Enhancing Survival after Ionizing Radiation Exposure through Mitigation of Pyroptosis

Abstract

• Pyroptosis, an inflammatory cell death, has been implicated in the pathogenesis of total body irradiation (TBI) so we investigated time course and cell type involvement of key mediators in a murine model.

• Pyroptotic mediators were most highly expressed at day 3 post TBI with immune cells from ileum being preferentially activated.

• We also investigated the effectiveness of MCC950, a potent pyroptosis inhibitor, in our murine model showing a survival benefit at 50 mg/kg regardless of sex.

• Treatment with MCC950 showed elevations in full-length downstream mediator IL-1β in ileum and decreased levels of cleaved IL-1β in splenic tissue.

Increasing threats of terrorist attacks and trafficking of radioactive materials necessitate development of new therapies for acute ionizing radiation [1]. Ionizing radiation causes radiolysis of water and production of a variety of reactive oxygen species (ROS) leading to DNA damage and apoptotic death of fast dividing hematopoietic stem cells and gastrointestinal cells 1–2 days after exposure[2]. This causes activation and recruitment of the immune cells which enhance the inflammatory response, and instigate a second wave of necrotic cell death due to increased production of not only ROS but also pro-inflammatory cytokines, chemotactic factors, and lipid mediators culminating in the translocation of bacteria from intestine to bloodstream [3]. This second wave provides a potential therapeutic window for intervention. NACHT, LRR and PYD domains containing protein 3 (NLRP3) has been implicated to have a role in the necrotic death. NLRP3 inflammasome is a multiprotein complex activated during infection or stress. Activation of this complex leads to development of canonical pyroptosis through caspase-1 yielding key mediators such as cleaved IL-1β, IL-18, and gasdermin D (Gsd-D)[4]. Cleavage of gasdermin-D by caspase-1 yields the active N-terminal form which, through the intermediary binding to phosphoinositides in the plasma membrane, gets oligomerized to form pores ultimately resulting in cell death [5,6]. Total body irradiation (TBI) was shown to activate the inflammasome in a dose, cell-type, and time dependent manner in all immune cells, epithelial cells, and bone marrow derived macrophages[7]. In addition to the production of the inflammatory cytokines IL-1β and IL-18, pyroptosis can cause release of extracellular inflammatory mediators IL-1, HMGB-1, heat-shock proteins and ATP, which further enhance the generation of proinflammatory cytokines via the activation of pattern-recognition receptors [6]. Inhibition of the NLRP3 inflammasome is an emerging strategy for targeting inflammatory diseases. Among the small molecule inhibitors is MCC950 shown to be a potent and specific NLRP3 inhibitor directly interacting with its Walker B motif within the NACHT domain, and blocking ATP hydrolysis, NLRP3 activation and inflammasome formation [8]. Thus, MCC950 serves as a prime treatment to determine its effectiveness in a murine ionizing radiation model.

Our main objectives included characterizing the time course and cell type involvement of pyroptotic mediators in mouse ileum as well as the efficacy/mechanism of MCC950 post-TBI. Complete methodology is provided in the supplementary material. To study the time course, mice were irradiated at a dose of 9.25 Gy and the ileum was harvested at specified - time points to determine expression of pyroptosis related proteins NLRP3 and caspase-1 with naïve mice ileum as a control. We observed a time dependent increase - of both NLRP3 and caspase-1 expression in ileum particularly on day 3 post-TBI (Figure 1A,B). While ileal whole tissue homogenates provided information on the global expression of NLRP3 and caspase-1, the specific cell types undergoing pyroptosis were not evident. To investigate these cell types, we isolated ileal CD326+ epithelial cells and F4/80+ macrophages using flow cytometry on day 2 and day 5 post-irradiation (9.25 Gy, Figure S1) with naïve mice ileal cells as a control. Of note, CD326+ epithelial cells did not have significant elevations of NLRP3, cleaved caspase-1, cleaved IL-_*1β, or cleaved gasdermin D levels (Figure S1). In contrast, F4/80+ macrophages displayed significant elevations of pyroptotic markers with NLRP3 most elevated on day 2 alongside cleaved caspase-1 being significantly elevated on day 5 with a general elevation trend over time. These results coincided with elevations in cleaved IL-1β and gasdermin D which require enzymatic cleavage by caspase-1 making this consistent with the known canonical pyroptosis pathway [4] (Figure 1C–F).

Figure 1. Total body irradiation elicits pyroptosis in time and cell dependent manner with inhibition via MCC950 enhancing survival in mouse model.

(A-B) Time course of A) NLRP3 (n=3–5/group) and B) caspase-1 (n=3–5/group) expression in ileum harvested from C57BL/6J female mice after 9.25 TBI. Data are mean + SD. *p < 0.05 vs Day 0. (C-F) Time course (day 2 [IRD2] and 5 [IRD5]) of pyroptotic markers in F4/80+ macrophages of ileum harvested from C57BL/6J female mice after 9.25 TBI acquired by flow cytometry. Expression of C) NRLP3, D) active caspase-1, E) cleaved IL-1β, and F) cleaved gasdermin D (GSDMDC) markers presented as percentage of CD45+ leukocytes (n=5/group). Data are mean + SD. *p < 0.05 vs nonirradiated control (Cntr). (G-J) Survival curves demonstrating that MCC950 (50 mg/kg i.p.) significantly improved survival vs vehicle when given 24h after G-H) 9.25 Gy (n=20/group, HR=0.45 and n=10/group, HR=0.38 respectively) and I) 9.50 Gy total body irradiation in female C57BL/6J mice (n=10/group, HR=0.28) as well as being the lowest effective dose amongst all tested concentrations. (J) A similar survival benefit also seen in male C57BL/6J mice (n=20/group, HR=0.50). Statistical significance with p-values provided on the plots as determined through log-rank test comparing treated and untreated groups. (K-M) Western blots showing MCC950 (50 mg/kg) (K) enhances levels of full-length IL-1β in ileum and (M) diminishes cleaved IL-1β in the spleen after 9.25 Gy TBI. (K) Expression of full-length IL-1β in ileum harvested from male C57BL/6J mice at day 3 after 9.25 Gy TBI followed by administration of vehicle or MCC950 24 hours post irradiation (n=5/group). (L) Expression of full-length IL-1β and (M) cleaved IL-1β in spleen harvested on day 3 from the same mice (n=5/group). Data are mean + SD. *p < 0.05 vs vehicle (9.25 Gy). (N-O) MCC950 administration (50 mg/kg) at 24h after 9.25 Gy TBI attenuates accumulation of oxidized cardiolipin (CL) species, N) CL (72:5+4[O]) and O) CL (72:7+4[O]), in the ileum of irradiated mice vs vehicle. Data are mean + SD. *p < 0.05 vs vehicle (9.25 Gy). Statistical significance for all other experiments determined by Student’s t-test unless otherwise specified. Additional western blots for A-B and flow cytometry plots for C-F are provided in the supplementary material. HR = Hazard Ratio

As NLRP3 was particularly elevated post-TBI, we decided to utilize MCC950, in survival studies to determine if inhibition has radiomitigative effects using a log-rank test to determine statistical significance and hazard ratios (Figure G-J). To determine the most effective dose of MCC950, we performed a survival analysis with C57BL/6NTac female mice irradiated to 9.25 Gy with concentrations of MCC950 ranging from 0 mg/kg-50mg/kg administered in vehicle (50:50 vol cremophorel: ethanol to 1:3 saline) at 24h after TBI. We found that the lowest effective dose of MCC950 is 50 mg/kg (Figure 1G). In confirmation experiments, female mice irradiated to 9.25 Gy and 9.50 Gy were administered MCC950 at a concentration of 50 mg/kg 24h after TBI demonstrating a significant survival benefit in the treated group (Figure 1H/I). To elucidate if this was a sex specific effect, male C57BL/ 6NTac were treated in the same manner showing a similar benefit regardless of sex (Figure 1J). These results suggest that targeting canonical pyroptosis through NLRP3 may be an effective strategy for mitigating the effects of TBI.

To explore the radiomitigative mechanisms of MCC950, we irradiated two groups of male C57BL/ 6NTac mice to 9.25 Gy, then administered one group with vehicle and the other with MCC950 (50 mg/kg) 24h post TBI. Since NLRP3 and caspase-1 were most significantly elevated on day 3 in ileum, we sacrificed the mice at that time point and harvested their ileum and spleen to determine changes in their downstream pyroptotic mediator, IL-1β. A Western blot of the harvested ileum showed a significant increase in the expression of full-length IL-1β in MCC950 treated mice (despite being unable to probe cleaved IL-1β) (Figure 1K). Further, we observed a significant decrease in the expression of cleaved IL-1β with MCC950 treatment in the spleen (Figure 1L/M). We next examined oxidized species of mitochondrial phospholipid, cardiolipin (CLox), which has been shown to be a marker of pyroptosis [98], in ileum of mice treated with MCC950 vs vehicle post-TBI using redox lipidomics approach. We found that the levels of two individual CLox molecular species containing four oxygens were significantly elevated in the ileum of mice exposed to TBI. Treatment of mice with MCC950 24h post-TBI resulted in a significant decrease in TBI-induced accumulation of CLox - in line with observed robust radiomitigative effect (Figure 1N and Figure S2).

In conclusion, we found that canonical pyroptosis is an important cell death pathway involved in the pathogenesis of ionizing radiation. Many of these pyroptotic mediators such as NLRP3 and caspase-1 were found to be highly expressed in a delayed manner peaking between 3–4 days. Our flow cytometry experiments demonstrated that NLRP3 activation, elevation in catalytically active caspase-1 and increases in cleaved IL-1β and cleaved GsdD downstream products occur in immune cells as early as day 2. Interestingly, TBI at 9.25 Gy has been shown to decrease levels of full-length IL-1β in intestine and bone marrow at the 1- and 2-day mark respectively which would be supported by our study [3].

To our knowledge, we are the first to show that MCC950 serves as a radiomitigator after exposure to TBI regardless of sex.. The efficacy of MCC950 has already been established in several inflammatory disease models (traumatic brain injury, diabetes, and inflammatory bowel disease). MCC950 and the next generation NLRP3 inhibitors currently in development could prove to have clinical utility in inflammatory disease states [10–12]. They may also have clinical utility in an emergency setting of an ionizing radiation attack to protect against the delayed necrotic cell death and improve outcomes for victims. More practically, NLRP3 inhibitors may also protect against radiation enteritis during cancer treatment since we have shown elevation of pyroptotic mediators in ileum.

In contrast to our study, a recent report on the knockdown of various inflammasome components (Nlrp3, Casp1/11, Aim2) indicated a potential protective role upon ionizing radiation exposure in male mice[13]. Here, we report that inhibition of NLRP3 and presumedly the downstream mediators of caspase-1/GsdD/IL-1β confers a radiomitigative role in both male and female mice which would seemingly be in partial contradiction with previous reports. This may be explained by gene knockdowns exacerbating the translocation of microbial components as well as components of the pathway serving in a dual protective/sensitizing role in TBI [14]. From a mechanistic standpoint, knockout of the NLRP3 radiosensitizing effect in mice through the upregulation of the cGAS-STING pathway has been linked to the development of inflammatory disease[15]. Therefore, it is possible that attenuation rather than eradication of NLRP3 function with MCC950 provides the protective effect. Differences in the microbiota between mice may be - crucial in the response to irradiation due to the role pyroptosis plays in the defense against pathogens[13]. Findings consistent with our study is that the knockdown of AIM2 which mediates caspase-1 dependent pyroptosis through sensing double stranded DNA release was radioprotective in a similar mouse model[16].

While it is known that ileum is sensitive to TBI, we did not see a decrease in cleaved IL-1β in response to MCC950 treatment. However, there was an increase in full-length IL-1β in mouse ileum potentially due to inhibition of its cleavage. It is possible that the cleaved IL-1β was diluted due to the more numerous epithelial cells and predilection for pyroptosis in immune cells. Of note, lipidomics experiments showed significant attenuation of TBI-induced CLox accumulation in ileum after MCC950 treatment suggesting that pyroptosis and subsequent survival benefit can be attributed to MCC950’s effect in ileal tissue despite our inability to probe cleaved IL-1β [9]. Interestingly, we found a significant decrease of cleaved IL-1β in the spleen suggesting that this is a likely site of action for MCC950 as it is principally a lymphoid organ.

Among the limitations, we note that we utilized young 8–12 weeks mice thus our findings cannot be generalized to juvenile or old mice and future studies will be necessary to evaluate radiomitigative potential of MCC950 in these age groups. Furthermore, evaluation of full-time course of pyroptosis markers in spleen and other immune tissues and the effect of MCC950 on these markers post-TBI might be revealing. We were limited in our assessment of pyroptosis markers and the effect of MCC950 within the acute post-TBI period and potential late effects of TBI were not assessed.

In conclusion, this study provides further insight into exploration of the mechanism through which the delayed pyroptotic phenotype occurs thus identifying potential therapeutic targets that can be used in the aftermath of an ionizing radiation injury.