Heat Killed Salmonella typhimurium protects intestine against radiation injury through Wnt signaling pathway

doi:10.21203/rs.3.rs-96695/v1

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Heat Killed Salmonella typhimurium protects intestine against radiation injury through Wnt signaling pathway

https://doi.org/10.21203/rs.3.rs-96695/v1

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published 18 Jun, 2021

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Purpose: Gastrointestinal (GI) toxicity caused by ionizing radiation (IR) appears to be a limited factor in radiotherapy and a great threat for soldiers on nuclear-related military missions. However, there are currently no approved medical strategies to effectively prevent or mitigate the damage on intestine induced by IR. The present study aimed to elucidate the protective activity of Heat Killed Salmonella typhimurium (HKST) on intestine against ionizing radiation exposure.

Materials and Methods: Mouse intestinal crypts and intestinal organoids were isolated from C57BL/6J mice. Mice and HIEC cells were exposed to ⁶⁰Co γ‐radiation. The dosage was set as following: whole body radiation of 8 Gy; mouse intestinal organoids were irradiated with 0 Gy, 4 Gy, and 6 Gy of γ-rays at dose rate of 1 Gy/min. Cells or mice were pretreated with Heat Killed Salmonella typhimurium (HKST) (10⁷ cells/mice) at 12 hours prior to irradiation.

Results: By culturing mouse intestinal organoids and employing whole body irradiation of mice, we found that the radiation-induced damage on intestine was dramatically mitigated by pretreating with HSKT in vitro and in vivo, in which the structure of intestinal organoids and small intestine of the HKST-treated group was more integrated than of the radiation damage group, and HKST pretreatment remarkably promoted the proliferation of intestinal cells post IR exposure (Figure 1 and 2). Further study showed that the radio-protective effects of HKST was involved in DNA damage response (DDR) signaling. Moreover, the stimulation of DDR signaling by HKST upon radiation damage was mediated by Wnt signaling, in which the inhibition of Wnt signaling diminished the radio-protective effects of HKST.

Conclusion: Our study demonstrated that Heat Killed Salmonella typhimurium (HKST) could significantly alleviate the damage on intestine induced by IR, and the radio-protective effect of HKST was depended on DDR mediated by Wnt signaling pathway. These findings demonstrated that HKST is a potential bacterium using for the prevention of IR-induced GI toxicity in clinical practice.

Oncology

Heat Killed Salmonella typhimurium

intestinal injury

ionizing radiation

DNA damage response

Wnt signaling

Patients received radiotherapy and victims from nuclear accident unavoidably suffer from the damage induced by ionizing radiation (IR) [1]. Acute radiation sickness, which is also known as acute radiation syndrome (ARS), often occurs after a sudden high dose of IR exposure. At a dose less than 6 Gy, the fatal injuries are primarily as a result of hematopoietic syndrome that can be prevented by the bone marrow shielding or cured by transplant of bone marrow [2, 3]. Gastrointestinal (GI) tract represents other susceptible organ to IR injury. GI toxicity (the GI syndrome) primarily induced by a higher dose of radiation, in which a mass of intestinal stem cells (ISCs) are irreparably killed, the regeneration of villi is severely impaired and the epithelial integrity along the entire GI tract is compromised [4]. Victims with GI syndrome usually bear the pain of fluid loss, malabsorption, and electrolyte imbalances [5]. Besides, the invasion of enteric pathogens and flora into the bloodstream due to the epithelial integrity damage could lead to sepsis and death [6]. Unfortunately, there are currently no medical countermeasures approved to prevent or ameliorate GI syndrome [7]. Although a handful of Food and Drug Administration (FDA)–approved radioprotectors were reported to eliminate internally ingested radiation or scavenge free radical species, the unfavorable side effect profiles limit the treatment of patients on a large scale [8, 9]. There is a great need for agents to diminish radiation-induced injury on the intestine.

Toll-like receptors (TLRs) family agonists represent a series of effective agents in facilitating the nullification of the IR-induced injuries. Since the activation of TLR5 was demonstrated to counteract radiation-induced damage on mice and monkey [10], the activation of TLRs family, including TLR2/6, TLR4 and TLR9, have been reported to effectively alleviate irradiation-induced damage [11–13]. Nevertheless, as different TLRs distribute among various organs and initiate different signaling pathways, the radio-protective effects by co-activating multiple TLRs may be stronger than the stimulation of single TLRs. The evidence comes from that Escherichia coli O111:B4 LPS, the co-agonist of TLR4 and TLR2, exerts stronger immune stimulation effects than that TLR2 or TLR4 agonist used alone or the combined use of them [14].

Heat Killed Salmonella typhimurium (HKST) represents an potent co-agonist of TLR2 and TLR4 receptor, and immune system of mammalian cells can be stimulated as Salmonella typhimurium is recognized by multiple TLRs [15, 16]. Previous study have shown that the pretreatment with attenuated Salmonwlla typhimurium could induce protective cell-mediated immunity or facilitate the conversion of immunosuppressive myeloid-derived suppressor cells into TNF-α-secreting neutrophil-like myeloid cells thereby protecting mice against malaria or tumor [17, 18]. In our preliminary work, damages on bone marrow, spleen and testis which were induced by γ-irradiation could be significantly eased by the pretreatment of HKST [19]. However, the radio-protective effects of HKST on small intestine has remained unclear. In the current study, HKST was demonstrated to alleviate the radiation-induced injury on small intestine in vitro and in vivo, which was further confirmed to be in a Wnt signaling dependent manner.

Medium and reagentsHKST was purchased from InvivoGen Asia (Hong Kong, China) and suspended in phosphate buffered saline (PBS). PBS and RMPI1640 medium was obtained from Hyclone (Logan, UT). ICG-001 was obtained from Selleck Chemicals (Shanghai, China). IntestiCult Organoid Growth Medium (Mouse) was purchased from STEMCELL Technologies Inc. (Canada). Growth factor-reduced Matrigel was purchased from Corning Incorporated (USA) and stored in -20 ℃.

Mouse intestinal organoids culture and treatment

Mouse intestinal crypts isolation and intestinal organoids culture were performed as previously described with modest modification [20]. Briefly, small intestine was obtained after mice was euthanized with CO₂ and then washed with PBS followed by being longitudinally opened. Intestinal crypts were dissociated with PBS plus EDTA (15 mM). 250 crypts/well were suspended in the mixture composed of 75% Growth factor-reduced Matrigel and 25% IntestiCult Organoid Growth Medium. Enough medium was supplement after the Matrigel polymerized at cell incubator (37 ℃, 5% CO₂). For HKST treatment, HKST was dissolved in IntestiCult Organoid Growth Medium (10⁷ cells/ml) and the medium was replaced every 3 days. Optical image was taken after 7 days of cultivation.

Cell treatment

Cells (Human intestinal epithelial cells (HIEC), ATCC) were maintained in 1640 medium with 1% antibiotics from Gibco and 10% fetal bovine serum (Gibco, Australia) at 37 ℃ in a 5% CO₂ cell incubator. Cells were pretreated with HKST (10⁷ cells/ml) alone or in combined used with ICG-001 (25 µM) at 12 hours prior to IR.

Mice and treatment

Animals experiments were approved by the Ethic Committee of The Second Military Medical University according to the instruction about Care and Use of Laboratory Animals published by the US NIH (Publication No.96 − 01). Mice (C57BL/6J, Male, 6–8 weeks, Jihui Experimental Animal Breeding Co., Ltd) were housed in a room with a 12 h light/dark cycle, in which water and food were obtained ad libitum. HKST (Invivogen, US, Lot: HST-39-01) resuspended in PBS (10⁷/mice) was administrated through gavage 12 hours prior to irradiation.

Irradiation

Mice (in transparent boxes) and HIEC cells were exposed to γ-radiation (⁶⁰Co source, Second Military Medical University, China). The dosage was set as following: whole body radiation of 8 Gy, a dose rate of 1 Gy/min. Mouse intestinal organoids were irradiated with 0 Gy, 4 Gy, and 6 Gy.

Histopathology and immunohistochemistry

Mice were randomly divided into Control group, ionizing radiation (IR) group, IR plus HKST (IR + HKST) group, IR plus ICG-001 (IR + ICG-001) group, and IR plus HKST in combined with ICG-001 (IR + HKST + ICG-001) group, each group contained at least 3 mice. Mice were anesthetized with pentobarbital sodium (60 mg/kg) through intraperitoneal injection and pretreated with HKST (10⁷ cells/mice), ICG-001 (10 mg/kg), HKST (10⁷ cells/mice) in combined with ICG-001 (10 mg/kg) or placebo at 12 hours before 8 Gy IR exposure. At 3 days post irradiation, mice were sacrificed; small intestine was then isolated and fixed in paraformaldehyde and subjected to histological and immunohistochemical examination. Hematoxylin and eosin (H&E) staining was employed for the pathological morphology evaluation, in which the tissue slices were 3 µm. To detect the apoptosis rate in the small intestine, TdT-mediated dUTP Nick-End Labeling (tunel) assay was employed according to the manufacture instructions of the tunel kit (Roche, Basel, Switzerland). For the immunohistochemistry examination, tissue slices were stained with Olfm4 antibody (Cell Signaling Technology, Inc), and then the biotinylated secondary antibodies and DAB substrate kit were employed for immunohistochemistry.

Immunofluorescence assay

Cell-Light EdU DNA cell proliferation kit (C103102, RiboBio) was employed for the EDU staining. Briefly, intestinal organoids were culture with EdU (25 mM ) for 1.5 h at 37 ℃, after which the organoids were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. And then the organoids were reacted with 1 × Apollo cocktail (RiboBio). For immunofluorescence analysis, tissue slices were stained with antibody against ki67 (Abcam, USA) and phosphorylated P65 (Proteintech Group, China), and then the secondary antibodies were employed. The images of tissues were obtained using a fluorescent microscope (Olympus BX60, Center Valley, PA, USA) equipped with a digital camera (Retiga 2000R, Surrey, BC, Canada).

Western blot analysis

M-PER Mammalian Protein Extration Reagent was employed to extract proteins from irradiated cells at 8-hour post IR exposure. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The antibodies used to probe the membranes were listed as following: GAPDH (1:1000) was purchased from Proteintech (China); phosphorylated‐ATR (1:1000), ATR (1:1000), ATM (1:1000) and phosphorylated‐ATM (1:1000) was purchased from Cell Signaling Technology (USA); CHK1 (1:1000), phosphorylated‐CHK1 (1:1000), phosphorylated‐CHK2 (1:1000), CHK2 (Abcam, 1:1000) and p21 (1:1000) was purchased from Abcam (USA). Levels of proteins were by an ECL western blotting detection system (Thermo Fisher Scientific, Waltham) after the specific secondary antibodies (Servicebio, China) were probed.

Statistical analysis

Data of each experiment are presented as means ± the standard error of mean (SEM). Student’s t test was employed to evaluate the significant differences, in which P values less than 0.05 were considered significant. All the experiments were repeated for at least 3 independent times and the quantification were conducted in a blinded fashion.

HKST protected radiation-induced intestinal injury in mice

When exposed to a high dose of radiation, gastrointestinal toxicity remains a critical clinical issue to be settled. Thus, the radio-protective of HKST on intestine in vivo was investigated using whole body irradiation of mice. As is shown in Fig. 1A, after radiation exposure, the structure of intestine was heavily damaged when compared with the control group, in which the villus became shorter and the villus cells of the small intestine became discontinuous. HKST pretreatment effectively preserved the structure of intestine upon IR exposure, in which the length, density and the distribution uniformity of villus were remarkably improved (Fig. 1A). Accordingly, high apoptosis rate in the small intestine was induced upon IR exposure, and HKST pretreatment remarkably decreased the apoptosis rate as compared to the IR group (Fig. 1B).

HKST promoted proliferation of intestinal cells in vitro and in vivo post irradiation

Intestinal organoids were primarily established to mimic the regeneration of intestinal crypts in vitro [20, 21], we therefore detected whether the pretreatment of HKST could protect the intestinal organoids against IR-induced injury. As our result showed that the structure of intestinal organoids was destroyed by IR, in which the surface area of organoids and the height of organoids’ buds were dramatically decreased upon IR exposure as compared to the control group, and the destructive effect on organoids was elevated as the dosage of IR increased (Fig. 2A, B and C). Whereas, the structure of intestinal organoids was effectively preserved by HKST pretreatment upon IR damage, in which the budding number and surface area of organoids in the HKST-treated group were more striking than that of the IR group (Fig. 2A, B and C), indicating the protective effect of HKST on intestinal organoids against IR injury. With immunofluorescence staining, we found that the proliferation of cells in organoids and crypts of mice were inhibited in IR group, and the HKST pretreatment significantly promoted the proliferation of intestinal cells as compared to IR group (Fig. 2D, E and F).

Pretreatment with HKST stimulates DNA damage response upon IR exposure

DNA damage response (DDR) appears to be an essential mode for mammalian cells to maintain and modulate the genome integrity thereby nullifying IR-induced toxicity [22, 23], HIEC cells was employed to detect the effect of HKST on DDR upon radiation exposure. Our study showed that without IR exposure, there were minor differences of the levels of the kinases phosphorylated CHK2 (P-CHK2), phosphorylated ATM (P-ATM), phosphorylated ATR (P-ATR), phosphorylated CHK1 (P-CHK2) and p21 between the untreated group and the HKST-treated group (Fig. 3). Whereas, upon radiation damage, levels of P-ATR, P21, P-CHK1 were significantly increased in HKST-treated cells as compared to the IR damaged group (Fig. 3). With selective inhibitor of Wnt signaling named ICG-001 [24], the mechanism involved in the stimulation of DDR by HKST after IR exposure was determined. As is shown in Fig. 3, when compared with HKST-treated cells, there were minor changes on the levels of the kinases P-ATR, P-CHK1, P-ATM, P-CHK2 and p21 when combined used of HKST and ICG-001. However, after radiation exposure, the increased levels of these proteins by HKST were diminished with ICG-001, indicating the inhibition of DDR when the Wnt signaling was blocked.

Radioprotective effects of HKST on intestine was blocked by Wnt signaling inhibitor

The intestinal stem cells that are highly susceptive to IR damage represent the sources of intestine regeneration. By immunohistochemistry, we detected the levels of Olfm4 that is the marker of crypt base columnar (CBCs) stem cells in mice intestine [25]. In our result, level of Olfm4 in intestine was notably decreased upon IR injury as compared to the control group (Fig. 4), whereas the HKST pretreatment preserved the levels of intestinal stem cells, which again confirmed the protective effect of HKST on intestine against radiation damage. However, after adding ICG-001 into the HKST-treated group, the radio-protective activity of HKST on intestinal stem cells was significantly blocked (Fig. 4), indicating the radio-protective of HKST on intestine was blocked by Wnt signaling inhibitor.

HKST activated target cells in liver and spleen besides intestine

Besides intestine, we wonder whether HKST activates other cells and whether there is any correlation between the whole body radioprotection and intestinal protection. As most TLRs activates NF-kB as a downstream signaling pathway. To identify target cells of HKST, we perform a p65 staining in liver, spleen, bone marrow, which are tissues with high level of TLRs. Our data showed that upon HKST treatment, p65 expression and translocation was strongly activated in liver and spleen (Fig. 5). These findings suggests that the protective effects of HKST on intestine is not just a local activation of DNA damage repair in intestine, but also a whole body protective effects.

Since the intestinal epithelium is one of the fastest self-renewing tissue in mammals and therefore sensitive to IR damage, IR-induced GI toxicity is the limited factor in abdominal pelvic radiotherapy and a great threat to public health in the potential nuclear accident [26]. Nonetheless, there are no effective strategies approved to alleviate the pain of victims with GI syndrome, and the currently reported agents have unfavorable side effect profiles [8, 9]. The development of novel agents protecting against IR-induced intestinal damage is of great significance. Currently, the radio-protective effects of HKST on small intestine was investigated. As is shown in our results, the radiation-induced damage on intestine was dramatically mitigated by pretreating with HSKT in vitro and in vivo, in which the structure of intestinal organoids and small intestine of the HKST-treated group was more integrated than of the radiation damage group, and HKST pretreatment remarkably promoted the proliferation of intestinal cells post IR exposure (Figs. 1 and 2). HKST is the potent co-agonist of TLRs family which has been reported to alleviate the radiation-induced injury on bone marrow, spleen and testis in our previous study [19]. It has been demonstrated that the attenuated Salmonwlla typhimurium could be employed to stimulate immune system or developed into vaccine thereby protecting against malaria or tumor [17, 18]. Besides, the therapeutic use of bacteria in preventing IR-induced GI toxicity has begun to receive more attention [27–29]. Our study demonstrates that HKST is of great potential alleviating injury on intestine induced by radiation and facilitating the relief of victims with GI syndrome in clinical practice.

Generally, the critical target of IR is considered to be DNA [30]. Upon IR exposure, the irreparable DNA damage is induced and the unrepaired or error-repaired DNA damage subsequently leads to cellular damage or death [31]. DNA double-strand breaks (DSBs) is regarded as the most deleterious form of DNA damage, which can rise chromosomal aberrations, loss of genetic materials, the cell death, or other detrimental consequences [32]. Homologous recombination (HR) and non-homologous end joining (NHEJ) and are the two main modes to repair DSBs in mammalian cells, in which ATR and ATM are confirmed to be the key components of DNA damage response (DDR) pathway [22, 23]. As DDR is critical for the maintenance and modulation of genome integrity in intestinal cells after radiation exposure [33, 34], we speculated that the radio-protective effects of HKST might be involved in DDR. As our results show that, after radiation exposure, levels of P-ATM, P-ATR, P-CHK1 and p21 were significantly increased in cells pretreated with HKST (Fig. 3). The dysfunction of genome integrity caused by radiation damage requires DDR to prevent the transmission of incompletely replicated or damaged chromosomes. DNA damage repair reactions in mammalian cells require the cell cycle arrest by the activation of a DNA damage checkpoint [35]. ATM and ATR are the main regulators of two major checkpoint pathways, in which ATR-CHK1 checkpoint signaling is essential for HR repair pathway while the conversion of ATM to monomers by autophosphorylation and then phosphorylates CHK2 appear to be essential for the checkpoint response [36, 37]. In some condition, the cell-cycle arrest is initiated by ATR-CHK1 signaling but maintained by ATM-CHK2 signaling [38]. Cell cycle arrest will activate the tumor suppressor p53 which then targets its transcriptional protein named cyclindependent kinase inhibitor (p21) [39]. As the elevated levels of P-ATR, P-CHK1 and p21 after radiation exposure in HKST-treated cells were observed, and there were minor changes on P-ATM and P-CHK2, between HKST treatment and IR group, the ATR pathway might be more crucial in facilitating the protective effects of HKST against IR.

The maintenance and self-renew of intestinal cells, especially the stem cells, in adult mammalian requires Wnt signaling pathway, whereas the inactivation of the essential components of Wnt signaling leads to the deficiency in intestine development and regeneration [40, 41]. Previous study have demonstrated that DDR activation was amplified by Wnt signaling in mammalian cells, whereas the inhibition of Wnt signaling fail to resolve DSBs after radiation [42–44]. As the protective activity of HKST against radiation injury was confirmed to be related to DDR, and the pretreatment of HKST effectively preserved intestinal stem cells when exposed to IR (Fig. 4), the radio-protective effect of HKST might be mediated by Wnt signaling pathway. As expected, the stimulation of ATR and ATM signaling upon IR in HKST-treated cells and the radio-protective effects of HKST on intestinal stem cells in mice were diminished by the selective inhibitor of Wnt signaling pathway (Figs. 3 and 4). Previous study have shown that the kinases ATM/ATR and CHK1/2 were involved in the inflammatory responses induced by TLRs signaling [45], and TLRs signaling was reported to modulate the DNA repair reaction in cells [46]. As HKST is a co-agonist of TLRs, the role of TLRs signaling in DDR mediated by Wnt signaling pathway upon IR damage remains further investigation. As HKST is the co-agonist of TLRs family, and TLRs family are expressed in different organs. We presumed that HKST might activates other cells and the protective effects of HKST against IR is a whole body protective effects. As the results showed that, besides small intestine, HKST pretreatment significantly activates NF-kB signaling pathway in liver and spleen.

Gastrointestinal (GI) toxicity caused by ionizing radiation (IR) appears to be a limited factor in radiotherapy and a great threat for soldiers on nuclear-related military missions. However, there are currently no approved medical strategies to effectively prevent or mitigate the damage on intestine induced by IR. Our study demonstrated that Heat Killed Salmonella typhimurium (HKST) could significantly alleviate the damage on intestine induced by IR, and the radio-protective effect of HKST was depended on DDR mediated by Wnt signaling pathway. As HKST is a vaccine model employed to protect against facultative intracellular bacteria, our study provides a bacterium potentially used in clinical practice for IR-induced GI toxicity prevention.

GI: Gastrointestinal；IR: ionizing radiation；HKST: Heat Killed Salmonella typhimurium; DDR: DNA damage response; ARS: acute radiation syndrome; ISCs: intestinal stem cells; TLRs: Toll-like receptors; HIEC: Human intestinal epithelial cells;

HR: Homologous recombination; NHEJ: non-homologous end joining.

Ethics Approval and consent to participate

All the experiments were approved by the Second Military Medical University of China in accordance with the Guide for Care and Use of Laboratory Animals published by the US NIH (Publication No.96-01).

Consent for publication

Not applicable.

Availability of supporting data

Not applicable.

Competing interests

The data used to support the findings of this study are available from the corresponding author upon request. The authors declare that they have no conflicts of interest.

Funding

This study was supported in part by the grants from National Natural Science Foundation of China (No. 11605289, No. 31700739, No. 81892559), and Shanghai association for science and technology (No. 20184Y0167). The authors have no conflicts of interest to disclose.

Authors’ contributions

Cai and Gao designed the study; Chen, Cao and Hu performed the experiment; Liu, Li and Cui analyzed data and edited the figures; Chen wrote the paper and Yang modified the article. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Authors' information

¹Department of Radiation Medicine, Faculty of Naval Medicine, Second Military Medical University, Shanghai, P.R. China. ²Department of Gastrointestinal Surgery, Second Military Medical University First Hospital, Shanghai, P. R. China.

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published 18 Jun, 2021

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Heat Killed Salmonella typhimurium protects intestine against radiation injury through Wnt signaling pathway

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Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

Mouse intestinal organoids culture and treatment

Cell treatment

Mice and treatment

Irradiation

Histopathology and immunohistochemistry

Immunofluorescence assay

Western blot analysis

Statistical analysis

Results

HKST protected radiation-induced intestinal injury in mice

HKST promoted proliferation of intestinal cells in vitro and in vivo post irradiation

Pretreatment with HKST stimulates DNA damage response upon IR exposure

Radioprotective effects of HKST on intestine was blocked by Wnt signaling inhibitor

HKST activated target cells in liver and spleen besides intestine

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Journal Publication

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