Stromal cells from perinatal and adult sources modulate the inflammatory immune response in vitro by decreasing Th1 cell proliferation and cytokine secretion

Abstract Many immune‐mediated conditions are associated with a dysregulated imbalance toward a Th1 response leading to disease onset, severity, and damage. Many of the therapies such as immunomodulators or anti‐TNF‐α antibodies often fall short in preventing disease progression and ameliorating disease conditions. Thus, new therapies that can target inflammatory environments would have a major impact in preventing the progression of inflammatory diseases. We investigated the role of human stromal cells derived from the amniotic fluid (AFSCs), the placenta (PLSCs), and bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) in modulating the inflammatory response of in vitro‐stimulated circulating blood‐derived immune cells. Immune cells were isolated from the blood of healthy individuals and stimulated in vitro with antigens to activate inflammatory responses to stimuli. AFSC, BM‐MSCs, and PLSCs were cocultured with stimulated leukocytes, neutrophils, or lymphocytes. Inflammatory cytokine production, neutrophil migration, enzymatic degranulation, T cell proliferation, and subsets were evaluated. Coculture of all three stromal cell types decreased the gene expression of inflammatory cytokines and enzymes such as IL‐1β, IFN‐γ, TNF‐α, neutrophil elastase, and the transcription factor NF‐κB in lipopolysaccharide‐stimulated leukocytes. With isolated phytohemagglutinin‐stimulated peripheral blood mononuclear cells, cells coculture leads to a decrease in lymphocyte proliferation. This effect correlated with decreased numbers of Th1 lymphocytes and decreased secreted levels of IFN‐γ.

implicated in allergic reactions. Th2 also produce IL-10, thus, potentially having some anti-inflammatory properties. A balance between Th1 and Th2 responses and their associated cytokines are thus important to maintain a balanced immune response. [2][3][4][5] Dysfunction in the regulation of Th1/Th2-mediated inflammation can lead to disease and tissue damage. 6 For example, in chronic obstructive pulmonary disease, lung tissue-derived elastin is responsible for the activation of CD4 + Th1 cell-mediated inflammation, causing tissue injury and emphysema. 7 Crohn's disease (CD) is also closely related to a Th1 skewed immune response, in which enhanced IFN-γ expression and release have been shown. 8,9 Th1-mediated inflammation is also implicated for many types of autoimmune diseases, including rheumatoid arthritis (RA), multiple sclerosis (MS), corneal transplant rejection, and type I diabetes. [10][11][12][13] Current treatments for these types of diseases include the use of Th1 pre-inflammatory cytokine antagonists, such as antibodies to TNF-α or IFN-γ. 14 However, these treatments, even when used in combination, appear to have limited effects on the more chronic and severe forms of disease and there is a pressing need for the development of new treatment strategies. In this study, we investigated the potential role of stromal cells derived from different tissue sources to modulate the inflammatory response of immune cells in vitro, thus identifying a potential anti-inflammatory therapy for a range of diseases.
Stromal cells present in cell populations derived from perinatal tissues such as the placenta and amniotic fluid, offer an advantage over other sources like bone marrow (BM) because of their ease of collection, ready availability, high abundance, and high proliferation rates. 15,16 Human amnion-derived mesenchymal stromal cells (hAMSC) have been shown to skew macrophage polarization toward M2, inhibit monocyte differentiation into dendritic cells, and reduce the expression of Th1, Th2, and Th17 associated markers and their corresponding subset cytokines including TNF-α, IFN-γ, IL-1β, IL-5, IL-9, and IL-22. 17 In another study, human umbilical cord-derived MSCs (UC-MSCs) inhibited the proliferation of lymphocytes stimulated with phytohemagglutinin (PHA) under coculture setting in vitro, and enhanced the abundance of T regulatory (Tregs) cells, while also increasing IL-10 levels. 18 Thus, perinatal cells exhibit a wide range of therapeutic properties that make them the ideal candidates to treat inflammatory diseases. In this study, we com-   Biotec, Auburn, California) as previously described. 19 This study highlights the immunosuppressive properties of   perinatal cells on Th1 cells and their associated cytokines   thus providing further understanding of the role of perinatal   cells as a potential therapy to target Th1 mediated diseases. AmnioMax media respectively, and were supplied with 18% fetal bovine serum and 1% penicillin/streptomycin. All cells were grown at 37 C and 5% CO 2 in a humidified atmosphere. Cells between passages 10-15 were used. Prior to use, cryopreserved cells were thawed and cultured for 4 days in their respective media. TrypLE was used for the initial isolation and expansion of the cell stocks, whereas 0.05% trypsin was used for routine cell culture. So, cells were detached using 0.05% or 0.25% (for BM-MSCs) trypsin and viability was assessed using trypan blue.

| Leukocyte isolation from peripheral blood
Total white cells (leukocytes) were isolated by HetaSep (StemCell Technologies, Vancouver, Canada) density gradient centrifugation.
Briefly, 1-part HetaSep was added to five parts blood and the sample was centrifuged at 90g for 3 minutes at room temperature. The supernatant rich in total white cells was then collected and washed three times with Roswell Park Memorial Institute (RPMI) 1640 media +1% bovine serum albumin (BSA). Red blood cell (RBC) lysis was performed by incubating the leukocyte pellet in 10 mL of RBC Lysing buffer Hybri-Max (Sigma-Aldrich) at room temperature for 10 minutes with frequent vortexing. Cell count and viability was determined using the trypan blue exclusion method.

| Isolation of peripheral blood mononuclear cells (PBMCs)
PBMCs were isolated from blood by density centrifugation using Histopaque (Sigma). Briefly, the total volume of blood was laid over an equivalent volume of Histopaque and the sample was centrifuged at 450g for 30 minutes. PBMCs were then collected from the buffy coat phase and transferred into a fresh tube and washed with PBS and centrifuged at 770g for 10 minutes. RBC lysis was performed by incubating the PBMC pellet in 10 mL of RBC Lysing buffer Hybri-Max (Sigma-Aldrich) at room temperature for 10 minutes with frequent vortexing. Cell count and viability was determined using the trypan blue exclusion method.

| Neutrophil isolation
Neutrophils were isolated by means of negative selection magnetic isolation using the Human Neutrophil Enrichment Kit (StemCell Technologies) according to the manufacturer's guidelines. Briefly, total white cells were incubated with EasySep Human Neutrophil Enrichment cocktail for 10 minutes at 4 C. EasySep nanoparticles were then added to the mixture and were incubated for 10 minutes at 4 C. The suspension was then mixed and brought to a total volume of 2.5 mL.
The tube was then inserted into the EasySep magnet, and with one continuous motion, the tube was inverted, and the contents were poured into a fresh 5 mL polystyrene tube. Cell count and viability were determined using the trypan blue exclusion method.

| Lymphocyte proliferation assay
PBMCs were stimulated with 10 μg/mL PHA and seeded at 20 × 10 4 cells in a 96-well plate in the presence or absence of 20 × 10 3 mitomycin-C (25 μg/mL) treated AFSCs, BM-MSCs, or PLSCs. Stromal cells were plated into a 96-well plate and allowed to adhere overnight.
The coculture system was incubated for 6 days before cell proliferation was assessed using the Cell proliferation ELISA bromodeoxyuridine (BrDU) kit (Roche, Basel, Switzerland). An equivalent of 0.1 mM BrDU was added on day 5 for an overnight incubation.

| Flow cytometry T cell subset analysis
PBMCs were collected and stained with cell surface markers CD3   2.9 | Th1/Th2/Th17 cytokine bead assay Supernatants collected from cells cultured under the system described above were used to measure the levels of seven cytokines by flow cytometry, IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A using the Human Th1/Th2/Th17 Kit (BD Biosciences), according to the manufacturer's protocol. Analysis was acquired using a BD Accuri C6 flow cytometer (BD Biosciences).

| In vitro stimulation of total white cells
Total white cells (2 × 10 6 cells) were stimulated with 10 ng/mL lipopolysaccharide (LPS) for 24 hours in a 12-well plate, supplied with RPMI +1% BSA media. LPS stimulated cells were exposed to 20 × 10 4 stromal cells in a transwell culture system using cell culture inserts (Millicell cell culture inserts 0.4 μm, 12 mm diameter). Cells were used to perform the assays detailed below.

| Cytokine ELISA
Cell supernatants from the in vitro stimulated and unstimulated total white cells were collected by centrifuging the total white cells at 433g for 5 minutes. NE, IL-6, and TNF-α protein levels were then assessed using the Human PMN Elastase Platinum ELISA (Invitrogen, Carlsbad, California), IL-6 and TNF-α PicoKine ELISA Kits (Boster).

| Nuclear extraction and NF-kB ELISA
Cells from the above cell culture system were collected and centrifuged at 433g for 5 minutes. The cell pellet was used for nuclear and cytoplasmic extraction using the NE-PER nuclear and cytoplasmic extraction reagents kit (Abcam) following the manufacturer's protocol. Briefly, cells were incubated with cytoplasmic extraction reagent (CER) and vortexed vigorously for 5 seconds. Tubes were then centrifuged at maximum speed (16 000g) for 5 minutes to obtain the supernatant containing the cytoplasmic extract. The remaining pellets were incubated with the nuclear extraction reagent (NER) included in the kit and vortexed vigorously for 15 seconds. After centrifugation at maximum speed (16 000g) for 10 minutes, nuclear extracts in the supernatant were collected and stored at −80 C until use. Protein quantification was performed on nuclear extracts using the Pierce BCA Protein Assay Kit (Thermo Fisher).
Nuclear extracts were then used to perform the NF-kB p50 transcription factor assay kit (Abcam) following the kit's protocol.

| Statistical analysis
In vitro assays were performed with triplicate technical replicates, and data from the groups were pooled and displayed as mean ± SEM. Data were analyzed using GraphPad Prism version 6.0. One-way ANOVA tests were used to determine statistical significance between the groups of immune cells at baseline (unstimulated), exposed to stimulus and/or perinatal cells. Tuckey's test was performed to correct for multiple comparisons. A confidence limit of 95% was considered significant.

| Assessment of stromal cells' effect on gene expression profile of inflammatory cytokines and enzymes
A total of 81 samples (obtained from 21 females and 60 males, average age: 33, age range: 23-57) were used in this study. We first compared the gene expression profile of healthy leukocytes following LPS stimulation with that of the LPS-stimulated leukocytes cultured with the three stromal cell types located in the upper chamber of a transwell system for indirect coculture conditions. RNA isolation was performed 24 hours after initiation of LPS stimulation for all groups.
We found that BM-MSCs (P < .05) and PLSCs (P < .01) coculture decreased IFN-γ gene expression in LPS-stimulated leukocytes F I G U R E 2 Secreted levels of cytokines and enzymes from unstimulated (US) leukocytes and following stimulation with lipopolysaccharide (LPS) alone, and in the presence of amniotic fluid-derived cells (AFSCs), bone marrow-derived mesenchymal stromal cells (BM-MSCs), and placenta-derived cells (PLSCs). A, Interleukin-6 (IL-6); B, tumor necrosis factor-alpha (TNF-α); C, interleukin-1 beta (IL-1 β); and D, neutrophils elastase (NE). Bars represent SEM *P < .05; **P < .01; ***P < .001; #P < .05 compared to AFSCs, ##P < .01 compared to AFSCs ( Figure 1A). A significant decrease in the NE ( Figure 1B) gene expression was also observed with the transwell coculture of AFSCs (P < .05), BM-MSCs (P < .01) as well as PLSCs (P < .001). We also observed a significant decrease in NF-κB gene expression only in the presence of PLSCs (P < .05) ( Figure 1C). AFSCs and BM-MSCs coculture also showed a trend of decreased NF-κB gene expression; however, this was not statistically significant. We observed decreased levels of IL-6 and TNF-α with the transwell coculture of all stromal cell types, however these changes were not statistically significant. We did not observe any changes in IL-8, IL-10, or CCL2 expression levels with any of our stromal cell types ( Figure 1G Although stromal cell coculture appeared to decrease secreted TNF-α levels, the observed increase in IL-6 may be due to increased secretion from monocytes, and/or secretion from the stromal cells themselves, which have been shown to secrete IL-6 under various conditions. 23

| Effect of stromal cells on neutrophil migration and enzymatic activities
Our qPCR data indicated a significant decrease of NE gene expression following transwell coculture with all three stromal cell types.
To explore potential mechanisms of interaction of stromal cells with neutrophils, we isolated neutrophils by means of negative-selection magnetic isolation and subjected them to an IL-8 concentration gradient. Neutrophils exhibited a significant increase in migration following exposure to the IL-8 gradient however; none of the three stromal cell populations had any significant effect on this phenomenon ( Figure 3A).
Similarly, stromal cells did not seem to affect the degranulation of MPO or NE ( Figure 3B,C). These data suggest that there are no significant direct effects of stromal cells coculture on clinically relevant functions of neutrophils under these in vitro conditions.

| Lymphocyte proliferation and subset analysis
Given that our data showed that coculture with stromal cells had modulatory effects on the gene expression related to T lymphocytes such as NF-κB, IL-1β, and TNF-α, we investigated the effect of all three stromal cell types on lymphocyte proliferation and T cell subsets We also measured the levels of other Th-associated cytokines using the Th1/Th2/Th17 detection beads. Our findings further con- In our study, we found that PLSCs showed the greatest level of suppression of NF-κB and NE gene expression. As shown in Figure 1 Figure S1). We also observed significantly lower levels of secreted TNF-α when leukocytes were cocultured with PLSCs.
During the early stages of the immune response, IL-1β, TNF-α, and IL-6 are secreted by macrophages and monocytes into the inflammatory milieu, and trigger downstream inflammatory activity including increased vascular permeability and recruitment of neutrophils and lymphocytes. [27][28][29] Macrophages also promote Th1 mediated immunity by secreting the cytokine IL-12. IL-12, along with TNF-α and other inflammatory cytokines, then, stimulate the production of IFN-γ. 30 32 suggesting potential differences between these interactions between human and mouse cells. In another study, human MSCs derived from Wharton's jelly, trabecular bone and BM were capable of suppressing human neutrophil recruitment to TNF-α-stimulated endothelial cell monolayers. 33 As distinct stimulation approaches can be used for neutrophil activation and recruitment, these differences may be useful in evaluating differential responses to stromal cells. In addition, cells derived from several tissue sources such as adipose, perinatal tissues exhibit distinct therapeutic properties and immunomodulatory effects that could impact the interactions with the immune system. Studies have shown that MSCs derived from the umbilical cord exhibit a significantly different paracrine profile when compared to adipose derived MSCs suggesting that the tissue source of MSCs strongly impacts the biological diversity potential of the derived cells and thus influence their immunomodulatory properties. 34,35 Similarly, the presence of multiple interacting cell types appears to be essential for appropriate modeling of neutrophil functions. Our neutrophil isolation protocol eliminated other immune cell populations such as macrophages and monocytes, suggesting these cell interactions may be important for neutrophil activation and interactions with stromal cells. 36 40 During an infection, antigen presenting cells present antigen derived proteins to naïve T cells (Th0) which recognize these signals using their T cell receptors and become activated. CD4 + naïve T cells then differentiate into multiple T helper subsets including Th1.
Th0 give rise to Th1 cells in the presence of IL-12 and IFN-γ, which trigger the expression of high levels of STAT4 and T-bet transcription factors in naïve T cells, favoring Th1 differentiation. 41 Th1 have been implicated in many illnesses as a potential mechanism of disease dysfunction and progression. Studies have shown that increased levels of IFN-γ, associated with a predominant Th1 imbalanced inflammatory response, play a major role in the progression of autoimmune disease in lupus prone MRL mice. 42 Other studies investigating the role of Th1 in RA in rat models revealed that polyclonal expansion of Th1 cells was necessary for the onset of disease. 43 Patients with CD exhibited high infiltration of Th1 cells in the gastric and intestinal mucosa, accompanied by increased levels of IFN-γ and TNF-α which have been implicated in the progression of the disease. 44 In autoimmune demyelinating diseases, such as MS, patients showed elevated serum levels of TNF and IFN-γ derived from Th1 cells. 45,46 More recently, Zhou et al reported that patients with psoriasis exhibited increased levels of IFN-γ as well as IL-17 in the serum, which were mainly mediated by increased NF-κB expression. 47 Current treatments for Th1 mediated diseases include anti-inflammatory and immunomodulatory drugs to reduce the Th1 mediated immune response, such as anti-TNFα antibodies. [48][49][50][51] However, these therapies have been associated with major side effects such as infusion reactions especially in nonspecific cells of the body, and is some cases, the development of eczema and tuberculosis due to neutralization of TNF-α. 52,53 Thus, the potential to modulate Th1 responses in patients with autoimmune diseases may be beneficial for controlling or preventing disease onset. PLSCs' suppression of one axis of the pro-inflammatory response might come at the risk of allowing upregulation of other inflammatory pathways that could be just as deleterious to disease severity.

| CONCLUSION
Our study showed that PLSCs have a robust effect on the suppression and function of Th1 subsets. These findings confirm the anti-inflammatory properties of PLSCs on Th1, provide further understanding approximately the interaction of PLSCs with the different components of the immune system, and offer a potential anti-inflammatory effect on Th1 cell subset, suggesting a potential use of these cells to target Th1-related diseases.

CONFLICT OF INTEREST
The authors indicated no potential conflicts of interest.

AUTHOR CONTRIBUTIONS
O.K.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of the manuscript; A.A., S.V.M.: financial support, conception and design, data analysis and interpretation, manuscript writing, final approval of the manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.