Maria Mouktaroudi

TLR4 polymorphisms, infectious diseases, and evolutionary pressure during migration of modern humans.

Infectious diseases exert a constant evolutionary pressure on the genetic makeup of our innate immune system. Polymorphisms in Toll-like receptor 4 (TLR4) have been related to susceptibility to Gram-negative infections and septic shock. Here we show that two polymorphisms of TLR4, Asp299Gly and Thr399Ile, have unique distributions in populations from Africa, Asia, and Europe. Genetic and functional studies are compatible with a model in which the nonsynonymous polymorphism Asp299Gly has evolved as a protective allele against malaria, explaining its high prevalence in subSaharan Africa. However, the same allele could have been disadvantageous after migration of modern humans into Eurasia, putatively because of increased susceptibility to severe bacterial infections. In contrast, the Asp299Gly allele, when present in cosegregation with Thr399Ile to form the Asp299Gly/Thr399Ile haplotype, shows selective neutrality. Polymorphisms in TLR4 exemplify how the interaction between our innate immune system and the infectious pressures in particular environments may have shaped the genetic variations and function of our immune system during the out-of-Africa migration of modern humans.

Altered innate and adaptive immune responses in patients with hidradenitis suppurativa

Background  The clinical improvement of hidradenitis suppurativa reported in a small number of patients with antitumour necrosis factor (anti‐TNF)‐α therapies supports the hypothesis for an altered immune response in these patients.

Functional and genetic evidence that the Mal/TIRAP allele variant 180L has been selected by providing protection against septic shock.

Adequate responses by our innate immune system toward invading pathogens were of vital importance for surviving infections, especially before the antibiotic era. Recently, a polymorphism in Mal (Ser180Leu, TIRAP rs8177374), an important adaptor protein downstream of the Toll-like receptor (TLR) 2 and 4 pathways, has been described to provide protection against a broad range of infectious pathogens. We assessed the functional effects of this polymorphism in human experimental endotoxemia, and we demonstrate that individuals bearing the TIRAP 180L allele display an increased, innate immune response to TLR4 and TLR2 ligands, but not to TLR9 stimulation. This phenotype has been related to an increased resistance to infection. However, an overshoot in the release of proinflammatory cytokines by TIRAP 180L homozygous individuals suggests a scenario of balanced evolution. We have also investigated the worldwide distribution of the Ser180Leu polymorphism in 14 populations around the globe to correlate the genetic makeup of TIRAP with the local infectious pressures. Based on the immunological, clinical, and genetic data, we propose that this mutation might have been selected in West Eurasia during the early settlement of this region after the out-of-Africa migration of modern Homo sapiens. This combination of functional and genetic data provides unique insights to our understanding of the pathogenesis of sepsis.

Crystals of monosodium urate monohydrate enhance lipopolysaccharide-induced release of interleukin 1 beta by mononuclear cells through a caspase 1-mediated process.

OBJECTIVE Recent studies suggest that crystals of monosodium urate (MSU), deposited in joints of patients with acute gouty arthritis, activate the NACHT domain, leucine-rich repeat and pyrin domain-containing protein (NALP)3 inflammasome. In the present study we have investigated whether production of proinflammatory cytokines by crystals was exacerbated during costimulation with Toll-like receptor (TLR) ligands. METHODS Mononuclear cells of 22 healthy donors were stimulated by various concentrations of MSU crystals in the absence or presence of lipopolysaccharide (LPS), Pam3Cys and flagellin. Production of tumour necrosis factor alpha (TNFalpha), interleukin (IL)1 beta and IL6, as well as the intracellular concentrations of proIL1 beta were measured by ELISA. mRNA transcripts of TNFalpha and IL1 beta were assessed by real-time PCR. Stimulation experiments were also performed with peripheral blood mononuclear cells (PBMCs) of one patient carrying a NALP3 mutation. RESULTS MSU induced a moderate release of IL1 beta and IL6, but not of TNFalpha. Urate crystals amplified IL1 beta production stimulated by the TLR4 ligand LPS, while no synergy was apparent for IL6 production. In addition, no synergy between urate crystals and Pam3Cys (TLR2 ligand) or flagellin (TLR5 ligand) was apparent. The synergy between urate crystals and LPS was directed at the level of the NALP3 inflammasome, as it was present only when active IL1 beta was measured, but not at the level of IL1 mRNA or proIL1 beta. The synergy between LPS and MSU crystals ceased to exist in the presence of a caspase 1 inhibitor. CONCLUSIONS MSU crystals act in synergy with LPS for the induction of enhanced release of IL1 beta. Increased cleavage of proIL1 beta by urate-activated caspase 1 is proposed as the underlying mechanism.

Immunomodulatory clarithromycin treatment of experimental sepsis and acute pyelonephritis caused by multidrug-resistant Pseudomonas aeruginosa.

ABSTRACT Clarithromycin was administered intravenously to 55 rabbits to evaluate its effect on experimental sepsis caused by multidrug-resistant Pseudomonas aeruginosa. Acute pyelonephritis was induced after ligation of the right ureter and injection of 108 CFU of the test isolate per kg of body weight into the renal pelvis. The animals were divided into six groups: group A, controls; group B, rabbits that received one intravenous dose of 80 mg of clarithromycin per kg concomitantly with bacterial challenge; group C, rabbits that received two doses of clarithromycin, the second one of which was given 2 h after the first one; group D, rabbits that received 15 mg of amikacin per kg; group E, rabbits that received one dose of clarithromycin and amikacin; and group F, rabbits that received two doses of clarithromycin and amikacin. Serum endotoxin levels were estimated by the QCL-1000 Limulus amoebocyte lysate assay, tumor necrosis factor alpha (TNF-α) levels were measured by a bioassay, and malondialdehyde (MDA) levels were measured by the thiobarbiturate assay. Viable bacterial counts in various tissue samples were also assessed. The mean survival times of the animals in groups A, B, C, D, E, and F were 4.50, 7.69, 4.07, 4.55, 11.55, and 11.60 days, respectively (P = 0.033 for group D versus group F, P = 0.006 for group D versus group E, P = not significant for group B versus group E, P = 0.042 for group C versus group F). Serum endotoxin levels were similar between groups at all sampling times; TNF-α and MDA levels in groups B, C, E, and F decreased significantly over follow-up. The numbers of viable bacterial cells in the infected kidney were similar among the groups; those in the liver, spleen, lungs, and mesenteral lymph nodes were significantly decreased in groups B, E, and F compared to those in groups A and D. It is concluded that a prolongation of survival in animals with experimental sepsis caused by multidrug-resistant P. aeruginosa was achieved after coadministration of clarithromycin and amikacin and that the increased survival was probably attributable to the immunomodulatory properties of clarithromycin.

Effect of the Novel Influenza A (H1N1) Virus in the Human Immune System

Background The pandemic by the novel H1N1 virus has created the need to study any probable effects of that infection in the immune system of the host. Methodology/Principal Findings Blood was sampled within the first two days of the presentation of signs of infection from 10 healthy volunteers; from 18 cases of flu-like syndrome; and from 31 cases of infection by H1N1 confirmed by reverse RT-PCR. Absolute counts of subtypes of monocytes and of lymphocytes were determined after staining with monoclonal antibodies and analysis by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from patients and stimulated with various bacterial stimuli. Concentrations of tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-18, interferon (FN)-alpha and of IFN-gamma were estimated in supernatants by an enzyme immunoassay. Infection by H1N1 was accompanied by an increase of monocytes. PBMCs of patients evoked strong cytokine production after stimulation with most of bacterial stimuli. Defective cytokine responses were shown in response to stimulation with phytohemagglutin and with heat-killed Streptococcus pneumoniae. Adaptive immune responses of H1N1-infected patients were characterized by decreases of CD4-lymphocytes and of B-lymphocytes and by increase of T-regulatory lymphocytes (Tregs). Conclusions/Significance Infection by the H1N1 virus is accompanied by a characteristic impairment of the innate immune responses characterized by defective cytokine responses to S.pneumoniae. Alterations of the adaptive immune responses are predominated by increase of Tregs. These findings signify a predisposition for pneumococcal infections after infection by H1N1 influenza.

Influence of genetic variations in TLR4 and TIRAP/Mal on the course of sepsis and pneumonia and cytokine release: an observational study in three cohorts.

IntroductionIt has been proposed that individual genetic variation contributes to the course of severe infections and sepsis. Recent studies of single nucleotide polymorphisms (SNPs) within the endotoxin receptor and its signaling system showed an association with the risk of disease development. This study aims to examine the response associated with genetic variations of TLR4, the receptor for bacterial LPS, and a central intracellular signal transducer (TIRAP/Mal) on cytokine release and for susceptibility and course of severe hospital acquired infections in distinct patient populations.MethodsThree intensive care units in tertiary care university hospitals in Greece and Germany participated. 375 and 415 postoperative patients and 159 patients with ventilator associated pneumonia (VAP) were included. TLR4 and TIRAP/Mal polymorphisms in 375 general surgical patients were associated with risk of infection, clinical course and outcome. In two prospective studies, 415 patients following cardiac surgery and 159 patients with newly diagnosed VAP predominantly caused by Gram-negative bacteria were studied for cytokine levels in-vivo and after ex-vivo monocyte stimulation and clinical course.ResultsPatients simultaneously carrying polymorphisms in TIRAP/Mal and TLR4 and patients homozygous for the TIRAP/Mal SNP had a significantly higher risk of severe infections after surgery (odds ratio (OR) 5.5; confidence interval (CI): 1.34 - 22.64; P = 0.02 and OR: 7.3; CI: 1.89 - 28.50; P < 0.01 respectively). Additionally we found significantly lower circulating cytokine levels in double-mutant individuals with ventilator associated pneumonia and reduced cytokine production in an ex-vivo monocyte stimulation assay, but this difference was not apparent in TIRAP/Mal-homozygous patients. In cardiac surgery patients without infection, the cytokine release profiles were not changed when comparing different genotypes.ConclusionsCarriers of mutations in sequential components of the TLR signaling system may have an increased risk for severe infections. Patients with this genotype showed a decrease in cytokine release when infected which was not apparent in patients with sterile inflammation following cardiac surgery.

Early changes of CD4-positive lymphocytes and NK cells in patients with severe Gram-negative sepsis

IntroductionOur aim was to define early changes of lymphocytes and of NK cells in severe sepsis and to correlate them with serum levels of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1).MethodsBlood was sampled from 49 patients with proven highly suspected infection by Gram-negative pathogens, within 12 hours of the advent of severe sepsis, and was also sampled from six healthy volunteers. White blood cells were targeted with monoclonal antibodies and were analyzed by flow cytometry. The concentrations of sTREM-1 were estimated by ELISA.ResultsThe presence of CD3/CD4 cells was significantly lower (P < 0.0001) and that of NK cells significantly higher among patients with sepsis compared with controls (P = 0.011). The proportions (median ± standard error) of ANNEXIN-V/CD4/CD3-positive cells, of ANNEXIN-V/CD8/CD3-positive cells and of ANNEXIN-V/CD14-positive cells of the patient population were 7.41 ± 2.26%, 7.69 ± 3.42% and 1.96 ± 4.22%, respectively. Patients with NK cells >20% survived longer compared with those patients with NK cells ≤20% (P = 0.041), and patients with sTREM-1 concentrations >180 pg/ml survived longer compared with those patients with sTREM-1 concentrations ≤180 pg/ml (P = 0.042). A negative correlation was found between the percentages of ANNEXIN-V/CD4/CD3-positive cells and of CD3/CD4 cells (rs = -0.305, P = 0.049), and a positive correlation was found between the serum sTREM-1 concentration and the percentage of NK cells (rs = +0.395, P = 0.014). NK cells isolated from two healthy volunteers released sTREM-1 upon triggering with endotoxins.ConclusionEarly severe sepsis is characterized by CD4-lymphopenia and increased NK cells, providing a survival benefit for the septic patient at percentages >20%. The survival benefit resulting from elevated NK cells might be connected to elevated serum levels of sTREM-1.

Inhibition of caspase-1 activation in gram-negative sepsis and experimental endotoxemia

IntroductionDown-regulation of ex-vivo cytokine production is a specific feature in patients with sepsis. Cytokine downregulation was studied focusing on caspase-1 activation and conversion of pro-interleukin-1β into interleukin-1β (IL-1β).MethodsPeripheral blood mononuclear cells were isolated from a) 92 patients with sepsis mainly of Gram-negative etiology; b) 34 healthy volunteers; and c) 5 healthy individuals enrolled in an experimental endotoxemia study. Cytokine stimulation was assessed in vitro after stimulation with a variety of microbial stimuli.ResultsInhibition of IL-1β in sepsis was more profound than tumour necrosis factor (TNF). Down-regulation of IL-1β response could not be entirely explained by the moderate inhibition of transcription. We investigated inflammasome activation and found that in patients with sepsis, both pro-caspase-1 and activated caspase-1 were markedly decreased. Blocking caspase-1 inhibited the release of IL-1β in healthy volunteers, an effect that was lost in septic patients. Finally, urate crystals, which specifically induce the NLPR3 inflammasome activation, induced significant IL-1β production in healthy controls but not in patients with sepsis. These findings were complemented by inhibition of caspase-1 autocleavage as early as two hours after lipopolysaccharide exposure in volunteers.ConclusionsThese data demonstrate that the inhibition of caspase-1 and defective IL-1 β production is an important immunological feature in sepsis.

Risk assessment in sepsis: a new prognostication rule by APACHE II score and serum soluble urokinase plasminogen activator receptor.

IntroductionEarly risk assessment is the mainstay of management of patients with sepsis. APACHE II is the gold standard prognostic stratification system. A prediction rule that aimed to improve prognostication by APACHE II with the application of serum suPAR (soluble urokinase plasminogen activator receptor) is developed.MethodsA prospective study cohort enrolled 1914 patients with sepsis including 62.2% with sepsis and 37.8% with severe sepsis/septic shock. Serum suPAR was measured in samples drawn after diagnosis by an enzyme-immunoabsorbent assay; in 367 patients sequential measurements were performed. After ROC analysis and multivariate logistic regression analysis a prediction rule for risk was developed. The rule was validated in a double-blind fashion by an independent confirmation cohort of 196 sepsis patients, predominantly severe sepsis/septic shock patients, from Sweden.ResultsSerum suPAR remained stable within survivors and non-survivors for 10 days. Regression analysis showed that APACHE II ≥17 and suPAR ≥12 ng/ml were independently associated with unfavorable outcome. Four strata of risk were identified: i) APACHE II <17 and suPAR <12 ng/ml with mortality 5.5%; ii) APACHE II < 17 and suPAR ≥12 ng/ml with mortality 17.4%; iii) APACHE II ≥ 17 and suPAR <12 ng/ml with mortality 37.4%; and iv) APACHE II ≥17 and suPAR ≥12 ng/ml with mortality 51.7%. This prediction rule was confirmed by the Swedish cohort.ConclusionsA novel prediction rule with four levels of risk in sepsis based on APACHE II score and serum suPAR is proposed. Prognostication by this rule is confirmed by an independent cohort.