In the present study, we have evaluated the basic immunological characteristics of BTBR mice. The BTBR strain has been reported to have abnormal behaviors that resemble autism 
, and our behavioral analysis supports the BTBR strain's previously described lack of sociability. The lack of sociability displayed by male and female BTBR mice was lost with the F1 offspring; however, the heterzygosity of the F1 strains results in an intermediate behavioral phenotype. The F1 offspring spent less time with the novel mouse and more time with the novel object than B6 mice. The intermediate behavioral phenotype is apparent with the behavior index (time with novel mouse minus time with novel object). With regard to the differential strain phenotypes, this time differential (behavior index) correlated with a number of immune parameters, especially IgG deposited in the brain and the amount of cytokines (e.g., IL-33) present in the brain (). Thus, the elevated IgG, which includes Abs deposited in the brain, and neuroinflammation inversely correlates with degree of sociablity. Functional alterations of the peripheral or central immune system have been studied in some individuals with ASD, and the implication of brain inflammation or generation of AutoAbs against brain antigens has been discussed, in the context of the background mechanisms of autism development 
. Beyond the documented autism-like behavior of BTBR mice 
, our present investigation demonstrates that certain immunologic characteristics of BTBR mice compare well with the immune modulations reported from autistic subjects. Unlike B6 mice, an inbred strain with highly social behavior, 3 week old BTBR mice exhibited signs of neuroinflammation. BTBR mice expressed higher levels of brain-deposited IgG, and IgE, brain cytokines, and MHC class II expressing microglia than these levels in B6 mice; the peripheral level of CD4+
T cells also was higher in BTBR mice. The neuroinflammation could be due to the activated microglia or increased presence of mast cells, which were prominent within the meninges and brains of BTBR mice, especially at circumventricular organs, particularly the IIIrd ventricle, the hippocampal fissure, and perivascular spaces in the posterior lateral thalamus; these areas may play a role in the BTBR behavioral abnormalities.
Correlation analysis of brain IgG and IL-33 of the strains with their behavior index.
BTBR mice also appear to have a predominant Th2 profile, in that they have high humoral immune responses to KLH, low immunity to LM, and significantly higher levels of serum IgE. If all of our data are taken together, BTBR inbred mice appear to be a strain highly susceptible to neuropathology, due to the elevated levels of cells, antibodies, and cytokines in their brains. Most interestingly, the two types of offspring from F1 crosses, BCF1 and CBF1 mice, have phenotypes intermediate between those of the parental BTBR and B6 mice, although the BCF1 mice seem to show slightly increased signs of neuroinflammation than do the CBF1 mice. These immunologic differences suggest that the BTBR strain could be a useful model to further study the developmental influences of immunity on aberrant behaviors.
There is overall consensus on male predominance for autism prevalence 
. Even though various genetic variations in X chromosome of autistic subjects have been reported, the relationship of such variance with pathogenesis of autism or ASD, especially with regard to a greater percentage of males having ASD (4
female, ratio), remains in doubt 
. Likewise, two quantitative trait loci found in X chromosome of BTBR strain were suggested to modulate the corpus callosum abnormalities associated with autism 
, but these chromosomal variations did not give rise to a gender difference in the anatomical brain defects 
. Among autistic subjects, no striking difference between genders has been reported for immune abnormalities 
. Interestingly, for monozygotic male and female twins, concordance was reported to be 86 and 100%, respectively, for an ASD outcome, and for dizygotic male-male and female-male twins, the respective concordance values were 40% and 20% 
. Thus, yet undefined gender influences appear to exist. Gender influences were not evident in our analysis of the central and peripheral levels of IgG in BTBR mice; there also were no gender differences with regard to expression of proinflammatory cytokines and immune cell composition. Most published studies of the autism-like behaviors of BTBR mice have employed male mice only; no published data have indicated a significant behavioral difference between male and female BTBR mice. Therefore, the current assumption seems to be that sex-linked genetic differences do not influence the development of proinflammatory and autoimmune-prone immunologic characteristics of BTBR mice. Nevertheless, some genetic contribution, especially maternal nuclear or mitochondrial genes likely contribute to the immune modulations, given that BCF1 mice born of BTBR dams showed higher autoreactive IgG level, microglial cell percentage, and expression of proinflammatory cytokines than did B6 mice. Genetically, the two F1 strains differ in terms of mitochondrial inheritance as well as X- and Y-linked genes and potentially early maternal epigenetic influences.
With respect to maternal influence on the autistic phenotype of offspring, two published experimental approaches provide a hint as to the role of maternal IgG against fetal brain antigens, in the induction of behavioral alterations after birth 
. Both of the studies used serum IgG purified from the blood of mothers of autistic children, which was injected intravenously into pregnant Rhesus monkeys or was injected intraperitoneally into pregnant B6 mice. Reactivity of serum IgG against fetal brain proteins was confirmed through Western blot analysis or immunohistochemical staining. Rhesus monkey infant offspring and adolescent/adult B6 offspring that had been prenatally exposed to IgG from mothers of autistic children displayed higher levels of stereotypic and hyperactive motor activity than did controls 
; the prenatally exposed mice, as adults, displayed less social interactions with strangers than did controls 
. The two studies provided clear evidence for involvement of antibody in the development of autistic behaviors in offspring. Our present findings indicate that the level of anti-brain IgG is higher in BTBR or BCF1 mice born of BTBR dams than in B6 or CBF1 mice born of B6 dams. Analyses in the literature of serum from autistic children or mothers of autistic children have detected Abs against various CNS Ags, including myelin basic protein, serotonin receptor, glial fibrillary acidic protein, brain-derived neurotrophic factor, and heat shock proteins 
. A role of AutoAbs in the pathogenesis of autism is not proven, nor is the involvement of autoimmune mechanisms in ASD. However, clearly, identifying the specificities and etiologic roles of AutoAbs should be further investigated.
Cytokines affect the development of neuronal or glial cells, as well as behavioral phenotypes 
. In autistic subjects, immune alterations, particularly cytokine modulations, that affect the pathogenesis of autism development, are not agreed upon 
. Vargas et al. 
provided evidence for an ongoing process of neuroinflammation in the CNS of their autistic subjects. Macrophage chemoattractant protein-1, a proinflammatory cytokine, was the typical cytokine prominently expressed in both brain and cerebrospinal fluid of autistic patients. Marked activation of microglial cells and astrocytes was also evident across the brain regions, especially in the cerebellum. An anti-inflammatory cytokine, tumor growth factor-β1, was simultaneously elevated in brain regions indicative of chronic neuroinflammation. In addition to the usual analysis of IL-1β and TNFα, IL-33 and IL-18 were assayed in our study. Overall, levels of several cytokines, including IL-33, IL-18, IL-1β, and IL-6, were elevated, whereas IFNγ and TNFα were decreased in the brains of PND21 BTBR mice, compared to brains of B6 or CBF1 pups. IL-33, IL-18, and IL-1β share many traits: synthesis as pro-forms lacking activity, production of mature/active forms through caspase-1 cleavage, secretion via endoplasmic reticulum/Golgi-independent pathway, and biological activity for inflammation 
. Glial cells are a major source of IL-33, IL-18, and IL-1β production in the brain. The percentage of microglial cells was higher in the BTBR mice than in B6 mice, perhaps related to the higher levels of these cytokines in the brains of the former strain. Furthermore, when we compared the mean fluorescence intensity of MHC-II expression on brain microglial cells, the expression intensity was higher (although the difference was not significant) in the BTBR (670±204) than in the B6 mice (498±314). The greater MHC-II expression, a phenomenon also observed in Parkinson's disease 
, suggests an elevated state of activation of microglia in the BTBR mice. Among the above three proinflammatory cytokines, IL-1β is the only one to have been reported in previous investigations of immune alterations in autistic subjects 
; the level of IL-1β from stimulated or unstimulated peripheral mononuclear cells in vitro
was higher in autistic children than in controls. Elevation of IL-18 has been reported for rodents and humans with psychiatric disorders 
. Among the brain regions that we examined, the cerebellum was one that demonstrated significantly higher expression of both IL-33 and IL-18 in BTBR mice than in B6 mice; the cerebellum could thus be a “hot” region for neuroinflammation in the BTBR mice and for ASD in humans. Loss of Purkinjie cells has been frequently observed in the cerebellum, and neuronal degeneration and glial cell activation have predominantly been found in the cerebellum of autistic patients 
The Th1-Th2 paradigm, that is immune skewing toward type-1 cell-mediated immunity (interferon-gamma promoted immunity) vs.
type-2 humoral immunity (IL-4, IL-5 and IL-13 promoted immunity), may not be properly applicable to immune alterations in autism. Conflicting results have been derived from human studies on a Th1 or Th2 shift: several investigations reported Th1 skewedness, but other studies suggested a Th2 shift 
. Here, we found a substantial downregulation of the Th1 response in BTBR mice, as compared to B6 mice; IL-12 and IFNγ expression was similar or lower, whereas IL-33 and IL-10, cytokines promoting Th2-mediated response 
, were elevated. The greater IL-6 expression in the brain of BTBR mice may be worth noting since IL-6 is involved in sickness behavior and also in modulation of brain development (probably mediated by neuronal inflammation) 
. In addition, IL-6 has been reported to mediate the behavioral abnormalities of adult offspring from dams that experienced maternal immune activation during pregnancy 
. Since type-2 immunity appears to predominate in BTBR mice and IL-33, a type-2 immune promoter, is the only cytokine significantly elevated in all brain regions, IL-33 may be especially important to consider for its affects on behavior, in that it has been suggested that IL-33-producing glia are critical regulators of innate immune responses in the CNS 
The existence of an animal model will be very valuable in the investigation of mechanistic pathways and therapeutic interventions of psychiatric diseases, especially for neurodegenerative or neurodevelopmental disorders. The present study is the first report of the involvement of neuroinflammation, accompanied by elevation of IgG and IgE and IgG-secreting B cells, in BTBR mice, a strain with abnormal behaviors. Considering the elevated amount of IgG accumulated in the brain and serum anti-brain antibodies of BTBR mice, specific brain Ag(s) need to be identified, and the involvement of humoral immunity in the aberrant behaviors of the BTBR mice needs to be explored. The genetic basis for the constitutively high levels of IgG and IgE also need to be investigated. Although higher levels of several proinflammatory cytokines, including IL-33, IL-18, and IL-1β, were shown in BTBR in the present study, brain cellular sources of these cytokines need to be delineated. It also is necessary to evaluate the individual contribution of each cytokine to the development of the aberrant BTBR behaviors, as well as the cross-talk between proinflammatory cytokines and certain anti-inflammatory cytokines, such as TGFβ1. Since BCF1 mice (born of BTBR dams, sired by B6), especially the males, demonstrated immunologic characteristics closer to BTBR mice than did the CBF1 mice, these combinations may be useful for dissection of the genetic and environmental influences on development of the aberrant behaviors.