Sjogren’s syndrome (SS) is a common progressive autoimmune disease that affects females predominantly [1
]. The prevalence of SS is variable worldwide; ranging from 0.1% to 0.72% of the population [4
]. SS progresses slowly and patients exhibit clinical symptoms years after the disease onset [12
]. The immune system targets epithelial tissues, infiltrates it with lymphocytes, and later forms autoantibodies against glands antigens [3
]. The aberrant immune dysregulation leads to the destruction of epithelial tissues, especially salivary and lacrimal glands, and to several extra-glandular manifestations. The secretory function of the glands diminishes gradually resulting in dryness of the mouth (xerostomia), eyes (keratoconjunctivitis sicca), and organs containing exocrine glands, such as the nose and vagina [17
]. The current SS management is symptomatic-based to alleviate the dryness severity and complications [21
]. However, patients with systemic involvement and serious complications are prescribed immunosuppressant and disease-modifying antirheumatic drugs [23
]. Unfortunately, the current management is not adequate nor satisfactory, leading to a compromised quality of life [26
MSCs are multipotent cells that can self-renew and give rise to specialized cell types such as bone, cartilage, and muscles [29
]. They were firstly isolated from the bone marrow, and later were extracted from various tissues, including peripheral blood, umbilical cord, adipose tissue, periodontal ligaments, and dental pulp [30
]. Under normal homeostasis, MSCs are actively involved in the connective tissue maintenance. During tissue repair, they are responsible for secreting bioactive molecules that result in tissue regeneration and restoration [36
]. MSCs are hypoimmunogenic because they lack the expression of MHC II and they express low levels of MHC I [37
]. MSCs have demonstrated promising therapeutic potentials when used in different diseases and in tissue regeneration. They were successfully deployed in the management of neural injuries, GVHD (graft versus host disease), cardiac regeneration, and most importantly autoimmune diseases [39
]. MSCs display a unique combination of immunoregulatory/immunosuppression, tissue regeneration/repair, and anti-fibrotic properties [59
] which make them a suitable therapeutic modality for autoimmune diseases.
MSCs are well documented for their immunomodularity and anti-inflammatory properties [61
]. Several studies have reported that MSCs suppressed T and B cells proliferation when injected at the peak or at the onset of the disease [65
]. However, Xu et al. have reported a defective MSCs immunoregulatory function in SS patients and NOD mice [50
]. They incubated BM-derived MSCs from NOD mice and SS patients with PBMCs (Peripheral Blood Mononuclear Cells), a significantly higher proliferation rate of PBMCs was found in comparison to MSCs isolated from healthy donors. Thus, the previous findings rationalize the replacement of MSCs in NOD mice or SS patients with adequately functioning ones from healthy donors to compensate for the defective immunoregulation function. Nonetheless, the utilization of MSCs in treatments is not risk-free. These cells possess an attractive self-renewal and unlimited proliferation capacities, but these characteristics can be unpredictable and uncontrollable in vivo. MSCs might form tumors or enhance the progression of an existing one [68
]. Therefore, transforming MSCs into extract/lysate can eliminate, theoretically, the tumorigenic risk. Our group and others have reported the therapeutic potentials of bone marrow cell extract (soup) in the management of irradiation-induced and SS damage of salivary glands and myocardial infraction, respectively, indicating the success of the concept [70
]. Yet, to the best of our knowledge, the efficacy of MSCs extract (MSCsE) has not been tested in any field.
The main aim of this study is to evaluate the efficacy of MSCsE in preserving the exocrine function of the salivary and lacrimal glands in NOD mice in comparison to MSCs. We hypothesized that MSCsE treatment executes this task via two mechanisms. Firstly, through their trophic and regenerative capacities, and secondly, by re-establishing peripheral tolerance which protects the glands against the autoimmune attack and eventually preserving the tissues from the autoimmune destruction.
The findings of our study were:
(1) MSCs/MSCsE treatments were successful in preserving the exocrine function of salivary and lacrimal glands in female NOD mice.
(2) Specialized cell subpopulations were preserved in the MSCs-/MSCsE-treated groups along with higher proliferation rates and higher EGF serum levels.
(3) MSCs/MSCsE treatments upregulated expression levels of multiple genes responsible for tissue regeneration, proliferation, and saliva/tears secretion, such as EGF, FGF2, LYZ1 and AQP5 genes and lowered CASP3 a gene involved in the apoptosis cascade.
(4) MSCs/MSCsE treatments promoted the formation of extracellular matrix by upregulation of BMP7 gene expression and prevented fibrosis by down-regulation of TGF-β1 gene expression.
(5) MSCs/MSCsE treatments preserved the corneal integrity by maintaining the epithelial thickness.
(6) Peripheral tolerance in salivary/lacrimal tissues was somewhat restored in MSCs-/MSCsE-treated groups; evidenced by less lymphocytic infiltration (less and smaller foci), selective suppression against B cells, inhibition of anti-SSA/Ro autoantibodies production, and down-regulated levels of pro-inflammatory genes like TNF-α, TGF-β1, and IL-1β.
(7) MSCs/MSCsE treatments influenced immunomodulation via inducing more T-regulatory cells peripherally and upregulation of IL-10.
We have reported in previous studies that BM cells and compact bone-derived MSCs have successfully preserved the salivary gland function when injected into female NOD mice [49
]. Other researchers have also reported the effectiveness of BM-derived MSCs in treating SS in NOD mice [50
]. We have also reported that treatment with bone marrow cell extract (BM Soup) has preserved the salivary gland function, up-regulated the expression of certain critical proteins and genes in female NOD mice [77
]. This indicates that the active protein ingredients from BM, including the MSCs subpopulation in it, were preserved and employed successfully when extracted and injected into NOD mice. Hence, combining both principles into MSCsE (mesenchymal stem cells extract) is a unique, safe and a practical treatment modality. The MSCs population in BM is quite small, 0.0017–0.0201% [78
]; therefore, their expansion will enable us to enrich our MSCs population pool and accordingly, enrich the extract with more therapeutic proteins [70
]. Previous reports have emphasized on the fact that MSCs exert their therapeutic and immunoregulatory capacity via secreting soluble factors in a paracrine mode [79
]. However, MSCs possess a unique self-renewal and unlimited proliferation capacity that can be unpredictable in vivo [68
]. In addition, results from MSCs utilization in clinical trials have been inconsistent and success rates were variable [80
]. It was found that the efficacy of these cells is vastly affected by their ability to sense the environment in which they exist [80
]. In conclusion, MSCs cell therapy although promising and has been used extensively, but several external and internal factors affect and limit their use. Hence, the development of a safer cell-free biological therapy that compromises the therapeutic capacities of MSCs proteins and avoids their potential risks was our goal. Additionally, the concept of formulating cells into extract is more practical in terms of storage and transfer [81
The progression of Sjogren’s Syndrome-like disease (SSLD) in NOD mice is divided into three phases. Phase 1 (initiation of glandular pathology) extends from 0–8 weeks of age. It is characterized by cellular disruption in the exocrine glands and initiation of the immune dysregulation. Phase 2 (onset of autoimmunity) extends from 8–16 weeks of age. At this stage, lymphocytic infiltration and autoantibodies formation start. Phase 3 (onset of clinical disease) starts around 16 weeks onwards. In this phase, the secretory loss is very prominent and worsens with time [82
]. In our study, we designed the timing and frequency of the treatments to serve several purposes. Regarding the timing, we injected MSCs/MSCsE at 8 weeks of age which is critical in SSLD development in NOD mice. At this age, phase two of SSLD, lymphocytic infiltration and autoantibodies production take place [82
]. Therefore, the treatments will combat the immune dysregulation just around the time it starts glandular infiltration and formation of antibodies against its antigens. Moreover, we hypothesized that the MSCsE will reinstate the peripheral tolerance same as the parental cells, MSCs, hoping to ameliorate or at least slow down the disease progression toward the glandular dysfunction (phase three). We tried to choose a time point that is realistic to the pathogenesis timing in SS patients. Yet, it would be more rational to test the treatment at an earlier age in NOD mice, at birth for example (phase one); however, the exact initiation of SS in humans, equivalent to phase 1, is simply unknown to us and it is extremely difficult to investigate. As per the frequency, we aimed at keeping the concentration of MSCs/MSCsE in the blood as high as possible during this phase to allow for a continuous immunomodulation and immunosuppression effects of the treatment while the immune system is actively attacking the glandular tissues. In addition, several studies have reported that MSCs exert a short-lived paracrine effect and this might applies to their extract as well; therefore, repetitive and extended treatment injections is preferred to keep their therapeutic functions for as long as possible [83
MSCs are well documented for promoting tissue repair in addition to their immunosuppression and immunoregulation capacities [60
]. MSCs-/MSCsE-treated groups showed higher SFR/TFR, higher protein intensity for AQP5, AQP4, CK5, α-SMA, and c-Kit, markers for acinar, ductal, myoepithelial, and progenitor/stem cells populations, respectively, in the submandibular and lacrimal glands. Proliferation, detected by Ki-67 antibody, was also upregulated and accompanied by higher serum EGF level, upregulated gene expression of EGF (submandibular and lacrimal glands), FGF2 (submandibular glands), BMP7 (submandibular and lacrimal glands), LYZ1 (lacrimal glands) and MMP2 (lacrimal glands) and down-regulated MMP2 (submandibular glands) and CASP3 (Caspase-3) in the submandibular glands. AQP5 and AQP4 are water channels that are critically involved in the formation of saliva and tears. AQP5 is located at the apical membrane of acinar cells in salivary glands, whereas in the lacrimal glands, it is located apically in the acinar and ductal cells [86
]. AQP4 is located at the basolateral membrane of acinar cells in salivary glands and laterally in acinar cells of the lacrimal glands [88
]. Several studies have reported a defective localization of AQP5 in SS patients and SS mouse models [86
]. NOD mice express AQP5 weakly in the lacrimal glands but not the ICR mice, and similar results were found in SS human patients [87
]. In the salivary glands, AQP5 tends to be primarily located basolaterally instead of the normal apical location. As assumed, we have found that in the submandibular and lacrimal glands of MSCs-/MSCsE-treated groups the apical expression of AQP5 was upregulated as measured by the immunofluorescence staining whereas in the control group, AQP5 was expressed partially at the apical surface with very low intensity, and the same applies to AQP4. The upregulation of AQPs in the treated mice explains the preservation of the SFR and TFR. Moreover, gene analysis results have confirmed our immunofluorescence staining results. In both glands, AQP5 gene expression was comparable to ICR group, especially in submandibular glands. We have also investigated the expression of several salivary and lacrimal glands markers involved in regeneration and proliferation, including CK5, c-Kit, and Ki-67. CK5 (cytokeratin 5) is an intermediate filament that is widely expressed at birth and considered a marker for ductal/progenitor cells, and CK5+
cells are considered a reservoir for the gland regeneration [92
]. C-Kit or CD117 (Type III receptor tyrosine kinase) is a marker for stem/progenitor cells in salivary and lacrimal glands [93
]. Ki-67 is a nuclear protein used for detecting actively proliferating cells [96
]. MSCs-/MSCsE-treated groups expressed higher CK5+
, and Ki-67+
cells than the control group but slightly less than the ICR group. Lysozyme, secreted by the acinar cells, is a bacteriolytic enzyme responsible for direct defense against bacteria [97
]. Lysozyme along with lipocalin and lactoferrin compose 80% of the tears proteins [98
]. We measured the lysozyme mRNA gene transcripts to evaluate the health and activity of the lacrimal gland. Our results showed an upregulation of lysozyme gene; 4 folds in the MSCs-treated and almost 1.3 in the MSCsE-treated groups, which supports the effectiveness of these treatments in reviving the lacrimal gland. Caspase-3 which is encoded by CASP3 gene, plays an important role in the execution phase of cell apoptosis. Our results showed a down-regulation of CASP3 (submandibular gland) in both treated groups, especially MSCsE, indicating a lower apoptosis. We believe that the general tissue restoration/preservation, the upregulated proliferation, and the downregulated apoptosis are in fact the function of systemic increase of EGF protein and the local upregulation of EGF and FGF2 gene expression levels.
SS patients suffer from dry eyes (keratoconjunctivitis sicca) due to tear secretion loss from lacrimal glands. The chronic dryness leads to loss of the corneal epithelium, erosions, and a possible perforation if left unmanaged [99
]. Dry eyes patients displayed thinner central cornea in comparison to healthy subjects [101
]. Preservation of the tear secretion is crucial for the health of the ocular apparatus and most importantly, the cornea. Our treatment has successfully preserved the TFR which led to the preservation of the corneal epithelium from the damaging effect of dryness.
The percentage of B cells in the lymphocytic infiltrates is very crucial for SS patients [102
]. Their ratio is higher in advanced cases and in patients with higher focus score. Several studies have reported the ability of MSCs in suppressing B cells, preventing their differentiation, and decreasing their secretion of autoantibodies [67
]. In our study, we assessed the percentage of B cells in the glandular infiltration by measuring the intensities of B220+
(a pan B cell marker in mice) B cells and the intensity of BAFF using immunohistochemical staining. We also measured the serum levels of the autoantibodies anti-SSA by ELISA. BAFF is important for maturation and homeostasis of B cells; however, uncontrolled secretion leads to autoimmunity [104
]. Excess BAFF will enable autoreactive B cells to overcome apoptotic signal in negative selection. It is expressed at high levels in several autoimmune diseases, including SS. A positive correlation was found between the levels of BAFF and autoantibodies especially anti-SSA in SS patients [105
]. Our analysis showed a significantly lower intensities for B220+
B cells and BAFF in the submandibular and lacrimal glands foci of the MSCs-/MSCsE-treated groups. BAFF is produced by several immune cells, including T lymphocytes and dendritic cells [106
]. We believe that the immunosuppressive action of MSCs-/MSCsE on these cells has resulted in less production of several factors, including BAFF. The reduction of BAFF expression and/or blocking its action have led to the reduction of B cells survival signals. The later eventually steered the reduction in autoantibodies secretion by plasma cells [107
]. In conclusion, because we achieved comparable results from both treatments, we believe that the extract contains enough factors and cytokines that are necessary and efficient for the immunomodulation needed in SS.
TNF-α and IL-1β genes were upregulated in SS patients and NOD mice [108
]. Activated macrophages secretion of TNF-α and IL-1β was attenuated by MSCs [110
]. We have previously reported that MSCs down-regulated TNF-α gene expression in treated NOD mice [49
]. Our results showed a down-regulation of gene expression for the pro-inflammatory cytokines: TNF-α, and IL-1β in the MSCs-/MSCsE-treated groups in comparison to the control group. However, MSCsE-treated group showed lower expression than MSCs treated group. We believe that the protein composition in the extract was more efficient in the suppression mechanism, probably, due to the direct bioavailability of the proteins in more significant quantities than what the MSCs can secret to achieve the same results.
Several studies, including ours, have reported that MSCs-based treatment has increased the percentage of FoxP3+
cells are essential in self-tolerance, tissue repair and proliferation [111
enforces immune suppression either by direct effect on antigen presenting cells like dendritic cells, or via anti-inflammatory cytokines secretion, such as IL-10 and TGF-β1 [113
]. IL-10 is a potent immunosuppressive cytokine that can act through different channels to block immune dysregulation, such as inhibition of pro-inflammatory cytokines TNF-α and IL-1β, antigen presentation and immune cells proliferation [115
]. IL-10 is secreted by various T cell populations most importantly FoxP3+
and it is also secreted by MSCs to target specific cells, including Treg
]. Our results showed a significant increase in the percentage of FoxP3+
within the lymphocytic infiltration in the submandibular and lacrimal glands of MSCs-/MSCsE-treated groups in comparison to the control group. We have also found an upregulation of IL-10 serum levels and its mRNA, down-regulation of TNF-α mRNA and IL-1β in submandibular and lacrimal glands. Aggarwal et al. 2005 have reported that hMSCs have induced a more anti-inflammatory and tolerogenic environment [118
]. They found that hMSCs have negatively influenced DC1 secretion of TNF-α, prompted DC2 to secrete more IL-10, and caused an increased in Treg
cell number. Thus, the high increase in IL-10 and its mRNAs was orchestrated by MSCs. MSCs-secreted IL-10 has positively influenced Treg
which in its turn secreted more that has led to immune tolerance induction via a cascade of regulatory steps. Although IL-10 serum levels in MSCsE-treated group was significantly higher than that of the control but it was lower than the MSCs-treated group levels. We think that this difference is due to the constant release of Il-10 from the MSCs via a paracrine mode from their engraftment site. However, our quantitative RT-PCR results showed lower levels of TGF-β1 in MSCs and even much lower in MSCsE-treated group in comparison to the control group. TGF-β play an important role in the salivary glands morphogenesis, extracellular matrix deposition, and controls the immune homeostasis as well [119
]. Mice that were overexpressing TGF-β suffered hyposalivation due to the excessive deposition of fibrous tissue in the gland [119
]. The therapeutic level achieved by our treatments has played its role in the anti-inflammatory aspect; orchestrated by inducing IL-10 from DC2 and Treg
and prevented the over formation of extracellular matrix and eventual fibrosis that might have occurred if excessive TGF-β1 was secreted. MSCs/MSCsE have delivered a balanced treatment that managed the immune dysregulation and promoted tissue restoration and regeneration. In agreement with our finding, Park et al. have reported that conditioned media from human umbilical cord blood-MSC down-regulated TGF-β1 and upregulated BMP7 levels in renal epithelial cells [121