Human Ifn Gamma Peprotech

[Nayra Waddles]

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Mesenchymal stem cells (MSCs) exert their anti-inflammatory and anti-fibrotic effects by secreting various humoral factors. Interferon-gamma (IFN-γ) can enhance these effects of MSCs, and enhancement of regulatory T (Treg) cell induction is thought to be an underlying mechanism. However, the extent to which Treg cell induction by MSCs pretreated with IFN-γ (IFN-γ MSCs) ameliorates renal fibrosis remains unknown. In this study, we investigated the effects of Treg cell induction by IFN-γ MSCs on renal inflammation and fibrosis using an siRNA knockdown system. Administration of IFN-γ MSCs induced Treg cells and inhibited infiltration of inflammatory cells in ischemia reperfusion injury (IRI) rats more drastically than control MSCs without IFN-γ pretreatment. In addition, administration of IFN-γ MSCs more significantly attenuated renal fibrosis compared with control MSCs. Indoleamine 2,3-dioxygenase (IDO) expression levels in conditioned medium from MSCs were enhanced by IFN-γ pretreatment. Moreover, IDO1 knockdown in IFN-γ MSCs reduced their anti-inflammatory and anti-fibrotic effects in IRI rats by reducing Treg cell induction. Our findings suggest that the increase of Treg cells induced by enhanced secretion of IDO by IFN-γ MSCs played a pivotal role in their anti-fibrotic effects. Administration of IFN-γ MSCs may potentially be a useful therapy to prevent renal fibrosis progression.

The prevalence of acute kidney injury (AKI) has been increasing in recent decades and is recognized as a worldwide health problem1,2. AKI is associated with subsequent development of chronic kidney disease (CKD)3. Therefore, mechanisms underlying AKI-to-CKD progression have been extensively investigated over the past decade. The main pathological feature of AKI-to-CKD progression is interstitial fibrosis, which can result from one or more pathological mechanisms, such as hypoxia4, endothelial dysfunction, microvascular rarefaction5, inflammation6, transforming growth factor (TGF)-β1 production7, and epithelial-mesenchymal transition (EMT)8. Notably, among these mechanisms, inflammation plays a major role in fibrosis progression9.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells20 isolated from various tissues, such as bone marrow, adipose tissue, and umbilical cord21. We elucidated that the anti-fibrotic effects of xenogeneic and allogeneic MSCs are almost equal22. Moreover, many studies have revealed that xeno therapy using human MSCs is effective for mouse models of renal injury23,24. MSCs contribute to tissue regeneration by secreting various soluble factors25,26. MSCs are known to favor the generation of Treg cells, producing anti-inflammatory effects27.

We previously demonstrated that MSCs pretreated with IFN-γ (IFN-γ MSCs) potently ameliorate renal fibrosis. Several studies have demonstrated that pretreatment with interferon-gamma (IFN-γ) promotes Treg cell induction by enhancing indoleamine 2,3-dioxygenase (IDO) secretion from MSCs28,29. These findings led us to hypothesize that IFN-γ MSCs attenuate renal fibrosis by enhancing regulatory T cell induction.

In this study, we investigated the effects of Treg cell induction by IFN-γ MSCs on renal inflammation and fibrosis in a unilateral renal ischemia reperfusion injury (IRI) rat model with contralateral nephrectomy using a siRNA knockdown system.

Upregulation of vascular endothelial growth factor (VEGF) in IRI rats is critical to prevent AKI progression. Therefore, we investigated the effects of IFN-γ MSCs on VEGFA expression in IRI rats at 7 days post-injection. The protein level of VEGFA was upregulated in the control MSCs group and further upregulation was observed in the IFN-γ MSCs group (Fig. 1c, Additional File 1A: Fig. S1a).

Previous studies demonstrated that IDO expression plays a direct role in inducing the conversion of nave CD4 T cells into Treg cells30,31. Therefore, we examined IDO expression levels in MSCs and CM collected from MSCs. We found that mRNA and protein levels of IDO were more significantly upregulated in IFN-γ MSCs compared with those in control MSCs (Fig. 3a,b, Additional File 1B: Fig. S1d). Additionally, ELISA results show that the concentration of IDO in IFN-γ MSCs-CM was higher than in control MSCs-CM (Fig. 3c).

To investigate whether IFN-γ MSCs mediated the induction of FOXP3-positive Treg cells, we isolated nave CD4 T cells from PBMCs and cultured them with control MSCs-CM or IFN-γ MSCs-CM. IFN-γ MSCs-CM more significantly increased FOXP3 protein levels compared with control MSCs-CM (Fig. 3d, Additional File 1B: Fig. S1e).

Finally, we investigated the extent to which Treg cell induction by IFN-γ MSCs ameliorated renal fibrosis. Western blotting revealed that protein levels of α-SMA and TGF-β1 were increased in the PBS group and significantly ameliorated by injection of NC siRNA/IFN-γ MSCs, while these reductions were completely abrogated by injection of IDO1 siRNA/IFN-γ MSCs (Fig. 5a,b, Additional File 1C: Fig. S1f,g). Similarly, immunostaining showed that α-SMA- and collagen type I-positive areas were expanded in the PBS group and markedly ameliorated by injection of NC siRNA/IFN-γ MSCs, whereas these improvements were abolished by injection of IDO1 siRNA/IFN-γ MSCs (Fig. 5c,d).

The present study clarified that administration of IFN-γ MSCs induced Treg cells and ameliorated inflammation and tubulointerstitial fibrosis in IRI rats more drastically than unstimulated MSCs. Mechanistically, IFN-γ MSCs enhanced IDO secretion, while IFN-γ MSCs-CM strongly induced conversion of nave CD4 T cells into Treg cells. In addition, IDO1 knockdown in IFN-γ MSCs diminished numbers of FOXP3-positive cells and reduced the therapeutic effect of IFN-γ MSCs on renal inflammation and fibrosis in IRI rats.

Advances in MSC biology have confirmed that sufficient inflammatory signals are required to activate the immunosuppressive properties of MSCs32,33. IFN-γ, a cytokine expressed in activated lymphocytes, reportedly modifies the immunomodulatory function of MSCs34,35. As a mechanism by which MSCs exert an anti-inflammatory effect, we previously showed that IFN-γ MSCs increase prostaglandin E2 (PGE2) secretion to induce immunosuppressive M2 macrophage polarization, leading to enhanced anti-inflammatory and anti-fibrotic effects36. The present study demonstrates that IFN-γ MSCs enhanced IDO secretion, thereby inhibiting infiltration of inflammatory cells by inducing Treg cells, leading to pronounced anti-fibrotic effects. Taken together, our findings indicate that IFN-γ MSCs enhanced the suppression of inflammatory cell infiltration through two pathways: induction of M2 macrophages by increasing PGE2 secretion and induction of Treg cells by upregulating IDO secretion.

MSCs exert immunosuppressive and therapeutic effects by a Treg cell-induced mechanism in various diseases models, such as transplantation37,38, autoimmune disorders39,40, and systemic inflammatory diseases41,42. MSCs remain dormant under normal conditions and exert anti-inflammatory effects when activated by cytokines released from immune cells in damaged tissues. This activation may require several days and some MSCs may be unable to change to the active form36, so normal MSCs may not exert sufficient therapeutic effects. In fact, our current study also revealed that control MSCs mediate only a few Treg cell induction in in vitro experiments and exert only slight anti-fibrotic efficacy in IRI rats. Therefore, we believe that pretreatment with IFN-γ is important when performing MSC therapy. Although IFN-γ MSCs reportedly increase Treg cell induction in in vitro28,43, the extent to which induction of Treg cells by IFN-γ MSCs ameliorates renal injury remains unknown. In this study, we observed that downregulation of MSC-induced Treg cells worsened renal inflammation and fibrosis in IRI rats injected with IFN-γ MSCs. Therefore, induction of Treg cells by IFN-γ MSCs was a central contributor to the suppression of renal fibrosis.

The biologic function of the IDO pathway is both counter-regulatory (controlling inflammation) and tolerogenic (creating acquired antigen-specific tolerance in T cells)44. IDO helps create a tolerogenic milieu by directly suppressing T cells and enhancing Treg-mediated immunosuppression45. In fact, in our experiments, IDO1 knockdown in MSCs pretreated with IFN-γ reduced Treg cell induction and attenuated their anti-inflammatory effects. An, et al. reported that PGE2 secreted from MSCs is involved in Treg cell induction46. We previously demonstrated that pretreatment with IFN-γ promoted PGE2 secretion from MSCs. Therefore, IFN-γ MSCs likely enhanced induction of Treg cells by enhancing secretion of both IDO and PGE2.

MSCs were pretreated with or without recombinant human IFN-γ (PeproTech, Cranbury, NJ, USA) by the following method. When MSCs reached 70% confluence, IFN-γ was added to the medium to achieve a final concentration of 10 ng/mL. After 48 h, cells were collected and subjected to in vivo and in vitro analyses.

Sample collection and western blotting were performed as previously reported36,47 with the following primary antibodies: anti-VEGFA antibody (Abcam), mouse monoclonal anti-α-SMA (Sigma-Aldrich), rabbit monoclonal anti-TGF-β1 (Abcam), IDO1 polyclonal antibody (Proteintech, Rosemont, IL, USA), mouse monoclonal anti-Foxp3 (Abcam), rabbit polyclonal anti-CD4 (Abcam), and mouse monoclonal anti-GAPDH (Sigma-Aldrich). Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Dako) or goat anti-mouse immunoglobulin G (Dako) were used as secondary antibodies. SuperSignal West Dura or Pico Systems (Thermo Fisher Scientific, Waltham, MA, USA) were used to detect signals. The intensity of each band was analyzed by ImageJ software and standardized to the level of GAPDH.

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