Galectin-3 binding protein stimulated IL-6 expression is impeded by antibody intervention in SARS-CoV-2 susceptible cell lines

doi:10.21203/rs.3.rs-1458420/v1

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Galectin-3 binding protein stimulated IL-6 expression is impeded by antibody intervention in SARS-CoV-2 susceptible cell lines

https://doi.org/10.21203/rs.3.rs-1458420/v1

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COVID-19 is the global pandemic that affected our population in the past two years. Considerable research has been done to better understand the pathophysiology of this disease and to identify new therapeutic targets, especially for severe cases. Galectin-3 (Gal-3) is a receptor present at the surface of different cell types, namely epithelial and inflammatory cells, which has been described as a severity marker in COVID-19. The activation of Gal-3 through its binding protein (Gal-3BP) is directly linked to the production of pro-inflammatory cytokines that contribute for the cytokine storm (CS) observed in severe COVID-19 patients. Here, we show that D2, a recombinant fragment of the lectin-binding region of Gal-3BP was able to stimulate the expression of IL-6 in colon and lung epithelial cell lines in β-galactoside dependent manner. We further show that D2-induced IL-6 augmentation was reduced by the anti-Gal-3BP monoclonal antibody 1959. Our data confirm and extend prior findings of Gal-3BP mediated IL-6 induction, enlightening the potential of its antibody-mediated s blockage for the prevention and treatment of CS and severe disease in COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the strain of coronavirus responsible for the novel coronavirus disease 2019 (COVID-19), which is the respiratory illness responsible for the global COVID-19 pandemic. By March 7, 2022, nearly 450 million infections and over 6.0 million deaths have been reported globally¹. COVID-19 clinical course ranges from asymptomatic cases to very severe disease and septic status which may ultimately lead to multi organ failure^2,. Evidence accumulated so far showed that a complex interplay involving several components of the immune system, including immune-active infiltrating cells and soluble factors such as cytokines and chemokines, is mandatory for the onset of an acute severe systemic hyperinflammatory response known as 'cytokine storm' (CS), which is associated with serious adverse outcomes in patients infected with SARS-CoV-2³. Particularly, exaggerated production of IL-6, through induction of pro-inflammatory chemokines and cytokines, is the pioneer of the hyperinflammatory condition and CS in severe COVID-19⁴. Unfortunately, there is still no effective approach for prevention or treatment of ARDS and severe COVID-19 disease.

The cellular glycoprotein Galectin-3-binding protein (Gal-3BP, Uniprot ID – Q08380), also known as 90K, Mac-2 BP is a multidomain, multi-functional secreted protein that has been originally identified and characterized by our group⁵ and others⁶ as a tumor associated antigen and is involved in neoplastic transformation and cancer progression^7,8. Also, upregulation of Gal-3BP has been documented in infectious diseases, including Human Immunodeficiency Virus infection^9,10 and Human Hepatitis C virus infections¹¹. Functionally, Gal-3BP induces IL-6 expression and secretion in multiple different cell types, through carbohydrate-mediated interaction with Galectin-3 (Gal-3) at the cell surface^12,13. Therefore, Gal-3BP could represent a target for modulation of CS and treatment of severe COVID-19 patients.

Here, we show that a recombinant fragment of the lectin-binding region of Gal-3BP stimulates the expression of IL-6 in SARS-CoV-2 sensitive cells, which is reduced by anti-Gal-3BP monoclonal antibody.

Cell culture and treatment

Caco-2 (epithelial cell line from human colorectal adenocarcinoma), HCC827 and A549 (epithelial cell lines from human lung adenocarcinoma) were purchased to ATCC and cultured using Dulbecco's Modified Eagle's medium supplemented with 10% Fetal Bovine Serum (Gibco), 2mM Glutamine, 100U/mL penicillin plus 100 µg/mL streptomycin mixture and 20mM HEPES buffer (Thermo Fisher). Cells were seeded at a concentration of 1x10⁵ cells/mL in a 24-well plate and incubated overnight to adhere. After adhesion, different combinations of D2 (5, 10 and 20µg/mL), 1959 (40µg/mL) antibodies, and D-lactose (50mM) were added. After 24h and 48h, culture medium was collected for cytokine quantification. Cells were collected 24h after treatment for RNA extraction.

Galectin-3 expression

The human epithelial cell lines Caco-2, HCC827 and A549 were detached and stained with anti-human Gal-3 (Clone eBioM3/38, eBioscience, USA). Samples were acquired on LSRII flow cytometer (BD Biosciences) using the DIVA Software and data was analyzed using the FlowJo V10 software.

RNA isolation and quantitative real-time PCR (qPCR)

Total RNA was isolated using TripleXtractor (Grisp) according to manufacturer’s instructions. RNA was quantified using NanoDrop (Thermo Scientific) with ND-1000 software. RNA was converted to cDNA using Xpert cDNA Synthesis Mastermix kit (Grisp) according to manufacturer’s instructions. qRT-PCR was performed using NZYSpeedy qPCR Green Master Mix (NZYTech) in an Applied Biosystems 7500 Fast qPCR system (Applied Biosystems, Thermo Fisher Scientific). The specific primers used for IL-6 were AGTGAGGAACAAGCCAGAGC (forward) and GGTCAGGGGTGGTTATTGCA (reverse) and for 18s were GTAACCCGTTGAACCCCATT (forward) and CCATCCAATCGGTAGTAGCG (reverse). IL-6 expression levels were normalized to 18s expression and relative expression was determined as described in Gaifem et al¹⁴.

IL-6 measurement

IL-6 was quantified using an ELISA kit (reference 430504, BioLegend, CA, USA), according to the manufacturer's instructions.

Expression and purification of the recombinant Gal-3BP fragment D2

The mature Gal-3BP consists of 585 amino acids (Uniprot ID – Q08380)¹⁵. After cleavage of the first 18 amino acids (signal peptide), four putative regions could be distinguished in the cDNA sequence: (i) a scavenger receptor cysteine-rich domain (D1); (ii) a BTB/POZ (Broad-Complex, Tramtrack and Bric a brac/Poxvirus and Zinc finger) domain; (iii) a BACK (BTB and C-terminal Kelch) domain and (iv) an approximately 200 amino acid C-terminus with no significant similarity to other human proteins¹⁶. For convenience, we have named the 28 kDa DNA fragment corresponding to amino acid residues 134–288, encompassing BTP and about the first half of the BACK regions as domain 2 (D2).

MNFGLRLIFLVLTLKGVQCTRSTHTLDLSRELSEALGQIFDSQRGCDLSISVNVQGEDALGFCGHTVILTANLEAQALWKEPGSNVTMSVDAECVPMVRDLL RYFYSRRIDITLSSVKCFHKLASAYGARQLQGYCASLFAILAWSHPQFEKRGS

This is a highly glycosylated mucin-like region which binds Gal-3¹⁶. D2 was generated by the polymerase chain reaction (PCR). The fragment was subcloned into an Evitria’s proprietary vector system (EVITRIA, Schlieren, Switzerland) allowing the fusion in-frame to the 6xHis tag sequence that was located at the C-terminus of the domain.

D2 was transiently expressed by transfection into Chinese Hamster Ovary (CHO) cells. Concentrated culture supernatant was applied to a column of Ni-NTA Superflow (Qiagen) and D2 was eluted with increasing concentrations of imidazole according to the manufacturer’s instructions.

Anti-Gal-3BP antibodies

Antibody 1959, a humanized variant of the murine monoclonal antibody SP-2 recognizing the human Gal-3BP was generated by identifying murine complementary determinant regions (CDRs) that were grafted onto a human antibody framework (patent reference WO2019197651A1). The antibody was purified on a Protein-G column (Biovision) and dialyzed against PBS.

Statistical analysis

Data are expressed as mean ± SD. For multiple group comparisons, one-way ANOVA test with a Tukey multiple-comparison posttest was applied. All statistical analysis were carried out with GraphPad Prism (version 7.01). Statistically significant values are as follows: ∗p < 0.05; ∗∗p < 0.01.

Previous reports have shown that Gal-3BP induces IL-6 expression and secretion in multiple different cell types^12,13. To evaluate whether induction of IL-6 also occurs in cells permissive to SARS-CoV-2 infection, human colon adenocarcinoma Caco-2 cells were cultured with different concentrations of D2 and the levels of IL-6 in the cellular supernatant was collected for IL-6 quantification. Treatment of the cells with D2 (5, 10 or 20 µg/mL) dose-dependently increased the level of IL-6 at 24 hours (p = 0.0435 and 48 hours (p = 0.0020) compared to untreated cells (Fig. 1A). The stimulatory effect of D2 was also observed when IL-6 expression was analysed by real-time PCR (p = 0.0254; Fig. 1B).

IL-6 expression induced by Gal-3BP requires β-galactoside-mediated interaction between Gal-3BP and Gal-3¹⁷. Then, we analyzed IL-6 secretion in the presence of lactose (Galβ1-4glucose) in Caco-2 cells and two additional SARS-CoV-2 susceptible cell lines derived from human lung adenocarcinoma, A549 and HCC827. As expected, treatment of the cells with 20 µg/ml of D2 for 48 hours elicited a significant increase of IL-6 in the supernatant of all three cell lines compared to untreated controls (Fig. 1C). On the contrary, treatment with D2 in the presence of lactose (25 mM) leads to a significant decrease of IL-6 secretion compared to cells treated with D2 alone (p = 0.0222 for Caco-2 cells; p = 0.0260 for A549 cells; p = 0.0008 for HCC827 cells; Fig. 1C). The extent of inhibitory effect of lactose was proportional to the levels of Gal-3 expressed by the cells, being greater in HCC827 than A549 and Caco-2 cells (Fig. 1D),

We later verified whether the ability of D2 to stimulate IL-6 secretion could be blocked by an anti-Gal-3BP monoclonal antibody (1959). To this end, Caco-2, HCC827 and A549 cells were pre-treated with 40 µg/ml of 1959 for 20 min before adding 10 µg/ml of D2 and continuing incubation for 48 hours. As shown in Fig. 1D, the augmentation of IL-6 secretion upon D2 stimulation was significantly reduced by treatment with 1959 (p = 0.0222 for Caco-2 cells; p = 0.0026 for A549 cells; p = 0.0488 for HCC827 cells; Fig. 1E).

Several studies revealed increased levels of Gal-3BP in the plasma of patients with COVID-19^18–20. Consistently, these studies showed that Gal-3BP was significantly overexpressed in all symptomatic COVID-19 cohorts, with highest levels associated with severe disease. Thus, this increased production of Gal-3BP may induce a higher activation of its endogenous ligand Gal-3, promoting the release of inflammatory cytokines, which may contribute or even exacerbate pathologic proinflammatory signalling cascades in the lung and other organs²¹. Indeed, Gal-3 was recently recognized as a potential prognostic biomarker of severe COVID-19 and its inhibition has been suggested as a potential therapeutic approach to attenuate the hyperinflammation observed in these patients^22,23.

In conclusion, we showed that D2, a highly glycosylated, lectin binding fragment of Gal-3BP stimulated secretion of IL-6 in SARS-CoV-2 susceptible epithelial cell lines in a Gal-3 dependent manner. Further, we showed that the stimulatory effect could be partially reverted by the anti-Gal-3BP monoclonal antibody 1959.

Overall, these findings show that targeting Gal-3BP-Gal-3 interaction with a specific antibody may represent a potential approach to reduce massive inflammation and organ damage in severe COVID-19 patients.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This work has been funded by Italian National Funding for Centers of Excellence (Science Department, Roma Tre University—MIUR, Articolo 1, Commi 314–337 Legge 232/2016).

Authors' contributions

(I) SI and RS designed and supervised the project; (II) M-FA, VG, VI and GR performed the experiments; (III) GA, RS and SI analyzed and interpreted data; (IV) Manuscript writing: all authors; (VI) All authors read and approved the final manuscript.

Data Availability statement

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

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You are reading this latest preprint version

Galectin-3 binding protein stimulated IL-6 expression is impeded by antibody intervention in SARS-CoV-2 susceptible cell lines

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Cell culture and treatment

Galectin-3 expression

RNA isolation and quantitative real-time PCR (qPCR)

IL-6 measurement

Expression and purification of the recombinant Gal-3BP fragment D2

Anti-Gal-3BP antibodies

Statistical analysis

Results And Discussion

Declarations

References

Additional Declarations

Status:

Version 1