Regulating Bcl2L12 expression in mast cells inhibits food allergy

Rationale: Mast cells play a crucial role in allergic diseases. Yet, the regulation of mast cell bioactivities is not fully understood. This study aims to elucidate the role of B cell lymphoma 2 like protein 12 (Bcl2L12), one of the anti-apoptosis proteins, in regulating mast cell apoptosis. Methods: A food allergy (FA) mouse model was developed to establish mast cell over population in the intestinal tissue. Either compound 48/80 (C48/80) or specific antigens were used to activate mast cells in the intestinal mucosa. Results: After treating with C48/80, apoptosis was induced in mast cells of the intestine of naive control mice, but not in FA mice. The expression of Fas ligand (FasL) was lower in the mast cells of FA mice. Interleukin (IL)-5 was responsible for the suppression of FasL by upregulating the expression of Bcl2L12 in mast cells. Bcl2L12 prevented c-Myc, the major transcription factor of FasL, from binding the FasL promoter to inhibit the expression of FasL in mast cells. Inhibition of Bcl2L12 restored the apoptosis machinery of mast cells in the FA mouse intestine. Conclusions: The apoptosis machinery in mast cells is impaired in an allergic environment. Inhibition of Bcl2L12 restores the apoptosis machinery in mast cells in the FA mouse intestine.


Isolation of LPMC from intestine
Intestinal segments were excised immediately after the sacrifice. The tissues were cut into small pieces (2 × 2 × 2 mm) and incubated with collagenase IV (1 mg/ml) at 37 °C for 2 h with mild agitation. Single cells were passed through a cell strainer (70 µm first, then 40 µm). The mononuclear cells were isolated by the Percoll gradient density centrifugation and cultured in RPMI1640 medium. The viability of the isolated cells was greater than 98% as assessed by Trypan blue exclusion assay. 4

Cell culture
Immune cells were cultured in RPMI1640 medium supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine and 10% fetal bovine serum. The medium was changed in 1-2 days. The viability of the cells was greater than 99% as determined by Trypan blue exclusion assay.

Enzyme-linked immunosorbent assay (ELISA)
Cytokine levels in the serum and culture supernatant were determined by ELISA with commercial reagent kits following the manufacturer's instructions.

Real-time quantitative RT-PCR (RT-qPCR)
Cells were obtained from relevant experiments. The total RNA was extracted from the cells with the TRIzol reagents. The cDNA was synthesized from the RNA with a reverse transcription kit following the manufacturer's instructions. Samples were amplified in a qPCR device with the SYBR Green Master Mix and the presence of relevant primers as presented in Table 1. The results were presented as fold change against the housekeeping gene β-actin.

Western blotting
The total proteins were extracted from cells obtained from relevant experiments, fractioned by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), 5 and transferred onto a PVDF membrane. After blocking with skim milk solution (5%) for 30 min, the membrane was incubated with the primary antibodies of interest overnight at 4 °C, washed with Tris-buffered saline containing 0.1% Tween 20 (TBST) 3 times, incubated with the second antibodies (conjugated with peroxidase) for 1 h at room temperature, washed with TBST 3 times. The immunoblots on the membrane were developed with the enhanced chemiluminescence and photographed with an imaging station.

Preparation of protein extracts
Cells were collected from relevant experiments. The cells were incubated with a lysing buffer for 30 min. The lysates were centrifuged for 10 min at 13,000 rpm. The supernatant was collected and used as the cytosolic extracts. The pellets were incubated with a nuclear lysing buffer for 30 min. The lysates were centrifuged for 10 min at 13,000 rpm. The supernatant was collected and used as the nuclear extracts.
All the procedures were performed at 4 °C.

Co-immunoprecipitation (co-IP)
The protein samples were precleared by incubating with protein G agarose beads for 30 min to remove pre-existing immune complexes. After centrifugation for 3 min at 13,000 rpm, the supernatant was collected and incubated with antibodies of interest or isotype IgG overnight. The immune complexes in the samples were precipitated by incubating with protein G agarose beads for 30 min with mild agitation. The beads 6 were collected by centrifugation of the samples at 13,000 rpm for 3 min. The immune complexes on the beads were eluted with an eluting buffer. The proteins were analyzed by Western blotting. All the procedures were performed at 4 °C.

Chromatin IP (ChIP)
Cells were collected from relevant experiments and fixed with 1% formalin for 15 min to cross-link DNA and surrounding proteins. The cells were lysed with a lysing buffer containing protease inhibitor. The DNA in the samples was sheared to small pieces by sonication. The samples were centrifuged at 13,000 rpm for 10 min. The supernatant was collected and processed with the procedures of IP. DNA was extracted from the protein/DNA complexes using a DNA extracting kit following the manufacturer's instructions. The DNA was analyzed by qPCR in the presence of a pair of FasL promoter primers (aggcagagtggtcggtttta and cctatccatcccacttcccc). The results were normalized as fold change against the input. All the procedures were performed at 4 °C.

Assessment of intestinal epithelial barrier permeability in mice
Sixteen hours after treating with C48/80 (ip), mice were treated with oral gavage       Figure 2) and protein of Th2 cytokines (presented in Figure   S1E-G). Each dot represent data from one mouse. Mast cells were counted in 20 randomly selected microscope windows per sample. 13 The summarized counts of mast cells are presented in Fig 6E. Original magnification: ×400.

Figure S9. Generation of mouse model of mast cell specific BcL2L12 deletion
To avoid affect the role of Bcl2L12 in other cells, Bcl2L12 was specifically deleted from mast cells with c-kit as the specific marker. Following published procedures (Elife. 2014;3:e01949 and J Clin Invest. 1996;98(3):600-3), we generated mice with loxP-flanked Bcl2L12 gene (flox). In the first step, we constructed a gene targeting vector containing three loxP sites, in which two of them flanking the neomycin resistance gene. The genomic locus was modified between vector and Bcl2L12 gene in embryo stem (ES) cells by homologous recombination. The loxP-flanked neo gene was deleted by transient c-kit-Cre expression in ES cells with two loxP sites remained in target gene. Using the modified ES cells, a loxP-Bcl2L12 containing mouse line was generated. Then, we crossed the mouse strain harboring two loxP sites in the Bcl2L12