Lyme & Autism Connection = Incompetence to TLR2 Agonists
Mort Zuckerman
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Subject: Lyme & Autism Connection = Incompetence to TLR2 Agonists
Date: Sep 3, 2009 8:35 AM
:"Thus our findings indicate that children in the ASD test group may
be
less capable of controlling microbial infection in the initial stages,
leading
to ineffective signalling to the brain."
Welp., I knew I would be right about that too, since I
*NEVER* *LIE.*
There ya have it. More brain damaged children because
of the Yale pimps and their DCF whories.
Kathleen M. Dickson
http://www.actionlyme.org
http://www.actionlyme.org/PHILLIPS_JE_PERVERT.htm
==============================
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=19025588
J Neuroinflammation. 2008; 5: 52.
Published online 2008 November 21. doi: 10.1186/1742-2094-5-52.
PMCID: 2625336
Copyright © 2008 Jyonouchi et al; licensee BioMed Central Ltd.
Impact of innate immunity in a subset of children with autism spectrum
disorders: a case control study
Harumi Jyonouchi,corresponding author1 Lee Geng,1 Agnes Cushing-Ruby,1
and Huma Quraishi2
1Division of Allergy/Immunology and Infectious Diseases, Department of
Pediatrics, University of Medicine and Dentistry of New Jersey (UMDNJ)-
New Jersey Medical School (NJMS), Newark, NJ, USA
2Department of Surgery, University of Medicine and Dentistry of New
Jersey (UMDNJ)-New Jersey Medical School (NJMS), Newark, NJ, USA
corresponding authorCorresponding author.
Harumi Jyonouchi: jyan...@umdnj.edu; Lee Geng: gen...@umdnj.edu;
Agnes Cushing-Ruby: cush...@umdnj.edu; Huma Quraishi:
qura...@umdnj.edu
Received September 23, 2008; Accepted November 21, 2008.
This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/
licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly
cited.
Top
>Abstract
Background
Methods
Results
Discussion
Conclusion
Abbreviations
Competing interests
Authors' contributions
References
Abstract
Background
Among patients with autism spectrum disorders (ASD) evaluated in our
clinic, there appears to be a subset that can be clinically
distinguished from other ASD children because of frequent infections
(usually viral) accompanied by worsening behavioural symptoms and/or
loss/decrease in acquired skills. This study assessed whether these
clinical features of this ASD subset are associated with atopy,
asthma, food allergy (FA), primary immunodeficiency (PID), or innate
immune responses important in viral infections.
Methods
This study included the ASD children described above (ASD test, N =
26) and the following controls: ASD controls (N = 107), non-ASD
controls with FA (N = 24), non-ASD controls with chronic
rhinosinusitis/recurrent otitis media (CRS/ROM; N = 38), and normal
controls (N = 43). We assessed prevalence of atopy, asthma, FA, CRS/
ROM, and PID. Innate immune responses were assessed by measuring
production of proinflammatory and counter-regulatory cytokines by
peripheral blood mononuclear cells (PBMCs) in response to agonists of
Toll-like receptors (TLRs), with or without pre-treatment of
lipopolysaccharide (LPS), a TLR4 agonist.
Results
Non-IgE mediated FA was equally prevalent in both ASD test and ASD
control groups, occurring at higher frequency than in the non-ASD
controls. Allergic rhinitis, atopic/non-atopic asthma, and atopic
dermatitis were equally prevalent among the study groups except for
the CRS/ROM group in which non-atopic asthma was more prevalent
(52.6%). CRS/ROM and specific polysaccharide antibody deficiency
(SPAD) were more prevalent in the ASD test group than in the ASD
control, FA, and normal control groups: 23.1% vs. < 5% for CRS/ROS and
19.2% vs. < 1% for SPAD. However, CRS/ROM patients had the highest
prevalence of SPAD (34.2%). When compared to ASD and normal case
controls, PBMCs from 19 non-SPAD, ASD test group children produced: 1)
less IL-1β with a TLR7/8 agonist, less IL-10 with a TLR2/6 agonist,
and more IL-23 with a TLR4 agonist without LPS pre-treatment, and 2)
less IL-1β with TLR4/7/8 agonists with LPS pre-treatment. These are
cytokines associated with the neuro-immune network.
Conclusion
Clinical features of the ASD test group were not associated with
atopy, asthma, FA, or PID in our study but may be associated with
altered TLR responses mediating neuro-immune interactions.
Top
Abstract
>Background
Methods
Results
Discussion
Conclusion
Abbreviations
Competing interests
Authors' contributions
References
Background
Autism spectrum disorder (ASD) is a complex developmental disorder
encompassing a heterogeneous patient population. It is generally
agreed that there are at least two types of ASD with regard to disease
development; abnormal cognitive development evident from birth
(classical autism) and developmental regression, usually between 18–24
months of age, following apparent normal development (regressive
autism) [1].
In addition to behavioral symptoms, co-morbid clinical conditions such
as gastrointestinal (GI) symptoms are frequently noted in ASD
children. A high prevalence of GI symptoms, which often improve after
dietary intervention, has been reported by parents. This has led to
speculation that there may be a high prevalence of food allergy (FA)
in ASD children. Although IgE-mediated FA does not appear to be
prevalent in ASD children, our previous study indicated a higher
prevalence of non-IgE mediated FA (NFA) in ASD children. Specifically,
our results revealed increased tumour necrosis factor-α (TNF-α)
production against cow's milk protein along with correlating clinical
features consistent with NFA [2]. In these ASD children with NFA, we
also observed excessive production of TNF-α in response to LPS, an
agonist of Toll-like receptor 4 (TLR4) [3].
Apart from FA, we have also encountered a number of ASD children who
suffer from recurrent infection (typically viral syndromes)
accompanied by exacerbations of behavioral symptoms (hyperactivity,
temper tantrums, irritability and self-stimulatory behaviors) despite
good responses to dietary intervention. Such behavioral changes were
pointed out by teachers/therapists/care takers independent of parents.
Immune insult via microbial infection caused by various pathogens
appears to counter-act beneficial effects of behavioral, dietary, and
other intervention measures in these ASD children. However, atopy or
primary immunodeficiency (PID) does not appear to be a major factor in
these children. The above-described clinical observations led us to
hypothesize that in these ASD children, antigen non-specific (innate)
immune responses are altered, leading to dysregulated neuro-immune
interactions apart from PID or atopy.
Among 133 ASD children evaluated in our clinic, we identified 26 ASD
children with the above-described clinical features (ASD test group).
Interestingly, they are all documented to have shown regression at the
onset of symptoms. In this study, we assessed prevalence of atopy, FA,
CRS/ROM, and PID in the ASD test and control groups as well as in the
non-ASD control groups (FA, CRS/ROM, and normal controls). In
addition, we assessed responses to a panel of TLR agonists in 19 ASD
test group children and compared these to responses for ASD and normal
case controls.
Top
Abstract
Background
>Methods
Results
Discussion
Conclusion
Abbreviations
Competing interests
Authors' contributions
References
Methods
Study Subjects
Study subjects included children with ASD, CRS/ROM, and FA that were
referred to the Pediatric Allergy/Immunology Clinic at our institution
from April, 2005 through May, 2008. These children were evaluated for
atopic disorders, FA, and PID as medically indicated. They were also
enrolled in institutional review board- (IRB-) approved study
protocols that involved additional immunological evaluation of innate/
adaptive immune responses. Control children were recruited from the
same clinic and also from the General Pediatrics Clinic, and were
evaluated for innate immune responses and atopy through their
participation in established study protocols. Control children were
children with no major medical issues affecting major organ systems,
no documented behavioural problems, and age-appropriate cognitive
activity as determined by his/her primary physician's evaluations and
reported school performances obtained from chart review and from
histories taken from their parents.
Demographic data of the study subjects are summarized in Table 1.
Consistent with previous reports, the male/female ratio is
significantly high in ASD children [4]. Normal control children also
included higher numbers of males, reflecting our efforts to recruit
normal control subjects matching the test group ASD children in age
and sex. None of our study subjects were diagnosed with celiac disease
or inflammatory bowel diseases.
Table 1
Table 1
Demographics of the study subjects
ASD diagnosis
ASD diagnosis was made or ascertained by DSM-IV (Diagnostic and
Statistical Manual of Mental Disorders IV) criteria, ADI-R (Autism
Diagnostic Interview-Revised), and/or ADOS (Autism Diagnostic
Observational Schedules). ASD diagnosis was made by board-certified
developmental pediatricians and/or pediatric neurologists/
psychiatrists. The ADI-R and ADOS were administered by certified
examiners from various autism centers including the ones at our
institution.
The ASD test group was defined by having 1) frequent infections (more
than 6/year documented by a physician) with poor responses to
treatment measures such as the first line antibiotics and 2) at least
3 occurrences of changes in behavioral symptoms and/or loss of
cognitive skills following infection, documented independently by
caretakers, teachers, and therapists. Parents of the ASD test group
reported repeated set backs of their children's progress at home and
at school due to loss of acquired skills and interference from
worsening behavioral issues. It is of note that all the ASD test group
subjects were documented to have normal growth and development that
was followed by major regression between 15–30 months of age. In most
cases, this regression followed some type of immune insult (typically
a viral syndrome.)
Diagnosis of atopic disorders
Allergic rhinitis (AR), allergic conjunctivitis (AC), and atopic
dermatitis (AD) were diagnosed with positive skin-prick test
reactivity and/or presence of allergen-specific IgE accompanied by
clinical features consistent with AR, AC, or AD [5-7]. Asthma
diagnosis was based on NIH guideline criteria [8]. Asthma without skin
test reactivity and/or allergen-specific IgE antibody was categorized
as non-atopic asthma[5]
Diagnosis of NFA
NFA to common dietary proteins (cow's milk protein, wheat, and soy)
was diagnosed using the following criteria: 1) presence of objective
GI symptoms (diarrhea, loose stool, and constipation) that resolved
with avoidance of causative DPs, 2) delayed (more than 6 h) recurrence
of GI symptoms following exposure to offending food after resolution
of GI symptoms, and 3) cellular immune reactivity to offending dietary
proteins defined as production of TNF-α and/or IL-12 greater than 1
standard deviation above control mean value by PBMCs after stimulation
with causative dietary proteins [2].
Evaluation of ROM and CRS
ROM was defined as physician-diagnosed OM occurring more than 6 times/
year. Most of the ROM patients referred to our clinic were
unresponsive to surgical interventions (installment of pressure-
equalizing tube and tonsillectomy/adenoidectomy). CRS was diagnosed
with documentation of sinus inflammation by imaging studies
(computerized tomography) despite prior prolonged/repeated courses of
antibiotic therapy (more than 12 weeks) [9].
Evaluation of primary immunodeficiency
Presence of PID was evaluated in those patients with clinical features
suggesting immune abnormalities such as recurrent or prolonged
infections unresponsive to conventional treatment. Our routine
immunological workup includes the following: serum levels of
immunoglobulin (Ig), IgG subclass, and antibody titers to recall
antigens; enumeration of peripheral blood T and B cell subsets, and T
cell responses to mitogens and recall antigens.
For evaluation of innate immune responses, blood samples were
collected following obtainment of the IRB-approved signed consent
forms. At the time of venipuncture, all the study subjects were
afebrile, were not on antibiotics, and had no evidence of acute
microbial illnesses by physical examination.
Cell cultures
PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation.
Innate immune responses were assessed by incubating PBMCs (106 cells/
ml) overnight with TLR4 agonist (LPS; 0.1 μg/ml, GIBCO-BRL,
Gaithersburg, MD), TLR2/6 agonist (zymosan; 50 μg/ml, Sigma-Aldrich,
St. Luis, Mo), and TLR7/8 agonist (CL097, water-soluble derivative of
imidazoquinoline, 20 μM, InvivoGen, San Diego, CA) in RPMI 1640 with
additives as described before [10]. We then measured the levels of
proinflammatory (TNF-α, IL-1β, IL-6, IL-12p40, IL-23) and counter-
regulatory (IL-10, sTNFRII, and TGF-β) cytokines in the culture
supernatant. When testing the effects of LPS pre-treatment, PBMCs were
incubated overnight with LPS (0.1 μg/ml), the medium replaced, and
then re-stimulated with the above-described TLR agonists.
All cytokine levels were measured by an enzyme-linked immunosorbent
assay, using OptEIA™ Reagent Sets (BD Pharmingen, San Diego, CA) for
TNF-α, IL-1β, IL-6, IL-10, IL-12p40 and ELISA reagent set (R & D,
Minneapolis, MN) for sTNFRII and TGF-β. IL-23 ELISA kit was purchased
from eBioscience (San Diego, CA). Intra- and inter-variations of
cytokine levels were less than 5%.
Statistics
For comparison of test values with control values, a Wilcoxon signed
rank test was used. For comparison of values of multiple groups, a
Kruskall-Walls test was used. For case control studies, a paired
Wilcoxon signed rank test was used. Assessment of difference of
frequency was tested with a Chi square (χ2) test. These tests were
performed using R.2.5.1 (R-Development Core Team 2007). A p value of <
0.05 was considered to be statistically significant.
Top
Abstract
Background
Methods
>Results
Discussion
Conclusion
Abbreviations
Competing interests
Authors' contributions
References
Results
Prevalence of atopy, FA, CRS/ROM, and PID in the study subjects
Table 2 summarizes the prevalence of atopy, FA, CRS/ROM, and PID in
all of the study groups.
Table 2
Table 2
Prevalence of atopy, food allergy, CRS/ROM and primary
immunodeficiency in study subjects
Despite the fact that many parents of ASD children reported that their
children suffered from 'allergies', the prevalence of atopic disorders
(AR+AC, AD, and atopic asthma) in the ASD test and ASD control groups
were similar to that of the general population and to our normal
controls [11]. CRS is a major trigger for asthma [12] and, not
surprisingly, CRS/ROM patients were distinguished by a high frequency
of atopic and non-atopic asthma as compared to other study groups
(Table 2). In FA children, prevalence of atopy was not high but this
may reflect their young age. AR+AC, AD, and asthma frequency in
control children were equivalent to that in the general population
[11]. These results indicate that atopy is not closely associated with
clinical features of the ASD test group.
Consistent with the high prevalence of GI symptoms in ASD children,
non-IgE-mediated, but not IgE-mediated, FA was observed at a high
frequency in both ASD test and ASD control groups (Table 2). In
contrast, despite frequent antibiosis, which causes GI symptoms by
disrupting commensal flora and may subsequently predispose the
subjects to FA, NFA was not as prevalent in CRS/ROM children as in the
ASD children (p = 4.048e-11 by χ2 test) (Table 2). These results
indicate that FA is not closely associated with the clinical
characteristics of the ASD test group.
The ASD test group children showed higher frequency of CRS/ROM and PID
than did the ASD control group (Table 2). However, CRS/ROM patients
showed the highest frequency of PID [mainly specific polysaccharide
antibody deficiency (SPAD)] (Table 2). More importantly, diagnosis of
these diseases alone does not explain the frequent infections and
subsequent changes in behavioral symptoms in the majority of ASD test
group children (Table 2).
Evaluation of innate immune responses in the test group ASD children
We evaluated innate immune responses by assessing responses to TLR
agonists that are important for airway and gut mucosal immunity in the
ASD test group, excluding subjects diagnosed with SPAD. Results were
compared with those obtained in ASD and normal case controls.
Demographic summaries of ASD test-group subjects and of ASD and normal
case controls are shown in Table 3. Since the ASD test group patients
were all documented to have an initial regression, we selected ASD
case controls who also had documented initial regression. We were not
able to obtain such case controls in the FA and CRS/ROM study groups
mainly due to age-limitations.
Table 3
Table 3
Summary of matched cases
When PBMCs were stimulated with TLR agonists without LPS pre-
treatment, PBMCs from the ASD test group revealed lower IL-1β
production with a TLR7/8 agonist and lower IL-10 production with a
TLR2/6 agonist than did ASD control cells (Figs. (Figs.1,1Figure 1, ,
2).2Figure 2). In contrast, the ASD test group revealed higher IL-23
production with the TLR4 agonist than control groups (Fig. (Fig.
33Figure 3).
Figure 1
Figure 1
Figure 1
Lower IL-1β production with a TLR7/8 agonist in the ASD test group
without LPS pre-treatment. In all experiments shown in Figures
Figures1,1Figure 1, ,2,2Figure 2, ,3,3Figure 3, ,4,4Figure 4, ,
55Figure 5 and and6,6Figure 6, PBMCs were incubated (more ...)
Figure 2
Figure 2
Figure 2
Lower IL-10 production with a TLR2/6 agonist in the ASD-test group
without LPS pre-treatment. IL-10 production with a TLR2/6 agonist was
lower in the ASD-test group than in the ASD case controls.
Figure 3
Figure 3
Figure 3
Higher IL-23 production with a TLR4 agonist in the ASD test group
without LPS pre-treatment. IL-23 production with a TLR4 agonist was
lower in the ASD test group than in the ASD and normal case controls.
Following LPS pre-treatment, the ASD test group revealed lower IL-1β
production with TLR4 and TLR7/8 agonists (Fig. (Fig.4,4Figure 4, ,5).
5Figure 5). The ASD control group did not reveal such differences.
sTFNRII production with a TLR7/8 agonist following LPS pre-treatment
was lower in the ASD test group than in normal controls, and ASD
controls also revealed this tendency except for 2 outliers (Fig. (Fig.
66Figure 6)
Figure 4
Figure 4
Figure 4
Lower IL-1β production with a TLR4 agonist in the ASD test group
following LPS pre-treatment. In experiments shown in Figures
Figures4,4Figure 4, ,55Figure 5 and and6,6Figure 6, PBMCs were
treated with LPS (0.1 μg/ml) overnight and re-stimulated (more ...)
Figure 5
Figure 5
Figure 5
Lower IL-1β production with a TLR7/8 agonist in the ASD test group
following LPS pre-treatment. IL-1β production was lower in the ASD
test group following LPS pre-treatment in the ASD test group than in
the normal case controls.
Figure 6
Figure 6
Figure 6
Lower sTNFRII production with a TLR7/8 agonist in the ASD test group
following LPS pre-treatment. sTNFRII production with a TLR7/8 agonist
was lower in the ASD test group than in normal case controls.
Top
Abstract
Background
Methods
Results
>Discussion
Conclusion
Abbreviations
Competing interests
Authors' contributions
References
Discussion
Subtle immune abnormalities in ASD children have been reported by many
researchers [13-15]. However, results are rather variable partly due
to the small number of study subjects, heterogeneous patient
populations, and lack of careful immunological characterization of the
study subjects in these studies. Moreover, reported results are
conflicting; some studies indicate type 1 T-helper (Th1)-skewed
responses while other studies indicate Th2-skewed responses [13-16]. A
high prevalence of GI symptoms in autistic children also raised
possibilities of GI autoimmune conditions and FA. However, to date,
the role of the immune system in the onset and progression of ASD
remains unclear.
The effects of environmental factors in genetically vulnerable
individuals have also been implicated in the development of autism.
There is ample evidence indicating that there is increased oxidative
stress in autistic children [17]. The 'redux/methylation hypothesis'
postulates that environmental factors and genetic factors play
pathogenic roles in some autistic children [17]. It has been
postulated that exposure to xenobiotics in subjects with a genetic
predisposition for impaired methylation and/or increased
susceptibility to oxidative stress may result in the neurological
deficits observed in certain ASD children [18,19]. Oxidative stress
also activates innate immunity. Interestingly, the presence of low-
grade chronic inflammation has been reported in brain tissue of
individuals with autism [20,21]. This 'redux/methylation hypothesis'
may also be associated with skewed Th1 or Th2 responses in certain
conditions [22]. However, how such genetic/environmental factors
modulate the immune system in autistic children is not well
understood.
For a number of ASD children, parents report frequent infections
[recurrent pharyngitis and viral syndromes, ROM, and CRS] and an
unusually prolonged course of such illnesses. Following microbial
infection, parents repeatedly describe exacerbations of behavioral
symptoms and even loss of previously acquired cognitive skills, an
occurrence also documented by care-takers/teachers/therapists. This
study focused on this subset of ASD children (ASD test group) as
defined in the Methods section. Our first hypothesis is that clinical
features of the ASD test group are not associated with atopy, asthma,
FA, or PID.
To test this hypothesis, we retrospectively reviewed 133 ASD children
referred to the Pediatric Allergy/Immunology Clinic for evaluation of
atopy/immune abnormalities. Given the nature of our clinic, the ASD
children reviewed may have had more medical issues than ASD children
in general, as evidenced by their need to visit the clinic.
Nevertheless, it is advantageous to analyze these children, since
extensive data had already been generated by thorough allergy and
immune workups. We also have the advantage of having control non-ASD
children with chronic airway or GI inflammation who underwent a
similar workup (control CRS/ROM and FA groups) that enabled us to
address the presence of any distinguishing clinical features in the
ASD test group.
Prevalence of atopic disorders and asthma was equivalent to that
reported in general population in both test and control ASD children.
A lower prevalence of atopy in the control FA patients may reflect
their young age, since atopic disorders develop with age. Likewise, a
younger median age of the control ASD children indicates that atopic
diseases may develop with age. Nevertheless, given the frequency of
atopic disorders in the ASD test group, who are slightly older in
median age than the ASD controls, it is very unlikely that atopy is
associated with the clinical features of the ASD test group.
NFA was highly prevalent in both ASD groups, consistent with the high
prevalence of GI symptoms in ASD children (Table 2). As summarized in
Table 2, a major difference in the clinical features of the test vs.
control ASD children was the higher frequency of CRS/ROM and SPAD in
the ASD test group children. However, diagnoses of CRS/ROM and SPAD
were only seen in 6/26 and 5/26 of the ASD test group children,
respectively. All of the ASD test group children with SPAD suffered
from CRS/ROM (Table 2). SPAD was most prevalent in CRS/ROM children
(Table 2). These findings taken together suggest that the
distinguishing clinical features of the ASD test group are unlikely to
be associated with FA, asthma, or SPAD in most of the ASD test group
children.
In CRS patients, it was postulated that transmission of inflammatory
mediators via post-nasal dripping may be associated with chronic GI
irritation and predispose them to FA [23]. It was also speculated that
frequent antibiosis and resultant dysbiosis can disrupt gut mucosal
immune homeostasis, resulting in sensitization to food proteins.
However, our results revealed a higher prevalence of NFA in ASD
children than in CRS/ROM children, who were likely to have had more
frequent antibiosis than ASD children (Tables 2). Asthma was prevalent
in CRS/ROM children, which is not surprising since CRS is one of the
major triggers of asthma [12]. Our findings indicate CRS/ROM children
do not display clinical features similar to those observed in ASD test
or ASD control children, making it unlikely that CRS/ROM account for
the high prevalence of NFA in ASD children in our study.
It is of note that we did not find a high prevalence of known PID in
control ASD children, although the test group ASD children revealed a
few cases of SPAD (Table 2). Therefore, extensive immune workup
focusing on PID is likely to yield negative results in most ASD
children, especially those without concerning clinical features, as
reported previously [24].
In summary, our results support our initial hypothesis that the
distinct clinical features of our ASD test group are not associated
with atopy, asthma, FA, or presence of known PID. However, our results
have limitations. The number of the subjects and a potentially skewed
population due to the fact that the ASD study subjects were those
evaluated in the Pediatric Allergy/Immunology Clinic have some bearing
on our results. A larger population study will be informative to
further elaborate our initial findings.
In this study, we identified 19 ASD test group children without SPAD
among 133 ASD children (14.3%). Pathogens affecting these ASD test
group children appeared various, indicating the possibility of
impaired antigen-independent immune responses – innate immunity.
Innate immunity mounts the first line, antigen-independent immune
defense by recognizing microbial by-products or those from damaged
tissue cells via pattern recognition receptors including TLRs [25-27].
TLR-mediated responses lead to the production of various soluble
mediators that can signal the brain [28-31]. Such signalling events
help the central nervous system restore autonomic homeostasis and
provide inhibitory regulatory signals to prevent excessive immune
responses [28]. Subtle changes in innate immune responses can affect
the intricate neuro-immune network that is mediated by innate
immunity. Since NFA prevalence was similar in both the ASD groups, it
was the frequent occurrence of viral syndromes in the ASD test group
children that led us to our 2nd hypothesis. Namely, we theorized that
TLR responses, especially those sensing viral by-products, are altered
in the ASD test group.
Among TLRs, TLR2 is important for sensing encapsulated bacteria and
intracellular pathogens such as mycobacterium [32,33]. TLR4 is
important for sensing gram negative bacteria, common found in the GI
tract [32]. TLR7/8 are important for sensing single stranded RNA
derived from RNA viruses, which are common causes of childhood viral
infections including measles, rhinovirus (the most common cause of
'cold'), and influenza [34]. This study assessed responses to TLR2/6,
4, and 7/8 agonists by measuring production of a panel of cytokines in
the test group ASD children without SPAD and comparing those to
children with ASD and to normal case controls. It is important to
mention that since many FA patients outgrow this condition with age,
the FA control children are younger than the ASD children evaluated in
the study. In addition, the number of non-ASD controls with FA or CRS/
ROM is low due to the fact that the study was limited to patients
evaluated/treated in our clinic. Thus the study is lacking non-ASD
case controls with FA or CRS/ROM for evaluation of innate immune
responses.
Several abnormalities were found in the ASD test group: lower
production of IL-6 with the TLR2/6 agonist, lower production of IL-1β
with the TLR7/8 agonist, and higher production of IL-23 with the TLR4
agonists than case controls in the absence of LPS pre-treatment. These
cytokines which were found to be altered in production in the ASD test
group are the key regulators in the neuro-immune network [28,35,36].
Thus our findings indicate that children in the ASD test group may be
less capable of controlling microbial infection in the initial stages,
leading to ineffective signalling to the brain. It is also intriguing
to find increased production of IL-23 with the TLR4 agonist in the ASD
test, since IL-23 is associated with development and maintenance of
Th17 cells, a recently defined T-helper cell subset [37,38]. It is of
note that Th17 cells are implicated in various autoimmune and chronic
inflammatory diseases including multiple sclerosis and inflammatory
bowel diseases [37-39]. Thus the ASD test group children may be more
prone to Th17-mediated inflammatory responses.
When immune cells were pre-treated with LPS, a TLR4 agonist,
subsequent TLR responses were suppressed; this phenomenon is called
LPS tolerance [40]. This is thought to be important for immune
homeostasis in the gut, and especially in the colon where bacteria
exist at high density and hence innate immune cells in the mucosa are
likely to have frequent exposures to endotoxins (LPS) [40]. We found
lower IL-1β production by PBMCs with TLR4 or TLR7/8 agonists following
LPS pre-treatment in the ASD test group. IL-1β is one of the major
mediators of the neuro-immune network for maintenance of homeostasis
[35,36,41]. Thus suboptimal production of IL-1β may impair neuro-
immune signaling, while excessive IL-1β can be toxic to the brain
[35,41,42]. Our finding may indicate a risk of suboptimal neuro-immune
signalling in the ASD test group.
sTNFRII is an important counter-regulatory cytokine for TNF-α as made
evident by the marked therapeutic effects of exogenous sTNFRII in the
treatment of certain autoimmune diseases [43]. We also found lower
sTNFRII production by PBMCs with TLR 7/8 agonist following LPS pre-
treatment in the ASD test group, indicating a possibility of blunted
counter-regulatory measures in the ASD test group at the time of viral
infection. However, sTNFRII production in this condition also appeared
to be lower in the ASD control group except for 2 high outliers. Thus
this finding may not be specific for the ASD test group children.
Taken together, our findings of altered TLR7/8 responses with or
without endotoxin (LPS) pre-treatment may indicate a predisposition to
recurrent viral infections in the ASD test group as compared to ASD
and non-ASD case controls. The above-described, altered TLR responses
may also affect neuro-immune interactions in the test group. Our
findings may be useful further defining this subset of ASD children in
larger scale studies.
Top
Conclusion
Our results indicate the presence of a distinct subset of ASD
children. This subset, the ASD-test group, is characterized by
frequent infections and by recurrent loss of previously acquired
cognitive skills with worsening behavioral symptoms following
infection. Clinical features of this subset were not associated with
atopy, asthma, FA, or PID in this study but may be associated with
altered TLR responses important for neuro-immune interactions.
Top
Abbreviations
AC: allergic conjunctivitis; AD: atopic dermatitis; AR: allergic
rhinitis; ASD: autism spectrum disorder; CRS: chronic rhinosinusitis;
FA: food allergy; GI: gastrointestinal; Ig: immunoglobulin; IL:
interleukin; LPS: lipopolysaccharide; NFA: non-IgE mediated food
allergy; PBMCs: peripheral blood mononuclear cells; PID: primary
immunodeficiency; ROM: recurrent otitis media; SPAD: specific
polysaccharide antibody deficiency; sTNFRII: soluble TNF receptor II;
TGF: transforming growth factor; Th: T-helper; TLR: toll like
receptor; TNF: tumor necrosis factor.
Top
Competing interests
The authors declare that they have no competing interests.
Top
Authors' contributions
HJ designed this project, obtained approval of IRB, recruited study
subjects and obtained blood samples. HJ also reviewed the clinical
data, analyzed the data of TLR responses, and prepared most of the
manuscript.
LG carried out all the assays of TLR responses and helped HJ to
analyze data of TLR responses.
AC helped HJ for obtainment of the IRB approval and recruitment of the
study subjects and sample obtainment as a clinical coordinator in the
Pediatric Allergy/Immunology Clinic. HQ participated in recruitment of
CRS/ROM patients and also helped HJ in retrospective review of the
study subjects with regard to CRS/ROM as a pediatric otolaryngologist.
Acknowledgements
This study was funded by The Jonty Foundation, St. Paul, MN and the
Autism Research Institute, San Diego, CA, USA. The Jonty Foundation is
a private non-profit organization funding biomedical research in
neuroimmune and neurodevelopmental disorders in children. The ARI is a
public non-profit organization funding biomedical research in autism.
None of the authors has conflict of interest with The Jonty Foundation
or the ARI. Authors are thankful for critical review of the manuscript
by Dr. L Zussman-Huguenin.
Top
Abstract
Background
Methods
Results
Discussion
Conclusion
Abbreviations
Competing interests
Authors' contributions
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