We described previously our patient as having a long history of severe, recurrent bacterial infections and greatly diminished responses to LPS in vivo or to LPS and IL-1 in vitro (1
), and speculated that this phenotype was due to a defect early in the LPS signal transduction pathway. In addition to abnormal LPS and IL-1 signaling, we now show that this patient's T cells failed to respond to rIL-18 to secrete IFN-γ (). Our findings that LPS- or IL-1–induced NF-κB and AP-1 translocation, p38 phosphorylation, or LPS-induced gene expression were all impaired in this patient's PBMCs (–) provided strong evidence for an upstream signaling defect shared by all three receptor systems. This was further strengthened by the observation that this patient exhibited dysregulated neutrophil IRAK-1 kinase activity in response to LPS. Rather than being increased by LPS, IRAK-1 kinase activity was transiently down-regulated, suggesting an inhibitory effect of this mutation on IRAK-1 kinase activity. Although sequence analysis of this patient's IRAK-1
gene revealed a heterozygotic polymorphism (F196S) located in a critical region of the protein (i.e., just upstream of the region that encodes the kinase domain; reference 19), it fully reconstituted signaling in IRAK-1–deficient cells (Fig. S1).
However, this patient also possesses a “compound heterozygous” genotype in IRAK-4
. The expression of distinct mutations on each of the patient's IRAK-4
alleles results in the failure to express a normal gene product (i.e., a “compound heterozygous genotype” that results in a recessive phenotype). Generally, inheritance of a compound heterozygous genotype leading to a recessive phenotype is more common than expression of homozygous recessive mutations, particularly in diseases where many different mutations have been reported (e.g., β-globin; references 24, 25). The two distinct mutations in this patient encode proteins that, when expressed, are truncated within the IRAK-4 kinase domain, yet retain the entire DD. Although overexpression of the WT IRAK-4 vector in HEK293T cells augmented IRAK-1 kinase activity induced by IL-1, overexpression of the mutant forms inhibited endogenous IRAK-1 kinase activity modestly, indicating that these molecules do not function normally and can interfere with WT IRAK-4 (i.e., exhibit dominant negative activity) when coexpressed with a normal IRAK-4
gene. These data are consistent with those of S. Li et al. (8
) in which a 193 amino acid form of IRAK-4, or one that expresses mutations within the kinase domain, were found to inhibit IL-1–mediated signaling in a subline of HEK293 cells. Sequencing analysis revealed that the patient's father was heterozygous for the point mutation (i.e., +/C877T genotype), whereas her mother and sister were heterozygous for the deletion mutation (i.e., +/620–621del genotype), yet both parents and her sibling are fully LPS responsive, based on a history of normal febrile response to bacterial infections, no history of repeated infections, and normal LPS-inducible gene expression in vitro. One can only assume that compared with overexpression systems in vitro, the levels of expression of each of the mutated forms by the parents is insufficient to block activity of the their WT IRAK-4 such that LPS responsiveness is not measurably impaired. Therefore, gene therapy may be a viable approach for this patient.
The central role of IRAK-4 in LPS- and IL-1 signaling has only recently emerged. Work by S. Li et al. (8
), published within the past year, indicates that IRAK-4 activation is required for phosphorylation of IRAK-1. This is consistent with earlier work of X. Li et al. (18
), who showed that the IRAK-1 kinase domain is unnecessary for reconstitution of phosphorylation of IRAK-1 and signaling in IRAK-1–deficient cells. These authors postulated the existence of an upstream kinase that elicits the initial phosphorylation of IRAK-1 in response to IL-1. Although other IRAK family members (e.g., IRAK-2 and IRAK-M) can substitute for IRAK-1 (for review see reference 6), the necessity of IRAK-4 is further supported by the strong LPS- and IL-1–resistant phenotypes in mice with a targeted mutation in IRAK-4
). Burns et al. (7
) demonstrated an interaction of MyD88, IRAK-4, and IRAK-1; IRAK-1 interacts with MyD88 through homotypic DD interactions, whereas the region of MyD88 between the TIR and DD is necessary for recruitment of IRAK-4, although the region on IRAK-4 that engages MyD88 was not reported. In coimmunoprecipitation experiments, we have found that overexpression of either truncated form (M #1 or M #2) in HEK293T cells results in a stronger association with MyD88 than WT IRAK-4 (unpublished data). This suggests that the DD of IRAK-4 enables it to bind to the region of MyD88 between its TIR and DD (7
), and suggests the possibility that dominant negative inhibition may be facilitated through preferential or sustained binding of truncated forms. When coupled with the failure of the truncated form to compete for signaling components leading to inhibition of NF-κB reporter activity ( A; WT IRAK-4), their failure to augment IRAK-1 kinase activity, and their capacity to inhibit endogenous IRAK-1 kinase activity when overexpressed ( B and 9 C), a model of interaction between MyD88 and truncated IRAK-4, but not between truncated IRAK-4 and downstream signaling molecules, is suggested.
Quite recently, three unrelated patients with homozygous recessive IRAK-4 deficiencies were identified (26
). Like our patient, these patients had recurrent pyogenic infections. Our patient expresses a unique pattern of IRAK-4 insufficiency as a consequence of her compound heterozygosity, further supporting the essentiality of IRAK-4 in the response to bacterial infection. Unquestionably, the most fascinating aspect of these two independent papers is that the substitution of T for C at nucleotide 877 of the IRAK-4 cDNA (C877T) is common to our patient and two of the patients described in the other paper. Because these patients are, to the best of our knowledge, unrelated, the data suggest that this site may represent a hypermutable site or “hot spot” in this gene. Coupled with the fact that our patient expresses a compound heterozygous genotype, these data suggest that there are likely to be additional patients that fail to express functional IRAK-4 and present with recurrent bacterial infections.
The data in this report also provide compelling new evidence for an important role for IRAK-4 in mobilizing the acute inflammatory response. The observation that our patient also fails to mount a normal inflammatory response in response to blister formation suggests that the role for IRAK-4 in inflammation is not restricted to pyogenic stimuli, as suggested by Picard et al. (26
). Specifically, in response to skin blister induction, our patient had normal acute (3–5 h) cellular inflammation with normal levels of C5a, but impaired delayed (8–24 h) cellular inflammation with depressed levels of IL-8 and other proinflammatory cytokines. Normal levels of C5a, and presumably of other chemoattractants not measured (i.e., leukotriene B4), that do not require the TLR pathway for synthesis, likely account for the normal, early phase of inflammation. Our patient's diminished capacity to produce IL-8 and other proinflammatory cytokines likely explains the defective later phase of acute inflammation in the blister model. The defective production of inflammatory mediators identified in our patient as a result of mutations in IRAK-4
provides strong evidence for the importance of IRAK-4 and products of the TLR pathway in mobilizing the inflammatory response in vivo. It is also possible that the release of endogenous, cell-derived TLR agonists released as a consequence of the inflammatory response to blister formation provides the necessary stimulus for this later inflammatory response. In this regard, Kuhns et al. (27
) and Smiley et al. (28
) reported that fibrinogen is a potent TLR4 agonist, leading to the induction of chemokine expression; our patient also fails to respond to fibrinogen in vitro (unpublished data). Finally, the absence of increased susceptibility to viral and fungal infections in our patient further suggests that IRAK-4–independent pathways must also be triggered in host defense against these infections.