Prosurvival Pathway Protects From Clostridioides difficile Toxin-Mediated Cell Death

Abstract

There is an urgent need for new nonantibiotic-based treatment strategies for Clostridioides difficile infection. C. difficile toxin B (TcdB) is a virulent factor that is essential for causing disease. Here, we investigated whether a survival-signaling pathway could protect against TcdB. We found significant increase in caspase-3 apoptotic activity in intestinal epithelial cells of mice exposed to TcdB. Subsequently, activation of the MIF-CD74-Akt prosurvival signaling pathway blocked TcdB-induced caspase-3 activity and intestinal epithelial cell death. This brief report provides proof-of-concept that targeting prosurvival pathways may represent a unique antibiotic-independent strategy for protecting against C. difficile toxin-mediated cell death.

New approaches are urgently needed for Clostridioides difficile infection. This study provides proof-of-concept for a nonantibiotic strategy against C. difficile by targeting cell survival pathway.

There is an urgent need for new nonantibiotic-based treatment strategies for Clostridioides difficile infection. C. difficile toxin B (TcdB) is a virulent factor that is essential for causing disease. Here, we investigated whether a survival-signaling pathway could protect against TcdB. We found significant increase in caspase-3 apoptotic activity in intestinal epithelial cells of mice exposed to TcdB. Subsequently, activation of the MIF-CD74-Akt prosurvival signaling pathway blocked TcdB-induced caspase-3 activity and intestinal epithelial cell death. This brief report provides proof-of-concept that targeting prosurvival pathways may represent a unique antibiotic-independent strategy for protecting against C. difficile toxin-mediated cell death.

Clostridioides difficile is the leading cause of infectious diarrhea in hospitalized patients. C. difficile infections (CDI) occurs after antibiotic use and almost all antibiotics increase the risk for CDI. Approximately 25% of patients experience recurrent CDI after initial infection and reinfected individuals have a 50% chance of another CDI. Recurrent infections are more difficult to treat and are associated with more hospitalizations and severe outcomes, including mortality [1]. For these reasons, there is an urgent need for new nonantibiotic approaches for the treatment and prevention of CDI.

C. difficile produces exotoxins, which are disease-causing virulence factors. C. difficile toxin B (TcdB) alone is capable of causing the full spectrum of diseases and is essential for disease pathogenesis [2–4]. TcdB causes apoptotic cell death of intestinal epithelial cells resulting in breakdown of the intestinal barrier with subsequent gut inflammation and damage [2–4]. Targeting TcdB-mediated cell death represents a promising alternative nonantibiotic strategy to combat CDI.

Akt, also known as protein kinase B, is a serine/threonine kinase that promotes cell survival by blocking apoptosis [5]. CD74 is the cell membrane receptor for the macrophage migration inhibitory factor (MIF) cytokine. MIF stimulation of CD74 on intestinal epithelial cells leads to Akt activation [6–8]. The aim of this study was to determine whether activating the prosurvival Akt pathway could protect against C. difficile toxin. We found that the MIF-CD74-Akt pathway protected intestinal epithelial cells from C. difficile toxin-mediated cell death.

METHODS

Mice

Experiments were performed with genotype-confirmed wild-type (WT) C57BL/6 and CD74 knockout (KO) mice in a C57BL/6 background [7].

Immunohistochemical Staining

TcdB (15 μg) in phosphate-buffered saline (PBS) or PBS control was injected into the cecum of WT and CD74 KO mice in vivo by laparotomy and the cecum tissues were harvested from mice 8 hours later as described previously [9]. Immunohistochemical staining was performed by the Biorepository and Tissue Research Facility at the University of Virginia. Mouse tissue was probed with cleaved (active) caspase 3 antibody (No. 9661; Cell Signaling Technology) at 1:500 dilution.

Generation of Primary Colon Epithelial Monolayers

Mouse primary colonic epithelial cells were created and maintained in culture with IntestiCult Organoid Growth Medium Mouse (Stemcell Technologies) as previously described [10].

MIF Stimulation and TcdB-Induced Cell Death

WT and CD74 KO colonic epithelial cells were stimulated with MIF at 100 ng/mL [7]. Cells were treated with TcdB (1 µg/mL) for 16 hours as previously described [11]. Propidium iodide incorporation is an effective method for detecting late apoptotic cells, including C. difficile-toxin mediated apoptotic cell death, and was used for cell death assays [11–13]. Cells were stained with propidium iodide at 1 μg/mL. Plates were incubated at room temperature for 10 minutes. Fluorescence was measured at room temperature with an excitation and emission of 544 and 620 nm, respectively.

Immunoblotting

For immunoblotting, samples were resolved on polyacrylamide gels and transferred to nitrocellulose membrane (Bio-Rad). Blots were blocked with bovine serum albumin and then probed for proteins with the following antibodies: pan-Akt (No. 4691S; Cell Signaling Technology), phospho-Akt (No. 4060S; Cell Signaling Technology), procaspase 3 and cleaved (active) caspase 3 (No. 9662S and No. 9664S, respectively; Cell Signaling Technology), and actin (Sigma).

Statistical Analysis

Statistical differences were determined using Mann-Whitney U test and analysis of variance (ANOVA) followed by post hoc test. P values of less than .05 were considered statistically significant.

Study Approval

All animal procedures were approved by the University of Virginia Institutional Animal Care and Use Committee (IACUC). All animal studies were performed in compliance with the federal regulations set forth in the Animal Welfare Act, the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the guidelines of the University of Virginia IACUC.

RESULTS

TcdB causes death of intestinal epithelial cells (IECs) by inducing apoptosis. However, these findings are based mainly on in vitro studies [11, 14, 15]. Therefore, we investigated whether TcdB could induce IECs apoptosis in vivo. Caspase 3 is a key mediator of apoptotic cell death that executes the cell death pathway. We found significantly higher expression of activated caspase 3 in intestinal epithelium of mice exposed to TcdB as analyzed by immunohistochemical staining (Figure 1). This is in keeping with prior findings that caspase 3-mediated apoptosis plays an essential role in TcdB-induced cell death. Given that Akt activation is antiapoptotic and MIF-CD74 interaction activates Akt, we hypothesized that the MIF-CD74-Akt pathway would result in the inhibition of TcdB-induced apoptotic cell death. We observed reduction in TcdB-mediated caspase 3 activation and cell death in MIF-stimulated intestinal epithelial cells expressing CD74. This protective effect was not found in knockout cells that lack CD74 (Figure 2), suggesting that the pathway blocks TcdB-induced cell death.

Figure 1.

Clostridioides difficile toxin B (TcdB) induces apoptosis of intestinal epithelial cells. A and B, Immunohistochemical analysis of intestinal active caspase 3 expression in mice after intraluminal injection with TcdB or control (n = 7 per group). Scale bar: 100 μm. ***P < .001.

Figure 2.

MIF-CD74-Akt prosurvival pathway protects against TcdB-induced intestinal epithelial cell death. A, Schematic illustration showing MIF-CD74-Akt pathway. B, MIF-CD74 interaction activates Akt pathway. Immunoblot analysis of Akt phosphorylation in WT and CD74 KO intestinal epithelial cells stimulated with MIF. Actin served as a loading control. C and D, Inhibition of TcdB-mediated cell death. Immunoblot analysis of caspase 3 activation and evaluation of cell death of MIF-stimulated WT and CD74 KO intestinal epithelial cells treated with TcdB. Immunoblots are representative of 3 independent experiments, cell death data represent mean and SD of triplicates from 1 experiment and are representative of 3 independent experiments. ***P < .001. Abbreviations: KO, knockout; MIF, macrophage migration inhibitory factor; ns, not significant; TcdB, C. difficile toxin B; WT, wild type.

DISCUSSION

According to the Centers for Disease Control and Prevention (CDC), C. difficile is an immediate public health threat that requires urgent action. Current therapies are associated with unacceptably high treatment failure, recurrence, and re-recurrence rates. Hence, new treatment strategies are needed, especially nonantibiotic approaches to fight this pathogen. In this study, we provide proof-of-concept that activating prosurvival pathway protects intestinal epithelial cells from TcdB-mediated damage. A limitation is that administration of MIF has the potential to lead to an excessive inflammatory state. This is because in addition to the prosurvival effects, MIF is capable of stimulating receptors other than CD74, such as chemokine receptors that have proinflammatory effects [6]. Generating a selective agonist that will stimulate CD74-mediated prosurvival effects with minimal or no unwanted side effects might be a promising approach to overcome this limitation. C. difficile toxin-mediated cell death leads to extensive epithelial erosion, compromising the gut epithelial barrier, resulting in inflammation and tissue damage. Preserving the gut barrier by targeting prosurvival pathways may represent a unique therapeutic strategy for improving C. difficile colitis outcomes and presents an opportunity to reduce our dependence on antibiotics.

Notes

Author contributions. W. T., I. S., H. O., and S. M. conducted experiments. W. T. and S. M. wrote the article.

Acknowledgments. We thank Hanping Feng, PhD (University of Maryland), Cirle Warren, MD and William Petri, MD, PhD (University of Virginia), and Richard Bucala, MD, PhD (Yale University) for the support; and the University of Virginia's Research Histology, and Biorepository and Tissue Research facilities.

Financial support. This work was supported by the National Institutes of Health (grant number R01DK131313); and the Hartwell Foundation (to S. M.).