anti-mouse cd52 treatment ameliorates colitis through
TRANSCRIPT
Vol. 41, No. 9 1423Biol. Pharm. Bull. 41, 1423–1429 (2018)
© 2018 The Pharmaceutical Society of Japan
Regular Article
Anti-mouse CD52 Treatment Ameliorates Colitis through Suppressing Th1/17 Mediated Inflammation and Promoting Tregs Differentiation in IL-10 Deficient MiceJianhui Liu,a,b,# Honggang Wang,c,# Yi Li,a Peiliang Shi,d Jianfeng Gong,a Lili Gu,a Weiming Zhu,*,a and Jieshou Lia
a Department of General Surgery, Jinling Hospital, Medical School of Nanjing University; No. 305 East Zhongshan Road, Nanjing, Jiangsu Province 210002, China: b Department of General Surgery, The Second Affiliated Hospital of Nanjing Medical University; 121 Jiangjiayuan Road, Nanjing, Jiangsu Province 210011, China: c Department of General Surgery, Taizhou People’s Hospital, Taizhou Clinical Medical College of Nanjing Medical University; No. 366 Taihu Road, Taizhou, Jiangsu Province 225300, China: and d Model Animal Research Center of Nanjing University; No. 12 Xuefu Road, Nanjing, Jiangsu Province 210089, China.Received April 4, 2018; accepted June 4, 2018; advance publication released online June 13, 2018
Recent studies suggested that excessive T helper (Th)1/17 cells concomitant with regulatory T cell defi-ciency might play important roles in Crohn’s disease. Anti-cluster of differentiation 52 (CD52) monoclonal antibody (mAb), which aims on CD52 antigen on mature immunocytes, has both T cell depletion and im-munosuppressive activities. In this study, we evaluated the therapeutic effects and possible mechanisms of anti-CD52 treatment on interleukin-10 (IL-10) deficient mouse. Anti-mouse CD52 mAb was administered to C3H/HeJBir.IL-10−/− (C3H.IL-10−/−) mice intraperitoneally 20 µg per week for 2 weeks. The disease activity index, body weight, the histological grading of colitis, and levels of tumor necrosis factor (TNF)-α, inter-feron (IFN)-γ, IL-17 and IL-6 in colon were quantified after treatment. In addition, CD25, Forkhead box P3 (Foxp3) and transforming growth factor (TGF)-β gene as well as the percentage of CD25+Foxp3+ T cells in colon were also measured. The severity of colitis in IL-10−/− mice was significantly decreased by the treat-ment, with improvement of colon histological grade. The treatment also decreased the TNF-α, IFN-γ, IL-17 and IL-6 levels in colon. Furthermore, the treatment up-regulated the mRNA expression of CD25, Foxp3 and TGF-β gene as well as the percentage of CD25+Foxp3+ T cells in colon lamina propria mononuclear cells (LPMCs) of IL-10−/− mice. Our data might indicate that anti-CD52 treatment could ameliorate the colitis of C3H.IL-10−/− mice and it might be related to the suppression of Th1/17 related inflammation and the promo-tion of regulatory T cell differentiation. Thus, our data reveals that anti-CD52 treatment may hold potential for clinical applications for Crohn’s disease treatment.
Key words T cell; interleukin-10 (IL-10) deficient mouse; Crohn’s disease; anti-cluster of differentiation 52 (CD52) monoclonal antibody
Crohn’s disease (CD), one of the two major clinically de-fined forms of inflammatory bowel disease, is an immune-mediated relapsing systemic inflammatory disease manly affecting the gastrointestinal tract with extra-intestinal manifestations.1) Although the exact pathogenesis of CD is complex, it’s believed that CD might be triggered by en-vironmental factors and resulted in a disturbed innate and adaptive immune response towards a diminished diversity of commensal microbiota.2) Researches declare that CD is a T-helper (Th)1/Th17 type inflammatory reaction, with exorbitant secretion of numbers of effector cytokines such as interferon (IFN)-γ, interleukin (IL)-17 and tumor necrosis factor (TNF)-α as well as the unmatched changes of the T cell response.3,4) The imbalance of effector T cells versus regulatory T cells (Tregs) in gastrointestinal tract mucosa is a hallmark of CD.5) And genome-wide association studies support the imbalance model by linking loci that are crucially involved in Tregs and Th1/Th17 differentiation to CD.6)
Cluster of differentiation 52 (CD52) is a cell-surface gly-coprotein mainly expresses on mature lymphocytes. Anti-CD52 monoclonal antibody (known as campath-1) can rapidly
produce a profound lymphopenia mainly by the cytotoxicity, leading to long-lasting changes in adaptive immunity. The humanized Campath-1H has been licensed for chronic lym-phocytic leukemia (CLL)7) and been used successfully in several autoimmune diseases, such as arthritis, multiple scle-rosis, vasculitis8) as well as in organ transplantation.9) And in several trials, Campath-1H was found that it could promote increasing of Tregs.10–12)
Our previous studies demonstrated that anti-CD52 treat-ment could improve histological inflammation score,13) inhibit epithelial apoptosis and tight junction permeability14) in IL-10 deficient mice. In this study, we mainly investigated the thera-peutic effects on colitis and potential mechanisms relating to Th1/17 related inflammation and regulatory T cells of treat-ment with anti-mouse CD52 monoclonal antibody in IL-10 deficient mouse model which mimics human CD.
MATERIALS AND METHODS
Animals and Immunotherapy C3H/HeJBir.IL-10−/− (C3H.IL-10−/−) mice and C3H/HeJ (wild type) mice were ob-tained from the Jackson Laboratory (Bar Harbor, ME, U.S.A.). All the mice were maintained at the Model Animal Research
* To whom correspondence should be addressed. e-mail: [email protected]
# These authors contributed equally to this work.
1424 Vol. 41, No. 9 (2018)Biol. Pharm. Bull.
Center of Nanjing University (Nanjing, China) in a specific-pathogen-free (SPF) animal facility. All mice received humane care in accordance with the law concerning the protection and control of animals in China. C3H.IL-10−/− mice were used be-cause they develop colitis more severely than other strains.15) Age and sex matched C3H.IL-10−/− mice at 12 weeks of age with established colitis and wild type mice were used for the experiments of anti-mouse CD52 administration. The mice were assigned to the anti-mouse CD52 treated IL10−/− mice group (anti-mouse CD52 group), phosphate buffered saline (PBS) treated IL10−/− mice group (Control group) and PBS treated wild type group (normal group). There were 6 mice per group. The drug administration of anti-CD52 monoclonal antibody was performed as previously described.13) Briefly, each mouse in anti-mouse CD52 group was injected intra-peritoneally with 20 µg anti-mouse CD52 monoclonal antibody (clone BTG-2G) (MBL, Nakaku Nagoya, Japan) dissolved with PBS. One week later, the drug was administrated again with the same dose. The normal and control group mice re-ceived an equivalent quantity of PBS at the same time. The clinic symptoms were observed after the treatment. One and three weeks after the final drug administration, the mice were sacrificed for the following tests.
Disease Activity Index (DAI) The clinical manifestation of colitis was quantified with the DAI according to the meth-ods described previously.16) Briefly, the ruffled fur, occult fecal blood determined by the Fecal Occult Blood Card (Jiancheng Biotechnology Co., Nanjing, China), rectal prolapsed (<1 cm for one point or >1 cm for two points), soft stool and diarrhea were the score points of DAI. All the points were added to a final DAI score with a range from 0 to 6.
Histopathology The colon samples were collected and fixed in paraformaldehyde (PFA). Samples were paraffin-embedded, sectioned, and stained with hematoxylin and eosin. Grading of intestinal inflammatory score was determined as described previously,16) with the scores ranged from 0 to 4.
Cytokine-Specific Enzyme-Linked Immunosorbent Assay (ELISA) Cytokine determination in colon mucosa was performed as previously described,17) briefly, protein ex-tracts were obtained by homogenization of colonic segments (0.5 mg tissue/mL) in homogenization buffer consisting of a protease inhibitor (Roche Diagnostics, Mannheim, Germany). Samples were centrifuged at 20000×g for 30 min at 4°C. Supernatant was aliquoted and stored at −80°C for cytokine examination. TNF-α, IFN-γ, IL-17 and IL-6 concentrations in colonic protein extracts were measured using ELISA Kit (Invitrogen, Vienna, Austria) according to the manufacturer’s recommendations. Values were expressed as pg/mg protein.
Isolation of Mononuclear Cells from Colon Lamina Pro-pria Lamina propria mononuclear cells (LPMCs) of colon were isolated according to the methods described previously with some modifications.18) Briefly, the colon of each mouse was immediately harvested after sacrifice, cut longitudinally and washed with cold RPMI1640 containing 5% fetal calf serum (FCS), then cut into pieces to incubate in the solution contains 1 mM ethylenediaminetetraacetic acid (EDTA) (Sig-ma-Aldrich, St. Louis, MO, U.S.A.) and 1 mM dithiothreutol (Sigma) for 30 min at 37°C. Subsequently, the remained tissues were digested for 30 min in the presence of 40 U/mL collage-nase II (Sigma) and 10 µg/mL deoxyribonuclease (DNase) I (Boehringer Mannheim, Mannheim, Germany). The suspen-
sions were passed through a 40-µm nylon mesh separately and then centrifuged through a 40/70% discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient at 400×g and 20°C for 25 min without braking. The cells from the interface were collected. All the collected mononuclear cells were washed and resuspended with RPMI1640 for the following FACS analysis.
FACS Analysis Fluorescein isothiocyanate (FITC)-con-jugated anti-mouse CD4, Allophycocyanin (APC)-conjugated anti-mouse CD25 and Phycoerythrin (PE)-conjugated anti-mouse Foxp3 (eBioscience, San Diego, CA, U.S.A.) were used for the stain of Tregs according to the protocols supplied by manufacturer. Briefly, 1×106 cells were suspended in 100 µL flow staining buffer with saturating amounts of fluorochrome-conjugated anti-CD4/CD25 for the staining of cell surface antigens, then the cells were incubated with Fixation/Permea-bilization buffer and stained with PE-conjugated anti-mouse Foxp3. All the flow cytometric analyses were acquired using the FACScan flow cytometer (Becton Dickinson, San Jose, CA, U.S.A.), and the data were analyzed using the Flowjo software (Tree Star, U.S.A.). Unstained cells and cells stained with isotype-matched control antibodies served as controls.
Quantitative Real-Time PCR (qPCR) Total RNA of colon tissue was isolated with RNAiso Plus (TaKaRa, Dalian, China) and reversed transcribed by RT reagent Kit (TaKaRa) according to manufacturer’s instructions. The primers were as follows: Foxp3, 5′-CCC AGG AAA GAC AGC AAC CTT-3′ and 5′-TTC TCA CAA CCA GGC CAC TTG-3′, CD4, 5′-AAG TGA CCT TCA GTC CGG GTA-3′ and 5′-GGG TTA GAG ACC TTA GAG TTG CT-3′, CD25, 5′-CTC CCA TGA CAA ATC GAG AAA GC-3′ and 5′-ACT CTG TCC TTC CAC GAA ATG AT-3′, transforming growth factor (TGF)-β, 5′-CCG CAA CAA CGC CAT CTA TG-3′ and 5′-CCC GAA TGT CTG ACG TAT TGA AG-3′, Beta-actin, 5′-GAG AAG ATC TGG CAC CAC ACC-3′ and 5′-GCA TAC AGG GAC AGC ACA GC-3′. The qPCR was performed with the Step One Real-Time PCR system (ABI, Carlsbad, CA, U.S.A.) using the SYBR Premix Ex Taq II kit (TaKaRa). All the process was according to manufacturer’s direction and PCR reactions were done in duplicate and rela-tive expression was calculated using the 2−ΔΔCt method after normalizing beta-actin expression for each sample.
Statistical Analysis SPSS version 17.0 software (SPSS, Inc., Chicago, IL, U.S.A.) was used to perform the statistical analyses. The Mann–Whitney U test with Bonferroni cor-rection was used for multiple comparisons of non-parametric data (the DAI score and histopathology score) for statistical significance. And for the parametric data, Student’s t test was used to compare two groups, and one-way ANOVA was used for multiple comparisons followed by Tukey post-hoc test. Statistical significance was defined as p<0.05. The GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, U.S.A.) software was used for figures drawing.
RESULTS
Anti-CD52 Treatment Reduced Disease Activity in IL-10−/− Mice The onset and severity of colitis of IL-10−/− mice were dramatically different among strains and laborato-ries. We chose the 12 weeks old C3H/HeJ background mice as animal model for the severe and established colitis. The body weight of mice was measured every week from the first drug
Vol. 41, No. 9 (2018) 1425Biol. Pharm. Bull.
administration, the result showed that the IL-10−/− mice spon-taneously developed chronic colitis with weight loss, while the anti-CD52 mAb treated group grew more steadily. One, 2 and 3 weeks after the final administration, the body weights of an-ti-mouse CD52 group mice were significantly higher than the control mice, while the normal group got the similar outcome. (Fig. 1). DAI, which is defined by ruffled fur, fecal blood, rectal prolapse, stool form and diarrhea, was used to analyze the therapeutic benefits of anti-CD52 treatment. Compared to normal group, the control group had higher mean DAI values (Fig. 2b), while the anti-mouse CD52 group showed a sig-nificant reduction of DAI compared to the control group three weeks after the treatment (Fig. 2b).
Anti-CD52 Treatment Lowered Histopathology Scores of Colitis in IL-10−/− Mice Remarkable findings in the his-topathologic effect of anti-CD52 treatment were found three weeks after the administration on mouse colon. The anti-mouse CD52 group mice which received anti-CD52 treatment showed notable depletion of inflammatory cells in the lamina propria of colon mucosae (Fig. 3c), while the control group mice showed the massive infiltration with inflammatory cells, disruption of mucosal and muscular architecture and ulcers penetrated the whole intestinal wall (Fig. 3b). The histopathol-ogy scores were calculated and confirmed the effect of the anti-CD52 treatment. Compared to control group, the normal
group mice showed a significant low score (Fig. 3d), and the score of anti-mouse CD52 group was also lower than the con-trol (Fig. 3d).
The Depletion of CD4+ among Colon LPMCs by Anti-CD52 Treatment in IL-10−/− Mice It has showed that CD4+ T-helper type 1 and T-helper type 17 cells and the inflammation mediated by them are important in CD, while the mature lymphocyte lysis effect of the anti-CD52 mAb was reported in many former studies. Next we tested the depletion of CD4+ T cells in the mononuclear cells in colon tissue three weeks after the anti-CD52 treatment using the flow cytom-etry. The FACS results showed that after the development of colitis, the percentage of CD4+ T cell increased in the control group when compare to the normal group (21.80±0.87% ver-sus 13.02±0.58%, p<0.01). However, in the anti-mouse CD52 group, significant decreases in the percentage of CD4+ T cell were observed compared with control group (8.03±1.64% ver-sus 21.80±0.87%, p<0.01) after the treatment (Fig. 4).
Anti-CD52 Treatment Decreased the Pro-inflammatory Cytokine Expressions in Colon of IL-10−/− Mice CD is usually considered as a Th1/Th17 type of inflammatory reac-tion, the enhanced secretion of pro-inflammatory cytokines is the key process and of CD. To determine the therapeu-tic effect of anti-CD52 mAb in IL-10−/− mouse model, the TNF-α, IFN-γ, IL-17 and IL-6 expressions in colon tissue were examined by ELISA. Remarkable increase expres-sions were observed in control group compared to normal group (TNF-α 88.84±12.18 vs. 16.01±3.87 pg/mg, p<0.01; IFN-γ 59.92±19.58 vs. 5.75±1.43 pg/mg, p<0.01; IL-17 171.56±18.82 vs. 12.48±4.30 pg/mg, p<0.01; IL-6 64.33±13.45 vs. 7.18±3.21 pg/mg, p<0.01), and as ex-pected, the anti-CD52 treatment dramatically decreased the level of these cytokines compared to the control (TNF-α 26.25±5.54 vs. 88.84±12.18 pg/mg, p<0.01; IFN-γ 25.66±8.09 vs. 59.92±19.58 pg/mg, p<0.05; IL-17 14.86±4.96 vs. 171.56±18.82 pg/mg, p<0.01; IL-6 19.84±3.21 vs. 64.33±13.45 pg/mg, p<0.01) (Fig. 5).
Anti-CD52 Treatment Promoted the CD25+Foxp3+ Regulatory T Cell Response in IL-10−/− Mice Th17 and Treg cells exist primarily in the intestinal mucosa, where they have a significant role in T cell-mediated immune responses, and excessive Th17 cells concomitant with a regulatory T cell deficiency might play important roles in the inflammation
Fig. 1. The Body Weight Changes of Mice from the First Anti-CD52 Treatment in Interleukin-10 Deficient Mouse ModelOne, 2 and 3 weeks after the final administration, the body weights of anti-
mouse CD52 group mice were significantly higher than the control mice, while the normal group got the similar outcome. Values were presented means±S.E.M. (n=6 per group). NS p>0.05, * p<0.05, ** p<0.01, compared to the Control group.
Fig. 2. The Changes of DAI Three Weeks after Anti-CD52 Treatment in IL-10 Deficient Mouse Model(a) Rectal prolapse and stool form of mice in each group. (b) Compared to normal group, the control group had higher DAI values, while the anti-mouse CD52 group
showed a significant reduction of DAI compared to the Control group three weeks after the treatment. ** p<0.01.
1426 Vol. 41, No. 9 (2018)Biol. Pharm. Bull.
of CD.19) Foxp3-expressing Tregs are believed to have anti-inflammatory activity through the suppression of the Th17 response.20) And CD25+Foxp3+ Tregs are believed to attenu-ate the severity of ileitis in a Crohn’s-like mouse model.21) So we tested the CD25+Foxp3+ Tregs in colon LPMC by FACS, additionally the mRNA expressions of CD4, CD25, Foxp3 and TGF-β were also examined. The result showed that the percent of CD25+Foxp3+ Tregs in anti-mouse CD52 group was much higher compared to the control group (8.65±0.33 vs. 3.77±0.27%, p<0.01) after receiving the anti-CD52 treatment. (Fig. 6) And the mRNA expressions levels of CD25, Foxp3 and TGF-β in anti-mouse CD52 group were increased, while the mRNA expression of CD4 decreased as expected com-pared to the control group (Fig. 7).
DISCUSSION
CD is one of the two major forms of inflammatory disease (IBD) and it may affect any part of the whole gastrointestinal tract from mouth to anus. Although it is believed to be due to a combination of abnormal gut microbiota, immune response dysregulation, environmental changes and gene variants, a full understanding of CD pathogenesis is still unclear.22) However, the exaggerated immune responds to the gut microbial anti-
Fig. 3. Histological Sections of the Colon from the Each Group Three Weeks after the Anti-CD52 Treatment(a) HE staining of colon from a wild-type mouse, (b) HE staining of a control group mouse showed significant lymphocyte infiltration and distortion of colon wall struc-
ture, while (c) anti-CD52 treated mouse showed markedly decreased infiltration of inflammatory cells. (d) The histopathology scores of these three groups in the colon are presented. Representative sections from three separate groups are shown. ** p<0.01.
Fig. 4. The Depletion of CD4+ T Cells among Colon LPMCs Three Weeks after the Anti-CD52 Treatment(A) The percentage of CD4+ T cell in side scatter-height (SSC-H), (B) the percentage of CD4+ T cell among colon LPMCs in each group was Control 21.80±0.87%,
normal 13.02±0.58% and anti-mouse CD52 8.03±1.64%. Values were presented means±S.E.M. (n=6 per group). ** p<0.01.
Fig. 5. The Th1/17 Related Pro-inflammatory Cytokine Expressions in Colon Tissue of Each Group Mice Three Weeks after the Final Admin-istration
The concentration of the cytokines in each groups (normal vs. Control vs. anti-mouse CD52) were as follows: TNF-α: 16.01±3.87 vs. 88.84±12.18 vs. 26.25±5.54 pg/mg, IFN-γ: 5.75±1.43 vs. 59.92±19.58 vs. 25.66±8.09 pg/mg; IL-17 12.48±4.30 vs. 171.56±18.82 vs. 14.86±4.96 pg/mg; IL-6 7.18±3.21 vs. 64.33±13.45 vs. 19.84±3.21 pg/mg. Values were presented means±S.E.M. (n=6 per group). * p<0.05, ** p<0.01.
Vol. 41, No. 9 (2018) 1427Biol. Pharm. Bull.
gens have been reported to play important roles in beginning and development of CD. Usually, CD was assumed to be me-diated mainly by Th1 cells, and the concentration has recently moved to the differentiation and balance of Th17/Tregs which has recently been showed to affect in CD.23,24) Th17 and Treg cells play an important role in the immune homeostasis, IL-17-releasing Th17 cells are well known for its primarily pro-inflammatory function, while Tregs show anti-inflammatory activity by the suppression of Th17 response, and the TGF-β and IL-10 secreted by Tregs are the main regulatory factors which are aslo believed could suppress the differentiation of pro-inflammatory T cells.25) As Th17 cells are important in intestinal inflammation, it has been aimed as therapeutic tar-
get to interfere the intestinal inflammatory. Zhang et al. found IL-17R knockout mice were significantly protected against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis.26) Sandborn et al.27–29) got promised results in patients with CD with anti-IL12/23p40 antibody (ustekinumab), which inhibits IL-23 mediated Th17 responses. The efficiency of vidofludi-mus in IBD patients was separately confirmed by Fitzpatrick et al.30) and Herrlinger et al.,31) which could ameliorate ex-perimental colitis by decreasing IL-17A and IL-17F levels via down-regulation of nuclear factor (NF)-κB and signal trans-ducer and activator of transcription 3 (STAT3) pathway. Al-though the secukinumab, a mAb directed against IL-17A, was ineffective and associated with more adverse events than pla-
Fig. 6. The Percentage of CD25+Foxp3+ Regulatory T Cell among Colon LPMCs Three Weeks after the Anti-CD52 Treatment(A) FACS results of the CD25+Foxp3+ regulatory T cell in colon LPMCs. (B) The percentage of CD25+Foxp3+ regulatory T cell among colon LPMCs of IL-10 deficient
mice, the anti-mouse CD52 group was 8.65± 0.33% and the Control group was 3.77±0.27%. Values were presented means±S.E.M. (n=6 per group). ** p<0.01.
Fig. 7. The mRNA Expression Levels of CD4, CD25, Foxp3 and TGF-β in Colon Tissues Three Weeks after the Anti-CD52 TreatmentThree weeks after the treatment, the mRNA transcription levels of CD4, CD25, Foxp3 and TGF-β were evaluated by qPCR. The results showed that the levels of CD25,
Foxp3 and TGF-β in anti-mouse CD52 group were up-regulated by the anti-CD52 administration compared to Control group, while the CD4 mRNA expression level de-creased after the treatment. Values were presented means±S.E.M. (n=6 per group). * p<0.05, ** p<0.01.
1428 Vol. 41, No. 9 (2018)Biol. Pharm. Bull.
cebo,32) combined blockade of IL-17A and IL-17F may prevent the development of experimental colitis.33,34) And in another trial, the treatment to inhibit STAT3, which is important in the differentiation of Th17 cells, was proved to attenuate intestinal inflammation.35) And in an experimental colitis model, Treg cells were declared to prevent intestinal inflammation and re-duced the expression of Th17-related cytokines.36) Meanwhile the regulatory T cell therapy in vitro and in vivo for CD was carried out to pave the way for future clinical trials.37,38) These positive outcomes suggest that the treatment upon suppres-sion of Th17-mediated inflammation and promotion of Tregs response holds potential therapeutic utility in T cell-mediated intestinal inflammation in CD.The anti-CD52 monoclonal antibody (marketed as Cam-
path-1), which specifically targets CD52, a most abundant gly-coprotein presents on the surface of all T and B lymphocytes, monocytes and eosinophils, but not hematological precursors, can lyse the cells after binding to its receptor through numer-ous mechanisms and rapidly produce a profound lympho-penia, particularly the low levels of CD4+ T cells.39,40) And Campath-1 was also found that it could promote increasing of Tregs in kidney transplantation and the treatment of mul-tiple sclerosis.10–12) In this study, we aimed on T1/17 mediated inflammation and Tregs and tried to validate the efficacy of anti-mouse CD52 in the treatment of IL-10−/− mouse model.C3H.IL-10−/− mice could develop colitis spontaneously
under a SPF condition as a result of their extreme bias toward a Th1/17-type environment and have many common features with the pathogenesis of human CD patients. In this study, our data showed that the administration of anti-mouse CD52 mAb significantly reduced disease activity, lowered histopathol-ogy scores of colitis and decreased the percentage of CD4+ T cell in colon lamina propria, as well as the expressions of TNF-α, IFN-γ, IL-17 and IL-6 in IL-10−/− mice. Furthermore anti-CD52 treatment also could increase the percentage of CD25+Foxp3+ Tregs in colon LPMCs and up-regulate the mRNA expression of CD25, Foxp3 and TGF-β. Current results provide evidence that anti-CD52 treatment ameliorates the intestinal inflammation of IL-10−/− mice and it might relate to the suppressing Th1/17 type inflammation and promoting regulatory T-Cell response. However, the exact mechanisms of Th1/Th17 inhibition and Tregs promotion is still unclear, we hypothesize that the directly depletion of mature lymphocytes by CD52 mAb might play the role like chemotherapy but not to the stem cell, then the redifferentiation from naive T cell might be something like the stem cell transplants. Because of the empty of pro-inflammatory cells and cytokines by CD52 mAb, a normalizing T-cell balance might reach in the re-dif-ferentiation, the TGF-β secreted by Tregs could suppress the differentiation of Th1/17 cells in IL-10−/− mice and it might be important in this procedure. However, further studies focus on IL-12/23 pathway, TGF-β pathway and other pathways related to T cell regulation should been carried out to certify the roles which CD52mAb plays in the process.In summary, the current study, has suggested anti-CD52
therapy may reverse the clinic symptoms and tissue patho-logical process in IL-10−/− mouse model. It may relate to the depletion of CD4+ T cells in colon tissue, inhibition of Th1/Th17 mediated inflammation and the promotion of Tregs re-sponse. Therefore, the anti-CD52 mAb may be a firenew way for the treatment of CD patients. However, further studies are
required to determine the long-term maintaining effects and adverse reactions of anti-CD52 treatment.
Acknowledgments This study was supported in part by funding from the National Science Foundation of China (Grant Nos. 81170365, 81200263 and 81600434), Jiangsu Natural Science Foundation (Grant No. BK20160572), Jiangsu Provincial Medical Youth Talent (Grant No. QNRC2016514), and the China Postdoctoral Science Foundation (Grant No. 2018M630581). The present study was also partly supported by the Model Animal Research Center, Nanjing University (Nanjing, China). The authors would like to acknowledge the expert technical assistance of Professor Xiang Gao and the members of his lab (the Model Animal Research Center of Nanjing University, China).
Conflict of Interest The authors declare no conflict of interest.
REFERENCES
1) Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annu. Rev. Immunol., 28, 573–621 (2010).
2) Baumgart DC, Sandborn WJ. Crohn’s disease. Lancet, 380, 1590–1605 (2012).
3) Brand S. Crohn’s disease: Th1, Th17 or both? The change of a para-digm: new immunological and genetic insights implicate Th17 cells in the pathogenesis of Crohn’s disease. Gut, 58, 1152–1167 (2009).
4) Olsen T, Rismo R, Cui G, Goll R, Christiansen I, Florholmen J. TH1 and TH17 interactions in untreated inflamed mucosa of inflam-matory bowel disease, and their potential to mediate the inflamma-tion. Cytokine, 56, 633–640 (2011).
5) Weaver CT, Hatton RD. Interplay between the TH17 and TReg cell lineages: a (co-)evolutionary perspective. Nat. Rev. Immunol., 9, 883–889 (2009).
6) Franke A, McGovern DP, Barrett JC, Wang K, Radford-Smith GL, Ahmad T, Lees CW, Balschun T, Lee J, Roberts R, Anderson CA, Bis JC, Bumpstead S, Ellinghaus D, Festen EM, Georges M, Green T, Haritunians T, Jostins L, Latiano A, Mathew CG, Montgomery GW, Prescott NJ, Raychaudhuri S, Rotter JI, Schumm P, Sharma Y, Simms LA, Taylor KD, Whiteman D, Wijmenga C, Baldassano RN, Barclay M, Bayless TM, Brand S, Buning C, Cohen A, Colombel JF, Cottone M, Stronati L, Denson T, De Vos M, D’Inca R, Dubin-sky M, Edwards C, Florin T, Franchimont D, Gearry R, Glas J, Van Gossum A, Guthery SL, Halfvarson J, Verspaget HW, Hugot JP, Karban A, Laukens D, Lawrance I, Lemann M, Levine A, Libioulle C, Louis E, Mowat C, Newman W, Panes J, Phillips A, Proctor DD, Regueiro M, Russell R, Rutgeerts P, Sanderson J, Sans M, Seibold F, Steinhart AH, Stokkers PC, Torkvist L, Kullak-Ublick G, Wilson D, Walters T, Targan SR, Brant SR, Rioux JD, D’Amato M, Weers-ma RK, Kugathasan S, Griffiths AM, Mansfield JC, Vermeire S, Duerr RH, Silverberg MS, Satsangi J, Schreiber S, Cho JH, Annese V, Hakonarson H, Daly MJ, Parkes M. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease suscepti-bility loci. Nat. Genet., 42, 1118–1125 (2010).
7) Alinari L, Lapalombella R, Andritsos L, Baiocchi RA, Lin TS, Byrd JC. Alemtuzumab (Campath-1H) in the treatment of chronic lymphocytic leukemia. Oncogene, 26, 3644–3653 (2007).
8) Reiff A. A review of Campath in autoimmune disease: biologic therapy in the gray zone between immunosuppression and immuno-ablation. Hematology, 10, 79–93 (2005).
9) Morris PJ, Russell NK. Alemtuzumab (Campath-1H): a system-atic review in organ transplantation. Transplantation, 81, 1361–1367 (2006).
Vol. 41, No. 9 (2018) 1429Biol. Pharm. Bull.
10) Bloom DD, Chang Z, Fechner JH, Dar W, Polster SP, Pascual J, Turka LA, Knechtle SJ. CD4+CD25+FOXP3+regulatory T cells increase de novo in kidney transplant patients after immunodeple-tion with Campath-1H. Am. J. Transplant., 8, 793–802 (2008).
11) Hester J, Mills N, Shankar S, Carvalho-Gaspar M, Friend P, Wood KJ. Th17 cells in alemtuzumab-treated patients: the effect of long-term maintenance immunosuppressive therapy. Transplantation, 91, 744–750 (2011).
12) Hersh CM, Cohen JA. Alemtuzumab for the treatment of relapsing-remitting multiple sclerosis. Immunotherapy, 6, 249–259 (2014).
13) Zhu WM, Li Y, Yu C, Li N, Li JS. Antimouse CD52 monoclonal antibody inhibits established spontaneous colitis in IL-10-deficient mice. Inflamm. Bowel Dis., 17, E72–E73 (2011).
14) Wang H, Dong J, Shi P, Liu J, Zuo L, Li Y, Gong J, Gu L, Zhao J, Zhang L, Zhang W, Zhu W, Li N, Li J. Anti-mouse CD52 mono-clonal antibody ameliorates intestinal epithelial barrier function in interleukin-10 knockout mice with spontaneous chronic colitis. Im-munology, 144, 254–262 (2015).
15) Bristol IJ, Farmer MA, Cong Y, Zheng XX, Strom TB, Elson CO, Sundberg JP, Leiter EH. Heritable susceptibility for colitis in mice induced by IL-10 deficiency. Inflamm. Bowel Dis., 6, 290–302 (2000).
16) Spencer DM, Veldman GM, Banerjee S, Willis J, Levine AD. Dis-tinct inflammatory mechanisms mediate early versus late colitis in mice. Gastroenterology, 122, 94–105 (2002).
17) Wei XW, Gong JF, Zhu J, Wang P, Li N, Zhu WM, Li JS. The suppressive effect of triptolide on chronic colitis and TNF-alpha/TNFR2 signal pathway in interleukin-10 deficient mice. Clin. Im-munol., 129, 211–218 (2008).
18) Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat. Rev. Immunol., 3, 133–146 (2003).
19) Lee SH, Kwon JE, Cho ML. Immunological pathogenesis of inflam-matory bowel disease. Intest. Res., 16, 26–42 (2018).
20) Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the gen-eration of pathogenic effector TH17 and regulatory T cells. Nature, 441, 235–238 (2006).
21) Collins CB, Aherne CM, McNamee EN, Lebsack MD, Eltzschig H, Jedlicka P, Rivera-Nieves J. Flt3 ligand expands CD103+ dendritic cells and FoxP3+ T regulatory cells, and attenuates Crohn’s-like murine ileitis. Gut, 61, 1154–1162 (2012).
22) Colombel J, Watson AJM, Neurath MF. The 10 remaining mysteries of inflammatory bowel disease. Gut, 57, 429–433 (2008).
23) Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Reciprocal developmental pathways for the gen-eration of pathogenic effector TH17 and regulatory T cells. Nature, 441, 235–238 (2006).
24) Xu L, Kitani A, Fuss I, Strober W. Cutting edge: regulatory T cells induce CD4+ CD25− Foxp3− T cells or are self-induced to become Th17 cells in the absence of exogenous TGF-β. J. Immunol., 178, 6725–6729 (2007).
25) Chaudhry A, Samstein RM, Treuting P, Liang Y, Pils MC, Heinrich JM, Jack RS, Wunderlich FT, Bruning JC, Muller W, Rudensky AY. Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation. Immunity, 34, 566–578 (2011).
26) Zhang Z, Zheng M, Bindas J, Schwarzenberger P, Kolls JK. Criti-cal role of IL-17 receptor signaling in acute TNBS-induced colitis. Inflamm. Bowel Dis., 12, 382–388 (2006).
27) Sandborn WJ, Feagan BG, Fedorak RN, Scherl E, Fleisher MR, Katz S, Johanns J, Blank M, Rutgeerts P, Ustekinumab Crohn’s Disease Study Group. A randomized trial of Ustekinumab, a human interleukin-12/23 monoclonal antibody, in patients with moderate-to-severe Crohn’s disease. Gastroenterology, 135, 1130–1141 (2008).
28) Sandborn WJ, Gasink C, Gao LL, Blank MA, Johanns J, Guzzo C, Sands BE, Hanauer SB, Targan S, Rutgeerts P, Ghosh S, de Villiers WJ, Panaccione R, Greenberg G, Schreiber S, Lichtiger S, Feagan BG, CERTIFI Study Group. Ustekinumab induction and mainte-nance therapy in refractory Crohn’s disease. N. Engl. J. Med., 367, 1519–1528 (2012).
29) Feagan BG, Sandborn WJ, Gasink C, Jacobstein D, Lang Y, Fried-man JR, Blank MA, Johanns J, Gao LL, Miao Y, Adedokun OJ, Sands BE, Hanauer SB, Vermeire S, Targan S, Ghosh S, de Vil-liers WJ, Colombel JF, Tulassay Z, Seidler U, Salzberg BA, Des-reumaux P, Lee SD, Loftus EV Jr, Dieleman LA, Katz S, Rutgeerts P, UNITI–IM-UNITI Study Group. Ustekinumab as induction and maintenance therapy for Crohn’s disease. N. Engl. J. Med., 375, 1946–1960 (2016).
30) Fitzpatrick LR, Small JS, Doblhofer R, Ammendola A. Vidofludi-mus inhibits colonic interleukin-17 and improves hapten-induced colitis in rats by a unique dual mode of action. J. Pharmacol. Exp. Ther., 342, 850–860 (2012).
31) Herrlinger KR, Diculescu M, Fellermann K, Hartmann H, Howaldt S, Nikolov R, Petrov A, Reindl W, Otte JM, Stoynov S, Strauch U, Sturm A, Voiosu R, Ammendola A, Dietrich B, Hentsch B, Stange EF. Efficacy, safety and tolerability of vidofludimus in patients with inflammatory bowel disease: the ENTRANCE study. J. Crohn’s Co-litis, 7, 636–643 (2013).
32) Hueber W, Sands BE, Lewitzky S, Vandemeulebroecke M, Reinisch W, Higgins PD, Wehkamp J, Feagan BG, Yao MD, Karczewski M, Karczewski J, Pezous N, Bek S, Bruin G, Mellgard B, Berger C, Londei M, Bertolino AP, Tougas G, Travis SP, Secukinumab in Crohn’s Disease Study Group. Secukinumab, a human anti-IL-17A monoclonal antibody, for moderate to severe Crohn’s disease: un-expected results of a randomised, double-blind placebo-controlled trial. Gut, 61, 1693–1700 (2012).
33) Wedebye SEG, Larsen HL, Kristensen NN, Poulsen SS, Lynge PAM, Claesson MH, Pedersen AE. TH17 cell induction and effects of IL-17A and IL-17F blockade in experimental colitis. Inflamm. Bowel Dis., 19, 1567–1576 (2013).
34) McLean LP, Cross RK, Shea-Donohue T. Combined blockade of IL-17A and IL-17F may prevent the development of experimental colitis. Immunotherapy, 5, 923–925 (2013).
35) Fitzpatrick LR, Stonesifer E, Small JS, Liby KT. The synthetic triterpenoid (CDDO-Im) inhibits STAT3, as well as IL-17, and improves DSS-induced colitis in mice. Inflammopharmacology, 22, 341–349 (2014).
36) Ogino H, Nakamura K, Ihara E, Akiho H, Takayanagi R. CD4+CD25+ regulatory T cells suppress Th17-responses in an experimental colitis model. Dig. Dis. Sci., 56, 376–386 (2011).
37) Danese S, Fiorino G, Rutella S. Regulatory T-cell therapy for Crohn’s disease: in vivo veritas. Gastroenterology, 143, 1135–1138 (2012).
38) Canavan JB, Scotta C, Vossenkamper A, Goldberg R, Elder MJ, Shoval I, Marks E, Stolarczyk E, Lo JW, Powell N, Fazekasova H, Irving PM, Sanderson JD, Howard JK, Yagel S, Afzali B, MacDon-ald TT, Hernandez-Fuentes MP, Shpigel NY, Lombardi G, Lord GM. Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn’s disease. Gut, 65, 584–594 (2016).
39) Coles AJ, Cox A, Le Page E, Jones J, Trip SA, Deans J, Seaman S, Miller DH, Hale G, Waldmann H, Compston DA. The window of therapeutic opportunity in multiple sclerosis: evidence from mono-clonal antibody therapy. J. Neurol., 253, 98–108 (2006).
40) Coles AJ, Compston DA, Selmaj KW, Lake SL, Moran S, Margolin DH, Norris K, Tandon PK, CAMMS223 Trial Investigators. Alem-tuzumab vs. interferon beta-1a in early multiple sclerosis. N. Engl. J. Med., 359, 1786–1801 (2008).