Host chitinase 3-like-1 is a universal therapeutic target for SARS-CoV-2 viral variants in COVID-19
- Department of Molecular Microbiology and Immunology, Brown University, United States
- Brown University, United States
Figures
Figure 1
CHI3L1 stimulation of pseudovirus uptake.
Calu-3 cells were incubated with recombinant human (rh) CHI3L1 (CHI3L1, 250 ng/ml) or vehicle (PBS) control for 48 hr. Pseudoviruses (PS) that contain S proteins with the (A) G614, (B) alpha, (C) beta, (D) gamma, or (E) delta mutations were added, and GFP was quantitated by fluorescence-activated cell sorting (FACS). The percentage of GFP-positive cells was evaluated by flow cytometry. The noted values are representative of a minimum of three similar evaluations.
Figure 2
Effects of FRG on G614, alpha, beta, gamma, and delta pseudovirus infection.
Calu-3 cells were incubated with rhCHI3L1 (CHI3L1, 250 ng/ml) or vehicle control for 48 hr in the presence of anti-CHI3L1 (FRG) or its isotype control (IgG). Pseudoviruses (PS) that contain S proteins with the (A) G614, (B) alpha, (C) beta, (D) gamma, or (E) delta mutations were added, and GFP was quantitated by fluorescence-activated cell sorting (FACS). The percentage of GFP-positive cells was evaluated by flow cytometry. The noted values are representative of a minimum of three similar evaluations.
Figure 3
Immunocytochemical evaluation of delta pseudovirus infection of Calu-3 cells.
Calu-3 cells were incubated in the presence and/or absence of rhCHI3L1 (CHI3L1) in the presence of FRG or its isotype control. (A) Pseudoviruses with delta S proteins (PS-δ) were added, and ACE2 and GFP viral infection were evaluated using double-labeled immunocytochemistry (ICC). DAPI (blue) was used to evaluate nuclei, red label was used to evaluate ACE2, and the pseudoviruses contained GFP. (B, C) The quantification of ACE2 can be seen in panel (B), and the quantification of GFP is illustrated in panel (C). These evaluations were done using fluorescent microscopy (×20 of original magnification). In these quantifications, five randomly selected fields were evaluated. The values in panels (B, C) are the mean ± SEM of the noted five evaluations. **p<0.01; ***p<0.001, ****p<0.0001; ns, not significant (one-way ANOVA with multiple comparisons). Scale bar:10 μm (applies to all subpanels in A).
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Figure 3—source data 1
Composite images of immunocytochemical evaluation of delta pseudovirus infection of Calu-3 cells.
- https://cdn.elifesciences.org/articles/78273/elife-78273-fig3-data1-v2.pdf
Figure 4
Kasugamycin inhibition of CHI3L1-induced signaling.
Calu-3 cells were stimulated with rhCHI3L1 (250 ng/ml) or its vehicle control for 2 hr in the presence of kasugamycin (250 ng/ml) and vehicle control (PBS). (A, B) Western blotting and densitometry analysis were then employed to evaluate the levels of activated (phosphorylated) (p) and total ERK (A) and AKT (B). The noted figure is representative of a minimum of three similar experiments. ***p<0.0001 (t-test).
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Figure 4—source data 1
Uncut full gel photo for Western blots used in Figure 4.
- https://cdn.elifesciences.org/articles/78273/elife-78273-fig4-data1-v2.pdf
Figure 5
Effects of kasugamycin on alpha, beta, gamma, and delta pseudovirus infection.
Calu-3 cells were incubated with rhCHI3L1 (250 ng/ml) or vehicle control for 48 hr in the presence of kasugamycin or its vehicle control. Pseudoviruses that contain S proteins with the (A) G614, (B) alpha, (C) beta, (D) gamma, or (E) delta mutations were added, and GFP was quantitated by fluorescence-activated cell sorting (FACS) analysis. The percentage of GFP-positive cells was evaluated by flow cytometry. The noted values are representative of a minimum of three similar evaluations.
Figure 6
Immunocytotochemical evaluation of delta pseudovirus infection of Calu-3 cells.
Calu-3 cells were incubated in the presence and/or absence of CHI3L1 (250 ng/ml) in the presence or of kasugamycin (250 ng/ml) or its vehicle control. (A) Pseudoviruses with delta S proteins were added, and ACE2 and GFP viral infection were evaluated using double-labeled immunocytochemistry (ICC). DAPI (blue) was used to evaluate nuclei, red label was used to evaluate ACE2, and the pseudoviruses contained GFP. (B, C) The quantification of ACE2 can be seen in panel (B), and the quantification of GFP is illustrated in panel (C). These evaluations were done using fluorescent microscopy (×20 of original magnification). In these quantifications, five randomly selected fields were evaluated. The values in panels (B, C) are the mean ± SEM of the noted five evaluations. *p<0.05, **p<0.01; ***p<0.001; ns, not significant (one-way ANOVA with multiple comparisons). Scale bar:10 μm (applies to all subpanels in A).
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Figure 6—source data 1
Composite images of immunocytotochemical evaluation of delta pseudovirus infection of Calu-3 cells.
- https://cdn.elifesciences.org/articles/78273/elife-78273-fig6-data1-v2.pdf
Figure 7
Effects of FRG and kasugamycin on omicron pseudovirus infection.
Calu-3 cells were incubated with rhCHI3L1 (CHI3L1, 250 ng/ml) or vehicle control for 48 hr in the presence or absence of anti-CHI3L1 (FRG) or its isotype control (IgG) or kasugamycin or vehicle control. Pseudoviruses that contain S proteins with the omicron mutations (PS-ο) were added, and GFP was quantitated by fluorescence-activated cell sorting (FACS). The percentage of GFP-positive cells was evaluated by flow cytometry. The noted values are representative of a minimum of three similar evaluations.
Additional files
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Supplementary file 1
Pseudoviruses containing S protein mutations of COVID variants used in this study.
- https://cdn.elifesciences.org/articles/78273/elife-78273-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/78273/elife-78273-transrepform1-v2.pdf
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Host chitinase 3-like-1 is a universal therapeutic target for SARS-CoV-2 viral variants in COVID-19
eLife 11:e78273.
https://doi.org/10.7554/eLife.78273
