Known mutations in human IRAK4

Within two years of the first report of IRAK-4 deficiency, mutations in the human IRAK-4 gene were identified in 13 patients within nine unrelated families suffering from repeated, life-threatening bacterial infections early in life who also exhibit hyporesponsiveness to LPS, IL-1, and IL-18 [87, 100, 101]. These individuals are highly susceptible to Gram-positive infections, although one patient had a documented N. meningitidis infection [101], show no developmental abnormalities, and manifest this pattern of life-threatening bacterial infections in childhood. Interestingly, if the IRAK-4-deficient child survives the early life-threatening bacterial infections, this phenotype dissipates with age, suggesting that repeated immunization and exposure to pathogens elicits a level of adaptive immunity that is sufficient to elicit, at least partially, a protective immune response.

Picard et al. [100] first described autosomal recessive mutations in exons 7 and 8 of the IRAK-4 gene in three unrelated patients suffering from infections caused by pyogenic bacteria and hyporesponsiveness to IL-1, IL-18, LPS, as well as to TLR2, TLR3, TLR5, and TLR9 agonists. These defects were found to be specific for the TLR pathway as stimulation of patients' cells with either TNF-a or PMA and ion-omycin resulted in normal levels of cytokine production [100]. One patient had a homozygous deletion of thymidine in exon 7 (821delT in mRNA), whereas two other patients had a point mutation in exon 8 (C877T substitution in mRNA), both of which resulted in premature stop codons and the lack of expression of functional full-length IRAK-4 mRNA and protein [100]. The health state of two of these patients gradually improved with age, whereas the third patient has had recurring bacterial and fungal infections, requiring IVIG therapy. In a follow-up study of this patient, Day et al. [102] reported that booster immunizations with diphtheria/ tetanus toxoid, pneumococcal polysaccharide and bacteriophage 0X174 led to an impaired antibody response with low titers that were not sustained, suggesting an association of IRAK-4 deficiency with impaired antibody response and failure to sustain it. Thus, it appears that if IRAK-4 is mutated, thereby blocking the TLR pathway, B lymphocytes fail to be properly activated, resulting in inadequate maturation to antibody-secreting plasma cells. Alternatively, the mutation of IRAK-4 may lead to decreased co-stimulatory molecule expression and, thus, improper T-lymphocyte activation, resulting in insufficient T-helper function.

Our studies have focused on a now 23-year old patient with a history of repeated bacterial infections, without fever [101], who failed to become febrile or produce cytokines in response to LPS challenge in vivo. In vitro, patient's PBMC and neu-trophils showed hyporesponsiveness to LPS and IL-1 stimulation, as evidenced by suppressed NF-kB activation and p38 phosphorylation, yet TNF-a responsiveness remained intact [87, 88, 101]. Subsequent cloning and sequencing revealed that the patient expresses a compound heterozygous genotype (i.e., a point mutation on one allele, "M1", and a two nucleotide deletion, "M2", on the other allele of the IRAK-4 gene) ([87]; Fig. 3), in contrast to autosomal recessive genotypes expressed by all but one of the other patients who have been genotyped to date. The IRAK-4 mutations expressed by our patient were inherited in a strictly Mendelian fashion: the two nucleotide deletion mutation was carried heterozygously by the maternal grandfather, mother, and only sibling, while the point mutation was carried het-erozygotically by the paternal grandmother and father. Our recent studies have underscored a failure of LPS to upregulate expression of a number of co-stimulatory molecules, including CD18 and CD67, in patients' polymorphonuclear cells (PMNs), in contrast to strong responses observed in patient's parents and an unrelated control volunteer (Fig. 4). Again, stimulation of patient's PMNs with a non-TLR-specific stimulus, f-Met-Leu-Phe (fMLP), induced an increase in CD18 and CD67 expression that was similar to responses observed in her parents or a normal healthy volunteer (Fig. 4 and data not shown). Interestingly, our patient also had an abnormal inflammatory response to a non-microbial stimulus in vivo, e.g., blister formation, manifested by depressed influx of neutrophils into the blister base, and diminished cytokine and chemokine production in the region of the blister when compared to controls. Yet, under these conditions, complement activation was normal [87]. Thus, even a non-microbial stimulus revealed the deficiency in IRAK-4 in this patient, supporting previous observations that endogenous proteins (e.g., heat shock proteins, fibrinogen, etc.) may serve as TLR agonists (reviewed in [22]). Most interestingly, the point mutation expressed by our patient (Q293X) has been observed in five unrelated families ([103], and D. Speert, personal communication at the time of this report). Although a founder effect has not been formally exclud-

A. CD18 Expression

Healthy Volunteer-| |-Patient

A. CD18 Expression

Healthy Volunteer-| |-Patient

Fluorescence Intensity

Fluorescence Intensity

B, CD67 Expression

Healthy Volunteer-1 |-Patient

B, CD67 Expression

Healthy Volunteer-1 |-Patient

0.1 1 to too 1000 0.1 1 10 100 1000 Fluorescence Intensity

Figure 4

LPS-mediated regulation of expression of CD18 and CD67 in PMNs obtained from IRAK-4-deficient patient versus a normal volunteer

PMNs obtained from IRAK-4-deficient patient or normal volunteer were left unstimulated (resting) or treated with LPS (top panels) or fMLF (bottom panels). Cells were subjected to FACS analysis with either isotype control Abs (dotted lanes) or anti-CD18 (A) and anti-CD67 Abs (B) (depicted in bold lanes).

0.1 1 to too 1000 0.1 1 10 100 1000 Fluorescence Intensity

Figure 4

LPS-mediated regulation of expression of CD18 and CD67 in PMNs obtained from IRAK-4-deficient patient versus a normal volunteer

PMNs obtained from IRAK-4-deficient patient or normal volunteer were left unstimulated (resting) or treated with LPS (top panels) or fMLF (bottom panels). Cells were subjected to FACS analysis with either isotype control Abs (dotted lanes) or anti-CD18 (A) and anti-CD67 Abs (B) (depicted in bold lanes).

ed, these patients are from eight different countries and differ in their ethnicities. However, the sequence surrounding the C877T point mutation does not, from inspection, appear to be a typical hypermutable sequence or "hot spot" [104]. Studies are currently in progress to evaluate these possibilities.

The point mutation and the small, two nucleotide deletion expressed heterozy-gously on distinct alleles by our patient were predicted to encode truncated forms of the IRAK-4 protein, with both truncations occurring within the kinase domain. Unfortunately, we were unable to detect IRAK-4 protein (either WT or mutant) by Western analysis using a polyclonal antiserum obtained from Tularik. Endogenous IRAK-4 protein is expressed at extremely low levels and requires a very large number of cells (~200 x 106 cells per treatment; e.g., see [55]), and, in all probability, even more cells would be required to detect this protein in primary PBMC, the main source of cells from our patient. Unfortunately, this large number of cells was not available from our patient. Despite extensive efforts to detect WT and mutant forms of endogenous IRAK-4 in PBMC cell lysates and IRAK-1 immune complexes obtained from our patient versus healthy volunteers, the amount of endogenous IRAK-4 protein was below the level of detection by Western analysis. However, when we introduced these two mutations into the pRK7 IRAK-4 expression vector encoding epitope (flag)-tagged WT IRAK-4 and transfected them in HEK293 cells, the predicted sizes for the truncated forms were readily observed by Western analysis using anti-flag antibody [87].

Importantly, when overexpressed in HEK293 cells, both mutant forms act as dominant negative inhibitors of WT, endogenous IRAK-4, as evidenced by suppression of IL-1-induced IRAK-1 kinase activity, with the highest level of inhibition achieved upon transfection of the vector encoding the C877T point mutation [87]. Analysis of the molecular mechanisms by which these kinase-defective, short forms of IRAK-4 block signaling has demonstrated that mutations in the kinase domain of IRAK-4 impair its IL-1-inducible association with the IL-1RI and IRAK-1 [88]. In addition, we have shown that overexpressed mutant IRAK-4 proteins failed to be recruited to TLR4 upon stimulation of HEK293/TLR4/MD-2 cells with LPS (Fig. 5). We have also found that overexpression of truncated IRAK-4 variants inhibited recruitment of endogenous IRAK-1 and MyD88 to the IL-1RI in response to IL-1 stimulation, and result in constitutive association of kinase-defective IRAK-4 with endogenous or overexpressed cytoplasmic MyD88 [88]. Since the heterozygous parents, grandparents, and sibling of this patient are phenotypically normal with respect to LPS sensitivity and exhibit normal resistance to infection, we can only surmise that insufficient amounts of the "truncated" forms of IRAK-4 are produced by these individuals to block the activity of the normal IRAK-4 protein. In other words, only a single copy of wild-type IRAK-4 is necessary to render an individual phenotypically "normal." In contrast, upon overexpression, a large proportion of kinase-defective IRAK-4 variants are likely to be present in the cells relative to the amount of WT IRAK-4, leading to an inhibitory effect. These results demon-

Figure 5

Mutations in the kinase domain impair LPS-inducible recruitment of IRAK-4 mutant proteins to TLR4

HEK293/TLR4 cells were transfected with pRK7 expression vectors encoding Flag-tagged WT, M1, or M2 IRAK-4. After recovery for 48 h, cells were treated for 15 min with medium or 100 ng/ml LPS. The top panel shows total expression of TLR4 detected by Western analysis of cell lysates with anti-TLR4 Ab prior to immunoprecipitation. Cell lysates were immunoprecipitated with anti-Flag Ab, and immune complexes subjected to Western analysis with anti-TLR4 Ab (middle panel, detection of TLR4 association with IRAK-4) or anti-Flag Ab (bottom panel; total expression of transfected IRAK-4 species).

Figure 5

Mutations in the kinase domain impair LPS-inducible recruitment of IRAK-4 mutant proteins to TLR4

HEK293/TLR4 cells were transfected with pRK7 expression vectors encoding Flag-tagged WT, M1, or M2 IRAK-4. After recovery for 48 h, cells were treated for 15 min with medium or 100 ng/ml LPS. The top panel shows total expression of TLR4 detected by Western analysis of cell lysates with anti-TLR4 Ab prior to immunoprecipitation. Cell lysates were immunoprecipitated with anti-Flag Ab, and immune complexes subjected to Western analysis with anti-TLR4 Ab (middle panel, detection of TLR4 association with IRAK-4) or anti-Flag Ab (bottom panel; total expression of transfected IRAK-4 species).

strate that hyporesponsive phenotype of our IRAK-4-defective patient may result from the failure of mutant IRAK-4 species to form functional signaling complexes with components of the IL-1R/TLR4 pathways in response to stimulation with IL-1 and LPS. They also suggest that small molecular weight mimetic compounds may be designed to diminish IRAK-4-dependent signaling in people with hyperinflam-matory syndromes [88].

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