Otelo Rigato

TLR signaling pathway in patients with sepsis.

The pathogenesis of sepsis involves complex interaction between the host and the infecting microorganism. Bacterial recognition and signaling are essential functions of the cells of innate immune systems and drive a coordinated immune response. One of the more intriguing aspects of sepsis is the fact that the protective and damaging host response are part of the same process, that is, the inflammatory response that is aimed to control the infectious process also underscores many of the pathophysiological events of sepsis. The discovery of Toll-like receptors (TLRs) in humans, and the early recognition of TLR-4 as the receptor that signals LPS bioactivity were major breakthroughs not only in the field of sepsis but also in immunology as a whole. In this article, we aimed to review TLR expression and signaling in the context of sepsis. The results obtained by our group show that TLR and other cellular surface receptors may be differently regulated on mononuclear cells and neutrophils, and that they are dynamically modulated across the stages of sepsis. Toll-like receptor signaling gene expression in mononuclear cells is decreased in more severe forms of the disease. In contrast, up-regulated genes are seen along the clinical spectrum of sepsis in neutrophils.

Impaired production of interferon-gamma and tumor necrosis factor-alpha but not of interleukin 10 in whole blood of patients with sepsis.

It has been demonstrated that lipopolysaccharide (LPS)-induced cytokine response in patients with sepsis differ from the normal host, yet this has not been controlled for the presence of underlying disease. We studied the ability of LPS and killed gram-negative bacteria (GNB) to induce tumor necrosis factor (TNF)-&agr; and interleukin (IL) 10, and of phytohemagglutinin (PHA) to induce interferon (IFN)-&ggr;, in whole blood from patients with sepsis (SP, n = 20), patients with matched underlying disease and without sepsis (control patients, n = 20), and healthy volunteers (HV, n = 20). LPS-induced TNF-&agr; production was lower in SP (median = 638 pg/mL) compared with control patients (4060 pg/mL;P = 0.003), and control patients production was lower compared with HV (5329 pg/mL;P < 0.001). Pseudomonas aeruginosa-induced TNF-&agr; production was lower in SP (1443 pg/mL) than in control patients (7319 pg/mL;P < 0.05), and was not different between control patients and HV (6612 pg/mL;P = 0.6). IFN&ggr; production was lower in SP (948 pg/mL) compared with control patients (5516 pg/mL;P < 0.001), and the control patients production was lower compared with HV (11,282 pg/mL;P < 0.001). IL-10 production was not different among the three groups. Down-regulation of TNF-&agr; production in patients with sepsis, although not restricted to them, was more pronounced with LPS than with GNB. Although the presence of underlying disease may be involved in the regulatory mechanisms of host response, the use of controls with matched underlying diseases provides strong evidence for the septic condition in the down-regulation of inflammatory response in patients with sepsis.

Generation of nitric oxide and reactive oxygen species by neutrophils and monocytes from septic patients and association with outcomes.

ABSTRACT In this study, our aims were to evaluate the reactive oxygen species (ROS) and nitric oxide (NO) generation by monocytes and neutrophils from septic patients and to correlate their levels with clinical outcomes. Forty-nine septic patients and 19 healthy volunteers were enrolled in the study. The ROS and NO production was quantified in monocytes and neutrophils in whole blood by flow cytometry, constitutively, and after stimulation with Staphylococcus aureus and Pseudomonas aeruginosa. Nitric oxide production by monocytes was higher in septic patients compared with healthy volunteers for all conditions and by neutrophils at baseline, and ROS generation in monocytes and neutrophils was higher in septic patients than in healthy volunteers for all conditions. Nitric oxide production by monocytes and neutrophils was decreased at day 7 compared with that at admission (day 0) in survivors at baseline and after stimulation with S. aureus. Reactive oxygen species production by the monocytes and neutrophils was decreased in survivors at day 7 compared with day 0 under all conditions, except by neutrophils at baseline. No difference was found in NO and ROS generation by monocytes and neutrophils between day 7 and day 0 in nonsurvivors. Generation of NO and ROS by neutrophils and monocytes is increased in septic patients, and their persistence is associated with poor outcome.

Toll-like receptor pathway signaling is differently regulated in neutrophils and peripheral mononuclear cells of patients with sepsis, severe sepsis, and septic shock.

Objectives:Up- and down-regulation of inflammatory response was described in blood cells from septic patients, according to the stage of sepsis and the cells evaluated. This study aimed to evaluate the Toll-like receptor (TLR) signaling pathway gene expression in peripheral blood mononuclear cells (PBMC) and neutrophils in patients throughout the different stages of sepsis. Design:Prospective, observational study. Settings:Two emergency rooms and two intensive care units in one university and one teaching hospital. Patients and Controls:A total of 15 septic patients, five with sepsis, five with severe sepsis, and five with septic shock, in addition to five healthy volunteers were enrolled. Interventions:None. Measurements and Main Results:The Human-TLR Signaling Pathway, which comprises 84 genes related to TLR-mediated signal transduction, was evaluated by real time polymerase chain reaction in PBMC and neutrophils obtained from patients and controls. The fold change for each gene (2(−&Dgr;&Dgr;Ct)) was compared between the groups. Genes with fold changes greater than 2 and significant changes in &Dgr;CT are reported as differently expressed. The fold change ratios in PBMC gene expression between septic patients and healthy controls revealed a dynamic process according to the stage of sepsis, tending toward down-regulation of the TLR signaling pathway in PBMC in the more severe forms of the disease. However, the differential gene expression was restricted to five down-regulated genes in septic shock patients, which are found in the effector and downstream pathways. Neutrophils showed a different pattern of adaptation. Patients with sepsis, severe sepsis, and septic shock presented a broad gene up-regulation, which included all functional groups evaluated and persisted throughout the stages of the disease. Conclusions:TLR-signaling pathway genes are differently regulated in PBMC and neutrophils of septic patients, and are dynamically modulated throughout the different stages of sepsis.

Increased Percentages of T Helper Cells Producing IL-17 and Monocytes Expressing Markers of Alternative Activation in Patients with Sepsis

Background A shift from Th1 to Th2 as well as an increase in Treg CD4+T cell subsets has been reported in septic patients (SP). Furthermore, these patients display modulation of monocyte function, with reduced production of pro-inflammatory cytokines upon LPS stimulus, which resembles the phenotype of alternatively activated macrophages. In this study, we evaluated the percentages of T cells differentiated into Th1, Th17 and Treg subsets, as well as the percentage of monocytes expressing markers of alternatively activated monocytes/macrophages (AAM) in SP. Methodology/Principal Findings Peripheral blood mononuclear cells (PBMC) were obtained from 32 healthy volunteers (HV) and from SP at admission (D0, n = 67) and after 7 days of therapy (D7, n = 33). Th1 and Th17 (CD3+CD8−) lymphocytes were identified by the intracellular detection of IFN-γ and IL-17, respectively, spontaneously and after PMA/Io stimulation, and Treg cells were identified by Foxp3+CD127- expression. Monocytes were evaluated for CD206 and CD163 expression. Absolute numbers of CD4+T lymphocytes were measured in whole blood samples by flow cytometry. The Mann-Whitney or Wilcoxon test was applied, as appropriate. The percentage of Th1 cells was lower in SP than in HV at admission after PMA/Io stimulation, whereas the percentage of Th17 cells was higher. In patients’ follow-up samples, a higher percentage of Th1 cells and a lower percentage of Th17 cells were observed on D7 compared with the D0 samples. Treg cells remained unchanged. Septic patients showed a markedly increased proportion of monocytes expressing CD163 and CD206. Conclusions/Significance Upon in vitro stimulus, the percentage of T helper lymphocytes producing IL-17 was higher in SP than in HV at admission, and the percentage producing IFN-γ was lower, a pattern that was reversed during follow-up. The increased expression of CD163 and CD206 indicates that monocytes may acquire the AAM phenotype during sepsis.

Tumor necrosis factor alpha (TNF-α) and sepsis: Evidence for a role in host defense

Serum levels of TNF-alpha were evaluated in 29 patients with sepsis, using TNF-alpha sensitive L929 cells (sensitivity=15 pg/ml). Blood samples were collected serially at the first 24–36 h of symptoms. Seventeen patients had severe underlying disease and 12 patients had mild or no underlying disease. Shock was present in 25 patients. Overall mortality was 62.1%. TNF-alpha was detected in nine patients (range: 57.7–3,169 pg/ml). There was a tendency to detect TNF-alpha in patients with mild or no underlying disease (p=0.07). Detection of TNF-alpha was associated with survival (p=0.0003) even when adjusted for severity of underlying disease (p=0.005), shock (p=0.0005), coagulation abnormality (p=0.002) and immunosuppressive therapy (p=0.005), using a bivariate analysis. In this investigation, detection of circulating TNF-alpha was predictive of good outcome in septic patients, suggesting a role for this cytokine in host-defense against this kind of infection. Bei 29 Patienten mit Sepsis wurden die Serumspiegel von TNF-alpha mittels Bioassay mit TNFalpha-sensitiven L929 Zellen (untere Nachweisgrenze 15 pg/ml) bestimmt. In den ersten 24–36 Stunden nach Beginn der Symptome wurden seriell Blutproben entnommen. Bei 17 Patienten bestand eine schwere Grundkrankheit und bei 12 eine leichtere oder keine Grundkrankheit. Bei 25 der 29 Patienten war ein Schock festzustellen. Die Gesamtmortalität lag bei 62,1%. Bei neun Patienten war TNF-alpha (Bereich 57,7–3 169 pg/ml) nachzuweisen. Tendenziell war TNF-alpha eher bei Patienten mit leichten oder ohne Grundkrankheit nachzuweisen (p=0,07). Der Nachweis von TNF-alpha war mit Überleben verbunden (p=0,0003) auch wenn eine gepaarte Analyse unter Berücksichtigung des Schweregrades der Grundkrankheit (p=0,005), Schock (p=0,0005), einer Gerinnungsstörung (p=0,002) oder immunsuppressiven Therapie (p=0,005) durchgeführt wurde. In dieser Studie erwies sich der Nachweis von TNF-alpha als prädiktiver Faktor für einen günstigen Verlauf bei Sepsispatienten, so daß angenommen werden muß, daß dieses Zytokin bei dieser Infektionsform eine Funktion in der Wirtsabwehr hat.SummarySerum levels of TNF-alpha were evaluated in 29 patients with sepsis, using TNF-alpha sensitive L929 cells (sensitivity=15 pg/ml). Blood samples were collected serially at the first 24–36 h of symptoms. Seventeen patients had severe underlying disease and 12 patients had mild or no underlying disease. Shock was present in 25 patients. Overall mortality was 62.1%. TNF-alpha was detected in nine patients (range: 57.7–3,169 pg/ml). There was a tendency to detect TNF-alpha in patients with mild or no underlying disease (p=0.07). Detection of TNF-alpha was associated with survival (p=0.0003) even when adjusted for severity of underlying disease (p=0.005), shock (p=0.0005), coagulation abnormality (p=0.002) and immunosuppressive therapy (p=0.005), using a bivariate analysis. In this investigation, detection of circulating TNF-alpha was predictive of good outcome in septic patients, suggesting a role for this cytokine in host-defense against this kind of infection.ZusammenfassungBei 29 Patienten mit Sepsis wurden die Serumspiegel von TNF-alpha mittels Bioassay mit TNFalpha-sensitiven L929 Zellen (untere Nachweisgrenze 15 pg/ml) bestimmt. In den ersten 24–36 Stunden nach Beginn der Symptome wurden seriell Blutproben entnommen. Bei 17 Patienten bestand eine schwere Grundkrankheit und bei 12 eine leichtere oder keine Grundkrankheit. Bei 25 der 29 Patienten war ein Schock festzustellen. Die Gesamtmortalität lag bei 62,1%. Bei neun Patienten war TNF-alpha (Bereich 57,7–3 169 pg/ml) nachzuweisen. Tendenziell war TNF-alpha eher bei Patienten mit leichten oder ohne Grundkrankheit nachzuweisen (p=0,07). Der Nachweis von TNF-alpha war mit Überleben verbunden (p=0,0003) auch wenn eine gepaarte Analyse unter Berücksichtigung des Schweregrades der Grundkrankheit (p=0,005), Schock (p=0,0005), einer Gerinnungsstörung (p=0,002) oder immunsuppressiven Therapie (p=0,005) durchgeführt wurde. In dieser Studie erwies sich der Nachweis von TNF-alpha als prädiktiver Faktor für einen günstigen Verlauf bei Sepsispatienten, so daß angenommen werden muß, daß dieses Zytokin bei dieser Infektionsform eine Funktion in der Wirtsabwehr hat.

Patterns of Gene Expression in Peripheral Blood Mononuclear Cells and Outcomes from Patients with Sepsis Secondary to Community Acquired Pneumonia

Mechanisms governing the inflammatory response during sepsis have been shown to be complex, involving cross-talk between diverse signaling pathways. Current knowledge regarding the mechanisms underlying sepsis provides an incomplete picture of the syndrome, justifying additional efforts to understand this condition. Microarray-based expression profiling is a powerful approach for the investigation of complex clinical conditions such as sepsis. In this study, we investigate whole-genome expression profiles in mononuclear cells from survivors (n = 5) and non-survivors (n = 5) of sepsis. To circumvent the heterogeneity of septic patients, only patients admitted with sepsis caused by community-acquired pneumonia were included. Blood samples were collected at the time of sepsis diagnosis and seven days later to evaluate the role of biological processes or genes possibly involved in patient recovery. Principal Components Analysis (PCA) profiling discriminated between patients with early sepsis and healthy individuals. Genes with differential expression were grouped according to Gene Ontology, and most genes related to immune defense were up-regulated in septic patients. Additionally, PCA in the early stage was able to distinguish survivors from non-survivors. Differences in oxidative phosphorylation seem to be associated with clinical outcome because significant differences in the expression profile of genes related to mitochondrial electron transport chain (ETC) I–V were observed between survivors and non-survivors at the time of patient enrollment. Global gene expression profiles after seven days of sepsis progression seem to reproduce, to a certain extent, patterns collected at the time of diagnosis. Gene expression profiles comparing admission and follow-up samples differed between survivors and non-survivors, with decreased expression of genes related to immune functions in non-survivors. In conclusion, genes related to host defense and inflammatory response ontology were up-regulated during sepsis, consistent with the need for a host response to infection, and the sustainability of their expression in follow-up samples was associated with outcomes.

Lipopolysaccharide-cell interaction and induced cellular activation in whole blood of septic patients.

We used biotinylatedLPS (LPSb) and flow cytometry to study LPS—monocyte interaction and LPS-induced cellular activation in whole blood from septic patients (SP). Expression of surface activation markers was evaluated on monocytes (HLA-DR) and T lymphocytes (CD69 and CD95), and intracellular TNF-α on monocytes. Saturating curve and kinetics of LPSb detection on monocytes were similar in SP and healthy volunteers (HV). LPSb bound to monocytes was detected after 5 min of incubation in both groups, with a more pronounced decay in SP. Monocytes from SP had a lower expression of HLA-DR as compared to HV, both constitutive and upon LPS stimulation. The proportion of monocytes producing TNF-α after LPS stimulus was higher in HV than SP (mean ± SD = 25.2 ± 14.2% and 2.2 ± 2.6%, respectively, P < 0.001). LPS-induced CD69 on T CD8+ and CD8— lymphocytes was similar for patients and controls. Expression of CD95 on T lymphocytes was higher in SP as compared to HV on T CD8+ cells (GMFI, mean ± SD = 22.3 ± 14.6 and 8.6 ± 5.0, respectively, P = 0.01) and CD8— cells (GMFI, mean ± SD = 28.3 ± 7.7 and 14 ± 4.3 respectively, P < 0.001). Thus, monocytes and lymphocytes seem to respond differently to LPS in septic patients. Monocyte hyporesponsiveness appears not to be related to a decreased binding capacity of LPS, but rather to an impaired signal transduction.

Bacterial Recognition and Induced Cell Activation in Sepsis

The pathogenesis of sepsis involves complex interaction between the host and the infecting microorganism. Recognition and processing of microorganism antigens are essential functions of the cells of innate immune systems, and will ultimately, through the antigen presentation to the cells of adaptive immunity and the synthesis and secretions of mediators, such as cytokines, drive a coordinated immune response. Neutrophils and monocytes will therefore function as sensing and effectors cells. Fundamental in this process is the ability to discriminate self from non-self molecules. Of major interest in sepsis is that the protective and damaging host responses are part of the same process, that is, the inflammatory response that controls the infection process also underscores many of the pathophysiological events of sepsis. Moreover, this is a dynamic process according to the continuum of sepsis and its complications; up and down regulation of cellular activities may be differently regulated in different tissues, different cells and even in different functions of the same cell. This review will focus on microorganism recognition and signalization in sepsis, with emphasis on the neutrophils and monocytes adaptation during the ongoing disease.

Pathogenetic Aspects of Sepsis and Possible Targets for Adjunctive Therapy

The outcome of patients with sepsis arises from multiple factors affecting both the host and the invading microorganisms. Age, presence of underlying disease, source of infection and some specific etiological agents have been related to prognosis. Appropriateness of antimicrobial therapy, considering the in vitro susceptibility tests for the infecting bacteria, has been strongly associated with the outcome. Therefore even after the cascade of sepsis has been triggered, the control of bacteria growth is still fundamental for the outcome of the infection. This is a major distinction point from experimental studies in which whole killed bacteria and their products are used as model of sepsis. However, even within the setting of adequate antimicrobial use, patients still die of sepsis. Thus, strategies focusing on further therapy targets are an important area of interest for basic and clinical research. Although such adjunctive sepsis therapy has failed to achieve consistent better survival rates so far, nevertheless, it improved our understanding of the pathophysiological events seen in sepsis that the possibility that a new and effective treatment may arise has been warmly considered. In this paper we aim to review some aspects of the pathogenesis of sepsis, focusing on recent advances and on possible targets for adjunctive therapy. Published clinical trials and experimental data supporting such trials are commented on.