Naturally-occurring genetic mutations in humans, causing rare extreme immunodeficiency phenotypes, present powerful opportunities to determine the relationship between specific defects and human disease processes in vivo and to study the underlying mechanisms involved in primary human cells.
Here we present novel findings, using primary cells (PBMC, dermal fibroblasts, MDDC) from a child with autosomal recessive homozygous Q293X IRAK-4 mutation, to demonstrate that IRAK-4-dependent mechanisms control innate immune responses at both transcriptional and post-transcriptional levels, with IRAK-4-deficiency differentially affecting NF-κB activation and cytokine production. We demonstrate that Q293X IRAK-4 mutant primary cells displayed NF-κB activation with functional MyD88-independent pathways, but defective MAPK activation. We report cell type-specific and stimulus-specific signalling dysfunction in IRAK-4-deficiency, including demonstration of IRAK-4-dependent LPS responses in primary dermal fibroblasts. We also describe the first definitive documentation of fatality from pneumococcal disease attributable to autosomal recessive homozygous Q293X IRAK-4 mutation in a sibling of our patient. Our data suggest that this mutation is likely to be an unrecognized primary immunodeficiency in apparently healthy children succumbing to lethal pyogenic bacterial infections. We offer important considerations for diagnostic approaches, and provide key insights into the mechanisms underlying innate immune dysfunction in IRAK-4-deficiency and the normal role of this protein.
Primary dermal fibroblasts from our patient displayed defective IκB-α phosphorylation and cytokine production in response to IL-1β, but intact TNF-α responses, consistent with observations from immortalised fibroblast lines from other homozygous Q293X patients (10
). These data indicate that IRAK-4 is not required for TNF-α responses in fibroblasts, and suggest a simple signalling block upstream of TRAF6 inhibiting IL-1β responses. However, assessment of LPS-induced responses introduces further complexity. Conflicting published literature concerning the potential for LPS-responsiveness in fibroblasts (24
) may reflect heterogeneous fibroblast subsets (25
), differences between primary and immortalised cells (26
), and potential LPS contaminants. Primary human dermal fibroblasts have not been extensively characterised, although reports indicate potential for LPS-responsiveness (25
). We demonstrated expression of CD14 and TLR4, with IκB-α phosphorylation and the production of IL-6 and IL-8 in response to ultra-pure LPS in our adult and child control primary dermal fibroblasts. In contrast, although our patient’s primary dermal fibroblasts clearly demonstrated intact I-κBα phosphorylation () and transcription of IL-6
-8 (), these IRAK-4-deficient cells demonstrated a profound failure to produce IL-6 and IL-8 in response to LPS. These data demonstrate a functional LPS-activation pathway in both wild-type and IRAK-4-deficient dermal fibroblasts and likely confirm TLR4 signalling by well characterised MyD88-independent pathways (2
), which we demonstrated are intact in our patient’s PBMC and MDDC, and which would not require functional IRAK-4 for LPS-stimulated NF-κB activation. However, critical to these studies, our patient’s primary dermal fibroblasts and MDDC demonstrated a failure of cytokine production in response to LPS, despite NF-κB signalling and cytokine transcription, and activation, respectively. These data indicate that MyD88-independent signalling is insufficient to generate the normal inflammatory response in these cells, and that even in the presence of NF-κB activation, functional IRAK-4 is required for post-transcriptional processing and cytokine production in these cells.
Intriguingly, our data demonstrate cell-type specificity when comparing the defective responses secondary to IRAK-4 Q293X mutation in primary dermal fibroblasts as compared to PBMC. The molecular basis for these observations remains unclear. However, recent studies have demonstrated key signalling differences between myeloid and non-myeloid cells, with differential use of adaptor molecules, and demonstrated MyD88-dependent LPS responses in synovial fibroblasts that may not utilise TLR4 (26
). These observations suggest that the precise role for IRAK-4 in different cell types may vary, and introduce a complexity that may be critical to understanding the clinical consequences of IRAK-4-deficiency.
In our patient’s PBMC, neither LPS, IL-1β, or TNF-α induced production of IL-6. However, LPS induced NF-κB translocation and IL-8 transcription, but induced 10 fold less IL-8 protein expression than control cells, reiterating the likely significance of IRAK-4 dependent post-transcriptional mechanisms, as observed in the dermal fibroblasts. Nevertheless, the induction of IL-8 protein production (albeit a significantly reduced level) contrasted with the absolute deficiency demonstrated in the patient’s primary dermal fibroblasts. Our recent quantitative RT-PCR studies on purified monocytes from this patient demonstrate a more severe defect in LPS-induced IL-8 transcription in these cells (albeit retaining a low level transcriptional response), when compared to the PBMC (K. L. Brown et al., manuscript in preparation). Future studies will be required to determine the relative significance of responses of each the different cell types present in PBMC preparations, and the possible significance of interaction between these cells.
IL-1β stimulation of our patient’s PBMC was surprisingly able to repeatedly induce low level NF-κB translocation and stimulate a diminished level of IL-8
(but not IL-6
) transcription, but was unable to induce IL-8 protein production. IRAK-4 has been demonstrated to be essential for IL-1R-stimulated NF-κB activation (9
). Thus, these data might represent IL-1β-induced transcription of IL-8
by an undetermined, primarily NF-κB-independent mechanism in our patient’s PBMC (but not dermal fibroblasts), or partial substitution of IRAK-4 function in NF-κB signalling by alternative molecules or a truncated translation product of IRAK-4
. Indeed IRAK-4 is capable of transmitting signals both dependent on and independent of its kinase activity (28
), and truncated IRAK-4 protein has been shown to retain DD interactions (29
). Chain-termination mutations resulting in mRNAs that contain premature stop-codons rarely produce truncated proteins, as a consequence of nonsense-mediated mRNA decay (22
). However, although it could simply reflect an “overwhelming” of the nonsense-mediated mRNA decay system, a truncated IRAK-4 protein has been detected in transfected cells over-expressing IRAK-4
with the C877T mutation, (29
), raising the possibility of its expression in patient cells. Additionally, translation of the alternatively spliced IRAK-4
transcript described would produce a protein unaffected by the Q293X mutation, with a disrupted kinase region, but intact DD and undetermined domain (UD). While low level expression in IRAK-4 competent cells might have no effect, this could be significant in the absence of full length IRAK-4. Thus, although truncated IRAK-4 proteins have not been detected in patient cells, we cannot exclude the possibility that low level expression could influence IL-1β signalling in IRAK-4-deficient PBMC.
Our data demonstrate that the innate immune dysfunction in IRAK-4-deficiency is both stimulus-specific, and determined differentially for specific cytokines. This is consistent with near-complete deficiency in LPS-induced TNF-α production (10
), and profound transcriptional defect in GM-CSF
, but partial defect in Cox-2
) in PBMC from other IRAK-4-deficient individuals. In addition, this is consistent with defective LPS-induced IL-6
transcription, but post-transcriptional defects in IL-8, TNF-α, and IL-12p35 observed in our patient’s MDM (15
). These data also indicate a critical role for IRAK-4 in both transcriptional and post-transcriptional control of cytokine production, even in the presence of intact NF-κB signalling. In this regard, the severe defects in our patient’s MAPK responses may be highly significant.
The MAPK p38 can affect transcription (30
) via chromatin restructuring (5
) or transactivation of p65 (8
). However, the core transcriptional regulator for IL-8
has an NF-κB element required for activation in all cell types studied, with additional transcription factors required for maximal expression being largely dispensable (31
). Thus, IL-8
transcription was expected where NF-κB activation was observed, regardless of other factors. This was clearly evident in our patient’s cells. However, translocation of NF-κB subunits in addition to p65 remains to be assessed. Critically however, activation of p38 by MKK-3, -4 and -6 also has an essential post-transcriptional function in the stabilisation of mRNAs with 3′ AU rich elements, including IL-8
), and p38 inhibition can have profound post-transcriptional effects without affecting transcription rates (7
). In our patient’s PBMC, p38 activation was undetectable in response to IL-1β, and substantially diminished in response to LPS, correlating with the defect in production of IL-8 protein. This suggests a critical role for p38-dependent post-transcriptional mechanisms in IL-8 production, defective in our patient as a consequence of IRAK-4
transcription is additionally affected by the p38-activated CHOP (30
) via inhibition of negative transcriptional regulators (34
). Furthermore, inhibition of Jun N-terminal kinase (JNK), a MAPK activated downstream of TAK-1 by MAPK Kinase (MKK)-4, -7, inhibited IL-6
transcription through failure of an undefined interaction between JNK pathways and other signalling pathways such as NF-κB (35
). Our patient’s PBMC demonstrated a complete absence of JNK activation in response to LPS and IL-1β, correlating with defective IL-6
transcription, suggesting a critical role for IRAK-4 in IL-6
transcription, through activation of JNK.
In addition to TLR/IL1-R signalling defects, significantly impaired responses to TNF-α were also observed in IRAK-4-deficient PBMC. In contrast, no significant defect was observed in the TNF-α-induced cytokine response in IRAK-4-deficient dermal fibroblasts (despite slightly diminished TNF-α-induced phosphorylation of I-κBα). These data suggest an as yet undetermined role for IRAK-4 in TNF-α signalling, and cell type-specific differences in the TNF-R signalling pathways in PBMC as compared to fibroblasts. In addition, our data revealed more substantial impairment in the production of IL-6 in comparison to IL-8, in TNF-α stimulated IRAK-4-deficient PBMC. Although TNF-α-induced IL-6 responses in PBMC can vary significantly between individuals, our patient was the only complete non-responder we observed, in contrast to ten normal controls with significant IL-6 responses (all p<0.05). Interestingly, defective TNF-α-induced activation of MAPK was demonstrated in our patient’s PBMC, with JNK activation more notably deficient than p38, correlating with more substantial impairment of IL-6 protein production in comparison to IL-8. In addition, low level transcription of IL-6
was observed in response to TNF-α, the only stimulus to which any JNK activation was observed. Although IRAK-4 is upstream of the proposed convergence of TLR/IL-1R and TNF-R signalling pathways at TRAF6 (36
), a role for IRAK-1 has been demonstrated in TNF-R signalling (37
) and in the absence of MyD88 function MDM (but not fibroblasts) have been shown to have impaired cytokine production in response to TNF-α, despite intact NF-κB activation (26
). Our data suggest a critical role for IRAK-4 in the activation of MAPK downstream of TNF-R activation in PBMC.
We propose that dysfunctional accessory pro-inflammatory signals, rather than NF-κB activation alone, underpins the cellular phenotype in this patient. TLR/IL-1R stimulation of NF-κB via activated TAK-1 is relatively well characterised. Hyperphosphorylation of IRAK-1 by activated IRAK-4, results in Pellino-IRAK-4-IRAK-1-TRAF6 complex (Complex 1) formation and receptor release. Complex 1 interacts with membrane-bound TAK-1-TAB-1-TAB-2, with resultant cytosolic translocation of TRAF6-TAK-1-TAB-1-TAB-2, and TAK-1 activation of IKK complex (38
). In addition to NF-κB pathways, TAK-1 also activates MAPK via MKK (3
). However, despite the common requirement for TRAF6 and TAK-1, divergence of these pathways occurs at, or perhaps upstream of IRAK-1 (40
), with the relative overlap of proximal signalling components unclear. Different IRAK-1 regions mediate TRAF6 and TAB-2 translocation (41
), and despite inability to interact with TRAF6, the UD of IRAK-1 is sufficient (but not required) for activation of JNK, but not NF-κB (42
). This suggests that IRAK-1 mediates interaction of other undefined signalling components with TRAF6 to activate JNK signalling. Thus, Complex 1 dysregulation in IRAK-4-deficient individuals could disrupt MAPK pathways independently from intact TAK-1/IKK/NF-κB signalling, as observed in our patient’s cells. Interestingly, we have observed IRAK-1 kinase function perturbation in our patient’s neutrophils, with high baseline activity diminishing after LPS exposure, in contrast to LPS-enhanced kinase activity in controls (data not shown), consistent with findings in an IRAK-4 compound heterozygote (11
). In addition, our patient’s PBMC cultured in M-CSF generated multinucleated giant cells, positive for tartrate-resistant acid phosphatase, in addition to MDM (data not shown). This is suggestive of osteoclastic differentiation, a process normally requiring additional RANKL stimulation, acting via TRAF6 signalling, and also suggesting dysregulated Complex 1 function (43
). Intriguingly, both this patient and his deceased IRAK-4
Q293X homozygote sibling were above 95th
percentile for height, in contrast to their heterozygotic siblings (between 50 and 75th
percentiles for age). Thus, we propose that dysfunctional Complex 1 activity in response to TLR/IL-1R activation, resulting in defective MAPK activation, underpins the innate immunodeficient phenotype in IRAK-4 deficient primary cells.
Ongoing studies utilising cells from IRAK-4-deficient individuals are expected to further elucidate the precise nature of the selective signalling defect downstream of Complex 1, and the specific nature of the susceptibility to infection in IRAK-4–deficient individuals. It is interesting to note the relative selectivity of infecting organisms in untreated IRAK-4–deficient individuals, particularly prior to diagnosis, with an apparent particular susceptibility to S. pneumoniae
and to Gram positive organisms (although not exclusively)(45
). Prophylactic antibiotic therapy after diagnosis clearly precludes longitudinal characterisation of the range of infections which IRAK-4–deficient individuals might have suffered from if left untreated. Nevertheless, in the absence of antibiotics our patient and others were highly susceptible to infection, and in the case of our patient’s eldest brother, this condition was fatal. This clearly indicates the importance of TLR signalling to innate immunity, but the phenotype perhaps contrasts with the acute, broad-spectrum immunodeficiency that might have been predicted for a severe TLR signalling deficiency. The specific reasons for this remain uncertain. However, our new data indicates that the major IRAK-4
mutation described (Q293X) does not result in a complete block to TLR signalling, but rather results in cell type-specific, and ligand-specific defects, differentially affecting different cytokines at transcriptional and or post-transcriptional levels, and in which defects in MAPK pathways may be more significant than the largely intact NF-κB pathway. This suggests levels of complexity that may contribute to the relative selectivity in increased susceptibility to infection in these patients. It is also interesting to note that some older IRAK-4-deficient patients have been taken off prophylactic antibiotics and remained healthy (45
), suggesting the possibility that the immunodeficiency associated with IRAK-4 deficiency can be compensated for in those who survive childhood and may be relatively less critical in adult life.
In conclusion, we demonstrate that IRAK-4 mutation in primary human cells from a naturally-occurring, clinically-relevant human “model” of disease does not result in an absolute null phenotype for TLR/IL-1R signalling. Rather, it establishes dysfunctional cellular signalling with partially intact NF-κB pathways, but defective MAPK signalling and dysregulated Complex 1 function, affecting transcriptional and post-transcriptional control of TLR/IL-1R responses. Specificity of accessory pro-inflammatory pathways affected, combined with cytokine-specific mechanisms regulating expression could explain the complex cell type-specific, stimulus-dependent, and cytokine-specific defects observed and further illuminate the unusual pattern of disease susceptibility in IRAK-4–defective patients.