BPIFB4 Circulating Levels and Its Prognostic Relevance in COVID-19

Abstract

Aging and comorbidities make individuals at greatest risk of COVID-19 serious illness and mortality due to senescence-related events and deleterious inflammation. Long-living individuals (LLIs) are less susceptible to inflammation and develop more resiliency to COVID-19. As demonstrated, LLIs are characterized by high circulating levels of BPIFB4, a protein involved in homeostatic response to inflammatory stimuli. Also, LLIs show enrichment of homozygous genotype for the minor alleles of a 4 missense single-nucleotide polymorphism haplotype (longevity-associated variant [LAV]) in BPIFB4, able to counteract progression of diseases in animal models. Thus, the present study was designed to assess the presence and significance of BPIFB4 level in COVID-19 patients and the potential therapeutic use of LAV-BPIFB4 in fighting COVID-19. BPIFB4 plasma concentration was found significantly higher in LLIs compared to old healthy controls while it significantly decreased in 64 COVID-19 patients. Further, the drop in BPIFB4 values correlated with disease severity. Accordingly to the LAV-BPIFB4 immunomodulatory role, while lysates of SARS-CoV-2-infected cells induced an inflammatory response in healthy peripheral blood mononuclear cells in vitro, the co-treatment with recombinant protein (rh) LAV-BPIFB4 resulted in a protective and self-limiting reaction, culminating in the downregulation of CD69 activating-marker for T cells (both TCD4+ and TCD8+) and in MCP-1 reduction. On the contrary, rhLAV-BPIFB4 induced a rapid increase in IL-18 and IL-1b levels, shown largely protective during the early stages of the virus infection. This evidence, along with the ability of rhLAV-BPIFB4 to counteract the cytotoxicity induced by SARS-CoV-2 lysate in selected target cell lines, corroborates BPIFB4 prognostic value and open new therapeutic possibilities in more vulnerable people.

Keywords: Immunity function; Longevity; Plasma; SARS-CoV-2.

Figure 1.

Circulating levels of BPIFB4 protein from long-living individuals (LLIs), SARS CoV-2-negative and SARS CoV-2-positive subjects. ELISA quantification of BPIFB4 levels in plasma from n = 49 LLIs (median age 96, range 95–99), n = 58 SARS CoV-2 negative (median age 55, range 42–62) and n = 64 SARS CoV-2-positive subjects (median age 65, range 20–91) expressed in mean ± SD. Statistical evaluation was carried out with 1-way analysis of variance corrected for multiple comparisons by false discovery rate using 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (GraphPad Prism). The individual p values are shown.

Figure 2.

BPIFB4 levels are reduced in severe SARS-CoV-2 patients. Significant differences in laboratory levels of C-reactive protein, lactate dehydrogenase, and platelets count in n = 32 severe (high grade) (median age 62, range 88–32) and n = 32 nonsevere (low grade) (median age 68, range 20–91) COVID-19 patients (A–C). ELISA quantification of BPIFB4 levels in plasma from the 2 groups of SARS-CoV-2 positive patients (high grade and low grade) compared with the group of n = 58 SARS-CoV-2-negative patients (D). Statistical evaluation was carried out with 1-way analysis of variance corrected for multiple comparisons by false discovery rate using 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (GraphPad Prism). The individual p values are shown.

Figure 3.

Recombinant human LAV-BPIFB4 tunes inflammatory response to lysates from SARS-CoV-2-infected cells in peripheral blood mononuclear cells (PBMCs) in vitro. After a 2-hour LAV-BPIFB4 pretreatment, healthy PBMCs were incubated with SARS-CoV-2 lysate (50 μg/mL) or control lysate for 48 hours and 72 hours. In order to avoid the “activation-induced cell death” (AICD) in vitro, after the first 2 hours, RPMI-FBS medium (with or without SARS-CoV-2 lysate) was discarded and fresh RPMI-FBS medium with rhLAV-BPIFB4 (18 ng/mL) was added for the following 46 hours or 70 hours. At the end of the cell culture, supernatants were collected and PBMCs assayed by FACS analysis after staining with anti-CD3, anti-CD4, anti-CD8, and anti-CD69 mAb. (A) A representative FACS dot plot is presented. Bar graphs report the percentage ± SD of CD69+ of both CD8+ gated TCD3+ cells and CD4+ gated TCD3+ cells from 3 independent experiments using different donors (analysis of variance [ANOVA] with correction for multiple comparisons using the Holm-Sidak method). (B) Bar graphs report the percentage ± SD of CD69+ of both CD8+ gated TCD3+ cells and CD4+ gated TCD3+ cells among peripheral blood lymphocytes obtained after CD14+ immunomagnetic depletion from PBMCs in 3 independent experiments using different donors (ANOVA with correction for multiple comparisons using the Holm-Sidak method). (C–E) Multiplex ELISA assay of cytokines’ release in medium after 72 hours of treatment. Results were expressed as the mean ± SD of all sample determinations conducted in triplicate. p-values indicate significance levels comparing average cytokines’ release among different groups (ANOVA).

Figure 4.

Protective effects of recombinant human LAV-BPIFB4 against SARS-CoV-2 lysate-induced cytotoxicity in vitro. Bar graph showing average cytotoxicity (±SD) determined using LDH assay in (A) BEAS-2B (human bronchial epithelial cells), (B) A549 (human alveolar basal epithelial cells), and (C) HUVEC (human umbilical vein endothelial cells) following 3 days treatment with SARS CoV-2 lysate or control lysate (50 μg/mL) in presence or absence of rhLAV-BPIFB4 (18 ng/mL; n = 3). p-values indicate significance levels comparing average lactate dehydrogenase release among different groups (analysis of variance).