Background
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) [
1]. Current knowledge suggests that the disease is maintained by auto-reactive T cells that target proteins expressed predominantly in myelin and, to a lesser extent on axons, which ultimately results in CNS tissue injury [
2]. A number of therapeutic approaches using immunomodulatory or immunosuppressive drugs such as interferon-β, glatiramer acetate, natalizumab, and Fingolimod (FTY720) have been designed to target the immune component of the disease process [
3]. While these treatments are beneficial in halting the disease in approximately 30 % of relapsing-remitting (RR)-MS patients, they are only partially effective and have little impact on disease progression [
4]. For this reason, there is a desperate need for alternative therapies to improve the outcomes for the majority of MS patients. Improved therapeutic outcomes will require the suppression of the inflammatory response, restoration of immunological tolerance, and the incorporation of neuroprotective strategies. For these reasons, stem cell therapy has gained momentum over the past decade as a potential treatment for MS.
One proposed stem cell source is human amnion epithelial cells (hAECs). These cells are isolated from the epithelial layer of the amniotic membrane, the innermost layer of the fetal membranes that surround the fetus [
5]. The amnion is originally derived from embryonic ectoderm [
6,
7] with differentiation of hAECs from the epiblast occurring around day 8 of human pregnancy, before gastrulation, at a time when the cells are still pluripotent. As a result of this early divergence, hAECs retain a high level of pluripotency as evidenced by the expression of several embryonic stem cell (ESC) markers including OCT-4, nanog, SSEA-3, SSEA-4, TRA 1-60, and c-kit [
8‐
11]. hAECs are claimed to be immune privileged in so far as they do not express human leukocyte antigen (HLA) class II or co-stimulatory molecules [
12,
13], theoretically making them potential candidates in allogeneic settings. Given that, on average, about 100–200 million hAECs can be isolated from a term placenta [
13], these cells present an abundant source of potential regenerative tissue. Moreover, their collection does not hold ethical constraints in comparison with other stem cell sources such as ESCs. In vitro studies have shown that hAECs can generate clinically relevant cell types from ectoderm, mesoderm, and endoderm, such as cardiomyocytes, myocytes, osteocytes, adipocytes, pancreatic cells, hepatocytes, as well as neural and astrocytic cells [
9,
10,
14]. More poignantly, investigations into their immunomodulatory properties have shown that hAECs inhibit cells of the innate and adaptive immune system, as shown by the inhibition of neutrophil and macrophage migration by secrete factors [
8,
15] and reduction of both T and B cell proliferation [
5,
16] in vitro.
The potential of hAECs for the treatment of MS has recently been highlighted by transplantation studies in experimental autoimmune encephalomyelitis (EAE) by us and others [
17,
18], which links the amelioration of EAE with the capacity of hAECs to suppress inflammation. However, the mechanisms behind the suppression of disease are not well understood. It has also been claimed that hAECs are capable of homing to sites of inflammation [
19], including the brain [
20]. It is noteworthy that amnion has been used clinically for over 30 years for the treatment of dural defects [
21], burns [
22,
23], ocular surface disease [
24], and other numerous eye diseases [
25‐
27], thus establishing their strong safety profile. The current study extends on previous findings by showing that hAECs can significantly suppress in vitro T cell activation, proliferation, and cytokine production. When administered at the time of disease onset in a RR-EAE model, hAEC transplantation significantly attenuated disease through increasing the number of T regulatory cells, increasing the pool of peripheral naïve CD4+ T cells, and promoting a shift towards a Th2 dominant environment. This study demonstrates that hAECs are highly immunomodulatory and may have potential as a therapy for MS.
Discussion
hAECs are a novel source of stem cells that can be obtained in large quantities [
13] and possess potent immunosuppressive properties [
5,
15]. In the current study, we examined whether the administration of hAECs in a RR-EAE model could modulate the immune response and suppress MOG-induced EAE development. Our results show that transplantation of hAECs at the time of disease onset significantly ameliorates RR-EAE by inhibiting pathogenic T cell responses, namely Th17 and Th1 cells within the periphery and CNS. We propose that this effect is mediated by an increase in the number of peripheral T regulatory cells, an increase in the pool of naïve CD4+ T cells, and a shift towards a Th2 dominant environment.
Given that hAECs are routinely isolated from different donors, it is important for their future clinical application that donor-specific variations are assessed. We confirmed that hAECs do not express HLA class II antigen (HLA-DR) or co-stimulatory factors CD80 and CD86 [
12], and no variation between donors was noted. However, we did observe expression of HLA class I antigens in cells from all of our donors, which is in contrast to previous reports [
9,
37] and may lead to an increased risk of transplant rejection. The expression of HLA-G, a non-classical class I antigen, is most commonly expressed on placental tissues [
38,
39], including amnion [
32]. We found that there was variation in HLA-G expression between donors. While all were positive, some had threefold higher expression than others. It has previously been reported that preterm amnion expresses less HLA-G than term amnion [
32]. It is possible that HLA-G expression is related to proximity to the timing of birth. Furthermore, upon analysis of CD90, we found discrepancies to previous literature that have stated that hAECs are negative for this marker [
13]. In this current study, we did not directly compare the in vivo efficacy between the different donors, but given the differences in HLA-G and CD90 expression, these future studies are warranted. The phenotypic differences we observed between the donors in this study suggest that the pre-screening of amnion donors may be required in order to identify those most suitable for use in specific clinical applications, such as the treatment of MS patients.
As shown here, hAECs posses potent immunomodulatory properties capable of suppressing antigen-specific, non-specific, and allogeneic T cell responses in vitro, including human PBMC proliferation. While it has previously been shown that hAECs do not stimulate allogeneic proliferation when cultured with PBMCs [
12], our observations further demonstrate that these cells do not elicit a xenogeneic response from mouse splenocytes. The immune suppressive activity of hAECs in vitro was associated with a significant decrease in Th1 cytokines, including IFN-γ and TNF-α. It is interesting to note that hAECs did not influence secretion of IL-17A and IL-4 in in vitro assays, while in vivo, hAEC treatment led to decreased IL-17A and increased IL-4 secretion. This may be explained by differences in mouse strain (C57BL/6 versus NOD/Lt) and stimulating antigen (MOG35-55 versus recombinant MOG protein) used in the in vitro and in vivo experiments. While these results are not directly comparable, it is noteworthy that hAECs suppress proliferation and pro-inflammatory cytokines in both settings, thus demonstrating their broad immunosuppressive properties. In examining the in vitro suppressive effect of hAECs, we show here for the first time that hAECs restrain naïve CD4+ T cells from developing into an activated phenotype. This was demonstrated by inhibition of the upregulation of two markers, the T cell activation marker CD25, and the adhesion receptor CD44. This is important in the context of MS pathogenesis, since MS patients have more myelin-reactive effector/memory T cells compared to healthy controls [
40]. In the EAE mice, CD44 has been shown to be involved in both the differentiation of Th1/Th17 cells [
41] and the extravasation of effector/memory CD62LloCD44hi T cells into the CNS [
42,
43].
The ability of a single dose of two million hAECs to ameliorate EAE has previously been shown in the C57BL/6 mice [
18], and our earlier work has also demonstrated that a single dose of one million hAECs can suppress EAE in the NOD/Lt mice [
17]. Results presented here extend these findings by demonstrating that low-dose (1 × 10
6) and high-dose (5 × 10
6) hAECs injected intraperitoneally were effective in significantly ameliorating clinical and pathological signs of RR-EAE in the NOD/Lt mice. We also demonstrate for the first time that hAECs ameliorate EAE in a dose-dependant manner and importantly, as little as 100,000 hAECs suppressed clinical signs of the disease. We have previously investigated the efficacy of different sources of MSCs [
29] and neural stem cells (NSCs) [
28] in EAE and have never observed significant amelioration of disease when less than one million cells were transplanted. While not directly comparable, the current results suggest that hAECs are more powerful in suppressing EAE than MSCs and NSCs.
Liu et al. [
18] have previously suggested that hAECs work via a Th2 shift based on the single observation that IL-5 was increased in the EAE mice treated with hAECs. However, no change in other cytokines, including IL-4, IL-17, IFN-γ, TNF-α, or IL-10, was reported. Moreover, no investigation was performed on the individual T helper cell phenotypes. In that study, EAE was induced in the C57BL/6 mice resulting in chronic progressive paralysis, a model that displays different immune responses compared to the relapsing-remitting EAE in the NOD/Lt mice [
44]. Furthermore, Liu et al. [
18] transplanted cells intravenously, whereas we delivered cells into the intraperitoneal cavity. While we cannot exclude the possibility that the variability seen in the cytokine secretion between these two studies is due to the strain of mice used for EAE, as well as the route of hAECs injection, it is noteworthy that we have previously shown that both administration routes are effective for reducing clinical signs [
29]. In our study, we unequivocally show that, together with the decrease in clinical scores, we observed a significant decrease in MOG-specific secretion of IL-17A and a significant increase in IL-4, a Th2 cytokine that has been associated with spontaneous EAE recovery [
45] and reduction in disease severity [
46]. Importantly, the decreased secretion of IL-17A was observed in spite of the significant increase in the proportion of Th17 and Th1 cells within the spleen. Collectively, these data suggest that the increased proportion of Th1 and Th17 cells in the spleens of the hAEC-treated mice produce less pro-inflammatory mediators and are under the tight regulatory control of either Th2 or other regulatory cell types. This was verified by examining the Th1 to Th2 and Th17 to Th2 ratios, whereby the mice receiving five million hAECs displayed a significant Th2 shift.
Regulatory T cells play a key role in controlling immune responses and protect against the development of EAE [
47]. In our study, increased numbers of Treg cells were observed in the peripheral lymphoid tissues of the mice that received hAECs. Using a model of fibrotic lung injury, we have recently shown that hAECs induce Treg polarisation and these Tregs were critical for macrophage polarisation and subsequent injury resolution [
48]. Taken together, it is clear that hAECs have the ability to influence Tregs and this functional property is critical to their overall therapeutic efficacy. Studies using other placental-derived cells have found similar effects with increased Treg polarisation and downregulation of Th1 and Th17 responses in normal PBMCs and collagen reactive T cells from arthritis patients [
49,
50]. Given the plasticity of CD4 cell subsets, further studies are required to define how hAECs regulate the differentiation of T helper cells and Tregs. We showed in vitro that hAECs had the ability to restrain naïve CD4+ T cells from acquiring a memory phenotype upon activation. Therefore, in our in vivo studies, we examined the naïve CD4+ T cell pool and similarly found a significant increase in the number of naïve CD4+ T cells in the mice treated with the low dose of one million hAECs compared to the PBS controls. This demonstrates that hAECs also have the ability to suppress the activation of T cells in vivo, which has not been shown previously.
While our findings show that hAECs act via modulation of peripheral immune responses, administration of hAECs also modulates the immune response directly within the CNS. This was demonstrated by a decrease in microglia and macrophages within both the gray and white matters of the CNS. Not only were there less overall numbers of macrophages and microglia, but the activation state of microglia was decreased, with more resting microglia present following hAEC administration (data not shown). Furthermore, an increase in the proportion of CD4 + IL-4+ T cells directly within the CNS was also observed in the mice that received hAECs. Increased IL-4 levels within the CNS of the EAE mice have been associated with decreased demyelination and axonal pathology [
51] as well as promoting the recruitment of Tregs [
52]. Th2 cytokines have also been found to have a positive effect on neuronal cultures by upregulating arginase, which can aid in neuroprotection by decreasing NO generation and enhancing neurite outgrowth [
53]. The mechanism behind the increase in Th2 cells observed in the periphery and within the CNS following hAEC administration has not yet been elucidated. It has been shown that MSCs derived from adipose have the ability to upregulate chemokine receptor expression, such as CCR4, on peripheral lymphocytes [
54]. It may be that hAECs are programming Th2 cells in the peripheral lymphoid organs to increase their expression of CCR4, which has been implicated in increased Th2 trafficking to the CNS during disease [
55].
Although we did not use a control cell type in the current study, previous work, including our own, has clearly demonstrated that not all mouse and human cell types are capable of significantly attenuating clinical and pathological disease in EAE mice [
28,
29,
56]. Based on this current literature, we believe that the beneficial effect observed in the current setting is due to the immunosuppressive and reparative properties of hAECs rather than a non-specific response to cell transplantation. Moreover, even though our results suggest that hAECs do not elicit a xenogeneic T cell response in vitro, we cannot rule out the possibility that the induction of an innate or humoral immune response to the injected cells may cause their subsequent rejection. Nevertheless, there is very strong evidence in current literature that shows long-term engraftment of transplanted cells is not required for therapeutic benefit [
57‐
59].
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CM participated in the design of the study; performed and analyzed the phenotypic characterisation, in vitro proliferation assays, and T cell activation assays; performed all EAE studies; performed and analyzed all the T cell and Treg analysis from the EAE mice; and drafted the manuscript. NP participated in the design of the study, performed the in vitro proliferation assays, performed all EAE studies, performed all the T cell and Treg analysis from the EAE mice, and drafted the manuscript. GS performed the in vitro proliferation assays, performed all EAE studies, and performed all the T cell and Treg analysis from the EAE mice. LM injected all the animals for the EAE studies and performed the T cell and Treg analysis from the EAE mice. CS participated in the design of the study, analyzed the phenotypic characterisation and T cell activation assays, and drafted the manuscript. RL isolated and provided the cells for the study and participated in drafting of the manuscript. EW participated in the design of the experiment, provision of the cells, and drafting of the manuscript. GJ participated in the original conception of the project, design of the experiment, and drafting of the manuscript. CCB conceived the idea, participated in the design of the experiment and drafting of the manuscript, and obtained funding for the work. All authors read and approved the final manuscript.