Journal of Investigative Medicine:
EB Symposium Manuscripts
Genetic Factors Impacting Therapy in Acute Lung Injury/Acute Respiratory Distress Syndrome
Maloney, James Peter MD
From the Division of Pulmonary and Critical Care Medicine, University of Colorado and Denver VA Medical Center, Denver, CO.
Received June 18, 2008, and in revised form July 22, 2008.
Accepted for publication July 27, 2008.
Reprints: James P. Maloney, MD, Associate Professor, Division of Pulmonary and Critical Care Medicine, University of Colorado, 4200 E 9th Ave, C-272, Denver, CO 80262. E-mail: firstname.lastname@example.org.
Supported by NHLBI grant HL071618.
The proceedings of a symposium presented at the Experimental Biology Meeting in San Diego, CA on Monday, April 7, 2008.
This symposium was supported in part by a grant from the National Center for Research Resources (R13 RR023236).
Acute lung injury (ALI) and its most severe form the acute respiratory distress syndrome occur in patients who have a predisposing severe inflammatory insult to the lung. Most often ALI is due to sepsis from bacterial infection, but ALI can occur with any infection and with noninfectious insults such as severe trauma, acute pancreatitis, aspiration, and near-drowning. After any of these insults, the interindividual risk of progression to ALI and the risk of death remain difficult to predict. Our inability to predict an individual's susceptibility to acute lung injury has long suggested that genetic factors influence ALI risk. There is substantial evidence for heritable predispositions to severe infections and an emerging body of literature implicating genetic factors in ALI pathogenesis. A paradigm is emerging that the genetic risk for ALI can be best understood in terms of factors that control 3 overlapping stages of ALI pathogenesis: risk for the acquisition of a predisposing condition (such as a severe pneumonia), risk for progression to lung injury during systemic inflammatory states (such as severe sepsis), and risk for failure of endogenous mechanisms to resolve the lung injury. The evidence supporting this paradigm is herein reviewed, along with potential treatment strategies that could be directed by knowledge of specific genetic factors in an individual patient.
There is substantial evidence that genetic factors influence death rates from infection as well as the occurrence, severity, and survival rates of acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS).1,2 The great interest in the identification of these genetic factors arises in part out of our clinical frustration to understand why a healthy young patient can develop severe sepsis and die of multiorgan failure, whereas an elderly patient in the next bed with multiple comorbidities and the same infection can quickly resolve their sepsis from an identical organism/site with minimal to no organ failure. Elucidating the genetic factors that influence ALI risk and outcome is an important step needed to develop therapies targeted to the specific genes or pathways and to better apply existing prevention and therapeutic strategies to the specific groups most likely to see benefit. Implementation of this strategy has many thresholds to cross including the validation of genetic risk factors in the heterogeneous populations of patients at risk for ALI, development of new drugs or approaches based on genetic studies, and proving in clinical trials that genetic risk-based treatment and prevention strategies are of benefit.
Most work in the field of ALI susceptibility has been in patients with sepsis as the predisposing condition. Although sepsis is the most common cause of ALI, an understanding of the genetic factors that influence sepsis-related ALI is of great importance. A paradigm is likely where genetic risk for ALI can be best understood in terms of factors that control 3 overlapping stages of its pathogenesis: risk for the acquisition of the predisposing condition such as a severe pneumonia; risk for progression to lung injury during systemic inflammatory states such as severe sepsis; and risk for failure of endogenous homeostatic mechanisms to counter inflammation and resolve the lung injury (Fig. 1). Because sepsis is a common disease, those variants which are common (carrier rate >1%) in the healthy population are most relevant as risk factors for complications such as ALI. The existing studies supporting the existence of common genetic variants as risk factors for ALI have limitations typical for the initial genetic studies of any disease, being often confounded by small sample size, bias, lack of evidence for meaningful functional effects of implicated polymorphisms, examination of only whites, lack of control for baseline differences between groups (such as comorbidities), lack of statistical rigor, failure to examine all relevant polymorphisms for a gene of interest, and the lack of validation in multiple populations.3,4 The use of hypothesis-independent methods of identifying ALI genetic risk factors is only recent5,6; and the field awaits the performance of powerful and expensive hypothesis-independent, bias-free approaches such as whole genome studies, which have been helpful in identifying genetic risk factors in more common diseases like diabetes.7
This review focuses on illustrative studies that provide compelling data for implicated polymorphisms based on the following: functional effects in pathways of clear relevance to ALI, large sample size, use of hypothesis-independent strategies, and replication in more than 1 population or by more than 1 investigative group. In this brief review, the evidence for variants in 3 distinct genes as ALI risk factors is discussed, each having a principal role in 1 stage of the ALI genetic risk paradigm detailed in Figure 1. The scope of this article prevents discussion of the many other relevant and interesting publications in this field, though most of the genes evaluated in those studies are shown in Figure 2.
Genetic Factors That Increase Susceptibility to Infection and Mortality During Sepsis
The heritability of death because of infection in the preantibiotic era appeared to be much higher than the heritability of death from heart disease or cancer.2 Evidence also suggests that minorities experience discordant population rates of sepsis and of death from ALI.8,9 Such data on the overall risk of infection in families and ethnic groups are consonant from what we know of how an individual's genetic background can influence the risk and outcome of sepsis from a specific microorganism. Sickle cell disease in people of African descent is the best example of a common polymorphism that influences the risk of acquiring and/or dying of an infection with a specific organism-malaria. The single nucleotide polymorphism (SNP) that causes sickle cell anemia is common, with a minor allele frequency of 4% in African Americans and is the result of persistence of an ancient A to G nucleotide mutation, resulting in alteration of glutamic acid to valine in the beta chain of hemoglobin (Hb S). Sickle cell hemoglobin (Hb S) alters red blood cell shape and in heterozygous carriers leads to decreased susceptibility to plasmodium infection, decreased severity of acute malaria, and improved survival during chronic infection. Heterozygous individuals have rare ill effects from carrying a single Hb S allele and thus benefit from Hb S carriage, whereas homozygotes for Hb S (SS) have a shorter life span because of sickle cell disease despite protection from malaria. Thus, Hb S in the heterozygous state is a balanced polymorphism and has undergone positive selection in African populations.10 Mutations in the late phase of complement, although rare compared with Hb S, are well-established causes of heritable susceptibility to infection and sepsis from Neisseria species.11 Such examples demonstrate that both common and rare genetic variants can provide protection from sepsis or alternatively predispose to it.
Mannose-binding lectin 2 (MBL2) is an opsonic protein of the innate immune system. Mannose-binding lectin 2 recognizes mannans and other ligands on the surface of microorganisms. Mannose-binding lectin 2 is liver-secreted, present constitutively in serum in μg/l quantities, and undergoes increased secretion during acute infections.12 After binding to the surface of a microorganism, MBL2 is then able to participate in host defense by activation of the complement pathway.11 Ligand binding and complement activation functions are highly dependent on a complex quarternary structure of 9 to 18 MBL2 monomers; MBL2 oligomerization and function diminish greatly for each of the 3 well-described missense variants of MBL2.13 These 3 missense isoforms all result from SNP and occur in exon 1 in the oligomerization region: Arg52Cys (rs5030737), Gly54Asp (rs1800450), and Gly57Glu (rs1800451). All of these SNP are common enough to be of importance as risk factors for infection in the general population. The 52Cys variant is common in whites (19% are carriers) and less so in African Americans (4% are carriers); 54Asp is very common in whites (2-4% homozygotes, 21% heterozygotes) and Asians (2-4% homozygotes, 26-38% heterozygotes) but not in African Americans (2-4% heterozygotes); the 57Glu variant is very common in African Americans (36% heterozygotes; 6% of sub-Saharan Africans are actually homozygotes), but rare in whites and Asians (heterozygotes <1%). Carriers of missense SNP all have low MBL2 levels and dysfunctional protein. Observational studies have shown that these missense MBL2 variants are associated with increased susceptibility to infection with various organisms, and thus a potential role for MBL2 as a genetic factor that predisposes to sepsis has been explored.12,14,15 In a case-control study of genetic risk factors for ALI, Gong et al.16 evaluated 654 white subjects at risk for ARDS (>80% had sepsis) for the 3 missense MBL2 SNP and 1 promoter SNP. In the 212 cases of ARDS, the 7 subjects who were 54Asp homozygotes had a statistically significant worse severity of illness on admission, a greater likelihood of septic shock, and a 6.7-fold increased risk of ARDS when compared with heterozygotes and noncarriers (of the ten 54Asp homozygotes in the entire study, 7 developed ARDS). Furthermore, 54Asp homozygotes had an 80% mortality; only 2 of the 10 survived the past 20 days. None of the other MBL2 polymorphisms were associated with ARDS risk or outcomes. The 57Glu polymorphism was too rare in this white study population (variant allele frequency 1.5%) to detect an association with outcome, as it is common only in populations with African ancestry (as described previously). The lack of association of 52Cys with outcome may also have been due to insufficient statistical power in this population (as the variant allele frequency was only 8% in the population, half that of the 54Asp allele) or a milder phenotypic effect on MBL2 protein. Although this study was limited by the at-risk group being small, it is highly consistent from what we know of other diseases characterized by mutations in other serum proteins such as hemophilia and α-1 antitrypsin deficiency-it is the homozygote carriers who get the disease, not the heterozygotes. Like α-1 antitrypsin and clotting factors in other diseases, MBL2 is ideally suited for replacement therapy in sepsis patients. If recombinant wildtype human MBL2 were pursued by the biotechnology industry as a potential therapy, and if it passed animal and early clinical human trials, it could be tested in a multicenter trial of sepsis patients who are MBL2 deficient because of homozygosity for missense SNP. It is rational to expect that MBL2 replacement therapy in such a scenario would improve recovery from an acute infection in variant homozygotes, even if just by augmenting host defense against subsequent nosocomial infections, thus improving survival and decreasing the risk for ALI and other organ failures. Moreover, the 2% to 10% of Asians, African Americans, and whites who are genetically deficient in MBL2 may benefit from long-term replacement strategies to prevent infection (secondary prevention), creating a niche for drug development.
Genetic Factors That Predispose to ALI/ARDS During Severe Infections-A Role for Pre-B-Cell Colony-Enhancing Factor
Once severe sepsis has occurred, genetic variants that predispose to an initial infection may be less relevant to the potential for downstream organ injury (including ALI) than genetic factors which permit an overexuberant systemic inflammatory response during the acute infection. In evidence of a second phase of genetic risk in ALI are the profound differences in the interindividual response to bacterial cell wall lipopolysaccharide (LPS, endotoxin) demonstrated by several investigative groups. Using whole blood cells and/or neutrophils isolated from healthy donors, profound and consistent interindividual differences in cytokine release, gene expression, and nuclear factor-κB (NF-κB) activity in response to endotoxin were shown.17,18 Activation of circulating neutrophils is a key response to acute infection and leads to killing and phagocytosis of microorganisms. Yet, neutrophil-induced collateral damage to tissues can occur. Such collateral damage by rogue neutrophils is a cardinal feature of ALI, acute renal failure, and other organ failures in sepsis.19 The potential of neutrophils to cause collateral tissue injury is balanced by an intrinsic apoptotic pathway such that neutrophils undergo apoptosis within days of release from bone marrow or within hours of activation.20 Thus, any genetic factors that influence activation and apoptosis of neutrophils would be expected to have an important role in ALI risk.
A prominent role for pre-B-cell colony-enhancing factor (PBEF) in the regulation of neutrophil apoptosis has recently emerged. Pre-B-cell colony-enhancing factor was first described as a B-cell mitogen. Studies on LPS-activated neutrophils revealed PBEF to be one of the most differentially expressed genes during expression profiling.21 Jia et al found that PBEF was a potent inhibitor of the neutrophil apoptotic pathway, that neutrophils expressed and secreted PBEF after stimulation with LPS and IL-1β, and that neutrophils from septic patients had profound defects in apoptosis versus control neutrophils.22 This septic apoptotic defect could be reversed by PBEF inhibition in vitro. Unaware of these findings, Ye et al. contemporaneously reported results from a hypothesis-independent project designed to identify genetic risk factors for ALI using multiple animal models of ALI and in vitro human cell injury models.6 Pre-B-cell colony-enhancing factor was one of the top 10 dysregulated genes on microarray experiments across experimental platforms.6 They went on to describe the common genetic variation in PBEF in humans by resequencing healthy controls and sepsis subjects. Finding no missense SNP, they tested for association of 2 common promoter SNP (T-1001G, C-1543T) with mortality and the risk for ALI in 187 subjects with severe sepsis, 87 of whom had ALI. They found an 8-fold increased risk of ALI in carriers of the GC haplotype, with −1543T having an ALI protective effect. The −1543T allele also was associated with less transcriptional activity of the PBEF promoter, suggesting that the ALI protective effect of −1543T might be via a decrease in PBEF expression, thereby enhancing neutrophil apoptosis. In a replication study, Bajwa et al. evaluated these 2 PBEF SNP in a cohort of 1162 white subjects at risk for ARDS, 375 of whom developed ARDS.23 This is the largest case-control study of ALI genetics published to date. Like Ye et al.,6 they found a protective effect of the −1543T allele in septic shock (0.66 odds ratio (OR) of ARDS) and better outcomes (decreased ARDS mortality, more ventilator-free days). They also reported that −1001G increased the risk of developing ARDS (OR 1.35). Thus, genetic variation in PBEF has been validated as an ALI risk factor in multiple populations and has a strong functional rationale via control of neutrophil apoptosis. Temporary inhibition of PBEF during severe sepsis after initial antibiotic therapy and resuscitation may have a role in the future care of severe sepsis and other ALI predisposing states. Such inhibition could effectively reinstitute the normal apoptotic pathway in neutrophils in the 40% to 58% of at-risk subjects who carry at least one of the implicated PBEF genotypes.
Genetic Factors That Affect ALI Resolution
Once ALI and other organ injuries are established after sepsis and other insults, recovery requires a concerted activation of antiinflammatory, antioxidant, and antifibrotic pathways by the host. These pathways likely have little to do with acquisition of the initial insult but could overlap with factors that influence the initial progression to organ injury. Failure to reverse ongoing systemic inflammatory states days to weeks after the initial injury is a well described as a factor in ALI persistence and mortality. The antioxidant pathway appears to be quite important in this phase of ALI pathogenesis. Acute inflammation and oxidant stress induce a number of antioxidant gene responses through the transcription factor NF-E2-related factor 2 (NRF2). In mice, NRF2 has been linked to genetic sensitivity to hyperoxic lung damage and to protection from pulmonary fibrosis after bleomycin.24,25 Marzec et al resequenced the human NRF2 gene and found a functional polymorphism in the promoter (C-617A) that alters an NRF2 binding site.26 The variant allele −617A decreased baseline transcription activity of NRF2 by over 50%. They evaluated −617A and 1 other SNP in a cohort of 90 patients (45 whites, 45 African Americans) at risk for ALI from severe trauma; 30 of these subjects had ALI. They found that −617A carriers had a 6.4-fold elevated risk of developing ALI relative to wildtype subjects. Although this was a small genetic association study, the relevance of NRF2 to ALI pathogenesis and the marked functional effects of the variant −617A are noteworthy. Further study and replication of NRF2 as a genetic risk factor for ALI is needed. Large cohorts, evaluation of sepsis subjects, and evaluation of outcomes such as ventilator-free days, survival, and other organ failures that reflect the resolution phase of ALI are needed.
Strategies for the Future Application of Genetic Testing in ALI and Its Predisposing Conditions
The implementation of genetic testing for genetic risk in ALI and its predisposing conditions will require further confirmation of emerging risk factors in large cohorts, animal studies, and phase III clinical trials of agents that manipulate the genes and pathways involved in genetic subgroups at risk. Genetic testing of the population at large, for example, before the development of specific illnesses, is an area fraught with cultural, legal, and ethical dilemmas. Acute testing at the time of illness will be limited by the return time of the genetic assay in the face of an often rapid progression to organ failure. This will limit our ability to implement any genetic-risk strategies before organ failure occurs. In the end, secondary prevention using genetic information may emerge as the most successful strategy by preventing recurrent insults (for instance, prevention of nosocomial infections in MBL2 deficient patients), closely followed by agents that can facilitate resolution of organ injury in at-risk subgroups (such as by inhibition of profibrotic factors). Although a paradigm of 3 progressive stages of ALI based on genetic risk factors for specific pathways is presented here, clearly these stages overlap; genes such as PBEF likely have a role in the resolution of ALI, genes such as NRF2 are important in limiting initial tissue injury, and genes such as MBL2 encode proteins that may have less appreciated roles in the control of inflammation, as indicated in Figure 2. Only further research and clinical trials will tell us if treatment of ALI based on at-risk genetic factors can be a successful strategy.
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acute lung injury; ARDS; genetics; pre-B-cell colony-enhancing factor; mannose-binding lectin 2
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