Interleukin-33 improves local immunity during Gram-negative pneumonia by a combined effect on neutrophils and inflammatory monocytes.

Pneumonia represents a major health care burden and Gram-negative bacteria provide an increasing therapeutic challenge at least in part through the emergence of multidrug-resistant strains. IL-33 is a multifunctional cytokine belonging to the IL-1 family that can affect many different cell types. We sought here to determine the effect of recombinant IL-33 on the host response during murine pneumonia caused by the common Gram-negative pathogen Klebsiella pneumoniae. IL-33 pretreatment prolonged survival for more than 1 day during lethal airway infection and decreased bacterial loads at the primary site of infection and distant organs. Postponed treatment with IL-33 (3 h) also reduced bacterial growth and dissemination. IL-33-mediated protection was not observed in mice deficient for the IL-33 receptor component IL-1 receptor-like 1. IL-33 induced a brisk type 2 response, characterized by recruitment of type 2 innate lymphoid cells to the lungs and enhanced release of IL-5 and IL-13. However, neither absence of innate lymphoid cells or IL-13, nor blocking of IL-5 impacted on IL-33 effects in mice infected with Klebsiella. Likewise, IL-33 remained effective in reducing bacterial loads in mice lacking B, T, and natural killer T cells. Experiments using antibody-mediated cell depletion indicated that neutrophils and inflammatory monocytes were of importance for antibacterial defense. The capacity of IL-33 to restrict bacterial growth in the lungs was strongly reduced in mice depleted of both neutrophils and inflammatory monocytes, but not in mice selectively depleted of either one of these cell types. These results suggest that IL-33 boosts host defense during bacterial pneumonia by a combined effect on neutrophils and inflammatory monocytes. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

A genome-wide association study of survival in patients with sepsis.

BACKGROUND: Sepsis is a severe systemic inflammatory response to infections that is accompanied by organ dysfunction and has a high mortality rate in adult intensive care units. Most genetic studies have identified gene variants associated with development and outcomes of sepsis focusing on biological candidates. We conducted the first genome-wide association study (GWAS) of 28-day survival in adult patients with sepsis. METHODS: This study was conducted in two stages. The first stage was performed on 687 European sepsis patients from the GEN-SEP network and 7.5 million imputed variants. Association testing was conducted with Cox regression models, adjusting by sex, age, and the main principal components of genetic variation. A second stage focusing on the prioritized genetic variants was performed on 2,063 ICU sepsis patients (1362 European Americans and 701 African-Americans) from the MESSI study. A meta-analysis of results from the two stages was conducted and significance was established at p < 5.0 × 10-8. Whole-blood transcriptomic, functional annotations, and sensitivity analyses were evaluated on the identified genes and variants. FINDINGS: We identified three independent low-frequency variants associated with reduced 28-day sepsis survival, including a missense variant in SAMD9 (hazard ratio [95% confidence interval] = 1.64 [1.37-6.78], p = 4.92 × 10-8). SAMD9 encodes a possible mediator of the inflammatory response to tissue injury. INTERPRETATION: We performed the first GWAS of 28-day sepsis survival and identified novel variants associated with reduced survival. Larger sample size studies are needed to better assess the genetic effects in sepsis survival and to validate the findings.

Intracellular expression of granzymes A, B, K and M in blood lymphocyte subsets of critically ill patients with or without sepsis.

Sepsis is a complex syndrome related to an infection-induced exaggerated inflammatory response, which is associated with a high mortality. Granzymes (Gzm) are proteases mainly found in cytotoxic lymphocytes that not only have a role in target cell death, but also as mediators of infection and inflammation. In this study we sought to analyse the intracellular expression of GzmA, B, M and K by flow cytometry in diverse blood lymphocyte populations from 22 sepsis patients, 12 non-infected intensive care unit (ICU) patients and 32 healthy controls. Additionally, we measured GzmA and B plasma levels. Both groups of patients presented decreased percentage of natural killer (NK) cells expressing GzmA, B and M relative to healthy controls, while sepsis patients showed an increased proportion of CD8+ T cells expressing GzmB compared to controls. Expression of GzmK remained relatively unaltered between groups. Extracellular levels of GzmB were increased in non-infected ICU patients relative to sepsis patients and healthy controls. Our results show differential alterations in intracellular expression of Gzm in sepsis patients and non-infected critically ill patients compared to healthy individuals depending on the lymphocyte population and on the Gzm.

Should MASP-2 Deficiency Be Considered a Primary Immunodeficiency? Relevance of the Lectin Pathway.

Mannose-binding lectin (MBL)-associated serine protease-2 (MASP-2) is an indispensable enzyme for the activation of the lectin pathway of complement. Its deficiency is classified as a primary immunodeficiency associated to pyogenic bacterial infections, inflammatory lung disease, and autoimmunity. In Europeans, MASP-2 deficiency, due to homozygosity for c.359A > G (p.D120G), occurs in 7 to 14/10,000 individuals. We analyzed the presence of the p.D120G mutation in adults (increasing the sample size of our previous studies) and children. Different groups of patients (1495 adults hospitalized with community-acquired pneumonia, 186 adults with systemic lupus erythematosus, 103 pediatric patients with invasive pneumococcal disease) and control individuals (1119 healthy adult volunteers, 520 adult patients without history of relevant infectious diseases, and a pediatric control group of 311 individuals) were studied. Besides our previously reported MASP-2-deficient healthy adults, we found a new p.D120G homozygous individual from the pediatric control group. We also reviewed p.D120G homozygous individuals reported so far: a total of eleven patients with a highly heterogeneous range of disorders and nine healthy controls (including our four MASP-2-deficient individuals) have been identified by chance in association studies. Individuals with complete deficiencies of several pattern recognition molecules of the lectin pathway (MBL, collectin-10 and collectin-11, and ficolin-3) as well as of MASP-1 and MASP-3 have also been reviewed. Cumulative evidence suggests that MASP-2, and even other components of the LP, are largely redundant in human defenses and that individuals with MASP-2 deficiency do not seem to be particularly prone to infectious or autoimmune diseases.

Systemic Effects Induced by Hyperoxia in a Preclinical Model of Intra-abdominal Sepsis.

Supplemental oxygen is a supportive treatment in patients with sepsis to balance tissue oxygen delivery and demand in the tissues. However, hyperoxia may induce some pathological effects. We sought to assess organ damage associated with hyperoxia and its correlation with the production of reactive oxygen species (ROS) in a preclinical model of intra-abdominal sepsis. For this purpose, sepsis was induced in male, Sprague-Dawley rats by cecal ligation and puncture (CLP). We randomly assigned experimental animals to three groups: control (healthy animals), septic (CLP), and sham-septic (surgical intervention without CLP). At 18 h after CLP, septic (n = 39), sham-septic (n = 16), and healthy (n = 24) animals were placed within a sealed Plexiglas cage and randomly distributed into four groups for continuous treatment with 21%, 40%, 60%, or 100% oxygen for 24 h. At the end of the experimental period, we evaluated serum levels of cytokines, organ damage biomarkers, histological examination of brain and lung tissue, and ROS production in each surviving animal. We found that high oxygen concentrations increased IL-6 and biomarkers of organ damage levels in septic animals, although no relevant histopathological lung or brain damage was observed. Healthy rats had an increase in IL-6 and aspartate aminotransferase at high oxygen concentration. IL-6 levels, but not ROS levels, are correlated with markers of organ damage. In our study, the use of high oxygen concentrations in a clinically relevant model of intra-abdominal sepsis was associated with enhanced inflammation and organ damage. These findings were unrelated to ROS release into circulation. Hyperoxia could exacerbate sepsis-induced inflammation, and it could be by itself detrimental. Our study highlights the need of developing safer thresholds for oxygen therapy.

Sepsis Patients Display a Reduced Capacity to Activate Nuclear Factor-κB in Multiple Cell Types.

OBJECTIVES: Sepsis is a complex clinical condition associated with high morbidity and mortality. A distinctive feature of sepsis is the reduced capacity of leukocytes to release proinflammatory cytokines in response to ex vivo stimulation. Cellular signaling events leading to immunosuppression in sepsis are not well defined. We investigated cell-specific signaling events underlying the immunosuppressed phenotype in sepsis. DESIGN: Ex vivo study. SETTING: ICU of an academic hospital. PATIENTS: Nineteen patients with sepsis and 19 age-matched healthy controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The phosphorylation state of p38 mitogen activated protein kinase and nuclear factor kappa-light-chain-enhancer of activated B cells were determined in ex vivo stimulated CD4 T cells, CD8 T cells, B cells, monocytes, and neutrophils. Messenger RNA expression levels of p38 mitogen activated protein kinase and nuclear factor kappa-light-chain-enhancer of activated B cells and negative regulators tumor necrosis factor-α-induced protein 3 (A20) and mitogen activated protein kinase phosphatase-1 were determined in neutrophils and peripheral blood mononuclear cells. Upon ex vivo stimulation, monocytes of sepsis patients were less capable in phosphorylating nuclear factor kappa-light-chain-enhancer of activated B cells. Sepsis was also associated with reduced phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells in stimulated B cells, CD4 and CD8 T cells. Messenger RNA expression levels of nuclear factor kappa-light-chain-enhancer of activated B cells and A20 were diminished in peripheral blood mononuclear cells of sepsis patients, whereas p38 mitogen activated protein kinase messenger RNA was up-regulated. In neutrophils of sepsis patients, mitogen activated protein kinase phosphatase-1 messenger RNA levels were down-regulated. CONCLUSIONS: Sepsis-induced immunosuppression associates with a defect in the capacity to phosphorylate nuclear factor kappa-light-chain-enhancer of activated B cells in lymphoid cells and monocytes.

Expression and Function of Granzymes A and B in Escherichia coli Peritonitis and Sepsis.

Escherichia (E.) coli is the most common causative pathogen in peritonitis, the second most common cause of sepsis. Granzymes (gzms) are serine proteases traditionally implicated in cytotoxicity and, more recently, in the inflammatory response. We here sought to investigate the role of gzms in the host response to E. coli-induced peritonitis and sepsis in vivo. For this purpose, we used a murine model of E. coli intraperitoneal infection, resembling the clinical condition commonly associated with septic peritonitis by this bacterium, in wild-type and gzmA-deficient (gzmA-/- ), gzmB-/- , and gzmAxB-/- mice. GzmA and gzmB were predominantly expressed by natural killer cells, and during abdominal sepsis, the percentage of these cells expressing gzms in peritoneal lavage fluid decreased, while the amount of expression in the gzm+ cells increased. Deficiency of gzmA and/or gzmB was associated with increased bacterial loads, especially in the case of gzmB at the primary site of infection at late stage sepsis. While gzm deficiency did not impact neutrophil recruitment into the abdominal cavity, it was accompanied by enhanced nucleosome release at the primary site of infection, earlier hepatic necrosis, and more renal dysfunction. These results suggest that gzms influence bacterial growth and the host inflammatory response during abdominal sepsis caused by E. coli.

Granzyme K synergistically potentiates LPS-induced cytokine responses in human monocytes.

Granzymes are serine proteases released by cytotoxic lymphocytes to induce apoptosis in virus-infected cells and tumor cells. Evidence is emerging that granzymes also play a role in controlling inflammation. Granzyme serum levels are elevated in patients with autoimmune diseases and infections, including sepsis. However, the function of extracellular granzymes in inflammation largely remains unknown. Here, we show that granzyme K (GrK) binds to Gram-negative bacteria and their cell-wall component lipopolysaccharide (LPS). GrK synergistically enhances LPS-induced cytokine release in vitro from primary human monocytes and in vivo in a mouse model of LPS challenge. Intriguingly, these extracellular effects are independent of GrK catalytic activity. GrK disaggregates LPS from micelles and augments LPS-CD14 complex formation, thereby likely boosting monocyte activation by LPS. We conclude that extracellular GrK is an unexpected direct modulator of LPS-TLR4 signaling during the antimicrobial innate immune response.

Long-Term Treatment with Alcaligenes faecalis A12C Improves Host Resistance to Pathogens in Septic Rats: Possible Contribution of Curdlan-Like Immune Trainer.

Curdlan, a ß-1,3/1,6-glucan found in Alcaligenes faecalis (A. faecalis) wall, activates innate and humoral immunity. The aim of this study is to evaluate whether pretreated rats with A. faecalis A12C could prevent sepsis disturbances and identify the immunomodulatory mechanisms involved. Experiments occurred in two stages: a survival study with 16 rats randomly divided into septic (SC) (n = 8) and septic pretreated (SA) (n = 8) groups and 45 rats divided into four groups: healthy (AGUSAN) (n = 9), septic (AGUIC) (n = 13), septic pretreated (AGUIA) (n = 14), and healthy pretreated (AGUSTO) (n = 9). Sepsis was induced by cecal ligation and puncture after 30 days of A. faecalis A12C pretreatment or without. SA group had a higher survival rate of 58% vs. 16% for SC group (P < 0.05). Overall, AGUIA showed better status than AGUIC (P < 0.01). Higher monocytosis was found in AGUIA and AGUSTO vs. AGUIC and AGUSAN, respectively (P < 0.05). A gradual increase in curdlan fecal concentration was observed in AGUIA during pretreatment. Fecal concentrations of Escherichia coli significantly decreased in AGUIA and AGUSTO. Bacterial load in urine, peritoneal lavage fluid (PLF), and bronchoalveolar lavage fluid (BALF) decreased (P < 0.05) in AGUIA vs. AGUIC. Finally, lower inflammation was observed in serum, BALF, and PLF, with reduced IL-6, IL-10, IL-1ß, and TNF-α, along with less damage in lungs and peritoneum in AGUIA vs. AGUIC. These findings suggest the connection between curdlan-produced by A. faecalis A12C-with the immune system and the reduction in severity of experimental sepsis.

Partial recessive IFN-γR1 deficiency: genetic, immunological and clinical features of 14 patients from 11 kindreds.

We report a series of 14 patients from 11 kindreds with recessive partial (RP)-interferon (IFN)-γR1 deficiency. The I87T mutation was found in nine homozygous patients from Chile, Portugal and Poland, and the V63G mutation was found in five homozygous patients from the Canary Islands. Founder effects accounted for the recurrence of both mutations. The most recent common ancestors of the patients with the I87T and V63G mutations probably lived 1600 (875-2950) and 500 (200-1275) years ago, respectively. The two alleles confer phenotypes that are similar but differ in terms of IFN-γR1 levels and residual response to IFN-γ. The patients suffered from bacillus Calmette-Guérin-osis (n= 6), environmental mycobacteriosis (n= 6) or tuberculosis (n= 1). One patient did not suffer from mycobacterial infections but had disseminated salmonellosis, which was also present in two other patients. Age at onset of the first environmental mycobacterial disease differed widely between patients, with a mean value of 11.25 ± 9.13 years. Thirteen patients survived until the age of 14.82 ± 11.2 years, and one patient died at the age of 7 years, 9 days after the diagnosis of long-term Mycobacterium avium infection and the initiation of antimycobacterial treatment. Up to 10 patients are currently free of infection with no prophylaxis. The clinical heterogeneity of the 14 patients was not clearly related to either IFNGR1 genotype or the resulting cellular phenotype. RP-IFN-γR1 deficiency is, thus, more common than initially thought and should be considered in both children and adults with mild or severe mycobacterial diseases.

The role of mannose-binding lectin in pneumococcal infection.

The role of mannose-binding lectin (MBL) deficiency (MBL2; XA/O and O/O genotypes) in host defences remains controversial. The surfactant proteins (SP)-A1, -A2 and -D, other collectins whose genes are located near MBL2, are part of the first-line lung defence against infection. We analysed the role of MBL on susceptibility to pneumococcal infection and the existence of linkage disequilibrium (LD) among the four genes. We studied 348 patients with pneumococcal community-acquired pneumonia (P-CAP) and 2,110 controls. A meta-analysis of MBL2 genotypes in susceptibility to P-CAP and to invasive pneumococcal disease (IPD) was also performed. The extent of LD of MBL2 with SFTPA1, SFTPA2 and SFTPD was analysed. MBL2 genotypes did not associate with either P-CAP or bacteraemic P-CAP in the case-control study. The MBL-deficient O/O genotype was significantly associated with higher risk of IPD in a meta-analysis, whereas the other MBL-deficient genotype (XA/O) showed a trend towards a protective role. We showed the existence of LD between MBL2 and SP genes. The data do not support a role of MBL deficiency on susceptibility to P-CAP or to IPD. LD among MBL2 and SP genes must be considered in studies on the role of MBL in infectious diseases.

The Fcγ receptor IIA-H/H131 genotype is associated with bacteremia in pneumococcal community-acquired pneumonia.

OBJECTIVE: To assess the potential association of the functional polymorphism rs1801274 in the receptor IIa for the Fc portion of immunoglobin G (FcγRIIa) gene (FCGR2A-H131R) with the susceptibility to and the severity of community-acquired pneumonia (CAP). DESIGN: Multicenter prospective and observational study. SETTING: Four university hospitals in Spain. PATIENTS: FCGR2A-H131R polymorphism was determined in 1,262 patients with CAP and in 1,224 in the subject control group. MEASUREMENTS AND MAIN RESULTS: Severe sepsis was recorded in 366 patients. No significant differences in genotype or allele frequencies were seen among patients with CAP or pneumococcal CAP (PCAP) and controls. Patients with bacteremic PCAP (B-PCAP) had significantly higher frequencies of FCGR2A-H/H131 genotypes than those with nonbacteremic PCAP (p = .00016, odds ratio = 2.9, 95% confidence interval 1.58-5.3). The differences remained significant when adjusting for pneumonia severity index, hospital of origin, and intensive care unit admission (p = .0012, odds ratio = 2.83, 95% confidence interval 1.51-5.32). B-PCAP was associated with a significantly higher severity of the disease, evaluated as sepsis severity (p = .000007, odds ratio = 4.40, 95% confidence interval 2.31-8.39), multiorgan dysfunction syndrome (0.00048, odds ratio = 3.29, 95% confidence interval 1.69-6.41), intensive care unit admission, acute renal failure, and acute respiratory distress syndrome. CONCLUSIONS: Our results do not support a role of FCGR2A-H131R polymorphism in susceptibility to CAP or PCAP. However, we provide the insight that homozygosity for FCGR2A-H131 predisposes B-PCAP, which was associated with higher severity in our study.

Influence of genetic variability at the surfactant proteins A and D in community-acquired pneumonia: a prospective, observational, genetic study.

INTRODUCTION: Genetic variability of the pulmonary surfactant proteins A and D may affect clearance of microorganisms and the extent of the inflammatory response. The genes of these collectins (SFTPA1, SFTPA2 and SFTPD) are located in a cluster at 10q21-24. The objective of this study was to evaluate the existence of linkage disequilibrium (LD) among these genes, and the association of variability at these genes with susceptibility and outcome of community-acquired pneumonia (CAP). We also studied the effect of genetic variability on SP-D serum levels. METHODS: Seven non-synonymous polymorphisms of SFTPA1, SFTPA2 and SFTPD were analyzed. For susceptibility, 682 CAP patients and 769 controls were studied in a case-control study. Severity and outcome were evaluated in a prospective study. Haplotypes were inferred and LD was characterized. SP-D serum levels were measured in healthy controls. RESULTS: The SFTPD aa11-C allele was significantly associated with lower SP-D serum levels, in a dose-dependent manner. We observed the existence of LD among the studied genes. Haplotypes SFTPA1 6A(2) (P = 0.0009, odds ration (OR) = 0.78), SFTPA(2) 1A(0) (P = 0.002, OR = 0.79), SFTPA1-SFTPA2 6A2-1A(0) (P = 0.0005, OR = 0.77), and SFTPD-SFTPA1-SFTPA(2)C-6A2-1A(0) (P = 0.00001, OR = 0.62) were underrepresented in patients, whereas haplotypes SFTPA2 1A(10) (P = 0.00007, OR = 6.58) and SFTPA1-SFTPA2 6A(3)-1A (P = 0.0007, OR = 3.92) were overrepresented. Similar results were observed in CAP due to pneumococcus, though no significant differences were now observed after Bonferroni corrections. 1A(10) and 6A-1A were associated with higher 28-day and 90-day mortality, and with multi-organ dysfunction syndrome (MODS) and acute respiratory distress syndrome (ARDS) respectively. SFTPD aa11-C allele was associated with development of MODS and ARDS. CONCLUSIONS: Our study indicates that missense single nucleotide polymorphisms and haplotypes of SFTPA1, SFTPA2 and SFTPD are associated with susceptibility to CAP, and that several haplotypes also influence severity and outcome of CAP.

Oesophageal squamous cell carcinoma in a young adult with IL-12R beta 1 deficiency.

Genetic defects in the IL-12-IL-23/IFN-gamma circuit confer Mendelian susceptibility to mycobacteria and salmonella. The IL-12/IFN-gamma axis is essential for anti-tumoral immunity in mice. Cancer susceptibility has not been recognised in these patients so far. We report three relatives with IL-12R beta 1 deficiency. At the age of 25 years old, one patient presented with oesophageal squamous cell carcinoma (OSCC). The patient had no previous risk factors for OSCC. He died at the age of 29 years. OSCC is exceedingly rare in individuals under 30 years and frequently relates to alcohol intake and smoking. Disorders of the IL-12-IL-23/IFN-gamma axis may predispose to cancer.

Clinical and biological markers for predicting ARDS and outcome in septic patients.

Sepsis is a common cause of acute respiratory distress syndrome (ARDS) associated with a high mortality. A panel of biomarkers (BMs) to identify septic patients at risk for developing ARDS, or at high risk of death, would be of interest for selecting patients for therapeutic trials, which could improve ARDS diagnosis and treatment, and survival chances in sepsis and ARDS. We measured nine protein BMs by ELISA in serum from 232 adult septic patients at diagnosis (152 required invasive mechanical ventilation and 72 had ARDS). A panel including the BMs RAGE, CXCL16 and Ang-2, plus PaO2/FiO2, was good in predicting ARDS (area under the curve = 0.88 in total septic patients). Best performing panels for ICU death are related to the presence of ARDS, need for invasive mechanical ventilation, and pulmonary/extrapulmonary origin of sepsis. In all cases, the use of BMs improved the prediction by clinical markers. Our study confirms the relevance of RAGE, Ang-2, IL-1RA and SP-D, and is novel supporting the inclusion of CXCL16, in BMs panels for predicting ARDS diagnosis and ARDS and sepsis outcome.

Probiotic Properties of Alcaligenes faecalis Isolated from Argyrosomus regius in Experimental Peritonitis (Rat Model).

A strain of Alcaligenes faecalis A12C (A. faecalis A12C) isolated from Argyrosomus regius is a probiotic in fish. Previous experiments showed that A. faecalis A12C had inhibitory effects on the growth of multidrug-resistant bacteria. We aimed to confirm whether A. faecalis A12C is safe and has adequate intestinal colonization in experimental rats, and evaluate its efficacy in an animal model of peritonitis. We used 30 male rats, randomly divided into 6 groups (n = 5): three groups (HA7, HA15, HA30) received A. faecalis A12C in drinking water (6 × 108 CFU/mL) for 7 days, and three control groups received drinking water only. All groups were evaluated at 7, 15, and 30 days. Survival after A. faecalis A12C administration was 100% in all groups. Mild eosinophilia (1.5%, p < 0.01) and increased aspartate aminotransferase (86 IU/L, p < 0.05) were observed in HA7, followed by progressive normalization. No histological signs of organ injury were found. We observed significant E. coli decline in faeces, parallel to an increase in A. faecalis A12C at 7 days. E. coli had a tendency to recover initial values, while A. faecalis A12C disappeared from the intestinal microbiota at 30 days. To evaluate its efficacy against peritonitis, we studied two additional groups of animals: IA group pretreated with A. faecalis A12C before E. coli intra-abdominal inoculation, and IC group inoculated with no A. faecalis A12C. We found an increase in C-reactive protein, alanine aminotransferase, urea, and eosinophils in IC animals when compared with IA. Peritonitis was more evident in IC than in IA animals. Our findings suggest that A. faecalis A12C altered clinically relevant parameters in sepsis and was associated with a lesser spread of infection.

Sepsis-associated acute respiratory distress syndrome in individuals of European ancestry: a genome-wide association study.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a lung inflammatory process caused mainly by sepsis. Most previous studies that identified genetic risks for ARDS focused on candidates with biological relevance. We aimed to identify novel genetic variants associated with ARDS susceptibility and to provide complementary functional evidence of their effect in gene regulation. METHODS: We did a case-control genome-wide association study (GWAS) of 1935 European individuals, using patients with sepsis-associated ARDS as cases and patients with sepsis without ARDS as controls. The discovery stage included 672 patients admitted into a network of Spanish intensive care units between January, 2002, and January, 2017. The replication stage comprised 1345 individuals from two independent datasets from the MESSI cohort study (Sep 22, 2008-Nov 30, 2017; USA) and the VISEP (April 1, 2003-June 30, 2005) and MAXSEP (Oct 1, 2007-March 31, 2010) trials of the SepNet study (Germany). Results from discovery and replication stages were meta-analysed to identify association signals. We then used RNA sequencing data from lung biopsies, in-silico analyses, and luciferase reporter assays to assess the functionallity of associated variants. FINDINGS: We identified a novel genome-wide significant association with sepsis-associated ARDS susceptibility (rs9508032, odds ratio [OR] 0·61, 95% CI 0·41-0·91, p=5·18 × 10-8) located within the Fms-related tyrosine kinase 1 (FLT1) gene, which encodes vascular endothelial growth factor receptor 1 (VEGFR-1). The region containing the sentinel variant and its best proxies acted as a silencer for the FLT1 promoter, and alleles with protective effects in ARDS further reduced promoter activity (p=0·0047). A literature mining of all previously described ARDS genes validated the association of vascular endothelial growth factor A (VEGFA; OR 0·55, 95% CI 0·41-0·73; p=4·69 × 10-5). INTERPRETATION: A common variant within the FLT1 gene is associated with sepsis-associated ARDS. Our findings support a role for the vascular endothelial growth factor signalling pathway in ARDS pathogenesis and identify VEGFR-1 as a potential therapeutic target. FUNDING: Instituto de Salud Carlos III, European Regional Development Funds, Instituto Tecnológico y de Energías Renovables.

Mannose-binding lectin and mannose-binding lectin-associated serine protease 2 in susceptibility, severity, and outcome of pneumonia in adults.

BACKGROUND: Community-acquired pneumonia (CAP) is the leading cause of death from infection in developed countries. Mannose-binding lectin (MBL) and MBL-associated serine protease 2 (MASP-2) deficiencies are common primary immunodeficiencies the clinical penetrance of which remains controversial. MBL is a serum lectin that mediates phagocytosis and activates the lectin pathway of complement involving MASP-2. OBJECTIVE: We sought to evaluate the significance of MBL deficiency (O/O genotypes) and insufficiency (O/O plus XA/O genotypes), as well as MASP-2 deficiency (D105G mutation), in the susceptibility to and severity and outcome of CAP in adults. METHODS: MBL and MASP-2 serum levels, as well as lectin pathway activity with regard to MBL2 and MASP2 genotypes, were measured in healthy control subjects. For susceptibility, 848 patients with CAP, 1447 healthy control subjects, and a control group of 519 patients without relevant infectious diseases were studied in a case-control study. Severity and outcome were evaluated in a prospective study of the 848 patients. RESULTS: We found similar frequencies of MBL2 and MASP2 alleles and genotypes among patients and control subjects. However, in a multivariate analysis MBL insufficiency was associated with the development of the most severe forms of sepsis (P = .007), acute respiratory failure (P = .009), multiorgan dysfunction syndrome (P = .036), intensive care unit admission (P = .020), and death (P = .003). CONCLUSION: Our large study suggests that MBL plays a redundant role in human defenses against primary infection, at least in adults with CAP, and provides, for the first time, evidence that MBL insufficiency predisposes to higher severity and fatal outcome in patients with CAP, irrespective of the causal microorganisms.

Influence of mannose-binding lectin on HIV infection and tuberculosis in a Western-European population.

Mannose-binding lectin (MBL) is a serum lectin that mediates phagocytosis and activates complement. Its deficiency has been associated with increased susceptibility to infectious diseases, mainly in childhood. However, non-producer mbl-2 alleles are common in most populations, suggesting a selective advantage of these alleles. We have analysed the association of mbl-2 structural and promoter polymorphisms with HIV infection and tuberculosis (TBC) in a white Spanish population, including 615 HIV patients with and without TBC, 127 no-HIV TBC patients, 142 TBC household contacts and 344 controls. The frequency of low or non-producer mbl-2 genotypes was lower in HIV patients than in controls. HIV-TBC patients presented lower frequencies of low or non-producer alleles and genotypes than HIV no-TBC patients and controls. Additionally, we found a significantly positive correlation between the incidence of TBC and the frequency of non-producer mbl-2 alleles in Western Europe. Therefore, MBL deficiency may be associated with a lower risk of HIV infection, and also of active TBC, at least in HIV patients. The protective role of low-producer mbl-2 genotypes against TBC together with the positive correlation observed between non-producer mbl-2 alleles and TBC incidence, suggest a balancing selection: in spite of an increased susceptibility to respiratory infections associated with MBL deficiency, mbl-2 deficient alleles would have been selected along different populations as a consequence of its selective advantage against intracellular pathogens, such as M. tuberculosis.

Biomarkers for the acute respiratory distress syndrome: how to make the diagnosis more precise.

The acute respiratory distress syndrome (ARDS) is an acute inflammatory process of the lung caused by a direct or indirect insult to the alveolar-capillary membrane. Currently, ARDS is diagnosed based on a combination of clinical and physiological variables. The lack of a specific biomarker for ARDS is arguably one of the most important obstacles to progress in developing novel treatments for ARDS. In this article, we will review the current understanding of some appealing biomarkers that have been measured in human blood, bronchoalveolar lavage fluid (BALF) or exhaled gas that could be used for identifying patients with ARDS, for enrolling ARDS patients into clinical trials, or for better monitoring of patient's management. After a literature search, we identified several biomarkers that are associated with the highest sensitivity and specificity for the diagnosis or outcome prediction of ARDS: receptor for advanced glycation end-products (RAGE), angiopoietin-2 (Ang-2), surfactant protein D (SP-D), inteleukin-8, Fas and Fas ligand, procollagen peptide (PCP) I and III, octane, acetaldehyde, and 3-methylheptane. In general, these are cell-specific for epithelial or endothelial injury or involved in the inflammatory or infectious response. No biomarker or biomarkers have yet been confirmed for the diagnosis of ARDS or prediction of its prognosis. However, it is anticipated that in the near future, using biomarkers for defining ARDS, or for determining those patients who are more likely to benefit from a given therapy will have a major effect on clinical practice.