Figure 1. Caspase-1 expression in immune cells. Caspase-1 expression is shown for (A-C) total (CD3+) T-cells, CD4+ T cells, non-CD3+ T cell lymphocytes, and myeloid cells. (D-F) Caspase 1 expression in CD4+ T cells is correlated with γ/δ T cells, NK cells (CD3-CD56/16+) and CRTH2+ CD4+ T cells. Individual patient data are shown and the frequency of caspase-1+ CD4+ T cells are significantly elevated at baseline in COVID-19 patients (n =29) compared to healthy (n =28) participants with and without nigericin stimulation. All p-values are by unpaired and 2-tailed Student’s t  test or linear regression analysis. *p<0.05, ***p<0.001, ****p<0.0001
Figure 2. Caspase-1 expression in CD4+ T cells of non-COVID-19 patients (unexposed and uninfected adult and pediatric patients with allergic/immunological disorders). Open symbols are resting non-stimulated CD4+ T cells. Closed symbols represent caspase-1 expression in nigericin stimulated CD4+ T cells. Different symbols represent different disease states. Adults (>18 years) are represented with an A (n =65) and pediatric subjects (<18 years) represented with a P (n =39) in the bottom of the graph. CRS; chronic rhinosinusitis, CVID; common variable immune deficiency, and CIU; chronic idiopathic urticaria. The diagnosis and T-cell caspase-1 data are retrospective data from medical records of patients presenting to an Allergy Immunology Clinic for an immunological evaluation. Control patient data was generated during clinical assay validation of T-helper cell caspase-1 assay (n =45 for adults and n =39 for pediatrics). All p-values are by unpaired and 2-tailed Student’s t  test. *p<0.05, ***p<0.001, ****p<0.0001.
Figure 3. Caspase-1 expression on CD4+ T cells in post-COVID-19 Health Care Workers. Blood samples were analyzed at least 90 days after SARS-CoV-2 exposure in healthcare workers. Patients with no exposure history and negative PCR to SARS-CoV-2 were used as controls. Solid black circles represent symptomatic, green circles represent non-symptomatic patients. Exposure indicates being in close proximity to SARS-CoV-2 infected patients in the absence of personal protection equipment. Persistent post-COVID19 symptoms correlated with elevated caspase-1 expression in T-helper cells (p< 0.05).
Figure 4. Effect of caspase inhibition on CD4+ T cells in COVID-19 patients. Samples from healthy and COVID-19 subjects incubated with caspase inhibitors: EMR or VX765. Activated caspase-1 was measured by flow cytometry using a Fam-FLICA probe. Emricasan at 1μM concentration induces the strongest suppression of CD4+ T cell caspase-1 in unstimulated cells (p< 0.01), whereas the selective caspase-1 inhibitor VX-765 does not induce a similar effect. Krustal-Wallis ANOVA test with Tukey multiple comparisons for >2 group comparisons were used. For P values are as follows: *p<0.05, **p<0.01. Experiments represent n =3.
Figure 5. Caspase 3/7 activity in red blood cells (RBC) derived from COVID-19 patients. Blood samples were analyzed from hospitalized patients with SARS-CoV-2 infection. A) RBC contamination of the PBMC layer after Ficoll separation. B) Analysis of caspase 3/7 activity in COVID-19 patients and healthy controls. Some experiments were done using plasma from COVID-19 or subjects with influenza with incubated with RBCs from health uninfected donors as indicated. COVID-19 patients RBCs show elevated caspase 3/7 (p< 0.01) and EMR has a significant suppressive activity on this expression (p< 0.05). Plasma from hospitalized COVID-19 patients induces caspase 3/7 in health RBCs on overnight incubation (p< 0.01). Krustal-Wallis ANOVA test with Tukey multiple comparisons for >2 group comparisons were used. For p values are as follows: *p<0.05, **p<0.01. Experiments representn =3.
Figure 6. Emricasan mechanism of action.
FIGURE 1