Heterogeneous NLRP3 inflammasome signature in circulating myeloid cells as a biomarker of COVID-19 severity

Johan Courjon, Océane Dufies, Alexandre Robert, Laurent Bailly, Cédric Torre, David Chirio, Julie Contenti, Sébastien Vitale, Céline Loubatier, Anne Doye, Christelle Pomares-Estran, Géraldine Gonfrier, Romain Lotte, Patrick Munro, Orane Visvikis, Jean Dellamonica, Valérie Giordanengo, Michel Carles, Laurent Yvan-Charvet, Stoyan Ivanov, Patrick Auberger, Arnaud Jacquel, Laurent Boyer, Johan Courjon, Océane Dufies, Alexandre Robert, Laurent Bailly, Cédric Torre, David Chirio, Julie Contenti, Sébastien Vitale, Céline Loubatier, Anne Doye, Christelle Pomares-Estran, Géraldine Gonfrier, Romain Lotte, Patrick Munro, Orane Visvikis, Jean Dellamonica, Valérie Giordanengo, Michel Carles, Laurent Yvan-Charvet, Stoyan Ivanov, Patrick Auberger, Arnaud Jacquel, Laurent Boyer

Abstract

Dysregulated immune response is the key factor leading to unfavorable coronavirus disease 2019 (COVID-19) outcome. Depending on the pathogen-associated molecular pattern, the NLRP3 inflammasome can play a crucial role during innate immunity activation. To date, studies describing the NLRP3 response during severe acute respiratory syndrome coronavirus 2 infection in patients are lacking. We prospectively monitored caspase-1 activation levels in peripheral myeloid cells from healthy donors and patients with mild to critical COVID-19. The caspase-1 activation potential in response to NLRP3 inflammasome stimulation was opposed between nonclassical monocytes and CD66b+CD16dim granulocytes in severe and critical COVID-19 patients. Unexpectedly, the CD66b+CD16dim granulocytes had decreased nigericin-triggered caspase-1 activation potential associated with an increased percentage of NLRP3 inflammasome impaired immature neutrophils and a loss of eosinophils in the blood. In patients who recovered from COVID-19, nigericin-triggered caspase-1 activation potential in CD66b+CD16dim cells was restored and the proportion of immature neutrophils was similar to control. Here, we reveal that NLRP3 inflammasome activation potential differs among myeloid cells and could be used as a biomarker of a COVID-19 patient's evolution. This assay could be a useful tool to predict patient outcome. This trial was registered at www.clinicaltrials.gov as #NCT04385017.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

© 2021 by The American Society of Hematology.

Figures

Graphical abstract
Graphical abstract
Figure 1.
Figure 1.
Caspase-1 activation level in myeloid cells in the blood of COVID-19 patients. Whole peripheral blood cells of healthy donors or COVID-19 patients with mild to critical symptoms were stained for active caspase-1 (detected using the FAM-FLICA probe) and for CD45, CD14, CD16, and CD66b markers. Cells were immunophenotyped by flow cytometry. Leukocytes were defined as CD45+ and were analyzed for monocyte and granulocyte surface markers. (A-D) Monocytes were defined as CD14+ and subpopulations were gated as indicated in panel A using CD14 and CD16 markers. The indicated monocyte subsets were analyzed for the mean fluorescence intensity (MFI) of FAM-FLICA corresponding to the activation of caspase-1 (B-D). (E-G) Granulocytes were defined as CD66b+ and the different subsets were gated as indicated using CD66b and CD16 markers (E). (F-G) The indicated granulocyte subsets were analyzed for the FAM-FLICA MFI. **P ≤ .01.
Figure 2.
Figure 2.
Nonclassical monocyte disappearance and increased nigericin-triggered caspase-1 activation in nonclassical monocytes are associated with COVID-19 severity. Whole peripheral blood cells of healthy donors or COVID-19 patients were analyzed by flow cytometry using CD45, CD14, and CD16 markers. Whole peripheral blood was treated with vehicle (control) or nigericin (5 µM) for 30 minutes and monocyte subsets were analyzed for FAM-FLICA MFI (caspase-1 activation) (A,D,G) and nigericin-induced fold of FAM-FLICA MFI compared with control (B,E,H). Leukocytes were defined as CD45+ (C,F,I) and the frequency of monocyte subsets among leukocytes was analyzed: CD14dimCD16+ nonclassical monocytes (C) CD14highCD16− classical monocytes (F) and CD14highCD16+ intermediate monocytes (I). **P ≤ .01; ***P ≤ .001.
Figure 3.
Figure 3.
CD66b+CD16highgranulocytes display increased nigericin-triggered caspase-1 activation in severe COVID-19 whereas CD66b+CD16dimgranulocytes of severe COVID-19 lost their capacity to respond to the NLRP3 stimulation. Whole peripheral blood cells of healthy donors or COVID-19 patients were analyzed by flow cytometry using CD45, CD66b, and CD16 markers. (A-E) Whole peripheral blood was treated with vehicle (control) or nigericin (5 µM) for 30 minutes and granulocyte subsets were analyzed for FAM-FLICA MFI (caspase-1 activation) (A-B) and nigericin-induced fold of FAM-FLICA MFI compared with control (C-D). (E) Histogram of FAM-FLICA signal in CD66b+CD16dim cells. Light colors, FAM-FLICA in the control condition; dark colors, FAM-FLICA in the nigericin-treated condition. The dotted line represents the gate used to determine the percentage of FAM-FLICA+ cells. (F-G) Leukocytes were defined as CD45+ and the frequency of granulocyte subsets among leukocytes was analyzed. *P ≤ .05; **P ≤ .01; ****P < .0001.
Figure 4.
Figure 4.
Myeloid cell response to NLRP3 inflammasome stimulation in recovered COVID-19 patients. Peripheral blood cells of recovered COVID-19 patients were collected 30 to 50 days after the first analysis. Whole peripheral blood cells of recovered COVID-19 patients were analyzed by flow cytometry using CD45, CD16, and CD66b markers. (A) Whole peripheral blood was treated with vehicle (control) or nigericin (5 µM) for 30 minutes and CD66b+CD16dim granulocytes were analyzed for FAM-FLICA MFI (caspase-1 activation). (B) Histogram of FAM-FLICA signal in CD66b+CD16dim cells. Light colors, FAM-FLICA in the control condition; dark colors, FAM-FLICA in the nigericin-treated condition. The dotted line represents the gate used to determine the percentage of FAM-FLICA+ cells. (C) Whole peripheral blood was treated with vehicle (control) or nigericin (5 µM) for 30 minutes and CD14dimCD16+ were analyzed for FAM-FLICA MFI (caspase-1 activation).
Figure 5.
Figure 5.
Severe COVID-19 is associated with eosinophil disappearance and accumulation of immature neutrophils with impaired nigericin-triggered caspase-1 activation. Whole peripheral blood cells of healthy donors or COVID-19 patients were analyzed by flow cytometry using CD45, CD66b, CD16, and Siglec-8 or CD45, CD66b, CD16, CD10, and CD15 markers. (A) CD66b+CD16dim cells were analyzed for CD45 expression. (B) CD66b+CD16dim cells were analyzed for CD66b and Siglec-8 (eosinophil marker) and the frequency of CD66b+CD16dimSiglec8+ eosinophils among leukocytes was determined. (C) CD66b+CD16dim cells were analyzed for CD10 (marker of mature neutrophil) and CD15 (neutrophil marker) and the frequency of CD66b+CD16dimCD15+CD10− immature neutrophils among leukocytes was determined. (D-G) Whole peripheral blood was treated with vehicle (control) or nigericin (5 µM) for 30 minutes and eosinophils or immature neutrophils were analyzed for FAM-FLICA MFI (caspase-1 activation) (D,F) and nigericin-induced fold of FAM-FLICA MFI compared with control (E,G). (H) Peripheral blood cells of recovered COVID-19 patients were collected 30 to 50 days after the first analysis. Whole peripheral blood cells of recovered COVID-19 patients were analyzed by flow cytometry using CD45, CD16, and CD66b markers. CD66b+CD16dim cells were analyzed for CD45 expression. *P ≤ .05; **P ≤ .01; ****P < .001. D, diseased; R, recovered.
Figure 6.
Figure 6.
C1B score is associated with the final outcome of the patient and predicts patients’ evolution. (A-F) Correlation between (A,D) the percentage of CD14dimCD16+ nonclassical monocytes of live CD45+ cells, (B,E) the nigericin-triggered fold of FAM-FLICA in CD66b+C16dim cells compared with control, (C,F) the C1B score (defined as the percentage of CD14dimCD16+ × fold FAM-FLICA in CD66b+CD16dim cells) and the Spo2/Fio2 ratio at (A-C) day 1 and (D-F) day 3 of inclusion. The line represents the linear regression. Each dot represents a COVID-19 patient and the color its condition at the day of inclusion (blue, moderate; orange, severe; and red, critical). (G-I) Nigericin-triggered fold of FAM-FLICA in CD66b+C16dim cells compared with control (H), percentage of CD14dimCD16+ nonclassical monocytes of live CD45+ cells (G) and C1B score (I) as previously defined, observed in healthy donors or COVID-19 patients with favorable or unfavorable outcome. *P < .05; **P < .01; ***P < .001; ****P < .0001. r, Spearman coefficient.

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