CDT induces a proinflammatory signature related to type I interferon
To determine whether chronic exposition to CDT induces an inflammatory response in the host, a model human cancer cell line (HeLa) was chronically exposed to 0.25 ng/ml of E. col CDT, inducing more than 95% cell death after 10 days (Supp Fig. 1). The surviving fraction was cultured for 40 more days in presence of CDT and individual clones were selected as well as a pool of resistant cells (Fig. 1a). Compared to a short-term exposure, cells chronically treated to CDT (55 days total) do not show significant increase of γH2AX level, used as a marker of DNA damage signaling (Fig. 1b). In addition, these cells were unresponsive to the CDT-mediated G2/M checkpoint (Fig. 1c), suggesting an adaptation to the CDT toxin. However, chronically exposed cells exhibit a higher proportion of micronucleated cells, indicative of important chromosomal instability (Fig. 1d). These cells were subjected to transcriptomic analyses and compared to two control groups, i.e. cells without treatment or chronically exposed to the CDT catalytic dead mutant, bearing the H153A substitution on CdtB (H153A), which cannot induce DNA damage nor activate DDR [33, 34]. As depicted in the heatmap resuming expression profile of 9703 significantly regulated genes between these three conditions, individual clones and the pool of cells chronically exposed to active wild-type (WT) CDT share a common transcriptional adaptation, whereas the two control groups (non-treated and treated with CDT H153A) cannot be distinguished (Fig. 1e). The majority of the most upregulated genes, when comparing the three groups, depends on the catalytic activity of CDT rather than the presence of the toxin solely (Fig. 1f). Strikingly, the most upregulated biological processes in cells chronically exposed to WT CDT mainly rely on immune responses, more particularly to type I IFN signaling (Fig. 1g). To confirm that CDT exposure elicits a proinflammatory signature, mRNA expression level of IL-1β, IL-6, and IL-8 cytokines was determined after only 2 days of CDT WT or after repeated treatment with CDT WT or H153A during 55 days (Fig. 1h). In cells chronically exposed to CDT WT, proinflammatory cytokines mRNA expression level statistically increases, around tenfold compared to non-treated cells. This depends on the CdtB catalytic activity, as H153A mutation abolishes expression profile modification. In the same way, short exposure to 0.25 ng/ml of CDT WT during 2 days does not significantly alter cytokines expression level. We next tested a panel of ISGs related to type I IFN response (OAS1, MX1, ISG15, IFIT1, IFIT2, IFI6 and IFI44). While ISGs expression level does not significantly increase after 2 or 10 days of CDT treatment, a chronic 55 days exposure strongly enhances their expression with CDT WT but not H153A (Fig. 1i). Taken together, these results show that cells chronically exposed to CDT accumulate MN and modulate their immune response through increase of proinflammatory response and type I IFN signaling.
cGAS binds CDT-mediated micronuclei and promotes type I IFN response
MN recognition by cGAS triggers innate immune activation related to type I IFN signature [27, 28]. We thus questioned whether cGAS could bind to CDT-induced MN (Fig. 2a) and examined the proportion of MN recognized by cGAS or stained with a γH2AX antibody. After 24 h of CDT exposure, a dose-dependent increase of γH2AX-positive MN can be observed (Fig. 2b). In contrast, the proportion of cGAS-positive MN increases only after 72 h, with MN progressively accumulating γH2AX staining by increasing CDT concentration. Therefore, cGAS recognizes CDT-induced MN, but this binding is delayed in time.
To better understand the role of cGAS in response to CDT injury, cGAS knockout HeLa cells (cGAS−/−) were generated (Fig. 2c). cGAS−/− cells are more resistant to low CDT concentrations (0.025 and 0.25 ng/ml) than their WT cGAS counterpart (Fig. 2d) and accumulate less MN (Fig. 2e). Moreover, cGAS-deficient cells exhibit less cell distention (Fig. 2f). In the same way, the increase of β-Gal staining, a marker of cellular senescence, is less important in cGAS-depleted cells (Fig. 2g, h). These results indicate that cGAS might regulate MN formation and proliferation of HeLa cells exposed to CDT, at least partly by promoting senescence.
Then, we examined the mRNA expression level of proinflammatory cytokines and ISGs in cGAS−/− HeLa cells chronically exposed to CDT (Fig. 2i). Only IL-1β and IL-6 expression was significantly upregulated after 40 days of CDT exposure, whereas ISGs were not affected. Although IL-6 mRNA upregulation is less marked compared to cGAS+/+ cells (Fig. 1h), the increased concentration of secreted IL-6 cytokine is similar with or without cGAS, all along CDT exposure (Fig. 2j). Of note, only an active CDT toxin can stimulate IL-6 production, as revealed by chronic exposure to the H153 mutant. Thus, cGAS does not seem to impact IL-6 production during chronic exposure to CDT. Conversely, we found that ISG15 protein level was only enhanced in presence of cGAS, and could not be observed during the first 5 days of CDT treatment (Fig. 2k). Furthermore, this late ISG15 increase was accompanied by STAT1 phosphorylation at Y701 (pSTAT1), a surrogate marker for type I IFN signaling , that is not induced in cGAS−/− cells. Finally, we monitored inflammasome activation through Caspase-1 self-cleavage and did not observe any defect in cGAS−/− cells, but rather an increased Caspase-1 cleavage. Altogether, these results establish that CDT chronic exposure in HeLa cells triggers a cGAS-dependent type I IFN signaling that is delayed in time and a cGAS-independent proinflammatory response.
To confirm that these immunomodulatory effects represent a general response to CDT, these experiments were reproduced on cGAS+/+ and cGAS−/− HeLa cells treated with CDT from H. duc (H. duc CDT). Both cell lines exhibited similar enhanced proportion of micronucleated cells after a chronic exposure to H. duc CDT (Supp Fig. 2a). In contrast to E. col CDT, the proinflammatory response was higher in cGAS-defective cells than their wild-type counterpart (Supp Fig. 2b, c). Conversely, ISGs gene induction in response to H. duc CDT is less effective than with E. col CDT but still cGAS-dependent (Supp Fig. 2d). Finally, this was confirmed at the protein level for ISG15 (Supp Fig. 2e).
Similar experiments were also conducted on primary MEFs. MEFs chronically exposed to CDT still present MN after 40 days (Fig. 2l). These cells were co-exposed or not with the STING inhibitor H-151 (STINGi) or the cGAS inhibitor RU.521 (cGASi) and analyzed for IL-6 and ISGs mRNA expression (Fig. 2m). After 7 or 40 days of CDT, IL-6 mRNA expression shows a slight increase that is not statistically significant and is not affected by STING or cGAS inhibition. On the contrary, ISGs expression was enhanced after 40 days but not 7 days of CDT treatment, reaching up to 72.7-fold increase for IFI44 compared to non-treated cells. ISGs gene overexpression in response to CDT was not found upon exposure to the inactive H153 CDT mutant, and was impaired by STING or cGAS inhibition. This confirms the crucial role of cGAS-STING axis in CDT-induced type I IFN signaling. Finally, IL-6 and TNFα production was elevated in response to 3 days of CDT exposure in a STING-dependent manner (Fig. 2n). However, IL-6 and TNFα concentrations dropped back to the basal level after 40 days, indicating that the proinflammatory signature elicited by CDT is not maintained upon chronic exposure.
CDT-exposed cells reach mitosis despite active G2 cell cycle checkpoint
As cGAS-mediated type I IFN response depends on MN recognition, we next asked whether MN formation is the direct consequence of CDT intoxication. Previous studies from our lab and others showed that CDT-induced DNA damage activate the G2/M checkpoint . However, G2/M checkpoint arrest is inconsistent with MN formation that requires mitosis completion. To explain the accumulation of MN following CDT treatment, we first monitored DNA damage markers. As DNA damage checkpoints rely on DDR activation, phosphorylation of H2AX at S139 (γH2AX), CHK1 at S345 (pCHK1) and CHK2 at T68 (pCHK2) were measured after a 24 h treatment with CDT (Fig. 3a). Strong DDR activation is only observed at high concentration of CDT (2.5 and 25 ng/ml). This result supports that at low concentrations, the proliferation defects induced by CDT (Supp Fig. 1) is unlikely the consequence of a rapid DDR activation and immediate checkpoint-induced cell cycle arrest. Indeed, the CDT-mediated cell cycle arrest significantly increases from 24 to 72 h (Fig. 3b), implying that at least a part of CDT-exposed cells reach mitosis before to block their cell cycle during the next rounds of cell division. In contrast, exposure to etoposide (etop), camptothecin (campto) or mitomycin C (MMC), three other genotoxic compounds, induces a rapid and stable cell cycle block over time (Supp Fig. 3). To demonstrate that CDT-exposed cells can complete a first mitotic division prior efficient cell cycle arrest at the next G2, Hela cells were pulse-labeled with EdU in early S phase after a double thymidine cell synchronization, either being exposed to CDT after the first (“CDT 36 h” condition) or the second (“CDT 10 h” condition) thymidine bloc (Fig. 3c). In absence of CDT, around 90% of cells incorporated EdU, demonstrating that cell synchronization was effective (Fig. 3d). Moreover, 68.5% of EdU-positive cells proceeded to G1, implying they passed through G2 and mitosis. For the “CDT 10 h” condition, cells exposed to 0.025 and 0.25 ng/ml of CDT do not show any significant increase of the G2 cell population, demonstrating they did not activate the G2 checkpoint at the first cell cycle during CDT treatment (Fig. 3e). However, cells exposed to 0.25 ng/ml of CDT during the previous cell cycle (“CDT 36 h” condition) exhibit 42.8% of G2 cells compared to 20.8% without CDT, indicating that the G2 checkpoint was activated in response to CDT after one cell division. In contrast, a dose-dependent G2 arrest of the EdU-positive population is observed from 2.5 ng/ml of CDT for the “CDT 10 h” condition. This demonstrate that CDT-treated cells arrest in the first G2 phase only at high doses. Moreover, a significant increase of EdU-negative cells occurs in the “CDT 36 h” condition at 2.5 and 25 ng/ml of CDT, representing cells that did not reached S phase at the time of EdU labeling, therefore, cells that were arrested at the previous G2. To conclude, these results represent a direct evidence that CDT-exposed cells pass through mitosis before blocking at the next G2, except at highest concentrations.
To test whether DDR does not effectively abrogate cell proliferation during early phase of CDT intoxication, cells were co-exposed during 24 h to CDT and the ATR inhibitor VE-821 (ATRi), given that the G2/M checkpoint mostly depends on ATR rather than ATM under these conditions (Supp Fig. 4). Contrary to other genotoxic treatments, ATR inactivation does not sensitize HeLa cells to CDT during the first 24 h of exposure (Fig. 3f), further supporting a minor G2 checkpoint activation during early phase of CDT intoxication. Moreover, while exposure to control genotoxicants or high CDT concentration (25 ng/ml) block mitotic entry in an ATR-dependent manner, indicative of active G2 checkpoint, lowest CDT concentrations significantly increase the mitotic index, confirming that cells do progress through mitosis (Fig. 3g). This result demonstrates that ATR is only crucial after high treatment with CDT to protect cells from mitotic catastrophe by inducing a G2/M arrest, at least during the first 24 h of exposure. Finally, in contrast to high CDT concentrations or DNA damaging agents, low CDT concentrations (0.025 and 0.25 ng/ml) induce MN formation that is not aggravated by the presence of ATRi (Fig. 3h). Altogether, these data demonstrate that except for high concentrations, CDT exposure allows mitotic entry and MN generation, despite the presence of active cell cycle checkpoints.
CDT induces mitotic delay and cell death
The increased mitotic index observed in CDT-exposed cells (Fig. 3g) is accompanied by a dose-dependent diminution of the anaphase population (Fig. 3i). To gain insight into the mitotic phenotype of CDT-treated cells, live-cell imaging has been performed on HeLa cells stably expressing the chromatibody fused to GFP, enabling real-time chromatin visualization . When measuring the timing needed to complete metaphase, we found that unperturbed mitosis takes an average of 64 min that significantly increases to 109 and 164 min after treatment with 0.25 ng/ml and 2.5 ng/ml of CDT, respectively (Fig. 3j). Moreover, monitoring cell death during the course of live imaging revealed that an important fraction of CDT-exposed cells preferentially dies at metaphase (Fig. 3k). In conclusion, mitotic cells are particularly affected during CDT intoxication, as evidenced by a prolonged metaphase duration that eventually results in cell death.
CDT-exposed cells experience DNA damage at mitosis
To better understand the relationship between CDT-mediated DNA damage and cell cycle defects, cell cycle analyses were conducted after immunostaining with antibodies directed against γH2AX and H3 histone phosphorylated at S10 (pH3) to identify mitotic cells. Cells treated with CDT for 24 h present a dose-dependent augmentation of pH3 and γH2AX positive cells, representing a 12-fold increase at 2.5 ng/ml of CDT compared to control cells (Fig. 4a). In contrast, after exposure with moderate concentration of control genotoxic compounds, only cells without γH2AX staining do progress to mitosis. Thus, CDT-exposed cells progress through mitosis with damaged DNA, representing a unique feature over other genotoxic insult. Strikingly, asynchronous cells exposed to CDT display an intense γH2AX signal in mitosis compared to interphase (Fig. 4b). This staining is clearly distinguishable from the basal DNA damage-independent γH2AX signal described in unchallenged mitotic cells , that is diffuse all along the condensed chromosomes from prometaphase to anaphase, or from few γH2AX foci observed in mitotic cells exposed to other DNA damaging agents (Supp Fig. 5). The huge γH2AX increase at mitosis is observed with CDT from other bacterial origins or with other cell lines (Supp Fig. 6), thus representing a general cellular response to CDT. Moreover, the fraction of γH2AX-positive cells is more important in mitosis compared to interphase, after 24 h or even a shorter incubation of 8 h with CDT (Fig. 4c), demonstrating that mitotic cells represent the first population to be damaged during the course of CDT treatment. The strong γH2AX signal after CDT can be observed all along the mitotic phases (Fig. 4d). Finally, CDT exposure induces a dose-dependent increase of chromosome fragments that does not properly align during metaphase or segregate at anaphase (Fig. 4e), therefore, explaining the high level of MN observed after CDT treatment.
CDT induces DNA double-strand breaks during mitosis
To exclude the possibility that mitotic γH2AX signal originates from DNA damage induced before mitotic entry, HeLa cells were enriched in mitosis by a 22 h nocodazole block and then co-exposed during the last 6 h to CDT or genotoxic control agents. Similar to observations made on asynchronous cells, cells treated with CDT during mitosis exhibit a strong γH2AX level compared to etop, campto or MMC (Fig. 5a). To confirm that the γH2AX level increase in mitosis depends on DSB induction, cells arrested in mitosis were exposed to CDT before to be subjected to neutral comet assay (Fig. 5b). Mitotic cells treated with 0.25 or 2.5 ng/ml of CDT show a significant increase of comet tail moment (Fig. 5c). Taken together, these data demonstrate that CDT induces DSB during mitosis leading to chromosome fragmentation and missegregation.
Immortalized normal colonic epithelial cells chronically exposed to CDT display an altered type I IFN response associated to cGAS protein loss
We next assessed whether non-transformed colonic cells exhibit similar immune response to CDT genotoxic activity. Immortalized normal human colonic epithelial cells (HCECs), previously shown to be susceptible to CDT intoxication , present similar mitotic defects than HeLa cells (Supp Fig. 7). HCECs were exposed for 3 days to 0.25 ng/ml of CDT, with or without STING or cGAS inhibitors, and type I IFN signaling or inflammasome activation were assessed through pSTAT1 and cleaved Caspase-1, respectively (Fig. 6a). Contrary to the inactive H153A toxin, CDT induced an increase in pSTAT1 and cleaved Caspase-1 level. Cleaved Caspase-1 also increased in response to LPS and was not dependent on STING or cGAS activity. Intriguingly, STINGi but not cGASi could decrease pSTAT1 in CDT-exposed cells. To further analyze HCECs behavior, we measured the proportion of MN-containing cells and found that CDT-induced MN formation observed after 1 day is lost after 35 days (Fig. 6b). During the course of long-term CDT treatment, HCECs enter a senescence state associated to cell distention that is independent on STING or cGAS activities (data not shown) and maintained for at least 15 days (Fig. 6c). At these time points, p21 and pSTAT1 level increased with CDT but not with the H153A inactive mutant (Fig. 6d). These cells then escape from senescence and restart to proliferate with a basal level of micronucleated cells as seen after 35 days of chronic exposure, while p21 and pSTAT1 decreased. Strikingly, the level of cGAS protein was continuously disrupted all along the chronic exposure to CDT but not the inactive mutant. Furthermore, a low cGAS signal is observed in MN after 3 days of CDT treatment (Fig. 6e), and the proportion of cGAS-positive MN is not enhanced compared to untreated cells (Fig. 6f). Taken together, these results suggest that CDT exposure impedes cGAS-mediated response in HCECs by affecting cGAS protein stability and MN recognition. Moreover, the low pSTAT1 and MN levels of HCECs that escaped senescence also imply that the type I IFN response is not maintained during long-time exposure to CDT. We next monitored mRNA expression level of proinflammatory cytokines and ISGs in HCECs after 7 days (senescence state) or 35 days of CDT exposure (Fig. 6g, h). As observed in MEFs, proinflammatory cytokines mRNA level increased after 7 days independently of STING or cGAS activities, but decreased after 35 days with CDT. ISGs mRNA expression only shows a slight increase compared to HeLa or MEFs (Figs. 1i and 2m), and was not affected by STING or cGAS inhibitors, confirming that the cGAS-STING axis is not activated in HCECs after chronic exposure to CDT. Finally, kinetics of proinflammatory cytokine mRNA expression is reflected by the low IL-6 concentration measured in the culture medium after 35 days of CDT treatment compared to 15 days (Fig. 6i). Similar to MEFs, IL-6 increase at 3 days partially depends on STING, but not after 15 days. In the same way, STINGi decreases pSTAT1 level induced by 3 days of CDT exposure but not after 15 days (Fig. 6j). ISG15 protein increased until 15 days but decreased at 35 days of CDT treatment, and the CDT-induced cleavage of Caspase-1 also diminishes at latter time point. To conclude, proinflammatory and type I IFN responses are poorly activated in HCECs during long-time exposure to CDT, which is probably correlated to cGAS protein downregulation and loss of MN induction after senescence escape.