Neutrophils Display Novel Partners of Cytosolic Proliferating Cell Nuclear Antigen Involved in Interferon Response in COVID-19 Patients

Abstract

Introduction: Neutrophils are key players in the hyperinflammatory response during SARS-CoV-2 infection. The cytosolic proliferating cell nuclear antigen (PCNA) is a scaffolding protein highly dependent on the microenvironment status and known to interact with numerous proteins that regulate neutrophil functions. This study aimed to examine the cytosolic protein content and PCNA interactome in neutrophils from COVID-19 patients.

Methods: Proteomic analyses were performed on neutrophil cytosols from healthy donors and patients with severe or critical COVID-19. In vitro approaches were used to explore the biological significance of the COVID-19-specific PCNA interactome.

Results: Neutrophil cytosol analysis revealed a strong interferon (IFN) protein signature, with variations according to disease severity. Interactome analysis identified associations of PCNA with proteins involved in interferon signaling, cytoskeletal organization, and neutrophil extracellular trap (NET) formation, such as protein arginine deiminase type-4 (PADI4) and histone H3, particularly in critical patients. Functional studies of interferon signaling showed that T2AA, a PCNA scaffold inhibitor, downregulated IFN-related genes, including STAT1, MX1, IFIT1, and IFIT2 in neutrophils. Additionally, T2AA specifically inhibited the secretion of CXCL10, an IFN-dependent cytokine. PCNA was also found to interact with key effector proteins implicated in NET formation, such as histone H3, especially in critical COVID-19 cases.

Conclusion: The analysis of the PCNA interactome has unveiled new protein partners that enhance the interferon pathway, thereby modulating immune responses and contributing to hyperinflammation in COVID-19. These findings provide valuable insights into interferon dysregulation in other immune-related conditions.

Introduction: Neutrophils are key players in the hyperinflammatory response during SARS-CoV-2 infection. The cytosolic proliferating cell nuclear antigen (PCNA) is a scaffolding protein highly dependent on the microenvironment status and known to interact with numerous proteins that regulate neutrophil functions. This study aimed to examine the cytosolic protein content and PCNA interactome in neutrophils from COVID-19 patients.

Methods: Proteomic analyses were performed on neutrophil cytosols from healthy donors and patients with severe or critical COVID-19. In vitro approaches were used to explore the biological significance of the COVID-19-specific PCNA interactome.

Results: Neutrophil cytosol analysis revealed a strong interferon (IFN) protein signature, with variations according to disease severity. Interactome analysis identified associations of PCNA with proteins involved in interferon signaling, cytoskeletal organization, and neutrophil extracellular trap (NET) formation, such as protein arginine deiminase type-4 (PADI4) and histone H3, particularly in critical patients. Functional studies of interferon signaling showed that T2AA, a PCNA scaffold inhibitor, downregulated IFN-related genes, including STAT1, MX1, IFIT1, and IFIT2 in neutrophils. Additionally, T2AA specifically inhibited the secretion of CXCL10, an IFN-dependent cytokine. PCNA was also found to interact with key effector proteins implicated in NET formation, such as histone H3, especially in critical COVID-19 cases.

Conclusion: The analysis of the PCNA interactome has unveiled new protein partners that enhance the interferon pathway, thereby modulating immune responses and contributing to hyperinflammation in COVID-19. These findings provide valuable insights into interferon dysregulation in other immune-related conditions.

Keywords: COVID-19; Histone 3; Interferon; Neutrophils; PAD4; Proliferating cell nuclear antigen.

Fig. 1.

Proteomic analysis of neutrophil cytosol in HD compared to patients with COVID-19. a PCA of the 3 groups of samples: HDs (black; n = 4); severe COVID-19 patients (green, n = 4); and critical COVID-19 patients (red, n = 4). The age of each individual is indicated. b Heat map showing the clustering of proteins in the COVID-19 samples versus HD. Data are Z-scored and normalized. c Pathway enrichment analyzed using the PANTHER software in the two groups of COVID-19 patients versus HD. d, e Immunofluorescence labeling of p-STAT1 on isolated neutrophils from critical COVID-19 patients (n = 2) and from HD treated or not with IFN-α (n = 3). d Representative experiment showing the cytosolic fluorescence of p-STAT1. This experiment has been performed with neutrophils from three different HDs and with two different COVID-19 patients. e Quantification of the intensity of the cellular fluorescence observed in d was performed as described in the Materials and Methods section. Data are means of corrected fluorescence/cell ± SEM. Statistical analysis has been performed using the nonparametric Mann-Whitney test. **p < 0.01.

Fig. 2.

DE proteins identified in cytosols from COVID-19 patients. Quantitative comparison of DE proteins in controls (n = 4) versus severe COVID-19 patients (n = 4) (a, b) and versus critical COVID-19 patients (n = 4) (c, d). Signaling pathways enrichment in neutrophil cytosols that are differentially regulated in severe patients (a) (in yellow) and in critical patients (c) (in red) versus HD have been analyzed using the PANTHER software. The volcano plot represents relative protein expression changes between COVID-19 patients and HD. Blue and green dots indicate proteins upregulated in COVID-19 in severe and critical, respectively, versus HD with a multicomparison adjusted −log10 (p value) ≥1.3 and a Log2(fold change) ≥1; yellow dots indicate proteins downregulated in COVID-19 versus HD with a multicomparison adjusted −log10 (p value) ≥1.3 and a Log2(fold change) <−1; gray dots indicate proteins that do not meet either criterion.

Fig. 3.

Differential analysis of PCNA interactome in cytosols from patients with COVID-19 volcano plot showing proteins that interact with PCNA in neutrophils from patients with (a) severe COVID-19 and (b) critical COVID-19. Quantitative comparison of differentially expressed proteins in HD (n = 4) versus severe COVID-19 patients (n = 4) (a, b) and versus critical COVID-19 patients (n = 4) (c, d). The volcano plot represents relative protein expression changes. Blue and green dots indicate proteins upregulated in severe and in critical COVID-19 samples, respectively, with a multicomparison adjusted −log10 (p value) ≥1.3 and Log2(fold change) ≥1; gray dots indicate proteins downregulated with a multicomparison adjusted −log10 (p value) ≥1.3 and a Log2(fold change) ≤−1; gray dots also indicate proteins that do not meet either criterion. See online supplementary Tables S1 and S2 for the complete list of proteins differentially expressed in the cytosol of patients either severe or critical COVID-19 versus HD, respectively.

Fig. 4.

Analysis of the PCNA interactome. Representation of the network of proteins that are differentially identified in the PCNA interactome in neutrophils from severe (a) and critical (b) COVID-19 patients versus HD and represented as functional clusters using the STRING online software. PCNA (red dot) has been identified in both severe and critical immunoprecipitated material. Among the common partners of PCNA between severe and critical patients are PADI4 (purple dot), the proteins involved in cytoskeleton (green circle), and IFN response (blue circle). Interaction with histone proteins has been identified only in the PCNA interactome of critical patients (red circle). See online supplementary Tables S3 and S4 for the complete list of partner proteins differentially identified in the PCNA interactome from patients either severe or critical COVID-19 versus HD. c Duolink proximity ligation assay of p-STAT1 and PCNA in neutrophils of HD without treatment (1) and treated with IFN-γ in the presence (3) or absence of T2AA (2). This representative experiment has been performed two times with neutrophils from two distinct HDs with similar results. d Quantification of the intensity of the cellular fluorescence observed in c was performed in samples (1-HD-untreated; 2-HD + IFN-γ; 3-HD + IFN-γ + T2AA) as described in the Materials and Methods section. Data are means of corrected fluorescence/cell ± SEM. Statistical analysis has been performed using the unpaired nonparametric Mann-Whitney test. **p < 0.01.

Fig. 5.

Role of PCNA in regulating IFN-induced gene expression in HD neutrophils. a Scheme illustrating the experimental protocol used: ultrapure HD neutrophils were pretreated with 1,000 U/mL IFN-α or 200 U/mL IFN-γ, then incubated for additional 1 h with 10 μM T2AA. After 1 h, 10 ng/mL TNF-α was added and neutrophils cultured for up to 18 h. Then RNA was extracted for the assessment of STAT1 (b), IFIT2 (c), IFIT1 (d), and MX1 (e) gene expression by RT-qPCR. These experiments have been performed twice using neutrophils from two independent HDs. Data are the mean values of normalized expression (MNE) ± SEM for each condition as indicated in the Material and Methods section.

Fig. 6.

Role of PCNA in regulating STAT1-dependent cytokine production by IFN-stimulated HD neutrophils. a Scheme illustrating the experimental protocol used: ultrapure HD neutrophils were pretreated for 3 h with 1,000 U/mL IFNα or 200 U/mL IFN-γ, before the addition of 10 µm T2AA. After 1 h, 10 ng/mL TNFα was added, and the neutrophil cultures were prolonged up to the 18-h time-point. The release of CXCL10 (b) and BAFF (c) was then assessed by ELISA in culture supernatants. Results are expressed as the mean value ± SEM of n = 3 independent experiments. Analysis was performed by a two-way ANOVA with Sidak’s multiple-comparison test. *p < 0.05.

Fig. 7.

Investigation of PADI4-PCNA interaction in HD neutrophils. a Immunofluorescence labeling of NET in resting (left panels) versus ionomycin-stimulated (right panels) HD neutrophils. NETs were stained for DNA (Sytox, green), neutrophil elastase (NE) (red), and total histones (purple). Under basal conditions, the localization of either NE or histones is intracellular, while both can be detected on extracellular DNA after ionomycin stimulation. This representative experiment has been performed two times with neutrophils from 2 different HDs with similar results. b Assessment of the colocalization between PCNA and PADI4 using the Duolink proximity ligation assay in basal and ionomycin-stimulated HD neutrophils in the absence or presence of T2AA (this latter condition is not shown since no effect of T2AA could be observed). This representative experiment has been performed two times with neutrophils from two distinct HDs with similar results. c Quantification of the intensity of the cellular fluorescence observed in b was performed in all the conditions (HD-basal; HD + ionomycin, HD + ionomycin + T2AA 10 μm) as described in the Materials and Methods section. Data are means of corrected fluorescence/cell ± SEM. Statistical analysis has been performed using the unpaired nonparametric Mann-Whitney test. **p < 0.01.

Fig. 8.

Contrasting effect of T2AA on the interaction between PCNA and Cit-H3 and on ionomycin-induced NET release in HD neutrophils. a Immunofluorescence labeling of PCNA and Cit-H3 in basal and ionomycin-stimulated neutrophils. This representative experiment has been performed two times with neutrophils from two distinct HDs with similar results. b Assessment of the colocalization between PCNA and Cit-H3 using the Duolink proximity ligation assay in basal and ionomycin-stimulated HD neutrophils in the absence or presence of T2AA. This representative experiment has been performed with neutrophils from two distinct HDs with similar results. c Quantification of the intensity of the cellular fluorescence observed in b was performed. Data are means of corrected fluorescence/cell ± SEM. *p < 0.05; **p < 0.01. d Measurement of released DNA after NET induction. HD neutrophils were untreated or stimulated with 8 µm ionomycin for 4 h, in the presence/absence of 25 µm T2AA, and DNA from soluble NET was quantified by fluorescence using PicoGreen. This representative experiment has been performed with neutrophils from three different HDs with similar results. e Live imaging of NET formation. Neutrophils were untreated or stimulated with 8 µm ionomycin in the presence/absence of 25 µm T2AA for 5 h and analyzed every 3 min. Mean and SEM of triplicates of each time point are shown. A representative picture is depicted. Nuclear DNA is stained with Hoechst 33342 (blue), while extracellular DNA is stained with Sytox green (green). This representative experiment has been performed with neutrophils from three distinct HDs with similar results.