Pattern recognition receptor expression is not impaired in patients
with chronic mucocutanous candidiasis with or without autoimmune
polyendocrinopathy candidiasis ectodermal dystrophy
M. Hong,*1 K. R. Ryan,*2
P. D. Arkwright,† A. R. Gennery,‡
C. Costigan,§ M. Dominguez,§
D. W. Denning,¶ V. McConnell,††
A. J. Cant,‡ M. Abinun,‡
G. P. Spickett,‡‡ D. C. Swan,§§
C. S. Gillespie,¶¶ D. A. Young* and
D. Lilic*
*Institute for Cellular Medicine, Faculty of
Medical Sciences, §§Bioinformatics Support Unit,
and ¶¶School of Mathematics and Statistics,
Newcastle University, ‡Department of Paediatric
Immunology, and ‡‡Regional Immunology
Department, Newcastle upon Tyne Hospitals
NHS Foundation Trust, Newcastle upon Tyne,
†Booth Hall Children’s Hospital, University of
Manchester, ¶Education and Research Centre,
Wythenshawe Hospital, Manchester, ††Northern
Ireland Regional Genetics Service, Belfast City
Hospital, Belfast, Northern Ireland, UK, and
§Our Lady’s Hospital for Sick Children, Dublin,
Republic of Ireland
Summary
Patients with chronic mucocutaneous candidiasis (CMC) have an unknown
primary immune defect and are unable to clear infections with the yeast
Candida. CMC includes patients with AIRE gene mutations who have autoim-
mune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), and
patients without known mutations. CMC patients have dysregulated cytokine
production, suggesting that defective expression of pattern recognition recep-
tors (PRRs) may underlie disease pathogenesis. In 29 patients with CMC
(13 with APECED) and controls, we assessed dendritic cell (DC) subsets and
monocyte Toll-like receptor (TLR) expression in blood. We generated and
stimulated monocyte-derived (mo)DCs with Candida albicans, TLR-2/6
ligand and lipopolysaccharide and assessed PRR mRNA expression by poly-
merase chain reaction [TLR-1–10, Dectin-1 and -2, spleen tyrosine kinase
(Syk) and caspase recruitment domain (CARD) 9] in immature and mature
moDCs. We demonstrate for the first time that CMC patients, with or without
APECED, have normal blood levels of plasmocytoid and myeloid DCs and
monocyte TLR-2/TLR-6 expression. We showed that in immature moDCs,
expression levels of all PRRs involved in anti-Candida responses (TLR-1, -2,
-4, -6, Dectin-1, Syk, CARD9) were comparable to controls, implying that
defects in PRR expression are not responsible for the increased susceptibility
to Candida infections seen in CMC patients. However, as opposed to healthy
controls, both groups of CMC patients failed to down-regulate PRR mRNA
expression in response to Candida, consistent with defective DC maturation,
as we reported recently. Thus, impaired DC maturation and consequent
altered regulation of PRR signalling pathways rather than defects in PRR
expression may be responsible for inadequate Candida handling in CMC
patients.
Keywords: APECED, Candida, DCs, PRRs, TLR
Accepted for publication 10 December 2008
Correspondence: D. Lilic, Musculoskeletal
Research Group (MRG), Institute for Cellular
Medicine, Faculty of Medical Sciences,
Newcastle University, Newcastle upon Tyne NE2
4HH, UK.
E-mail: desa.lilic@ncl.ac.uk
Present addresses: 1Nanjing University of
Chinese Medicine, Nanjing, China; 2Boehringer
Ingelheim Pharmaceuticals, Ridgefield, CT,
USA.
Introduction
Pattern recognition receptors (PRRs) on cells of the innate
immune system recognize core structures specific to
microorganisms (pathogen-associated molecular patterns:
PAMPs) that are alien to mammals [1,2]. Toll-like receptors
(TLR), particularly TLR-1, -2, – and –6, and C-type lectin
receptors such as Dectin-1 and -2, mannose receptor and
dendritic cell (DC)-specific intercellular adhesion molecule-
3-grabbing non-integrin, are known to be important PRRs
in fungal recognition, initiating immune responses to these
pathogens [3]. More recently, complex interactions and col-
laboration of these receptors through formation of het-
erodimers have been demonstrated to modulate immune
responses to different fungi [4].
In mice, activation of TLR-4 initiates interleukin (IL)-12
production by DCs generating a protective T helper type 1
(Th1) response. In contrast, activation of TLR-2 mediates a
Clinical and Experimental Immunology ORIGINAL ARTICLE doi:10.1111/j.1365-2249.2009.03873.x
40 © 2009 British Society for Immunology, Clinical and Experimental Immunology, 156: 40–51

non-protective Th2 response via IL-10 production and gen-
eration of T regulatory cells [5]. TLR-1 and TLR-6 form
heterodimers with TLR-2, modulating the Th1/Th2 balance
in response to fungi [6], while Dectin-1 synergizes with
TLR-2 and TLR-4 for cytokine production [7]. However,
humans with inborn errors of the IL-12/interferon (IFN)-g
pathway do not show increased susceptibility to Candida or
other fungal infections [8], suggesting that this pathway
might not have the same importance in fungal defences in
humans as in mice. Recent evidence suggests that the Th17
pathway is involved crucially in immune response to fungi
[9]. Dectin-1 signalling through a non-TLR signalling
pathway, that involves the spleen tyrosine kinase (Syk) and
adaptor caspase recruitment domain (CARD) 9 [10], primes
DCs to ‘instruct’ differentiation of CD4+ IL-17 producing
effector T cells, demonstrating a direct link between the
innate and adaptive immune system [11] in generating pro-
tective fungal responses.
Chronic mucocutaneous candidiasis (CMC) is a primary
immunodeficiency disorder (PID) with selective susceptibil-
ity to recurring and/or persistent debilitating infections
with the yeast Candida [12], and includes several subtypes.
The APECED syndrome (autoimmune polyendocrinopathy
candidiasis ectodermal dystrophy), also known as APS1
(autoimmune polyendocrinopathy type 1), is associated
with organ-specific autoimmune involvement of particularly
the endocrine glands and an underlying mutation of the
AIRE gene (online mendelian inheritance in man – OMIM
240300). Other subgroups of CMC include patients with
associated thyroid disease but no other autoimmunity
(OMIM 606415), isolated CMC with various modes of
inheritance (OMIM 11458, OMIM 212050) and sporadic
CMC. In these CMC patients the diagnosis remains clinical,
given that a genetic or biochemical marker is not yet
available.
Very little is known about the immune defect underlying
increased susceptibility to Candida infections in CMC
patients. Previously we have demonstrated dysregulated
cytokine production in response to Candida [12], suggesting
that the immune defect might be at the level of orchestrating
appropriate cytokine responses, rather than the effector T
cell level itself.
Our assumption that defects of PRRs, which can recognize
moeities from many different organisms, could specifically
cause an immune defect against Candida is based on the
recent recognition of similar scenarios underlying non-
conventional primary immune deficiencies (PIDs), defined
as a selective susceptibility to a single weakly pathogenic or
opportunistic organism. Surprisingly, in these PIDs a predis-
position to a single type of infection is caused by immune
defects affecting pathways central to the immune response.
Examples include disorders of the IL-12/IFN-g circuit,
resulting in selective susceptibility to infections with myco-
bacteria and Salmonella; defects in the TLR-3 pathway
resulting in selective predisposition to Herpes simplex infec-
tion; MyD88 deficiency and susceptibility to pyogenic bac-
terial infections. The infectious phenotype that the above
disorders confer in humans is much narrower than those of
corresponding mutant mice, suggesting that there is much
redundancy in human host defence in nature [13,14]. In
analogy, given that DCs are central initiators of immune
responses, while PRRs and TLRs are known to be involved in
production of cytokines in protective Candida immunity, we
hypothesized that reduced DC numbers and/or a defect of
PRRs involved in immune responses to Candida might
underlie a selective susceptibility to this microorganism in
patients with CMC.
In CMC patients and age- and gender-matched healthy
controls we investigated the proportion of DCs and DC
subsets in peripheral blood, as well as TLR receptor expres-
sion on blood monocytes; we then isolated monocytes
from peripheral blood and cultivated them in vitro into
monocyte-derived DCs (moDCs), which we stimulated/
matured with Candida and other relevant antigens to assess
expression of TLR-1–10, Dectin-1 and 2, CARD9 and Syk.
We demonstrate for the first time that CMC patients, with or
without APECED, have normal blood levels of plasmocytoid
and myeloid DCs and monocyte TLR-2/TLR-6 expression.
In immature moDCs, expression levels of all PRRs involved
in anti-Candida responses (TLR-1, -2, -4, -6, Dectin-1, Syk,
CARD9) were comparable to controls. However, as opposed
to healthy controls, both groups of CMC patients failed
to down-regulate PRR mRNA expression in response to
Candida, suggesting that impaired DC maturation and con-
sequently altered regulation of PRR signalling pathways
rather than defects in PRR themselves, may be responsible
for inadequate Candida handling in CMC patients.
These results complement our previous report of
impaired DC maturation, presented in a separate publica-
tion [15], where activation and function of these same DCs
was investigated in parallel through cytokine production and
cell surface marker expression.
Materials and methods
General conditions
The moDCs were used as representatives of skin and
mucosal myeloid-DCs involved in Candida recognition,
because obtaining skin biopsies from CMC patients for
purely research purposes was not deemed acceptable for
ethical reasons.
We stimulated moDCs with Candida albicans hyphae
(CH) rather than yeasts, as several studies suggest that
hyphae are the invasive morphotype of Candida in clinical
infections [16]. With the aim of investigating putative
impaired Candida binding to DCs, we assessed moDC
stimulation with a TLR ligand 2/6 (MALP-2) that engages
selectively the same TLRs that are known to bind Candida
and other yeasts [17]. Lipopolysaccharide (LPS) was used as
PRR expression in CMC
41© 2009 British Society for Immunology, Clinical and Experimental Immunology, 156: 40–51

a ‘positive’ non-Candida control, in order to assess moDC
functionality in response to other potent stimuli. Repeat
assessments and inclusion of additional stimuli were limited
by the quantity of blood we could draw from each patient,
particularly children.
Subjects
Patients. We studied 29 patients with CMC, 13 APECED
with a confirmed AIRE gene mutation and 16 non-APECED
without. All patients were screened for the two most
common AIRE gene mutations: p.R257X (a non-sense muta-
tion in exon 6) and c.964del13 (a 13 base pairs deletion in
exon 8) (Huch-Laboratory Diagnostics, Helsinki University
Hospital, Finland and Northern Molecular Genetics Service,
Institute of Human Genetics, Newcastle Upon Tyne, UK).
Thirteen patients were found to have an AIRE gene mutation
and the APECED syndrome, of whom nine had the
c.964del13 deletion and four had the p.R257X mutation. In
the remaining 16 non-APECED patients an AIRE mutation
was not detected. None of the patients without the AIRE
gene mutation had any clinical signs suggestive of APECED
that would justify investigations of additional AIRE gene
mutations. All patients were screened for autoantibodies to
type 1 IFNs, which were shown recently to be highly specific
for APECED patients [18]; these autoantibodies were
present in all APECED patients and none of the non-
APECED patients and controls.
In the APECED group, 10 patients had affected siblings
and nine patients in the non-APECED group, who are all
included in this study. Three patients from the latter group
had hypothyroidism, two with thyroid peroxidase anti-
bodies. No more than three patients from any one family
were studied. At the time of sampling, patients did not have
any other serious infections, were not on systemic antibiotic
treatment or receiving steroids. All patients suffered from
recurrent mucocutaneous Candida infection (mouth, nails,
skin, oesophagus and perineum), but no other fungal
infections including dermatophyte (e.g. tinea corporis)
infections. Patients were screened for systemic autoantibod-
ies including anti-nuclear factor (NF), smooth muscle, liver–
kidney microsomal, mitochondrial and gastric parietal cell
antibodies. Systemic autoantibodies were found in four of 13
APECED patients and five of 16 non-APECED patients.
Organ-specific autoantibodies and/or endocrinopathy were
observed in one or more of the following organs: parathy-
roid, thyroid, adrenal cortex, gonads and pancreas. Autoan-
tibodies were evaluated in patients’ sera using indirect
immunofluorescence on commercial rodent tissue (Euroim-
mune, Lubeck, Germany) for systemic autoantibodies and
monkey organ tissue (The Binding Site, Birmingham, UK)
for organ-specific autoantibodes. Endocrinopathy was diag-
nosed if/when there was clinical and laboratory evidence of
glandular hypofunction.
Controls. A total of 25 age- and sex-matched controls was
recruited for the study. Adults were healthy laboratory vol-
unteers, while control children were undergoing general ana-
esthesia for surgery to treat non-infectious causes (eye
squints, circumcision, hernia, etc.).
Both patients and healthy controls – parents on behalf of
children – received verbal and written explanations of the
study and signed informed consent forms. Ethical approval
was obtained from the Newcastle and North Tyneside Local
Research Ethics Committee.
Generation of moDCs from patient blood
Peripheral blood mononuclear cells were isolated from
patient blood by density centrifugation over a layer of lym-
phoprep (Axis-Shield, Oslo, Norway). CD14-positive cells
were then purified by magnetic separation on an LS column
following labelling of the cells with anti-CD14-coated mag-
netic beads (Miltenyi Biotec, Bergisch Gladbach, Germany),
which regularly yielded a purity of 94–98% CD14+ cells,
excluding T cell contamination. Purified monocytes were
then seeded into a 24-well plate at 0·75 ¥ 106 monocytes per
well in 1 ml total volume of RF10 media. RF10 media con-
sists of RPMI-1640 (BioWhittaker, Lonza, Wokingham, UK)
media supplemented with 10% fetal calf serum (FCS)
(PAA Laboratories, Pasching, Austria), 2 mM l-glutamine
(Sigma Aldrich, St Louis, MO, USA), and 1% penicillin–
streptomycin (Gibco, Carlsbad, CA, USA). Fifty ng/ml IL-4
and granulocyte–macrophage colony-stimulating factor
(GM-CSF) (Immunotools, Friesoythe, Germany) were
added to each well. Cells were incubated at 37°C with 5%
CO2. On day 3, the addition of 50 ng/ml IL-4 and GM-CSF
to the wells was repeated.
Dendritic cell maturation
On day 6 of the DC culture, immature DCs were divided
into four groups and activated as follows: no treatment
(unstimulated); addition of 1 : 10 000 final dilution of heat-
killed CH [American Type Culture Collection (ATCC)
#18804, Manassas, VA, USA]; 1 mg/ml of LPS (Invivogen, San
Diego, CA, USA); and 10 ng/ml of the purified TLR-2/6
ligand, MALP-2 (Apotech, Epalinges, Switzerland). Cells or
cytokines were harvested for analysis after 24 h on day 7.
Flow cytometry
To assess TLR expression on monocytes and quantitate DC
subsets in the blood, antibodies to various cell surface mol-
ecules and appropriate isotype controls were added directly
to 40 ml aliquots of patient blood. After a 15-min incubation
at room temperature, red blood cells were lysed by the addi-
tion of 1·5 ml of BD FacsLyse Buffer (BD Biosciences, San
Jose, CA, USA) for 10 min. Samples were then washed 2¥
in 1 ml fluorescence activated cell sorter (FACS) wash
(phosphate-buffered saline + 0·1% bovine serum albumin)
M. Hong et al.
42 © 2009 British Society for Immunology, Clinical and Experimental Immunology, 156: 40–51

and fixed in 1% paraformaldehyde (Sigma-Aldrich). The
following antibodies were used to stain cells in whole
blood: CD11c-fluorescein isothiocyanate (FITC), CD3-
phycoerythrin (PE), CD14-FITC, CD14-PE (iImmunoTools,
Friesoythe, Germany), CD19-PE, human leucocyte antigen
D-related (HLA-DR)-peridinin chlorophyll (BD Bio-
sciences), CD123-allophycocyanin (Miltenyi Biotec), TLR-1-
PE, TLR-2-PE, TLR-4-PE (eBioscience, San Diego, CA,
USA), TLR-6-FITC (Imgenex, San Diego, CA, USA),
immunoglobulin (Ig)G1-FITC, IgG1-PE, IgG2a-PE (BD
Pharmingen, San Jose, CA, USA) and appropriate isotype
controls (BD Biosciences). DCs were identified as HLA-DR/
CD11c (myDCs) or CD123 (pDCs) positive, CD3, CD14,
CD19 negative cells. CD14+ cells were stained for TLR
expression.
All stained cells were acquired using a FACScan (BD Bio-
sciences) equipped with a 488 nm laser and a 633 nm laser
upgrade. Acquired events were analysed using FlowJo soft-
ware (Tree Star, Inc., Ashland, OR, USA).
Real-time polymerase chain reaction
RNA was purified using the RNeasy Mini kit, including an
on-column DNAse I digestion (Qiagen, Crawley, UK). cDNA
was synthesized from 0·4 mg of total RNA, using Superscript
II reverse transcriptase and random hexamers in a total
volume of 20 ml according to the manufacturer’s instruc-
tions (Invitrogen). cDNA was stored at -20°C until used in
downstream real-time polymerase chain reaction (PCR).
Oligonucleotide primers were designed using Primer3 as
part of the Universal Probelibrary package (Roche; http://
www.roche-applied-science.com).
Primers for PRRs were as follows: TLR-1 forward
5′-CCTAGCAGTTATCACAAGCTCAAA-3′, reverse 5′-TCT
TTTCCTTGGGCCAT TC-3′; TLR-2 forward 5′-CGTTC
TCTCAGGTGACTGCTC-3′, reverse 5′-CCTTT GGATCC
TGCTTGC-3′; TLR-3 forward 5′-AGTTGTCATCGAATC
AAATTAAA GAG-3′, reverse 5′-AATCTTCCAATTGCG
TGAAAA-3′; TLR-5 forward 5′-GA CACAATCTCGGCT
GACTG-3′, reverse 5′-GCCAGGAACATGAACATCAA-3′;
TLR-6 forward 5′-TGAGGTTAGCCTGCCAGTTAG-3′,
reverse 5′-GCATTTACT CAAAAGAGACTGTTTCA-3′;
TLR-7 forward 5′-GCTAGACTGTCTCAAAAGA ACAAA
AA-3′, reverse 5′-GCCCACACTCAATCTGCAC-3′; TLR-8
forward 5′-GGGAGAATGAAGGAGTCATCTTT-3′, reverse
5′-TCAGCATTGACGACTGAA GG-3′; TLR-9 forward 5′-
TGTGAAGCATCCTTCCCTGT-3′, reverse 5′-GAGAGACA
GCGGGTGCAG-3′; TLR-10 forward 5′-TCTCAGCCCA
TCTCTGG ATT-3′, reverse 5′-TGGATTTCTTCCCGCAT
TTA-3′; Dectin-1 forward 5′-CTTT CTCGGCCCCAGA
CT-3′, reverse 5′-TTGGGTAGCTGTGGTTCTGA-3′; Syk
forward 5′-CGTCCACAACTTCCAGGTTC-3′, reverse 5′-
AGGGGAGGACTTTC TGTGG-3′; Dectin-2 forward 5′-TT
CAAGTCTCACCTGCTTCAGT-3′, reverse 5′-TCCAAGAA
GCTGGGCAAC-3′.
Relative quantitation of genes was performed using the ABI
Prism 7900HT sequence detection system (Applied Biosys-
tems, Foster City, CA, USA). TLR-1, -2, -3, -5–10, Dectin-1,
Syk and Dectin-2 expressions were determined using SYBR
Green (Takara Bio Inc., Shiga, Japan), in accordance with the
manufacturer’s suggested protocol. PCR mixtures contained
50% SYBR-Green PCR mix (Takara Bio Inc.); 50 nM (except
500 nM for TLR-3 and -10) of each primer and 3·2 ng cDNA
in a total volume of 20 ml. Conditions for PCR were as follows:
10 s at 95°C, then 40 cycles each consisting of 5 s at 95°C and
30 s at 60°C, followed by a dissociation plot. To confirm that
the amplification product was a single amplicon, products
were analysed by agarose gel electrophoresis.
The TLR-4 and CARD9 expression was determined using
TaqMan gene expression assays. PCR mixtures contained
50% TaqMan mastermix reagents (Sigma-Aldrich), 1 ml of
primer and probe mixture (assay ID Hs00152939_m1 for
TLR-4, Hs00364485_m1 for CARD9; Applied Biosystems),
1 ml H2O and 3·2 ng cDNA in a total volume of 20 ml. Con-
ditions for PCR were as follows: 2 min at 50°C, 10 min at
95°C, then a following 40 cycles, each consisting of 15 s at
95°C and 1 min at 60°C.
The GAPDH gene was used as an endogenous control to
normalize for differences in the amount of total RNA present
in each sample; GAPDH TaqMan primers and probe were
purchased from Applied Biosystems. TaqMan mastermix
reagents (Sigma-Aldrich) were used according according to
the manufacturer’s protocol. Where data are presented, the
2
–(CT gene – CT GAPDH)
(2–?CT) calculation was used as an approximate
measure of expression to allow comparison of expression
levels between genes.
The C. albicans
Freeze-dried C. albicans was purchased from ATCC (#18804)
and rehydrated according to the supplier’s instructions.
C. albicans was grown in autoclaved 1¥ broth [67 g/l yeast
nitrogen base (YNB) and 10% D-glucose] at 30°C. Candida
yeasts were propagated into hyphal forms in RPMI-1640
with 10% FCS at 37°C and heat-killed before use in DC
cultures. The concentration of Candida yeasts in YNB was
determined to be 24·5 ¥ 106 by counting in a haemocytom-
eter (10 mg/ml protein content). Transformation of > 98%
yeasts into hyphae was confirmed by visual inspection under
the microscope. Dense flasks of hyphae were decanted into
newly purchased sterile glass bottles and heated in a pressure
cooker for 30 min at 120°C, based on previous titration
experiment. Heat-killed C. albicans was centrifuged at 400 g
for 10 min, and the supernatant was removed without dis-
rupting the pellet. The pelleted material was used in cell
cultures at a final concentration of 1:10 000.
Statistical analysis
Results were analysed separately for groups of patients with
a confirmed AIRE gene mutation (APECED), patients
PRR expression in CMC
43© 2009 British Society for Immunology, Clinical and Experimental Immunology, 156: 40–51

without a confirmed AIRE gene mutation (non-APECED)
and healthy controls. Statistical analyses were performed
using the freely available software package r [19]. For each
gene, we fitted a random effects model with treatment
type, subgroups, age and patient as covariates. Interactions
between treatment type and subgroups were also considered.
Where necessary we transformed the expression level via a
log or a square root transformation. Variables that were not
statistically significant at the 0·05 level (based on the anova
results) were removed and the model was refitted. At each
point of model fitting the residuals were examined to check
that our assumptions were valid. To explore individual dif-
ferences among subgroups and treatments we calculated
adjusted P-values to control the family-wise error rate.
Average values are presented as means  standard errors of
the mean (s.e.m.); level of significance was set at P < 0·05.
The graphic network in Fig. 4 was calculated using graphic
Gaussian models and shows partial correlations which took
into account all available variables. Inclusion and exclusion
of edges was determined using the method described by
Drton and Perlman [20].
Expression analysis and clustering were assessed with
GeneSpring, Agilent Technologies software. The data analy-
sis for the clusters involved importing the data into Gene-
Spring and genes were clustered using a 5-cluster k-means
with 100 iterations using a Pearson correlation for the simi-
larity measure.
Results
Percentages of monocytes, plasmocytoid and myeloid
DC subsets in peripheral blood (ex vivo) from patients
with CMC and controls
In peripheral blood, monocytes, identified as CD14+ cells,
showed significantly higher percentages in APECED and
non-APECED patients compared with controls (Table 1).
There was no significant difference in percentages of myeloid
and plasmocytoid DCs between controls, APECED and non-
APECED patients (Fig. 1).
Expression of TLR-2 and TL-R6 on monocytes ex-vivo
The percentage and intensity of TLR-2 and TLR-6 expression
was assessed on peripheral blood monocytes, defined as
CD14+ cells. The percentage of TLR-2+ CD14+ cells did not
differ between patient groups and controls, but median fluo-
rescence intensity (MFI) was significantly higher in APECED
patients compared with controls and non-APECED patients.
Interestingly, non-APECED patients had a significantly
higher frequency of TLR-6+ CD14+ monocytes compared
with both APECED patients and controls, although the MFI
was comparable (Table 1).
The moDC mRNA expression of TLR-1, -4 and -6
(involved in protection against Candida)
All groups had similar TLR-1 mRNA expression in immature
DCs. Healthy controls significantly down-regulated TLR-1
expression in response to all stimuli, in particular to LPS
(Fig. 2a). As opposed to this, APECED and non-APECED
CMC patients failed to down-regulate TLR-1 mRNA expres-
sion following stimulation with either CH or TLR-2/6 ligand,
although they did respond to LPS (Fig. 2a). APECED and
non-APECED patients expressed lower levels of TLR-4
mRNA in immature DCs compared with controls, albeit not
significantly. As opposed to controls where TLR-4 was down-
regulated vigorously to all stimuli used, non-APECED
patients did not respond to CH, while APECED patients did
not respond to either CH or TLR-2/6 ligand. They did
respond, however, to LPS (Fig. 2b). An identical pattern to
TLR-1 was seen for TLR-6 (Fig. 2c), i.e. controls down-
regulated mRNA expression in response to all stimuli used
Table 1. Whole blood myeloid (my) and plasmocytoid (p) dendritic cell (DC) percentages and expression of Toll-like receptor (TLR)-2 and TLR-6 on
CD14+ monocytes.
APECED Non-APECED Controls APECED Non-APECED Controls
Percentage(%) Medianfluorescenceintensity(MFI)
CD14+ 7·6** 8·0** 6·3 n.a. n.a. n.a.
(0·4) (0·7) (0·6)
TLR-2 97 95 93 193* 160 142
(1·5) (1·3) (2·4) (21) (9) (19)
TLR-6 83 96** 74 27 16 14
(6) (1) (8) (8) (2) (2)
myDCs 0·54 0·46 0·52
(011) (0·01) (0·06)
pDCs 0·10 0·07 0·10
(0·02) (0·01) (0·01)
*P = 0·03; **P = 0·02. DCs in whole blood were identified as human leucocyte antigen D-related (HLA-DR) positive, CD11c (myDCs) or CD123
(pDCs) positive, CD3, CD14, CD19 negative cells. Monocytes, identified as CD14+ cells, were stained for TLR expression. Numbers are
means  standard error of the mean; n.a., not applicable. APECED, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy.
M. Hong et al.
44 © 2009 British Society for Immunology, Clinical and Experimental Immunology, 156: 40–51

while APECED and non-APECED patients responded only to
LPS.
In summary, healthy controls down-regulated TLR-1, -4
and -6 mRNA expression significantly in response to all
stimuli used. In contrast, APECED and non-APECED CMC
patients failed to down-regulate TLR-1, -4 and -6 expression
following stimulation with CH and/or TLR-2/6 ligand,
although the response to LPS stimulation was similar to
controls.
The moDC mRNA expression of TLR-2 (with
controversial role in protection against Candida)
The TLR-2 mRNA in immature moDCs was readily detect-
able and comparable in both patient groups and controls.
Interestingly, upon stimulation/maturation, healthy controls
down-regulated TLR-2 expression only in response to LPS.
CMC patients, both APECED and non-APECED failed to
down-regulate TLR-2 in response to any of the stimuli (data
not shown).
The moDC mRNA expression of TLR-3, -5 and -10 (not
known to have a role in protection against Candida)
The TLR-3 and TLR-10 mRNA were detectable, but did not
change in response to any of the stimuli used (data not
shown). An unusual finding was the down-regulation of
TLR-5 mRNA expression in healthy controls in response to
all stimuli – CH, TLR-2/6 ligand and LPS. Non-APECED
patients also responded to all three stimuli while APECED
patients did not respond to any (data not shown).
Dectin-1, Syk and CARD9 moDC mRNA expression
Immature moDCs expressed readily detectable and compa-
rable levels of mRNA in patients and controls. Dectin-1
mRNA levels in moDCs did not change in either healthy
controls or CMC patients (both APECED and non-
APECED) in response to CH or TLR-2/6-ligand stimulation,
but decreased significantly following LPS stimulation
(Fig. 3a). In contrast, healthy controls responded by
104
*1·31%/15·80%
104
103
103
102
102
HLA-DR
myDC(a)
C
D
11
c
101
101
100
100
104
**0·25%/3·01%
104
103
103
102
102
HLA-DR
pDC
C
D
12
3
101
101
100
100
100 92%
MFI: 184
104
80
103
60
102
TLR-2(b)
Fr
eq
ue
nc
y
40
101
0
20
100
100 79%
MFI: 27
104
80
103
60
102
TLR-6
TLR-2 TLR-6
Fr
eq
ue
nc
y
40
101
0
20
100
Fig. 1. Representative flow cytometry plots of whole blood stains. Whole blood was stained directly with antibodies to various cell surface
molecules and appropriate isotype controls, lysed, washed and fixed as described. Cells were acquired using a fluorescence activated cell sorter
(FACScan) (BD Biosciences, San Jose, CA, USA) and analysed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). (a, b) Myeloid (my) and
plasmocytoid (p) dendritic cells (DCs) flow cytometry dot-plots ex-vivo. *myDCs were identified as human leucocyte antigen (HLA)-DR/CD11c
positive, CD3/CD14/CD19 negative cells (1·31% of total lymphocyte population and 15·8% of CD11c/HLA-DR positive cells on graph shown).
**pDCs were identified as CD123/HLA-DR positive, CD3/CD14/CD19 negative cells (0·25% of total lymphocyte population and 3·01% of
CD123/HLA-DR positive cells on graph shown). (c, d) Flow cytometry histograms of frequency (%) and median fluorescence intensity (MFI) of
Toll-like receptor 2 (TLR-2+) or TLR-6+ monocytes in peripheral blood. CD14+ cells were stained for TLR expression (92% CD14/TLR-2 positive
and 79% CD14/TLR-6 positive on graphs shown). Negative peaks represent appropriate isotype controls.
PRR expression in CMC
45© 2009 British Society for Immunology, Clinical and Experimental Immunology, 156: 40–51

Controls APECED Non-APECED
0·00
0·05
0·10
0·15
Unstimulated
Candida hyphae
TLR 2/6 ligand
LPS
* *
*
(a)
TL
R
-1
m
R
N
A
re
la
tiv
e
ex
pr
es
si
on
** **
**
TLR-1
Controls APECED Non-APECED
0·0
0·3
0·4
0·5
* *
*
(b)
TL
R
-4
m
R
N
A
re
la
tiv
e
ex
pr
es
si
on
0·2
0·1 ** **
*
TLR-4
Controls APECED Non-APECED
0·00
0·10
0·15
** *
(c)
TL
R
-6
m
R
N
A
re
la
tiv
e
ex
pr
es
si
on
0·05
**
** **
TLR-6
Fig. 2. Messenger RNA (mRNA) expression of Toll-like receptors
(TLR)-1, -4 and -6 in monocyte-derived DCs (moDCs) from chronic
mucocutaneous candidiasis (CMC) patients with or without
autoimmune polyendocrinopathy candidiasis ectodermal dystrophy
(APECED) and controls following stimulation with Candida albicans
hyphae (CH), TLR-2/6 ligand and lipopolysaccharide (LPS)
(means  standard error of the mean). Immature moDCs from
patients and controls were either left unstimulated or treated with
CH, a specific TLR-2/6 ligand or LPS. Immature moDCs from
CMC patients demonstrate normal mRNA expression, whereas
down-regulation after stimulation with CH and TLR-2/6 is impaired.
Controls APECED Non-APECED
0
10
40
50
Unstimulated
Candida hyphae
TLR 2/6 ligand
LPS
(a)
D
ec
tin
-1
m
R
N
A
re
la
tiv
e
ex
pr
es
si
on
30
20
* * *
Dectin-1
Controls APECED Non-APECED
0
20
30
** **
(b)
S
ky
m
R
N
A
re
la
tiv
e
ex
pr
es
si
on
10
** **
*
Syk
Controls APECED Non-APECED
0·0
0·6
0·8(c)
C
A
R
D
9
m
R
N
A
re
la
tiv
e
ex
pr
es
si
on
0·4
0·2
**
**
**
CARD9
Fig. 3. mRNA expression of Dectin-1, Syk and CARD9 in
monocyte-derived dendritic cells (moDCs) from chronic
mucocutaneous candidiasis (CMC) patients with or without
autoimmune polyendocrinopathy candidiasis ectodermal dystrophy
(APECED) and controls following stimulation with Candida
albicans hyphae (CH), Toll-like receptor (TLR)-2/6 ligand and
lipopolysaccharide (LPS) (means  standard error of the mean).
Immature moDCs from patients and controls were either left
unstimulated or treated with CH, a specific TLR-2/6 ligand or LPS.
Immature moDCs from both CMC patients and controls have
detectable mRNA expression, whereas down-regulation after
stimulation with CH and TLR-2/6 is seen only with Syk.
M. Hong et al.
46 © 2009 British Society for Immunology, Clinical and Experimental Immunology, 156: 40–51

decreasing Syk mRNA expression in response to all three
stimuli. Further, APECED patients responded only to LPS
stimulation but not to either CH or TLR-2/6-ligand. Non-
APECED patients did not respond to TLR-2/6-ligand stimu-
lation but decreased their mRNA expression following
stimulation with both CH and LPS (Fig. 3b). Interestingly,
healthy controls did not alter CARD9 expression except in
response to LPS, as was also the case with APECED and
non-APECED patients (Fig. 3c). An additional finding was
that in healthy controls, the levels of Dectin-1 mRNA
increased significantly with age, which was not seen in either
APECED or non-APECED CMC patients (data not shown).
The moDCs TLR-7, -8, -9 and Dectin-2 mRNA
expression
mRNA levels of TLR-7, -8, -9 and Dectin-2 were undetect-
able or borderline low in both immature and mature moDCs
with all stimuli used.
A summary of changes in mRNA PRR expression follow-
ing stimulation in APECED, non-APECED patients and con-
trols is given in Table 2.
Based on the above results and data on cytokine produc-
tion by these same moDCs published previously [15], we
present a graphic dependency network (Fig. 4) demonstrat-
ing the interactions between cytokine production and PRR
expression by stimulated moDCs from CMC patients and
healthy controls. The network demonstrates the interdepen-
dence of cytokines and PRRs relevant for protective immu-
nity to Candida, and highlights differences between CMC
patients and healthy controls. It demonstrates that CMC
patients lack the interaction found in healthy controls
between TLR-1, TLR-4, TLR-6 and Th1 cytokines IL-12/IL-
23/IFN-g/IL-2, but have an interdependence of Dectin-1/
Syk/CARD9 that is not evident in healthy controls. These
findings suggest that the immune defect in CMC patients
may lie in the interaction of TLR-1, -4, -6 and Th1 cytokine
production, which may be compensated by the Dectin-Syk-
CARD9 pathway in CMC patients.
Clustering analysis of PRRS and cytokine levels, which
were reported previously [15], showed associations for
TLR-1, -4, -6, Dectin-1, Syk, CARD9 (set 1), TLR-2, -5, -10,
IL-2, -10, -13 (set 2) and TLR-7, 8, IL-6, IL-12, IL-23, IL-27
(set 3), which did not differ significantly between healthy
controls and patients (data not shown).
Discussion
Candida infections range from benign, commensal coloni-
zations of skin and mucous membranes in normal indi-
viduals to persistent, debilitating mucocutaneous infections
(as seen in CMC patients) or life-threatening systemic can-
didaemias with a significant mortality of more than 30% in
immunocompromised patients [16]. The role of the innate
immune system in protection against Candida has long
been recognized, but was thought to be limited to first-line
phagocytosis and killing. More recently, it has become clear
that the innate immune system not only distinguishes
between various microorganisms, but through interactions
with DCs initiates and guides subsequent adaptive immune
responses [21]. The crucial task of recognizing invading
pathogens and activating host responses is delivered by
PRRs, which recognize PAMPs on the microbe’s cell wall
[3].
Most components of the fungal cell wall are not found in
mammals and are thus ideal PAMPs for recognition of non-
self. The core of the C. albicans cell wall is made of polysac-
charide fibrils composed of b-glucan and chitin. The outer
layer is made of proteins that are glycosylated with mannose
– coined ‘mannan’ or ‘mannoprotein’. These structures differ
between fungi and are the major structures recognized by
PRRs such as TLRs and CTLs [22].
Table 2. Summary of Toll-like receptor/pattern recognition receptors (TLR/PRR) messenger RNA (mRNA) expression in APECED, non-APECED
patients and healthy controls.
APECED Non-APECED Controls APECED Non-APECED Controls APECED Non-APECED Controls
Candida hyphae TLR-2/6 ligand LPS
TLR-1 → → ↓ → → ↓ ↓ ↓ ↓
TLR-4 → → ↓ → ↓ ↓ ↓ ↓ ↓
TLR-6 → → ↓ → → ↓ ↓ ↓ ↓
TLR-2 → → → → → → → → ↓
Dectin-1 → → → → → → ↓ ↓ ↓
Syk → ↓ ↓ → → ↓ ↓ → ↓
CARD9 → → → → ↓ → ↓ ↓ ↓
TLR-5 → ↓ ↓ → ↓ ↓ → ↓ ↓
TLR-3 → → → → → → → → →
TLR-10 → → → → → → → → →
Immature monocyte-derived dendritic cells (moDC) were stimulated with Candida albicans hyphae, a specific TLR-2/6 ligand or lipopolysaccharide
(LPS). After 24 h, RNA was extracted from moDCs and processed for mRNA polymerase chain reaction analysis. Arrows indicate statistically significant
increases (!), decreases (") or no changes (,) of mRNA expression compared with levels in unstimulated moDCs. mRNA for TLR-7/8/9 and
Dectin-2 was undetectable (not shown). APECED, autoimmune polyendocrinopathy candidiasis ectodermal dystrophy.
PRR expression in CMC
47© 2009 British Society for Immunology, Clinical and Experimental Immunology, 156: 40–51

The role of TLRs in defence was first recognized in the
Drosophila model of fungal infection [23]. Subsequent
studies demonstrated that ligand recognition by TLRs
induces differential activation of signalling cascades and
leads to cytokine and chemokine production [24]. TLR-2
and TLR-6 were the first TLRs shown to be involved in
recognition of a fungal structure – zymosan, derived from
Saccharomyces cervisiae [17], and the adaptor MyD88, shared
by all TLRs (except TLR-3), was confirmed repeatedly to be
crucial for fungal defence in mice. At least four TLRs (TLR-2,
-4, -6) are involved in triggering cytokine production by C.
albicans. Recognition of Candida at the cellular level is medi-
ated by TLRs and CTL receptors [25]. TLR-4 induces mainly
proinflammatory signals through the MyD88-dependent
pathway, leading to activation of NF-kB, mitogen-activated
protein-kinases and interferon-releasing factor 3 (IRF-3),
stimulating Th1 responses through production of IL-12,
IFN-g, tumour necrosis factor (TNF-a) and type 1 IFNs,
resulting in efficient elimination of Candida infection. In
contrast, binding of Candida to TLR-2 leads to production of
transforming growth factor (TGF)-b and IL-10, leading to
tolerance, immunosuppression and generation of T regula-
tory cells [26]. However, TLR-2 is also able to associate with
Dectin-1 in the induction of proinflammatory responses
[27]. In CMC patients, we demonstrated previously low
IL-12 and very high IL-10 and IL-6 production by peripheral
blood mononuclear cells [28], with low IL-23 and high IL-6
responses by moDCs [15]. The results in our current study
show normal expression of TLRs and CTLs on immature
moDCs but inadequate down-regulation of TLR-1,
-4 and -6, suggesting defects in signalling pathways down-
stream of these receptors. More recent data suggest that
TLR-9 can recognize fungal DNA, but the importance of this
in fungal defence is still not clear [3]. Whether any of these
pathways is involved directly or impacts upon other signal-
ling pathways such as Dectin-1 is unclear, as would be sug-
gested by our ongoing studies which have demonstrated
markedly reduced IL-17 production in non-APECED (but
not APECED) patients [29]. AIRE gene mutations have also
been shown to down-regulate immunological pathways
critically in DCs from APECED patients [30], suggesting that
this mechanism may be involved in impaired immune
responses to Candida. However, AIRE gene mutations may
affect DC function in alternative ways, such as increasing
antigen-presentation to T cells [31] or interfering with T
regulatory cell induction/function, as has been demon-
strated [32,33].
Recently, patients with MyD88 deficiency were reported to
suffer with pyogenic infections, including invasive pneumo-
coccal diseases, but were otherwise healthy. Our patients
with CMC did not suffer with severe, life-threatening infec-
tions caused by pyogenic bacteria, although a previous
report in children with CMC demonstrated a high incidence
of bacterial infection and mortality from non-Candida
infections [34].
Mannose-binding lectin (MBL) is a soluble C-type lectin
receptor that binds Candida and other microorganisms,
promoting complement deposition. Low levels of MBL in
humans predispose to infections, although MBL-deficient
mice do not show increased susceptibility to infections with
Candida [35]. There is some evidence for a role of MBL in
recurrent vulvovaginal candidiasis [36], but its importance –
if any – in CMC is not clear.
In this study, we show for the first time that patients with
CMC, both APECED and non-APECED, have normal fre-
quencies of both myeloid and plasmocytoid DC subsets in
peripheral blood, indicating that a major lack of these cells
TLR-1
TLR-4
TLR-6
Syk
IL-12
IL23
CARD9
IL-2
Dectin1
IFN-γ
Fig. 4. Graphic dependency network describing interactions between
cytokine production and PRR expression on monocyte-derived DCs
(moDCs) from chronic mucocutaneous candidiasis (CMC) patients
and healthy controls, relevant for protective immunity against
Candida. The graphical network was calculated using graphical
Gaussian models. Each line indicates significant partial correlations
between genes. Black lines denote connections on networks for both
CMC patients and healthy controls. Red lines denote connections on
network for healthy controls only. Blue lines denote connections on
network for CMC patients only. The network shows that CMC
patients lack the interactions found in healthy controls between
Toll-like receptor (TLR)-1, TLR-4, TLR-6 and T helper 1 cytokines
interleukin (IL)-12/IL-23/interferon-g/IL-2 (marked in red), but
intriguingly demonstrate an interdependence of Dectin-1/Syk/CARD9
(marked in blue) that is not evident in healthy controls.
M. Hong et al.
48 © 2009 British Society for Immunology, Clinical and Experimental Immunology, 156: 40–51

is not responsible for their increased susceptibility to
Candida infections. The higher frequency of blood mono-
cytes in CMC patients (both APECED and non-APECED)
might be due to ongoing Candida-induced inflammation.
Almost all monocytes in both patients and controls
expressed TLR-2, which was up-regulated in APECED
patients. Interestingly, TLR-6 was expressed on almost all
monocytes in non-APECED patients but not in the other
groups. These findings argue against an intrinsic defect in
TLR-2 and TLR-6 expression on monocytes in CMC
patients as an underlying cause of their increased suscepti-
bility to Candida infections.
In our study, TLR-1, -4, -6, -2 and TLR-5 mRNA was
expressed in immature moDCs in all patients and controls
and the level of expression did not differ significantly
between groups. This is the first study to report that expres-
sion of these TLRs in immature myeloid DCs of APECED
and non-APECED CMC patients is normal.
Previous studies have demonstrated that when moDCs are
induced to mature by stimulation with pathogens or patho-
gen PAMPs, levels of TLR mRNA decrease or disappear, as
opposed to macrophages and monocytes [37] where TLR
mRNA increases. In healthy individuals, decreased mRNA
expression was already reported 2 h after stimulation, but
was most obvious after 24 h [38], suggesting that decreased
levels of TLR mRNA are consistent with normal DC matu-
ration dynamics. In our study, we found that healthy controls
down-regulated TLR-1, -4, -6 and -5 in response to all
stimuli used (CH, TLR-2/6 ligand and LPS). In contrast,
APECED patients responded only to LPS and did not down-
regulate TLR-1, -4, -6 and -5 mRNA expression when stimu-
lated with either CH or TLR-2/6 ligand. Non-APECED
patients failed similarly to down-regulate TLR-1 and TLR-6,
but they did down-regulate TLR-4 in response to TLR-2/6
ligand (but not CH) and down-regulated TLR-5 in response
to all stimuli, similar to controls. On the other hand, TLR-2
expression in healthy controls changed only in response to
LPS, but no change was seen following CH or TLR-2/6 ligand
stimulation. In APECED and non-APECED patients, TLR-2
mRNA expression did not change in response to any of the
stimuli used. In summary, when stimulated with Candida
and Candida-like stimuli, APECED patients did not down-
regulate any of the TLRs known to be involved in anti-
Candida responses (TLR-1, -4, -6, -2), while non-APECED
patients down-regulated only TLR-4 in response to TLR-2/6
ligand. These findings suggest that moDCs in both APECED
and non-APECED patients have normal patterns of mRNA
TLR expression in immature DCs, but that progression to
maturation is altered, particularly in response to Candida
and Candida-like stimuli, suggesting impaired maturation of
DCs in these patients.
The results reported in this paper are part of a larger
study, which includes assessment of a range of cytokines
produced (IL-12p70, IL-23, IFN-g, IL-2, TNF-a, IL-6,
TGF-b, IL-10, IL-5, IL-13) as well as cell-surface matura-
tion marker expression (CD83, CD86, HLA-DR) by the
same DCs on which PRR expression was assessed, pub-
lished recently [15]. These results demonstrate that
in both APECED and non-APECED CMC patients DC
function was impaired, as evidenced by altered cytokine
expression profiles and DC maturation/activation: (i) both
groups over-produce IL-2, IFN-g, TNF-a, IL-13 and dem-
onstrated impaired DC maturation; (ii) only non-APECED
patients showed markedly decreased Candida-stimulated
production of IL-23 and increased production of IL-6
markedly, suggesting impairment of the IL-6/IL-23/Th17
axis; and (iii) in contrast, only APECED patients showed
DC hyperactivation, which may underlie altered T cell
responsiveness, autoimmunity and impaired response to
Candida. The altered cytokine production may well be
linked to the altered PRR down-regulation in maturing
DCs, in particular TLR-1, -4 and -6, which are known to be
involved in Candida responses.
The interdependence of cytokines and PRRs relevant for
protective immunity to Candida in CMC patients and
healthy controls is also demonstrated in Fig. 4. Amazingly,
it shows that CMC patients lack the interaction found in
healthy controls between TLR-1, -4, -6 and Th1 cytokines
IL-12/IL-23/IFN-g/IL-2, but demonstrate an interdepen-
dence of Dectin-1/Syk/CARD9 that is not evident in healthy
controls, possibly as a compensatory mechanism.
An unexpected and unexplained finding is the down-
regulation of TLR-5 mRNA in both healthy controls and
non-APECED patients to CH, TLR-2/6, suggesting that
TLR-5 may be involved in recognition of Candida PRRs,
which has not been observed previously. Alternatively, ‘cross-
talk’ among TLRs and their ligands can be involved, where
microbial PAMPs are able to affect mRNA and protein
expression of different TLRs. It was reported that in addition
to flagellin, which is the known TLR-5 ligand, unrelated
PAMPs such as peptidogycan can exert a general stimulatory
activity on TLR-5 expression [39].
Dectin-1, Syk and CARD9 mRNA levels in immature
moDCs were readily detectable in all groups. Both CMC
patients and healthy controls showed a robust down-
regulation in response to LPS, but less so to Candida
or TLR-2/6 ligand stimulation, where patients actually
responded more robustly than controls. This was surprising,
given the important role of the Dectin-1, Syk and CARD9
pathways in fungal immune responses [40]. A possible expla-
nation is the lack of b-glucan on the surface of CH as
opposed to yeasts, as reported previously [27], which is nec-
essary for the engagement of Dectin-1. Dectin-1 binds
b-glucans on the Candida cell wall and activates the Syk–
CARD9 signalling pathway [41], leading to production of
IL-17, which is thought increasingly to be of major impor-
tance in protection against Candida infection [9]. Recently, a
homozygous mutation in the CARD9 gene was reported in a
family where patients suffered with recurrent oral and
vaginal candidiasis, but in contrast to classical CMC, suffered
PRR expression in CMC
49© 2009 British Society for Immunology, Clinical and Experimental Immunology, 156: 40–51

with other fungal (dermatophyte) infections as well [42].
The relevance of this finding for patients with classical CMC
(which were investigated in this study) is under investiga-
tion, although our results demonstrate that both APECED
and non-APECED CMC patients responded to Candida by
down-regulating CARD9. Interestingly, the network connec-
tivity analysis in our study (Fig. 4) actually suggests that the
Dectin-1–Syk–CARD9 pathway was of greater relevance for
Candida responses in our CMC patients than in healthy
controls.
In summary, we have demonstrated that CMC patients,
with or without APECED, do not lack plasmocytoid or
myeloid DCs in blood, nor do they harbour defects of
monocyte-TLR expression, relevant for anti-Candida
responses. In immature moDCs, levels of mRNA expression
for all PRRs involved in anti-Candida responses (TLR-1, -2,
-4, -6, Dectin-1, Syk, CARD9) were comparable to normal
controls, suggesting strongly that defects in PRR expression
are unlikely to be responsible for the increased susceptibil-
ity to Candida infections in these patients. However, both
groups of CMC patients (APECED patients in particular)
fail to down-regulate PRR mRNA expression in response to
Candida stimulation, implying defective DC maturation,
possibly involving defective TLR and/or CTL signalling,
which could be responsible for inadequate Candida han-
dling seen in these patients. Further investigation of TLR
and CTL signalling pathways will elucidate putative defects
in CMC patients which may underlie their inability to clear
Candida.
Acknowledgements
M. Hong and K. R. Ryan as first authors and D. Lilic and D.
A. Young as senior authors contributed equally to the paper.
We are very grateful to patients, healthy volunteers and their
families for supporting and participating in this research. We
thank Dr Idse Harrema, Dr Mike Clarke and the Medical and
Nursing staff in the Departments of Paediatric Surgery and
Paediatric Ophtalmology, Royal Victoria Infirmary, NUTH
NHS Foundation Trust for enabling and supporting collec-
tion of blood samples from healthy children. We thank Anne
Curtis, Head of the Molecular Genetic Diagnostics Labora-
tory, NUTH NHS Foundation Trust for screening for
AIRE gene mutations. This research was supported by a
Pfizer Independent Research Grant; the Primary Immuno-
deficiency Association (PIA), UK, Registered Charity
no. 803217; and UK/China Scholarships for Excellence
Programme.
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