Protein phosphatase 4 is an essential positive regulator for Treg development, function, and protective gut immunity
© Liao et al.; licensee BioMed Central Ltd. 2014
Received: 16 January 2014
Accepted: 21 March 2014
Published: 7 May 2014
Protein phosphates 4 (PP4), encoded by the ppp4c gene, is a ubiquitously expressed phosphatase that has been implicated in the regulation of cytokine signaling and lymphocyte survival; recent reports suggest that PP4 may be involved in pre-TCR signaling and B cell development. However, whether PP4 also modulates the functions of peripheral T cells has not been investigated due to the lack of a suitable in vivo model. Treg cells are a specialized subset of CD4 helper T cells that can suppress the proliferation of activated effector T cells. In the absence of this negative regulation, autoimmune syndromes and inflammatory diseases, such as human Crohn’s disease, will arise.
In this report, we generated mice with T cell-specific ablation of the ppp4c gene (CD4cre:PP4f/f) and a Foxp3-GFP reporter gene to examine the roles of PP4 in Treg development and function. Characterizations of the CD4cre:PP4f/f mice showed that PP4 deficiency induced partial αβ T lymphopenia and T cell hypo-proliferation. Further analyses revealed significant reductions in the numbers of thymic and peripheral Treg cells, as well as in the efficiency of in vitro Treg polarization. In addition, PP4-deficient Treg cells exhibited reduced suppressor functions that were associated with decreased IL-10, CTLA4, GITR and CD103 expression. More interestingly, the CD4cre:PP4f/f mice developed spontaneous rectal prolapse and colitis with symptoms similar to human Crohn’s disease. The pathogenesis of colitis required the presence of commensal bacteria, and was correlated with reduced Treg cells in the gut. Nevertheless, PP4-deficient Treg cells were still capable of suppressing experimental colitis, suggesting that multiple factors contributed to the onset of the spontaneous colitis.
While the molecular mechanisms remain to be investigated, our results clearly show that PP4 plays a non-redundant role for the differentiation, suppressor activity and gut homeostasis of Treg cells. The onset of spontaneous colitis in the CD4cre:PP4f/f mice further suggests that PP4 is essential for the maintenance of protective gut immunity. The CD4cre:PP4f/f mice thus may serve as a good model for studying the interactions between Treg cells and gut commensal bacteria for the regulation of mucosal immunity.
Protein phosphatase 4 (PP4/PPX) is a ubiquitously expressed serine/threonine phosphatase that belongs to the PP2A/PP4/PP6 family. Human and mouse PP4 nucleotide sequences, encoded by the ppp4c genes, are well-conserved with identical translated amino acid sequences, hinting an evolutionary pressure to preserve the function of PP4. Indeed, the embryonic lethality of ppp4c-knockout mice suggests that PP4 is essential for fetal development. Initially identified as a mediator of TNFα signalings through the activation of JNK, PP4 is now implicated in many biological processes such as apoptosis, microtubule organization and DNA double strand break repair[6, 7]. Nevertheless, while these reports convincingly identify possible functions of PP4, their conclusions are often shadowed by the use of siRNA and chemical inhibitors that may carry off-target effects, particularly on PP2A and PP6.
To more definitively interrogate the functions of PP4 in vivo, we generated mice carrying a floxed ppp4c allele (PP4f) by embryonic stem cell targeting, and introduced proximal Lck promoter-driven Cre recombinase transgene (Lckcre) to mediate T cell-specific deletion of ppp4c (Lckcre:PP4f/f). Analyses of the Lckcre:PP4f/f mice reveal that PP4 deficiency blocks pre-TCR signaling and induces apoptosis of immature thymocytes. Recent data also show that PP4 can regulate apoptosis in primary human T cells. These results thus suggest that PP4 may be an important mediator of T cell expansion and survival. Further analysis of the functions of PP4 in peripheral T cells, however, is prohibited by the absence of mature T cells in the Lckcre:PP4f/f mice.
A specialized subset of CD4 helper cells constitutively expresses CD25 on their surface, and is termed regulatory T (Treg) cells for their ability to suppress the proliferation of neighboring T cells. Treg cells develop in the thymus (known as nTreg), but can also be induced from naïve T cells in vitro under proper polarizing conditions (known as iTreg). The differentiation and function of Treg cells are critically enforced by the master transcription factor Foxp3 and its downstream genetic programs. Recent reports, however, suggest that the lineage stability and function of Treg cells are also critically controlled by epigenetic regulations on Foxp3 and other Treg-related genes[10, 11]. Regardless of how the Treg lineage is maintained, proper Treg function is pivotal for the establishment of a protective immune system, as the deficiency of foxp3 gene ablates Treg cells and causes multiple autoimmune syndromes; the deletion of foxp3 in adult Treg cells also induces catastrophic autoimmunity.
Inflammatory bowel disease (IBD) is one of the human disorders that are considered to have immunopathogenesis origin. IBD can be further categorized into Crohn’s disease and ulcerative colitis, in which Crohn’s disease is thought to be caused by deregulated Th1/Th17 inflammatory response, while imbalanced antibody reaction is considered to be upstream of the exacerbation of ulcerative colitis. Still, non-immune components, including alterations in commensal microbiota, epithelial barrier integrity, and gut exocrine function all contribute to the onset of IBD[14, 15]. With this complex nature of IBD in mind, it is not surprising that multi-pronged approaches are required to study the many aspects of IBD pathogenesis. In this regard, many spontaneous and inducible IBD mouse models have been developed to investigate the etiology of IBD in vivo, of which several reports have indicated Treg cells to be an important regulator of IBD[16–18].
To study the functions of PP4 in Treg cells, we crossed the PP4f allele with CD4cre transgene (CD4cre) and the Foxp3-GFP reporter knock-in gene to generate the CD4cre:PP4f/f:Foxp3-GFP+ mice. Characterizations of these mice revealed important modulatory roles of PP4 on Treg differentiation, homeostasis and function. Furthermore, the spontaneous rectal prolapse and colitis that developed in the CD4cre:PP4f/f:Foxp3-GFP+ mice further indicated an immune regulatory function of PP4 for maintaining the immunological balance in the gut.
PP4 deficiency induces partial αβ T cell lymphopenia
PP4 is essential for the differentiation, function and gut homeostasis of Treg cells
Treg cells are essential for maintaining a balanced immune system; in addition, their importance in gut immunity has been demonstrated in adoptive transfer models and in gene-deficient mice. To assess the roles of PP4 in Treg development and function, we crossed the CD4cre:PP4f/f mice with mice carrying the Foxp3-GFP reporter gene (hereon referred as CD4cre:PP4f/f:Foxp3-GFP+ mice). Flow cytometry analyses showed that the percentage of CD25+Foxp3-GFP+ cells in CD4 single-positive (CD4SP) thymocytes was reduced by 2-fold in the CD4cre:PP4f/f:Foxp3-GFP+ mice (Figure 1D; p < 0.001); similar reductions were also found in splenic CD4 T cells (Figure 1E; p = 0.037), but not in the lymph nodes (LNs) (Figure 1E). To test whether the deletion of ppp4c gene impacted Treg polarization in vitro, WT or CD4cre:PP4f/f CD4+CD62-L+ cells were activated under Treg-polarizing condition; the results showed that the induction of Foxp3-GFP+ cells was significantly reduced from ~70% in WT cells to ~30% in CD4cre:PP4f/f cells (Figure 1F; p < 0.001). Lastly, qPCR was performed using genomic DNA from sorted splenic CD4+Foxp3-GFP+ cells to assess the efficiency of ppp4c deletion in the CD4cre:PP4f/f:Foxp3-GFP+ mice; the results showed that the ppp4c gene was indeed deleted in >85% of the Treg cells (Figure 1G). Western analyses of purified T cells also confirmed the deficiency of PP4 in the CD4cre:PP4f/f mice (Additional file1: Figure S2A). These findings thus suggest that PP4 is essential for the differentiation and homeostasis of Treg cells in vivo.
Spontaneous rectal prolapse and colitis develop in the CD4cre:PP4f/f mice
The spontaneous prolapse is not preceded by accumulation of pro-inflammatory T cells in the gut
PP4-deficient T cells are hypo-responsive to antigen stimulation, ineffective for Th17 polarization, and incapable of inducing experimental colitis
The spontaneous colitis in the CD4cre:PP4f/f mice requires commensal bacteria and is heralded by systematic granulocyte infiltration
In this report, we have described the spontaneous prolapse and colitis in mice with T cell-specific ablation of PP4, the CD4cre:PP4f/f mice. What are the factors that might have contributed to the onset of colitis in these mice? Altered Treg cell functions (Figure 2) are likely a significant factor due to their immune-regulatory roles. Commensal bacteria are clearly a prerequisite, as indicated by the amelioration of colitis following antibiotic treatment (Figure 7B). The accumulation of granulocytes in prolapse-free CD4cre:PP4f/f mice (Figure 7A) and the increased number of pro-inflammatory IEL T cells in prolapsed CD4cre:PP4f/f mice (Figure 4G) further implicate the involvement of innate and adaptive, respectively, anti-commensal immune responses in the gut. Finally, the seemingly contradictory hypo-responsiveness of PP4-deficient T cell to antigen stimulation (Figure 6A-B) may actually contribute to the onset of colitis by preventing the clearance of infiltrating commensal bacteria. While it is difficult to quantify the relative contributions of the individual factors, these observations are consistent with the core scenario that uncontrolled activation of mucosal innate and adaptive immune cells, caused by defective Treg suppression, results in persistent, commensal-dependent gut inflammation that eventually breaches the mucosal barrier for the onset of colitis in the CD4cre:PP4f/f mice.
Although our data fit nicely into this working model, the clear disparity in the colitis incidence rates of Lckcre:PP4f/f (<0.5%) and CD4cre:PP4f/f (>60%) mice needs to be addressed. In this regard, a major difference between these mice is that the Lckcre:PP4f/f mice exhibit severe lymphopenia, while the CD4cre:PP4f/f mice contain reduced but substantial number of peripheral T cells (Figure 1B-C). The residual PP4-deficient T cells in the CD4cre:PP4f/f mice may thus be essential for the T cell component of the inflammatory response and tissue damage during the late phase of colitis pathogenesis. Without the induction of aggravated T cell inflammatory responses, innate immunity may be able to keep the commensal bacteria in check to prevent excessive tissue damage. Such a scenario has been observed when comparing the colitis incidence between TCRα-/-, TCRβ-/- and RAG1-/- mice housed in specific pathogen-free facility. Alternatively, it is possible that the difference in the timing of the deletion of the ppp4c gene may allow the generation of colitogenic cells in the CD4cre:PP4f/f thymus but not in the Lckcre:PP4f/f thymus. One such candidate is NKT cells, whose maturation begins during the DN-DP transition and is completed at the DP stage However, we did not find any significant alteration in the percentages of CD3ϵ+CD49b+ NKT cells in gated CD4+ and CD8+ populations in the CD4cre:PP4f/f mice (Additional file1: Figure S2C), although they did accumulate in the gut of prolapsed animal (Figure 5B). Results from IEL T cell analyses in prolapse-free CD4cre:PP4f/f mice (Figure 5 and S2B), from helper T cell polarization (Figure 6C), and from the induction of experiment colitis (Figure 6E) also helps rule out the possibility that PP4 deficiency induces novel pro-inflammatory, colitogenic effector CD4 T cells. Nevertheless, such a possibility remains viable for other T cell subsets.
Treg deficiency caused by the ablation of PP4 is fairly broad, encompassing defects in nTreg/iTreg differentiation, suppressor functions and gut homeostasis. In this context, PP4 may mediate these diverse effects either by functioning through a single master factor that regulates a complex network of downstream genes, or by acting individually on multiple target proteins to impact various signaling pathways, or both. For the former, monomeric regulation, the primary candidate that may be regulated by PP4 is Foxp3. Evidence supporting a potential role of PP4 comes indirectly from recent reports showing that the stability of Foxp3 is reduced when its serine 19 residue is phosphorylated by CDK2, and that the activity of Foxp3 is down-regulated when its serine 418 residue is dephosphorylated by protein phosphatase 1. However, in either case the constitutive phosphorylation of these residues should enhance Foxp3 activity, yet our data indicate that the loss of PP4 is manifested in the form of defective Treg functions. In this regard, Foxp3 contains several other potential serine/threonine phosphorylation sites on residues 13, 25, 114, 137 and 141 that may serve as the target of PP4-mediated dephosphorylation for the regulation of Foxp3 activity. Alternatively, the Ikaros family transcription factors, Eos and Helios, have been shown to regulate the transcription or activity of Foxp3, respectively. Our observation that the transcription and expression of Foxp3 are not significantly altered in PP4-deficient Treg cells (Figure 3B-C) argues against a dominant role for Helios, but potential PP4-mediated regulation on Eos remains a possibility.
Other than Foxp3, the reduced Treg cell numbers may also be caused by defective Treg cell survival or expansion in the absence of PP4. This possibility is supported by our previous report showing that the deletion of PP4 induces apoptosis in developing thymocytes and by the recent findings that the inhibition of PP4 blocks cell cycle progression. Alternatively, recent reports suggest that Treg cells require proper TCR activation to achieve optimal differentiation and homeostasis[35, 36]. Since PP4 is shown to be involved in TCR signaling and NFκB activation, altered TCR activation may also contribute to the Treg defects in the CD4cre:PP4f/f mice. We are currently investigating these possibilities.
In this report, we have described the defects in Treg differentiation, function and homeostasis caused by PP4 deficiency. These defects are associated with altered IL-10, CTLA4, GITR and CD103 expression in PP4-deficient Treg cells, and are accompanied by gut inflammation and spontaneous colitis in the CD4cre:PP4f/f mice. While the molecular mechanisms of PP4-mediated regulations on Treg cells remain to be elucidated, we believe that our characterizations of the CD4cre:PP4f/f mice provide important frameworks for future studies on how PP4, and potentially other phosphatases, may regulate Treg functions and gut immunity.
PP4f/f, CD4cre, Foxp3-GFP and RAG1-/- mice have been described. CD90.1 and CD45.1 C57/Bl6 congenic mice were obtained (Jackson laboratory). PP4f/f mice were crossed with CD4cre mice to generate the CD4cre:PP4f/f mice with T cell-specific deletion of the ppp4c gene. CD4cre:PP4f/f mice were further crossed with Foxp3-GFP mice to generate the CD4cre:PP4f/f:Foxp3-GFP mice. All mice were housed under specific pathogen-free condition at the Laboratory Animal Center of the National Health Research Institutes (NHRI). Mice with a loss of >20% body weight were removed by euthanasia. All animal experimental procedures followed the guidelines approved by the NHRI Institutional Animal Care and Use Committee.
Antibodies and flow cytometric analysis
Antibodies against mouse epitopes of B220, CD3ϵ, CD4, CD8, CD11b, CD25, CD39, CD45RB, CD49b, CD62-L, CD90, CD223, CTLA4, CXCR5, GITR, Gr1, TCRβ, TCRδ, TER119, IL-4, IL-6, IL-17A and IFNγ conjugated with various fluorescent dyes or biotin, 7AAD and AnnexinV-APC (all purchased from BioLegend or BD Biosciences) were used for surface and intracellular staining following standard protocols. CFSE (Invitrogen) was loaded into targets cells following the manufacturer’s suggestions. Flow cytometry results were obtained on 8-color FACSCanto II with FACSDiva software (BD Biosciences), were and analyzed by FlowJo software (Tree Star).
PCR and qPCR
For estimating the efficiency of ppp4c gene deletion, genomic DNA was extracted from sorted primary cells with standard protocols. Oligonucleotides for qPCR of exon 2 were 5′-GGGCGGTCCCAGAATCGAGT-3′ (primer a) and 5′-ATCAGCTCGCAGCGCCGTAG-3′ (primer b). For exon3, the oligonucleotides used were 5′-CCAGTTGGCAACAAGGAGCCAT-3′ (primer c) and 5′-CCAGCCCAATTCCTGACCTT-3′ (primer d) (see Figure 1G for primer locations). For gene transcription, total RNA was extracted from sorted LN CD4+Foxp3-GFP+ cells and converted into cDNA with standard protocol. The primers used are: Actin-1: 5′ AAGTGTGACGTTGACATCCGTAA-3′; Actin-2: 5′- TGCCTGGGTACATGGTGGTA-3′. CD103-1: 5′-CGTGGAGAAGAAGGCAGAGT-3′; CD103-2: 5′-TCGGGGGTAAAGGTCATAGAT-3′; CTLA4-1: 5′-CTCAACTGCAGCTGCCTTCTAGGA-3′; CTLA4-2: 5′-AAGCTGGCGACACCATGGCT-3′; Foxp3-1: 5′-GGCCCTTCTCCAGGACAGA-3′; Foxp3-2: 5′-GCTGATCATGGCTGGGTTGT-3′; IL-10-1: 5′-TGCAGGACTTTAAGGGTTACTTGGG-3′; IL-10-2: 5′-CCTTGCTCTTATTTTCACAGGGGAG-3′; TGFβ-1: 5′-GCTCGCTTTGTACAACAGCACCC-3′; TGFβ-2: 5′-GCTTCCCGAATGTCTGACGTATTG-3′; qPCR was performed using FastStart Universal Probe Master Rox (Roche Applied Science) on Realplex4 with Mastercycler ep realplex software (Eppendorf). Genotyping PCR for the CD4cre transgene was performed with oligonucleotides 5′-TCTCTGTGGCTGGCAGTTTCTCCA-3′ and 5′-TCAAGGCCAGACTAGGCTGCCTAT-3′. Genotyping of the PP4f allele was performed with oligonucleotides 5′-TGCTCTGGTGCAGGAGATGTGTG-3′, 5′-ACGTGATTTGCGAAAGCCTCTCA-3′, and 5′-CTTGGTAGAAGAGAGCAACGTGCAG-3′ in a three-primer reaction. PCR conditions are available upon request.
Cell sorting and culture
For qPCR, Treg suppression assays and adoptive transfer, cells were stained for surface markers and sorted on FACSAria (BD Biosciences) or enriched by magnetic-assisted cell sorting (MACS). All primary cells were cultured in DMEM supplemented with 1 x non-essential amino acid, 2 mM L-glutamine, 2 mM Glutamax, 1 mM sodium pyruvate, 10 mM HEPES (all from Invitrogen), 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin (all from Biological Industries) and 125 μM 2-mercaptoethanol (Sigma-Aldrich).
Colons were excised, flushed with PBS, and fixed in 10% formaldehyde for 1 hr before embedded in paraffin. Longitudinal or transverse sections were cut and stained with haematoxylin and eosin with standard protocols. Histological images were obtained on Olympus IX71 microscope with Olympus DP70 camera using Olympus DP controller software (Olympus).
Isolation of IEL and LPL cells
Small intestines were harvested and flushed with CMF solution (containing 2% FBS, 10 mM HEPES, Ca2+/Mg2+-free HBSS) before removing the Peyer’s patches. Residual small intestine was cut into 0.5 cm pieces and washed six more times with CMF solution, incubated in 10% FBS/0.1 mM EDTA/CMF at 37°C for 15 min with rotary shaking (220 rpm), transferred to a fresh tube, and vortexed for 15 sec at maximum setting. After the tissues settled, supernatant was saved in a fresh tube. The precipitated tissues were re-applied in the above incubation/transfer procedure for four more times. All supernatants were pooled for IEL and epithelial cells isolation via Percoll gradient separation. The remaining intestine pieces were washed four times with 10% FBS/5 mM EDTA/CMF solution at 37°C for 15 min with rotary shaking (220 rpm). After the last wash, the intestine pieces were incubated in 10% FBS/ RPMI containing 100 U/ml type VIII collagenase (Sigma-Aldrich) for 2 hr at 37°C with rotary shaking (220 rpm) and media change at 1 hr. The debris was allowed to settle, and the resulted supernatant was subjected to Percoll gradient separation for the isolation of LPL cells. Percoll (Sigma-Aldrich) gradient separation (for IEL: 44%/67%; for LPL: 40%/100%) was performed by loading the supernatant atop of appropriate Percoll gradients, followed by centrifugation at 400g for 20 min and collection of IEL or LPL cells at the interface.
Experimental colitis induction and antibiotics treatment
For adoptive transfer-induced experimental colitis, CD4 T cells were enriched from total splenocytes by MACS negative selection for B220, CD11b, CD49b, CD8, and Ter119. CD4+CD45RBhigh (upper 40% of CD45RB+ cells) or CD4+CD25+Foxp3-GFP+ cells were purified from these cells by sorting. Sorted cells were then transferred via tail vein into RAG1-/- recipients as indicated in the figure legend. For DSS-induced colitis, mice were administered 2% DSS dissolved in sterilized drinking ad libitum for 14 d. Animals were weighed daily and monitored for rectal bleeding, diarrhea, and general signs of morbidity. For antibiotics treatment, mice received drinking water containing 0.66 mg/ml ciprofloxacin, 2.5 mg/ml metronidazole (Sigma-Aldrich) and 1.5% fructose (to encourage consumption) for 3 weeks. Control animals were given drinking water containing 1.5% fructose only. Both DSS water and antibiotic solution were replaced 2-3 times weekly.
KLH immunization, T cell response and cytokine measurement
Mice at 6-8 wk age were immunized in the hind footpad with 0.1 ml of 1:1 emulsion of CFA (Difco) and 1 mg/ml KLH (Sigma-Aldrich). One wk later draining popliteal LN cells were harvested, labeled with CFSE, and restimulated with titrating doses of KLH in vitro for 3 d. The proliferation of responding cells was then measured by CFSE dye-dilution, while the cytokine production was assessed with FlowCytomix Mouse Th1/Th2 10plex kit (eBioscience) following the manufacturer’s procedure. Cytokine production from isolated IEL cells was assessed similarly.
Treg/Th1/Th2/Th17 polarization and suppression assays
For in vitro polarization of Treg cells, naïve CD4+CD62-L+ cells were purified by MACS from splenocytes and stimulated with 1.6 μg/ml soluble anti-CD28 and plate-bound anti-CD3ϵ in the presence of 5 ng/ml TGFβ, 10 μg/ml anti-IL-4 and 10 μg/ml anti-IFNγ for 3 d. Cells were fixed in 4% paraformaldehyde/PBS prior to surface staining and flow cytometry analyses with standard protocols. Th1 (5 ng/ml IL-2, 10 ng/ml IL-12 and 10 μg/ml anti-IL-4), Th2 (10 ng/ml IL-2, 4 ng/ml IL-4, 10 μg/ml anti-IFNγ and 10 μg/ml anti-IL-12) and Th17 (30 ng/ml IL-6, 1 ng/ml TGFβ, 10 μg/ml anti-IFNγ and 10 μg/ml anti-IL-4) cells were polarized and assessed similarly. For Treg suppression assays, CD4 T cells were enriched from pooled spleen and LN cells by MACS. CD4+Foxp3-GFP+ Treg cells were then purified from these cells by sorting. Irradiated APC were prepared from C57Bl/6 splenocytes following red blood cell lysis and 200 Gray irradiation. WT responder T cells were prepared from pooled spleen and LN cells from CD90.1 congenic mice by MACS, and were loaded with CFSE. Cell culture was set up in 96-well U-bottomed plates with 1 μg/ml soluble-anti-CD3ϵ at a final volume of 200 μl, and contained 5 × 104 WT responder cells, 2 × 105 irradiated APC, and titrating number of Treg cells to obtain 1:1 to 16:1 ratio of responder : Treg cells. The proliferation of WT responder T cells were assessed on d 3 by flow cytometry; division index was calculated using the FlowJo software (see Additional file1: Figure S2D for more detail).
When applicable, data were plotted as mean ± SEM with the p-values calculated using unpaired two-tailed Student’s t-test.
Availability of supporting data
Gating strategies of flow cytometry analyses and additional data are available as online supplemental materials in Additional file1.
CD4cre, CD4 promoter-driven Cre recombinase transgene CD4SP, CD4 single-positive; DSS, dextran sulfate sodium; E, number of independent experiment; IBD, inflammatory bowel disease; IEL, intra-epithelial lymphocyte; KLH, keyhole limpet hemocyanin; Lckcre, Lck proximal promoter-driven Cre recombinase transgene; LPL, lamina propria lymphocyte; LN, lymph node; MACS, magnetic-assisted cell sorting; MLN, mesenteric lymph node; NHRI, National Health Research Institutes; PP4, protein phosphatase 4; qPCR, quantitative PCR; Treg, regulatory T.
This work was supported in whole or in part by grants 99A1-IMPP01-014 (to T.-H. T.) and 100A1-IMPP02-014 (to C. H.) from the NHRI, Taiwan; grant 1R01-AI066895 (to T.-H. T.) from the National Institutes of Health, USA; and grant 98-2320-B-400 -006 -MY3 and 102-2321-B-400-017 (to C. H.) from the National Science Council, Taiwan. The authors thank the staffs at the Laboratory Animal Center, Pathology Core facility, and Flow Cytometry Core facility at NHRI for their assistance; the authors also thank Dr. Kuo-I Lin and Dr. Chuen-Miin Leu for their constructive comments on the manuscript.
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