key: cord-0796260-npzfvud4
authors: Duarte, Carolina; Akkaoui, Juliet; Ho, Anny; Garcia, Christopher; Yamada, Chiaki; Movila, Alexandru
title: Age-dependent effects of the recombinant spike protein/SARS-CoV-2 on the M–CSF– and IL-34-differentiated macrophages in vitro
date: 2021-02-03
journal: Biochem Biophys Res Commun
DOI: 10.1016/j.bbrc.2021.01.104
sha: cd7c3a028a55259787e14ecd8c9db4b4cf2c44b2
doc_id: 796260
cord_uid: npzfvud4

The SARS-CoV-2 virus causes elevated production of senescence-associated secretory phenotype (SASP) markers by macrophages. SARS-CoV-2 enters macrophages through its Spike-protein aided by cathepsin (Cat) B and L, which also mediate SASP production. Since M-CSF and IL-34 control macrophage differentiation, we investigated the age-dependent effects of the Spike-protein on SASP-related pro-inflammatory-cytokines and nuclear-senescence-regulatory-factors, and CatB, L and K, in mouse M–CSF– and IL-34-differentiated macrophages. The Spike-protein upregulated SASPs expression in young and aged male M–CSF–macrophages. In contrast, only young and aged male IL-34-macrophages demonstrated significantly reduced pro-inflammatory cytokine expression in response to Spike-protein in vitro. Furthermore, the S-protein elevated CatB expression in young male M–CSF–macrophages and young female IL-34-macrophages, whereas CatL was overexpressed in young male IL-34- and old male M–CSF–macrophages. Surprisingly, the S-protein increased CatK activity in young and aged male M–CSF–macrophages, indicating that CatK may be also engaged in the COVID-19 pathology. Altogether, we demonstrated the age- and sex-dependent effects of the Spike-protein on M-CSF and IL-34-macrophages using a novel in vitro mouse model of SARS-CoV-2/COVID-19.

The current COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is characterized by an overwhelming macrophage (Mφ)-induced cytokine storm, which leads to the mild-to-severe respiratory disease and death caused by pneumonia [1] . Severe symptomatology is more prevalent in males than in females, and both morbidity and mortality are aggravated by age [1] . These differences have been adjudicated to age-and gender-dependent expression of receptors that facilitate the entry of SARS-CoV-2 virus to the cell, along with the age-related co-morbidities [2] .

As a characteristic structural component of the virion membrane of SARS and MERS coronaviruses, the Spike protein (S-protein) is responsible for the viral entry into Mφ, by activation of cysteine protease cathepsins [3] . The activated Mφ produces a distinct panel of pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and IL-6, which are collectively termed senescence-associated secretory phenotype (SASP).

This Mφ polarization and activation is triggered by the ligation of Macrophage colony stimulating factor (M-CSF) and IL-34, with the CSF-1 receptor [4] . The M-SCF-induced Mφ are highly immunostimulatory, whereas the IL-34 activated Mφ possess immunosuppressive characteristics [5] . Furthermore, M-CSF differentiated Mφ are more susceptible to viral infection and correlate with higher mortality due to viral disease, whereas Il-34 differentiated Mφ is shown to possess elevated resistance to viral infection [6] . Incidentally, the SARS-CoV-2 S-protein binds to the surface proteins on peripheral blood mononuclear cells and induces the production of M-CSF, which then mediates the pro-inflammatory SASP cytokine storm that characterizes severe COVID-19 symptomatology [7] .

Although the production of SASPs, which includes pro-inflammatory cytokines and chemokines, senescence-regulatory proteins and lysosomal cathepsins (Cat), by Mφ is well established in various infectious and sterile experimental models of age-related inflammation [8] , the differential effect of the SARS-CoV-2 S-protein on M-CSF-and IL-34Mφ remains elusive in the context of age and gender. Thus, this study aimed to evaluate the age-and sex-dependent effects of the SARS-CoV-2 recombinant S-protein on the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, macrophage migration inhibitory factor (MIF)), nuclear senescence-regulatory proteins (Hmgb1, p53, p21), and cathepsins (CatB, CatL and CatK) using M-CSF and IL-34 primed bone marrow derived macrophages, as a potential in vitro experimental model of COVID-19/SARS-CoV-2.

Bone marrow derived macrophages (BMDMs) were isolated from the femurs and tibias of young 

The intracellular activities of cathepsin B, L and K were evaluated using the Magic Red™ Cathepsin (Immunochemistry Technologies, Bio-Rad) kits, following the manufacturer's recommended protocol. Images were acquired with a Zeiss LSM780 confocal microscope.

The collected data were analyzed using the Student's t-test for the comparison between two groups, or a one-way ANOVA with post hoc Tukey's test for the comparisons among different groups. A p < 0.05 was considered statistically significant. Data were analyzed using Sigma Plot v.14 software.

To the best of our knowledge, the S-protein significantly elevated expression of Tnf-α, Il-1β, Il-6

and Mif RNAs in aged male M-CSF-Mφ (Fig 1A-C, Suppl. Fig. 1 ), whereas only Tnf-α, Il-1β and Il-6 were overexpressed in young male Mφ (Fig 1A-C) . In contrast, IL34-Mφ from old male mice showed an anti-inflammatory effect, with inhibition of Tnf-α and Il-6 ( Fig 1G, I) , while inhibition of Il-1β and Il-6 were observed in IL-34-Mφ from young male mice (Fig 1H-I) . These data indicate that the S-protein increased the expression of SASP cytokines in M-CSF-Mφ while decreasing it J o u r n a l P r e -p r o o f in IL-34-Mφ, from male mice. For young and aged female MCSF-Mφ exposed to the S-Protein, no significant fluctuations in the expression of the Tnf-α, Il-1β, Il-6 and Mif genes were observed (Fig 2; Suppl. Fig. 1) . Furthermore, only a significant increase in the expression of Il-1β (Fig 2I) was detected in young female IL-34 Mφ, whereas no changes were observed in any of the reported cytokines in aged female IL34-Mφ. These data corresponded to a recently published observation showing that high serum levels of TNF-α and IL-6 have been identified in the COVID-19 patients, with IL-6 being significantly higher in critically ill patients [9] ; however, the gender or age distribution of these data have not been reported. Additionally, in COVID-19 patients, M-CSF, but not IL-34 expression, has been assessed, and found to be elevated [10] .

Although, M-CSF and IL-34 have been identified in chronic inflammation, their specific contribution to the inflammatory process has not yet been detailed [11] . Our sex and age specific observations in mouse Mφ correlated with statistical findings of high prevalence and severity of COVID-19 symptoms in senior male patients [2, 12] . Additionally, our data clearly demonstrated the expected inflammatory phenotype induced by the SARS-CoV-2 in M-CSF, but not in IL-34, differentiated Mφ.

Since the S-protein induced production of pro-inflammatory SASP cytokines from Mφ (Fig 1 and   2 ) and the increased susceptibility of senescent cells to viral infection [13] , we next evaluated the effect of the S-protein on the expression of key nuclear senescence-regulatory proteins, including Hmgb1, p53 and p21, in MCSF-and IL34-Mφ. Exposure to the S-protein induced the overexpression of Hmgb1, p53 and p21, by MCSF-Mφ (Fig 1D-F) , but not in IL34-Mφ, isolated from old and young male mice (Fig 1J-L) . In contrast, Hmgb1 and p21 were significantly overexpressed in young female IL34-Mφ (Fig 2D,F) , but not in MCSF-Mφ. Interestingly, no changes were observed in M-CSF-and IL-34-Mφ from old female mice, which may potentially be affected by the SARS-CoV-2 through different signaling pathways. HMGB1 is a damage associated molecular pattern (DAMP) alarmin, which amplifies senescence-associated J o u r n a l P r e -p r o o f inflammation [14] . Furthermore, HMGB1 is a mediator of the inflammatory cell infiltration to the lungs and contributes to the reprograming of Mφ towards a pro-inflammatory phenotype, which is upregulated in the aging lungs and kidneys [15] . Similarly, p53 and its downstream gene p21 are upregulated in fibrotic lung disease and are known to be activated in response to DNA damage [16, 17] . Furthermore, p53 intervenes in viral activation and has been identified in the bronchial lavage fluid of COVID-19 patients [18, 19] .

Numerous studies show cathepsins may facilitate the cellular senescence and aging-associated diseases, including osteoporosis and Alzheimer's disease [20] . Furthermore, CatB and L are activated by the S-proteins from the SARS and MERS coronaviruses to mediate membrane fusion and subsequent release of viral RNA into the host cell [3] . Importantly, CatK is not known to be directly associated with viral infection or replication; however, it does induce production of SASPs, including pro-inflammatory TNF-α, IL-6 and IL-1β cytokines, from Mφ and Mφ-like osteoclast precursors [21] [22] [23] [24] . Thus, we finally assessed the age-and sex-dependent mRNA expression of CatB, L and K, along with their intracellular activity in M-CSF-and IL-34-Mφ exposed to the S-protein using real time PCR and confocal microscopy assays, respectively.

In young males, S-protein was found to significantly elevate CatB and CatK genes expression and intracellular activities in M-CSF-Mφ (Fig 1 M, O) , whereas CatL and CatK expressions and activities were increased in IL-34-Mφ (Fig 1Q-R) . Conversely, the expression and activity of (Fig 2P,R) ; however, the expression and intracellular activities of CatB, CatL and CatK in response to S-protein were neither affected in young and old female M-CSF-Mφ nor in old female IL-34-Mφ (Fig. 2) . While the importance of CatB and CatL in the SARS coronavirus-induced inflammation has been reported in previous studies [25] , J o u r n a l P r e -p r o o f this study detected, for the first time, the increased expression and intracellular activity of CatK in M-CSF-Mφ and IL-34-Mφ exposed to the S-protein of SARS-CoV-2. Therefore, further investigations are warranted to elucidate the role of CatK in the age and sex associated severity of COVID-19 symptoms.

The mechanisms by which Spike-protein/SARS-CoV-2 induces the expression of inflammatory SASP cytokines, which characterize the severe COVID-19 clinical profile, are still to be determined. These inflammatory cytokines are largely produced by cells of the innate immune response, including Mφ. Hereby, we demonstrate the manner in which the specific age-and sex-dependent proliferation of Mφ induced by M-CSF or IL-34 leads to the distinct gene expression of various SASP markers in response to stimulus by the S-protein of SARS-CoV-2.

Significantly higher inflammatory and senescent phenotypes were observed in male M-CSF-Mφ, but not female MCSF-Mφ, which correlate with clinical data suggesting the male prevalence of COVID-19 [12] . Conversely, an anti-inflammatory effect was observed in IL-34 proliferated male but not female Mφ in vitro. IL-34 has been identified as a target of pro-inflammatory microRNA, miR-31-3p, which mediates inflammation in human lung fibroblasts, by inhibiting IL-34, which, in turn, causes flaring of IL-6 and IL-8 production [26] . Interestingly, a mild inflammatory effect and distinct gene expression phenotype was observed in the S-protein stimulated young female IL34-Mφ, which was not the case with old female Mφ. The expression of IL-34 in mild and severe COVID-19 cases should be assessed, with sex and age distinctions, to determine whether IL-34 correlates with SARS-CoV-2 susceptibility. Moreover, IL-34 may be considered a potential therapeutic target for COVID-19 in the male population. Sex and gender related variations in the immune response have been previously reported in SARS-CoV-2 positive J o u r n a l P r e -p r o o f patients [27] . In this context, the current study demonstrates that sex-and age-related variations to stimulus by the SARS-CoV-2 S-protein can also be obtained in mouse models of the disease. 

Racial and Gender-Based Differences in COVID-19

The Age-Related Risk of Severe Outcomes Due to COVID-19 Infection: A Rapid Review, Meta-Analysis, and Meta-Regression

Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research

IL-34 and M-CSF form a novel heteromeric cytokine and regulate the M-CSF receptor activation and localization

IL-34 induces the differentiation of human monocytes into immunosuppressive macrophages. antagonistic effects of GM-CSF and IFNgamma

Human interleukin-34-derived macrophages have increased resistance to HIV-1 infection

Monoclonal Antibodies Capable of Binding SARS-CoV-2 Spike Protein Receptor Binding Motif Specifically Prevent GM-CSF Induction. bioRxiv

Contribution of Porphyromonas gingivalis lipopolysaccharide to experimental periodontitis in relation to aging

Profiling serum cytokines in COVID-19 patients reveals IL-6 and IL-10 are disease severity predictors

Serum Cytokine and Chemokine Profile in Relation to the Severity of Coronavirus Disease 2019 in China

Colony-stimulating factor (CSF) 1 receptor blockade reduces inflammation in human and murine models of rheumatoid arthritis

Sex-Related Differences in COVID-19 Lethality

Exploring the Relevance of Senotherapeutics for the Current SARS-CoV-2 Emergency and Similar Future Global Health Threats. Cells

p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes

HMGB1 Facilitated Macrophage Reprogramming towards a Proinflammatory M1-like Phenotype in Experimental Autoimmune Myocarditis Development

DNA damage responses and p53 in the aging process

Serpine 1 induces alveolar type II cell senescence through activating p53-p21-Rb pathway in fibrotic lung disease

Reactivation of Epstein-Barr Virus by HIF-1alpha Requires p53

Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients

Cathepsin B and L activity in the serum during the human aging process: cathepsin B and L in aging

Odanacatib, A Cathepsin K-Specific Inhibitor, Inhibits Inflammation and Bone Loss Caused by Periodontal Diseases

Deficiency of cathepsin K prevents inflammation and bone erosion in rheumatoid arthritis and periodontitis and reveals its shared osteoimmune role

Essential mixed cryoglobulinemia as a cause of gastrointestinal hemorrhage

Cathepsin-k as a diagnostic marker in the identification of micro-granulomas in Crohn's disease. Pathologica

TMPRSS2 Contributes to Virus Spread and Immunopathology in the Airways of Murine Models after Coronavirus Infection

Circular RNA ANKRD36 attends to lipopolysaccharide-aroused MRC-5 cell injury via regulating microRNA-31-3p

In vivo antiviral host response to SARS-CoV-2 by viral load, sex, and age. bioRxiv

Development Grant and NIH Grants R01AG-064003, R15DE-027153, R03DE-028699.

The authors have no conflict of interest to declare.

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