Introduction

Autophagy, deriving from the Greek words auto “self” and phagein “to eat”, is a tightly regulated catabolic process present in all eukaryotic cells and indispensable for cell survival. During this process, organelles and other macromolecular cell debris are incorporated in double-membrane structures called autophagosomes, where they are degraded and recycled into nutrient products [1].

The process of autophagy is activated upon infection with certain intracellular bacteria, enabling pathogen engulfment in the autophagosome and subsequent killing via lysosomal fusion with the membrane-bound compartment [2]. Thus, this highly conserved machinery is recruited by host cells for the elimination of several pathogens, such as group A streptococci [3], Salmonella enterica serovar Typhimurium, Shigella spp., Listeria monocytogenes, Legionella pneumophila, and Mycobacterium tuberculosis [4].

One of the main proteins implicated in autophagy formation and regulation is autophagy-related 16-like 1 (ATG16L1). ATG16L1 is involved in a large protein complex together with ATG5 and ATG12, promoting the elongation of the autophagosome [5]. A non-synonymous single-nucleotide polymorphism (SNP) in the ATG16L1 (Thr300Ala c.898A > G, rs2241880) gene results in a functional impairment of autophagy and has been associated with increased susceptibility to Crohn’s disease and tuberculosis [6]. It has also been demonstrated that NOD-like receptors, NOD1 and NOD2, recruit ATG16L1 protein at the bacterial entry site, thus enabling autophagy [7]. In this context, carriage of the ATG16L1 SNP results in reduced protein production that subsequently induces some imbalance between autophagy and cytokine production after NOD2 stimulation [8]. Accordingly, autophagy inhibition in primary human peripheral blood mononuclear cells (PBMCs) potentiates the production of interleukin-1 beta (IL-1β) after stimulation with Μ. tuberculosis [9].

Sepsis is the result of an unbalanced pro- vs. anti-inflammatory host response to an invading pathogen, initiated by the sensing of pathogen-associated molecular patterns (PAMPs) by specific pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and the cytosolic NODs [10]. In intensive care units (ICUs), one of the major causes of severe sepsis is ventilator-associated pneumonia (VAP) [11, 12]. According to one recent meta-analysis, the incidence of VAP ranges from 2 to 16 episodes for 1,000 ventilator-days with an attributable mortality of 3–17 % [13]. Since no genetic association study has ever been conducted for the role of SNPs of genes implicated in the process of autophagy in sepsis, the aim of the present study was to genotype for the ATG16L1 SNP among a homogeneous patient population with sepsis due to VAP, hypothesizing that it could impact on disease severity and potential outcome.

Materials and methods

Study design

The DNA for conducting the study was derived from patients who were enrolled in a multicenter trial investigating the efficacy of intravenously administered clarithromycin in VAP and sepsis (http://clinicaltrials.gov/show/NCT00297674). Since the overall mortality did not differ between the two arms of treatment, it was considered that patients enrolled in both arms could be analyzed together for the purposes of the current study [14]. Patients intubated and mechanically ventilated in two ICUs were enrolled during the period from June 2004 to November 2005. The study protocol was approved by the Ethics Committees of both hospitals and a written informed consent form was provided for all patients by their relatives.

Inclusion criteria were: (i) written informed consent provided by first-degree relatives; (ii) patient in intubation and mechanical ventilation for at least 48 h prior to enrollment; (iii) age ≥18 years; (iv) Caucasian origin; (v) diagnosis of VAP; and (vi) at least two signs of the systemic inflammatory response syndrome [14]. Although there is no evidence that the frequency of SNPs of ATG16L1 differs between continents, the present study focused only on patients of Caucasian origin in order to be as homogeneous as possible.

Exclusion criteria were: (i) neutropenia, defined as any neutrophil count <500 cells/mm3; (ii) human immunodeficiency virus (HIV) infection; and (iii) oral intake of corticosteroids at a dose ≥1 mg/kg of equivalent prednisone for at least a period of 1 month.

VAP was diagnosed by all the following criteria [14–16]: (i) new or persistent consolidation on chest X-ray; (ii) purulent tracheobronchial secretions (TBS); and (iii) a Clinical Pulmonary Infection Score (CPIS) of more than 6, as assessed by Pugin et al. [17].

Organ failure was defined as follows: (a) acute respiratory distress syndrome as pO2/FiO2 < 200 and diffuse infiltrates in chest X-ray; (b) cardiovascular shock as systolic blood pressure (SBP) <90 mmHg or mean arterial pressure (MAP) <65 mmHg requiring the administration of vasopressors; (c) acute renal dysfunction as urine output <0.5 ml/kg/h for at least 2 h, despite adequate fluid resuscitation; (d) acute coagulopathy as platelet count <100,000/mm3 or international normalized ratio >1.5; and (e) metabolic acidosis as pH <7.30 and lactate levels twice the upper normal value [18].

Demographic and disease severity data were recorded, comprising the Acute Physiology and Chronic Health Evaluation (APACHE) II score, Sequential Organ Failure Assessment (SOFA) score, blood gases and the ratio of partial pressure of oxygen (pO2) to the fraction of inspired oxygen (FiO2), quantitative cultures of TBS and blood cultures, and complete laboratory work-out. Survival was monitored for 28 days.

Blood sampling and genotyping

Sixteen milliliters of whole blood drawn from patients were sampled after venipuncture of a forearm vein under sterile conditions; 3 ml were collected in an ethylene diamine tetraacetic acid (EDTA)-coated tube for genotyping analysis and stored at −70 °C until processed. Another 3 ml were collected into pyrogen-free tubes and 10 ml into heparin-coated tubes. The tubes were centrifuged and serum was kept refrigerated at −70 °C until assayed. Extraction of genomic DNA was performed using the Puregene Blood Core Kit C (Qiagen, Hilden, Germany), according to the instructions of the manufacturer. TaqMan® SNP Genotyping assays designed with two specific probes and primers for each variant were utilized for genotyping the ATG16L1 T300A, rs2241880 (C_9095577_20, Life Technologies, Carlsbad, CA, USA). Two microliters of genomic DNA were amplified by quantitative polymerase chain reaction (PCR) in a 7300 Real-Time PCR System (Life Technologies) and analysis of the results was done using the SDS Software version 1.4 (Life Technologies). Genotyping data were acquired by a technician blind to all clinical information. Quality control was performed by duplicating samples within and across plates and by the incorporation of positive and negative control samples.

Monocyte stimulation for tumor necrosis factor-α (TNF-α)

The collected heparinized venous blood was layered over Ficoll-Hypaque (Biochrom, Berlin, Germany) and centrifuged. Mononuclear cells were isolated, washed with ice-cold phosphate-buffered saline (PBS) (pH 7.2) (Merck, Darmstadt, Germany), and plated in 25-cm3 flasks with RPMI 1640 enriched with 10 % fetal bovine serum and 2 mM glutamine in the presence of 100 U/ml penicillin G and 0.1 mg/ml streptomycin (Sigma Co., St. Louis, MO, USA). Non-adherent cells were removed after 1 h of incubation at 37 °C in 5 % CO2, and adherent cells were collected with the use of 0.25 % trypsin–0.02 % EDTA solution (Biochrom) and counted in a Neubauer plate with trypan blue exclusion of dead cells. Their purity was more than 95 %, as determined after staining with anti-CD14-fluorescein isothiocyanate (FITC) (emission, 525 nm; Immunotech, Marseille, France) and reading through the EPICS XL/MSL flow cytometer (Beckman Coulter Co., Miami, FL, USA). Unstained cells served as negative controls.

Monocytes were then distributed in two wells of a 12-well plate and incubated with RPMI 1640 supplemented with 10 % fetal bovine serum and 2 mM glutamine for 24 h at 37 °C in 5 % CO2 in the absence/presence of 10 ng/ml purified lipopolysaccharide (LPS) of Escherichia coli O55:H5 (Sigma Co.). After incubation, the plate was centrifuged and cell supernatants were collected and kept refrigerated at −70 °C until assayed. The concentrations of TNF-α in supernatants were estimated by an enzyme immunoassay (lowest detection limit, 20 pg/ml; R&D Systems Inc., Minneapolis, MN, USA).

Measurements of procalcitonin (PCT)

PCT was estimated in serum in duplicate by an immuno-time-resolved amplified cryptate technology assay (Kryptor PCT; BRAHMS GmbH, Hennigsdorf, Germany), with a functional assay sensitivity of 0.06 ng/ml.

Statistical analysis

Statistical analysis was conducted using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). The Chi-square test was used for the assessment of the Hardy–Weinberg equilibrium (HWE) test in the studied SNP. Associations between the carriage of polymorphism and development of organ failure were estimated by the odds ratio (OR) and 95 % confidence interval (CI) using Mantel–Haenszel statistics. Comparisons among carriers and non-carriers of the studied polymorphism was done using Fisher’s test for qualitative and Student’s t-test for quantitative data. TNF-α was expressed as mean and standard error (SE). PCT was expressed as medians and 95 % CIs. Comparisons between groups were done by the Mann–Whitney U-test. All tests were two-sided and any p-value < 0.05 after adjustment for multiple comparisons was considered statistically significant.

Results

A total of 200 patients were enrolled in the study. DNA was available for only 155 of these patients to conduct analysis for the studied SNP. The demographic and clinical characteristics of these patients have already been described [18]. At the time of study enrolment, 41 (26.5 %) patients were classified with sepsis, 43 (27.7 %) with severe sepsis, and 71 (45.8 %) with septic shock. A total of 107 cases were microbiologically documented after quantitative cultures of the tracheobronchial secretions yielding more than 105 cfu/ml of the following isolates: Acinetobacter baumannii in 65 patients (41.9 %); Pseudomonas aeruginosa in 22 patients (14.2 %); Klebsiella pneumoniae in nine patients (5.8 %); Enterobacter cloacae in three patients (1.9 %); Enterobacter aerogenes in two patients (1.3 %); Stenotrophomonas maltophilia in two patients (1.3 %); Staphylococcus aureus in two patients (1.3 %); Proteus mirabilis in one patient (0.6 %); and Providencia stuartii in one patient (0.6 %).

The studied SNP was found in HWE in the patients group (Table 1). Minor allele carriers had a mean ± SD age of 60.0 ± 19.1 years; for the wild-type carriers, this was 61.5 ± 15.2 years (p: 0.651). The impact of the carriage of the minor allele of rs2241880 of ATG16L1 on disease severity was investigated. No differences were found between carriers and non-carriers concerning the APACHE II score, SOFA score, pO2/FiO2 ratio and the number of white blood cells/mm3 (WBC) in the serum. However, patients bearing at least one minor A allele presented with an increased risk for septic shock accompanied with at least another organ failure (OR: 2.40, 95 % CI: 1.06–5.60, p: 0.036). The results are presented in Table 2. A total of 38 patients had bacteremia; 24 were carriers of at least one minor A allele and 14 were carriers of only G alleles; ten (41.7 %) and five (35.7 %), respectively, developed septic shock with at least another organ failure.

Table 1 Genotype and allele frequencies of autophagy ATG16L1 polymorphism. The results are provided for 155 patients with ventilator-associated pneumonia (VAP)
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Table 2 Impact of the carriage of at least one minor allele of the ATG16L1 gene single-nucleotide polymorphism (SNP) on the severity of ventilator-associated pneumonia (VAP)
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Analysis within patients who presented with septic shock and at least one organ failure showed that the production of TNF-α from monocytes after stimulation with LPS was significantly greater among carriers of the A allele compared to carriers of only wild-type alleles of ATG16L1 (p: 0.040). No difference was found among patients without septic shock and organ dysfunction (Fig. 1). PCT levels were increased as a result of septic shock and failing organs only among patients who were carriers of A alleles; this was not the case for carriers of only the wild-type G alleles (Fig. 2).

Fig. 1
figure 1

Tumor necrosis factor-α (TNF-α) measurements in the supernatants of monocytes of patients with ventilator-associated pneumonia (VAP). TNF-α was measured in the supernatants of lipopolysaccharide (LPS)-stimulated monocytes from carriers of only wild-type G alleles or carriers of A alleles of the ATG16L1 gene. Production of TNF-α was below the limit of detection in the supernatants of non-stimulated monocytes. p-values represent comparisons between carriers of only wild-type alleles and carriers of A alleles

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Fig. 2
figure 2

Serum procalcitonin (PCT) levels of patients with ventilator-associated pneumonia (VAP). Patients are divided into subgroups of carriers of only wild-type G alleles and carriers of minor A alleles of ATG16L1. Comparisons between patients without and with septic shock and organ failures are shown

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Discussion

The link between autophagy and innate immune responses triggered by PAMPs and/or danger-associated molecular patterns (DAMPs) released by tissue injury is known. Autophagy is implicated in the signaling pathway of nearly all classes of PRRs and is enhanced or inhibited by cytokines and receptors that modulate innate and adaptive immunity [19]. Our investigation of a functional polymorphism in autophagy in a homogeneous cohort with VAP has proven the importance of this conserved mechanism in the protection against sepsis and multiple organ failure. This study showed, for the first time, that impaired autophagy caused by the carriage of SNP A alleles of the ATG16L1 gene led to increased susceptibility to septic shock and organ failure in patients with VAP. This is likely due to the fact that autophagy is an important innate mechanism activated for direct bacterial elimination. In cases of P. aeruginosa lung infection and S. enterica intestinal infection, knock-down of Atg16l1 resulted in impaired bacterial clearance; enhancement of autophagy by rapamycin or trifluoperazine (mTOR inhibitors) restored this defect [20, 21].

Additionally, the carriage of autophagy SNP modulates cytokine secretion and, subsequently, inflammation in infectious diseases. In mouse models of lethal endotoxemia and cecal ligation and puncture (CLP) polymicrobial sepsis, a defect in autophagy was correlated with increased mortality, accompanied by elevated IL-1β and IL-18 concentrations [22]. Accordingly, the secretion of pro-inflammatory cytokines, notably IL-1β, was significantly increased in the supernatants of human cell lines and human PBMCs stimulated with Borrelia burgdorferi [23] or with pathogenic adherent invasive E. coli with the concomitant use of autophagy inhibitors [24]. These findings underscore the importance of host autophagy in modulating cytokine responses to infection. However, in the case of sepsis, the inflammatory state of the patients is diverse, and, very often, patients with severe sepsis are in a state of immunoparalysis. Our results suggest that the carriage of A alleles of ATG16L1 predisposes to defective cytokine responses by circulating monocytes. This is further corroborated by the finding that PCT, which is considered a biomarker of sepsis severity [25], is increased in most severe cases of shock and organ failures among carriers of A alleles but not those of G alleles. Former publications have considered PCT as an index of sepsis-induced immunoparalysis linking circulating PCT with the expression of Cd11b on monocytes [26]. The observed findings on the impact of the studied SNP on PCT levels helps explain why PCT is not universally increased in all severe patients and corroborates the observations of defective cytokine responses with a role of ATG16L1 SNP in sepsis-induced immunoparalysis.

Our study demonstrates that carriers of the ATG16L1 gene, implicated in autophagy, present with an increased risk for septic shock and multiple organ failure development in the case of VAP. The results of our proof-of-principle study warrants that larger prospective investigations should be undertaken to definitively demonstrate these effects. If these results are replicated, genotyping for these polymorphism can not only identify patients who would benefit from an aggressive care management, but can additionally point to new treatment strategies by enhancing and restoring the mechanism of autophagy.