**Table 5.**

**91**

subjects and their variable behavioral symptoms.

**4. Discussion**

*1*

*2*

*3*

**Table 6.**

*subjects.*

*Associations between Monocyte Cytokine Profiles and Co-Morbid Conditions in Autism…*

**ASD without comorbid condition**

IL-6 (CL097) 6747.6 ± 1939.23 7795.3 ± 1824.6 6325.5 ± 2011.2 p < 0.005 IL-6 (ß-glucan) 5502.2 ± 1725.7 6211.1 ± 1731.7 5230.4 ± 1706.7 p < 0.05 IL-6 (ß-glucan+LPS) 6505.7 ± 1654.5 7661.9 ± 1943.0 5756.6 ± 1875.3 p < 0.00001 IL-1ß (ß-glucan+LPS)2 3176.5 ± 1278.1 3706.3 ± 1029.4 2545.7 ± 987.1 p < 0.001 sTNFRII (zymosan) 618.2 ± 350.1 723.0 ± 293.6 663.7 ± 368.1 p = 0.069 CCL2 (zymosan) 6717.1 ± 5609.0 8055.4 ± 4899.3 6138.5 ± 6351.9 p < 0.05

IL-10 (LPS)2 1289.6 ± 499.9 1497.9 ± 422.6 1200.0 ± 543.7 p < 0.05 sTNFRII (LPS)2 1228.6 ± 473.4 1410.4 ± 484.6 1172.5 ± 551.9 p < 0.05 TGF-ß (medium) 535.1 ± 469.6 685.1 ± 443.1 398.0 ± 372.1 p < 0.01 TGF-ß (LPS) 545.7 ± 498.1 689.8 ± 436.2 372.8 ± 340.1 p < 0.005 TGF-ß (zymosan) 429.2 ± 415.4 542.8 ± 356.7 285.9 ± 287.0 p < 0.001 TGF-ß (CLO97) 459.3 ± 442.9 613.7 ± 421.0 325.8 ± 299.2 p < 0.005 TGF-ß (ß-glucan) 370.4 ± 335.7 518.4 ± 345.7 259.7 ± 280.8 p < 0.0005

*Abbreviations used include: IL; interleukin, LPS, lipopolysaccharide, PANS; pediatric acute-onset neuropsychiatric syndrome, TGF, transforming growth factor, TNF; tumor nectrosis factor, sTNFRII; soluble TNF receptor II.*

*Changes in IL-1ß (ß-glucan+LPS) production with PANS like symptoms are affected with ASD severity. Changes in IL-10 and sTNFRII production with LPS was affected by ASD severity (p < 0.01) and Seizure disorder (p < 0.05). Changes in production of IL-10 (LPS) with sleep disorder is affected with the use of SSRIs (p < 0.005), and specific antibody deficiency and PANS like behaviors (p < 0.05) by co-variance analysis. sTNFRII production with sleep* 

*Differences of monocyte cytokine profiles with presence of PANS like symptoms and sleep disorders in ASD* 

**Non-ASD Control**

**Kruskal-Wallis test**

 **with comorbid condition**

**PANS like symptoms N = 73 N = 50 N = 2**

**Sleep disorders N = 56 N = 67 N = 26**

*disoders are affected with the use of SSRIs (p < 0.005) by co-variance analysis.*

*The results were expressed as a mean ± SD. Cytokine levels were shown as pg/ml.*

ASD subjects suffer from multiple co-morbid conditions. However, we know little about how the presence of co-morbid conditions are associated with ASD pathogenesis. Core ASD symptoms used for diagnosis such as irritability, hyperactivity, selfinjurious behaviors, etc. can be affected by discomfort and pain caused by co-morbid medical conditions. In addition, recently, mounting evidence indicates a pathogenetic association between GI symptoms and the onset/progress of ASD [5, 9]. This may also be true for other common co-morbid conditions such as seizure disorders. Unfortunately, impaired expressive language in ASD subjects make it more difficult to diagnose co-morbid medical conditions. For example, sinus headache caused by untreated sinusitis and AR can aggravate head banging and aggression (pinching others, etc.). Too often, such behaviors are dismissed as just being autistic, and diagnostic and treatment measures for common childhood diseases may not be properly sought in ASD children [30]. Considering the fact current ASD diagnosis is based on behavioral symptoms, the presence of co-morbid medical conditions may hold a key to assess pathogenesis in markedly heterogeneous ASD

When addressing the importance of co-morbid conditions frequently seen in ASD subjects, the role of immune mediated inflammation likely needs to be

*DOI: http://dx.doi.org/10.5772/intechopen.95548*

**Comorbid conditions ASD1**

*Differences in monocyte cytokine production in association with GI symptoms, seizures disorders, allergic rhinitis, and antibody deficiency in ASD subjects.*


*Associations between Monocyte Cytokine Profiles and Co-Morbid Conditions in Autism… DOI: http://dx.doi.org/10.5772/intechopen.95548*

*1 Abbreviations used include: IL; interleukin, LPS, lipopolysaccharide, PANS; pediatric acute-onset neuropsychiatric syndrome, TGF, transforming growth factor, TNF; tumor nectrosis factor, sTNFRII; soluble TNF receptor II. 2 Changes in IL-1ß (ß-glucan+LPS) production with PANS like symptoms are affected with ASD severity. Changes in IL-10 and sTNFRII production with LPS was affected by ASD severity (p < 0.01) and Seizure disorder (p < 0.05). Changes in production of IL-10 (LPS) with sleep disorder is affected with the use of SSRIs (p < 0.005), and specific antibody deficiency and PANS like behaviors (p < 0.05) by co-variance analysis. sTNFRII production with sleep disoders are affected with the use of SSRIs (p < 0.005) by co-variance analysis. 3 The results were expressed as a mean ± SD. Cytokine levels were shown as pg/ml.*

#### **Table 6.**

*Autism Spectrum Disorder - Profile, Heterogeneity, Neurobiology and Intervention*

**90**

**Comorbid conditions**

IL-1ß (medium)

IL-10 (medium)

IL-10 (LPS)2 IL-10 (zymosan)

IL-10 (CL097) IL-12 (zymosan)

*use of neuroleptics/SSRIs and PANS like symptoms.*

**Table 5.**

*3The results were expressed as a mean ± SD. Cytokine levels were shown as pg/ml.*

*Differences in monocyte cytokine production in association with GI symptoms, seizures disorders, allergic rhinitis, and antibody deficiency in ASD subjects.*

**ASD1 with comorbid condition**

214.5 ± 253.3 328.3 ± 336.4 1215.8 ± 539.2 506.3 ± 399.8 863.2 ± 570.6 302.2 ± 290.3 *1Abbreviations used: ASD; autism spectrum disorder, GI; gastrointestinal, IL; interleukin, LPS; lipopolysaccharide, TNF; tumor necrosis factor.*

*2Co-variance analysis revealed that changes in TNF-*α *and TNF-*α*/sTNFRII (zymosan) production with GI symptoms are affected by ASD severity (p < 0.05). Changes in sTNFRII production (ß-glucan)* 

*with allergic rhinitis was affected with the use of anti-seizure medications (p < 0.05). Changes in production of IL-6 (zymosan) and IL-10 production (LPS) with antibody deficiency was affected with the* 

**ASD without comorbid condition**

646.6 ± 503.0 1471.8 ± 417.9 620.1 ± 375.3 1150.0 ± 605.8

426.4 ± 354.6

**Non-ASD Control**

270.3 ± 185.9 597.1 ± 355.7 1200.0 ± 543.7

651.4 ± 380.2 1093.0 ± 579.5 360.7 ± 425.0

**Kruskal-Wallis test**

p < 0.05

p < 0.005

p < 0.05

p = 0.128

p = 0.07

p = 0.1206

*Differences of monocyte cytokine profiles with presence of PANS like symptoms and sleep disorders in ASD subjects.*

#### **4. Discussion**

ASD subjects suffer from multiple co-morbid conditions. However, we know little about how the presence of co-morbid conditions are associated with ASD pathogenesis. Core ASD symptoms used for diagnosis such as irritability, hyperactivity, selfinjurious behaviors, etc. can be affected by discomfort and pain caused by co-morbid medical conditions. In addition, recently, mounting evidence indicates a pathogenetic association between GI symptoms and the onset/progress of ASD [5, 9]. This may also be true for other common co-morbid conditions such as seizure disorders.

Unfortunately, impaired expressive language in ASD subjects make it more difficult to diagnose co-morbid medical conditions. For example, sinus headache caused by untreated sinusitis and AR can aggravate head banging and aggression (pinching others, etc.). Too often, such behaviors are dismissed as just being autistic, and diagnostic and treatment measures for common childhood diseases may not be properly sought in ASD children [30]. Considering the fact current ASD diagnosis is based on behavioral symptoms, the presence of co-morbid medical conditions may hold a key to assess pathogenesis in markedly heterogeneous ASD subjects and their variable behavioral symptoms.

When addressing the importance of co-morbid conditions frequently seen in ASD subjects, the role of immune mediated inflammation likely needs to be considered as a common denominator. The immune system has long been thought to play a role in neuroinflammation and is implicated with pathogenesis of ASD. One of the most extensively studied animal models of ASD is MIA, in which, ASD like behavioral changes in offspring are induced by sterile immune activation through stimuli of innate immunity given to pregnant rodents [13]. Discovery of IIM [15, 17] shed a light on the lasting effects of sterile, antigen non-specific inflammation generated in the MIA model. IIM is thought to be generated through epigenetic changes [17] and such changes created in fetal and early infancy could make such individuals more susceptible to common inflammatory conditions such as food induced enterocolitis syndrome (FPIES), a condition that were found frequently in ASD subjects in our clinic. Altered IIM skewed to pro-inflammatory responses may cause dysregulated responses to commensal microbiota in the gut, causing chronic GI inflammation, resembling inflammatory bowel diseases (IBD). Such changes in innate immune responses may lead to aberrant responses to respiratory microbes, resulting in altered clinical manifestations, as well. Such dysregulated innate immune responses to immune stimuli can also affect the brain, since many signaling pathways associated with innate immunity have roles in the nervous system [17].

Despite progress of our understanding of IIM, we do not know which innate immune parameters are associated with co-morbid medical conditions and how these parameters are associated with ASD severity. IIM is closely associated with changes in monocyte cytokine profiles [16]. Previously, we have found significant changes in monocyte cytokine profiles in a subset of ASD patients [10, 11]. Therefore, this study addressed whether the specific monocyte cytokine parameters are associated with ASD co-morbid conditions. In this study, we randomly screened monocyte cytokine profiles in ASD subjects recruited to the study. In our clinic, because of the allergy/immunology specialty, we likely recruited more ASD subjects with co-morbid medical conditions. However, we reasoned that such potentially skewed ASD study subjects may make it easier for us to find specific monocyte markers associated with co-morbid conditions.

We found changes in certain monocyte cytokine parameters had an association with ASD severity (**Table 4**). However, parameters associated with inflammatory responses (production of TNF-α and IL-1ß, and TNF-α/sTNFRII ratio) were also found to be affected by other clinical co-variables including GI symptoms, and PANS like behaviors (**Table 4**). This finding seems to support our initial assumption that associations between ASD behavioral symptoms and changes in monocyte cytokine profiles are affected by other clinical co-variables.

Therefore, we decided to assess changes of monocyte cytokine parameters in association with co-morbid conditions frequently found in ASD subjects. We found GI symptoms along with NFA or FPIES like conditions in ASD subjects at high frequency (>60%), which was consistent to our previous studies [10, 12, 29]. Most of the ASD patients with GI symptoms had a history of FPIES like symptoms (**Table 2**). In these patients, we found changes in production of TNF-α and TNF-α/sTNFRII ratios in association with GI symptoms, but to our surprise, we did not find any associations with other inflammatory markers typically associated with neuroinflammation. It may be that GI symptoms are mainly driven TNF-α mediated inflammation in these ASD subjects as seen in patients with IBD [31]. Our finding may indicate the possibility that treatment measures typically used for IBD patients may be applicable for treating GI symptoms in ASD. Interestingly, TNF-α production under zymosan mediated cultures was affected by ASD severity; this may provide further support of the gut-brain axis concept [5, 6].

As for seizure disorders, we found changes in IL-1ß production under the cultures stimulated with ß-glucan and CLO97 in ASD subjects with seizure disorders

**93**

likely required.

*Associations between Monocyte Cytokine Profiles and Co-Morbid Conditions in Autism…*

(**Table 5**). This association was independent of any other clinical co-variables by co-variance analysis. IL-1ß has been implicated with a major inflammatory component in febrile seizures and is also implicated in the pathogenesis of seizures associated with neuroinflammation [32, 33]. These results may indicate utility of IL-1ß blockers for controlling seizures in ASD subjects, if control is not well achieved by the 1st line anti-seizure medications. This finding is also intriguing because we have found better control of seizures with the use of IL-1ß blockers in some ASD subjects

The presence of AR appeared to be associated with an increase in sTNFRII levels

When we assessed associations between sleep disorders and changes in monocyte cytokine profiles, we expected to see changes in inflammatory monocyte cytokines, since ASD subjects with PANS like symptoms often suffer from sleep disorders. However, we mainly found lower production of TGF-ß which is considered to be a counter-regulatory cytokine and associated with tissue repair, promoting fibrotic changes [41]. Our results may indicate a decrease in counter-regulatory measures in neuroinflammation in sleep disorders in ASD subjects. The etiology of and the role neuroinflammation plays in sleep disorders in ASD are not well understood. Our finding may indicate that impairment of TGF-mediated pathways may play a role in

Our previous studies indicated that IL-1ß/IL-10 ratios can be general markers for dysregulatred innate immune responses in ASD subjects [12]. However, in this study, we did not find strong associations with this parameter to specific comorbid medical conditions, except for seizure disorder. This parameter may be associated with general inflammation caused by immune mediated inflammation. However, in order to more fully assess treatment options for co-morbid medical conditions in ASD subjects, detailed analysis of monocyte cytokine profiles is

which may be indicative of increase in counter-regulatory measures for allergic inflammation. However, since the numbers of AR patients in this study was rela-

Our ASD study subjects included a fair number of ASD subjects with SAD (**Table 2**). These ASD subjects revealed lower production of IL-6 and IL-10 under several culture conditions (**Table 5**). Two of these parameters were affected by the presence of PANS like symptoms. This may not be surprising, since in our experience, we often observe a high frequency of SAD in non-ASD PANS patients. Interestingly, ASD subjects with PANS like behavioral symptoms also revealed lower production of IL-6 (**Table 6**). IL-6 is associated with terminal differentiation of B cells and is reported to be lower in patients with antibody deficiency such as common variable immunodeficiency [35]. On the other hand, IL-6 has also be implicated with neuronal development, following neuronal insult during fetal and newborn periods [36, 37]. Reduced IL-6 production may reflect subsequent suppression, following prior IL-6 mediated neuroinflammation. If so, lowering IL-6 production may have evolved into impaired antibody production in some ASD subjects who had suffered from IL-6 mediated inflammation in their early years. In subjects with autoimmune encephalitis refractory to rituximab, IL-6 blockers such as tocilizumab which is an inhibitor of the IL-6 receptor, are reported to be effective [38, 39]. However, in ASD subjects with lower IL-6 production, the use of IL-6 blockers may not be effective, even if the PANS like behavioral symptoms are attributed to AE. IL-10 production was also lower in ASD patients with SAD, however this is not associated with the presence of PANS like symptoms. Changes in IL-10 production is reported in patients with common variable immunodeficiency (CVID) [40]. Therefore, this finding may be associated with the pathogenesis of

tively low, these results need to be validated in future studies.

*DOI: http://dx.doi.org/10.5772/intechopen.95548*

previously [34].

antibody deficiency.

sleep disorders in ASD.

*Associations between Monocyte Cytokine Profiles and Co-Morbid Conditions in Autism… DOI: http://dx.doi.org/10.5772/intechopen.95548*

(**Table 5**). This association was independent of any other clinical co-variables by co-variance analysis. IL-1ß has been implicated with a major inflammatory component in febrile seizures and is also implicated in the pathogenesis of seizures associated with neuroinflammation [32, 33]. These results may indicate utility of IL-1ß blockers for controlling seizures in ASD subjects, if control is not well achieved by the 1st line anti-seizure medications. This finding is also intriguing because we have found better control of seizures with the use of IL-1ß blockers in some ASD subjects previously [34].

The presence of AR appeared to be associated with an increase in sTNFRII levels which may be indicative of increase in counter-regulatory measures for allergic inflammation. However, since the numbers of AR patients in this study was relatively low, these results need to be validated in future studies.

Our ASD study subjects included a fair number of ASD subjects with SAD (**Table 2**). These ASD subjects revealed lower production of IL-6 and IL-10 under several culture conditions (**Table 5**). Two of these parameters were affected by the presence of PANS like symptoms. This may not be surprising, since in our experience, we often observe a high frequency of SAD in non-ASD PANS patients. Interestingly, ASD subjects with PANS like behavioral symptoms also revealed lower production of IL-6 (**Table 6**). IL-6 is associated with terminal differentiation of B cells and is reported to be lower in patients with antibody deficiency such as common variable immunodeficiency [35]. On the other hand, IL-6 has also be implicated with neuronal development, following neuronal insult during fetal and newborn periods [36, 37]. Reduced IL-6 production may reflect subsequent suppression, following prior IL-6 mediated neuroinflammation. If so, lowering IL-6 production may have evolved into impaired antibody production in some ASD subjects who had suffered from IL-6 mediated inflammation in their early years.

In subjects with autoimmune encephalitis refractory to rituximab, IL-6 blockers such as tocilizumab which is an inhibitor of the IL-6 receptor, are reported to be effective [38, 39]. However, in ASD subjects with lower IL-6 production, the use of IL-6 blockers may not be effective, even if the PANS like behavioral symptoms are attributed to AE. IL-10 production was also lower in ASD patients with SAD, however this is not associated with the presence of PANS like symptoms. Changes in IL-10 production is reported in patients with common variable immunodeficiency (CVID) [40]. Therefore, this finding may be associated with the pathogenesis of antibody deficiency.

When we assessed associations between sleep disorders and changes in monocyte cytokine profiles, we expected to see changes in inflammatory monocyte cytokines, since ASD subjects with PANS like symptoms often suffer from sleep disorders. However, we mainly found lower production of TGF-ß which is considered to be a counter-regulatory cytokine and associated with tissue repair, promoting fibrotic changes [41]. Our results may indicate a decrease in counter-regulatory measures in neuroinflammation in sleep disorders in ASD subjects. The etiology of and the role neuroinflammation plays in sleep disorders in ASD are not well understood. Our finding may indicate that impairment of TGF-mediated pathways may play a role in sleep disorders in ASD.

Our previous studies indicated that IL-1ß/IL-10 ratios can be general markers for dysregulatred innate immune responses in ASD subjects [12]. However, in this study, we did not find strong associations with this parameter to specific comorbid medical conditions, except for seizure disorder. This parameter may be associated with general inflammation caused by immune mediated inflammation. However, in order to more fully assess treatment options for co-morbid medical conditions in ASD subjects, detailed analysis of monocyte cytokine profiles is likely required.

*Autism Spectrum Disorder - Profile, Heterogeneity, Neurobiology and Intervention*

considered as a common denominator. The immune system has long been thought to play a role in neuroinflammation and is implicated with pathogenesis of ASD. One of the most extensively studied animal models of ASD is MIA, in which, ASD like behavioral changes in offspring are induced by sterile immune activation through stimuli of innate immunity given to pregnant rodents [13]. Discovery of IIM [15, 17] shed a light on the lasting effects of sterile, antigen non-specific inflammation generated in the MIA model. IIM is thought to be generated through epigenetic changes [17] and such changes created in fetal and early infancy could make such individuals more susceptible to common inflammatory conditions such as food induced enterocolitis syndrome (FPIES), a condition that were found frequently in ASD subjects in our clinic. Altered IIM skewed to pro-inflammatory responses may cause dysregulated responses to commensal microbiota in the gut, causing chronic GI inflammation, resembling inflammatory bowel diseases (IBD). Such changes in innate immune responses may lead to aberrant responses to respiratory microbes, resulting in altered clinical manifestations, as well. Such dysregulated innate immune responses to immune stimuli can also affect the brain, since many signaling pathways associated with innate immunity have roles in the

Despite progress of our understanding of IIM, we do not know which innate immune parameters are associated with co-morbid medical conditions and how these parameters are associated with ASD severity. IIM is closely associated with changes in monocyte cytokine profiles [16]. Previously, we have found significant changes in monocyte cytokine profiles in a subset of ASD patients [10, 11]. Therefore, this study addressed whether the specific monocyte cytokine parameters are associated with ASD co-morbid conditions. In this study, we randomly screened monocyte cytokine profiles in ASD subjects recruited to the study. In our clinic, because of the allergy/immunology specialty, we likely recruited more ASD subjects with co-morbid medical conditions. However, we reasoned that such potentially skewed ASD study subjects may make it easier for us to find specific monocyte

We found changes in certain monocyte cytokine parameters had an association with ASD severity (**Table 4**). However, parameters associated with inflammatory responses (production of TNF-α and IL-1ß, and TNF-α/sTNFRII ratio) were also found to be affected by other clinical co-variables including GI symptoms, and PANS like behaviors (**Table 4**). This finding seems to support our initial assumption that associations between ASD behavioral symptoms and changes in monocyte

Therefore, we decided to assess changes of monocyte cytokine parameters in association with co-morbid conditions frequently found in ASD subjects. We found GI symptoms along with NFA or FPIES like conditions in ASD subjects at high frequency (>60%), which was consistent to our previous studies [10, 12, 29]. Most of the ASD patients with GI symptoms had a history of FPIES like symptoms (**Table 2**). In these patients, we found changes in production of TNF-α and TNF-α/sTNFRII ratios in association with GI symptoms, but to our surprise, we did not find any associations with other inflammatory markers typically associated with neuroinflammation. It may be that GI symptoms are mainly driven TNF-α mediated inflammation in these ASD subjects as seen in patients with IBD [31]. Our finding may indicate the possibility that treatment measures typically used for IBD patients may be applicable for treating GI symptoms in ASD. Interestingly, TNF-α production under zymosan mediated cultures was affected by ASD severity; this may provide further support of the gut-brain axis

As for seizure disorders, we found changes in IL-1ß production under the cultures stimulated with ß-glucan and CLO97 in ASD subjects with seizure disorders

**92**

concept [5, 6].

nervous system [17].

markers associated with co-morbid conditions.

cytokine profiles are affected by other clinical co-variables.

Our study may be limited by the relatively small sample side of ASD subjects who had specific co-morbid conditions. Findings in this study need to be validated by a study using a larger numbers of study subjects, in association with responses to specific treatment measures targeted to each co-morbid condition.
