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Human adaptive immunity rescues an inborn error of innate immunity
Laura Israel1,2, Ying Wang3, Katarzyna Bulek4, Erika Della Mina1,2, Zhao Zhang3,
Vincent Pedergnagna1,2 Maya Chrabieh1,2, Nicole A. Lemmens5,
Vanessa Sancho-Shimizu1,2,6, Marc Descatoire2,7, Théo Lasseau1,2, Elisabeth Israelsson8,
Lazaro Lorenzo1,2, Ling Yun1,2, Aziz Belkadi1,2, Andrew Moran9, Leonard E. Weisman10,
François Vandenesh11,12, Frederic Batteux13, Sandra Weller7, Michael Levin6,
Jethro Herberg6, Avinash Abhyankar14, Carolina Prando14, Yuval Itan14,
Willem van Wamel5, Capucine Picard1,2,15,16, Laurent Abel1,2,14, Damien Chaussabel8,
Xiaoxia Li4, Bruce Beutler3, Peter D. Arkwright9,
Jean-Laurent Casanova1,2,14,15,17,# and Anne Puel1,2,14,#
1 Laboratory of Human Genetics of Infectious Diseases, Necker Branch, UMR 1163, Necker Medical School, Imagine Institute, Paris, France, EU 2 Paris Descartes University, Paris, France, EU 3Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA 4 Department of Immunology, Cleveland Clinic, Cleveland, OH 44195, USA 5 Medical Microbiology and Infectious Diseases, CN Rotterdam, The Netherlands, EU 6 Paediatric Infectious Diseases Group, Division of Medicine, Imperial College London, London, UK 7Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Sorbonne Paris Cité, Université Paris Descartes, Faculté de Médecine-Site Broussais, 75014 Paris, France, EU 8 Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA 9 University of Manchester, Royal Manchester Children’s Hospital, Manchester M13 9WL, UK 10 Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA 11 UMR U1111 INSERM Université de Lyon, Lyon, France, EU 12 Hospices Civils de Lyon, Groupement Hospitalier Est, Lyon, France, EU 13 EA 1833, Faculté de Médecine, Université Paris Descartes, Sorbonne Paris-Cité, Service d'Immunologie Biologique, Hôpital Cochin, Paris, France, EU 14 St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, New York, USA 15 Pediatric Hematology-Immunology Unit, Assistance Publique Hôpitaux de Paris, Necker Hospital, Paris, France, EU 16 Study Center for Primary Immunodeficiencies, Assistance Publique Hôpitaux de Paris, Necker Hospital, Paris, France, EU 17 Howard Hughes Medical Institute, USA # Equal contributions Correspondence: JLC ([email protected]) or AP ([email protected])
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Summary
The molecular basis of incomplete penetrance of monogenic disorders is unclear. We
describe here eight related individuals with autosomal recessive TIRAP deficiency. The
proband had life-threatening staphylococcal disease in childhood, unlike the other seven
homozygotes. Responses to all TLR1/2, TLR2/6, and TLR4 agonists are impaired in the
fibroblasts and leukocytes of all TIRAP-deficient individuals. However, whole blood
response to TLR2/6 agonist staphylococcal lipoteichoic acid (LTA), is abolished only in the
index case, who is the only family member lacking LTA-specific Abs. This defective
response is reversed in the patient, but not in IRAK-4-deficient individuals, by the addition
of anti-LTA mAb. Moreover, anti-LTA mAb rescues the response of macrophages from
mice lacking TIRAP, but not TLR2 or MyD88. Acquired anti-LTA Abs therefore rescue
TLR2-dependent immunity to staphylococcal LTA in individuals with inherited TIRAP
deficiency, accounting for incomplete penetrance. Combined deficiency of TIRAP and anti-
LTA Abs underlies staphylococcal disease in the patient.
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Graphical abstract
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Introduction
Staphylococcus aureus is a Gram-positive bacterium that colonizes the skin and
nostrils of most healthy individuals (Li et al., 2015; Mulcahy and McLoughlin, 2016). It is
also a common cause of human diseases, ranging from minor cutaneous infections to
invasive hematogenous infections, as observed in children and young adults with
necrotizing staphylococcal pneumonitis (Gillet et al., 2002). Most severe staphylococcal
diseases remain unexplained. Various forms of acquired immunodeficiencies, including
infection with human immunodeficiency virus, hemodialysis, chemotherapy, intravenous
drug use, central catheters and vascular lines increase the likelihood of severe
staphylococcal disease (Lowy, 1998). The pathogenesis of invasive staphylococcal disease
is complex in such settings, often involving neutropenia, skin breaches, or both.
Alternatively, severe staphylococcal disease may result from various single-gene inborn
errors of immunity, including disorders of phagocytes in particular (Notarangelo, 2010;
Picard et al., 2015; Zhang et al., 2015).These disorders include disorders of the TLR and
IL-1R pathway, inborn errors of NF-κB, severe congenital neutropenia, chronic
granulomatous disease, leukocyte adhesion deficiency, and autosomal dominant hyper IgE
syndrome (Bousfiha et al., 2015; Minegishi et al., 2007; Picard et al., 2015). All these
defects affect phagocytes, albeit in different ways. The clinical features differ between
patients with staphylococcal diseases, reflecting the diversity of the underlying inborn
errors.
Autosomal recessive MyD88 and IRAK-4 deficiencies selectively impair signaling
via the TLR and IL-1R pathway (Picard et al., 2003; von Bernuth et al., 2008). More than
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80 patients worldwide have been diagnosed (reviewed in (Picard et al., 2010)). These
patients are susceptible to invasive bacterial infections, particularly those caused by
Streptococcus pneumoniae, but also those caused by S. aureus. They also develop
superficial staphylococcal infections (Picard et al., 2011). Leukocytes from patients with
deficiencies of IRAK-4 or MyD88 do not respond to activation via IL-1Rs or TLRs other
than TLR3, which signals via TRIF but not MyD88, and TLR4, which signals via TRIF or
MyD88 (Picard et al., 2003; Picard et al., 2010; von Bernuth et al., 2008). The molecular
and cellular basis of staphylococcal disease in these patients remains unknown. We
investigated this aspect, by testing the hypothesis that severe staphylococcal disease in
otherwise healthy children may be caused by mutations in other genes of the TLR and IL-
1R pathway (Casanova, 2015b)
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Results
Homozygous TIRAP R121W mutation in a child with severe staphylococcal
infection
We investigated a nine-year-old girl admitted to the intensive care unit with
pneumonia and sepsis caused by Panton Valentine Leukocidin (PVL)-producing S. aureus,
at the age of three months (P1, V.1, Figure 1A). She was born to consanguineous parents of
Pakistani descent living in the United Kingdom. The presumed complete genetic tree was
assessed by inferring relationships with King software based on genome-wide SNP
genotyping (Manichaikul et al., 2010) (Figure 1A and Supplementary Figure S1). Known
genetic etiologies of PIDs associated with life-threatening staphylococcal diseases were
excluded by whole-exome sequencing (WES) and Sanger sequencing. We searched for rare
variants (MAF<1%), and identified a homozygous substitution, c.361C>T, in exon 5 of
TIRAP, which encodes a TIR domain-containing adapter of TLR2 and TLR4 (Fitzgerald et
al., 2001; Horng et al., 2001; Yamamoto et al., 2002a). We confirmed the TIRAP mutation
by Sanger sequencing of the P1’s genomic DNA extracted from whole blood cells (WBC),
Epstein-Barr virus-immortalized B lymphocytes (EBV-B cells), and SV40 immortalized
fibroblasts (SV40 fibroblasts) (Figure 1B). This missense variant results in the replacement
of the arginine residue in position 121 of the TIR domain of TIRAP with a tryptophan
residue (R121W) (Figure 1C). The R121 residue of TIRAP has been strongly conserved
throughout evolution (Figure 1D). It is also conserved in 21 of the 26 human molecules
identified to date containing a TIR domain, other than TIRAP (e.g. R196 in MyD88,
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Supplementary Figure S2). In silico analysis with CADD and MSC predicted that the
R121W variation would be deleterious (Itan et al., 2016; Kircher et al., 2014). The TIRAP
gene is well conserved in the human population, with a neutrality index of 0.2346
(McDonald and Kreitman, 1991) and a GDI of 5.46 (Itan et al., 2015). No other rare or
common variants of TIRAP were found in P1. No rare bi-allelic mutations were found in
other genes of the TIR pathway, including individual TLRs and the other three TLR
adaptors (TRAM, SARM and TRIF). Moreover, no other candidate variant was identified in
the linkage regions defined by parametric multipoint linkage analysis (methods section) or
among the other 107 non-synonymous rare homozygous variants found in P1
(Supplementary Table 1). The patient was therefore homozygous for a potentially
deleterious rare allele of TIRAP.
Homozygosity for the very rare R121W variant in seven healthy relatives
This variant was not detected in 140 Pakistani individuals from the CEPH-HGD
panel (1,050 individuals from 52 ethnic groups). Eight heterozygous, but no homozygous
individuals were reported among 57,085 individuals from the Exome Aggregation
Consortium (ExAc) database (MAF=0.00007). This variant was detected, in the
heterozygous state, in three of 3,033 individuals (6,066 chromosomes) in our in-house
exome database. This patient was therefore homozygous for a very rare TIRAP missense
allele, with a minor allele frequency (MAF) of less than 1/10,000. Surprisingly however,
seven relatives of P1 were also found to be homozygous for the R121W TIRAP mutation,
including the proband’s father (aged 33 years), four of the father’s siblings (aged 20 to 28
years) and both his parents (aged 53 and 54 years) (Figure 1A). None of these individuals
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carried TIRAP polymorphisms. The proband’s mother and younger sister were
heterozygous for the R121W allele. None of these relatives, whether heterozygous or
homozygous for the TIRAP allele, had ever suffered from any severe infection, including
staphylococcal infections in particular. Thus, eight individuals from a consanguineous
Pakistani kindred, including a child with severe staphylococcal infection and seven
asymptomatic adults, were homozygous for a very rare and probably deleterious TIRAP
allele. These data suggested that P1 had autosomal recessive TIRAP deficiency, with
incomplete penetrance for severe staphylococcal disease. We therefore investigated
whether the eight individuals had TIRAP deficiency, and the mechanisms underlying
incomplete clinical penetrance.
Normal mRNA and protein expression of the R121W TIRAP allele
Human TIRAP encodes two isoforms (Figure 1C) generated by four different
transcripts. Two transcripts (NM_001039661.1 and NM_001318777) encode isoform A,
which contains 221 amino acids (aa), with a molecular weight (MW) of 23.8 kDa, whereas
the other two transcripts (NM_001318776 and NM_148910) encode isoform B, which
contains 235 aa and has a MW of 25.3 kDa. The R121W mutation is located in exon 5 of
reference transcript NM_001039661.1 (encoding a sequence present in all isoforms). We
assessed the impact of the mutation, by inserting the WT and R121W alleles into
pCDNA3.1-V5 vectors encoding a C-terminally tagged version of each of the isoforms of
TIRAP. Following the transient transfection of HEK 293T cells, the R121W allele yielded
levels of both isoforms (A and B) about 50% lower than those obtained with the WT allele
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(Figure 1E). This suggested that both R121W TIRAP isoforms were somewhat less stable
than the WT isoforms, at least when tagged molecules were overproduced in HEK 293T
cells. We then evaluated TIRAP mRNA levels for both isoforms by qPCR and RT-PCR in
SV40 fibroblasts from P1, her father (R121W/R121W), and her mother (R121W/WT), and
by qPCR in EBV-B cells from P1, in PBMCs from P1 and her aunt (R121W/R121W)
(Figure 1F and Supplementary Figure S3 and S4). Both P1, her father, mother, and her aunt
had mRNA levels within the normal range of the controls, with transcripts encoding
isoform A being mostly expressed in the three cell types tested, and in all individuals
tested. We were unable to detect the endogenous expression of either of the two TIRAP
isoforms by western blotting (WB) in SV40 fibroblasts from healthy controls, whereas
TIRAP protein levels in control EBV-B cells varied considerably between individuals
correlating with mRNA levels variability in controls (data not shown, Figure 1F). In
PBMCs from healthy controls only a 24 kDa protein, probably corresponding to isoform A,
was detected, consistent with qPCR results (Figure 1G). Homozygous family members had
normal levels of this isoform of TIRAP in PBMCs (Figure 1G). Overall, these data
suggested that the R121W mutation had no detectable impact on mRNA or protein levels in
circulating leukocytes.
Impaired subcellular localization of the TIRAP R121W isoforms
We assessed the subcellular distribution of the mutant TIRAP molecules.
Fluorescence microscopy of HEK 293 T cells transiently transfected with a pcDNA3.1
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vector encoding the WT form of TIRAP V5-tagged and labeled with Alexa Fluor 488-
conjugated anti-V5 antibodies showed the WT TIRAP to be present in precise small dots in
the cytoplasm, whereas transfection with the R121W TIRAP construct resulted in diffuse
cytoplasmic staining (Figure 2A). For the quantification of this subcellular localization
phenotype, we cotransfected cells with constructs encoding V5-tagged WT TIRAP and
either myc-tagged WT or R121W TIRAP, and then assessed the colocalization of the
encoded proteins by determining Pearson’s correlation coefficient (PCC) (Figure 2B).
TIRAP WT had a PCC of 0.85 for colocalization with itself, whereas its PCC for
colocalization with R121W was 0.25 (p < 0.0001). These results indicated that the R121W
mutation impaired the subcellular localization of TIRAP. We then investigated whether this
cellular localization defect had an impact on the colocalization of TIRAP with its partner,
MyD88. We obtained a PCC of 0.6 for the colocalization of TIRAP WT and MyD88,
whereas the PCC for the colocalization of TIRAP R121W and MyD88 was 0.2 (Figure 2C)
(p < 0.0001). These data suggested that the R121W mutation impaired TIRAP trafficking
within cells, thereby reducing the accessibility of this molecule to MyD88.
Impaired interaction of the TIRAP R121W isoforms with TLR2 and MyD88
Following stimulation, TLR2 or TLR4 recruits TIRAP, which recruits MyD88 to
the receptor complex through TIR domain interactions (Horng et al., 2002; Horng et al.,
2001; Yamamoto et al., 2002a; Yamamoto et al., 2002b). The TIR domain interactions
between MyD88 and TLR2 or TLR4 have been characterized in structural studies (Nada et
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al., 2012; Ohnishi et al., 2009). Four residues of MyD88 have been identified as essential
for this interaction, including R196, which is mutated (R196C) in three MyD88-deficient
patients (Picard et al., 2010; von Bernuth et al., 2008) The R196 residue of MyD88
corresponded to the R121 residue of TIRAP (Supplementary Figure S2) (Valkov et al.,
2011). Like the R196C mutation of MyD88, the R121W mutation of TIRAP seems to have
at most a modest impact on protein levels (Figure 1G). The R121W mutation may impair
the interaction of TIRAP with MyD88, thereby preventing downstream signaling and the
activation of target gene transcription. Moreover, the R121 residue is part of a long AB
loop that mediates direct binding to the TIR domain of TLR4 (Lin et al., 2012). The
R121W mutation may, therefore, also impair TIRAP interaction with TLR2 and TLR4. We
tested these hypotheses, by first performing in vitro co-immunoprecipitation assays in HEK
293T cells transfected with the WT and R121W TIRAP isoforms A and B, together with
TLR2 or MyD88. Both WT TIRAP isoforms interacted with TLR2, whereas this interaction
was strongly impaired when HEK 293T cells were transfected with the R121W TIRAP
isoforms (Figure 2D). Similarly, both WT TIRAP isoforms interacted with MyD88,
whereas this interaction was strongly impaired when HEK 293T cells were transfected with
either R121W TIRAP isoform (Figure 2E). We also tested TLR2/MyD88 co-
immunoprecipitation after PAM2CSK4 stimulation, in fibroblasts from healthy controls,
TIRAP heterozygous P1’s mother, and TIRAP homozygous P1 and her father (Figure 2F).
TLR2 and MyD88 co-precipitated upon TLR2 stimulation in both control and R121W
heterozygous fibroblasts, but not in R121W homozygous fibroblasts, whereas MyD88-
mutated fibroblasts (L93P/R196C) displayed an intermediate pattern. These data suggested
that the R121W mutation disrupted the interaction of both TIRAP isoforms with their
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TLR2 (and presumably TLR4) and MyD88 partners. These data, therefore, suggested that
individuals homozygous for R121W had a complete functional deficiency of TIRAP.
The R121W TIRAP allele is loss-of-function
The transient transfection of SV40 fibroblasts from P1 and her father with a
pcDNA3 vector encoding WT TIRAP isoform A or B induced IL-6 production to levels
similar to those in transfected control cells (Figure 3A). Given this high background, the
increase observed in response to TLR2 or TLR4 stimulation was only modest, in both cell
types (Supplementary Figures S5, S6, S7). In contrast, transfection of these SV40
fibroblasts with constructs encoding either of the R121W TIRAP isoforms had no effect
(Figure 3A). Overexpression of the WT MyD88 allele in SV40 fibroblasts from the control,
P1, her father or a MyD88-deficient patient led to the constitutive activation of IL-6
production, but the overproduction of WT TIRAP in MyD88-deficient fibroblasts did not
induce IL-6 production. Moreover, stable transfection of fibroblasts from P1 or her father
with lentiviral particles encoding WT TIRAP isoform A or B, or both A and B, rescued
constitutive IL-6 production, whereas no such rescue was observed after stable transfection
with the R121W TIRAP construct or the empty vector (data not shown). Finally, the
overproduction of each TIRAP WT isoform in HEK293T cells, as already shown for the
TIRAP isoform A, induced constitutive activation of the NF-κB pathway and IL-6
production (Figure 3B and (Horng et al., 2001)), whereas no such activation was observed
following the overproduction of the TIRAP R121W isoforms (Figure 3B). Collectively,
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these data showed that the R121W TIRAP allele is loss-of-function, at least in terms of
constitutive and MyD88-dependent IL-6 induction following overexpression in fibroblasts.
Impaired responses via TLR2 and TLR4 in fibroblasts homozygous for R121W
TLR2 heterodimerizes with TLR1 for the recognition of triacylated lipopeptides (Jin
et al., 2007), which are present in most Gram-positive bacteria, including S. aureus
(Kurokawa et al., 2009), and with TLR6 for the recognition of diacylated lipopeptides, also
present in S. aureus (Buwitt-Beckmann et al., 2006). It also polymerizes with TLR6 and
CD36 for the recognition of staphylococcal lipoteichoic acid (LTA) from most Gram-
positive bacteria, including S. aureus (Jimenez-Dalmaroni et al., 2009), as reviewed by
(Zahringer et al., 2008). TLR4 recognizes LPS from Gram-negative bacteria. Macrophages
and dendritic cells from TIRAP-deficient mice display abolished responses to the
stimulation of TLR1/2, TLR2/6, and TLR4, and splenocyte proliferation in response to LPS
is impaired (Horng et al., 2002; Yamamoto et al., 2002a). Human SV40 fibroblasts express
TLR2 at their surface (data not shown). We thus assessed their production of cytokines
upon stimulation with TLR2 and TLR4 agonists (PAM2CSK4 and FSL-1, two TLR2/6
synthetic agonists; purified LTA from S. aureus, another TLR2/6 agonist; PAM3CSK4, a
synthetic TLR1/2 agonist; LPS and synthetic lipid A, two TLR4 agonists). We detected
cytokine production upon stimulation by all these agonists. The response to PAM2CSK4,
was both TLR2- and TIRAP-dependent, as demonstrated by the inhibition of IL-6 induction
by siRNAs specific for TLR2 and TIRAP (Supplementary Figure S8), respectively, but not
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by scrambled siRNA. The responses to LPS and lipid A were also TLR4- and TIRAP-
dependent (data not shown). No or impaired IL-6 induction was observed in response to
PAM2CSK4, LTA, FSL-1, PAM3CSK4, LPS, lipid A, and heat-killed S. aureus (HKSA), in
SV40 fibroblasts from P1 and her father (both R121W/R121W), or in IRAK-4- and
NEMO-deficient cells (Figure 3C). However, unlike IRAK-4- and NEMO-deficient cells,
SV40 fibroblasts from P1 and her father responded normally to IL-1β. All SV40 fibroblasts
other than NEMO-deficient cells tested responded to TNF-α and poly(I:C) (a TLR3 agonist
in human fibroblasts) (Zhang et al., 2007). These data suggested that homozygosity for the
R121W allele abolished TLR1/2, TLR2/6 and TLR4 responses in human fibroblasts,
consistent with the findings reported for TIRAP-deficient mouse macrophages and
dendritic cells (Horng et al., 2002; Yamamoto et al., 2002a).
Impaired responses of phagocytes via TLR2 and TLR4 in TIRAP R121W
homozygotes
We investigated P1’s leukocyte responses to TLR2 and TLR4 agonists. CD62L (L-
selectin) is a transmembrane protein cleaved by the metalloproteinase TACE upon TLR
stimulation (Peschon et al., 1998). CD62L shedding in response to the stimulation of all
TLRs other than TLR3 is abolished in granulocytes from IRAK4- and MyD88-deficient
patients (von Bernuth et al., 2006). We investigated the impact of the R121W mutation on
CD62L shedding in six family members homozygous for the R121W mutation, after the
stimulation of granulocytes for 1hr with four TLR2 agonists (PAM2CSK4, FSL-1, LTA,
and PAM3CSK4) and one TLR4 agonist (LPS). The responses were compared with those of
granulocytes from two healthy controls and P1’s heterozygous mother. The six
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R121W/R121W individuals tested displayed impaired CD62L shedding in response to
stimulation with all TLR2 and TLR4 agonists tested, as demonstrated by comparison with
healthy controls, but their response to PMA was normal (Figure 3D). In addition, the
stimulation of PBMCs with various doses of PAM2CSK4, FSL-1, LTA, PAM3CSK4, and
LPS for 3hrs resulted in the abolition of intracellular labeling for IL-6 and TNF-α in CD14+
cells, particularly at low agonist concentrations (Figure 3E and Supplementary Figure S9).
Similarly, transcriptome analysis after the stimulation of whole blood for 2hrs suggested
that the induction of gene expression in response to PAM2CSK4, FSL-1, or LTA was
abolished and that the induction of gene expression in response to PAM3CSK4 or LPS was
impaired but not abolished (Supplementary Figure S10). Collectively, these data suggest
that granulocytes from TIRAP R121W homozygotes did not respond to the stimulation of
TLR1/2, TLR2/6, and TLR4, whereas their monocytes did not respond to TLR2/6 and
responded poorly to TLR1/2 and TLR4.
Residual responses to high concentrations of TLR agonists in TIRAP R121W
homozygous monocytes
After 3hrs of stimulation, the responses of monocytes from R121W homozygous
PBMCs to low doses (10 ng/ml) of PAM3CSK4 were abolished, but the levels of production
of both IL-6 and TNF-α in response to agonist stimulation at concentrations above 1 µg/ml
were similar to those of healthy controls, suggesting that there were TIRAP-independent
responses to high concentrations of PAM3CSK4 and, possibly, other TLR2 agonists, at least
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in some cell types (Figure 3E and Supplementary Figure S9). An effect of contaminants is
unlikely, as the agonist used was synthetic. Moreover, low levels of TNF-α induction were
observed in response to LPS stimulation in CD14+ cells from the R121W PBMCs tested.
After 48 hrs of stimulation, IL-6 production by the PBMCs of all homozygous individuals
tested in response to PAM3CSK4 (1 µg/ml) was normal, whereas it was profoundly
impaired but not abolished in response to LPS (1 ng/ml) and lipid A (1µg/ml); responses to
PAM2SCK4 (1 µg/ml), FSL-1 (100 ng/ml), and LTA (1 µg/ml) were abolished (Figure 4A
and Supplementary Figure S11). These results confirmed that the R121W allele has a
deleterious effect on TLR2 and TLR4 responses in leukocytes. However, the normal
induction of IL-6 production observed in R121W/R121W monocytes in response to
TLR1/2 stimulation with PAM3CSK4, at late time points and high concentrations of agonist
(and, to a lesser extent, in response to stimulation with LPS and lipid A) further suggests
that TIRAP may be redundant in such circumstances, as we ruled out the possibility of the
TIRAP mutant protein having residual activity. Alternatively, PAM3CSK4 and LPS/lipid A
may also be recognized independently of TLR2 and TLR4, respectively. In the absence of
TLR2- or TLR4-deficient subjects, given that the R121W TIRAP allele is loss-of-function,
we can conclude that these late responses to high concentrations of TLR agonists were
probably either TIRAP- or TLR-independent.
Impaired whole-blood response to LTA and lack of anti-LTA Abs in P1
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In all individuals homozygous for the TIRAP R121W mutation tested, IL-6
production by whole blood in response to 48 hrs of stimulation of TLR2/6 with PAM2CSK4
and FLS-1 was impaired, whereas normal levels of IL-6 were produced in response to
stimulation of TLR1/2 with PAM3CSK4 and TLR4 with LPS and lipid A (Figure 4B and
Supplementary Figure S11). However, the induction of IL-6 via TLR2/6 in response to
LTA was reproducibly abolished in the whole blood of P1, whereas a normal response was
observed for the other TIRAP R121W homozygotes tested (Figure 4B). We investigated
this striking difference in cellular phenotype, which we considered to be of potential
clinical relevance as only P1 suffered from staphylococcal disease. The leukocyte response
to LTA response is mediated principally by TLR2/6 heterodimers acting in concert with
CD36 (Jimenez-Dalmaroni et al., 2009), but anti-LTA Abs have been shown to enhance
this response through the recognition of their invariant IgG domain by CD32 (Bunk et al.,
2010). The response to LTA was impaired in PBMCs, granulocytes, and monocytes from
all R121W homozygotes (including P1), but normal in the whole blood of all these
individuals other than P1. We therefore used enzyme-linked immunosorbent assays
(ELISA) to test for the presence of circulating anti-LTA Abs in the plasma of individuals
from the kindred of P1. P1 was the only member of this kindred for whom anti-LTA IgG
Abs were reproducibly undetectable, in 10 plasma samples taken between the ages of four
months and nine years. No maternal anti-LTA antibodies were thus detected in the plasma
of P1 when the infectious episode occurred. By contrast, the six homozygous TIRAP
R121W relatives tested and P1’s mother (R121W/WT) had high titers of anti-LTA Abs
(Figure 4C). As a control, P1 had high titers of Abs directed against H. influenzae,
pneumococcal polysaccharides, tetanus, diphtheria toxins, and against 68 other
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staphylococcal antigens, such as PVL and hemolysin-α (Supplementary Figure S12 and
Supplementary Table 2). Overall, P1 was unique among the eight R121W homozygotes in
three ways: her whole blood did not respond to LTA, she had no LTA-specific Abs in her
plasma, and she suffered from invasive staphylococcal disease.
Rescue of the whole-blood LTA response in P1 with LTA-specific mAbs
We investigated whether the lack of anti-LTA IgG was responsible for the lack of
whole-blood responses to LTA, by testing 26 healthy adults, 22 healthy children, 246 sick
children (67 with invasive staphylococcal infections and 179 with other infections), seven
patients with MyD88 or IRAK-4 deficiency (all of whom presented staphylococcal
disease), and 10 patients with severe PVL+ S. aureus pneumonia for the presence of
specific anti-LTA Abs (Figure 4C). All healthy adults and IRAK-4- and MyD88-deficient
patients, most of the children (62%), whether healthy (96%), with S. aureus infections
(72%) or with other types of infections (54%), and up to 60% of the patients with severe
PVL+ S. aureus pneumonia, had high levels of serum anti-LTA Abs. Three of the latter
patients had a normal whole-blood response to LTA stimulation, one to eight months after
infection (data not shown). These results showed that the age at which serum anti-LTA IgG
antibodies appear varies in the population, even after staphylococcal disease, consistent
with previous reports (Wergeland et al., 1989) . They also showed that a lack of anti-LTA
IgG does not influence the whole-blood response to LTA in TIRAP-expressing individuals.
We then restored P1’s PBMCs IL-6 production in response to LTA stimulation by addition
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of LTA-responsive control plasma, suggesting that circulating anti-LTA Abs could rescue
TIRAP deficiency (Supplementary Figure S13). To test this hypothesis, we added a
chimeric mouse anti-LTA mAb to whole blood from healthy controls, P1, her aunt, and an
IRAK-4-deficient patient, or to PBMCs from healthy controls and P1 (Figure 4D and 4E
and Supplementary Figure S14). This restored P1’s whole blood and PBMC IL-6
production in response to LTA stimulation, whereas an isotypic control mAb (directed
against RSV) had no effect. Monocytes were the main cells producing IL-6 upon LTA
stimulation in the presence of anti-LTA mAb (Supplementary Figure S15) (Bunk et al.,
2010). Among the R121W homozygotes, the specific lack of anti-LTA Abs in the plasma
of P1 therefore accounts for the abolition of IL-6 induction in the whole blood of this
specific individual in response to LTA stimulation via TLR2/6. The only TIRAP
homozygote to display catastrophic staphylococcal disease (P1) mounted no antibody
response to LTA and her whole blood was unable to respond to LTA. The LTA-specific
mAb did not rescue the response of IRAK-4-deficient cells, suggesting that the response of
these cells is mediated by the canonical TLR2-MyD88 pathway. No TLR2-and MyD88-
deficient individuals were however available for study.
Anti-LTA mAb restores the LTA response in TIRAP-deficient mouse macrophages
We tested this model in mice. Like MyD88- and TLR2-deficient human
macrophages, TIRAP-deficient mouse macrophages do not respond to TLR2 stimulation
(Horng et al., 2001; Yamamoto et al., 2002a). We investigated whether the addition of
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exogenous anti-LTA antibodies restored the response to TLR stimulation. We first
determined whether such Abs were present in mouse sera. No anti-LTA Abs were detected
by ELISA in the 15 mice used for the study (data not shown). Mouse peritoneal
macrophages from WT B6, Tirap-/-, Myd88-/- and Tlr2-/- mice (n>3) were then treated with
LTA or low doses of PAM3CSK4 for 24 hrs, and IL-6 and TNFα production were assessed
in the presence or absence of exogenous anti-LTA or isotype control Abs. As observed in
cells from P1, anti-LTA Abs restored the production of IL-6 and TNFα in response to LTA,
but not PAM3CSK4, in TIRAP-deficient mouse macrophages (Figure 4F and
Supplementary Figure S16), whereas no such restoration was observed with the isotype
control mAb. MyD88- and TLR2-deficient macrophages did not respond in this way to the
addition of anti-LTA mAb. These findings unequivocally demonstrate that the TIRAP-
independent mechanism compensating for TIRAP deficiency in the presence of anti-LTA
Abs in mice, and, by inference, in humans, is dependent not only on IRAK4 but also on
both TLR2 and MyD88. Collectively, these data establish a plausible mechanism for the
occurrence of staphylococcal disease in P1 but not in any of the other seven TIRAP-
deficient individuals. The lack of LTA IgG does not compensate for TIRAP deficiency,
preventing leukocyte responses to staphylococcal LTA via TLR2/6, thereby accounting for
invasive staphylococcal disease.
Discussion
21
We describe here a large family with autosomal recessive TIRAP deficiency. Eight
family members are homozygous for the rare TIRAP R121W mutation affecting the TIR
domain. TIRAP has been shown to play a crucial role in the TLR2- and TLR4-mediated
and MyD88-dependent pathways in mice, as an adapter between these two TLRs and
MyD88. Our data show that the R121W TIRAP allele is loss-of-function, as opposed to
hypomorphic, because it cannot interact with TLRs upstream and MyD88 downstream.
TIRAP-deficient mouse macrophages display impaired activation of NF-κB in response to
the stimulation of TLR2 and TLR4 by MALP2, PGN (peptidoglycans) (TLR2/6), BLP
(bacterial lipopeptides) (TLR1/2) and LPS, respectively. LPS-induced splenocyte
proliferation and cytokine production are also impaired in TIRAP-deficient mice, which are
resistant to LPS-induced septic shock (Horng et al., 2002; Yamamoto et al., 2002a).
Consistently, fibroblasts, granulocytes, and monocytes from TIRAP-deficient humans do
not respond to TLR2/6 agonists PAM2SCK4, FSL-1, and LTA. However, residual activation
of the TLR2 pathway was recently reported in mouse TIRAP-deficient macrophages, in
response to high concentrations of PAM3CSK4 (TLR1/2) and MALP2 (TLR2/6) (Kenny et
al., 2009). Moreover, prolongation or enhancement of the interaction between mouse TLR2
and its agonists PAM2CSK4 (TLR2/6) and PAM3CSK4 (TLR1/2) can induce TIRAP-
independent responses, whereas MyD88 remains strictly required for all TLR2-dependent
responses (Cole et al., 2010). These findings suggest that the response of R121W/R121W
monocytes to high concentrations of or prolonged stimulation with PAM3CSK4 may be due
to TIRAP-independent responses mediated via TLR1/2 and MyD88. Likewise, residual
responses to LPS/lipid A in the patients may be TLR4-and MyD88-dependent. Our study
thus provides the first comprehensive description of inherited TIRAP deficiency in humans,
22
highlighting the essential role of this protein for TLR2/6 signaling. Our results also confirm
that TIRAP is less critical than MyD88 and IRAK-4 for TLR1/2 and TLR4 responses, as it
can be bypassed by increasing agonist concentration or the duration of stimulation.
The mouse TLR2 pathway plays an important role in host defense against S. aureus
(Takeuchi et al., 2000). Humans with inherited IRAK-4 and MyD88 deficiencies are also
prone to staphylococcal disease (reviewed in (Picard et al., 2010)), indicating that the
human TIR signaling pathway is essential for protective immunity to S. aureus. The
specific contribution of individual human TLRs and IL-1Rs has remained unclear
(Casanova et al., 2011). We show that homozygosity for the loss-of-function TIRAP
R121W mutant allele severely impairs, but does not abolish responses to TLR1/2 and TLR4
stimulation, whereas it abolishes responses to TLR2/6 stimulation. Impaired responses to
TLR2 are much more likely than impaired responses to TLR4 to have contributed to the
development of staphylococcal disease in P1, as S. aureus is a Gram-positive bacterium
that contains both TLR1/2 and TLR2/6 agonists, including LTA in particular, but does not
produce LPS (Lowy, 1998). However, the TIRAP mutation alone is not sufficient to
account for P1’s staphylococcal disease, as none of the other seven affected relatives had
had any serious staphylococcal infections, despite frequent exposure to these commensal
bacteria. The bacterial load in the initial inoculum, or the nature of the staphylococcus, may
influence the ability of TIRAP-deficient individuals to mount a protective immune
response. In that regard, it may be relevant that P1 was infected by a PVL-positive strain of
S. aureus; these relatively rare staphylococci are known to be more pathogenic in humans
(Gillet et al., 2002). The observed clinical differences may also reflect the young age at
23
which P1encountered this pathogenic strain of S. aureus. The proband has been doing well
for the last nine years, perhaps in part thanks to her robust Ab response to all
staphylococcal antigens tested other than LTA. She is however on antibiotic prophylaxis
(without IgG substitution, which would provide LTA-specific Abs, Supplementary Figure
S17), making it impossible to draw firm conclusions concerning the efficacy of her current
immune response to staphylococci.
A more likely hypothesis is based on the observation that P1 was the only TIRAP-
deficient individual in the kindred to display a complete lack of whole-blood TLR2/6-
dependent responses to staphylococcal LTA. This lack of response was causally related to
the specific absence of circulating Abs against LTA in P1, as the phenotype was not only
specific to this patient and absent from the other seven TIRAP-deficient relatives, but also
complemented by the addition of LTA-specific mAbs in P1. Monocytes are responsible for
the response of TIRAP-deficient whole blood and PBMC to LTA in the presence of LTA-
specific Abs. Moreover, TIRAP-deficient mouse macrophages responded to LTA via TLR2
in the presence of LTA-specific Abs. This Ab defect may, again, reflect the young age of
P1, as LTA Ab levels gradually appear and rise during childhood and teenage, with great
inter-individual variability (Dryla et al., 2005; Wergeland et al., 1989). If this is, indeed, the
case, then the lack of staphylococcal disease during childhood in the other R121W
homozygotes may have resulted from the staphylococci encountered being insufficiently
virulent to cause disease, an earlier Ab response to LTA (triggered by any Gram-positive
bacterium), or both. The normal levels of anti-LTA Abs in IRAK-4- and MyD88-deficient
children (and the six TIRAP-deficient individuals other than P1 tested) indicate that a
24
deficiency of TLR signaling pathways does not account for the lack of anti-LTA Ab
production in P1. Furthermore, IRAK-4-deficient patients display a lack of whole-blood IL-
6 production in response to LTA (Supplementary Figure S14), despite presenting high titers
of endogenous anti-LTA Abs, and even in the presence of exogenous anti-LTA mAb,
indicating that the response to LTA mediated by anti-LTA Ab is TIRAP-independent but
IRAK-4-dependent. Moreover, mouse macrophages lacking MyD88 or TLR2 are not
rescued by LTA-specific Abs, unlike TIRAP-deficient macrophages, for their responses to
LTA. The TIRAP-independent pathway rescued by LTA-specific Abs is therefore also
TLR2- and MyD88-dependent. This rescue is probably mediated via the Fc receptor CD32
(Bunk et al., 2010), although CD32-deficient humans and mice were not tested.
The continued lack of anti-LTA Abs in P1 is probably intrinsic and remains
unexplained. However, this is not a unique feature, as approximately half of children under
the age of 9 years and with no history of staphylococcal disease tested here had no
detectable anti-LTA Abs. We did not test P1’s serum before disease onset, but the observed
Ab defect is unlikely to be a consequence of staphylococcal disease, as infection should
have triggered the Ab response and other children with staphylococcal disease (even with
an identical clinical phenotype to this patient) produce such Abs. This defect is not the sole
cause of the staphylococcal disease observed in P1, as children with various forms of Ab
deficiency, including agammaglobulinemia, are not prone to such staphylococcal diseases
(Conley et al., 2009). The combined effects of a lack of anti-LTA Abs and the inherited
TIRAP deficiency, i.e. of an acquired adaptive deficiency and an inborn innate deficiency,
are therefore the mechanism underlying staphylococcal disease in this single patient
25
(Casanova et al., 2014). In the other individuals, IgG-dependent, adaptive immunity to LTA
operates as a somatic modifier, selectively rescuing the inborn error of TIRAP, which
disrupts TLR2-, TIRAP-, and MyD88-dependent innate immunity to LTA and
staphylococci. This specific observation does not explain why staphylococcal (and
pneumococcal) diseases become less frequent from adolescence onward in patients with
inherited MyD88 or IRAK-4 deficiency, whose cells did not respond to LTA in the
presence of LTA-specific Abs. However, it suggests that other mechanisms of acquired
immunity may be at work. In these patients, adaptive immunity is more likely to
compensate (via pathways other than IRAK-4-MyD88) than to rescue (via the IRAK-4-
MyD88 pathway itself) innate immunity. Regardless, we have shown that the clinical
penetrance of an inborn error of innate immunity may be determined by adaptive immunity.
This is consistent with human twin studies, which revealed a poor heritability of adaptive
immunity, when compared with innate immunity (Brodin et al., 2015; Casanova, 2015a).
The somatic diversity of adaptive immune responses can compensate or even rescue inborn
errors of innate immunity.
Author contributions
L.I., A.P. and J.L.C. designed the study and wrote the manuscript. L.I., Y.W., K.B.,
E.D.M., Z.Z., M.C., T.L., E.L., L.L., L.Y., A.M. and S.W. performed the experiments.
V.P., A.B., A.A., C.Pr. and Y.I conducted exome, linkage and statistical analysis. P.D.A.,
L.E.W., M.L., and J.H. provided patient’s material and reagents. V.S.S, M.D., F.V., C.Pi.,
L.A., D.C., X.L., and B.B. provided expertise and feedbacks. J.L.C., A.P. and L.A secured
26
funding. P.D.A conducted the medical follow up, provided expertise, feedbacks and critical
reading. All authors critically reviewed the manuscript.
Acknowledgments
We thank the patient and her family for participating in the study and all the
members of both branches of the laboratory for discussions. We thank Jacinta Bustamante
and Shen-Ying Zhang for providing the plasma samples, Sylvanie Fahy, Jérome Flatot,
Martine Courat, Lahouari Amar and Yelena Nemiroskaya for their assistance. We also
thank the members of the imaging platform, Raphaëlle Desvaux and Nicolas Goudin for
their help. We thank Biosynexus Incorporated, Gaithersburg, MD USA for providing us
with pagibaximab (an anti-LTA monoclonal Ab). The Laboratory of Human Genetics of
Infectious Diseases is supported by grants from the European Research Council (ERC-
2010-AdG-268777), Institut National de la Santé et de la Recherche Médicale, Université
Paris Descartes, the Imagine Institute, l’Agence Nationale de la Recherche (grant ANR-
2012-BSV3-0003-02), the St. Giles Foundation, the National Center for Research
Resources, and the National Center for Advancing Sciences (NCATS) grant number
8UL1TR000043 from the National Institutes of Health, and The Rockefeller University.
This work was partly supported by the Innovative Medicines Initiative Joint Undertaking
under grant agreement n◦[115523], COMBACTE, resources of which are composed of
financial contributions from the European Union's 7th Framework Program (FP7/2007-
2013) and EFPIA companies in kind contribution. Laura Israel was supported by Fondation
27
pour la Recherche Médicale. Carolina Prando was supported by the Thrasher Research
Fund and Yuval Itan was supported by the AXA Research Fund.
28
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31
Figure legends
Figure 1: TIRAP R121W is a rare mutation located in the TIR domain of TIRAP with a
modest impact at the mRNA and protein level
A. Pedigree of the kindred, with allele segregation. Genotypes are reported on the
family tree: the patient (P1), her father, four aunts and uncles, and the two paternal
grandparents were sequenced and carry the homozygous R121W mutation of TIRAP.
Each generation is identified with a roman numeral (from I to V), and each
individual with an Arabic numeral (from left to right). The patient (P1) with clinical
infectious disease is shown in black and indicated with an arrow. The relationships
between individuals in gray were extrapolated according to the estimated kinship
coefficients (supplementary figure S1)
B. Electrophoregram of TIRAP genomic DNA sequences from a healthy control and
the patient (III.1). The R121W mutation replaces a cytosine with a thymine residue
in exon 5 (c.C797T).
C. Schematic representation of TIRAP, showing the PIP2 domain, the PEST domain,
the TIR domain and the coding exons. The R121W mutation in the TIR domain is
indicated in red.
D. Conservation of the R121 amino acid in the TIR domain of TIRAP in 16 other
species.
E. Expression of the WT and mutant TIRAP genes in transfected cell lines. HEK293T
cells were transfected with both TIRAP isoforms (A and B), as the WT and with the
R121W mutation. Whole-cell extracts were obtained 48 hrs after transfection and
32
evaluated by western blotting with a mouse monoclonal anti-TIRAP antibody and an
antibody raised against the V5 tag. (n=3)
F. Relative quantitative PCR performed to measure the levels of the transcripts
encoding TIRAP isoforms A and B in SV40 fibroblasts (five controls, P1’s, P1
father’s and mother’s cells), PBMCs (9 controls, P1, and P1 aunt’s cells) and EBV-B
cells (5 controls and P1). SV40 fibroblasts stably transfected with the cDNA
encoding either isoform A or B were used as controls to test probe specificity.
TIRAP mRNA levels encoding isoform A or B were expressed relative to GAPDH
mRNA levels using the 2ΔΔC(t) method. For each probe, normalization was made to
one of the controls which had been set to 1. Error bars represent SD (n=2).
G. TIRAP protein levels in PBMCs from two controls, P1’s father and P1, as
determined by western blotting using an anti-TIRAP mouse mAb. Anti-GAPDH Ab
was used as a protein load control. Quantification was performed with the Image J
software (n=1).
Figure 2: The R121W mutation impairs TIRAP cellular localization and interaction with its
partners MyD88 and TLR2
A. HEK293T cells were transfected with a construct encoding a V5-tagged WT or
mutant isoform A of TIRAP for 36 hrs and then labeled with an Alexa Fluor 488-
conjugated anti-V5 antibody. Analyses were carried out by ApoTome fluorescence
microscopy. The images shown are representative of three independent experiments
and correspond to >95% of all the images obtained (30 images acquired pre
experiment)
33
B. Cells were cotransfected with constructs encoding TIRAP WT-V5 and either myc-
tagged WT TIRAP or myc-tagged R121W TIRAP. V5-tagged proteins were labeled
with Alexa Fluor 488-conjugated anti-V5 antibodies and myc-tagged proteins were
labeled with Alexa Fluor 555-conjugated anti-myc antibodies. Colocalization
analysis was performed with Image J software, by calculating Pearson’s correlation
coefficient (PCC). Three independent experiments were performed and 30 cells
were analyzed in each experiment. Error bars represent SD.
C. Cells were cotransfected with constructs encoding myc-tagged MyD88 and either
V5-tagged WT TIRAP or V5-tagged R121W TIRAP. V5-tagged proteins were
labeled with Alexa Fluor 488-conjugated anti-V5 antibodies and myc-tagged
proteins were labeled with Alexa Fluor 555-conjugated anti-myc antibodies.
Colocalization analysis was carried out with Image J software, by calculating
Pearson’s correlation coefficient (PCC). Three independent experiments were
performed and 30 cells were analyzed in each experiment. Error bars represent SD.
D. HEK293T cells were cotransfected with constructs encoding both the TIRAP A and
B isoforms with a V5 tag, WT or mutated, and with a construct encoding HA-
tagged TLR2. HA immunoprecipitation and V5 immunoblotting were then
performed on total cell extracts (n=2).
E. Cells were cotransfected with constructs encoding both TIRAP isoforms (A and B
isoforms), with a V5 tag, WT or mutated, and with a construct encoding M2-tagged
MyD88. M2 immunoprecipitation and V5 immunoblotting were then performed on
total cell extracts (n=2).
34
F. Co-immunoprecipitation between TLR2 and MyD88 without or after stimulation
with PAM2CSK4 (10µg/ml) for 10 and 30min, in SV-40 fibroblasts from a control
(TIRAP WT/WT), the heterozygous P1’s mother (TIRAP R121W/WT), a MyD88-
mutated patient (MyD88 L93P/R196C), and TIRAP homozygous P1 and her father
(TIRAP R121W/R121W) (n=1)
Figure 3: TIRAP R121W allele is loss of function
A. IL-6 induction in SV40 fibroblasts from a healthy control, P1, her father, and
MyD88-deficient cells, upon transfection with WT or mutant TIRAP isoforms. IL-6
production was measured by ELISA in cells transfected with an empty vector, WT
or mutant isoforms A plus B, or MyD88-deficient cells transfected with WT
MyD88 (n=3). Error bars represent SEM.
B. NF-κB luciferase assay in HEK293T cells expressing stable overexpression of NF-
κB driven luciferase reporter plasmid upon transfection with WT TIRAP isoform A
or B, or mutant TIRAP isoforms A or B. (n=3). Error bars represent SEM
C. SV40 transformed fibroblasts from healthy controls, P1, her father, an IRAK-4-
deficient patient and NEMO-deficient fibroblasts were stimulated with TLR1/2
(PAM3CSK4), TLR2/6 (PAM2CSK4, purified LTA-SA, FSL-1), TLR3 (poly(I:C))
and TLR4 (LPS and lipid A) ligands, in addition to heat killed S. aureus (HKSA)
IL-1β (IL-1R) and TNF-α (TNF-αR). Supernatants were collected 24 hrs after
stimulation and IL-6 production was assessed by ELISA. (n=3). Error bars represent
SEM.
35
D. Granulocyte L-selectin shedding assessed in several family members (III.1, IV.1-5
and V.1) after one hour of stimulation with PAM2CSK4, PAM3CSK4, FSL-1,
purified LTA, LPS and PMA as an activation control. Mean fluorescence intensity
was determined by flow cytometry after the extracellular staining of CD62L. * p
values < 0.05 (unpaired t-test). (n=1)
E. PBMCs from several family members (III.1, IV.1 to 5 and V.1) were stimulated
with various doses of PAM2CSK4, FSL-1, LTA, PAM3CSK4 and LPS for 3 hrs,
subjected to extracellular staining with anti-CD3, anti-CD19 and anti-CD14
antibodies and intracellular staining for IL-6. The proportion of CD14+ cells
positive for IL-6 is shown. Acquisition was performed on 10,000 CD14+ cells. Each
data point represent the mean of all individuals +/- SEM
Figure 4: The lack of anti-LTA Abs in P1’s plasma accounts for defective LTA whole
blood response
A. PBMCs from controls, P1, her father (II.1) and aunt (II.2) were stimulated with
PAM3CSK4 (1 µg/ml), PAM2CSK4 (1 µg/ml), FSL-1 (100 ng/ml), LTA (1 µg/ml),
and LPS (10 ng/ml) for 48 hrs. The supernatant were tested, by ELISA, for IL-6
production. Representative results, from one of two experiments carried out, are
shown.
B. IL-6 production by whole blood was assessed by ELISA in I.1, I.2, II.2, II.3, II.4,
II.5, II.6 and III.1 after 48 hrs of stimulation with PAM2CSK4, FSL-1, LTA,
PAM3CSK4 and LPS. The assay was performed several times and each point
represents the mean for one individual. Whole blood from P1 was tested five times
for FSL-1 stimulation, six for LTA, seven for PAM3CSK4 and LTA and eight times
36
for PAM2CSK4. Whole blood from other individuals was tested once (I.1), twice
(I.2, II.2, II.3, II.4) or three times (II.1) for each stimulus.
C. Tests for the presence of specific anti-LTA antibodies in plasma detected by ELISA,
dilution 1/100 is shown but tests were performed at 1/20, 1/100, 1/500 plasma
dilution.
D. Complementation of the defect of IL-6 production in response to LTA stimulation in
whole blood from the patient, by the addition of monoclonal anti-LTA antibodies
(pagibaximab) (0,5 or 1 mg/ml). Comparison with the isotypic control (0,5 or 1
mg/ml), (palivizumab, anti-RSV). Results from one representative experiment of the
two carried out are shown. (p=0.001, unpaired t-test). Error bars represent SEM.
E. Complementation of the impaired IL-6 production in response to LTA stimulation in
PBMCs from P1 by the addition of anti-LTA mAbs. IL-6 production by controls’ or
P1’s PBMCs after 48hrs of stimulation with 1µg/ml of PAM3CSK4 or 1 µg/ml of
LTA in the presence of 10% of FCS, 1mg/ml of exogenous isotype control mAbs or
1mg/ml of anti-LTA mAbs. Error bars represent SEM (n=4).
F. Peritoneal mouse macrophages from WT B6, Myd88-/-, Tiraptor/tor and TLR2lan/lan
(n>3) were stimulated with PAM3CKS4 (40ng/ml) or LTA (1ug/ml) or left
unstimulated and complemented with 0,5mg/ml of either anti-LTA Abs
(Pagibaximab) or isotype control (anti RSV, Synagis). IL-6 production was assessed
by ELISA. Error bars represent SEM
37
Supplementary legends : Figure S1: Familial relationship. A. Within-family relationships estimated with KING. X-
axis represents the expected kinship coefficient for a non-consanguineous family and y-axis
represents the estimated kinship coefficient in this family. Each dot represents the kinship
coefficient between two members of the family. Black dots represent an estimated kinship
coefficient concordant with the expected kinship coefficient for this kind of relationship.
Red dots represent an estimated kinship coefficient discordant with the expected kinship
coefficient for this kind of relationship. Pedigree constructed according to the estimated
kinship coefficients.
Figure S2: Alignment of the human TIRAP protein with other human TIR domain-
containing molecules
Figure S3: TIRAP RT-PCR ranging from 27 to 33 cycles, on RNA extracted from SV40-
fibroblasts from P1 and her father, compared to healthy control. Primer specific for
transcripts encoding isoform A or B were used.
Figure S4: Quantitative PCR performed to measure the levels of the transcripts encoding
TIRAP isoforms A and B in SV40 fibroblasts (five controls, P1’s, P1 mother’s and father’s
cells), EBV-B cells (5 controls and P1) and PBMCs (9 controls, P1, and P1 aunt’s cells).
SV40 fibroblasts stably transfected with either the cDNA encoding isoform A or B were
used as controls to test probe specificity. The percentages of the transcripts encoding
isoform A or B were normalized to the total amount of all transcripts (encoding for both
isoforms A and B, set at 100%.) Error bars represent SD.
Figures S5-S7: Complementation study in SV40 fibroblasts from P1 (S5), her father (S6)
and a control (S7), after stable transfection with lentiviral particles encoding the WT or
38
mutant A or B isoform of TIRAP, or both. Particles containing luciferase instead of TIRAP
were used for mock transfection. IL-6 production was assessed by ELISA.
Figure S8: IL-6 mRNA levels assessed by RT-qPCR and expressed relative to GUS mRNA
levels by the 2ΔΔC(t) method, in SV40 fibroblasts from a healthy control. Cells were
stimulated for 4 hrs with PAM2SCK4 or TNF-α, 24 hrs after transfection with siRNAs
against TLR2 and TIRAP.
Figure S9: PBMCs from several family members (III.1, IV.1-5 and V.1) were stimulated
with various doses of PAM2CSK4, PAM3CSK4, LTA, FSL-1, and LPS for 3 hrs, subjected
to extracellular staining with anti-CD3, anti-CD19 and anti-CD14 antibodies and
intracellular staining for TNF-α. The proportion of CD14+ cells positive for TNF-α is
shown. Acquisition was performed on 10,000 CD14+ cells. Each data point represents the
mean of all individuals +/- SEM
Figure S10: Transcriptome analysis performed on whole blood after 3 hrs of stimulation
with PAM2CSK4, FSL-1, LTA, PAM3CSK4, LPS or PMA ionomycin. The results for P1
and her mother (R121W/WT) are compared with those for one healthy control and one
IRAK-4-deficient patient.
Figure S11: PBMCs and whole blood from healthy controls, P1 and her father were
stimulated with lipid A (1µg/ml), LPS (10ng/ml) and PMA/ionomycin for 48 hrs. IL-6
production was assessed by ELISA. Error bars represent SD.
Figure S12: Assessment by luminex of antibodies against 68 staphyloccocal antigens, (6
only are shown in the figure, all results are summarized on supplementary table 2), in 6
different plasma from P1 (between 1 to 8 years old), 5 homozygous relatives, 6 IRAK-4 or
MyD88 deficient patients and 11 sex and aged matched controls. Bonferroni’s multiple
39
comparison testing indicated that for none of the antigen tested, the patient showed
significantly lower level of antibodies than controls.
Figure S13: PBMCs from P1 and two controls were left non-stimulated or stimulated by
PAM3CKS4 or LTA in the presence of fetal calf serum (FCS) or plasma from each
individual. IL-6 production was assessed by ELISA. Error bars represent SD.
Figure S14: IL-6 production by whole blood was assessed in a healthy control and an
IRAK4-deficient patient upon 48 hrs of stimulation with PAM3CSK4 or LTA supplemented
with 0.5mg/ml of either a control isotype antibody (anti-RSV, Synagis) or with anti-LTA
(Pagibaximab) monoclonal antibodies.
Figure S15: IL-6 production by controls’ PBMCs, monocytes, and monocyte-depleted
PBMCs after 24 hrs of stimulation with 1µg/ml of PAM3CSK4 or 1µg/ml of LTA in the
presence of medium alone, or medium supplemented with 1 mg/ml of either anti-LTA mAb
or with a isotype control mAb (anti-RSV).
Figure S16: Peritoneal mouse macrophages from WT B6, Myd88-/-, Tiraptor/tor and
TLR2lan/lan (n>3) were stimulated with PAM3CKS4 (40ng/ml) or LTA (1ug/ml) or left
unstimulated and complemented with 0,5mg/ml of either anti-LTA Abs (Pagibaximab) or
isotype control (anti RSV, Synagis). TNF-α production was assessed by ELISA. Error bars
represent SEM.
Figure S17: Assessment by ELISA of levels of anti-LTA antibodies in 16 different batches
of commercially available IVIG compared to healthy controls (n=10) and P1. Error bars
represent SEM.
1
Supplementary tables legends:
Supplementary table 1: List of genes presenting a non-synonymous rare homozygous
variation in P1 exome data.
Supplementary table 2 : IgG levels against 68 recombinant S. aureus proteins were
measured in serum samples using a bead-based flow cytometry technique in 6 different
plasma from the patient (between 1 to 8 years old), 5 homozygous relatives, 6 IRAK-4
or MyD88 deficient patients and 11 aged matched controls. The table summarize mean
and SD for each antigen in each plasma category. Bonferroni’s multiple comparison
testing was performed and p value for significant differences compared to controls are
indicated.
Supplementary table 3: Metrics and coverage data for the three exomes sequenced
2
Supplementary tables:
Supplementary table 1 :
nomenclature chr allele gene AA
protein
pos
1 c.542G>A 1 G/A ENSG00000132681 (ATP1A4) R/Q 181
2 c.1150A>G 1 T/C ENSG00000137992 (DBT) S/G 384
3 c.125G>A 1 G/A ENSG00000213088 (DARC) G/D 42
4 c.1693T>C 1 A/G ENSG00000134242 (PTPN22) W/R 565
5 c.2009G>A 1 G/A ENSG00000169174 (PCSK9) G/E 670
6 c.344G>A 1 C/T ENSG00000143278 (F13B) R/H 115
7 c.1601A>G 1 T/C ENSG00000198734 (F5) Q/R 534
8 c.7480T>C 1 A/G ENSG00000066279 (ASPM) Y/H 2494
9 c.49A>G 2 A/G ENSG00000163599 (CTLA4) T/A 17
10 c.380T>C 2 A/G ENSG00000135899 (SP110) L/S 127
11 c.733G>A 2 C/T ENSG00000170820 (FSHR) A/T 245
12 c.1853G>A 2 C/T ENSG00000170820 (FSHR) S/N 618
13 c.3448T>C 2 A/G ENSG00000169432 (SCN9A) W/R 1150
14 c.41C>G 3 C/G ENSG00000173402 (DAG1) S/W 14
15 c.743T>C 3 T/C ENSG00000145192 (AHSG) M/T 248
16 c.767G>C 3 G/C ENSG00000145192 (AHSG) S/T 256
17 c.163A>G 4 T/C ENSG00000145384 (FABP2) T/A 55
18 c.53C>A 4 C/A ENSG00000154277 (UCHL1) S/Y 18
19 c.1805G>T 4 C/A ENSG00000174125 (TLR1) S/I 602
20 c.3568G>A 5 G/A ENSG00000095015 (MAP3K1) A/T 1190
21 c.46G>A 5 G/A ENSG00000169252 (ADRB2) G/R 16
22 c.79G>C 5 G/C ENSG00000169252 (ADRB2) E/Q 27
23 c.412G>A 5 G/A ENSG00000168685 (IL7R) V/I 138
24 c.197T>C 5 T/C ENSG00000168685 (IL7R) I/T 66
3
25 c.2893G>A 5 C/T ENSG00000138829 (FBN2) V/I 965
26 c.10411G>A 5 G/A ENSG00000164199 (GPR98) E/K 3471
27 c.122C>A 5 G/A ENSG00000258864 (CTC-554D6.1) A/E 41
28 c.5465T>A 5 T/A ENSG00000134982 (APC) V/D 1822
29 c.1630A>C 5 A/C ENSG00000112964 (GHR) I/L 544
30 c.989C>T 6 G/A ENSG00000204525 (HLA-C) A/V 330
31 c.982G>A 6 C/T ENSG00000204525 (HLA-C) V/I 328
32 c.925A>G 6 T/C ENSG00000204525 (HLA-C) M/V 309
33 c.302G>A 6 C/T ENSG00000234745 (HLA-B) S/N 101
34 c.47G>C 6 C/G ENSG00000204525 (HLA-C) G/A 16
35 c.11T>C 6 A/G ENSG00000234745 (HLA-B) M/T 4
36 c.1136T>C 6 A/G ENSG00000146070 (PLA2G7) V/A 379
37 c.1105A>G 6 T/C ENSG00000204525 (HLA-C) T/A 369
38 c.991A>G 6 T/C ENSG00000204525 (HLA-C) M/V 331
39 c.872A>C 6 T/G ENSG00000204525 (HLA-C) Q/P 291
40 c.41C>G 6 G/C ENSG00000234745 (HLA-B) S/W 14
41 c.44C>G 6 G/C ENSG00000234745 (HLA-B) A/G 15
42 c.49C>G 6 G/C ENSG00000234745 (HLA-B) L/V 17
43 c.292G>T 6 C/A ENSG00000234745 (HLA-B) D/Y 98
44 c.610G>C 6 C/G ENSG00000234745 (HLA-B) E/Q 204
45 c.363C>G 6 G/C ENSG00000234745 (HLA-B) S/R 121
46 c.355C>T 6 G/A ENSG00000234745 (HLA-B) L/F 119
47 c.5T>G 6 A/C ENSG00000234745 (HLA-B) L/R 2
48 c.560A>T 6 T/A ENSG00000234745 (HLA-B) E/V 187
49 c.22G>A 6 C/T ENSG00000204525 (HLA-C) A/T 8
50 c.559G>C 6 C/G ENSG00000234745 (HLA-B) E/Q 187
51 c.910C>G 6 G/C ENSG00000112619 (PRPH2) Q/E 304
52 c.206A>C 6 T/G ENSG00000234745 (HLA-B) E/A 69
53 c.929G>A 6 C/T ENSG00000112619 (PRPH2) R/K 310
4
54 c.594G>T 6 G/T ENSG00000078401 (EDN1) K/N 198
55 c.829C>G 6 G/C ENSG00000204525 (HLA-C) Q/E 277
56 c.142T>G 6 A/C ENSG00000234745 (HLA-B) S/A 48
57 c.205G>A 6 C/T ENSG00000234745 (HLA-B) E/K 69
58 c.71C>T 7 C/T ENSG00000184408 (KCND2) S/L 24
59 c.553A>C 7 T/G ENSG00000188050 (RNF133) I/L 185
60 c.1642G>A 7 G/A ENSG00000257743 (RP11-1220K2.2) E/K 548
61 c.236T>C 7 T/C ENSG00000211746 (TRBV19) I/T 79
62 c.785C>T 7 G/A ENSG00000257138 (TAS2R38) A/V 262
63 c.894T>G 7 T/G ENSG00000164867 (NOS3) D/E 298
64 c.1388T>C 7 A/G ENSG00000004948 (CALCR) L/P 463
65 c.1408C>T 7 G/A ENSG00000122512 (PMS2) P/S 470
66 c.1739T>C 7 A/G ENSG00000105929 (ATP6V0A4) M/T 580
67 c.2200T>G 8 T/G ENSG00000042832 (TG) S/A 734
68 c.5995C>T 8 C/T ENSG00000042832 (TG) R/W 1999
69 c.2564C>G 9 G/C ENSG00000165271 (NOL6) A/G 855
70 c.494G>T 9 G/T ENSG00000137124 (ALDH1B1) G/V 165
71 c.1091G>A 9 C/T ENSG00000095397 (DFNB31) R/H 364
72 c.6011C>G 10 C/G ENSG00000107736 (CDH23) T/S 2004
73 c.244G>C 10 G/C ENSG00000107736 (CDH23) G/R 82
74 c.457T>C 10 T/C ENSG00000165730 (STOX1) Y/H 153
75 c.145A>G 10 A/G ENSG00000043591 (ADRB1) S/G 49
76 c.1165G>C 10 G/C ENSG00000043591 (ADRB1) G/R 389
77 c.361C>T 11 C/T ENSG00000150455 (TIRAP) R/W 121
78 c.328A>C 11 A/G ENSG00000109861 (CTSC) I/L 110
79 c.775A>G 11 T/C ENSG00000172638 (EFEMP2) I/V 259
80 c.388C>T 12 G/A ENSG00000121318 (TAS2R10) P/S 130
81 c.242C>G 12 G/C ENSG00000197870 (PRB3) P/R 81
82 c.1751C>T 12 C/T ENSG00000182326 (C1S) A/V 584
5
83 c.603G>T 13 G/T ENSG00000123171 (CCDC70) E/D 201
84 c.7397T>C 13 T/C ENSG00000139618 (BRCA2) V/A 2466
85 c.1120T>G 13 A/C ENSG00000123191 (ATP7B) S/A 374
86 c.1270G>C 13 C/G ENSG00000123191 (ATP7B) V/L 424
87 c.2129T>C 13 A/G ENSG00000123191 (ATP7B) V/A 710
88 c.1565G>A 13 C/T ENSG00000123191 (ATP7B) R/K 522
89 c.2126G>A 14 G/A ENSG00000100714 (MTHFD1) R/Q 709
90 c.259G>A 15 C/T ENSG00000159337 (PLA2G4D) V/I 87
91 c.1061T>C 15 T/C ENSG00000140463 (BBS4) I/T 354
92 c.1331A>C 16 A/C ENSG00000179588 (ZFPM1) E/A 444
93 c.538G>A 16 C/T ENSG00000121270 (ABCC11) G/R 180
94 c.1066A>G 17 T/C ENSG00000108578 (BLMH) I/V 356
95 c.154G>T 17 G/T ENSG00000132518 (GUCY2D) A/S 52
96 c.188A>G 19 T/C ENSG00000131398 (KCNC3) D/G 63
97 c.7211G>C 20 C/G ENSG00000130702 (LAMA5) R/P 2404
98 c.385A>G 20 A/G ENSG00000171867 (PRNP) M/V 129
99 c.2339A>C 20 T/G ENSG00000075043 (KCNQ2) N/T 780
100 c.112A>G 21 T/C ENSG00000180509 (KCNE1) S/G 38
101 c.185A>G 22 A/G ENSG00000211647 (IGLV5-48) E/G 62
102 c.226A>G 22 A/G ENSG00000211647 (IGLV5-48) S/G 76
103 c.227G>A 22 G/A ENSG00000211647 (IGLV5-48) S/N 76
104 c.1049A>G 22 T/C ENSG00000100299 (ARSA) N/S 350
105 c.1172C>G 22 G/C ENSG00000100299 (ARSA) T/S 391
106 c.466A>G X A/G ENSG00000101981 (F9) T/A 156
107 c.97T>C X T/C ENSG00000147100 (SLC16A2) S/P 33
6
Supplementary table 2 :
Controls (n=11) IRAK4-/- or MyD88-/- (n=6) Patient Tirap-/- Tirap -/- healthy relatives (n=6)
ANTIGEN Mean SD Mean SD diff from controls Mean SD diff from
controls Mean SD diff from controls
lukS 7858 4788 10201 4213 ns 16101 1685 ** 15158 4463 * fnbpB5 1603 2194 643 387 ns 2586 1952 ns 1002 1339 ns CHIPS 13332 7139 18752 2287 ns 15310 3108 ns 17858 2357 ns eapH2 8513 6242 17449 4792 * 15821 5272 ns 12783 6814 ns clfBN2,3 7252 4927 7166 4749 ns 3198 1055 ns 7484 3602 ns fnbpA1 3719 4580 2577 2803 ns 259 219 ns 5206 3337 ns flip-R 6752 7105 7395 2962 ns 30.8 41.3 ns 4300 2863 ns SEM 979 1617 1827 1274 ns 128 132 ns 2606 2558 ns ETA 928 1560 3251 4680 ns 589 919 ns 6189 6222 ns SEH 1666 2642 4729 6702 ns 120 88.3 ns 1604 2893 ns sdrD 993 1515 2348 2668 ns 829 270 ns 1183 623 ns fndpA2 625 673 2850 3697 ns 2468 576 ns 4533 2618 ** SCIN 6051 5072 9487 6094 ns 14700 2490 ** 10059 4400 ns TSST-1 5464 7542 11846 9159 ns 857 1426 ns 12967 3838 ns fnbpA3 1771 2428 3706 3870 ns 850 438 ns 4711 3087 ns hlgB 9996 4257 12800 2716 ns 11915 2507 ns 13878 2583 ns Nuc 4187 4733 12337 6345 ns 14860 3065 * 10325 8195 ns fnbpA5 1095 1540 3063 3626 ns 2579 2280 ns 2592 2670 ns SEB 2315 3328 3566 3790 ns 11940 9488 * 11248 8091 ns SEA 2333 2946 7571 6999 ns 6581 3120 ns 12030 4061 *** lukE 11775 6025 15911 4041 ns 12915 2182 ns 17219 3311 ns lukD 10921 5598 11314 3694 ns 9710 1359 ns 14191 3657 ns eap 6709 3166 14967 2424 *** 13178 2035 *** 13154 1843 *** clfAN2,3 2260 2512 3886 5438 ns 8120 4415 * 5536 2257 ns A-tox 8050 5053 13513 5196 ns 13210 2040 ns 13791 4941 ns ssl-1 5833 5231 11074 8742 ns 1980 1616 ns 9521 5242 ns sasH 406 516 415 584 ns 213 138 ns 269 338 ns ssl-3 9652 6770 12954 3787 ns 6048 4741 ns 14085 3514 ns ssl-5 3274 2055 4918 2768 ns 2344 1132 ns 4184 1800 ns ssl-9 10101 5194 15332 2994 ns 9048 4923 ns 14444 5669 ns fnbpA6 561 432 1458 2632 ns 134 47.8 ns 1024 629 ns ssl-10 6309 4233 8170 3254 ns 2594 1361 ns 7962 1364 ns ssl-11 1589 1999 1792 2021 ns 85.7 79.7 ns 1125 1401 ns sec 7475 8773 14402 6989 ns 9714 7676 ns 15402 4819 ns etb 182 246 2672 5129 ns 79 60.7 ns 454 745 ns seo 608 720 2156 1622 * 338 266 ns 1246 536 ns fnbpB 562 847 285 255 ns 2300 1506 ** 946 948 ns fnbpB1 699 1123 472 531 ns 158 85.2 ns 372 310 ns isdA 14551 4645 17305 2466 ns 18029 1641 ns 13815 8321 ns
7
fnbpB2 2021 1901 2132 1722 ns 3835 1315 ns 4857 2757 ns fnbpB4 1504 2328 1809 2445 ns 543 239 ns 1235 1139 ns fnbpB6 438 596 704 477 ns 167 54.4 ns 1089 1081 ns fnbpB7 1448 2504 1716 2539 ns 440 177 ns 1194 1273 ns fnbpA7 741 832 1542 2410 ns 483 721 ns 1367 1279 ns sasC 154 124 329 416 ns 140 74.7 ns 310 181 ns flipR-L 5760 5261 9998 4771 ns 97 72.4 ns 4849 3099 ns esxB 259 299 247 225 ns 148 191 ns 159 147 ns prsA 621 908 2336 2516 ns 2727 3676 ns 572 308 ns atl2 7643 4486 14766 3511 * 17075 4651 ** 14688 3973 * lipase 11684 7289 17179 3296 ns 4671 3633 ns 10058 6773 ns sed 1528 2142 1757 684 ns 703 276 ns 516 308 ns fnbpA 2609 3450 3057 4941 ns 849 863 ns 2685 1722 ns sdrE 1541 1876 2450 2785 ns 1008 507 ns 2158 1399 ns SEE 253 620 3777 4716 ns 2646 1587 ns 5403 2913 ** SEJ 2491 3455 5448 1919 ns 1825 868 ns 2573 1698 ns isdH 695 920 796 623 ns 911 349 ns 740 728 ns sasG 850 1456 777 1042 ns 177 159 ns 1608 1932 ns sdrC 1478 1642 3970 3871 ns 897 835 ns 4118 3403 ns SEI 511 669 1499 1007 ns 298 145 ns 3369 3106 ** SEQ 745 1709 1484 1646 ns 74.7 41.6 ns 2098 1742 ns isaA 7943 5917 17129 6758 * 2210 1136 ns 6631 4743 ns lyt M 1678 2595 8325 6795 ** 900 631 ns 2280 1613 ns SER 312 573 725 832 ns 132 136 ns 1274 1705 ns Aly 1391 1447 3361 1938 ns 3427 1824 ns 2198 1016 ns pro Atl 1361 2225 4501 6461 ns 6402 3651 ns 3597 1229 ns SEG 493 596 1733 1107 ns 303 322 ns 3606 2476 *** eapH1 827 1128 2255 2254 ns 1471 1009 ns 1204 1083 ns SA2097 4.02 6.71 15.6 14.9 ns 25.6 18.8 * 8.42 8.23 ns
8
Supplementary table 3 :
P1 (III.1) II.5 I.1
Single/Paired-end Paired-end Paired-end Paired-end
Bait-set 50Mb 50Mb 71Mb
Total reads 164,759,220 184,566,394 132,090,076
Total reads aligned in the targeted regions 22,485,016 26,512,246 75,999,552
Mean coverage 44.97 53.02 107.04
Target bases covered by > 2X 99.4 99.6 99.7
Target bases covered by > 10X 92.3 94.6 98.5
Target bases covered by > 20X 75.8 81.5 95.2
Target bases covered by > 30X 58.4 66.1 90
A. B. C.
D.
Fig. 1
F.
C1 C2 Father P1
TIRAP
PBMCs
GAPDH
TIR
AP
norm
. to
GA
PD
H
G.
TIRAP
V5
GAPDH
HEK 293T cells E.
Ct r
+ A
Ct r
+ B
Ct r
+ A
&B
Ct r
P1
Fa t h
e r
Mo t h
e rC
t rP
1A
u n tC
t rP
1
0 . 1
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0
TIR
AP
is
ofo
rms
A o
r B
mR
NA
le
ve
l
no
rma
liz
ed
to
GA
PD
H
I s o A
S V 4 0
f i b r o b l a s t s
P B M C s
I s o B
B E B V
A.
B.
IB: HA
IB: V5
IP: HA IB: V5
TLR2-HA + + + + +
TIRAP A WT-V5 + - - - -
TIRAP A mut-V5 - + - - -
TIRAP B WT-V5 - - + - -
TIRAP Bmut-V5 - - - + -
MyD88-M2 + + + + - - + -
TIRAP A WT-V5 + - - - + - - -
TIRAP A mut-V5 - + - - - + - -
TIRAP B WT-v5 - - + - - - - -
TIRAP B mut-v5 - - - + - - - -
IB: V5
IB: M2
IP: M2 IB: M2
IP: M2 IB: V5
TIRAPWT
TIRAPmut
C.
D. E.
TIRAPWT TIRAPWT Merge
TIRAPWT TIRAPmut Merge
TIRAPmut MyD88 Merge
TIRAPWT MyD88 Merge
Fig. 2
F.
A.
B.
Fig. 3
C.
MFI
NS PAM2 LTA FSL-1 LPS PMA
Granulocytes, 1h
102
103
PAM3
Controls Mother P1 R121W/R121W healthy
E.
D.
A W T B W T A R 1 2 1 W B R 1 2 1 W M o c k
0
2 1́ 0 0 4
4 1́ 0 0 4
6 1́ 0 0 4
8 1́ 0 0 4
NF
-kB
lu
cif
era
se
(u
nit
s)
2 9 3 T c e l l s
0 0 , 0 1 0 , 1 1
0
1 0
2 0
3 0
P A M 2 C S K 4 ( u g / m l )
% o
f IL
-6+
ce
lls
0 0 , 0 1 0 , 1 1 1 0
0
2 0
4 0
6 0
P A M 3 C S K 4 ( u g / m l )
C t r
R 1 2 1 W / R 1 2 1 W
0 0 , 1 1
0
2 0
4 0
6 0
8 0
L T A ( u g / m l )
0 0 , 0 1 0 , 1
0
1 0
2 0
3 0
4 0
5 0
F S L - 1 ( u g / m l )
% o
f IL
-6+
ce
lls
0 0 , 1 1
0
2 0
4 0
6 0
L P S ( n g / m l )
CD14+ cells, 3h
C o n t r o ls P 1 F a t h e r M y D 8 8 - / -
0
2 1́ 0 0 3
4 1́ 0 0 3
6 1́ 0 0 3
IL-6
(p
g/m
l)
E m p t y v e c t o r
T I R A P A + B W T
T I R A P A + B R 1 2 1 W
M y D 8 8
S V - 4 0 f i b r o b l a s t s
N S P A M 2
T L R 2
L T A
T L R 2
F S L 1
T L R 2
P A M 3
T L R 2
P o ly ( I: C )
T L R 3
L P S
T L R 4
L ip id A
T L R 4
H K S A IL - 1 b
IL 1 - R
T N F a
T N F R
1 0 0
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
1 0 6
IL-6
(p
g/m
l)
C T R
I R A K 4 - / -
N E M O - / -
P 1 ( R 1 2 1 W / R 1 2 1 W )
F a t h e r ( R 1 2 1 W / R 1 2 1 W )
S V 4 0 f i b r o b l a s t s
Fig. 4
B.
PBMCs, 48h A. D.
C.
Whole blood, 48h
N S P A M 3 P A M 2 F S L - 1 L T A L P S
1 0 0
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
IL-6
pg
/ml
C t r
R 1 2 1 W / R 1 2 1 W h e a l t h y
P 1
E.
N S P A M 3 P A M 2 F S L - 1 L T A L P S
1 0 0
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
1 0 6
IL-6
(p
g/m
l)
C t r
R 1 2 1 W / R 1 2 1 W h e a l t h y
P 1
****
ns
C +
( a d u l t s )
C +
( c h i ld r e n )
C h i ld r e n
w i t h o t h e r
in f e c t io n
C h i ld r e n
w i t h S t a p h
in f e c t io n s
IR A K 4 - / -
M y D 8 8 - / -
P 1 R 1 2 1 W / R 1 2 1 W
h e a l t h y
0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
OD
a n t i - L T A i n p l a s m a
N S P A M 3 L T A N S P A M 3 L T A N S P A M 3 L T A
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
4 0 0 08 0 0 0
W T
M y D 8 8 - / -
T L R 2 - / -
T I R A P - / -
m e d i a + a n t i - L T A A b + C o n t r o l I s o t y p e A b
p = 0 . 0 1 4
n sIL
-6 (
pg
/ml)
F.
C t r A u n t P 1 C t r A u n t P 1 C t r A u n t P 1
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
IL-6
(p
g/m
l)
N S
P A M 3 C S K 4
L T A
W h o l e b l o o d + a n t i - L T A A b s + i s o t y p e
* *
C t r P 1 C t r P 1 C t r P 1
1 0 0
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
IL-6
(p
g/m
l)
F C S + a n t i - L T A A b s + i s o t y p e
N S
P A M 3 C S K 4
L T A
Supp Fig.
S1.
S2.
S3.
Ct r
+ A
Ct r
+ B
Ct r
+ A
&B
Ct r
P1
Fa t h
e r
Mo t h
e rC
t rP
1A
u n tC
t rP
1
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
% m
RN
A o
f e
ac
h p
rob
e
no
rma
liz
e t
o G
AP
DH
I s o A
I s o B
S V - 4 0
f i b r o b l a s t s
P B M C s B E B V
C+
P1
Father
27 30 33 cycles 27 30 33
Isoform A Isoform B
SV-40 fibroblasts
S4.
S5.
Non transfected Mock Iso A Iso B Iso A+B Iso A mut Iso B mut Iso A+B mut
IL-6
(pg/
ml)
Patient's SV-40 fibroblasts NS PAM2 LPS TNFα
102
103
104
105
101
100
S7.
Non transfected
Mock Iso A Iso B Iso A+B Iso A mut Iso B mut Iso A+B mut
IL-6
(pg/
ml)
Father's SV-40 fibroblasts
102
103
104
105
101
100
Non transfected Mock Iso A Iso B Iso A+B Iso A mut Iso B mut Iso A+B mut
IL-6
(pg/
ml)
Control’s SV-40 fibroblasts
102
103
104
101
100
S6.
Supp Fig.
Supp Fig.
S10.
PAM2
C+
IRA
K4-
/- P1
Mot
her
FSL-1
C+
IRA
K4-
/- P1
Mot
her
LTA
C+
IRA
K4-
/- P1
Mot
her
PAM3
C+
IRA
K4-
/- P1
Mot
her
LPS
C+
IRA
K4-
/- P1
Mot
her
C+
IRA
K4-
/- P1
Mot
her
PMA
S9. S8.
0
5
10
15
20
Scrambled siTIRAP siTLR2
IL-6
rela
ted
to G
US
SV-40 fibroblasts NS PAM2 TNFα
Supp Fig.
S11.
S12.
C t r P 1 F a t h e r C t r P 1 F a t h e r
1 0 0
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
N S
l i p i d A
L P S
P M A i o n o
IL-6
(p
g/m
l)
P B M C s W h o l e b l o o d
Supp Fig.
S13.
S14.
1 0 0
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
N SP A M 3 C S K 4
L T A
plasma FCS P1 Ctr1 Ctr2 FCS P1 Ctr1 Ctr2PBMCs Ctr P1
IL-6
(p
g/m
l)
Whole blood +Anti-LTA Abs +isotype Whole blood +Anti-LTA Abs +isotype
C+ IRAK4-/-
IL-6
(pg
/ml)
WB, 48H NS PAM3 LTA
102
100
101
103
104
105
Supp Fig.
S15.
m e d ia a n t i - L T A is o m e d ia a n t i - L T A is o m e d ia a n t i - L T A is o
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
1 0 0 0 0
1 5 0 0 0
IL-6
(p
g/m
l)
N S
P A M 3
L T A
P B M C s M o n o c y t e s M o n o - d e p l e t e d P B M C s
S16.
N S P A M 3 C S K 4 L T A N S P A M 3 C S K 4 L T A N S P A M 3 C S K 4 L T A
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
4 0 0 0
8 0 0 0W T
M y D 8 8 - / -
T L R 2 - / -
T I R A P - / -
m e d i a + a n t i - L T A A b + C o n t r o l I s o t y p e A b
p = 0 . 0 0 4
n s
TN
Fa
(p
g/m
l)
S17.
C o n t r o l s
( n = 1 0 )
P a t i e n t
( R 1 2 1 W / R 1 2 1 W )
IV Ig s
( n = 1 6 )
0
1
2
3
4
OD