Report

Pyogenic Bacterial Infections in Humans with IRAK-4 Deficiency

See allHide authors and affiliations

Science  28 Mar 2003:
Vol. 299, Issue 5615, pp. 2076-2079
DOI: 10.1126/science.1081902

Abstract

Members of the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) superfamily share an intracytoplasmic Toll–IL-1 receptor (TIR) domain, which mediates recruitment of the interleukin-1 receptor–associated kinase (IRAK) complex via TIR-containing adapter molecules. We describe three unrelated children with inherited IRAK-4 deficiency. Their blood and fibroblast cells did not activate nuclear factor κB and mitogen-activated protein kinase (MAPK) and failed to induce downstream cytokines in response to any of the known ligands of TIR-bearing receptors. The otherwise healthy children developed infections caused by pyogenic bacteria. These findings suggest that, in humans, the TIR-IRAK signaling pathway is crucial for protective immunity against specific bacteria but is redundant against most other microorganisms.

The members of the mammalian Toll-like/interleukin-1 receptor superfamily characteristically have a TIR domain (1). This superfamily contains two classes of membrane receptors: TLRs, seven of which recognize known ligands derived from microorganisms (2, 3) and interleukin-1 receptor and related receptors (IL-1Rs), two of which recognize known host cytokines, IL-1 (4) and IL-18 (5). Upon ligand binding, the receptor complex recruits, via its intracytoplasmic TIR domain, the TIR-containing cytosolic adapter proteins MyD88 (6) and TIRAP/Mal (7–9). These adapters in turn recruit the IRAK complex. Four IRAK molecules have been identified: IRAK-1 (10), IRAK-2 (6), IRAK-M (11, 12), and IRAK-4 (13–15). IRAK-1 and IRAK-4 are active kinases, dissociating from the receptor-adapter complex upon phosphorylation and activating tumor necrosis factor receptor-associated factor-6 (TRAF-6) (16). TRAF-6 then activates at least two pathways, leading to the activation of NF-κB and MAPK (16). In the mouse, the TIR-IRAK signaling pathway plays an extensive role in immunity to infections (1–5).

We investigated three unrelated children (P1, P2, and P3) with recurrent infections and poor inflammatory response (for case reports, see supporting online text). Extracellular, pyogenic bacteria were the only microorganisms responsible for infection. Gram-positive Streptococcus pneumoniae andStaphylococcus aureus were the most frequently found and were the only pathogens identified in two patients. The infections began early in life but became less frequent with age, and the patients (now aged 6, 11, and 7 years) are well with no treatment. All known primary immunodeficiencies were excluded. In particular, the patients had normal serum antibody titers against protein and polysaccharide antigens, including those from S. pneumoniae. However, one of our three patients (P3) had previously been shown not to respond to lipopolysaccharide (LPS) and Staphylococcus aureus(17). The phenotype of the three patients was similar to that of another child described elsewhere, with impaired responses to lipopolysaccharide (LPS) and IL-1β, but not to tumor necrosis factor–α (TNFα) (18). This suggested that our three patients might be suffering from impaired TIR pathway signaling.

We first tested the response of the patients' monocytes to LPS, which is predominantly detected via TLR4 (19,20). As P3 did, neither P1 nor P2 produced TNFα in response to LPS (Fig. 1A) (17). In contrast, P1 and P2 had a normal IL-10 response to stimulation with TNFα (Fig. 1B). We also primed polymorphonuclear neutrophils with low doses of TNFα or LPS then stimulated these cells with formyl-methionyl-leucyl-phenylalanine (fMLP), which resulted in the production of H2O2, which converts intracellular 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) to DCF. Priming with TNFα, but not with LPS, was effective in P1 and P2 (Fig. 1C). We then measured IL-6 secretion by monocytes stimulated with IL-1β; neither P1 nor P2 responded to IL-1β (Fig. 1D). We also measured interferon-γ (IFN-γ) secretion by lymphocytes after stimulation by IL-12, alone or in combination with IL-1β or IL-18, both of which up-regulate production of the β2 subunit of the IL-12 receptor. Neither P1 nor P2 responded to IL-12 plus IL-1β or IL-18 (Fig. 1E). Thus, a variety of blood cells from our patients responded normally to TNFα, but not to LPS or IL-1/IL-18, consistent with our hypothesis that TIR signaling was impaired in these individuals.

Figure 1

Impaired responses of blood cells to LPS, IL-1β, and IL-18. (A to E) are representative of two independent experiments each with cells from a healthy control (labeled C), and patients P1 and P2. (A) TNFα production in the supernatants of cultured whole blood cells stimulated for 24 hours with LPS (10 μg/ml) or phorbol 12-myristate 13-acetate (PMA)–ionomycin (10−7 M and 10−5 M), as measured by enzyme-linked immunosorbent assay (ELISA). (B) IL-10 production in the supernatants of cultured whole blood cells treated with TNFα (20 ng/ml), as measured by ELISA. (C) DCF production in polymorphonuclear neutrophils first treated with LPS (5 μg/ml), TNFα (500 U/ml), or phosphate-buffered saline (PBS) for 30 min, and then with fMLP (10−6 M) or PBS for 5 min, as determined by fluorescence-activated cell sorting (FACS) analysis. The results are expressed as a stimulation index, the ratio of the mean fluorescence intensity of stimulated cells to that of unstimulated cells. (D) IL-6 production in the supernatants of cultured whole blood cells treated with IL-1β (50 ng/ml) or PMA-ionomycin (10−7 M and 10−5 M), as measured by ELISA. (E) IFN-γ production in the supernatants of cultured whole blood cells treated with IL-12 (20 ng/ml), IL-12 + IL-1β (50 ng/ml), IL-12 + IL-18 (50 ng/ml), or PMA-ionomycin (10−7 M and 10−5 M), as measured by ELISA.

We then investigated the patients' responses to a broad set of TLR ligands (20). Whole blood from P1 and P2 displayed no secretion of TNFα (Fig. 2A), IL-1β, IL-12p40, or IL-6 (21), in response to any of the TLR1-6 ligands tested. Peripheral blood mononuclear cells from P1 and P2 did not secrete IL-6 in response to TLR9 ligand (Fig. 2B). We further investigated the impact of impaired TLR signaling on the recognition of whole microorganisms by stimulating the patients' blood cells with three bacteria: S. aureus, Escherichia coli andMycobacterium tuberculosis. Levels of production of TNFα (Fig. 2C), IL-1β, IL-12p40, and IL-6 (21) in the two patients were considerably lower than those in a healthy control. Overall, our patients failed to respond to the stimulation of at least five TLRs (TLR2, TLR3, TLR4, TLR5, TLR9) and two IL-1Rs (IL-1R and IL-18R), and they had a poor response to whole bacteria. This impaired response to several TLRs and IL-1Rs, but not to the TNFα receptor, suggested that the patients had a defect in the common TIR signaling pathway upstream from TRAF-6 and downstream from individual TIR membrane receptors.

Figure 2

Impaired responses of blood cells to TLR ligands and bacterial stimuli. (A to C) are representative of two independent experiments with cells from a healthy control (labeled C) and patients P1 and P2. (A) TNFα production in the supernatants of cultured whole blood cells stimulated for 24 hours with TLR ligands PAM3CSK4, PAM2CSK4, zymosan, PGN, LTA, Poly(I:C), LPS, and flagellin (see methods for the respective concentra- tions), as measured by ELISA. (B) IL-6 production in the supernatants of peripheral blood mononuclear cells stimulated for 24 hours with CpG DNA, as measured by ELISA. (C) TNFα production in the supernatants of cultured whole blood cells treated with several bacterial stimuli (heat-killedS. aureus, E. coli, and H37Rv M. tuberculosis) or PMA-ionomycin, as measured by ELISA.

No mutations were found in the coding regions of MYD88,TIRAP, IRAK1, IRAK2, andIRAKM in the patients (20). P1 had a homozygous deletion of thymidine 821 in exon 7 of IRAK4 (designated 821delT), resulting in a premature stop codon at position 287 (Fig. 3, A and B). P2 and P3 had a homozygous substitution of thymidine for cytidine at position 877 in exon 8. This resulted in the replacement of a glutamine residue (codon CAG) at amino acid 293 by a stop codon (TAG) (designated Q293X). These two mutations were not found in 60 healthy controls. Both parents of P1 were heterozygous for the corresponding mutant allele, whereas only the mother of P2 was heterozygous, and the parents of P3 could not be investigated (17). P2 had two copies of the maternal mutant IRAK4 gene, owing to segmental uniparental isodisomy (21). The healthy siblings tested were heterozygous or homozygous for the wild-type allele. Full-length IRAK4 mRNAs were detectable in a lymphoblastoid B cell line derived from a healthy individual (positive control, C), but not in cells from P1, P2, or P3, by Northern blotting (Fig. 3C) and cDNA-PCR (21), presumably because of nonsense-mediated mRNA degradation. No IRAK-4 protein was detected by Western blotting in cell lines derived from the three patients (Fig. 3D). These data suggested that our three patients had complete recessive IRAK-4 deficiency.

Figure 3

Inherited IRAK-4 deficiency. (A) Schematic representation of the IRAK-4 protein, with the death domain (DD) and kinase domain (KD). The positions of the two mutations found in the three IRAK-4–deficient patients are also indicated: 821delT in P1 (A.II.4) and Q293X in P2 (B.II.2), and P3 (C.II.1). (B) Pedigrees of the three families. Each kindred is identified by a capital letter (A to C), each generation by a roman numeral (I or II), and each individual by an arabic numeral (from left to right). Patients with clinical infectious diseases, homozygous for IRAK4mutations, are shown in black. An arrow indicates probands. Individuals for whom genetic analysis was not possible are indicated by “E?”. (C) IRAK4 and GAPDH mRNAs in lymphoblastoid B cells from a healthy positive control (labeled C), and patients P1, P2, and P3, as detected by Northern blotting. (D) IRAK-4 and STAT2 proteins in lymphoblastoid B cells from a control and patients, as detected by Western blotting.

Fibroblast lines from the patients and a healthy individual (positive control, C) were stimulated with IL-1β or TNFα (20). We first showed by Western blotting that the degradation of IRAK-1 after IL-1β stimulation was impaired in P1 and P2, consistent with the function of IRAK-4 as an IRAK-1–kinase (Fig. 4A) (14). We then investigated the pathways downstream from TRAF-6. In P1, P2, and P3, IκBα degradation [Fig. 4B (21)] and the DNA binding activity of NF-κB complexes [Fig. 4C (21)] in response to IL-1β were completely abolished, whereas the response to TNFα was unaffected. TIR-containing receptors also normally activate MAPKs, such as p38-MAPK, which was activated in P1 and P2 fibroblasts stimulated with TNFα, but not with IL-1β (Fig. 4D). Fibroblasts from P1, P2, and P3 also produced IL-6 in response to TNFα, but not in response to IL-1β [Fig. 4E (21)]. Transfection of fibroblasts from P1 and P2 with various numbers of wild-typeIRAK4 copies rescued the defects in a dose-dependent manner, restoring IRAK-4 expression (21) and IL-6 response to IL-1β [Fig. 4F; (21)]. IRAK-4 deficiency and impaired IL-6 response to IL-1β in our patients thus result from the inheritance of two loss-of-function IRAK4 alleles. Thus, the patients' fibroblasts behaved similarly to their blood cells, with defective IL-1β responses and maintained TNFα responses, as shown by activation of both NF-κB and p38-MAPK.

Figure 4

Impaired responses of fibroblasts to IL-1β. The healthy control is labeled “C.” (A) IRAK-1 and p38-MAPK in SV40-transformed fibroblast lines from a control and patients P1 and P2, unstimulated or stimulated with IL-1β (10 ng/ml) for 15 min, as detected by Western blotting. (B) IκBα and STAT2 in SV40-transformed fibroblast lines from a control and P1, unstimulated or stimulated with TNFα (20 ng/ml) or IL-1β (10 ng/ml) for various periods of time (min), as detected by Western blotting. (C) DNA binding activity of nuclear extracts from SV40-transformed fibroblast lines from a control, P1, and P2, unstimulated or stimulated with TNFα (20 ng/ml) or IL-1β (10 ng/ml) for 20 min, as detected by electrophoretic mobility shift assay, using a κB DNA probe. “E” indicates an excess of unlabeled probe. (D) Total and phosphorylated p38-MAPK in SV40-transformed fibroblast lines from a control, P1, and P2, unstimulated or stimulated with TNFα (20 ng/ml) or IL-1β (10 ng/ml) for 20 min, as detected by Western blotting. (E) IL-6 secretion by SV40-transformed fibroblast lines from a control, P1, and P2, unstimulated or stimulated with TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 18 hours, as measured by ELISA. (F) IL-6 secretion by SV40-transformed fibroblast lines from a control nontransfected, P1 and P2 either transfected with an insertless vector (mock) or a vector containing the wild-type IRAK4 gene, unstimulated or stimulated with TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 18 hours, as measured by ELISA.

We report the first characterization of human patients with inherited IRAK-4 deficiency and functional defects in the common TIR-IRAK signaling pathway. These patients do not respond to IL-1β, IL-18, or any of the TLR1–6 or 9 ligands tested, as assessed by activation of NF-κB and p38-MAPK, and induction of IL-1β, IL-6, IL-12, TNFα, and IFN-γ. We have not yet investigated their responses to TLR7 stimulation, or to other TIR receptors with unknown ligands (1), or their induction of IFN-β in response to TLR3–4 stimulation (22, 23). The cellular phenotype of IRAK-4–deficient patients differs from that of previously reported patients with anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID), who carry hypomorphic NEMO/IKKγ mutations (24–26). The clinical phenotypes of the two disorders overlap, but are not identical: like NEMO-deficient patients, IRAK-4–deficient patients suffer from infections caused by pyogenic bacteria, with minimal inflammatory responses; unlike patients with EDA-ID, patients with IRAK-4 deficiency present no overt developmental defect and are resistant to other ubiquitous microorganisms, such as Mycobacterium avium andPneumocystis carinii (25).

The narrow spectrum of infections in IRAK-4–deficient children is surprising, because TLRs, IL-1, and IL-18 were all thought to play crucial roles in defense against a wide range of microorganisms (1–5) (for additional references, see supporting online material). Mice with defects in the common TIR signaling pathway or in individual TIR receptors show broad susceptibility (references in supporting online material). Like the patients, mice deficient for IL-1R, IL-18, TLR2, TLR4, MyD88, or IRAK-4 are susceptible to S. pneumoniae and S. aureus. Such mice are also susceptible to various other microorganisms. In contrast, IRAK-4–deficient patients are resistant to most ubiquitous microorganisms, including viruses, fungi, and parasites, as well as a number of bacteria. Exposed to the same microbial flora, patients with an impaired response to IL-12 or IFN-γ (27, 28) are susceptible to Mycobacteria and Salmonella, and patients with an impaired response to IFN-α/β (28,29) are susceptible to viral infections. TIR adapters other than MyD88 and TIRAP, TRIF/TICAM-1 (22, 23) for example, may connect TIR receptors with kinases other than IRAK-4, thereby controlling microorganisms other than pyogenic bacteria. Nevertheless, our study of IRAK-4–deficient patients shows that protective immunity to most infectious agents is conferred without the action of IL-1R-, IL-18R, and TLR to induce key cytokines such as IL-1β, IL-6, IL-12, TNFα, and IFN-γ. The human TIR-IRAK signaling pathway confers specific protection against pyogenic bacteria.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1081902/DC1

SOM Text

Materials and Methods

References

  • * To whom correspondence should be addressed. E-mail: casanova{at}necker.fr

REFERENCES AND NOTES

View Abstract

Navigate This Article