CDT induces a proinflammatory signature related to type I interferon
To determine wether 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 (Fig. S1). 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 (50 days total) do not show significant increase of gH2AX level, used as a surrogate 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, which cannot induce DNA damage nor activate DDR [31, 32]. As depicted in the heatmap resuming expression profile of 9703 significantly regulated genes between the 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 mutant CDT) 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 proinflammatory responses, more particularly to type I IFN signaling (Fig. 1G). To confirm this result, mRNA expression level of different cytokines (IL1b, IL6, IL8 and IL10) and a panel of type I IFN signaling genes (OAS1, MX1, ISG15, IFIT1, IFIT2, IFI6 and IFI44) were determined after only 2 days of CDT WT or after repeated treatment with CDT WT or H153A during 40 days (Fig. 1H and 1I). In cells chronically exposed to CDT WT, all tested genes showed a strong mRNA expression enhancement, from 8.8-fold for IL6 to 232-fold for MX1, except the anti-inflammatory mediator IL10 for which the increase is less than 2-fold. This depends on the CdtB catalytic activity, as the H153A mutation abolishes expression profile modification. In the same way, short CDT WT exposure during 2 days does not significantly alter the expression level of any tested mRNA. Taken together, these results show that cells chronically exposed to CDT accumulate MN and display a proinflammatory response related to type I IFN signaling.
cGAS binds CDT-mediated micronuclei to promotes senescence and type I IFN signaling
Recent reports have demonstrated that MN recognition by cGAS triggers innate immune activation related to type I IFN signature [28, 29]. We thus questioned whether cGAS could bind to CDT-induced MN (Fig. 2A). In absence of treatment, 8.6 % of HeLa cells are micronucleated (Fig. 2B). At 0.025 ng/ml of CDT, the proportion of cells with MN reaches 16.4 % after 24 h and remains stable after 72 h. Increasing CDT concentration to 0.25 or 2.5 ng/ml does not clearly impact the percentage of MN-containing cells at 24 h. However, after 72 h, the proportion of micronucleated cells increases to 34.7 % with 0.25 ng/ml of CDT and 37.5 % with 2.5 ng/ml. These data demonstrate that prolonged CDT exposure favorizes MN formation. Then, we examined the proportion of MN recognized by cGAS or stained with a gH2AX antibody. After 24 h of CDT exposure, a dose dependent increase of gH2AX-positive MN can be observed (Fig. 2C). In contrast, the proportion of cGAS-positive MN increases only after 72 h, with MN progressively accumulating gH2AX 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. 2D). cGAS-/- cells are more resistant to low CDT concentrations (0.025 and 0.25 ng/ml) than their WT cGAS counterpart (Fig. 2E). Moreover, despite similar viability loss between cGAS+/+ and cGAS-/- cells at 2.5 ng/ml of CDT, cell distention is not observed in cGAS mutant cells (Fig. 2F). In the same way, the increase of β-Gal staining, a marker of cellular senescence, is abolished in cGAS-depleted cells (Fig. 2G and 2H). Finally, mRNA expression of three type I IFN signaling target genes (MX1, ISG15 and IFI44) was compared between cGAS+/+ and cGAS-/- cells after 10 days of CDT intoxication. An approximately 3-fold increased expression level of each tested gene after chronic CDT exposure was observed in cGAS+/+ but absent in cGAS-/- cells (Fig. 2I). Altogether, these results unravel the essential role of cGAS during CDT intoxication through MN recognition, eliciting cell distention, senescence and type I IFN signature.
CDT-exposed cells reach mitosis despite active G2 cell cycle checkpoint
As cGAS-mediated proinflammatory 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 relies on DDR activation, phosphorylation of H2AX at S139 (gH2AX), 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 (Fig. S1) is unlikely the consequence of a rapid DDR activation and a 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 (Fig. S2). 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 ATR inhibitor VE-821 (ATRi), given that the G2/M checkpoint mostly depends on ATR rather than ATM under these conditions (Fig. S3). Contrary to other genotoxic treatments, ATR inactivation does not sensitize HeLa cells to CDT during the first 24 h of exposure (Fig. 3C), further supporting that cells do not activate checkpoint 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. 3D). 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. 3E). 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. 3D) is accompanied by a dose-dependent diminution of the anaphase population (Fig. 3F). In order 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. 3G). Moreover, monitoring cell death during the course of live imaging revealed that an important fraction of CDT-exposed cells preferentially dies at metaphase (Fig. 3H). In conclusion, mitotic cells are particularly affected during CDT intoxication, as evidenced by a prolonged metaphase duration that eventually result 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 gH2AX and H3 histone phosphorylated at S10 (pH3) to identify mitotic cells. Cells treated with CDT for 24h present a dose-dependent augmentation of pH3 and gH2AX positive cells, representing a 12-fold increase at 2.5 ng/ml of CDT compared to control cells. In contrast, after exposure with moderate concentration of control genotoxic compounds, only cells without gH2AX staining do progress to mitosis (Fig. 4A). 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 gH2AX signal in mitosis compared to interphase (Fig. 4B). This staining is clearly distinguishable from the basal DNA damage-independent gH2AX signal described in unchallenged mitotic cells , that is diffuse all along the condensed chromosomes from prometaphase to anaphase, or from few gH2AX foci observed in mitotic cells exposed to other DNA damaging agents (Fig. S4). The huge gH2AX increase at mitosis is observed with CDT from other bacterial origins or with other cell lines (Fig. S5), thus representing a general cellular response to CDT. Moreover, the fraction of gH2AX-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 gH2AX 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 gH2AX 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 gH2AX level compared to etop, campto or MMC (Fig. 5A). To confirm that the gH2AX 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 in comet tail moment (Fig. 5C). Taken together, these data demonstrate that CDT induces DSB during mitosis leading to chromosome fragmentation and missegregation.
CDT promotes proinflammatory response and mitotic defects in normal colonic epithelial cells
We next assessed whether non-transformed cells exhibit similar inflammatory responses to CDT genotoxic activity. Immortalized normal human colonic epithelial cells (HCECs), previously shown to be susceptible to CDT intoxication , were exposed to CDT for 96 h and STAT1 phosphorylation at Y701 (pSTAT1) was measured as a surrogate for inflammatory pathway activation after DNA damage . HCECs cells show a dose dependent increase of pSTAT1 (Fig. 6A), confirming that CDT activates inflammatory pathway in non-transformed cells. CDT also induces senescence in HCEC cells, as evidenced by β-Gal staining (Fig. 6B and C). Importantly, the proportion of β-Gal positive cells decreases when adding CDK1 inhibitor RO-3306 (CDK1i), which blocks cells at G2/M boundary, supporting that CDT-treated cells must go through mitosis before entering a senescent state. Compared to control displaying only 4% of micronucleated cells, a 24 h exposure to CDT enables MN formation in approximately 16% of HCEC cells (Fig. 6D). This result indicates that normal cells do not stop at G2/M checkpoint during early course of CDT intoxication, but divide and form MN in daughter cells. Moreover, CDT-induced pSTAT1 increase is prevented by STING inhibitor H-151 (STINGi) (Fig. 6E), suggesting that similarly to HeLa cells, MN formed in HCEC cells in response to CDT activate the cGAS-STING axis to enhance proinflammatory responses. We further explored the mitotic phenotype of CDT-exposed HCEC cells and observed a decrease of the anaphase population in cells exposed to 0.25 ng/ml of CDT (Fig. 6F), as shown above (Fig. 3F). Moreover, mitotic HCEC cells displayed the same intense gH2AX signal as in cancer cell lines (Fig. 6G). Finally, the proportion of gH2AX positive cells after an 8 h treatment to 0.025 ng/ml of CDT is significantly higher in mitosis compared to interphase (Fig. 6H). Taken together, these data highlight the crucial role of cGAS-STING pathways in proinflammatory activation in normal HCEC cells, and demonstrate that this requires passage through mitosis where chromosomes accumulate DNA damage and form MN in daughter cells.