Bcl2l12 Contributes to Th2-Biased Inflammation in the Intestinal Mucosa by Regulating CD4+ T Cell Activities

Bcl2L12 Contributes to Th2-Biased Inflammation in the Intestinal Mucosa by Regulating CD4+ T Cell Activities

This information is current as Mao-Gang Li, Xiao-Yu Liu, Zhi-Qiang Liu, Jing-Yi Hong, of September 30, 2021. Jiang-Qi Liu, Cai-Jie Zhou, Tian-Yong Hu, Xiao-Jun Xiao, Pi-Xin Ran, Peng-Yuan Zheng, Zhi-Gang Liu and Ping-Chang Yang J Immunol published online 8 June 2018

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 8, 2018, doi:10.4049/jimmunol.1800139 The Journal of Immunology

Bcl2L12 Contributes to Th2-Biased Inﬂammation in the Intestinal Mucosa by Regulating CD4+ T Cell Activities

Mao-Gang Li,* Xiao-Yu Liu,* Zhi-Qiang Liu,*,†,‡ Jing-Yi Hong,* Jiang-Qi Liu,*,†,‡ Cai-Jie Zhou,x Tian-Yong Hu,*,† Xiao-Jun Xiao,* Pi-Xin Ran,{ Peng-Yuan Zheng,‖ Zhi-Gang Liu,* and Ping-Chang Yang*

The Th2-biased inflammation and immune deregulation play a critical role in the pathogenesis of ulcerative colitis (UC). Recent studies indicate that the Bcl2-like protein 12 (Bcl2L12) is associated with immune deregulation of UC. This study aims to investigate the role of Bcl2L12 in the induction of aberrant Th2-biased inflammation. In this study, peripheral blood samples were collected from patients with inflammatory bowel disease. The Th2 cell activities were analyzed by flow cytometry, real-time quantitative RT- PCR, and Western blotting. Mice with Bcl2L12-knockout CD4+ T cells were used in the experiments. The results showed that the expression of Bcl2L12 was detected in peripheral CD4+ T cells, which was significantly higher in UC patients than in healthy Downloaded from subjects. A positive correlation between the expression of Bcl2L12 and Th2 cytokines was detected in CD4+ T cells from UC patients. Naive CD4+ T cells with Bcl2L12 overexpression were prone to differentiate into Th2 cells. Mice with Bcl2L12 deficiency failed to induce the Th2-biased inflammation in the intestine. Bcl2L12 bound GATA3 to form a complex to enhance the binding between GATA3 and the Il4 promoter to enhance the expression of IL-4 in CD4+ T cells. CD4+ T cells with Bcl2L12 overexpression were resistant to apoptosis. In conclusion, the Bcl2L12 is a critical factor in the induction of aberrant Th2 polarization by upregulating Th2 responses and downregulating Th2 cell apoptosis. Bcl2L12 may be a novel therapeutic target in the management http://www.jimmunol.org/ of the disorders with Th2-biased inflammation. The Journal of Immunology, 2018, 201: 000–000.

nflammatory bowel disease (IBD) is a chronic inflammatory have been recognized in IBD patients. Published data show that disease in the intestine, including ulcerative colitis (UC) and both Th1 and Th2 responses play roles in the inflammation of I Crohn disease (CD), which are two subtypes. The patho- IBD. Profound infiltration of Th1 or Th2 cells can be found in the genesis of IBD is not fully understood (1). The skewed immune IBD intestinal mucosa (4). These Th1 or Th2 cells are highly responses to commensal microbes (2) or food components (3) activated, producing high amounts of Th1, Th2, or Th17 cytokines

(5). Yet, factors initiating the skewed immune cell differentiation by guest on September 30, 2021 and cytokine overproduction in the intestinal tissue of IBD pa- *Research Center of Allergy and Immunology, Shenzhen University School of Med- tients are not fully understood. icine, Shenzhen 518055, China; †Longgang ENT Hospital, Shenzhen ENT Institute, It is accepted that the dominant Th1 or Th17 response is involved Shenzhen 518116, China; ‡Brain-Body Institute, McMaster University, Hamilton, Ontario L8N 4A6, Canada; xLonggang Chinese Traditional Medical Hospital and in the pathogenesis of CD (6, 7). Dendritic cells in the intestinal Beijing University of Chinese Medicine Shenzhen Hospital, Shenzhen 518116, mucosa express TLR. Upon recognizing microbial stimuli by China; {State Key Laboratory of Respiratory Disease, Guangzhou Medical Univer- ‖ TLR, dendritic cells express IL-12 to initiate the Th1 response by sity, Guangzhou 510120, China; and Department of Gastroenterology, The Fifth + Affiliated Hospital of Zhengzhou University, Zhengzhou 470000, China enhancing the expression of IFN-g in CD4 T cells. Both Th1 and Received for publication January 31, 2018. Accepted for publication May 9, 2018. Th2 responses are associated with the immune phenotypes of UC. This work was supported by the Innovation of Science and Technology Commission of The skewed Th2-dominant immune response is an important Shenzhen Municipality (Grants JCYJ20150403091931195, JCYJ20160422101725667, factor in the pathogenesis of UC. A portion of UC patients is CXZZ20140902151802864, and JCYJ20160429091935720) and the National Natural sensitized to food allergens. Treatment with specific allergen im- Science Foundation of China (Grants 81503623, 31400856, 31570932, 81571790, and 81501573). munotherapy attenuated the inflammation of UC (8, 9). Yet, how M.-G.L., X.-Y.L., Z.-Q.L., J.-Y.H., J.-Q.L., C.-J.Z., T.-Y.H., and X.-J.X. performed the skewed Th2-dominant immune response initiated in the in- experiments, analyzed data, and reviewed the manuscript. P.-C.Y., Z.-G.L., P.-X.R., testine still remains elusive. Published data indicate a Th2- and P.-Y.Z. organized the study and supervised experiments. P.-C.Y. designed the project and wrote the paper. dominant immune response in UC patients (8–12). Based on the available knowledge, how the skewed Th2 response occurs in UC Address correspondence and reprint requests to Dr. Ping-Chang Yang, Dr. Zhi-Gang Liu, or Dr. Peng-Yuan Zheng, Room 509, Building A7, Xili Campus, Shenzhen patients is unclear. University School of Medicine, 1066 Xueyuan Boulevard, Shenzhen 518055, China In recent studies, we observed that the Bcl2-like protein 12 (P.-C.Y. and Z.-G.L.), or Department of Gastroenterology, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou 470000, China (P.-Y.Z.). E-mail addresses: (Bcl2L12) was associated with the skewed immune response in [email protected] (P.-C.Y.), [email protected] (Z.-G.L.), or [email protected] patients with UC (12) and allergic rhinitis (13). Bcl2L12 is a (P.-Y.Z.) member of the Bcl2 family belonging to the antiapoptotic mole- The online version of this article contains supplemental material. cules (14). Previous studies showed that Bcl2L12 could neutralize Abbreviations used in this article: AICD, activation-induced cell death; Bcl2L12, p53 to prevent glioma cell apoptosis (15). Based on the above Bcl2-like protein 12; CD, Crohn disease; IBD, inflammatory bowel disease; IP, immunoprecipitation; KO, knockout; LPMC, lamina propria mononuclear cell; information, we hypothesize that Bcl2L12 may be involved in the + MPO, myeloperoxidase; RNAi, RNA interference; RT-qPCR, real-time quantitative skewed Th2 response. In this study, we found that CD4 T cells PCR; shRNA, short hairpin RNA; SPT, skin prick test; UC, ulcerative colitis; WT, expressed Bcl2L12, which was higher in UC patients than in CD wild type. patients and healthy subjects. Bcl2L12 bound the GATA3 pro- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 moter to enhance the binding of GATA3 and the Il4 promoter.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800139 2 Bcl2L12 CONTRIBUTES TO ABERRANT Th2 RESPONSE

Mice with Bcl2L12-knockout (KO) CD4+ T cells failed to induce RT-qPCR + inflammation in the intestine. CD4 T cells with Bcl2L12 over- Cells were collected from relevant experiments. The total RNA was extracted expression were resistant to apoptosis. The results suggest that with the TRIzol reagents. The cDNA was synthesized with a reverse tran- Bcl2L12 may be a potential therapeutic target in the management scription kit following the manufacturer’s instructions. The samples were of Th2-biased inflammation. amplified in a quantitative PCR device with the SYBR Green Master Mix and the primers of IL-4 (59-AACGAGGTCACAGGAGAAGG-39 and 59-TCTG- CAGCTCCATGAGAACA-39), IFN-g (59-TTCTTCAGCAACAGCAAGGC- Materials and Methods 39 and 59-ACTCCTTTTCCGCTTCCTGA-39), and Bcl2L12 (59-TTCCGAG- Reagents TTCTATGCCCTGG-39 and 59-CCAGTTTACGATGCAGAGCC-39). The results were presented as fold change against the housekeeping gene b-actin The short hairpin RNA (shRNA) kit of Bcl2L12, Abs of Bcl2L12, IL-4, (59-GGAAATCGTGCGTGACATCA-39 and 59-GCCACAGGATTCCATA- CD3, and GATA3 were purchased from Santa Cruz Biotech (Santa Cruz, CCCA-39). CA). The fluorochrome-labeled Abs of CD3, CD4, IL-4, IL-5, IL-13, IFN-g, and TNF were purchased from BD Biosciences (Franklin Lakes, Western blotting NJ). The IL-2, ELISA kits of IL-4, IL-5, IL-13, and TNF were purchased from R&D Systems (Minneapolis, MN). The reagents for real-time The total proteins were extracted from the cells collected from relevant ex- quantitative PCR (RT-qPCR), Western blotting, molecular cloning, and periments, fractioned by SDS-PAGE, and transferred onto a PVDF mem- plasmid transfection were purchased from Invitrogen (Carlsbad, CA). The brane. The membrane was blocked with skim milk (0.5%) for 30 min and oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one), PMA, ion- incubated with the primary Abs of interest or isotype IgG overnight at 4˚C, omycin, reagents for cell culture, myeloperoxidase (MPO) assessment, followed by incubating with the second Abs (labeled with peroxidase) for immunoprecipitation (IP), and chromatin IP were purchased from Sigma- 1 h. The immune blots on the membrane were developed with the ECL and Aldrich (St. Louis, MO). The immune cell isolation reagent kits were photographed with an image device. The integrated density of the immu- purchased from Miltenyi Biotech (San Diego, CA). noblots was measured with Adobe Photoshop CC (2016) and presented as Downloaded from percentage against the immunoblots of housekeeping gene b-actin. Patients Flow cytometry Patients with either UC or CD at the remission stage were recruited into the current study at the Gastrointestinal Clinic of the Fifth Hospital of Zhengzhou For the surface staining, cells were stained with the fluorescence-labeled Abs or University. The diagnosis and treatment of IBD were carried out by our isotype IgG for 30 min at 4˚C. In the case of the intracellular staining, cells physicians. The demographic data of the patients are presented in Table I. were treated with fixative (1% paraformaldehyde) and permeable reagent Patients with any of the following conditions were excluded: cancer, severe (0.5% saponin) for 1 h. After washing with PBS, the cells were analyzed with http://www.jimmunol.org/ organ diseases, autoimmune diseases, and undergoing treatment with im- a flow cytometry. The results were analyzed with the software package of mune suppressors. The study was approved by the Human Ethics Committee Flowjo. The data from isotype IgG staining were used as a gating reference. at Zhengzhou University. A written informed consent was obtained from each human subject. Biopsy specimens were collected from IBD patients Immunoprecipitation under routine procedures by our physicians. One colon biopsy specimen was Cells were collected from relevant experiments and lysed with a lysing collected from the visually diseased mucosa of each IBD patient. One co- buffer. The lysates were precleared by incubating with Protein G Agarose lonic biopsy specimen was obtained from each control subject, which was for 2 h 4˚C. The supernatant was collected by centrifugation, and 100 mg subsequently confirmed as histologically normal. The control subjects were protein of each sample was incubated with the Abs of interest or isotype examined by colonoscopy for the purpose of ruling out colon cancer. IgG overnight at 4˚C. The immune complexes were precipitated by incu- Mice bating with Protein G Agarose for 2 h at 4˚C. The agarose beads were by guest on September 30, 2021 collected by centrifugation. The proteins on the beads were eluted by an Male BALB/c mice (6–8-wk-old) were purchased from the Guangdong eluting buffer. The proteins were analyzed by Western blotting. Experimental Animal Center (Guangzhou, China). The mice were main- tained in a specific pathogen-free facility at Shenzhen University. Chromatin IP Because BCL2L12 is broadly expressed in the body, a global KO may + Cells were collected from relevant experiments and fixed with 1% formalin affect other cell types. To study the role of BCL2L12 in CD4 T cells, for 15 min. After washing with PBS, the cells were then sonicated to shear models of lineage-specific deletion need to be used, such as the study of + the DNA into small pieces. The lysates were then processed with the IL-6 in CD4 T cells (16). More information about constructing this mouse procedures of IP. The DNA-protein complexes on the beads were eluted with an strain is presented in the supplemental material (Supplemental Fig. 1). The + eluting buffer. The DNA was recovered with a reagent kit following the mice (BALB/c background) with Bcl2L12 KO CD4 T cells were provided manufacturer’s instructions. The DNA was analyzed by quantitative PCR with by the Institute of Animal Sciences at the Chinese Academy of Agricul- the primers of the Il4 promoter primers (59-GGCCTCTCCCTTCTATGCAA- tural Sciences (Beijing, China). The animal experiments were approved by 39 and 59-GATTGTCAGTCACTTGGGGC-39; 21000 to 100). The results the Animal Ethics Committee at Shenzhen University. were presented as fold change against the input. Isolation of lamina propria mononuclear cells RNA interference The colon tissue was cut into small pieces and incubated with collagenase CD4+ T cells were isolated from the collected human blood samples by IV (1 mg/ml) for 2 h at 37˚C with mild agitation. The cells were first filtered MACS. The cells were transduced with shRNA of Bcl2L12 or control through a 70-mm cell strainer and then filtered through a 40-mm cell shRNA with purchased reagent kits following the manufacturer’s instruc- strainer. The cells were collected by centrifugation. + tions. Briefly, naive CD4 T cells were cultured in the presence of T cell Preparation of PBMCs activators (IL-2, PMA, and ionomycin) and incubated with Bcl2L12 shRNA lentivectors or control lentivectors. Three days later, the cells were Blood samples were collected from human subjects by the ulnar vein collected and washed with fresh medium, and puromycin was added puncture. PBMCs were isolated by gradient density centrifugation. (1.5 mg/ml) to select transduced T cells. Three days later, the expression of Bcl2L12 in the cells was analyzed by Western blotting. Cell culture Assessment of MPO activity The cells were cultured in an RPMI 1640 medium supplemented with FBS (10%), streptomycin (0.1 mg/ml), penicillin (100 U/ml), and L-glutamine (2 mM). The The MPO activity was assessed with the extracts of the colonic tissues with medium was changed in 2–3 d. The cell viability was .98% as checked by the an MPO assay kit following the manufacturer’s instructions. The MPO Trypan blue exclusion assay before handing over for further experiments. activity was measured with a spectrophotometer at 460 nm and expressed in units per gram of tissue. One U corresponds to the MPO activity re- Purification of immune cells quired to degrade 1 mM of hydrogen peroxide per minute at 25˚C. The immune cells used in relevant experiments were purified by magnetic Generation of CD4+ T cells with Bcl2L12 overexpression cell soring with purchased reagent kits following the manufacturer’s instructions. The purity of the isolated immune cells was 92–96% as The eukaryotic expression vector pcDNA3.1(+)-Bcl2L12 carrying a cDNA assessed by flow cytometry. copy of Bcl2L12 (mouse: NM_029410.3; human: NM_138639.1) was The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021 FIGURE 1. CD4+ T cells of UC patients express Bcl2L12. Peripheral blood samples were collected from healthy subjects (n = 30), UC patients (n = 30), and CD patients (n = 30). (A–D) The CD3+ CD4+ T cells were isolated from the blood samples by MACS. The RNA and protein extracts of the CD4+ T cells were analyzed by RT-qPCR and Western blotting. (A) The scatter dot plots show mRNA levels of Bcl2L12. (B and C) The immunoblots show Bcl2L12 protein; the scatter dot plots show the image analysis results of the immunoblots. (D) The bars indicate the mRNA levels of CD4+ T cell–derived cytokines (as shown on the x-axis). (E) The bars show the phenotypes of CD4+ T cells in PBMC (by flow cytometry). (F) The bars indicate the serum levels of CD4+ T cell–derived cytokines (by ELISA). (G–I) The scatter plots show the correlation between Bcl2L12 and Th2 cytokines. Bars indicate mean 6 SD. *p , 0.01 compared with the healthy group (Student t test). constructed by Sangong Biotech (Shanghai, China). The sequence of Bcl2L12 human) by electroporation using a Bio-Rad Gene Pulse set at 950 mFand was confirmed by DNA sequencing. Naive CD4+ T cells were isolated from 280 V. The cells were collected 48 h later for the examination of Bcl2L12 the wild type (WT) mouse spleen or PBMC of UC patients by MACS. The expression. The efficiency of the plasmid transfection was over 98% as cells were transfected with the plasmids of pcDNA3.1(+)-Bcl2L12 (mouse or checked by fluorescent microscopy (the plasmids have a GFP gene).

Table I. Demographic data

Characteristic UC (n = 30) CD (n = 30) Healthy (n =30) Age, y 32.2 6 8.9 31.5 6 7.2 28.5.5 6 4.1 Male, no. (%) 15 (50) 15 (50) 15 (50) Body weight, kg 53.5 6 4.6 54.1 6 4.8 55.2 6 5.9 Current smoker, no. (%) 1 (3.3) 1 (3.3) 1 (3.3) Duration of disease, y 5.5 6 3.9 5.1 6 4.8 — Mayo Clinic score (MCS) 8.7 6 1.3 8.5 6 1.4 — Partial MCS 6.3 6 1.1 6.2 6 1.1 — IBDQ score 123 6 36 126 6 33 — Fecal calprotectin, mg/g 1.3 6 1.2 1.1 6 1.4 0.12 6 0.06 Site of disease, no. (%) Rectum and sigmoid colon 9 (30) 12 (40) — Left side of colon 9 (30) 6 (20) — Proximal colon 6 (20) 6 (20) — All of the colon 4 (20) 6 (25) — Medication, no. (%) No medication 30 (100) 30 (100) — —, no data; IBDQ, Inﬂammatory Bowel Disease Questionnaire. 4 Bcl2L12 CONTRIBUTES TO ABERRANT Th2 RESPONSE

Table II. Results of SPT

Fish Egg Soy Hazelnut Shrimp Walnut Milk Peanut Healthy 1 0 0 0 0 0 1 0 UC 6 5 6 4 6 4 8 5 CD 1 0 1 1 1 1 5 1 Some patients were sensitized to more than one Ag. These foods are the most common food allergens in China.

Induction of the activation-induced cell death mononuclear cell infiltrate. The sections were coded. The observers were not aware of the code to avoid observer bias. CD4+ T cells were treated with activators (PMA [50 ng/ml]; ionomycin [0.5 mg/ml]) in the culture for 48 h, washed with culture medium, cultured Statistics with fresh medium for 8 h, and then stimulated with the activators in the culture overnight. The cells were collected and stained with annexin v The difference between two groups was determined by Student t test or reagents and propidium iodide. The cells were analyzed with a flow ANOVA followed by Bonferroni correction if there were more than two , cytometer. The annexin v+ cells or propidium iodide+ annexin v+ cells were groups. A p value 0.05 was set as the significance criterion. The cor- regarded as apoptotic cells. relation test was performed with the software package Prism GraphPad. Skin prick test Results

Skin prick test (SPT) was carried out in our clinic using commercial food extract Expression of Bcl2L12 is positively correlated with the Downloaded from reagents (Greer Company; Taipei, China). Food extracts included shrimp, eggs, expression of Th2 cytokines in CD4+ T cells of UC patients walnuts, hazelnuts, soy, peanuts, fish, and cow’s milk. Saline was used as a negative control. Histamine (1 mg/ml) was used as a positive control. SPT was Prompted by our recent finding that Bcl2L12 is associated with the defined as positive when the diameter of the wheal was 3 mm larger than a immune deregulation of UC patients (12), we further investigated negative control at 15 min. The SPT results are presented in Table II. the association between Bcl2L12 and Th2 response in peripheral Induction of Th2-type inflammation in the colon CD4+ T cells in IBD patients. The results showed that human peripheral CD4+ T cells expressed Bcl2L12, which was markedly http://www.jimmunol.org/ Following published procedures (16), mice were sensitized to oxazolone by topically applying 3% oxazolone in 100% ethanol (150 ml) on the shaved higher in patients with UC than in CD patients and healthy sub- abdomen. Seven days later, 1.5% oxazolone in 50% ethanol (150 ml) was jects (Fig. 1A–C, Tables I, II). Because the analysis of peripheral administered intrarectally with a polyethylene catheter. Control mice were blood samples showed a Th2-dominant profile in UC patients, treated with ethanol vehicle alone. Three days after rectal oxazolone ad- demonstrated as the higher expression of Th2 cytokines and ministration, mice were sacrificed. higher frequency of Th2 cells (Fig. 1D–F, Supplemental Fig. 2), Colon tissue inflammatory score we further analyzed the correlation between the expression of + Colon sections were stained with H&E stain analyzed by light microscopy Bcl2L12 and Th2 cytokines in CD4 T cells. The results showed that Bcl2L12 was positively correlated with the expression of Th2 using a 15-point scale (16), briefly, with three points each for enterocyte by guest on September 30, 2021 loss, crypt hyperplasia, crypt inflammation, neutrophil infiltrate, and cytokines (Fig. 1G–I) but not significantly correlated with the

FIGURE 2. Human intestinal CD4+ cells express Bcl2L12. Colon biopsy specimens were collected from patients with UC (n = 30), CD (n = 30), and controls (n = 6). The samples were analyzed by RT-qPCR, Western blotting, and immunohistochemistry. (A–C) The mRNA (A) and protein (B) levels of Bcl2L12 in colon tissue extracts. (C) The scatter dot plots show the image analysis results and the density of the immunoblots of (B). (D–F) The representative images show the Bcl2L12 staining (in red) in CD4+ cells (in green) of the colon mucosa. The isotype IgG staining is a negative control (G). The inset in (E) shows enlarged positively stained cells (indicated by arrows). *p , 0.01 (Student t test) compared with the healthy group. The Journal of Immunology 5 expression of Th1 cytokines (data not shown). The results indicate induced in the colon of WT mice, including tissue structure that human peripheral CD4+ T cells express Bcl2L12, which may damage and profound mononuclear cell infiltration (Fig. 3A, 3B); be associated with the Th2 polarization in patients with UC. a decrease in body weight (Fig. 3C); high levels of MPO, IL-4, IL- Next, we collected colon biopsy samples from patients with IBD and 5, and IL-13 but not IFN-g, IL-17, or TNF in colon protein ex- controls. The mRNA and protein of Bcl2L12 were detected in the tracts (Fig. 3D, 3E); and high frequency of Th2 cells, but not Th1 extracts of the colon samples, and there were significantly more de- or Th17 cells, among lamina propria mononuclear cells (LPMC) tected in the UC group as compared with the normal controls and the isolated from the colon (Fig. 3F–H). The results demonstrate that CD group (Fig. 2A, 2B). The results of immunohistochemistry showed the Th2-biased inflammation was induced in the colon of WT a high frequency of CD4-positive cells that were Bcl2L12 positive in mice but not Bcl2L12 KO mice. the UC group, but few CD4-positive cells were Bcl2L12 positive in Bcl2L12 facilitates Th2 cell development the CD group and normal controls (Fig. 2C–F). Bcl2L12 was localized + in both the cytoplasm and the nuclei (Fig. 2E). The data reported above show the Bcl2L12 overexpression in CD4 T cells of UC patients. To elucidate whether the Bcl2L12 over- Mice with Bcl2L12 deficiency fail to induce Th2-biased expression is associated with the skewed Th2-biased inflammation inflammation in the colon in UC patients, naive CD4+ T cells were prepared from PBMCs. Based on the data shown in Fig. 1, we inferred that Bcl2L12 might The cells were exposed to T cell activators (IL-2/PMA/ionomycin) be a critical molecule in the pathogenesis of UC. To test the in the culture for 3 d. The results showed that a much higher inference, mice with Bcl2L12-KO CD4+ T cells and WT mice frequency of IL-4+ CD4+ T cells was induced in the UC group were treated with oxazolone. The IBD-like inflammation was than in the healthy group or the CD group (Fig. 4A, 4B). The Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 3. Bcl2L12-deficient mice fail to induce Th2-biased inflammation in the intestine. The mice with Bcl2L12 KO CD4+ T cells and WT mice were treated with oxazolone. (A) Representative histology images (original magnification 3100) show inflammation in the colon of WT mice. (B) The inflammatory scores in the histology of (A). (C) The MPO levels in the colon tissue. (D) The body weight change. (E) The cytokine levels in the protein extracts of colon tissue (by ELISA). (F) The summary of the frequency of CD4+ T cell phenotypes in LPMC isolated from the colon (by flow cytometry); the flow cytometry histograms are presented in (G) and (H). Bars indicate mean 6 SD. *p , 0.01 (Student t test) compared with the WT group (each group consists of six mice). 6 Bcl2L12 CONTRIBUTES TO ABERRANT Th2 RESPONSE Downloaded from http://www.jimmunol.org/

FIGURE 4. Bcl2L12 promotes Th2 cell development. (A) PBMCs were collected from UC patients (n = 6), CD patients (n = 6), and healthy subjects (n = 6). Naive CD4+ T cells were isolated from PBMCs by MACS and treated with activators or saline in the culture for 3 d. The gated flow cytometry dot plots show the frequency of Th2 cells, which were summarized in the scatter dot plots. (B) The results of Bcl2L12 RNAi in CD4+ T cells. (C) Naive CD4+ by guest on September 30, 2021 T cells were collected from UC patients and treated with the procedures denoted above the dot plots. The gated flow cytometry dot plots indicate the frequency of induced Th2 cells, which was summarized in the scatter dot plots. (D) The results of Bcl2L12 overexpression in CD4+ T cells. (E) Naive CD4+ T cells were collected from healthy subjects and treated with the procedures denoted above the dot plot panels. The gated dot plots show the frequency of induced Th2 cells, which was summarized in the scatter dot plots. The data represent six independent experiments. *p , 0.01 (t test). results indicate that the naive CD4+ T cells from UC patients are collected from UC patients. The protein extracts of the CD4+ prone to differentiate into Th2 cells. T cells were analyzed by co-IP. A complex of Bcl2L12 and Because Bcl2L12 is positively correlated with the expression of GATA3 was detected in CD4+ T cells in UC patients (Fig. 5A), Th2 cytokines in CD4+ T cells, as shown by Fig. 1G–I, the Th2- and the data were corroborated by a finding of a complex of the differentiation tendency of CD4+ T cells in UC patients may be recombinant Bcl2L12 and GATA3 in HEK293 cells (Fig. 5B). attributed to the overexpression of Bcl2L12. To test this, naive Further analysis showed that GATA3 were detected at the Il4 CD4+ T cells were collected from UC patients; expression of promoter locus in peripheral CD4+ T cells; its levels were much Bcl2L12 was knocked down in these cells by RNA interference higher in the UC group than in the healthy group (Fig. 5C) and (RNAi) (Fig. 4C) and the cells were then exposed to the activators positively correlated with the expression of Bcl2L12 (Fig. 5D). in the culture for 3 d. Indeed, the cells showed low expression of The results suggest that Bcl2L12 may facilitate GATA3 to bind IL-4 as compared with the controls (Fig. 4D, 4E). The results the Il4 promoter. To test the inference, EL-4 cells were activated suggest that Bcl2L12 promotes the development of Th2 cells. In with PMA/ionomycin. In line with published data (17), the EL-4 contrast, naive CD4+ T cells were collected from healthy subjects. cells expressed higher levels of GATA3 after activation, which The cells were overexpressed with Bcl2L12 by transfecting with was also detected at the Il4 promoter locus. To test whether the Bcl2L12-expressing plasmids (Fig. 4F). The cells were treated Bcl2L12 was associated with the binding between GATA3 and with activators for 3 d. As analyzed by flow cytometry, the the Il4 promoter,EL-4cellsweretreatedwithBcl2L12RNAito overexpression of Bcl2L12 markedly enhanced the expression of knock down the Bcl2L12 (Fig. 5E). Very low levels of GATA3 IL-4 in the cells (Fig. 4G, 4H). The data support the inference that were detected at the Il4 promoter locus (Fig. 5F), whereas the Bcl2L12 promotes the expression of IL-4 in CD4+ T cells. total amount of GATA3 was not altered by the knockdown of

+ Bcl2L12 in the EL-4 cells (Supplemental Fig. 3A, 3B). To cor- Bcl2L12 enhances GATA3 binding to the Il4 promoter in CD4 roborate the results, EL-4 cells were overexpressed Bcl2L12 T cells of UC patients (Fig. 5G), which signiﬁcantly increased the amount of GATA3 at Because GATA3 is the essential transcription factor of Th2 cells, the Il4 promoter locus (Fig. 5H), whereas the expression of we next observed the relation between Bcl2L12 and GATA3 GATA3intheEL-4cellswasnotalteredbytheproceduresof activities in CD4+ T cells. Peripheral CD4+ T cells were Bcl2L12 overexpression (Supplemental Fig. 3C, 3D). The results The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. Bcl2L12 enhances GATA3 binding to Il4 promoter. The peripheral CD4+ T cells were isolated from PBMC collected from UC patients (n =30) A + B and healthy subjects (n =30).( ) The immunoblots show a complex of Bcl2L12 and GATA3 in peripheral CD4 T cells of UC patients. ( ) HEK293 cells by guest on September 30, 2021 were transfected with Bcl2L12-FLAG plasmids and GATA3-HIS plasmids. The cell extracts were analyzed by co-IP. The immunoblots show a complex of the recombinant GATA3 and Bcl2L12 in HEK293 cells. (C) The levels of GATA3 in peripheral CD4+ T cells of UC patients and healthy subjects. (D) The scatter plots show the correlation between the expression of Bcl2L12 and GATA3 in CD4+ T cells of UC patients. (E) The results of Bcl2L12 RNAi of EL-4 cells. (F) EL-4 cells were treated as denoted on the x-axis. The scatter dot plots indicate the GATA3 levels at the Il4 promoter locus of the EL-4 cells. (G) The results of Bcl2L12 overexpression in EL-4 cells. (H) EL-4 cells were treated as denoted on the x-axis. The scatter dot plots indicate the GATA3 levels at the Il4 promoter locus of the EL-4 cells. Bars indicate mean 6 SD. The samples from individual human subjects were analyzed separately in duplicate. The data shown in (E)–(H) represent three independent experiments. *p , 0.01 compared with the healthy group (C) or the saline group (F and H). *p , 0.01 (Student t test) compared with the saline group, #p , 0.01 (Student t test) compared with the activator alone group. demonstrate that Bcl2L12 enhances the binding between GATA3 patients is defective, in which Bcl2L12 overexpression plays a and Il4 promoter. critical role.

Bcl2L12 inhibits p53 to compromise apoptotic mechanism in + Discussion CD4 T cells of UC patients The present study revealed a previously unknown immune regu- In contrast, we collected peripheral CD4+ T cells collected from latory factor of Th2 response. We found that peripheral CD4+ UC patients. The cells were treated with the activation-induced T cells expressed Bcl2L12 and that the expression was greater in cell death (AICD) procedures. The results showed that after UC patients. The Bcl2L12 formed a complex with GATA3 to activation, the apoptotic cells (Fig. 6A) and the levels of p53 enhance the binding of GATA3 and the Il4 promoter in CD4+ were markedly less in the CD4+ T cells from UC patients than T cells. Overexpression of Bcl2L12 markedly enhanced the ex- those of healthy controls (Fig. 6B). A negative correlation was pression of IL-4 in CD4+ T cells. Knockdown of Bcl2L12 by identified between Bcl2L12 and p53 (Fig. 6C) in the CD4+ RNAi inhibited the expression of Th2 cytokines by CD4+ T cells T cells from UC patients. The results suggest that Bcl2L12 may collected from UC patients. Mice with Bcl2L12 KO CD4+ T cells inhibit apoptosis by suppressing p53 in CD4+ T cells of UC failed to induce the Th2-biased inflammation in the intestine. The patients. To test the inference, the naive EL-4 cells and EL-4 results also showed that Bcl2L12 inhibited p53 and compromised cells transfected with Bcl2L12-expressing plasmids or control the apoptosis machinery in CD4+ T cells. The data demonstrate plasmids were treated with doxorubicin, an inducer of apoptosis. that Bcl2L12 plays a critical role in the pathogenesis of the ab- The results showed that the overexpression of Bcl2L12 inhibited errant Th2 polarization in the intestine. the doxorubicin-induced apoptosis as well as decreased the UC is a chronic inflammation in the colon mucosa. The causative levels of p53 in EL-4 cells (Fig. 6D). The results demonstrate factors of UC are unknown. The skewed reactions to food allergens that the activation-induced apoptosis in CD4+ T cells of UC in the colon were observed by previous studies, which were called 8 Bcl2L12 CONTRIBUTES TO ABERRANT Th2 RESPONSE Downloaded from http://www.jimmunol.org/

FIGURE 6. Bcl2L12 suppresses the activation-induced apoptosis in CD4+ T cells of UC patients. (A and B) The peripheral CD4+ T cells were collected from UC patients (n = 30) and healthy subjects (n = 30). The cells were treated with the AICD-induction procedures. The gated dot plots show the apoptotic cells, which were summarized as the scatter dot plots. (B) The total RNA and proteins were extracted from the CD4+ T cells of UC patients and healthy

subjects. The samples were analyzed by RT-qPCR and Western blotting. The bars show the mRNA levels of p53, and the immunoblots show the protein by guest on September 30, 2021 levels of p53 in the CD4+ T cells. (C) The scatter dot plots show the correlation between p53 and Bcl2L12 in CD4+ T cells collected from UC patients. (D) EL-4 cells were treated as denoted above each subpanel. The gated dot plots show the apoptotic cells, which were summarized as the scatter dot plots. The immunoblots show the p53 protein levels. Doxorubicin (Dox) (10 mg/ml) was added to the culture. The samples from human subjects were analyzed separately. The data shown in (D) represent three independent experiments. *p , 0.01 compared with group a. the “food protein–induced enterocolitis syndrome or a non–IgE- the Bcl2 family and plays an important role in the regulation of the mediated food allergy” (18). Our previous study showed that UC apoptosis process. Early studies showed that glioma cells expressed patients had IgE-mediated food allergy, and the patients responded Bcl2L12 (23). Later studies showed that the Bcl2L12 expression well to the allergen-specific immunotherapy (9). It is accepted that was also found in the liver (24) and vascular endothelial cells (25). Th2-biased inflammation is one of the major features of allergic Our previous studies also found that Bcl2L12 interfered with the disorders. The present data are in line with this notion by showing expression of IL-10 in peripheral B cells (12, 13). It was revealed by high levels of Th2 cytokines in the serum and high frequency of Stegh et al. (23) that the expression of Bcl2L12 was enhanced in the Th2 cells in the peripheral blood of UC patients. Yet, various data glioma tissue and it was assumed that Bcl2L12 was associated with were also reported about the role of Th2 response in the patho- the tumor growth by suppressing p53, caspase 3 and 7, and inducing genesis of UC. Melgar et al. (19) found fewer Th2 cells in the defective apoptosis. The present data are in line with the previous colon mucosa of UC patients. West et al. (20) even found that the studies by showing that the expression of Bcl2L12 in CD4+ Tcells LPMCs of UC patients produced less IL-4 than healthy controls, also suppressed p53 and inhibited the AICD in T cells. AICD is an whereas Rosen et al. (21) found that the Th2 cytokines (IL-5 and important mechanism in the body to remove unnecessary cells. IL-13) were higher in UC patients and associated with mucosal Defective AICD has been recognized in a number of immune dis- healing with their diseases. Although the Th2 cytokines are pro- orders, such as asthma (26) and colitis (27). The present data also tective for the body at the proper levels, overproduction of Th2 show that CD4+ T cells from UC patients are less sensitive to the cytokines has been recognized in a large number of disorders (22). activation-induced apoptosis. Such a phenomenon may contribute to Our data also showed higher serum levels of Th2 cytokines in UC the pathogenesis of aberrant Th2 polarization. patients, which is in line with the data of Rosen et al. (21). The skewed Th cell hyper response is one of the factors involved Although the role of Th2 cytokines in the pathogenesis of chronic in the pathogenesis of IBD (28). Either Th1, Th17, or Th2 hyper inflammation (such as allergy and autoimmune diseases) is well response has been reported in IBD (28). The Th1 and Th17 hyper recognized, the causative factors of Th2 cytokine overproduction is responses to the stimuli of intestinal microbes in IBD have been unclear. The present data suggest that the Bcl2L12 overexpression in extensively studied (29). Yet, the causative factors inducing Th2 CD4+ T cells plays a role in the Th2-biased inflammation, which is hyper response in IBD are to be further investigated. In this study, associated with the pathogenesis of UC. Bcl2L12 is a member of the data show that Th2 hyper response was observed in UC The Journal of Immunology 9 patients. This is in line with previous studies; cumulative reports 10. Huang, Z., Y. Jiang, Y. Yang, J. Shao, X. Sun, J. Chen, L. Dong, and J. Zhang. 2013. 3,39-Diindolylmethane alleviates oxazolone-induced colitis through Th2/ indicate that the Th2 cytokines were significantly increased in Th17 suppression and Treg induction. Mol. Immunol. 53: 335–344. both UC patients and colitis mice (8–12). The present study re- 11. Seidelin, J. B., M. Coskun, P. H. Kvist, T. L. Holm, K. Holgersen, and veals a novel phenomenon that CD4+ T cell with Bcl2L12 over- O. H. Nielsen. 2015. IL-33 promotes GATA-3 polarization of gut-derived T cells in experimental and ulcerative colitis. J. Gastroenterol. 50: 180–190. expression is one of the factors contributing to the Th2-biased 12. Guo,X.,M.G.Li,S.S.Li,F.H.Liu,Z.J.Liu,andP.C.Yang.2017.Tumornecrosis inflammation in the colon. factor suppresses interleukin 10 in peripheral B cells via upregulating Bcl2-like protein It is well known that GATA3 is the essential transcription factor 12 in patients with inflammatory bowel disease. Cell Biochem. Funct. 35: 77–82. 13. Xue, J. M., L. T. Yang, G. Yang, X. R. Geng, Z. Q. Liu, S. Wang, H. L. Zhao, of the expression of IL-4 and Th2 cell differentiation. The present Z. G. Liu, C. Q. Zhao, and P. C. Yang. 2017. Protease-activated receptor-2 data show that the levels of GATA3 are higher in CD4+ T cells in suppresses interleukin (IL)-10 expression in B cells via upregulating Bcl2L12 in patients with allergic rhinitis. Allergy 72: 1704–1712. UC patients. Others also observed increased expression of GATA3 14. Thomadaki, H., and A. Scorilas. 2006. BCL2 family of apoptosis-related genes: by lamina propria T cells from mice with colitis (30); GATA3 functions and clinical implications in cancer. Crit. Rev. Clin. Lab. Sci. 43: 1–67. overexpression in T cells can accelerate the dextran sulfate so- 15. Stegh, A. H., C. Brennan, J. A. Mahoney, K. L. Forloney, H. T. Jenq, J. P. Luciano, A. Protopopov, L. Chin, and R. A. Depinho. 2010. Glioma oncoprotein Bcl2L12 dium–induced colitis (31). Elevation of GATA3 was also observed inhibits the p53 tumor suppressor. Genes Dev. 24: 2194–2204. in the colon mucosa of UC patients (32, 33). The present data are 16. Nish, S. A., D. Schenten, F. T. Wunderlich, S. D. Pope, Y. Gao, N. Hoshi, S. Yu, consistent with these studies by showing the expression of GATA3 X. Yan, H. K. Lee, L. Pasman, et al. 2014. T cell-intrinsic role of IL-6 signaling + in primary and memory responses. Elife 3: e01949. in CD4 T cells of UC patients. In addition, the data show a novel 17. Pyo, M. Y., S. J. Yoon, Y. Yu, S. 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Bcl2L12 expression may be capable of inhibiting the Th2-biased interleukin 4 in inflammatory bowel disease and mucosal immune reactivity. http://www.jimmunol.org/ Gastroenterology 110: 1683–1695. flammation. The profound infiltration of Th2 cells in the local tissue is 21. Rosen, M. J., R. Karns, J. E. Vallance, R. Bezold, A. Waddell, M. H. Collins, the basis of Th2-type inflammation. Published data indicate that the Y. Haberman, P. Minar, R. N. Baldassano, J. S. Hyams, et al. 2017. Mucosal frequency of Ag-specific Th2 cells is much higher in the intestine of expression of type 2 and type 17 immune response genes distinguishes ulcerative colitis from colon-only Crohn’s disease in treatment-naive pediatric patients. subjects with allergic inflammation; these Th2 cells produce high Gastroenterology 152: 1345–1357.e7. amounts of Th2 cytokines (34, 35). The present data provide a possible 22. Raphael, I., S. Nalawade, T. N. Eagar, and T. G. Forsthuber. 2015. T cell subsets and explanation for this phenomenon. Naive CD4+ T cells with Bcl2L12 their signature cytokines in autoimmune and inflammatory diseases. Cytokine 74: 5–17. 23. Stegh, A. H., and R. A. DePinho. 2011. Beyond effector caspase inhibition: overexpression are prone to differentiate into Th2 cells; these cells Bcl2L12 neutralizes p53 signaling in glioblastoma. Cell Cycle 10: 33–38. overproduce proinflammatory cytokines, such as IL-4, IL-5, and IL-13, 24. Real, L. M., A. Caruz, A. Rivero-Juarez, V. Soriano, K. Neukam, A. Rivero,

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Corrections

Li, M.-G., X.-Y. Liu, Z.-Q. Liu, J.-Y. Hong, J.-Q. Liu, C.-J. Zhou, T.-Y. Hu, X.-J. Xiao, P.-X. Ran, P.-Y. Zheng, Z.-G. Liu, and P.-C. Yang. 2018. Bcl2L12 contributes to Th2-biased inﬂammation in the intestinal mucosa by regulating CD41 T cell activities. J. Immunol. 201: 725–733.

The rightmost dot plot in Fig. 4C was inadvertently duplicated from the center dot plot in Fig. 4E in the published article. The corrected ﬁgure is shown below. The ﬁgure legend was correct as published and is shown below for reference. Fig. 4 has been corrected in the online version of the article, which now differs from the print version as originally published.

FIGURE 4. Bcl2L12 promotes Th2 cell development. (A) PBMCs were collected from UC patients (n 5 6), CD patients (n 5 6), and healthy subjects (n 5 6). Naive CD41 T cells were isolated from PBMCs by MACS and treated with activators or saline in the culture for 3 d. The gated ﬂow cytometry dot plots show the frequency of Th2 cells, which were summarized in the scatter dot plots. (B) The results of Bcl2L12 RNAi in CD41 T cells. (C) Naive CD41 T cells were collected from UC patients and treated with the procedures denoted above the dot plots. The gated ﬂow cytometry dot plots indicate the frequency of induced Th2 cells, which was summarized in the scatter dot plots. (D) The results of Bcl2L12 overexpression in CD41 T cells. (E) Naive CD41 T cells were collected from healthy subjects and treated with the procedures denoted above the dot plot panels. The gated dot plots show the frequency of induced Th2 cells, which was summarized in the scatter dot plots. The data represent six independent experiments. *p , 0.01 (t test). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900738

Corrections

The percentage on the Saline panel in Fig. 6D was incorrect as originally published. The correct percentage should have been 5.15%. The corrected ﬁgure is shown below. The ﬁgure legend was correct as published and is shown below for reference. Fig. 6 has been corrected in the online version of the article, which now differs from the print version as originally published.

FIGURE 6. Bcl2L12 suppresses the activation-induced apoptosis in CD41 T cells of UC patients. (A and B) The peripheral CD41 T cells were collected from UC patients (n 5 30) and healthy subjects (n 5 30). The cells were treated with the AICD-induction procedures. The gated dot plots show the apoptotic cells, which were summarized as the scatter dot plots. (B) The total RNA and proteins were extracted from the CD41 T cells of UC patients and healthy subjects. The samples were analyzed by RT-qPCR and Western blotting. The bars show the mRNA levels of p53, and the immunoblots show the protein levels of p53 in the CD41 T cells. (C) The scatter dot plots show the correlation between p53 and Bcl2L12 in CD41 T cells collected from UC patients. (D) EL-4 cells were treated as denoted above each subpanel. The gated dot plots show the apoptotic cells, which were summarized as the scatter dot plots. The immunoblots show the p53 protein levels. Doxorubicin (Dox) (10 mg/ml) was added to the culture. The samples from human subjects were analyzed separately. The data shown in (D) represent three independent experiments. *p , 0.01 compared with group a.