Diosmectite inhibits the interaction between SARS-CoV-2 and human enterocytes by trapping viral particles, thereby preventing NF-kappaB activation and CXCL10 secretion

Abstract

SARS-CoV-2 enters the intestine by the spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors in enterocyte apical membranes, leading to diarrhea in some patients. Early treatment of COVID-19-associated diarrhea could relieve symptoms and limit viral spread within the gastrointestinal (GI) tract. Diosmectite, an aluminomagnesium silicate adsorbent clay with antidiarrheal effects, is recommended in some COVID-19 management protocols. In rotavirus models, diosmectite prevents pathogenic effects by binding the virus and its enterotoxin. We tested the trapping and anti-inflammatory properties of diosmectite in a SARS-CoV-2 model. Trapping effects were tested in Caco-2 cells using spike protein receptor-binding domain (RBD) and heat-inactivated SARS-CoV-2 preparations. Trapping was assessed by immunofluorescence, alone or in the presence of cells. The effect of diosmectite on nuclear factor kappa B (NF-kappaB) activation and CXCL10 secretion induced by the spike protein RBD and heat-inactivated SARS-CoV-2 were analyzed by Western blot and ELISA, respectively. Diosmectite bound the spike protein RBD and SARS-CoV-2 preparation, and inhibited interaction of the spike protein RBD with ACE2 receptors on the Caco-2 cell surface. Diosmectite exposure also inhibited NF-kappaB activation and CXCL10 secretion. These data provide direct evidence that diosmectite can bind SARS-CoV-2 components and inhibit downstream inflammation, supporting a mechanistic rationale for consideration of diosmectite as a management option for COVID-19-associated diarrhea.

Figure 1

Trapping of viral particles by diosmectite. Diosmectite was incubated with spike protein RBD (a) and heat-inactivated severe acute respiratory syndrome coronavirus 2 (CoV-2) (b) at different concentrations. The suspension was then probed with the anti-spike protein RBD antibody. Diosmectite alone was used as a negative control (CTRL). Images are at × 1000 magnification and represent three separate experiments.

Figure 2

Spike-adhesion to the Caco-2 cell surface in the absence (a) or presence (b) of diosmectite, as shown by immunofluorescence microscopy. Spike protein RBD is shown after being probed with the primary and secondary fluorescein isothiocyanate-conjugated antibody (green). Nuclei were stained using Hoechst (blue). Images shown are × 1000 magnification. A detail of a single cell is shown in the square. (a) Caco-2 cells incubated with spike protein RBD. (b) Caco-2 cells pre-incubated with diosmectite and co-incubated with spike protein RBD.

Figure 3

NF-kappaB activation and preventive effect of diosmectite. (a) Western blot analysis of protein lysates from Caco-2 cells treated for 24 h with different concentrations of spike protein RBD (Spike) and heat-inactivated SARS-CoV-2 (CoV-2) as indicated. (b) Western blot analysis of protein lysates from Caco-2 cells treated with spike protein RBD (100 ng/mL) and heat-inactivated CoV-2 (100 ng/mL) for 24 h alone and after pretreatment with diosmectite (DS). The upper line was blotted with anti-pNF-kappaB antibodies and the lower line was blotted with anti-tubulin antibodies as a loading control. A representative image from three independent experiments is shown. The relative levels of pNF-kappaB were normalized to tubulin levels. Bars indicate the means and lines the standard deviations of the three independent experiments. Student’s t-test. *p ≤ 0.05. SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, DS diosmectite, NF-kappaB nuclear factor kappaB, NT not treated, p65 primary antibody for NF-kappaB, pNF-kappaB phosphorylated NF-kappaB. Full-length blots/gels are presented in Supplementary Fig. S3.

Figure 4

CXCL10 secretion and preventive effect of diosmectite. (a) Levels of CXCL10 measured by ELISA in supernatants of Caco-2 cell cultures treated for 24 h with different concentrations of spike protein RBD (Spike) and heat-inactivated SARS-CoV-2 (CoV-2) as indicated. (b) Levels of CXCL10 measured by ELISA in supernatants of Caco-2 cell cultures treated for 24 h with spike protein RBD (100 ng/mL) and heat-inactivated SARS-CoV-2 (100 ng/mL) alone and after pretreatment with diosmectite (DS). Bars indicate the means and lines the standard deviations of three independent experiments. Student’s t‑test. *p ≤ 0.05. ELISA enzyme-linked immunosorbent assay, DS diosmectite, NT not treated, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2.