key: cord-0899715-klzy18v5
authors: Bordoni, Veronica; Tartaglia, Eleonora; Sacchi, Alessandra; Fimia, Gian Maria; Cimini, Eleonora; Casetti, Rita; Notari, Stefania; Grassi, Germana; Marchioni, Luisa; Bibas, Michele; Capobianchi, Maria R.; Locatelli, Franco; Maeurer, Markus; Zumla, Alimuddin; Antinori, Andrea; Nicastri, Emanuele; Ippolito, Giuseppe; Agrati, Chiara
title: The unbalanced p53/SIRT1 axis may impact lymphocyte homeostasis in COVID-19 patients
date: 2021-02-10
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.02.019
sha: f990f6e2304ccd77c351a32d153910b60fa415ce
doc_id: 899715
cord_uid: klzy18v5

Background/Objectives Dysregulated inflammatory profile play an important role in COVID-19 pathogenesis. Moreover, depletion of lymphocytes is typically associated with an unfavorable disease course. We studied the role and impact of p53 and deacetylase Sirtuin 1 (SIRT1) on lymph-monocyte homeostasis and their possible effect on T and B cell signalling. Methods Gene expression analysis and flow cytometry were performed on peripheral blood mononuclear cells of 35 COVID-19 patients and 10 healthy donors (HD). Inflammatory cytokines, the frequency of Annexin + cells among CD3 + T cells and CD19 + B cell subsets were quantified. Results PBMC from COVID-19 patients had a higher p53 expression, and a higher concentrations of plasma proinflammatory cytokines (IL1β, TNF-α, IL8, IL6) than HD. Deacetylase Sirtuin 1 (SIRT1) expression was significantly decreased in COVID-19 patients, and was negatively correlated with p53 (p = 0.003 r=-0.48). A lower expression of IL-7R and B Cell linker (BLNK), key genes for lymphocyte homeostasis and function, was observed in COVID-19 compared with HD. Reduction of IgK and IgL chains was seen in lymphopenic COVID-19 patients. A significant increase of both apoptotic B and T cells were observed. Inflammatory cytokines correlated positively with p53 (IL-1β: r = 0.5, p = 0.05;IL-8: r = 0.5, p = 0.05) and negatively with SIRT1 (IL1-β: r=-0.5,p = 0.04;TNF-α: r=-0.4, p = 0.04). Conclusions Collectively, our data indicate that the inflammatory environment, the dysregulated p53/SIRT1 axis and low expression of IL7R and BLNK may impact cell survival, B cell signalling and antibody production in COVID-19 patients. Further studies are required to define the functional impact of low BLNK/IL7R expression during SARS-COV2 infection.

Gene expression analysis and flow cytometry were performed on peripheral blood mononuclear cells of 35 COVID-19 patients and 10 healthy donors (HD). Inflammatory cytokines, the frequency of Annexin+ cells among CD3+ T cells and CD19+ B cell subsets were quantified.

PBMC from COVID-19 patients had a higher p53 expression, and a higher concentrations of plasma proinflammatory cytokines (IL1, TNF-, IL8, IL6) than HD. Deacetylase Sirtuin 1 (SIRT1) expression was significantly decreased in COVID-19 patients, and was negatively correlated with p53 (p=0.003 r=-0.48). A lower expression of IL-7R and B Cell linker (BLNK), key genes for lymphocyte homeostasis and function, was observed in COVID-19 compared with HD. Reduction of IgK and IgL chains was seen in lymphopenic COVID-19 patients. A significant increase of both apoptotic B and T cells were observed. Inflammatory cytokines correlated positively with p53 (IL-1β: r=0.5, p=0.05;IL-8: r=0.5, p=0.05) and negatively with SIRT1 (IL1-β: r=-0.5,p=0.04;TNF-α: r=-0.4, p=0.04).

Collectively, our data indicate that the inflammatory environment, the dysregulated p53/SIRT1 axis and low expression of IL7R and BLNK may impact cell survival, B cell signalling and antibody production in COVID-19 patients. Further studies are required to define the functional impact of low BLNK/IL7R expression during SARS-COV2 infection. Table 1 . Quantitative variables were compared with nonparametric Mann-Whitney test. A p value lower than 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism v8.0 (GraphPad Software, Inc). Spearman rank test was used to determine correlations.

To evaluate the impact of COVID-19 on lymphocytes homeostasis, we first compared the level of p53 expression in PBMC from COVID-19 patients (n=35) and from healthy donors (HD, n=10).

PBMC from COVID-19 patients showed a significant higher p53 expression ( Figure 1A ) respect to HD, confirming previous data (Xiong et al., 2020) . The expression of the deacetylase SIRT1 was lower in COVID-19 patients (Figure 1B) , and was negatively correlated with p53 (p=0.003 r=-0.48, Figure 1C ). Furthermore, a significant higher level of p21, an essential mediator of p53-dependent cell-cycle arrest (Brugarolas et al., 1995) , was observed in COVID-19 patients compared to HD ( Figure 1D) . Figure   1E -F, a higher apoptotic T and B cell percentage were observed in COVID-19 patients compared to HD.

To evaluate the possible role of inflammation in modulating the apoptotic processes, we quantified the plasma level of IL1β, IL-6, IL-8 and TNF-α, confirming a higher levels of inflammation in COVID-19 than in HD (Figure 1G ). Of note, the inflammatory cytokines were correlated positively with p53 (IL-1β: r=0.5, p=0.05; IL-8: r=0.5, p=0.05, Figure 1H ), and negatively with SIRT1 (IL1-β: r=-0.5, p=0.04; TNF-α: r=-0.4, p=0.04, Figure 1I ).

We thus analysed the impact of p53 upregulation on the expression of important positive regulators of T and B lymphocytes development and homeostasis [IL-7/IL-7R and B cell linker (BLNK) respectively]. Although a high plasmatic IL-7 level (Figure 2A) , we found a significant lower IL-7R expression in PBMC from COVID-19 than HD (Figure 2B) , that was positively correlated with SIRT1 (p=0.0005, r=0.64, Figure 2C ). This result was confirmed by analysing the percentage of We finally explored the B cell signalling, and found a significant lower BLNK expression in COVID-19 patients compared to HD (Figure 2E) , that was negatively correlated with p53 (p=0.0004, r=-0.6, Figure 2F ), and with inflammatory cytokines ((IL1-β: r=-0.6, p=0.01; IL-6: r=-0.5, p=0.03, Figure   2G ), and positively with SIRT1 (p=0.0036, r=0.49, Figure 2H ). Since BLNK has effect on antibody production, we investigated a possible relationship between BLNK and Ig chains expression. The expression of both IgK and IgL was lower in B cells from COVID-19 patients compared to HD ( Figure 2I) , suggesting that the signal from BCR may be defective in COVID-19 patients.

Dysregulated inflammatory profiles, defective immune response and lymphopenia have been The significant higher p53 expression in PBMC from COVID-19 patients confirms previous observations by Xiong et al., (2020) . We showed that the high p53 expression correlates with a J o u r n a l P r e -p r o o f significant reduction in the expression of the deacetylase SIRT1, and was associated to a higher expression of p21 in COVID-19 patients. SIRT1 is a pleiotropic protein able to target several transcription factors, thus orchestrating several intracellular pathways associated with cell death/survival (Mendes et al. 2017) . A direct interaction between SIRT1 and p53 is well known, as the p53 deacetylation through SIRT1 can inhibit p53 activity (Yi J et al., 2010) . Therefore, our data suggest that COVID-19 can be characterized not only by an increase in p53 transcript in circulating lymphocytes but also by a persistently activated p53 form, possibly due to the low level of SIRT1.

Accordingly, the high level of p21 observed in COVID-19 patients may also participate to the cellcycle arrest (Brugarolas et al., 1995) , thus contributing to the lymphopenia of severe COVID-19.

The lymphopenia described in COVID-19 patients confirmed previous data (Huan C et al., 2020; 395: 497-506; Bordoni et al., 2020) and can be at least partially due to an increase of apoptotic processes. Accordingly, we found an increase of apoptotic T and B lymphocytes in COVID-19

patients. This observation was in line with reports on SARS-COV-1 infection during the 2003 SARS outbreak, and seems to be mediated by indirect mechanisms rather than by direct viral infection/replication pathways (Chan et al., 2008) . we found a lower IL-7R expression in PBMC from COVID-19 than HD, which was positively correlated to SIRT1, suggesting a reduced responsiveness to IL-7 that may contribute to the dysregulation of T-cell homeostasis and lymphopenia.

BLNK bridges B cell receptor-associated kinase activation with downstream signalling pathways. A lower BLNK expression in COVID-19 patients compared to HD was observed, that was negatively correlated with p53, suggesting an interplay between p53 and functional pathways in B cell response. 

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