Fig 2: CHI3L1 stimulates ACE2 and SPP in vitro and enhances S protein processing and cellular integration.(A) Calu-3 lung epithelial cells were incubated with the noted concentrations of recombinant CHI3L1 (rCHI3L1; ng/mL) or vehicle control (rCHI3L1 = 0) for 24 hours and were then subjected to RT-qPCR to quantitate the levels of mRNA encoding ACE2 and the SPP. (B) Western blot evaluations of the dose response and kinetics of CHI3L1 stimulation of ACE2 and SPP protein accumulation in Calu-3 cells. (C and D) Calu-3 cells were incubated with vehicle (PBS; CHI3L1–) or rCHI3L1 (500 ng/mL) for 24 hours, recombinant S protein of SC2 was added, and the incubation continued for an additional 2 hours. The cell lysates were prepared, Co-IP and immunoblot assays were undertaken (C), and the uncleaved S and cleaved S1 and S2 proteins were evaluated using Western immunoblotting (D). (E) Calu-3 cells were incubated with vehicle (rCHI3L1–) or the noted concentrations of rCHI3L1 for 24 hours. They were then transfected with a pseudovirus containing the S protein (PS; D614 variant) from SC2 and a GFP expression construct and incubated for additional 24 hours and then evaluated using fluorescence microscopy. Quantification of mean fluorescence intensity (MFI) can be seen in the dot plot on the right. (F) Calu-3 cells were incubated with rCHI3L1 or vehicle control for 48 hours in the presence or absence of antibody against CHI3L1 (FRG) or control antibody (IgG). Pseudovirus S (PS; D614 and G614 variants) that expressed GFP was added as noted, and GFP-positive cells (%) were evaluated by flow cytometry. Values in A and E are mean ± SEM. B–F are the representative data obtained from at least 3 independent experiments. ß-Actin or GAPDH was used as an internal control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA with post hoc Dunnett’s multiple comparison test). Scale bar: 50 µm.

Fig 3: S protein increases ACE2 expression in pericytes in a dose- and time-dependent manner. A) Western blot analysis shows that ACE2 expression in human brain vascular pericytes rapidly increases 6 h after exposure to 5 nM of S protein, reaching a peak at 15 nM. B) Western blot analysis shows that ACE2 expression in brain pericytes remains elevated 24 h after exposure to 10 nM of S protein compared to vehicle. C) XTT cell viability assay shows that exposure to 15 nM of S protein for 6 h attenuates the survival of brain pericytes. D) XTT cell viability assay shows that prolonged exposure to S protein for 24 h further attenuates the survival of brain pericytes. Data are mean ± SEM (n = 3 independent experiments/ condition; XTT cell viability assay n = 12 wells/ condition). *P < 0.05/ **P < 0.01 compared with vehicle-treated pericytes (two-tailed unpaired t-test). Vehicle, ddH2O; 6H, 6 hours; 24H, 24 hours; XTT, 2/3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.

Fig 4: SARS-CoV-2 intranasal infection induces pericyte reactivity in the brain. A) Representative images of immunofluorescence staining show that ACE2 (red) is co-localized with vascular pericytes labelled with desmin (green) in the brain of wildtype mice. B) Analysis of immunofluorescence intensity indicates that PDGFRß expression is induced in the brain of K18-hACE2 transgenic mice 4 days following intranasal infection with SARS-CoV-2, as well as in the ischemic ipsilateral hemisphere of C57BL6/J wildtype mice 3 days after cerebral ischemia induction via middle cerebral artery occlusion (MCAo). C) Analysis of immunofluorescence intensity indicates that ACE2 expression increases in the brain vasculature of K18-hACE2 transgenic mice 4 days following intranasal infection with SARS-CoV-2 as well as in the ischemic ipsilateral hemisphere of C57BL6/J wildtype mice 3 days after ischemic stroke. D) His-Tag ELISA analysis shows that the poly-histidine-tagged S protein is present in different brain regions 24 h after intranasal infusion. Data are mean ± SEM (n = 3 animals/ condition). **P < 0.01 compared with contralateral hemisphere (one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison post-hoc test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 5: Nicotine vapor inhalation increases ACE2 mRNA and protein expression in male, but not female, lung. (a-b) After five consecutive days of vapor exposure, lung tissue was analyzed for the expression of ACE2 mRNA. (a) Increased ACE2 mRNA expression was found in lung tissue of male mice exposed to nicotine vapor, as compared to control (no vapor exposure) and vehicle vapor exposure. (b) In females, no significant differences were found in ACE2 mRNA expression among exposure conditions. (c-d) Lung tissue was also examined for ACE2 protein expression. (c) Quantification of the number of ACE2-positive cells in lung tissue revealed that nicotine vapor significantly increased ACE2 protein expression in male mice, as compared to the control and vehicle vapor groups. (d) The number of ACE2-positive cells did not differ among groups in female lung tissue. Individual data points are represented on each graph, and graph bars express as the mean ± SEM. ##p < 0.01 vs vehicle vapor, ###p < 0.001 vs vehicle, **p < 0.01 vs control, ***p < 0.001 vs control. (e-j) Representative images of ACE2 immunoreactivity (red) in lung tissue following exposure. In males, high levels of ACE2 immunoreactivity can be evidenced in lungs exposed to nicotine vapor (i), as compared to control (e) and vehicle vapor (g). For females, representative images demonstrate similar levels of lung ACE2 protein expression for the control (f), vehicle vapor (h) and nicotine vapor (j) groups. DAPI: Blue. Scale bar =50 μm.