Mesenchymal Stem Cells: “Guardians of Inflammation”

Human mesenchymal stem cells (MSCs) are a unique progenitor cell that can be recovered from most vascularized tissues in the human body. In addition to their capability of differentiating into tissues of mesenchymal lineage, perhaps their most salient features are immunomodulation and trophic capabilities. A chronic wound is characterized by an overzealous immune response that leads to a corrosive extracellular matrix, deficiency of regulatory growth factors, hypoxia, and cellular senescence. Human MSCs are a potential therapeutic modality to recapitulate the healing paradigm through their capacity to modulate both the innate and the adaptive immune responses. They are capable of secreting specific proteins, as dictated by the microenvironment they are placed into, that will have anti-inflammatory and growth restorative effects on all cellular factions. Their low immunogenicity suggests MSCs can be transplanted without the need for matching between donor and recipient. The current understanding of the cellular mechanisms underlying their immunomodulatory effects is summarized in this review.

Effect of Stem Cells on the Lower Limb

Application of Viable Cryopreserved Human Placental Membrane Grafts in the Treatment of Wounds of Diverse Etiologies: A Case Series

Umbilical Cord Mesenchymal Stem Cells Combined With a Collagenfibrin Double-layered Membrane Accelerates Wound Healing

The chronic wound environment disturbs multiple molecular levels, leading to senescence of cellular components, destruction of the extracellular matrix (ECM) that provides ample reservoir for bacterial growth, and ultimately a state of chronic inflammation that perturbs the healing process. Characteristics of chronic wounds are innumerable and widely covered in the literature, which includes findings such as no clotting mechanism, excessive inflammation, leaking capillaries with accumulation of wound fluid, ECM-granulation tissue defects (including defective and degraded ECM), and an imbalance between proteases and their inhibitors. In addition, researchers see impaired cell-matrix interactions, round fibrotic edges, impaired keratinocyte migration, and excessive biofilm formation with possible infections that ultimately lead to excessive scarring and fibrosis. If human mesenchymal stem cells (MSCs) can modulate, reverse, and repair each one of these aforementioned deficiencies, then providers would have a tremendous weapon that might enable an expeditious wound closure before a devastating complication ensues.

The immunomodulatory function of an MSC is one of its most powerful characteristics. It can augment and modulate both the adaptive and innate immune responses as it pertains to the wound healing paradigm. 

Well documented in the literature, MSCs (perhaps all MSCs) are derived from perivascular cells called pericytes.^1,2 This explains how MSCs can be isolated from almost every vascularized tissue in the body.^3,4 The fact that MSCs possess the capacity to secrete immunomodulatory and trophic mediators strongly argues that their natural in vivo function is as medicinal signaling cells for sites of injury or inflammation^5,6 in all the tissues in which they are housed. Currently, on the US National Institutes of Health clinical studies website (https://clinicaltrials.gov), a search using “mesenchymal stem cells” shows more than 500 listed clinical trials that cover a surprisingly enormous array of clinical conditions. All of these clinical conditions have either 1 or both immunomodulatory or regenerative (trophic) aspects as central components to the therapeutic intent of using MSCs. 

When a blood vessel is injured or inflamed, the pericyte detaches from the basement membrane of that blood vessel or sinusoid to become an MSC. This newly formed MSC responds to the local environment by secreting site-specific bioactive molecules that have 2 very different functions. From the anterior side of the MSC (facing away from the damaged tissue), a curtain of molecules is secreted to stop the body’s overaggressive immune system from surveying the injured tissue behind the MSC.⁷ This is the body’s first line of defense against the establishment of a chronic autoimmune reaction. Thus, MSCs are naturally and intensely immunomodulatory; in the most primordial sense, MSCs have been called the “guardians of inflammation.”⁸

Caplan and Dennin⁹ demonstrated that this curtain of immunomodulatory proteins from human MSCs appears to function in models of disease without immunosuppression of the host. From the posterior side of the MSC (toward the damaged tissue), a different set of bioactive molecules are secreted that function as trophic agents,⁹ which are grouped into 4 categories that contribute to a regenerative microenvironment. The first group consists of molecules that inhibit ischemia-stimulated apoptosis (a broken blood vessel cannot deliver oxygen to the surrounding tissue, thus simulating ischemia-caused cell death). The second group consists of molecules that inhibit the formation of scar tissue and fibrosis. The third group consists of molecules such as the vascular endothelial growth factor that bring in endothelial cells to form new blood vessels. The MSCs can then revert to the pericyte phenotype and attach to these new fragile vessels to stabilize them. Lin et al¹⁰ found MSC engraftment into injured marrow involves the MSCs taking up a perivascular location. The final group includes molecules that function as mitogens for tissue-specific progenitors. In summary, it has been shown that the secretory or paracrine activities of MSCs will themselves change as a function of time and a local milieu.¹⁰ 

Now that clinicians know MSCs are intensely immunomodulatory, it is worthy to look at how they perform with regards to both the innate and the adaptive immune systems and the effect they have upon its constituent cells within each category. 

Mesenchymal stem cells are important immunoregulatory cells in the body, because they respond to inflammation by homing to affected tissues and then controlling inflammation locally at that site. An essential characteristic of MSCs is their expression of a variety of chemokine and cytokine receptors that can home to the sites of inflammation by migrating towards inflammatory chemokines and cytokines.^11-16 These MSCs carry out their immunomodulatory actions in several ways.  Bartholomew et al¹⁷ revealed that MSCs regulate T-cell function, both in vitro and in vivo. Mesenchymal stem cells can regulate an innate immune response by signaling dendritic cells to direct an anti-inflammatory T-cell response and by directly suppressing natural killer (NK) cell functions.¹⁸ Also, MSCs affect the adaptive immune response by the exertion of their immunoregulatory effects through direct interactions with T cells.¹⁸ These effects of MSCs occur in localized tissue environments,^19-21 and therefore are not systemic. This is unlike steroid therapy, where systemic suppression can lead to major clinical complications. In addition, by recruiting endogenous stem cells to sites of injury as well as signal local stem cell differentiation, MSCs can promote tissue regeneration.^22-24 

Because of the unique MSC properties of specific homing to damaged tissues, regulating immune and inflammatory responses at target sites, and facilitating repair of damaged tissues, MSCs have therapeutic potential for the treatment of inflammatory and immune-mediated adverse reactions such as organ transplant rejection, graft versus host disease, allergic reactions, and autoimmune diseases.

Mesenchymal stem cells are naturally immunoprivileged cells. Those found in children have persisted in mothers for the mother’s entire lifespan, suggesting these cells transfer from the fetus to the mother through the placenta and are able to escape immune surveillance for almost 40 years.²⁵ The MSCs immune-privileged status is at least partly due to their low immunogenicity profile. Human MSCs express low levels of major histocompatability complex (MHC) class I antigens, and they are negative for MHC class II and costimulatory molecules CD40, CD80, and CD86.²⁶

While lack of MHC class II is necessary for escaping immune surveillance, the presence of MHC class I may be important. Low levels of MHC class I protects cells from the NK cell-mediated cytotoxicity, whereas cells that do not express MHC class I are targeted and destroyed.²⁷

In a study by Nasef et al²⁸, the authors suggested human leukocyte antigen G (HLA-G), a nonclassical MHC class I antigen, is expressed by human MSCs and may be responsible for inhibiting an immune response against MSCs.

T cells (T lymphocytes) are major players in the adaptive immune system. Once activated by T-cell receptor engagement, these cells proliferate and release inflammatory cytokines and chemokines, thereby destroying allogeneic or pathogenic stimuli. Human MSCs can regulate the immune response by modulating cytotoxic or helper T-cell (Th1 or Th2) activity through modulating the release of various cytokines from effector T cells and promoting an anti-inflammatory environment. As an example, the addition of MSCs to differentiated effector T cells led to a decrease in the release of the pro-inflammatory cytokine interferon gamma (IFN-γ) from Th1 cells with a concomitant increase in the release of interleukin-4 (IL-4) from Th2 cells, which has anti-inflammatory activities in Th1-mediated diseases.¹⁸ However, in a murine model of ovalbumin-induced asthma,²⁹ a Th2-mediated inflammatory disease, an MSC-infusion attenuated airway hyperresponsiveness, reduced the number of eosinophils in bronchoalveolar lavage fluids and significantly decreased the release of Th2 cytokines. These findings suggest MSCs regulate T-cell immune responses dynamically.  

Another effector T-cell target of MSC immunomodulation is cytotoxic T cells (CTLs). Cells infected with a virus as well as allogeneic cells are targets for cytolytic attacks by activated CD8-positive T cells. Mesenchymal stem cells have been shown to inhibit the activation of naïve CTLs, resulting in decreased lysis of allogeneic cells.³⁰ However, activated CTLs are not inhibited in the presence of MSCs and are able to lyse allogeneic cells. Interestingly, allogeneic MSCs are not targets of CTL attacks.³⁰ 

It’s possible that MSCs may be protected from CTL attack due to their ability to inhibit IFN-γ and tumor necrosis factor alpha (TNF-α) production from CTLs.³¹

Regulatory T cells (Tregs) are important players in modulating immune response, because they are anti-inflammatory in nature and function to prevent hyperimmune responses. Accordingly, MSCs can exert immunoregulatory functions by inducing the recruitment and generation of Tregs. Upon IFN-γ stimulation, MSCs secrete the chemokine ligand 1 (CCL 1 [I-309]). It is the interaction of CCL1 with its receptor, CCR8, on T cells that has been shown to partially mediate MSC-induced Treg generation, thus producing CD4 + CD25 + high FoxP3 + Tregs.^18,32 Another mediator of Treg generation produced by MSCs is HLA-G5, a soluble form of nonclassical HLA class I molecules.³³ Secreted by MSCs upon intercellular contact between MSCs, HLA-G5 activates allostimulated T cells and is required for the expansion of Treg cells in lymphocyte-MSC cocultures.^28,33 Therefore, Tregs play a key role in inducing immune tolerance, and induction of new immune intolerance is integral for the successful treatment of inflammatory immune diseases and allogeneic cell, tissue, and organ transplantations.

The effect of MSCs on T-cell proliferation has been extensively documented both in vitro and in vivo.^34,35 A number of studies have demonstrated the immunosuppressive activity of MSCs is not mediated through induction of cell apoptosis,^26,36 but rather by arresting T cells in the Go/G1 phase of the cell cycle.³⁷ Markers of cell cycle Ki-67 and cyclin D2 in T cells are inhibited, whereas p27Kip1 expression is upregulated in the presence of MSCs.^29,38 The MSCs support survival of unstimulated T cells^26,39; however MSC-induced apoptosis of activated T cells has been reported.⁴⁰ Thus, depending on the cell status, MSCs may protect quiescent T cells from death, arrest T cells in Go/G1 phase of the cell cycle, or promote apoptosis of activated T cells. Benvenuto et al³⁹ showed that protection of T cells from apoptosis by MSCs involves down-regulation of Fas and Fas ligand expression and inhibition of endogenous apoptotic proteases.³⁹ Secretion of indoleamine 2,3-dioxygenase (IDO) by IFN-γ is linked to MSC-induced T-cell arrest and apoptosis.^40,41 

Although evidence is widely available describing the inhibitory role of MSCs and T-cell function, MSCs also stimulate T cells under varying conditions.⁴² In studies with low MSC-to-immune cell ratios, MSCs increase proliferation of allostimulated T cells by 40% to 190% as compared to control culture without MSCs.⁴² Although the underlying mechanism remains unknown, MSCs constitutively secrete low levels of cytokines (IL-1, IL-6) and chemokines (RANTES, MCP),⁴³ which may support the function of T cells and other immune cells in certain conditions.^44,45 Even though MSCs do not secrete IFN-γ, data suggest⁴⁶ viruses may trigger IFN-γ secretion in MSCs, which may shift the balance between stimulatory and inhibitory MSC-derived factors towards immune stimulation. With secretion of IFN-γ in response to viral antigens, MSCs can support expansion and cytotoxicity of CTLs.⁴⁶ Also, MSCs can support allostimulated T-cell proliferation by the secretion of IL-6.⁴⁷  

B cells (B lymphocytes) are another major type of adaptive immunity and integral to the human immune response. These cells are responsible for producing antibodies against antigens. In vitro studies have demonstrated that MSCs will inhibit the proliferation of B cells through arrest at the Go/G1 phase of the cell cycle. In addition, MSCs inhibit production of immunoglobulin M, immunoglobulin A, and immunoglobulin B cells.^47,48 However, other studies^49,50 found MSCs can stimulate immunoglobulin-G secretion and induce proliferation of B cells. The effects of MSCs on B-cell functions are mediated by both soluble factors and direct MSC-to-B cell contacts. Both the IL-6 and the intercellular adhesion molecule 1 receptor are proposed as potential mediators of the human MSC stimulatory effects on B cells.⁴⁹ Although the same MSC soluble factors that inhibit T-cell responses may play a role in the suppression of B cells, the nature of these factors as well as signaling molecules from mature B cells, which trigger the secretion of suppressive factors by MSCs, still remain to be investigated and elaborated.  

Dendritic cells (DCs) are derived from monocytes and are potent imaging-presenting cells that act by internalizing, shuttling, and presenting antigens to naïve T cells, which leads to T-cell activation. Mesenchymal stem cells inhibit differentiation of monocytes to DCs and downregulate the expression of several presentation molecules (HLA-DR, CD1a), costimulatory molecules (CD80 and CD86), and cytokine (IL-12).⁵¹ Human MSCs affect phenotype, cytokine secretion, and immunostimulatory activity of both mature and immature hematopoietic progenitor cell antigen and monocyte-derived DCs.⁵¹ The suppression of DC maturation is mediated by MSC-derived soluble factors such as IL-6 and macrophage-colony stimulating factor.^51,52 Furthermore, Aggarwal and Pittenger¹⁸ demonstrated that MSCs inhibit TNF-α expression as well as increase IL-10 expression from stimulated DCs through their expression of IL-1RA.

Natural killer cells are key players in the innate immune system and are important in targeting virus-infected cells and tumor cells. They act by releasing proinflammatory cytokines and directly destroying target cells. Natural killer cells are activated by cytokines released by target cells or by cell-surface receptors that bind to ligands expressed by target cells. Suppression of NK cell function by MSCs include inhibition of proliferation, cytokine secretion, and in some cases cytotoxicity. Inhibition of alloactivated NK cell proliferation is mediated by MSC-derived IDO activity, which is induced by IFN-γ secretion by activated NK cells.^41,53 In addition to IDO, HLA-G5, prostaglandin E2 (PGE2), and transforming growth factor beta (TGF-β) were identified as MSC-derived soluble factors that suppress NK cell proliferation and cytokine secretion.^33,38,53 It also should be noted that NK cells can also lyse MSCs, both allogenic and autologous. Spaggiari et al⁵⁴ discovered IFN-γ partially protects MSCs from NK cell cytolytic attack. The effect of MSCs on NK cells and NK cell phenotype and cytotoxicity requires direct intercellular contacts.³⁹  

Studies^55,56 suggest MSCs affect functions of neutrophils and macrophages. Interleukin 6 was identified as a key MSC-derived factor that protects neutrophils from apoptosis, while IL-IRA released by MSCs inhibits TNF-α production by activated macrophages.³⁵ 

In a noninflammatory environment, MSCs express low levels of cyclooxygenase 2, PGE2, TGF- β, IDO, and other factors; however, pro-inflammatory cytokines dramatically upregulate secretion of anti-inflammatory factors by MSCs. For example, IFN-γ upregulates the secretion of IDO, hepatocyte growth factor, and TGF-β; and TNF-α upregulates secretion of PGE2 by MSCs.^18,41,57,58 Cumulative data support a hypothesis of dynamic MSC response to inflammatory stimuli released from activated immune cells. Activated T cells release pro-inflammatory cytokine TNF-α that interact with the TNF receptors on MSCs and trigger the release of PGE2 from MSCs. Prostaglandin E2 acts back upon activated T cells and blocks the release of TNF-α.  

With any immunosuppressive agent, the question of whether the therapy increases the infection rate should be addressed. Cumulative data to date indicate that in contrast to the strong immunosuppressive effect of MSCs on alloreactive and mitogen-induced responses, MSCs do not appear to inhibit immune cell functionality against infectious agents.⁴⁶ In addition to the MSC-derived IFN-γ for antiviral CTL support,⁴⁶ a mechanism leading to the temporary inactivation of MSC immunosuppression has been described.⁵⁹ Activation of toll-like receptor (TLR) 3 and TLR4 by viral- and bacterial-derived antigens dramatically downregulate the expression of Jagged1 on MSCs, which has been shown to mediate MSC immunosuppressive activity via interaction with notch receptors on T cells. Decreased Jagged1 expression results in reversible inhibition of MSC immunosuppressive potential.⁵⁹ At the present time, it is clear TLRs play an important role not only during viral and bacterial infections, but also in orchestrating the activation of the innate immune response to protozoan parasites.⁶⁰ This suggests that similar to viruses and bacteria, interaction of parasites with TLRs expressed in MSCs will result in temporary inactivation of MSC immunosuppressive activity. Mesenchymal stem cells express a variety of TLRs including TLR2, TLR3, and TLR4,⁵⁹ which is activated by viral, bacterial, and parasitic antigens. Reversible inactivation of MSC immunosuppressive activity by these 3 antigens via TLRs may represent a mechanism that allows immune cells to fight infections in the body.  

When all considered in the literature, the therapeutic features of MSCs are many. The feature of biodistribution allows MSCs to targeted homing to inflammation instead of having the deleterious effects of systemic immunosuppressive drugs. In addition, immune and inflammatory responses occur in a very focal, localized area, not systemic like clinicians see with the harmful effects of steroids and immunosuppressive drugs. Mesenchymal stem cells can repair tissue damage as well as prevent it where systemic immunosuppressive drugs cannot. Also, the regulation of the extent of the immunosuppressive activity is dictated by the microenvironment, whereas systemic drugs do not have such regulation. Therefore, several conclusions can be reached about the powerful immunoregulatory functions that MSCs do afford human beings in the treatment of inflammatory and immunological diseases. The therapeutic potentials attributed to unique MSC properties such as specific homing to damaged tissue, inhibiting immune and inflammatory responses at these targeted sites, and facilitating repair of these damaged tissues are salient features of this powerful cell.   

Data support the hypothesis that MSCs can perform immunomodulatory functions to suppress an adverse immunological response. Even though MSCs possess immunosuppressive capabilities, there is no evidence of immunosuppressive toxicity globally or systemically throughout the body, suggesting MSCs restrict their immunomodulatory functions to areas where inflammation is present. It is likely that infused MSCs home along cytokine gradients in inflamed areas where they suppress inflammation in local microenvironments. Therefore, they would be ideal to use in the chronic wound environment as well as other diseases that show overregulated immune response because of these potent immunomodulatory functions that are exerted on the cellular level.

The important message herein is that physicians, clinicians, and scientists who manage the treatment of chronic wounds can apply this modality to correct the cellular dysfunction and soluble mediator discrepancies occurring within this highly inflamed chronic wound phenotype. Since the chronic wound bed has prolonged inflammation, MSCs can be ideal candidates to extinguish  chronic inflammation and correct the cellular derangement that is occurring. Currently known, there is an overproduction of a host of proteolytic enzymes produced by immune system cells. Mesenchymal stem cells will have the capability to extinguish this chronic inflammation and then allow for differentiation, recruitment, and engraftment into the wound bed to correct the abnormal cellular senescence that is occurring; thus it will allow proper dermal reconstruction and epithelialization to restore functional and structural integrity to this defect.  

From the Wound Institute of Ocean County, Toms River, NJ (Medical Director); and Ocean County Foot and Ankle Surgical Associates, Toms River, NJ (Partner) 

Address correspondence to:
Matthew J. Regulski, DPM, ABMSP, CMET, FAPWH(c), FASPM
1104 Seashell Avenue 
Manahawkin, NJ 08050 
mregulski@comcast.net

Disclosure: The author discloses no financial or other conflicts of interest.