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The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation
TRIM-01015; No of Pages 6 Transplant Immunology xxx (2015) xxx–xxx
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Transplant Immunology journal homepage: www.elsevier.com/locate/trim
The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation Yan He a, Jia Bei c,d, Hui Zeng c,d, Zhiqiang Pan b,⁎ a
Department of Ophthalmology, The Second Xiangya Hospital of Central South University, Eye Institute of The Second Xiangya Hospital of Central South University, Changsha 410010, China Beijing TongRen Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmic and Visual Science Key Laboratory, Beijing 100730, China Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China d Beijing Key Laboratory of Emerging Infectious Diseases, Beijing 100015, China b c
a r t i c l e
i n f o
Article history: Received 7 October 2015 Received in revised form 22 December 2015 Accepted 22 December 2015 Available online xxxx Keywords: MDSC Corneal–skin combined transplantation Adoptive transfer Allograft rejection
a b s t r a c t Purpose: To explore the effects of adoptive transferring sepsis induced myeloid-derived suppressor cells (iMDSCs) in mice corneal, skin, and combined corneal–skin survival. Methods: Allogeneic full-thickness corneal transplantation, fully mismatched skin transplantation, and corneal– skin combined transplantation (donor C57BL/6 to recipient Balb/c mice) were performed. Sepsis-induced infectious-MDSCs (iMDSCs), were purified from bone marrow of cecal ligated and punctured (CLP) Balb/c mice. Recipient-derived iMDSCs were adoptively transferred into different recipient groups by retro-orbital injection after surgeries. Corneal and skin grafts were examined and photographed routinely for a period of 45 days. Histopathology was performed to evaluate corneal-graft inflammation. Bone marrow and/or corneal grafts in each group were harvested from executed recipients on postoperative days 15, 25, 35. Corneal cells and bone marrow cells were stained with CD11b-PE and Gr1-FITC, analyzed by FACS. Results: iMDSCs were able to significantly prolong allograft survival in both corneal and corneal–skin combined transplant groups. A substantial expansion of MDSCs was observed in recipients' bone marrow, particularly in combined groups at an early stage postoperatively, and accordingly the concentration of MDSCs in corneal grafts increased significantly in adoptive transferred groups. Conclusions: Sepsis-induced MDSCs may suggest a novel cellular therapeutic approach for preventing various types of allograft rejection. © 2015 Elsevier B.V. All rights reserved.
1. Introduction A myeloid-derived suppressor cell (MDSC) is defined as a novel heterogeneous population of immature myeloid cells with immunosuppressive properties, including macrophages, granulocytes, dendritic cells (DC), and myeloid cells at earlier stages of differentiation [1,2]. In mice, they express the common myeloid cell markers CD11b (αM chain of β2 integrin) and Gr1 (Ly6G and Ly6C) [3]. In general, CD11b+Gr1+ myeloid cells represent about 30–40% of normal bone marrow cells and only 2–4% of all nucleated normal splenocytes [4,5]. Under pathological conditions, such as infection, tumor, and trauma, they accumulate in large numbers in bone marrow, blood, spleen and other lymphoid organs [6,7]. In our previous studies, a dramatic accumulation of CD11b+Gr1+ cells was detected in bone marrows of septic mice models and tumor-
Abbreviations: MDSC, Myeloid-derived suppressor cell; DC, Dendritic cell; CLP, Cecal ligated and punctured; iMDSC, Inflammation-induced myeloid-derived suppressor cell; tMDSC, Tumor-induced myeloid-derived suppressor cell. ⁎ Corresponding author at: Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Dongjiaominxiang, Beijing 100730, China. E-mail address: [email protected] (Z. Pan).
bearing mice models [8]. The functions of MDSCs are highly dependent on the circumstances in which their expansion occurs. Significantly cellmediated immunosuppressive capacities were observed in infectiousMDSCs (iMDSCs) and tumor-bearing MDSCs (tMDSCs) in vitro. However, CD11b+Gr1+ cells from naïve mice showed few immunosuppressive functions. The role of MDSCs in transplantation tolerance was wildly described in allograft models in recent years [9,10]. Our previous studies [8] were focused on the potential application of MDSCs from different sources to prolong allograft survival, and reported that MDSCs from septic mice models and tumor-bearing mice models were adoptively transferred to allogenic recipients after corneal transplantations, and significantly prolonged corneal allograft survival in vivo. Moreover, iMDSCs transferred significantly reduced neovascularization that was comparable to tMDSCs and naïve MDSC. However, the additional adoptive transfer of MDSCs did not further ameliorate corneal survival. Therefore, rather than a simplex immunosuppressive response, the function of MDSC is more than likely a complex balance between increased immune surveillance and decreased adaptive immune responses. Corneal allotransplant is an extreme situation, because the anterior chamber of the eye is an immunologically privileged site [11]. Corneal
http://dx.doi.org/10.1016/j.trim.2015.12.003 0966-3274/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
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Y. He et al. / Transplant Immunology xxx (2015) xxx–xxx
vascularization has been reported to be associated with a significantly increased risk of corneal graft rejection [12,13], which is primarily a cellmediated indirect T cell response mediated by CD4+ T cells. The fully mismatched allogenic skin transplant, as another kind of typical tissue transplantation, was reported to be recognized by recipients via both the direct and indirect pathways by CD4+ T cells in BALB/c recipient's lymphoid organs [14,15]. Moreover, both MHC and minor antigens induced an indirect alloresponse in skin transplantation. However, the entire indirect CD4+ T cell alloresponse in cornea-transplanted mice was directed exclusively to minor antigens [14–17]. As an immunomodulatory strategy to prevent corneal allograft rejection, MDSCs have been used in experimental corneal transplantation by suppressing the proliferation and cytokine production of effector T cells [1,18]. It is not clear whether there is the same positive effect when used in skin transplant, and even more interesting, in corneal– skin combined transplantation models. Moreover, the postoperative infiltration of MDSCs in graft remains unknown. Therefore, we utilized infectious MDSCs in the mice cornea, skin and cornea–skin combined transplant models, compared the therapeutic efficiency, and discussed the underlying mechanisms by analyzing the MDSC distribution and survival rate of allograft.
2. Materials and methods 2.1. Mice
2.4. Group and iMDSC adoptive transferring The recipient mice were divided randomly into 6 groups, as shown in Table 1 (18 recipients in each group), and 5 × 106 iMDSCs suspended in 150 μL PBS were transferred to the recipients' left eyes, or nonsurgery eyes, at the end of operation, via retro-orbital injection. The same volume of PBS was given to the Iso SCT, Allo CT, Allo ST, and Allo SCT groups as the control. 2.5. Assessment of graft survival Corneal grafts were assessed by slit lamp biomicroscopy every two days after surgery for 45 days. As described previously, the grafts were scored in a range for opacity (0 to 5), as shown in Table 2. Meanwhile, the extent of corneal edema and growth of neovessels were also brought under consideration. Edema and neovessels were scored within a range from 0 to 3, minimum to maximum. When the total scores of opacity, edema, and neovessels reached 6, or opacity score N 2, the grafts were rejected. Square skin grafts of 1 cm × 1 cm were excised from the backsides of C57BL/6 mice and sutured to the same regions of Balb/c mice with 12 interrupted 8–0 nylon sutures. Corneal and skin transplantation were performed at the same day in the corneal–skin combined transplantation group (SCT group). Skin grafts were examined at daily intervals. Rejection was defined as complete dissolution of graft, and wound completely healed by recipient skin.
C57BL/6(H-2b) and Balb/c(H-2d) mice were purchased from the Experimental Center of Capital Medical University (Beijing, China) and housed in a specific pathogen-free facility. Female C57BL/6 mice (8 to 12 weeks old) were used as donors and female Balb/c mice (8 to 12 weeks old) were used as recipients. Balb/c mice underwent cecal ligation and puncture, as described previously [19], and were prepared at the Institute of Infectious Diseases of Beijing Ditan Hospital. All procedures performed on animals were approved by the Animal Care Research Ethics Committee of the Capital Medical University of China.
2.6. Flow cytometric analysis of grafts
2.2. Flow cytometry and purification of MDSCs from bone marrow
2.7. Statistical analysis
Monoclonal antibodies used for fluorescence-activated cell sorting staining were: phycoerythrin (PE) conjugated anti-mouse CD11b and fluorescein isothiocyanate (FITC) conjugated anti-mouse Gr-1 (BD Bioscience, San Diego, CA, USA). Bone marrow cells from cecal ligated and punctured (CLP) Balb/c mice were stained with the above antibodies at 4 °C for 15 min and washed in PBS. The cells were isolated by fluorescence-activated cell sorter (FACSAria, BD, CA, USA). The purity of CD11b+Gr1+cells from CLP Balb/c mice (iMDSC) was routinely more than 94%. For FACS analysis, the data were acquired on a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and was analyzed using FlowJo Software v 5.7.2–2 (Tree Star, Ashland, OR, USA).
Results are expressed as means ± SD. Graft survival time was compared between various groups by the Mantel–Cox Log Rank Test and the Kaplan–Meier survival curve. P values less than 0.01 were considered to be statistically significant.
2.3. Corneal penetrating keratoplasty and skin transplantation Corneal penetrating keratoplasty was performed as described previously in the right eyes of the mice [20]. In brief, the central full thickness cornea with a diameter of 2.0 mm from the mice C57BL/6 were excised and secured in Balb/c mice graft beds with a diameter of 1.5 mm by eight interrupted 11–0 nylon sutures. 48 h later, the grafts were examined by a slit lamp. Grafts with severe complications, such as infection or hyphema, were excluded from the study. The sutures of the grafts were removed 7 days after surgery. Skin allografts of 1 cm × 1 cm in size were taken from hair removed from the dorsum of C57BL/6 mice, and were sutured in Balb/c mice graft beds with the same area by 12 interrupted 6–0 silk sutures.
On days 15, 25, and 35 after surgeries, 3 recipients for each group were executed. Corneal grafts were sheared into small pieces, and immersed in 1 mg/mL collagenase IV (Sigma) for 2 h at 37 °C in a rocking device. Bone marrow cells were harvested from executed recipients. The collected suspensions were separately forced through a 100-Μm nylon mesh. Corneal cells and bone marrow cells were stained with CD11b-PE and Gr1-FITC (BD Biosciences, USA). Samples were analyzed by FACS.
3. Results 3.1. Adoptive transfer of iMDSCs has similar inhibitory properties in corneal and corneal–skin combined transplantation Mice in different groups were treated as shown in Table 1 and iMDSC suspensions (5 × 105 cells/150 μL) and were adoptively transferred separately to iMDSC CT and iMDSC SCT groups by retrobulbar injection after corneal transplantation. The clinical characteristics of the graft, including edema, opacity and neovascularization, were examined and scored by slit lamp examination regularly up to 45 days after transplantation. Compared to the untreated allograft control group (Fig. 1A, Allo CT), no remarkable differences were observed in mice with corneal–skin combined transplantation (Fig. 1A, Allo SCT), while the grafts in the Iso SCT group remained clear and survived until the end of observation. By contrast, mice adoptively transferred with iMDSC (Fig. 1A, iMDSC CT, iMDSC SCT) exhibited reduced mean opacity, edema, and neovessel scores from the control group, particularly at an early period after surgeries. The most remarkable differences were observed in corneal– skin combined transplantation, which developed neovascularization
Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
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Table 1 Recipient group and postoperative survival time of skin and cornea grafts in each group. No.
1 2 3 4 5 6
Group name
Isograft skin & cornea transplantation Allograft cornea transplantation Allograft skin transplantation Allograft skin & cornea transplantation iMDSC cornea transplantation iMDSC skin & cornea transplantation
Group name in short
Transplantation Cornea
skin
Iso SCT Allo CT Allo ST Allo SCT iMDSC CT iMDSC SCT
√ √ – √ √ √
√ – √ √ – √
Survival time (skin)/days
Survival time (cornea)/days
N45@,# – 8.0 ± 0.94 8.80 ± 2.34 – 13.90 ± 1.20@,#
N45⁎,# 15.65 ± 2.06 – 15.02 ± 1.49 22.71 ± 3.01⁎,# 25.30 ± 8.22⁎,#
⁎ Compared with Allo CT group at the same period, P b 0.01. # Compared with Allo SCT group at the same period, P b 0.01. @ Compared with Allo ST group at the same period, P b 0.01.
more rapidly than corneal transplantation with or without iMDSC adoptive transferring (Fig. 1A, Allo CT, Allo SCT, iMDSC CT, iMDSC SCT). However, the extent of graft opacity and edema shows no difference between the corneal and corneal–skin combined transplantation groups. Graft survival analysis also demonstrated that cornea and skin allograft survival time was significantly prolonged in mice adoptively transferring iMDSCs compared to control groups (Table 1, P b 0.01, Fig. 2A, 2C). However, the differences in allograft survival time between single corneal/skin and corneal–skin combined groups were not statistically significant (Table. 1, P N 0.05). Interestingly, the formation of neovessels in SCT groups increased sharply compared to single corneal-transplantation groups (Fig. 2B).
3.2. Adoptive transfer of iMDSCs reduced histopathological changes in corneal allografts in corneal and corneal–skin combined transplantation Three different histopathological findings were evaluated in all specimens: severity of inflammatory reaction, vessels in the corneal grafts, and regularity of stroma fiber (Fig. 1B). The extent of inflammatory infiltrate associated with neovessels within the corneal stroma increased with time in all groups except the Iso SCT group. A severe inflammatory reaction and new blood vessels from the infiltrated limbal arcade reached even central grafts in the early stage of group Allo CT and Allo SCT (Fig. 1B, Allo CT, Allo SCT), while the stroma fibers appeared to be a mess. In contrast, corneal slides of iMDSC groups (Fig. 1B, iMDSC CT, iMDSC SCT) exhibited few changes as normal cornea in the first 25 days after transplantation, and a chronic slightly inflammatory infiltration and relatively regular fibers appeared in the late period. Compared to the corneal graft in corneal transplantation in histopathological slices, increases in neovascularity and extent of inflammatory infiltrate associated with neovessels within the corneal stroma seemed somewhat severe in corneal grafts of combined transplantation (Fig. 1B).
Table 2 Corneal grafts survival assessment. Standard
Score
Sign
Opacification
0 1 2 3 4 5 0 1 2 3 0 1 2 3
Clear graft Minimal superficial (nonstromal) opacity Minimal deep stromal opacity, iris striation sharp Moderate stromal opacity, iris striation blur Intense stromal opacity; only the pupil can be discerned Complete stromal opacity No edema Slight edema Moderate edema Severe edema No neovessels Neovessels appeared in rim of bed Neovessels in rim of graft Neovessels in centre of graft
Edema
Neovessels
3.3. Detection of postoperative distribution of MDSCs in bone marrow and corneal graft Next, we examined changes in MDSC distribution in recipients' corneal grafts and bone marrows after transplantation. Corneal grafts and bone marrows from each group were harvested, cells were double stained for CD11b and Gr-1, and then they were analyzed by flow cytometry. At an early stage, MDSC percentages in mouse bone marrow combined with skin transplantation were comparably higher than corneal transplant groups (Fig. 3A, P b 0.01). Meanwhile, a markedly high intensity of MDSC was found in corneal grafts of iMDSC adoptively transferring groups relative to normal corneal and corneal–skin combined transplant groups (Fig. 3D, P b 0.01). However, with the passage of time, the concentrating effect of MDSC in corneal grafts of iMDSC groups decreased, while levels of MDSCs in the cornea paralleled neovessel growth in those groups. Grafts in models with skin transplantation showed higher levels of MDSC than groups with corneal transplantation only at 25 days after surgeries (Fig. 3E, P b 0.01); interestingly, the expression of BM MDSC reached almost the same levels in all six groups (Fig. 3B, C, P N 0.05). 4. Discussion Corneal and skin transplants represent two extreme situations. Corneal transplants are routinely performed without HLA matching and systemic immunosuppressive drugs, and may even have a high acceptance rate, exceeding 90% [21]. In contrast, fully allogeneic skin transplants experience a 100% incidence of immune rejection without HLA matching and systemic immunosuppressive drugs. Recently, various studies have emphasized the intrinsic and extrinsic factors leading to differences between skin and corneal transplants. (1) The eye is a site with an absence of blood and lymph vessels. The existence of anterior chamber-associated immune deviation downregulates antigen-specific delayed-type hypersensitivity responses [11]. However, any factors that stimulate the growth of new blood and lymph vessels into grafts are considered to abolish the immune privilege of cornea allografts [12,13]. (2) Difference in recognition. Both MHC and minor antigens induce indirect alloresponse in skin transplantation, while cornea-transplanted mice are directed exclusively by minor antigens via indirect CD4 + T cell alloresponse [15,16]. (3) Different alloresponse. Either CD4+ or CD8+ T cells are activated both directly and indirectly in skin transplants [14,17]. On the other hand, with the lack of acute rejection, only CD4 + T cells are indirectly activated in the rejection process of cornea transplantation [12,15]. Thus, various immunomodulatory strategies have been used in kinds of experimental transplantation. The adoptive transfer of MDSC is a new attempt. Studies have shown that MDSC suppresses the cytokine production and proliferation by effector T cells. We previously confirmed that MDSC was easily able to be activated and expanded under inflammatory stimuli, and appears to display a significant cellmediated immunosuppressive capacity both in vitro and in vivo.
Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
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Fig. 1. Slit-lamp photographs and corresponding histopathological images (hematoxylin and eosin staining, ×400) of corneal grafts at various times after transplantation in all corneal transplantation groups. A. Graft edema, opacity, and neovascularization were evaluated from slit-lamp appearance. B. Three different histopathological findings were evaluated in all specimens: severity of inflammatory reaction, vessels in the corneal grafts, and regularity of stromal fibers.
Adoptive transfer of inflammation-induced MDSC (iMDSC) may effectively reduce corneal neovascularization and prolong corneal allograft survival [22]. In this study, the life span of transplants, both cornea and skin, were both equivalently contributed to adoptive transferred iMDSCs. Possible explanations were investigated by comparing mechanisms underlying the recognition and response of fully allogeneic skin and corneal allotransplants in mice. (1) iMDSCs significantly inhibited CD4+ [22] and CD8 + (unpublished data, submitted) T cell proliferation in an in vitro mixed lymphocyte reaction (MLR) with dosage association. (2) Of note, we observed that the speed of neovascularization was much faster in skin–corneal combination transplant groups than in groups with single transplants, which may be due to stronger immune responses induced by surgery and alloantigens in combination groups. We previously reported that adoptive transfer of iMDSCs significantly reduced neovascularization in allogeneic corneal grafts to inhibit antigen-presenting cells and lymphocyte migration to grafts [22]. However, the same dose of iMDSCs in combination and single transplant groups achieved the same effect, which implied that the one-off dose of iMDSCs we chose was sufficient or even excessive. Moreover, it is likely that for transplantation, the predominant mechanisms of iMDSCs are systemic immunosuppressive effects, such as cytokine secretion of arginase 1, INF-γ, or the induction of CD4+CD25+Foxp3+ regulatory T cells [1,10,23]. We detected changes in MDSC distribution in bone marrow and corneal graft in different postoperative periods for understanding the mechanisms of homing and proliferation. In combination transplant groups, a temporary but significant elevation of the MDSC rate was observed in an early stage after transplantation. No differences were observed among groups with or without MDSC adoptive transfer. It was
indicated that levels of MDSC expansion may be determined by the nature of soluble factors and cytokines produced by transplant or surgical stimuli. With the process of rejection, the proliferation of bone marrow MDSCs in SCT groups slowed and showed no difference in late periods following surgeries. However, survival time in SCT groups did not show any superiority compared to solo transplant groups. Possible causes may be: [1] newly induced MDSCs in bone marrow may have a low inhibition ability. It is more likely to represent a control condition to maintain immunological balance. [2] Alternately, the inhibition function of newly induced MDSCs may have been counteracted by stronger immunoreaction in SCT groups. It is not clear at this time which mechanisms should be attributed to this phenomenon. Moreover, our data demonstrated that in early postoperative stages, the elevation of corneal MDSC in adoptive transferred groups may indicate a concentrated effect to stimulating places, which seems to have a relationship with the local immune-suppression. Interestingly, corneal MDSC in SCT groups obviously increased in the middle and late periods. This may be caused by the supplement of bone marrow derived MDSCs, and the growth of neovessels and lymphangiogenesis, which may take more immune cells to the corneal grafts. We were not able to tell iMDSCs from recipient bone-marrow derived MDSCs in flow analysis of corneal grafts, as both MDSCs showed the same surface markers. However, this finding continues to show that MDSC distribution in vivo tends to maintain a steady balance and homeostasis. Collectively, survival of solo or combined allogeneic skin and corneal allotransplants may be supported by equivalent adoptive transferring of iMDSCs. This may indicate that the underlying mechanisms of iMDSCs are the suppressive effects in either CD4+ or CD8+ T cells activated directly or indirectly against MHC or minor antigens. Moreover, our results also showed a substantial expansion of MDSCs in recipients' bone
Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
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Fig. 2. A. Kaplan–Meier curves of corneal grafts to evaluate the effects of retro-orbital injected iMDSCs from different origins. Adoptively transferred infectious-induced myeloid-derived suppressor cells (iMDSCs) prolonged corneal allograft survival times in corneal transplantation (P b 0.01 vs. allogeneic). B. Evaluation of neovascularization in corneal allografts (Scored 0–3). Adoptive transferring of iMDSCs inhibited the growth of neovessels at the early stages after surgery, while the formation of neovessels in SCT groups increased sharply compared to single corneal transplantation groups. C. Kaplan–Meier curves of skin grafts to evaluate the effects of adoptive transferred iMDSCs. Adoptively transferred iMDSCs prolonged skin allograft survival times in both skin and skin–corneal combined transplant models (P b 0.01 vs. allogeneic).
marrow, particularly in combined groups at an early postoperative stage; accordingly, the concentration of MDSCs in corneal grafts increased significantly in adoptive transferred groups. It is possible that the proliferation
and gathering effect of MDSCs with or without suppressive effects are not restricted to one or several elements, but rather attributed to entire immune states that reflect biological changes in transplantation.
Fig. 3. MDSC distribution in recipients' bone marrow and corneal grafts at various times after transplantation. A, B, C. MDSC distribution in recipients' bone marrow at postoperative days 15, 25, 35 (*P b 0.01). D, E, F. MDSC distribution in recipients' corneal grafts at postoperative days 15, 25, 35 (*P b 0.01).
Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
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Conflict of interest statement The authors declare no conflict of interest. Acknowledgments Dr. Zhiqiang Pan and Dr. Yan He were supported by grants from the National Natural Science Foundation of China (No. 81070709). Dr. Hui Zeng was supported by grants from the National Natural Science Foundation of China (Nos. 81200316and 30872458). References [1] D.I. Gabrilovich, S. Nagaraj, Myeloid-derived suppressor cells as regulators of the immune system, Nat. Rev. Immunol. 9 (3) (2009) 162–174 (Epub 2009/02/07). [2] S. Ostrand-Rosenberg, P. Sinha, Myeloid-derived suppressor cells: linking inflammation and cancer, J. Immunol. 182 (8) (2009) 4499–4506 (Epub 2009/04/04). [3] E. Peranzoni, S. Zilio, I. Marigo, L. Dolcetti, P. Zanovello, S. Mandruzzato, et al., Myeloid-derived suppressor cell heterogeneity and subset definition, Curr. Opin. Immunol. 22 (2) (2010) 238–244 (Epub 2010/02/23). [4] K. Movahedi, M. Guilliams, J. Van den Bossche, R. Van den Bergh, C. Gysemans, A. Beschin, et al., Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity, Blood 111 (8) (2008) 4233–4244 (Epub 2008/02/15). [5] D. Wang, R.N. DuBois, Myeloid-derived suppressor cells link inflammation to cancer, Oncoimmunology 3 (2014), e28581 (Epub 2014/07/24). [6] J.I. Youn, S. Nagaraj, M. Collazo, D.I. Gabrilovich, Subsets of myeloid-derived suppressor cells in tumor-bearing mice, J. Immunol. 181 (8) (2008) 5791–5802 (Epub 2008/ 10/04). [7] I.H. Younos, F. Abe, J.E. Talmadge, Myeloid-derived suppressor cells: their role in the pathophysiology of hematologic malignancies and potential as therapeutic targets, Leuk. Lymphoma 1–13 (2015) (Epub 2014/11/20). [8] Y. He, B. Wang, B. Jia, J. Guan, H. Zeng, Z. Pan, Effects of adoptive transferring different sources of myeloid-derived suppressor cells in mice corneal transplant survival, Transplantation 99 (10) (2015) 2102–2108 (Epub 2015/08/14). [9] T. Wu, Y. Zhao, The roles of myeloid-derived suppressor cells in transplantation, Expert. Rev. Clin. Immunol. 10 (10) (2014) 1385–1394 (Epub 2014/08/15). [10] W. Gong, F. Ge, D. Liu, Y. Wu, F. Liu, B.S. Kim, et al., Role of myeloid-derived suppressor cells in mouse pre-sensitized cardiac transplant model, Clin. Immunol. 153 (1) (2014) 8–16 (Epub 2014/04/03).
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Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
Contents lists available at ScienceDirect
Transplant Immunology journal homepage: www.elsevier.com/locate/trim
The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation Yan He a, Jia Bei c,d, Hui Zeng c,d, Zhiqiang Pan b,⁎ a
Department of Ophthalmology, The Second Xiangya Hospital of Central South University, Eye Institute of The Second Xiangya Hospital of Central South University, Changsha 410010, China Beijing TongRen Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmic and Visual Science Key Laboratory, Beijing 100730, China Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China d Beijing Key Laboratory of Emerging Infectious Diseases, Beijing 100015, China b c
a r t i c l e
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Article history: Received 7 October 2015 Received in revised form 22 December 2015 Accepted 22 December 2015 Available online xxxx Keywords: MDSC Corneal–skin combined transplantation Adoptive transfer Allograft rejection
a b s t r a c t Purpose: To explore the effects of adoptive transferring sepsis induced myeloid-derived suppressor cells (iMDSCs) in mice corneal, skin, and combined corneal–skin survival. Methods: Allogeneic full-thickness corneal transplantation, fully mismatched skin transplantation, and corneal– skin combined transplantation (donor C57BL/6 to recipient Balb/c mice) were performed. Sepsis-induced infectious-MDSCs (iMDSCs), were purified from bone marrow of cecal ligated and punctured (CLP) Balb/c mice. Recipient-derived iMDSCs were adoptively transferred into different recipient groups by retro-orbital injection after surgeries. Corneal and skin grafts were examined and photographed routinely for a period of 45 days. Histopathology was performed to evaluate corneal-graft inflammation. Bone marrow and/or corneal grafts in each group were harvested from executed recipients on postoperative days 15, 25, 35. Corneal cells and bone marrow cells were stained with CD11b-PE and Gr1-FITC, analyzed by FACS. Results: iMDSCs were able to significantly prolong allograft survival in both corneal and corneal–skin combined transplant groups. A substantial expansion of MDSCs was observed in recipients' bone marrow, particularly in combined groups at an early stage postoperatively, and accordingly the concentration of MDSCs in corneal grafts increased significantly in adoptive transferred groups. Conclusions: Sepsis-induced MDSCs may suggest a novel cellular therapeutic approach for preventing various types of allograft rejection. © 2015 Elsevier B.V. All rights reserved.
1. Introduction A myeloid-derived suppressor cell (MDSC) is defined as a novel heterogeneous population of immature myeloid cells with immunosuppressive properties, including macrophages, granulocytes, dendritic cells (DC), and myeloid cells at earlier stages of differentiation [1,2]. In mice, they express the common myeloid cell markers CD11b (αM chain of β2 integrin) and Gr1 (Ly6G and Ly6C) [3]. In general, CD11b+Gr1+ myeloid cells represent about 30–40% of normal bone marrow cells and only 2–4% of all nucleated normal splenocytes [4,5]. Under pathological conditions, such as infection, tumor, and trauma, they accumulate in large numbers in bone marrow, blood, spleen and other lymphoid organs [6,7]. In our previous studies, a dramatic accumulation of CD11b+Gr1+ cells was detected in bone marrows of septic mice models and tumor-
Abbreviations: MDSC, Myeloid-derived suppressor cell; DC, Dendritic cell; CLP, Cecal ligated and punctured; iMDSC, Inflammation-induced myeloid-derived suppressor cell; tMDSC, Tumor-induced myeloid-derived suppressor cell. ⁎ Corresponding author at: Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Dongjiaominxiang, Beijing 100730, China. E-mail address: [email protected] (Z. Pan).
bearing mice models [8]. The functions of MDSCs are highly dependent on the circumstances in which their expansion occurs. Significantly cellmediated immunosuppressive capacities were observed in infectiousMDSCs (iMDSCs) and tumor-bearing MDSCs (tMDSCs) in vitro. However, CD11b+Gr1+ cells from naïve mice showed few immunosuppressive functions. The role of MDSCs in transplantation tolerance was wildly described in allograft models in recent years [9,10]. Our previous studies [8] were focused on the potential application of MDSCs from different sources to prolong allograft survival, and reported that MDSCs from septic mice models and tumor-bearing mice models were adoptively transferred to allogenic recipients after corneal transplantations, and significantly prolonged corneal allograft survival in vivo. Moreover, iMDSCs transferred significantly reduced neovascularization that was comparable to tMDSCs and naïve MDSC. However, the additional adoptive transfer of MDSCs did not further ameliorate corneal survival. Therefore, rather than a simplex immunosuppressive response, the function of MDSC is more than likely a complex balance between increased immune surveillance and decreased adaptive immune responses. Corneal allotransplant is an extreme situation, because the anterior chamber of the eye is an immunologically privileged site [11]. Corneal
http://dx.doi.org/10.1016/j.trim.2015.12.003 0966-3274/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
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Y. He et al. / Transplant Immunology xxx (2015) xxx–xxx
vascularization has been reported to be associated with a significantly increased risk of corneal graft rejection [12,13], which is primarily a cellmediated indirect T cell response mediated by CD4+ T cells. The fully mismatched allogenic skin transplant, as another kind of typical tissue transplantation, was reported to be recognized by recipients via both the direct and indirect pathways by CD4+ T cells in BALB/c recipient's lymphoid organs [14,15]. Moreover, both MHC and minor antigens induced an indirect alloresponse in skin transplantation. However, the entire indirect CD4+ T cell alloresponse in cornea-transplanted mice was directed exclusively to minor antigens [14–17]. As an immunomodulatory strategy to prevent corneal allograft rejection, MDSCs have been used in experimental corneal transplantation by suppressing the proliferation and cytokine production of effector T cells [1,18]. It is not clear whether there is the same positive effect when used in skin transplant, and even more interesting, in corneal– skin combined transplantation models. Moreover, the postoperative infiltration of MDSCs in graft remains unknown. Therefore, we utilized infectious MDSCs in the mice cornea, skin and cornea–skin combined transplant models, compared the therapeutic efficiency, and discussed the underlying mechanisms by analyzing the MDSC distribution and survival rate of allograft.
2. Materials and methods 2.1. Mice
2.4. Group and iMDSC adoptive transferring The recipient mice were divided randomly into 6 groups, as shown in Table 1 (18 recipients in each group), and 5 × 106 iMDSCs suspended in 150 μL PBS were transferred to the recipients' left eyes, or nonsurgery eyes, at the end of operation, via retro-orbital injection. The same volume of PBS was given to the Iso SCT, Allo CT, Allo ST, and Allo SCT groups as the control. 2.5. Assessment of graft survival Corneal grafts were assessed by slit lamp biomicroscopy every two days after surgery for 45 days. As described previously, the grafts were scored in a range for opacity (0 to 5), as shown in Table 2. Meanwhile, the extent of corneal edema and growth of neovessels were also brought under consideration. Edema and neovessels were scored within a range from 0 to 3, minimum to maximum. When the total scores of opacity, edema, and neovessels reached 6, or opacity score N 2, the grafts were rejected. Square skin grafts of 1 cm × 1 cm were excised from the backsides of C57BL/6 mice and sutured to the same regions of Balb/c mice with 12 interrupted 8–0 nylon sutures. Corneal and skin transplantation were performed at the same day in the corneal–skin combined transplantation group (SCT group). Skin grafts were examined at daily intervals. Rejection was defined as complete dissolution of graft, and wound completely healed by recipient skin.
C57BL/6(H-2b) and Balb/c(H-2d) mice were purchased from the Experimental Center of Capital Medical University (Beijing, China) and housed in a specific pathogen-free facility. Female C57BL/6 mice (8 to 12 weeks old) were used as donors and female Balb/c mice (8 to 12 weeks old) were used as recipients. Balb/c mice underwent cecal ligation and puncture, as described previously [19], and were prepared at the Institute of Infectious Diseases of Beijing Ditan Hospital. All procedures performed on animals were approved by the Animal Care Research Ethics Committee of the Capital Medical University of China.
2.6. Flow cytometric analysis of grafts
2.2. Flow cytometry and purification of MDSCs from bone marrow
2.7. Statistical analysis
Monoclonal antibodies used for fluorescence-activated cell sorting staining were: phycoerythrin (PE) conjugated anti-mouse CD11b and fluorescein isothiocyanate (FITC) conjugated anti-mouse Gr-1 (BD Bioscience, San Diego, CA, USA). Bone marrow cells from cecal ligated and punctured (CLP) Balb/c mice were stained with the above antibodies at 4 °C for 15 min and washed in PBS. The cells were isolated by fluorescence-activated cell sorter (FACSAria, BD, CA, USA). The purity of CD11b+Gr1+cells from CLP Balb/c mice (iMDSC) was routinely more than 94%. For FACS analysis, the data were acquired on a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and was analyzed using FlowJo Software v 5.7.2–2 (Tree Star, Ashland, OR, USA).
Results are expressed as means ± SD. Graft survival time was compared between various groups by the Mantel–Cox Log Rank Test and the Kaplan–Meier survival curve. P values less than 0.01 were considered to be statistically significant.
2.3. Corneal penetrating keratoplasty and skin transplantation Corneal penetrating keratoplasty was performed as described previously in the right eyes of the mice [20]. In brief, the central full thickness cornea with a diameter of 2.0 mm from the mice C57BL/6 were excised and secured in Balb/c mice graft beds with a diameter of 1.5 mm by eight interrupted 11–0 nylon sutures. 48 h later, the grafts were examined by a slit lamp. Grafts with severe complications, such as infection or hyphema, were excluded from the study. The sutures of the grafts were removed 7 days after surgery. Skin allografts of 1 cm × 1 cm in size were taken from hair removed from the dorsum of C57BL/6 mice, and were sutured in Balb/c mice graft beds with the same area by 12 interrupted 6–0 silk sutures.
On days 15, 25, and 35 after surgeries, 3 recipients for each group were executed. Corneal grafts were sheared into small pieces, and immersed in 1 mg/mL collagenase IV (Sigma) for 2 h at 37 °C in a rocking device. Bone marrow cells were harvested from executed recipients. The collected suspensions were separately forced through a 100-Μm nylon mesh. Corneal cells and bone marrow cells were stained with CD11b-PE and Gr1-FITC (BD Biosciences, USA). Samples were analyzed by FACS.
3. Results 3.1. Adoptive transfer of iMDSCs has similar inhibitory properties in corneal and corneal–skin combined transplantation Mice in different groups were treated as shown in Table 1 and iMDSC suspensions (5 × 105 cells/150 μL) and were adoptively transferred separately to iMDSC CT and iMDSC SCT groups by retrobulbar injection after corneal transplantation. The clinical characteristics of the graft, including edema, opacity and neovascularization, were examined and scored by slit lamp examination regularly up to 45 days after transplantation. Compared to the untreated allograft control group (Fig. 1A, Allo CT), no remarkable differences were observed in mice with corneal–skin combined transplantation (Fig. 1A, Allo SCT), while the grafts in the Iso SCT group remained clear and survived until the end of observation. By contrast, mice adoptively transferred with iMDSC (Fig. 1A, iMDSC CT, iMDSC SCT) exhibited reduced mean opacity, edema, and neovessel scores from the control group, particularly at an early period after surgeries. The most remarkable differences were observed in corneal– skin combined transplantation, which developed neovascularization
Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
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Table 1 Recipient group and postoperative survival time of skin and cornea grafts in each group. No.
1 2 3 4 5 6
Group name
Isograft skin & cornea transplantation Allograft cornea transplantation Allograft skin transplantation Allograft skin & cornea transplantation iMDSC cornea transplantation iMDSC skin & cornea transplantation
Group name in short
Transplantation Cornea
skin
Iso SCT Allo CT Allo ST Allo SCT iMDSC CT iMDSC SCT
√ √ – √ √ √
√ – √ √ – √
Survival time (skin)/days
Survival time (cornea)/days
N45@,# – 8.0 ± 0.94 8.80 ± 2.34 – 13.90 ± 1.20@,#
N45⁎,# 15.65 ± 2.06 – 15.02 ± 1.49 22.71 ± 3.01⁎,# 25.30 ± 8.22⁎,#
⁎ Compared with Allo CT group at the same period, P b 0.01. # Compared with Allo SCT group at the same period, P b 0.01. @ Compared with Allo ST group at the same period, P b 0.01.
more rapidly than corneal transplantation with or without iMDSC adoptive transferring (Fig. 1A, Allo CT, Allo SCT, iMDSC CT, iMDSC SCT). However, the extent of graft opacity and edema shows no difference between the corneal and corneal–skin combined transplantation groups. Graft survival analysis also demonstrated that cornea and skin allograft survival time was significantly prolonged in mice adoptively transferring iMDSCs compared to control groups (Table 1, P b 0.01, Fig. 2A, 2C). However, the differences in allograft survival time between single corneal/skin and corneal–skin combined groups were not statistically significant (Table. 1, P N 0.05). Interestingly, the formation of neovessels in SCT groups increased sharply compared to single corneal-transplantation groups (Fig. 2B).
3.2. Adoptive transfer of iMDSCs reduced histopathological changes in corneal allografts in corneal and corneal–skin combined transplantation Three different histopathological findings were evaluated in all specimens: severity of inflammatory reaction, vessels in the corneal grafts, and regularity of stroma fiber (Fig. 1B). The extent of inflammatory infiltrate associated with neovessels within the corneal stroma increased with time in all groups except the Iso SCT group. A severe inflammatory reaction and new blood vessels from the infiltrated limbal arcade reached even central grafts in the early stage of group Allo CT and Allo SCT (Fig. 1B, Allo CT, Allo SCT), while the stroma fibers appeared to be a mess. In contrast, corneal slides of iMDSC groups (Fig. 1B, iMDSC CT, iMDSC SCT) exhibited few changes as normal cornea in the first 25 days after transplantation, and a chronic slightly inflammatory infiltration and relatively regular fibers appeared in the late period. Compared to the corneal graft in corneal transplantation in histopathological slices, increases in neovascularity and extent of inflammatory infiltrate associated with neovessels within the corneal stroma seemed somewhat severe in corneal grafts of combined transplantation (Fig. 1B).
Table 2 Corneal grafts survival assessment. Standard
Score
Sign
Opacification
0 1 2 3 4 5 0 1 2 3 0 1 2 3
Clear graft Minimal superficial (nonstromal) opacity Minimal deep stromal opacity, iris striation sharp Moderate stromal opacity, iris striation blur Intense stromal opacity; only the pupil can be discerned Complete stromal opacity No edema Slight edema Moderate edema Severe edema No neovessels Neovessels appeared in rim of bed Neovessels in rim of graft Neovessels in centre of graft
Edema
Neovessels
3.3. Detection of postoperative distribution of MDSCs in bone marrow and corneal graft Next, we examined changes in MDSC distribution in recipients' corneal grafts and bone marrows after transplantation. Corneal grafts and bone marrows from each group were harvested, cells were double stained for CD11b and Gr-1, and then they were analyzed by flow cytometry. At an early stage, MDSC percentages in mouse bone marrow combined with skin transplantation were comparably higher than corneal transplant groups (Fig. 3A, P b 0.01). Meanwhile, a markedly high intensity of MDSC was found in corneal grafts of iMDSC adoptively transferring groups relative to normal corneal and corneal–skin combined transplant groups (Fig. 3D, P b 0.01). However, with the passage of time, the concentrating effect of MDSC in corneal grafts of iMDSC groups decreased, while levels of MDSCs in the cornea paralleled neovessel growth in those groups. Grafts in models with skin transplantation showed higher levels of MDSC than groups with corneal transplantation only at 25 days after surgeries (Fig. 3E, P b 0.01); interestingly, the expression of BM MDSC reached almost the same levels in all six groups (Fig. 3B, C, P N 0.05). 4. Discussion Corneal and skin transplants represent two extreme situations. Corneal transplants are routinely performed without HLA matching and systemic immunosuppressive drugs, and may even have a high acceptance rate, exceeding 90% [21]. In contrast, fully allogeneic skin transplants experience a 100% incidence of immune rejection without HLA matching and systemic immunosuppressive drugs. Recently, various studies have emphasized the intrinsic and extrinsic factors leading to differences between skin and corneal transplants. (1) The eye is a site with an absence of blood and lymph vessels. The existence of anterior chamber-associated immune deviation downregulates antigen-specific delayed-type hypersensitivity responses [11]. However, any factors that stimulate the growth of new blood and lymph vessels into grafts are considered to abolish the immune privilege of cornea allografts [12,13]. (2) Difference in recognition. Both MHC and minor antigens induce indirect alloresponse in skin transplantation, while cornea-transplanted mice are directed exclusively by minor antigens via indirect CD4 + T cell alloresponse [15,16]. (3) Different alloresponse. Either CD4+ or CD8+ T cells are activated both directly and indirectly in skin transplants [14,17]. On the other hand, with the lack of acute rejection, only CD4 + T cells are indirectly activated in the rejection process of cornea transplantation [12,15]. Thus, various immunomodulatory strategies have been used in kinds of experimental transplantation. The adoptive transfer of MDSC is a new attempt. Studies have shown that MDSC suppresses the cytokine production and proliferation by effector T cells. We previously confirmed that MDSC was easily able to be activated and expanded under inflammatory stimuli, and appears to display a significant cellmediated immunosuppressive capacity both in vitro and in vivo.
Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
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Fig. 1. Slit-lamp photographs and corresponding histopathological images (hematoxylin and eosin staining, ×400) of corneal grafts at various times after transplantation in all corneal transplantation groups. A. Graft edema, opacity, and neovascularization were evaluated from slit-lamp appearance. B. Three different histopathological findings were evaluated in all specimens: severity of inflammatory reaction, vessels in the corneal grafts, and regularity of stromal fibers.
Adoptive transfer of inflammation-induced MDSC (iMDSC) may effectively reduce corneal neovascularization and prolong corneal allograft survival [22]. In this study, the life span of transplants, both cornea and skin, were both equivalently contributed to adoptive transferred iMDSCs. Possible explanations were investigated by comparing mechanisms underlying the recognition and response of fully allogeneic skin and corneal allotransplants in mice. (1) iMDSCs significantly inhibited CD4+ [22] and CD8 + (unpublished data, submitted) T cell proliferation in an in vitro mixed lymphocyte reaction (MLR) with dosage association. (2) Of note, we observed that the speed of neovascularization was much faster in skin–corneal combination transplant groups than in groups with single transplants, which may be due to stronger immune responses induced by surgery and alloantigens in combination groups. We previously reported that adoptive transfer of iMDSCs significantly reduced neovascularization in allogeneic corneal grafts to inhibit antigen-presenting cells and lymphocyte migration to grafts [22]. However, the same dose of iMDSCs in combination and single transplant groups achieved the same effect, which implied that the one-off dose of iMDSCs we chose was sufficient or even excessive. Moreover, it is likely that for transplantation, the predominant mechanisms of iMDSCs are systemic immunosuppressive effects, such as cytokine secretion of arginase 1, INF-γ, or the induction of CD4+CD25+Foxp3+ regulatory T cells [1,10,23]. We detected changes in MDSC distribution in bone marrow and corneal graft in different postoperative periods for understanding the mechanisms of homing and proliferation. In combination transplant groups, a temporary but significant elevation of the MDSC rate was observed in an early stage after transplantation. No differences were observed among groups with or without MDSC adoptive transfer. It was
indicated that levels of MDSC expansion may be determined by the nature of soluble factors and cytokines produced by transplant or surgical stimuli. With the process of rejection, the proliferation of bone marrow MDSCs in SCT groups slowed and showed no difference in late periods following surgeries. However, survival time in SCT groups did not show any superiority compared to solo transplant groups. Possible causes may be: [1] newly induced MDSCs in bone marrow may have a low inhibition ability. It is more likely to represent a control condition to maintain immunological balance. [2] Alternately, the inhibition function of newly induced MDSCs may have been counteracted by stronger immunoreaction in SCT groups. It is not clear at this time which mechanisms should be attributed to this phenomenon. Moreover, our data demonstrated that in early postoperative stages, the elevation of corneal MDSC in adoptive transferred groups may indicate a concentrated effect to stimulating places, which seems to have a relationship with the local immune-suppression. Interestingly, corneal MDSC in SCT groups obviously increased in the middle and late periods. This may be caused by the supplement of bone marrow derived MDSCs, and the growth of neovessels and lymphangiogenesis, which may take more immune cells to the corneal grafts. We were not able to tell iMDSCs from recipient bone-marrow derived MDSCs in flow analysis of corneal grafts, as both MDSCs showed the same surface markers. However, this finding continues to show that MDSC distribution in vivo tends to maintain a steady balance and homeostasis. Collectively, survival of solo or combined allogeneic skin and corneal allotransplants may be supported by equivalent adoptive transferring of iMDSCs. This may indicate that the underlying mechanisms of iMDSCs are the suppressive effects in either CD4+ or CD8+ T cells activated directly or indirectly against MHC or minor antigens. Moreover, our results also showed a substantial expansion of MDSCs in recipients' bone
Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
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Fig. 2. A. Kaplan–Meier curves of corneal grafts to evaluate the effects of retro-orbital injected iMDSCs from different origins. Adoptively transferred infectious-induced myeloid-derived suppressor cells (iMDSCs) prolonged corneal allograft survival times in corneal transplantation (P b 0.01 vs. allogeneic). B. Evaluation of neovascularization in corneal allografts (Scored 0–3). Adoptive transferring of iMDSCs inhibited the growth of neovessels at the early stages after surgery, while the formation of neovessels in SCT groups increased sharply compared to single corneal transplantation groups. C. Kaplan–Meier curves of skin grafts to evaluate the effects of adoptive transferred iMDSCs. Adoptively transferred iMDSCs prolonged skin allograft survival times in both skin and skin–corneal combined transplant models (P b 0.01 vs. allogeneic).
marrow, particularly in combined groups at an early postoperative stage; accordingly, the concentration of MDSCs in corneal grafts increased significantly in adoptive transferred groups. It is possible that the proliferation
and gathering effect of MDSCs with or without suppressive effects are not restricted to one or several elements, but rather attributed to entire immune states that reflect biological changes in transplantation.
Fig. 3. MDSC distribution in recipients' bone marrow and corneal grafts at various times after transplantation. A, B, C. MDSC distribution in recipients' bone marrow at postoperative days 15, 25, 35 (*P b 0.01). D, E, F. MDSC distribution in recipients' corneal grafts at postoperative days 15, 25, 35 (*P b 0.01).
Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003
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Conflict of interest statement The authors declare no conflict of interest. Acknowledgments Dr. Zhiqiang Pan and Dr. Yan He were supported by grants from the National Natural Science Foundation of China (No. 81070709). Dr. Hui Zeng was supported by grants from the National Natural Science Foundation of China (Nos. 81200316and 30872458). References [1] D.I. Gabrilovich, S. Nagaraj, Myeloid-derived suppressor cells as regulators of the immune system, Nat. Rev. Immunol. 9 (3) (2009) 162–174 (Epub 2009/02/07). [2] S. Ostrand-Rosenberg, P. Sinha, Myeloid-derived suppressor cells: linking inflammation and cancer, J. Immunol. 182 (8) (2009) 4499–4506 (Epub 2009/04/04). [3] E. Peranzoni, S. Zilio, I. Marigo, L. Dolcetti, P. Zanovello, S. Mandruzzato, et al., Myeloid-derived suppressor cell heterogeneity and subset definition, Curr. Opin. Immunol. 22 (2) (2010) 238–244 (Epub 2010/02/23). [4] K. Movahedi, M. Guilliams, J. Van den Bossche, R. Van den Bergh, C. Gysemans, A. Beschin, et al., Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity, Blood 111 (8) (2008) 4233–4244 (Epub 2008/02/15). [5] D. Wang, R.N. DuBois, Myeloid-derived suppressor cells link inflammation to cancer, Oncoimmunology 3 (2014), e28581 (Epub 2014/07/24). [6] J.I. Youn, S. Nagaraj, M. Collazo, D.I. Gabrilovich, Subsets of myeloid-derived suppressor cells in tumor-bearing mice, J. Immunol. 181 (8) (2008) 5791–5802 (Epub 2008/ 10/04). [7] I.H. Younos, F. Abe, J.E. Talmadge, Myeloid-derived suppressor cells: their role in the pathophysiology of hematologic malignancies and potential as therapeutic targets, Leuk. Lymphoma 1–13 (2015) (Epub 2014/11/20). [8] Y. He, B. Wang, B. Jia, J. Guan, H. Zeng, Z. Pan, Effects of adoptive transferring different sources of myeloid-derived suppressor cells in mice corneal transplant survival, Transplantation 99 (10) (2015) 2102–2108 (Epub 2015/08/14). [9] T. Wu, Y. Zhao, The roles of myeloid-derived suppressor cells in transplantation, Expert. Rev. Clin. Immunol. 10 (10) (2014) 1385–1394 (Epub 2014/08/15). [10] W. Gong, F. Ge, D. Liu, Y. Wu, F. Liu, B.S. Kim, et al., Role of myeloid-derived suppressor cells in mouse pre-sensitized cardiac transplant model, Clin. Immunol. 153 (1) (2014) 8–16 (Epub 2014/04/03).
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Please cite this article as: Y. He, et al., The roles of sepsis-induced myeloid derived suppressor cells in mice corneal, skin and combined transplantation, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.12.003