PPARβ/δ activation of CD300a controls intestinal immunity

Macrophages are important for maintaining intestinal immune homeostasis. Here, we show that PPARβ/δ (peroxisome proliferator-activated receptor β/δ) directly regulates CD300a in macrophages that express the immunoreceptor tyrosine based-inhibitory motif (ITIM)-containing receptor. In mice lacking CD300a, high-fat diet (HFD) causes chronic intestinal inflammation with low numbers of intestinal lymph capillaries and dramatically expanded mesenteric lymph nodes. As a result, these mice exhibit triglyceride malabsorption and reduced body weight gain on HFD. Peritoneal macrophages from Cd300a−/− mice on HFD are classically M1 activated. Activation of toll-like receptor 4 (TLR4)/MyD88 signaling by lipopolysaccharide (LPS) results in prolonged IL-6 secretion in Cd300a−/− macrophages. Bone marrow transplantation confirmed that the phenotype originates from CD300a deficiency in leucocytes. These results identify CD300a-mediated inhibitory signaling in macrophages as a critical regulator of intestinal immune homeostasis.

washed with PBS three times for 5 min, and incubated with the secondary antibody.
The secondary antibody for F4/80, Podoplanin, and Insulin, was Simple Stain mouse MAX-PO (Rat), Simple Stain mouse MAX-PO (G), and Simple Stain MAX-PO (Mu) (NICHIREI BIOSCIENCE INC.), respectively. Secondary antibodies were incubated for 60 min at room temperature. Slides were washed with PBS three times for five minutes. Then, slides were immersed in 3,3'-diaminobenzidine (DAB) solution (pre-filtered 0.01% DAB in 0.05 M Tris-HCI, pH 7.4) for 10 min at room temperature.
H2O2 was added to a final concentration of 0.01%. Slides were washed with running tap water for 3 min, counterstained with Mayer's hematoxylin for 0.5 min, and washed again with running tap water. Slides were dehydrated using increasing concentration of ethanol (85%, 95%, 100%, and 100%) for 3 min. Finally, slides were washed three times for 3 min with xylene and mounted with mounting medium.
Immunohistochemistry for frozen sections. Small intestine, liver, and colon were fixed for 4 hours at 4°C in periodate-lysine-paraformaldehyde (PLP), washed for 6 hours in three changes of PBS solutions containing 10%, 15%, 20% sucrose, then for 1 hour in PBS containing 20% sucrose + 5% glycerin and embedded in OCT compound (SAKURA, Tissue-Tek). Tissue specimens were cut into 6-μm-thick sections by cryostat (LEICA, CM1850). Slides were washed 3 times in PBS in the slide chamber.
Endogenous peroxidase activity was blocked using freshly made 0.3% H2O2 in methanol for 20-30 minutes. Slides were washed three times for five minutes with PBS and blocked with normal serum for 10 min.
We performed immunohistochemistry using the antibodies for B220 (AbD), Thy1.2 (BD) and FA/11 (AbD). Antibodies were incubated overnight at 4°C, washed three times for 5 min with PBS, and incubated with the secondary antibody. The secondary antibody used for B220, Thy1.2 and FA/11 was ECL anti rat IgG horseradish peroxidase linked species-specific F(ab')2 fragment from goat (GE Healthcare). Slides were incubated with the secondary antibody for 60 min at room temperature and washed three times for five minutes with PBS. Then, slides were developed in 3,3'-diaminobenzidine (DAB) solution (pre-filtered 0.01% DAB in 0.05 M Tris-HCI, pH 7.4) for 10 min at room temperature. H2O2 was added to a final concentration of 0.01%. Slides were washed in running tap water for 3 min, then counterstained with methylene green for 10 min.
Slides were dehydrated with ethanol solutions of increasing concentration (85%, 95%, 100%, and 100%) for 3 min per wash. Finally, slides were washed in xylene three times for 3 min and mounted with mounting medium.
Microscope and PC software for photograph. Nikon ECLIPS Ci and NIS-ElementsD software were used for microscopic analysis.
Cell fractionation and immunoblot analysis. The isolation and immunoblot analysis of the nuclear extracts were performed as described previously 1,2 . To prepare the nuclear extracts, THP-1 or PMA-treated THP-1 macrophages were washed with PBS and allowed to swell at 4°C for 30 min in buffer A (10 mM HEPES-KOH (pH 7.6),

Transcriptome microarray analysis.
For genome-wide transcription analysis, GeneChip Human Genome U133 Plus 2.0 Array or GeneChip Mouse Genome 430 2.0 array were used as described previously 1,2 . Briefly, total RNA was extracted with ISOGEN (Nippon Gene Inc) from THP-1 macrophages, peritoneal macrophages, and small intestine.
After in vitro transcription (IVT) and cRNA fragmentation, fragmented IVT product was hybridized on array and stained with streptavidin phycoerythrin using the manufacturer's recommended protocol. The arrays were scanned using the Affymetrix GeneChip Scanner 3000 (Affymetrix). To calculate the average differences for each gene probe, the GeneChip Analysis Suite software version 5.0 was used.
Chromatin immunoprecipitation (ChIP). 100 million THP-1 macrophages were cross-linked with 1% formaldehyde for 10 min at room temperature. were incubated for 45 min at 37°C. These samples were treated with 60 mg Proteinase K at 55°C for 1.5 h. The immunoprecipitated DNA was extracted with phenol-chloroform-isoamylalcohol. The aqueous phase was transferred to a 1.5 ml tube and ethanol-precipitated in the presence of 20 mg glycogen. Precipitated DNA was dissolved in EB buffer and purified using Qiaquick PCR purification kit (Qiagen).
Prepared DNA was quantified using Q-bit (Invitrogen) and more than 10 ng of DNA was processed for qPCR or sequence. Quantitative real-time PCR (qPCR). The qPCR method has been described 7 . Specific primers for each gene were designed using the Primer Express software (Life Technologies). All primer sequences used in this paper are available on request.
Total RNA was isolated using an RNA preparation kit (Isogen; Nippon Gene Corp.).
First strand cDNA was synthesized from total RNA with oligo dT primers using
Blood samples were drawn from the tail vein at 0, 15, 30, 60 and 120 min and assayed for serum glucose and insulin.