Chronic inflammation had long been considered as a risk factor for cancer development and progression (Guerra, Collado et al. 2011, Shi and Xue 2019). In the pancreas, chronic inflammation is known to sustain an inflammatory milieu that can destabilise the acinar cell genome, hence encouraging the accumulation of oncogenic mutations (Kong, Sun et al. 2014). The central mechanism driving this inflammation in immune cells is proposed to be orchestrated by the inflammasome (Hausmann, Kong et al. 2014, Kolodecik, Shugrue et al. 2014). However, till date, it remains unresolved how the crosstalk, between PDAC and macrophages, regulates the NLRP3 inflammasome and pro-inflammatory cytokines in the tumour microenvironment and ultimately its effect on PDAC progression.
In this study, in the co-culture of PDAC cells and macrophages, elevated levels of IL-1β (about 200-300pg/mg) and TNF-α (about 100–150 pg/mg) were produced by PDAC cells and macrophages, in the presence of LPS (L and LA) (Fig. 2, Fig. 3). The increase in cytokines is in accordance to the reported ability of LPS to activate NF-κB in all cell lines, leading to downstream transcription of the cytokines and inflammasomes (Groslambert and Py 2018). Although increased level of cytokines were also reported in the monoculture of LPS-induced PDAC cells, the level of IL-1β was only approximately 6pg/mg (Yaw, Chan et al. 2020). This elevated production level of IL-1 β observed in the co-culture of PDAC cells and macrophages suggested that the crosstalk between macrophages and PDAC cells could positively regulate cytokines, particularly IL-1β production, contributing to the inflammatory microenvironment. Studies had shown that the stimulation of macrophage IL-1 receptors by IL-1β produced by PDAC cells, could lead to the activation of Casein Kinase I and II in macrophages, which in turn phosphorylated the p65 subunit of NF-κB heterodimer, resulting in the increased activation of NF- κB (Chantôme, Pance et al. 2004). The activated NF-κB might then trans-locate into the nucleus and stimulate the transcription of the mRNA of inflammatory components such as the NLRP3 inflammasome (Cornut, Bourdonnay et al. 2020).
The expressed cytokines could further contribute to the crosstalk between macrophages and PDAC cells by repressing PP2A in PDAC cells (Engström, Erlandsson et al. 2014, Tao, Liu et al. 2016). PP2A protein dephosphorylates and inactivates the IKK complex keeping NF-κB activation in check, hence the repression of PP2A leads to uninhibited activation of NF-κB in PDAC cells. As mentioned in previous reports, the reduced inhibition of NF-κB activation meant that along with the existing KRAS and STAT3 cytokine networks, a greater sustained activation of NF-κB ensues (Corcoran, Contino et al. 2011). PP2A repression also increases the activity of kinases including IKK, JNK, PKC and ERK, all of which have important roles in accelerating pancreatic cancer progression (Takahashi, Hirata et al. 2013, Storz 2015, Tao, Liu et al. 2016, Mollinedo and Gajate 2019). In accordance with these reports, this study showed that the cell proliferation of PDAC cells increased when treated with LPS and/or ATP (Fig. 1).
Following MCC950 treatment (M), the cytokine production by PDAC cells and macrophages were significantly reduced. This could be due to the role of MCC950 as a highly specific inhibitor of ATP hydrolysis by the NLRP3 inflammasome (Coll, Hill et al. 2019). The inhibition of ATP hydrolysis, prevents or reverses conformational changes in NLRP3 protein necessary for inflammasome assembly and activation. The addition of LPS however (LM) offsets the inhibition by MCC950 in the production of IL-1β by PDAC cells (Fig. 3.2). This could be due to the increase in NF-κB activation in the macrophages and, hence greater transcription and activation of NLRP3 inflammasome in both cell lines (He, Hara et al. 2016). ATP on its own did not massively increase the production of IL-1β in SW 1990 PDAC cells and RAW 264.7 macrophages as the two-step activation is required for NLRP3 inflammasome (Fig. 2, Fig. 3). Unlike the SW 1990 metastatic cancer cell line, in Panc 10.05, induction with LPS and ATP individually or together (L, A and LA groups) resulted in a similar increase in the production of IL-1β (Fig. 2). This is in accordance with previous studies, as primary PDAC cells (Panc 10.05) lack ASC meaning the NLRP3 inflammasome is not largely responsible for the production IL-1β in this cell line and instead other inflammasomes play a prominent role in the cleavage of pro-IL-1β into IL-1β (Yaw, Chan et al. 2020).
In addition, in this study, LPS-induced Panc 10.05 PDAC cells (L, LA and LM groups) demonstrated an increase in proliferation compared to their control of counterparts (Fig. 1). This confirms the ability of LPS to further repress PP2A expression and promote IL-1β production, effectively stimulating NF-κB activation, which in turn enhances mitogenic EGF receptor signalling (Liptay, Weber et al. 2003, Ling, Kang et al. 2012). This also correlated with the significant increase in the expression of NLRP3 in Panc 10.05 PDAC cells following treatment with LPS and/or ATP (L, A, LA, LM groups) (Fig. 4). The level of pro-inflammatory cytokines produced by Panc 10.05 PDAC cells and macrophages was also increased in the LPS-stimulated co-cultures (L, LA and LM groups) suggesting that the increased levels of pro-inflammatory cytokine in the tumour microenvironment also contributed to the above-mentioned PP2A repression mechanism, resulting in an increased cell proliferation. Treatment with MCC950 did not prevent the increase in pro-inflammatory cytokines in the tumour microenvironment, nor the increase in proliferation of Panc 10.05 PDAC cells in LPS-stimulated pro-inflammatory microenvironment.
Similarly, LPS-induced SW 1990 PDAC cells (L and LA groups) demonstrated a similar increase in cell proliferation. However, the addition of MCC950 to inflammatory stimuli LPS (LM and LAM groups) reduced the level of IL-1β in the tumour microenvironment by the SW 1990 PDAC cells and macrophages, and this could prevent the crosstalk-induced increase in NF-κB activation in the PDAC cells. Hence, despite the increase in NLRP3 expression in both SW 1990 PDAC cells and macrophages, in these groups, there was no increase in the proliferation of SW 1990 PDAC cells in the presence of MCC950 (Fig. 1, Fig. 2).
Interestingly, it was observed that NLRP3 levels were generally increased in PDAC cells when treated with LPS and/or ATP (Fig. 4) but not in macrophages (Fig. 5). Also, in the SW 1990 PDAC cells, there is no significant change in the expression of NLRP3 in the LA group (Fig. 4B), suggesting that the massive increase in pro-inflammatory cytokine IL-1β could significantly induce Tripartite Motif Containing 31 (TRIM31) and Growth Factor Independence 1 (GFI1) negative regulation, off-setting the initial increase in transcription. Studies have reported that IL-1β stimulates the mRNA transcription and protein expression of TRIM31 and the downstream sustained NF-κB activation also leads to the transcription of GFI1. TRIM31, which is also constitutively expressed in macrophages and PDACE cells, promotes the proteasomal degradation of the NLRP3 inflammasome and GFI1 represses the transcription of NLRP3 inflammasome (Zhu, Meng et al. 2014, Song, Liu et al. 2016). The results suggest that for a particular cell, IL-1β present in the microenvironment can stimulate both TRIM31 and GFI negative regulation. The effects of LPS and ATP on the regulation of NLRP3 expression in PDAC cells in co-culture are not similar to those observed in monocultures of the PDAC cells in a study by Yaw et al. This could be due to the additional input from the crosstalk between the PDAC cells and the macrophages, setting the inflammatory microenvironment.
Our observations led to the understanding that the levels of NLRP3 expression in cells are finely controlled by the crosstalk via the same regulators (e.g., IL-1β), with different effects at different concentrations. The differing effects of MCC950 in decreasing the expression of NLRP3 in the LA group of primary Panc 10.05 PDAC cells and increasing the expression of NLRP3 in LA group of metastatic SW1990 PDAC cells depicts that role of MCC950 in the crosstalk depends on whether the particular PDAC cell line relies on the NLRP3 inflammasome for background IL-1β production. The lesser the PDAC cell line relies on NLRP3 inflammasome for background IL-1β production, the greater the level of inflammatory cytokines in the tumour microenvironment and the greater the extent of negative feedback on NLRP3 expression, that could not be offset by MCC950 inhibition on cytokine production.
In both co-cultures, there is a significant decrease in the expression of NLRP3 inflammasome in RAW 264.7 macrophages in the background of an increased levels of pro-inflammatory cytokine in the tumour microenvironments stimulated by LPS and ATP (Fig. 5A and B). This highlights the prominent role of the negative regulators e.g., TRIM31 and GFI1, and the effect of crosstalk on these negative regulators (Song, Liu et al. 2016, Cornut, Bourdonnay et al. 2020). Despite the initial induction of NF-κB to stimulate transcription of NLRP3 inflammasome, the massive production of cytokines by both cell lines could increase the levels of TRIM31 and GFI1, which in turn decreased the expression of NLRP3. Our results also suggest that the negative regulation of NLRP3 inflammasome is relatively greater in RAW 264.7 macrophages compared to the PDAC cells.
In the co-culture of macrophages with Panc 10.05 PDAC cells, the addition of LPS or ATP to MCC950 (AM and LM), significantly reduced the NLRP3 inflammasome expression in the macrophages. On contrary, in the co-culture of SW 1990 PDAC cells with macrophages, a significant increase in NLRP3 expression in macrophages, was observed in the AM and LM groups. This variation between the co-cultures could be due to Panc 10.05 exerting greater negative feedback on its crosstalk, as it produces more IL-1β than the MCC950-inhibited SW 1990, leading to possibly increased TRIM31 induction in the macrophages. This highlights the impact of NLRP3 inflammasome activity in the crosstalk and tumour microenvironment.