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Autophagy-Related IRGM Polymorphism Is Associated with Mortality of Patients with Severe Sepsis

  • Tomonori Kimura,

    Affiliation Department of Emergency and Critical Care Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chu-ku, Chiba City, Chiba, Japan

  • Eizo Watanabe ,

    watanabee@faculty.chiba-u.jp

    Affiliation Department of Emergency and Critical Care Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chu-ku, Chiba City, Chiba, Japan

  • Teruo Sakamoto,

    Affiliation Department of Emergency and Critical Care Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume City, Fukuoka, Japan

  • Osamu Takasu,

    Affiliation Department of Emergency and Critical Care Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume City, Fukuoka, Japan

  • Toshiaki Ikeda,

    Affiliation Division of Critical Care and Emergency Medicine, Tokyo Medical University Hachioji Medical Center, 1163 Tate-machi, Hachioji City, Tokyo, Japan

  • Kazumi Ikeda,

    Affiliation Division of Critical Care and Emergency Medicine, Tokyo Medical University Hachioji Medical Center, 1163 Tate-machi, Hachioji City, Tokyo, Japan

  • Joji Kotani,

    Affiliation Department of Emergency, Disaster and Critical Care Medicine, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya City, Hyogo, Japan

  • Nobuya Kitamura,

    Affiliation Department of Emergency and Critical Care Medicine, Kimitsu Chuo Hospital, 1010 Sakurai, Kisarazu City, Chiba, Japan

  • Tomohito Sadahiro,

    Affiliation Department of Emergency and Critical Care Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chu-ku, Chiba City, Chiba, Japan

  • Yoshihisa Tateishi,

    Affiliation Department of Emergency and Critical Care Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chu-ku, Chiba City, Chiba, Japan

  • Koichiro Shinozaki,

    Affiliation Department of Emergency and Critical Care Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chu-ku, Chiba City, Chiba, Japan

  • Shigeto Oda

    Affiliation Department of Emergency and Critical Care Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chu-ku, Chiba City, Chiba, Japan

Autophagy-Related IRGM Polymorphism Is Associated with Mortality of Patients with Severe Sepsis

  • Tomonori Kimura, 
  • Eizo Watanabe, 
  • Teruo Sakamoto, 
  • Osamu Takasu, 
  • Toshiaki Ikeda, 
  • Kazumi Ikeda, 
  • Joji Kotani, 
  • Nobuya Kitamura, 
  • Tomohito Sadahiro, 
  • Yoshihisa Tateishi
PLOS
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Abstract

Objective

Autophagy is the regulated catabolic process for recycling damaged or unnecessary organelles, which plays crucial roles in cell survival during nutrient deficiency, and innate immune defense against pathogenic microorganisms. Autophagy has been also reported to be involved in various conditions including inflammatory diseases. IRGM (human immunity-related GTPase) has an important function in eliminating Mycobacterium tuberculosis from host cells via autophagy. We examined the association between genetic polymorphism and clinical course/outcome in severely septic patients.

Methods

The study included 125 patients with severe sepsis/septic shock (SS) and 104 non-sepsis patients who were admitted to the intensive care unit (ICU) of Chiba University Hospital between October 2001 and September 2008 (discovery cohort) and 268 SS patients and 454 non-sepsis patients who were admitted to ICUs of five Japanese institutions including Chiba University Hospital between October 2008 and September 2012 (multi-center validation cohort). Three hundred forty seven healthy volunteers who consented to this study were also included. Genotyping was performed for a single-nucleotide polymorphism (SNP) within the coding region of IRGM, IRGM(+313) (rs10065172). Lipopolysaccharide challenge of whole blood from randomly selected healthy volunteers (n = 70) was performed for comparison of IRGM mRNA expression among different genotypes.

Results

No significant difference in genotypic distributions (CC/CT/TT) at the IRGM(+313) locus was observed among the three subject groups (SS, non-sepsis, and healthy volunteers) in either cohort. When mortality were compared, no significant difference was observed in the non-sepsis group, while TT homozygotes exhibited a significantly higher mortality than the CC+CT genotype category in the SS group for both cohorts (P = 0.043, 0.037). Lipopolysaccharide challenge to whole blood showed a significant suppression of IRGM mRNA expression in TT compared with the CC+CT genotype category (P = 0.019).

Conclusions

The data suggest that the IRGM(+313), an autophagy-related polymorphic locus, influences outcome in severely septic patients, with the possible involvement of autophagy in sepsis exacerbation.

Introduction

Severe sepsis is the host inflammatory response to infection with presence of organ dysfunction [1]. It is a critical condition that is very difficult to survive, as indicated by high mortality rates ranging from 30% and 80% in various countries [2], [3]. After overcoming the acute inflammatory response phase and remission of SIRS, septic patients go into an anti-inflammatory phase, which is even more difficult to overcome (CARS; compensatory anti-inflammatory response syndrome). The subsequent development of immunoparalysis is believed to affect the prognosis of sepsis, particularly during the post-acute phase [4][6].

Apoptosis (type I programmed cell death) of immune effector cells plays a crucial role in the pathophysiology of CARS and immunoparalysis in sepsis, and the regulation of apoptosis is expected to improve survival in sepsis [7], [8]. On the other hand, the involvement of autophagy (type II programmed cell death) in the pathophysiology of sepsis has attracted the attention of both researchers and clinicians [9][11]. Autophagy (“self-eating”), a regulated catabolic process, has roles in the degradation of unnecessary organelles, elimination of pathogenic microorganisms, and tumor suppression [12]. Sepsis-induced organ failure is considered to be the summation of cellular dysfunction induced by cell death of different cells [13], and vital organ failure may be related to immune system abnormalities. Recently, the genome-wide association study (GWAS) approach has enabled systematic searches of disease susceptibility genes. Using this methodology, an autophagy-related gene, autophagy-related protein 16-like 1 (ATG16L1), was identified as a disease susceptibility gene closely involved in the pathophysiology of Crohn’s disease, an inflammatory bowel disease [14]. In addition, an association between IRGM (a human immunity-related GTPase) expression and the induction and execution of autophagy upon bacterial infections, such as tuberculosis, has been reported with regulation of autophagy formation in proportion to IRGM expression [15]. Furthermore, genetic polymorphism of IRGM has been confirmed to be involved in the development of inflammatory bowel disease and in the induction of autophagy [16].

To investigate how autophagy is involved in the pathophysiology of severe sepsis/septic shock, i.e., a highly systemic inflammatory disease, we examined the association between a single-nucleotide polymorphism (SNP) in the autophagy-related IRGM gene and clinical outcomes in severe sepsis for two cohorts (one single-center cohort and one multi-center cohort) in Japanese population. We also examined the mRNA expression of autophagy-related SNP under stimulus by lipopolysaccharide (LPS) ex vivo.

Materials and Methods

1. Discovery Cohort

The protocol was approved by institutional Ethic Committees at all the 5 participating institutes (the Ethics Committee of Chiba University School of Medicine (permission number 205), the Ethical Committee of Kurume University (bioethics permission number 49), the Medical Research Ethics Committee of Tokyo Medical University, the Ethics Review Board of Hyogo College of Medicine, and the Ethics Committee of Kimitsu Chuo Hospital). After approval by the institutional ethics committees, written informed consent from patients or their next of kin was obtained. In discovery cohort, 259 critically ill patients admitted to the general intensive care unit (ICU) of Chiba university hospital in Chiba, Japan, between October 2001 and September 2008. Inclusion criteria were admitted to the ICU, 18 years of age or older, patients capable of obtaining informed consent in writing own, family, or the legal representative. Exclusion criteria included pregnancy, treatment in the hematologic malignancies, patients receiving radiation treatment and chemotherapy, history of genetic therapy, and outside the scope of active treatment. Blood samples were obtained immediately after admission to the ICU. Centrifuged blood samples were kept separate blood cells and serum at −80°C. The genomic DNA was extracted from whole blood cells.

2. Multi-center Validation Cohort

In multi-center validation cohort, 793 critically ill patients admitted to the five general ICU including the five tertiary medical centers, or Kurume University Hospital, Tokyo Medical University Hachioji Medical Center, Hyogo College of Medicine, Kimitsu Chuo Hospital and Chiba University Hospital from October 2008 to September 2012. Inclusion and exclusion criteria were the same as the discovery cohort. On admission at the ICUs, blood samples were obtained. The blood cells were refrigerated and collected to Chiba University Hospital, and the genomic DNA was thereafter extracted.

3. Data Collection

Baseline characteristics (age, gender), as well as clinical data including length of ICU stay, Sequential Organ failure Assessment (SOFA) scores [17], Acute Physiology and Chronic Health Evaluation (APACHE) II scores [18], morbidity of severe sepsis and septic shock, ICU mortality were obtained after the patients were documented at study entry. The APACHE II scores and SOFA scores were calculated in the first 24 hours after admission.

The diagnosis of systemic inflammatory response syndrome (SIRS) and sepsis, severe sepsis and septic shock were based on the criteria presented at the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference in 1992 [19]. In the present study, both severe sepsis and septic shock is expressed together as SS group.

4. IRGM(+313) (rs10065172) Genotyping

Genomic DNA was extracted from ethylenediaminetetraacetic acid anticoagulated blood using Qiagen’s QIAamp DNA Minikit (Qiagen, Valencia, CA) according to manufacturer’s instructions. We amplified the target region of DNA by polymerase chain reaction (PCR) with primers (Applied Biosystems, Foster City, CA) specific for the sequence of IRGM(+313) (rs10065372), a SNP which presents at the position of +313 inside the exon 2 of IRGM in the chromosome 5q33.1 [20], [21]. Each 25 µL of PCR mixture contained 20 ng of genomic DNA, 900 nM primers, 250 nM probes, and 12.5 µL of TaqMan Univers