Disease severity and CTSL levels in COVID-19 patients with and without diabetes.

a Design and inclusion flowchart of the case-control study. Out of 207 COVID-19 patients from two hospitals, 62 were included in the study after matching for gender and age, 31 with diabetes and 31 without. b Comparison of symptom severity and prevalence between diabetic and non-diabetic COVID-19 patients. c Study design and timeline of the enrollment and follow-up study. After admitted to the hospital (Day 0), patients were hospitalized for a mean duration of 14 days, followed up 14 days (Day 14) and 28 days (Day 28) after discharge, and blood samples were collected at each time point. d-f Plasma levels of COVID-19 related proteins were measured in diabetic and non-diabetic COVID-19 patients on Day 0, Day 14, and Day 28. Statistical significance was assessed by unpaired t-test (b) and Mann-Whitney U-test (d-f). The data are presented as the means ± SEM. *P < 0.05, ***P < 0.001.

Impact of chronic and acute hyperglycemia on CTSL concentration and activity.

a-d Effects of chronic hyperglycemia on CTSL activity and concentration in 122 gender-and age-matched individuals without COVID-19, including 61 euglycemic volunteers and 61 diabetic patients. a Correlation between plasma CTSL activity and blood glucose level indicated by HbA1c. b Correlation between plasma CTSL concentration and HbA1c. The dashed line represents the 95% CI in a and b. c Comparison of plasma CTSL activity between the euglycemic and diabetic groups. d Comparison of plasma CTSL concentration between the euglycemic and diabetic groups. The data are presented as the median with 95% CI in c and d. e-g Hyperglycemic clamp study performed in 15 healthy male subjects. e Plasma glucose levels in subjects throughout the clamp study. The dashed lines represent the range of ± 5% of the hyperglycemic target level, i.e., basal blood glucose level + 6.9 mmol/L. f Insulin levels and g proinsulin C-peptide levels throughout the clamp study. h-j Effects of acute hyperglycemia on CTSL concentration and activity in 15 healthy male volunteers. h Plasma CTSL activity at the beginning and the end of the clamp study. i Plasma CTSL activity throughout the clamp study. j Plasma CTSL concentration at the beginning and the end of the clamp study. Statistical significance was assessed by Spearman correlation analysis (a and b), unpaired t-test (c and d) and paired t-test (h and j). The data are presented as the means ± SEM. **P < 0.01, ****P < 0.0001.

Hyperglycemia enhances SARS-CoV-2 infection through CTSL.

Huh7 cells were infected with SARS-CoV-2 pseudovirus. a Wildtype (WT) cells cultured in sera from healthy and diabetic individuals were infected with SARS-CoV-2 pseudovirus (1.3 × 104 TCID50/ml). Non-infected cells are used as control. The infection levels, as indicated by luciferase activities, were adjusted by cell viability, as indicated by CCK (n = 3). b The SARS-CoV-2 infection rate of WT cells after being cultured in different doses of glucose (5.6 mM, 15 mM and 25 mM) (n = 3). c The SARS-CoV-2 infection rate of WT cells after being cultured in different doses of insulin (0 nM, 20 nM, 100 nM and 500 nM) (n = 3). d Comparison of CTSL expression in WT and CTSL knockout (KO) cell lines. e-h The SARS-CoV-2 pseudovirus infection of WT and CTSL KO cells. e The SARS-CoV-2 infection rate of control, CTSL KO, and WT cells (n = 3). f The SARS-CoV-2 infection rate of WT and CTSL KO Huh7 cells cultured in different doses of glucose (5.6 mM and 25 mM) (n = 3). g The SARS-CoV-2 infection rate of CTSL KO Huh7 cells cultured in different doses of glucose (5.6 mM, 15 mM and 25 mM) (n = 3). h The SARS-CoV-2 infection rate of CTSL KO Huh7 cells cultured in different doses of insulin (20 nM, 100 nM and 500 nM) (n = 3). Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (a-c, g and h) and Mann-Whitney U-test (e and f). The data are presented as the means ± SEM. *P < 0.05, **P < 0.01.

Elevation of glucose levels enhance CTSL activity.

Effects of high glucose levels on CTSL activity in Huh7 cells, as well as in biopsy samples of mice and diabetic patients. a Intracellular CTSL activity was measured in Huh7 cells cultured in different pH as indicated (n = 4). b Intracellular CTSL activity was measured in Huh7 cells cultured in different glucose concentrations as indicated (n = 3). c Extracellular CTSL activity was measured in Huh7 cells cultured in different glucose concentrations as indicated (n = 6). d Intracellular CTSL activity was measured in Huh7 cells cultured in different insulin concentration as indicated (n = 3). e Intracellular CTSL activity was measured in Huh7 cells cultured in different glucose and insulin concentrations as indicated (n = 3). f Blood glucose levels during the intraperitoneal glucose tolerance test (IPGTT) in db/db diabetic and db/m control mice (n = 5). g Body weight of db/db and db/m mice was measured (n = 10). h Fasting insulin levels were measured in db/db and db/m mice (n = 5). i CTSL activity was measured in the lung and liver biopsy samples of db/db and db/m mice (n = 8). j CTSL activity was measured in human lung biopsy samples from diabetic (DM) and non-diabetic patients (n = 3). Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (a-e) and unpaired t-test (f-j). The data are presented as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

High glucose levels stimulate CTSL maturation.

a Schematic of the CTSL maturation process. Pro-cathepsin L (ProCTSL, 39 kDa) in endoplasmic reticulum (ER) and Golgi apparatus translocated to the lysosome and autoactivated into the single chain mature cathepsin L (sc-mCTSL, 31 kDa) and double chain mature cathepsin L (dc-mCTSL, 24KDa). b Western blot analysis of CTSL protein in Huh7 cells cultured with different doses of D-glucose and insulin as indicated (n = 5). c Western blot analysis of CTSL protein in Huh7 cells cultured with 5.6 mM D-glucose or D-glucose plus 19.4 mM L-glucose as indicated (n = 3). d Western blot analysis of CTSL protein in lung tissues from db/db and db/m mice (n = 3). e Western blot analysis of CTSL protein in human lung tissues from non-diabetic and diabetic patients (n = 3). Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (b) and Mann-Whitney U-test (c-e). The data are presented as the means ± SEM. *P < 0.05, ##P < 0.01.

High glucose promotes CTSL translocation from endoplasmic reticulum to lysosome and enhances SARS-CoV-2 infection.

a A diagram illustrating the process of CTSL translocation via the endoplasmic reticulum (ER)-Golgi-lysosome axis. b-d Immunofluorescent staining of Huh7 cells cultured in 5.6 mM or 25 mM glucose as indicated. Co-localization analysis of CTSL (labeled red) and different organelles (labeled green) was performed using CTSL and organelle marker protein antibodies. b calreticulin for ER, c GM130 for Golgi apparatus, and d lamp1 for lysosome. e-g Fluorescence co-localization intensity analysis of the dashed line on the 500 times enlarged immunofluorescence picture. Fluorescence co-localization intensity was calculated using the Plot Profile tool in Image J software (n = 3). Scale bars, 20 μm for 100 x and 4 μm for 500 x. h Proposed mechanisms of hyperglycemia drives CTSL maturation and enhances SARS-CoV-2 infection. (1) Blood glucose increased in diabetic patients. (2) Hyperglycemia promoted CTSL maturation through the ER-Golgi-lysosome axis. (3) CTSL activity increased and facilitated SARS-CoV-2 entry, by cleaving the spike protein (consist of S1 and S2 subunits), and enhanced COVID-19 severity in diabetic patients. All statistical significance was assessed using Mann-Whitney U-test. The data are presented as the means ± SEM. *P < 0.05, **P < 0.01.

CTSL mRNA levels remain unchanged under different glucose conditions.

a-c mRNA levels of CTSL in Huh7 cells cultured in media containing different concentrations of a D-glucose, b L-glucose, and c insulin, as indicated (n = 3-4). d mRNA levels of CTSL in db/db and db/m mouse lung and liver tissues (n = 5). e mRNA levels of CTSL in human lung tissues (n = 3). Significance was assessed by one-way ANOVA with Tukey’s post hoc test (a-c) and Mann-Whitney U-test (d and e). The data are presented as the means ± SEM.

CTSL protein expression in Huh7 cells under different D-glucose concentrations.

CTSL expression in Huh7 cells cultured in media containing different concentrations of D-glucose (n = 3). Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. The data are presented as the means ± SEM. *P < 0.05.

Immunofluorescent staining of organelle markers representing the endoplasmic reticulum (ER), Golgi apparatus and lysosome.

a Expression of calreticulin (endoplasmic reticulum marker protein) in WT Huh7 cells. b Expression of GM130 (Golgi apparatus marker protein) in WT Huh7 cells. c Expression of Lamp1 (lysosome marker protein) in WT Huh7 cells. d Mouse immunoglobulin G (IgG) was used as a negative control to show the specificity of the primary antibody binding to GM130 and Lamp1 antigen. e Rabbit IgG was used as a negative control to show the specificity of the primary antibody binding to calreticulin antigen.

Immunofluorescent staining of CTSL in Huh7 cells.

a CTSL expression in wild-type (WT) Huh7 cells. b CTSL expression in CTSL knockout (KO) Huh7 cells. c CTSL expression in overexpression (OE) Huh7 cells. d Goat IgG was used as a negative control to show the specificity of the primary antibody binding to CTSL antigen.

Comparation of clinical features of COVID-19 patients

Demographic and clinical characteristics of non-COVID-19 patients

Nonparametric correlations of parameters correlated with CTSL levels and diabetes in non-COVID-19 individuals

Demographic and clinical characteristics of human lung tissues donor

List of oligonucleotide primer pairs used in real time RT-PCR analysis

Specific Antibodies for Western blotting and Immunofluorescence assa