We found that the IRAK4 haplotype clade marked by 23338C/24472T/29429A (C/T/A) was associated with increased prevalence of positive bacterial cultures on admission to the ICU in a cohort of 775 critically ill Caucasians with SIRS. Specifically, the C/T/A clade was associated with increased prevalence of Gram-positive cultures and increased prevalence of positive blood cultures. No IRAK4 haplotype clade was associated with prevalence of sepsis or septic shock at admission, or with 28-day survival. Furthermore, we have demonstrated that the IRAK4 C/T/A clade is associated with decreased lymphoblastoid cell immune response to CpG (as measured by IL-6), and that fibroblasts transfected with the 29429A allele that marks the C/T/A clade show a trend to decreased IL-6 secretion in response to stimulation with LPS compared to IRAK4 29429G fibroblasts. To our knowledge, this is the first report of an association of genetic polymorphisms of IRAK4 with prevalence and type of bacterial cultures in a cohort of critically ill patients and supported by mechanistic evidence of decreased cellular immune response to TLR ligands.
Haplotypes within the C/T/A clade are uniquely distinguished from haplotypes in the other four clades of IRAK4 by 29429A. The substitution of an A for a G at position 29429 results in an alanine, a nonpolar aliphatic amino acid, being replaced by a threonine, a polar uncharged amino acid, at amino acid position 428 of the IRAK4 protein [21
]. The rs4251545 (Ala428Thr) IRAK 4 polymorphism could alter the kinase activity since it is located in the kinase domain of the protein (154–460) [35
]. This amino acid substitution may disrupt IRAK4 signaling and result in a less-effective immune response to invading pathogens so that critically ill patients carrying the C/T/A clade have an increased risk of positive bacterial cultures. Our finding that the C/T/A haplotype clade of IRAK4 is associated with increased prevalence of positive bacterial cultures at admission to the ICU is consistent with our previous studies [36
], and with recent animal studies [37
] which suggest that polymorphisms of innate immunity genes could be associated with impaired clearance of bacteria. A number of studies have shown that rare germline mutations in IRAK4 cause recurring bacterial infections in children and deficiencies in cytokine production in response to a range of microbial-derived TLR agonists and to recombinant IL-1β or IL-18 [10
]. Our finding that the IRAK4 C/T/A clade is associated with decreased lymphoblastoid cell immune response to CpG and with a trend to decreased immune response to LPS in IDFs transfected with the IRAK4 expression plasmid carrying the 29429A allele suggests that the replacement of Ala428 with a threonine decreases the cellular response to TLR ligands, perhaps impairing the host's ability to clear an infection.
There are several strengths of our gene association study that minimized limitations of genetic association studies. Firstly, our large cohort of critically ill patients (n = 775) reduced the risk of type I error (finding a spurious association) compared to other studies of smaller sample size. Our large sample size also ensured that we had adequate power to determine that there was truly no association of IRAK4 haplotype clades with prevalence of sepsis and septic shock, or 28-day survival in our cohort of critically ill patients. Second, we adjusted for confounders in our survival analysis (age, gender, APACHE II score, surgical diagnosis) and still found no association of IRAK4 haplotypes with 28-day survival. Third, to avoid spurious associations we included only Caucasians in our cohort of critically ill adults, thus limiting the risk of positive associations due to ethnic heterogeneity [17
]. Finally, by testing the immune response of cell lines of different IRAK4 haplotypes to TLR ligands, we have provided plausible mechanistic data to support our clinical association.
An important strength of the design of our association study is our use of haplotypes and haplotype clades as the unit of genetic variation. Haplotype analysis is more effective in determining association of genotype with phenotype than is individual SNP analysis [42
]. Haplotypes serve as markers of unidentified polymorphisms that may be the cause of phenotypic variation [42
]. The phylogenetic history of haplotypes within a population may be determined and used to group haplotypes into clades using cladistic analysis [16
]. Cladistic analysis has two unique strengths. First, grouping haplotypes into clades decreases the degrees of freedom, thereby increasing the statistical power to associate genotype with phenotype [43
]. Second, grouping haplotypes into clades facilitates identification of causal SNPs. Another practical strength of our approach is that a small number of ‘haplotype tag’ SNPs (htSNPs) can be used to distinguish haplotype clades, eliminating the need to genotype all SNPs within a gene [24
]. Associations of altered phenotypes with polymorphisms of key genes such as innate immune receptors, second messengers in signaling pathways and cytokines have been extensively studied in critically ill patients [36
]. Our study shows that variants of a second messenger, IRAK4, create a partial innate immune response defect leading to increased risk of Gram-positive infection while polymorphisms of receptors and signaling proteins show increased risk of sepsis (TLR1 [48
], TLR2, CD14 and MBL [36
]), and polymorphisms of receptors, second-messenger molecules and cytokines are associated with an increased risk of death (TLR1 [48
], TNF, IL-6 [49
], IL-10 [50
]). We did not find an association of IRAK4 294249G/A and altered risk of sepsis, perhaps because this IRAK4 variant causes only a partial defect in NF-κB pathway signaling as described in case reports of IRAK4 deficiency [14
]. Mutations that cause IRAK4 deficiency create dysfunctional signaling with partially intact NF-κB but defective MAPK signaling and dysregulated complex function that affects transcriptional and post-transcriptional control of TLR/IL-1R response in cell- and cytokine-specific ways. Thus, the IRAK4 294249G/A may alter the risk of acquiring Gram-positive infection, but not the risk of sepsis, because of a redundancy in the innate immune response.
There are several limitations of our gene association study. We have not examined how the 29429G/A (A428T) polymorphism affects the function or expression of the IRAK4 protein, so we do not know the functional consequences of the C/T/A haplotype clade in the systemic inflammatory response syndrome. We also do not know how the amino acid change at position 428 may affect the interaction of IRAK4 with other proteins in the MyD88-IRAK1-IRAK4 signaling pathway. Additionally, we were only able to show a trend to decreased IL-6 secretion in response to LPS stimulation of IRAK4-deficient fibroblasts transfected with the IRAK4 29429A allele (compared to the IRAK4 29429G allele). As this trend is in line with our finding of an association of the C/T/A clade with decreased IL-6 secretion in lymphoblastoid cells, we believe that it is an important result, although limited by the experimental design. The Ala428Thr amino acid change may only mildly disrupt IRAK4 function, and thus cause only minor changes in IL-6 secretion in response to stimulus with TLR ligands. Although we treated all cells identically at the same time, because we introduced IRAK4 into the IDFs using transient transfection, it is possible that varying numbers of cells in each well of the transfection and stimulation experiments were viable and expressing IRAK4. This experimental variability may affect our ability to accurately measure the genetic variability in the immune response due to the IRAK4 G29429A polymorphism. In the future, it may be appropriate to use stable transfection to introduce the IRAK4 gene into IDFs. Furthermore, although involved in wound repair, fibroblasts are not the ideal cells in which to be measuring variability of the innate immune response. The IRAK4-deficient fibroblasts were a generous gift to us from our collaborators and served to limit genetic heterogeneity at other loci. However, we may be able to observe greater genetic variability of the immune response to TLR ligands in monocytes or macrophages, cells that are much more sensitive to TLR ligands.
In summary, we have demonstrated a novel association between a haplotype clade of IRAK4 marked by G29429A (Ala428Thr) and increased risk of Gram-positive infection in a cohort of critically ill adults. We have shown that a possible explanation for this association is decreased cellular response to TLR ligands through the IRAK4 signaling pathway, potentially inhibiting the host's ability to clear an infection. Future studies in a separate clinical cohort and in other cellular models will be needed to confirm these findings.