Btbrt+Tf/J Mice Exhibit an Inflammatory Macrophage Cytokine Profile with Associations to Repetitive Grooming Behavior

Background:  Although autism is a behaviorally defined disorder, systemic associations are also noted with a number of studies reporting atypical myeloid cell phenotype and function including increased pro-inflammatory cytokine production.  Recent characterization of the BTBRT⁺tf/J (BTBR) inbred mouse strain revealed several behavioral characteristics including social deficits, repetitive behavior, and atypical vocalizations which may be relevant to autism.  We therefore hypothesized that BTBR mice may exhibit immune abnormalities similar to those observed in children with autism when compared to social C57BL/6J (C57) mice.

Objectives:  The objectives of this study were to characterize the immune profile of BTBR macrophages and associations with autism relevant behaviors including sociability and repetitive grooming.

Methods:  All procedures were performed with approval by the University of California, Davis Institutional Animal Care and Use Committee in accordance with the guidelines provided by the National Institutes of Health for the scientific treatment of animals. C57 mice n=9, and BTBR n=7 mice were tested for social preference using the automated three-chambered social approach apparatus, and scored for self-grooming behavior. Post behavioral testing, mice were sacrificed and bone-marrow derived macrophages were generated.  Macrophages were incubated for 24hr in growth media alone, 10 ng/ml LPS, 1 ng/ml recombinant IL-4/10 ng/ml LPS, or 150 ng/ml recombinant IFN-γ/10 ng/ml LPS.  Supernatants were analyzed by Milliplex® immunoassay

Results:  BTBR produced higher levels of inflammatory cytokines as compared to C57 macrophages including increased MCP-1 (p=0.0164) without stimulation, and increased IL-6 (p=0.0003), MCP-1 (p=0.0002), and MIP-1α (p=0.0002) and lower IL-10 (p=0.0003) after stimulation with LPS.   After exposure to the IL-4/LPS (p=0.0003), BTBR macrophages produced significantly less IL-10 than C57 macrophages while levels of IL-6 (p=0.0052), MCP-1 (p=0.0115), and MIP-1α (p=0.0021) were significantly higher. After exposure to IFN-γ/LPS, BTBR macrophages produced significantly less IL-12 (p40) (p=0.0002) than C57 macrophages, while levels of IL-6 (p=0.0021), MCP-1 (p=0.0002) remain higher.  We further observed a positive correlation between time spent grooming, and production of MCP-1 (p=0.0583) and MIP-1β (0.0583) in untreated BTBR macrophages, and production of IL-6 (p=0.0333), and TNF-α (p=0.0167) after treatment with IFN-g/LPS.

Conclusions:  BTBR mice display a trend to increased inflammatory cytokine production including IL-6, MCP-1 and MIP-1α, and decreased production of the ant-inflammatory cytokine IL-10, suggesting a more inflammatory macrophage phenotype in asocial BTBR mice compared to the social C57 strain.  In addition to this inflammatory phenotype, BTBR macrophages show a impaired ability to produce IL-10 in response to treatment with the M2 polarizing condition IL-4/LPS, and IL-12(p40) in response to the M1 polarizing condition IFN-γ/LPS,  suggesting that macrophages fail to polarize correctly in response to M1 or M2 polarizing cytokine signals.  We additionally observed positive association between increased inflammatory cytokine production and increased repetitive behavior, which may suggest a direct relationship between inflammatory phenotype and a behavioral phenotype that may have relevance to a core symptom of autism.