In this study, we demonstrate that exposing hAECs to inflammatory conditions increases their anti-inflammatory and immunomodulatory properties by affecting their phenotype and function. Moreover, we show that hAECs are capable to protect islets from inflammatory damage through the modulation of the inflammatory response. To the best of our knowledge, this is the first study that reports the effect of factors secreted by human hAECs on islet cell viability and function under inflammatory conditions in vitro.
We have previously reported that hAECs protect islet cells from ischemic injury in vitro via HIF-1α pathway (Fanny Lebreton et al., 2019). Furthermore, we showed that hAECs facilitate larger β-cell mass engraftment and improve in vivo function via acceleration of revascularization and reestablishment of cell-to-matrix contacts (Lebreton et al., 2020; F. Lebreton et al., 2019). In addition to these cytoprotective effects, hAECs have the potential to protect islets from immune destruction by inhibiting lymphocyte proliferation (Qureshi et al., 2015). However, underlyng mechanisms for these protective actions still need to be elucidated. Integrating the findings of this study with the relevant literature, we propose a mechanistic model for the enhancement of anti-inflammatory and immunomodulatory properties of hAECs and the protection they confer to islets grafts (Fig. 4).
Our results showed that hAECs exposed to pro-inflammatory cytokines exhibit increased secretion of anti-inflammatory factors, in particular IL6, IL8, IL10 and G-CSF. This was associated with overexpression of the transcription factor NF-κB1, suggesting an involvement of the NF-κB1 pathway due to the activation of IFN-γ, TNF-α and IL-1β receptors, as shown in Fig. 4 (left part).
NF-κB is a multi-functional transcription factor, activated under pro-inflammatory stimuli and involved in various important biological processes including survival, inflammation, apoptosis and immune regulation (Pires, Silva, Ferreira, & Abdelhay, 2018). At early stages of pregnancy release of IL-1β and TNF-α from endometrial cells activates NF-κB in fetal cells, which in turn facilitates trophoblast invasion and angiogenesis. At the time of delivery, NF-κB secreted by amnion has a leading role in stimulating uterine contraction during labor by inducing in particular pro-inflammatory gene expression (Lindstrom & Bennett, 2005). NF-κB is also linked to the activation of several anti-apoptotic genes, adhesion molecules and growth factors (Karin & Lin, 2002). The pro-survival role of NF-κB has been attributed to the production of IL6 and IL8 (Yu, Wan, & Huang*, 2009). Interestingly, our results demonstrating absence of apoptosis in hAECs exposed to pro-inflammatory conditions, correlated with elevation of IL6 and IL8. This can be explained by activation of NF-κB mediated anti-apoptotic signaling.
The granulocyte colony-stimulating factor (G-CSF) is a potent regulator of granulocyte production that is produced in response to the inflammatory stimuli by different hematopoietic and non-hematopoietic cells including placental tissue cells(Rahmati et al., 2015). Among its many biological effects, G-CSF has a cytoprotective effect on islets cells (Gomez, Diaz-Solano, Gledhill, Wittig, & Cardier, 2017). During pregnancy, G-CSF regulates embryo implantation and development via activation of the STAT3 signaling pathway(Robertson, 2007). Furthermore, it was shown that G-CSF enhances MMP-2 activity and VEGF secretion in a human trophoblast cell line through activation of PI3K/Akt and Erk signaling pathways (Furmento, Marino, Blank, & Roguin, 2014). Finally, G-CSF has been shown to have modulatory effects on immune cells. In particular, it suppresses pro-inflammatory cytokines in peripheral blood mononuclear cells, induces tolerant dendritic cells (DCs), increases IL4 but reduces IFN-γ in vivo(Martins, Han, & Kim, 2010), and promotes tolerance to the graft in islet transplantation experiments (Zoso et al., 2016).
Interestingly, we have observed increased G-CSF secretion by hAECs in response to pro-inflammatory cytokine exposure accompanied by upregulation of STAT1 and STAT3 genes, indicating involvement of G-CSF in the protective action of hAECs through the activation of STAT signaling.
Along with secretion of cytoprotective factors by primed hAEC, we observed a significant increase of immunomodulatory molecule expression. HLA-G expression increased with time, as well as in high concentrations of IFN-γ. In contrast, marked increase in HLA-E expression was detected in response to low concentrations of IFN-γ (10 U/mL). Expression of these markers is known to be regulated by pro-inflammatory conditions (Gustafson & Ginder, 1996; Lefebvre et al., 1999). Mechanistically, HLA-E expression is upregulated by IFN-γ, mediated by an upstream STAT1 binding site (Gustafson & Ginder, 1996). HLA-G expression is mainly regulated by IFN-γ through the JAK/STAT pathway, involving in particular STAT1 (Castelli, Veiga-Castelli, Yaghi, Moreau, & Donadi, 2014)In our studies we observed upregulation of STAT1 and STAT3, which suggests that overexpression of HLA-E and HLA-G is due to the activation of the IFNγ – JAK 1/2 – STAT1/3.
Expression of HLA-class Ib molecules, such as HLA-G and HLA-E, and anti-inflammatory molecules by hAECs exerts in turn a protective effect on islets against damage induced by inflammation. Indeed, in the immediate post-transplantation period, islets are exposed to a highly inflammatory liver microenvironment (Delaune et al., 2017), where pro-inflammatory cytokines, such as IFN-γ, TNF-α and IL-1β, are largely produced in response to ischemia reperfusion injury. These factors trigger β-cell apoptosis through NF-κB activation and endoplasmic reticulum stress(Cieslak et al., 2015 ) and impair insulin secretion through an excessive nitric oxide production affecting both ATP production by the mitochondria (Fig. 4, right part, bold arrows) (Cnop et al., 2005) and gap junction coupling between β-cells (Farnsworth, Walter, Hemmati, Westacott, & Benninger, 2016). Infiltration of leukocytes and macrophages during the peri-transplantation period, as well as recruitment of neutrophils, macrophages, Kupffer cells and CD4 + and CD8 + lymphocytes in the later stages of engraftment also contribute to islet cell death (Kanak et al., 2014).
In this context, the increased expression of HLA-G and HLA-E by hAECs under pro-inflammatory conditions appear of particular interest to protect islet grafts from inflammation-induced damage.
HLA-G and HLA-E belong to the nonclassical HLA Ib family, characterized by low polymorphism and immunomodulatory properties. HLA-G is mainly expressed by placental and embryonic tissues and participates in development of foeto-maternal tolerance (Ferreira, Meissner, Tilburgs, & Strominger, 2017). In contrast, HLA-E is ubiquitously expressed and acts as an inhibitor of NK-cell driven lysis (Wieten, Mahaweni, Voorter, Bos, & Tilanus, 2014). Both molecules induce immune tolerance by inhibiting DC proliferation, switching T lymphocytes to a Treg phenotype, inhibiting CD8 + and CD4 + T cells (Wassmer & Berishvili, 2020) and modulating the release of cytokines from mononuclear cells (MNCs) (Banas et al., 2008) (Fig. 4, bottom panels).
In addition, the immunomodulatory cytokines secreted by hAECs play a major role in suppressing inflammatory responses (Fig. 4, right panel, dashed lines). Expression of IL10 by amniotic cells has been well described, and is known to inhibit the release of pro-inflammatory mediators by monocytes and macrophages, reducing antigen presentation, and inhibiting CD4 + and CD8 + T cell differentiation and proliferation as well as B cell recruitment (Wassmer & Berishvili, 2020). Increased levels of IL10 have been associated to improved islet survival and function in allotransplantation experiments while artificial upregulation of IL10 expression decreased alloreactivity to human islets and increased rat islet allograft survival (Kim et al., 2008; Vaithilingam et al., 2017). IL6 is known to exert anti-inflammatory actions through STAT3 activation, and has been shown to protect islets and β-cells from pro-inflammatory cytokine-induced apoptosis and loss of function in vitro. Moreover, improved survival and graft function was demonstrated after transplantation of islets pre-treated with IL6 through the overexpression of anti-apoptotic genes (Choi et al., 2004). In addition, IL6 improves β-cell survival by stimulating autophagy and reducing cell oxidative stress (Marasco et al., 2018). Finally, IL6 exerts angiogenic effects by inducing expression of vascular endothelial growth factor (VEGF) in various cell lines (Cohen, Nahari, Cerem, Neufeld, & Levi, 1996). Another factor overexpressed in cytokine-exposed hAECs is IL8, a neutrophil-recruiting cytokine, which is also known to promote endothelial cell proliferation (Li, Dubey, Varney, Dave, & Singh, 2003) and angiogenesis (Norrby, 1996) and thus contribute to improved vascularization (F. Lebreton et al., 2019).