significantly sustained lower expression in comparison with survivors. This down-regulation was reproduced by incubation of cells from healthy individuals with LPS, whole bacteria (Escherichia coli and Staphylococcus aureus), and, to a lower extent, with corticosteroids–in accordance with the concept of LPS-induced monocyte deactivation. In addition, CX3CL1 serum concentrations were elevated in patients supporting the hypothesis of increased cleavage of the membrane-anchored form expressed by endothelial cells. As CX3CR1/CX3CL1 interaction preferentially mediates arrest and migration of proinflammatory cells, the present observations may contribute to patients’ inability to kill invading microorganisms. This could represent an important new feature of sepsis-induced immunosuppression.

Galectin-10 mRNA is overexpressed in peripheral blood of aspirin-induced asthma

Background:  In sensitive patients, aspirin is associated with nasal and bronchial inflammation, eliciting local symptoms. Although the disease is clinically well characterized, its physiopathology is incompletely understood and noninvasive procedures, allowing an effective distinction between aspirin‐induced asthma (AIA) and aspirin‐tolerant asthma (ATA) are missing.

The discriminative capacity of soluble Toll-like receptor (sTLR)2 and sTLR4 in inflammatory diseases.

BackgroundThe extracellular domains of cytokine receptors are released during inflammation, but little is known about the shedding of Toll-like receptors (TLR) and whether they can be used as diagnostic biomarkers.MethodsThe release of sTLR2 and sTLR4 was studied in in-vitro stimulations, as well as in-vivo during experimental human endotoxemia (n = 11, 2 ng/kg LPS), and in plasma of 394 patients with infections (infectious mononucleosis, measles, respiratory tract infections, bacterial sepsis and candidemia) or non-infectious inflammation (Crohn’s disease, gout, rheumatoid arthritis, autoinflammatory syndromes and pancreatitis). Using C-statistics, the value of sTLR2 and sTLR4 levels for discrimination between infections and non-infectious inflammatory diseases, as well as between viral and bacterial infections was analyzed.ResultsIn-vitro, peripheral blood mononuclear cells released sTLR2 and sTLR4 by exposure to microbial ligands. During experimental human endotoxemia, plasma concentrations peaked after 2 hours (sTLR4) and 4 hours (sTLR2). sTLR4 did not correlate with cytokines, but sTLR2 correlated positively with TNFα (rs = 0.80, P < 0.05), IL-6 (rs = 0.65, P < 0.05), and IL-1Ra (rs = 0.57, P = 0.06), and negatively with IL-10 (rs = -0.58, P = 0.06), respectively. sTLR4 had a similar area under the ROC curve [AUC] for differentiating infectious and non-infectious inflammation compared to CRP: 0.72 (95% CI 0.66-0.79) versus 0.74 (95% CI 0.69-0.80) [P = 0.80], while sTLR2 had a lower AUC: 0.60 (95% CI 0.54-0.66) [P = 0.0004]. CRP differentiated bacterial infections better from viral infections than sTLR2 and sTLR4: AUC 0.94 (95% CI 0.90-0.96) versus 0.58 (95% CI 0.51-0.64) and 0.75 (95% CI 0.70-0.80), respectively [P < 0.0001 for both].ConclusionssTLRs are released into the circulation, and suggest the possibility to use sTLRs as diagnostic tool in inflammatory conditions.

Decreased HLA-DR antigen-associated invariant chain (CD74) mRNA expression predicts mortality after septic shock

IntroductionSeptic syndromes remain the leading cause of mortality in intensive care units (ICU). Septic patients rapidly develop immune dysfunctions, the intensity and duration of which have been linked with deleterious outcomes. Decreased mRNA expressions of major histocompatibility complex (MHC) class II-related genes have been reported after sepsis. We investigated whether their mRNA levels in whole blood could predict mortality in septic shock patients.MethodsA total of 93 septic shock patients were included. On the third day after shock, the mRNA expressions of five MHC class II-related genes (CD74, HLA-DRA, HLA-DMB, HLA-DMA, CIITA) were measured by qRT-PCR and monocyte human leukocyte antigen-DR (mHLA-DR) by flow cytometry.ResultsA significant correlation was found among MHC class II related gene expressions. Among mRNA markers, the best prognostic value was obtained for CD74 (HLA-DR antigen-associated invariant chain). For this parameter, the area under the receiver operating characteristic curve (AUC) was calculated (AUC = 0.67, 95% confidence interval (CI) = 0.55 to 0.79; P = 0.01) as well as the optimal cut-off value. After stratification based on this threshold, survival curves showed that a decreased CD74 mRNA level was associated with increased mortality after septic shock (Log rank test, P = 0.0043, Hazard Ratio = 3.0, 95% CI: 1.4 to 6.5). Importantly, this association remained significant after multivariate logistic regression analysis including usual clinical confounders (that is, severity scores, P = 0.026, Odds Ratio = 3.4, 95% CI: 1.2 to 9.8).ConclusionDecreased CD74 mRNA expression significantly predicts 28-day mortality after septic shock. After validation in a larger multicentric study, this biomarker could become a robust predictor of death in septic patients.

mRNA-based approach to monitor recombinant gamma-interferon restoration of LPS-induced endotoxin tolerance

IntroductionIt is now well accepted that sepsis is associated with the development of a pronounced immunosuppressive state, characterized by severe immune alterations (e.g. reduced proliferative capacity, endotoxin tolerance, apoptosis) participating in increased mortality and susceptibility to nosocomial infections. Efforts are currently aimed at restoring a functional immune response in septic patients. Successful therapy depends on the identification of appropriate immunostimulatory drugs and on the development of suitable biomarkers that could be used to stratify patients and to follow response to treatment.MethodsIn this study, we evaluated the ex vivo effect of recombinant interferon gamma (rIFN-γ) in restoring monocyte functionality (endotoxin-induced Tumor Necrosis Factor-α production) in a two-hit model of endotoxin tolerance (ET) with peripheral blood mononuclear cells from healthy volunteers and in whole blood of septic shock patients. Importantly, we used quantitative-reverse transcription polymerase-chain reaction to monitor the effect of rIFN-γ on the expression of seven genes known to participate in ET (TNF-α, IL-10, HLA-DRA, CIITA, IRAK-M, ABIN-3 and LY64).ResultsExpression analysis of those genes confirmed the presence of an immunosuppression state and the ex vivo restoration of immune functions by rIFN-γ. We show for the first time that rIFN-γ is able to bypass, at the mRNA level, the effect of negative regulators of the LPS signalling pathway such as IRAK-M, ABIN-3 and LY 64.ConclusionsOverall, mRNA expressions of a panel of genes could represent promising candidates for the ex vivo evaluation of rIFN-γ effect on monocyte functionality. This ex vivo translational research study demonstrates the potential of a mRNA-based approach to successfully monitor drug efficacy.

S100A8/A9 mRNA Induction in an Ex Vivo Model of Endotoxin Tolerance: Roles of IL-10 and IFNγ

Objectives Septic syndromes are the leading cause of death in intensive care units. They are characterized by the development of immune dysfunctions such as endotoxin tolerance (ET), whose intensity and duration are associated with increased risk of nosocomial infections and mortality. Alarmins S100A8 and S100A9 have been shown to be increased after septic shock. Importantly, a delayed S100A9 mRNA increase predicts hospital-acquired infection in patients. The aim of this study was to investigate the regulation of S100A8 and S100A9 mRNA expression in an ex vivo model of ET. Subjects and Measurements ET was reproduced ex vivo by priming healthy peripheral blood mononuclear cells (number of donors  = 9 to 10) with low-dose endotoxin (2 ng/ml) before stimulation with high dose endotoxin (100 ng/ml). S100A8 and S100A9 mRNA levels were measured by quantitative real-time polymerase chain reactions. Main Results ET was established by observing decreased TNFα and increased IL-10 transcriptomic responses to two subsequent endotoxin challenges. Interestingly, ET was associated with increased S100A8 and S100A9 mRNA expression ex vivo. We showed that IL-10 played a role in this process, since S100A8 and S100A9 mRNA increases were significantly abrogated by IL-10 blockade in the model. Conversely, treatment with rIFN-γ, a pro-inflammatory and immunostimulating molecule known to block ET induction, was able to restore normal S100A8 and S100A9 mRNA in this model. Conclusions In this ex vivo model, we observed that S100A8 and S100A9 mRNA expression was significantly increased during ET. This reproduced ex vivo the observations we had previously made in septic shock patients. Interestingly, IL-10 blockade and rIFN-γ treatment partially abrogated S100A8/A9 mRNA increases in this model. Pending confirmation in larger, independent clinical studies, these preliminary results suggest that S100A8 and S100A9 mRNA levels might be used as surrogate markers of ET and as stratification tools for personalized immunotherapy in septic shock patients.