Novel evidence of Thymosin α1 immunomodulatory properties in SARS-CoV-2 infection: Effect on innate inflammatory response in a peripheral blood mononuclear cell-based in vitro model

Graphical abstract

Abstract

The peculiar property of Thymosin alpha 1 (Tα1) to act as master regulator of immune homeostasis has been successfully defined in different physiological and pathological contexts ranging from cancer to infection. Interestingly, recent papers also demonstrated its mitigating effect on the “cytokine storm” as well as on the T-cell exhaustion/activation in SARS-CoV-2 infected individuals. Nevertheless, in spite of the increasing knowledge on Tα1-induced effects on T cell response confirming the distinctive features of this multifaceted peptide, little is known on its effects on innate immunity during SARS-CoV-2 infection. Here, we interrogated peripheral blood mononuclear cell (PBMC) cultures stimulated with SARS-CoV-2 to disclose Tα1 properties on the main cell players of early response to infection, namely monocytes and myeloid dendritic cells (mDC). Moving from ex vivo data showing an enhancement in the frequency of inflammatory monocytes and activated mDC in COVID-19 patients, a PBMC-based experimental setting reproduced in vitro a similar profile with an increased percentage of CD16⁺ inflammatory monocytes and mDC expressing CD86 and HLA-DR activation markers in response to SARS-CoV-2 stimulation. Interestingly, the treatment of SARS-CoV-2-stimulated PBMC with Tα1 dampened the inflammatory/activation status of both monocytes and mDC by reducing the release of pro-inflammatory mediators, including TNF-α, IL-6 and IL-8, while promoting the production of the anti-inflammatory cytokine IL-10. This study further clarifies the working hypothesis on Tα1 mitigating action on COVID-19 inflammatory condition. Moreover, these evidence shed light on inflammatory pathways and cell types involved in acute SARS-CoV-2 infection and likely targetable by newly immune-regulating therapeutic approaches.

1. Introduction

In addition to the rapidly realized coronavirus disease 2019 (COVID-19) vaccination campaign, the development of multiple anti-SARS-CoV-2 antiviral and monoclonal antibody (mAb)-based therapies has also contributed to the expansion of a prophylactic and therapeutic armamentarium against COVID-19. The growing possibilities in this field gained particular importance not only to reduce mortality but also to prevent hospitalization of specific categories of individuals, such as fragile patients with mild or moderate disease, unvaccinated subjects or those who developed a breakthrough infection. Indeed, the activation of innate as well as cellular and humoral immunity by SARS-CoV-2 infection can lead in several individuals to a persistent inflammation, such as acute respiratory distress syndrome, characterized by cytokine storm and diffuse organ involvement [1]. Complex alterations of the immune system ranging from inhibition to activation and exhaustion, as well as modification of lymphocyte total number and functional status, have been associated with severe pathology [2]. Thus, being hyperinflammation and immune-mediated respiratory injury the distinctive traits of COVID-19 disease, the advantage of anti-inflammatory drugs has been extensively exploited [3]. Although in this context several molecules with inhibitory and immunomodulating activity have been characterized, only a portion of patients benefitted from anti-inflammatory therapies, opening a debate on their effectiveness and highlighting the need to identify alternative therapeutic approaches [4]. Moreover, COVID-19, in its numerous variant forms, is a rapidly changing landscape that requires a dynamic intervention for the development of new therapies for community, outpatient and inpatient settings. Thus, alternative therapeutic approaches are needed to face the changes in SARS-CoV-2-induced symptoms and infection severity due to the different immunopathological features of the existing and emerging variants of concern (VOC). Recently, host-directed therapies (HDTs) with antiviral efficacy against SARS-CoV-2 infection have been shown to be less prone to the development of drug resistance and, therefore, represent promising pharmacological options to combat emerging viral variants, since host genes possess a more controlled and much lower mutation rate as compared to viral genes. In particular, HDTs, targeting aberrant host immune and inflammatory responses, is an area of growing interest to mitigate COVID-19 pathophysiological and clinical manifestations, namely lymphopenia (including reduced counts of CD4⁺ T cells, CD8⁺ T cells and Natural Killer cells), excessive T cell activation, high expression of T cell inhibitory molecules and uncontrolled cytokine responses [5], [6].

In this context, thymosin alpha 1 (Tα1), a polypeptide hormone secreted by thymic epithelial cells, gained new attention in the clinical setting since it has been already and successfully used in many diseases involving immune dysfunctions (such as chronic B and C hepatitis, sepsis, and cystic fibrosis), as well as in some types of cancer [7]. Acting as a multifaceted and pleiotropic molecule able to restore immune homeostasis in different physiological and pathological conditions [8], Tα1 has been proposed as immunomodulatory therapy in COVID-19 and, indeed, was used in China during the SARS-CoV-2 outbreak [9], [10], [11], [12]. In particular, it was shown that Tα1 can reduce the mortality of severe COVID-19 patients (CP) by enhancing their immune responses and cooperating in the limitation of SARS-CoV-2 viral spreading [12]. Depending on the host inflammatory status or pre-existing immune dysfunction, Tα1 is able to activate the tolerogenic pathway of tryptophan catabolism via indoleamine 2,3-dioxygenase [13]. Tα1 can also promote immune tolerance mechanisms, thus limiting the activation of the chronic inflammation in response to various infectious settings [14], [15]. One emerging strategy to reduce infectivity and ameliorate clinical symptomatology considers as target for immune intervention the endogenous barriers and anti-inflammatory pathways that are defective in CP. Thus, being upper respiratory airways and the nasal barrier the entry site of SARS-CoV-2, their role in the prevention or limitation of infection is increasingly recognized as crucial for COVID-19 outcomes, together with the early innate response of the host. In line with this view, a combined treatment of recombinant human Interferon (IFN)-α nasal drops together with Tα1 has been shown to effectively prevent COVID-19 in medical personnel [16].

Having found in ex vivo peripheral blood mononuclear cells (PBMC) from severe CP a rearrangement in monocytes and myeloid dendritic cells (mDC) with a reduction in total cell count but an increase in cell subsets expressing activation and inflammatory markers, a human PBMC-based model was interrogated to investigate in vitro the impact of Tα1 on the pro-inflammatory status induced by SARS-CoV-2 stimulation. Interestingly, when Tα1 was added to SARS-CoV-2-stimulated PBMC cultures a decrease in pro-inflammatory cytokine production and an increase in IL-10 release was observed. Likewise, a reversion in the differentiation of inflammatory monocyte subsets as well as of the activation status of both monocytes and mDC induced by SARS-CoV-2 stimulation was elicited by Tα1 treatment. These findings confirm the capacity of Tα1 to act as homeostatic molecule also in the context of the innate immune response stimulated during SARS-CoV-2 infection.

2. Material and methods

2.1. Ethics statement

Istituto Superiore di Sanità Review Board approved the use of blood from Healthy Donors (HD) (AOO-ISS—14/06/2020–0020932) and from asymptomatic SARS-CoV-2 infected individuals (IN_COVID, AOO-ISS—22/03/2021–0010979). Policlinico Tor Vergata approved blood withdrawal from hospitalized CP (COVID-SEET, CE#46.20, 18/04/2020).

2.2. Patients

Six hospitalized COVID-19 [CP-H; 2 females/4 males; median age ± Standard Deviation (SD) 51.5 ± 23.3 years (yrs)] and eight asymptomatic [CP-AS; 4 females/4 males; 58.5 ± 10.4 yrs] patients matched to five HD [3 females/2 males; 53 ± 12.5 yrs] were enrolled and provided written informed consent. Main demographic, clinical and experimental data related to the subjects included in the study cohort [enrolled from March to May 2020] are listed in Supplementary Table 1.

2.3. Isolation of PBMC

PBMC were isolated from freshly collected buffy coats obtained from healthy volunteers (Blood Bank of University “La Sapienza”, Rome, Italy) by density gradient centrifugation using lympholyte-H (Cedarlane) as previously described [17].

2.4. Virus production and titration

Virus was produced and titrated as previously described [17]. Briefly, Vero E6 (Vero C1008, clone E6-CRL-1586; ATCC) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO) supplemented with non-essential amino acids (NEAA, 1x) (GIBCO), penicillin/streptomycin (P/S, 100 U/mL) (GIBCO), HEPES buffer (10 mM) (GIBCO) and 10% (v/v) Fetal bovine serum (FBS) (Euroclone). The clinical isolate of SARS-CoV-2.

hCoV-19/Italy/LOM-ASST-CDG1/2020 (GISAID accession ID: EPI_ISL_412973) was cultivated in Vero E6 cells, and viral titer was determined by 50% tissue culture infective dose (TCID50) and plaque assay for confirming the obtained titer. SARS-CoV-2 stocks were titrated using End-point Dilutions Assay (EDA, TCID50/mL). Vero E6 cells (4 × 10⁵ cells/mL) were seeded into 96 wells plates and infected with base 10 dilutions of collected medium, each condition tested in triplicate. After 1 hour (h) of adsorption at 37 ˚C, the cell-free virus was removed, and complete medium was added to cells. After 72 h, cells were observed to evaluate CPE. TCID50/mL was calculated by Endpoint Dilution Assay by using the Reed-Muench formula.

2.5. Cell stimulation and sample collection

PBMC were pre-incubated for 1 h at 37 ˚C with infectious SARS-CoV-2 at a multiplicity of infection (MOI) of 0.02 and then cultured at 2x10⁶ cells/ml in RPMI 1640 (GIBCO) in presence of P/S (100 U/mL) (GIBCO), L-glutamine (2 mM) (GIBCO) and 10% FBS (Euroclone) for 24 h. Furthermore, where indicated, 100 ng/ml Tα1 (SciClone Pharmaceuticals) were used to pre-treat PBMC for 3 h before SARS-CoV-2 stimulation. After treatments, cells were stained as detailed below (see flow cytometry section) while culture supernatants were harvested and treated for 30 min at 56 °C prior of their storage at -80 °C for later use.

2.6. Detection of cytokines and chemokines in culture supernatants

Release of cytokines (IL-6, TNF-α, IL-8 and IL-10) was quantified by specific cytometric bead array (BD Biosciences) on a FACS Canto (BD Biosciences) and analyzed by FCAP array software (BD Biosciences).

2.7. Flow cytometric analysis

Monoclonal Ab specific for human CD86, CD16, CD11c, CD14, HLA-DR, CD3, CD19, and CD56 were purchased from BD Biosciences. To establish viability of cells and to exclude dead cells from flow cytometry analysis Fixable Viability Dye eFluor450 (FvDye) (eBioscience) was used as previously described [18]. Briefly, 4x10⁵ PBMC were incubated with monoclonal Abs at 4 °C for 30 min and then fixed with 4 % paraformaldehyde before analysis on a Cytoflex LX flowcytometer (Beckman Coulter). Data were analyzed by CytExpert software (Beckman Coulter). The expression of cell surface molecules was evaluated using the median fluorescence intensity (MFI) after subtraction of the values of the isotype Ab controls. Only single cells present in the viable cell gate were considered for further analysis.

In the mixed cell population of PBMC, after excluding the DUMP channel (CD3, CD19, CD56 mAbs and FvDye), we considered the HLA-DR⁺ cells and defined in this gate the CD14⁺ monocytes as well as in the CD14^- cells, the CD11c⁺ mDC as exemplified in gating strategy (Supplementary Fig. 1). Moreover, classical, inflammatory, and atypical monocyte subsets were identified by the differential surface expression of CD16 marker.

2.8. Statistical analysis

Statistical analysis was performed using One or Two-way Repeated-Measures ANOVA when three or more stimulation conditions were compared. In case of significant ANOVA, the pairwise comparisons were carried out using post-hoc approaches for multiple comparisons, in order to test the significance of the difference between two stimulation effects. Results were shown as median values ± Interquartile range (IQR) or, where indicated, as mean values ± standard error of the mean (SEM). A p value ≤ 0.05 was considered statistically significant. In the figures, star scale was assigned as follows: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001. Data and statistical analyses were processed by Prism software version 9.4.1 (Graph Pad).

3. Results

3.1. Monocytes of asymptomatic and hospitalized COVID-19 patients show altered frequency and activation status

We and others previously reported a different frequency as well as phenotype of circulating plasmacytoid DC (pDC) in asymptomatic versus hospitalized COVID-19 patients (CP) [17], [19]. Here, we sought to investigate whether in the same study group also the frequency and the phenotype of monocytes were changing according to disease severity (Fig. 1 ). In particular, the immune-phenotype of PBMC from individuals with asymptomatic SARS-CoV-2 infection (CP-AS, n = 8), hospitalized CP (CP-H, n = 6) and healthy donor volunteers (HD, n = 5; matched for sex and age) was analyzed (see main demographic and clinical features in Supplementary Table 1 and gating strategy in Supplementary Fig. 1).

Fig. 1.

Scheme of ex vivo study conducted on healthy subjects and COVID-19 patients. A schematic representation of subjects enrolled in this study, sample type and data analysis are depicted. Peripheral blood mononuclear cells (PBMC) were collected from asymptomatic (CP-AS, n = 8) and hospitalized COVID-19 patients (CP-H, n = 6) as well as matched healthy donors (HD, n = 5). Cytofluorimetric analysis was conducted to evaluate phenotype and activation status of monocytes and myeloid dendritic cells (mDC).

Our comparative analysis highlighted that the frequency as well as the absolute number of CD14⁺ monocytes were strongly reduced in SARS-CoV-2 infected individuals with respect to HD. The amplitude of this reduction was consistent with the degree of COVID-19 severity since monocyte depletion was more pronounced in CP-H irrespective of their age and sex (Fig. 2 A and Supplementary Table 1).

Fig. 2.

Frequency and inflammatory/activation status of monocytes in asymptomatic and hospitalized COVID-19 subjects. Freshly isolated peripheral blood mononuclear cells (PBMC) from asymptomatic (CP-AS, n = 8) and hospitalized COVID-19 patients (CP-H, n = 6) as well as matched healthy donors (HD, n = 5) were stained with a cocktail of antibodies (CD16, CD14, CD86, HLA-DR, as well as CD3, CD19, CD56 and FvDye as DUMP channel) to study by flow cytometry monocyte frequency, abundance of the different subsets and activation status. (A) Monocyte frequency and absolute number in HD, CP-AS and CP-H. (B) Representative dot plots for HD, CP-AS and CP-H of monocyte subsets gated on CD14⁺ cells. Classical monocytes [CD14^hiCD16^neg] are indicated in blue, inflammatory monocytes [CD14⁺CD16^int] in red and atypical monocytes [CD14^low-negCD16^hi] in light blue. (C) Percentages of CD14^hiCD16^neg classical, CD14⁺CD16^int inflammatory and CD14^low-negCD16^hi atypical monocytes within the parental gate were measured in HD, CP-AS and CP-H patients. Surface expression of CD86 (D) and HLA-DR (E), was determined as mean fluorescence intensity (MFI) in monocyte subsets. Results were shown as median values ± Interquartile range and non-parametric Two-way ANOVA corrected for multiple comparisons with Tukey’s test was used to calculate significant statistical differences. Star scale was assigned as follows: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

It is known that the differential surface expression of the markers CD14 and CD16 can be used to distinguish among three different subsets of circulating monocytes, namely the classical [CD14^hiCD16^neg], the intermediate/inflammatory [CD14⁺CD16^int] and the non-classical/atypical [CD14^low-negCD16^hi] monocytes. In depth analysis of these subpopulations highlighted consistent differences among CP and HD (Fig. 2 B and 2C). Indeed, a robust reduction of classical CD14^hiCD16^neg monocytes and an increase of both inflammatory CD14⁺CD16^int and atypical CD14^low-negCD16^hi monocyte subsets was found in all SARS-CoV-2 infected subjects as compared to HD. Of note, these differences were more pronounced in CP-H respect to CP-AS indicating a correlation with disease severity. Interestingly, the costimulatory molecule CD86, which was significantly expressed in CP monocytes (Fig. 2 D), was further enhanced in CP-AS. A significant induction of the activation marker HLA-DR was also observed in total CD14⁺ as well as in classical and non-classical monocytes of CP (Fig. 2 E), further corroborating that monocytes are highly activated in response to SARS-CoV-2 infection. Accordingly, the levels of the pro-inflammatory cytokines IL-6, TNF-α and IL-1β well correlated with both cytofluorimetric data and disease severity displaying higher levels in sera of CP-H and CP-AS in respect to HD (Supplementary Table 1).

3.2. Myeloid DC of asymptomatic and hospitalized COVID-19 patients show altered frequency and activation status

Given the importance of mDC in protection against viral infections [20], the variation in the frequency and absolute number of circulating mDC was also investigated ex vivo in PBMC of CP and HD (see gating strategy in Supplementary Fig. 1 and Fig. 1).

As shown for monocytes (Fig. 2 A and B), both frequency and absolute number of circulating mDC were affected in SARS-CoV-2 infected individuals (Fig. 3 A and 3B). Indeed, as respect to HD, CP exhibit a consistent depletion of mDC in the bloodstream that was exacerbated in hospitalized patients (Fig. 3 A and B). Importantly, the expression of the maturation markers CD86 and HLA-DR indicated that mDC of CP were significantly activated with respect to HD. In particular, both markers were more expressed on mDC of CP-AS as compared to CP-H (Fig. 3 C and D).

Fig. 3.

Frequency and phenotype of myeloid DC in hospitalized and asymptomatic COVID-19 patients. Freshly isolated peripheral blood mononuclear cells (PBMC) from asymptomatic (CP-AS, n = 8) and hospitalized COVID-19 patients (CP-H, n = 6) as well as matched healthy donors (HD, n = 5) were stained with a cocktail of antibodies (CD14, CD11c, CD86, HLA-DR, as well as CD3, CD19, CD56 and FvDye as DUMP channel) to study by flow cytometry myeloid dendritic cells (mDC) frequency and activation status. (A) mDC frequency and absolute number in HD and CP-AS and CP-H. (B) Representative dot plots of mDC for HD, CP-AS and CP-H gated on CD14^-CD11c⁺ cells. Surface expression of CD86 (C) and HLA-DR (D) was determined as mean fluorescence intensity (MFI). Results were shown as median values ± Interquartile range and non-parametric Two-way ANOVA corrected for multiple comparisons with Tukey’s test was used to calculate significant statistical differences. Star scale was assigned as follows: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

3.3. Tα1 acts as modulator of monocyte inflammatory status during in vitro stimulation of PBMC with SARS-CoV-2

Having defined ex vivo that monocytes of CP harbor a highly inflammatory profile that correlates with the hyperinflammation observed during COVID-19 disease, we evaluated in vitro the immune phenotype of monocytes upon SARS-CoV-2 stimulation of human PBMC isolated from healthy volunteers. In this experimental setting, we firstly verified that data obtained here from PBMC of HD most likely immunized to SARS-CoV-2 by vaccination, infection, or both, matched with those obtained earlier in SARS-CoV-2 naïve HD [17] in term of intensity and quality of immune response to the clinical SARS-CoV-2 isolate (hCoV-19/Italy/LOM-ASST-CDG1/2020) isolated in 2020 in Italy. The addition of Tα1 was then evaluated on the immune profile of monocytes and mDC as well as on the balance of pro- and anti-inflammatory cytokines (Fig. 4 ).

Fig. 4.

Scheme of in vitro study conducted on SARS-CoV-2-stimulated peripheral blood mononuclear cells. A schematic representation of the experimental procedure and data analysis of the in vitro study. Peripheral blood mononuclear cells (PBMC) were collected from buffy coats obtained from healthy donors. PBMC were stimulated for 24 hours (h) with SARS-CoV-2 at 0.02 multiplicity of infection alone or in combination with a 3 h pre-treatment with 100 ng/mL of synthetic thymosin α1. After stimulation, flow cytometric analysis was conducted to evaluate the phenotype and the activation status of monocyte subsets and myeloid dendritic cells. Cytokine release was also analyzed in culture supernatants by using a cytometric bead array.

The relative abundance of the different monocyte subpopulations carrying differential CD16 expression levels was investigated in SARS-CoV-2 stimulated PBMC as done in the ex vivo study on CP and HD PBMC (see gating strategy in Supplementary Fig. 1, Fig. 5 and Supplementary Fig. 2). Flow cytometric analysis indicated that PBMC stimulation with SARS-CoV-2 significantly reduces the percentage of classical subset [CD14^hiCD16^neg], while promoting phenotypical modifications of monocytes towards a differentiation onto inflammatory [CD14⁺CD16^int] and atypical [CD14^low-negCD16^hi] subsets. Accordingly, the percentage of CD16^int inflammatory monocytes importantly increased in SARS-CoV-2 treated PBMC as compared to not-stimulated cells (NS) (Fig. 5 A, and B and Supplementary Fig. 2). Furthermore, following SARS-CoV-2 treatment the frequency of CD16^hi atypical monocytes doubled with respect to NS cultures (Fig. 5 A, and B and Supplementary Fig. 2). Interestingly, Tα1 pre-treatment compensates the SARS-CoV-2-mediated induction of inflammatory/atypical monocytes dampening the differentiation of CD16-expressing subsets and restoring the monocyte immune-phenotype found at the steady-state level (Fig. 5 A, and B and Supplementary Fig. 2).

Fig. 5.

Effect of Tα1 on frequency of monocyte subsets in in vitro SARS-CoV-2-stimulated peripheral blood mononuclear cells. Total peripheral blood mononuclear cells (PBMC) were left untreated (not stimulated, NS) or stimulated for 24 hours (h) with SARS-CoV-2 (CoV-2) at 0.02 multiplicity of infection in presence or absence of a 3 h pre-treatment with thymosin α1 (Tα1) (100 ng/ml). PBMC were studied by flow cytometry for monocyte subset differentiation. (A) A representative experiment, out of 4 independent experiments separately performed, is shown. Classical monocytes [CD14^hiCD16^neg] are indicated in blue, inflammatory monocytes [CD14⁺CD16^int] in red and atypical monocytes [CD14^low-negCD16^hi] in aqua blue. Numbers in the dot plots are the percentage of the different monocyte subpopulations in the parental single-live CD14⁺-gated cells. (B) Results shown in the pie charts are mean values of the different monocyte subsets analyzed in the 4 independent experiments. Non-parametric Two-way ANOVA corrected for multiple comparisons by controlling false discovery rate was used to calculate significant statistical differences. Star scale was assigned as follows: * p ≤ 0.05 for inflammatory monocytes CoV-2 vs CoV-2 + Tα1; ** p ≤ 0.01 for atypical and classical monocytes CoV-2 vs CoV-2 + Tα1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4. Tα1 dampens the expression of CD86 and HLA-DR on monocytes and mDC in SARS-CoV-2-stimulated PBMC

Next, we evaluated whether Tα1 treatment influences the maturation/activation status of both monocytes and mDC in SARS-CoV-2-treated PBMC (Fig. 4 and Fig. 6 ). The stimulation with SARS-CoV-2 consistently promoted the surface expression of CD86 in total as well as in the different monocyte subsets and its expression increases along with CD16 surface level (Fig. 6 A). Interestingly, the treatment of PBMC with Tα1 prior to SARS-CoV-2 stimulation importantly weakened CD86 expression in total CD14⁺ and monocyte subpopulations. This was especially true for the atypical subset [CD14^low-negCD16^hi], in which the highest level of CD86 was observed upon SARS-CoV-2 stimulation and Tα1-mediated reduction resulted to be statistically significant. Importantly, the effect exerted by Tα1 on CD86 expression was also found in SARS-CoV-2-stimulated mDC (Fig. 6 A).

Fig. 6.

Effect of Tα1 on immune-phenotype of monocyte subsets and mDC in in vitro SARS-CoV-2-stimulated peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMC) were untreated (not-stimulated, NS; grey color) or stimulated for 24 hours (h) with SARS-CoV-2 (CoV-2) at 0.02 multiplicity of infection without (green color) or with (purple color) a 3 h pre-treatment with 100 ng/mL synthetic Thymosin α1 (Tα1). PBMC stimulated with Tα1 alone (pink color) were included as experimental control. Surface expression of CD86 (A) and HLA-DR (B) was determined as mean fluorescence intensity (MFI) by flow cytometric analysis in classical (CD14^hiCD16^neg), inflammatory (CD14⁺CD16^int) and atypical (CD14^low-negCD16^hi) monocytes as well as in CD11c⁺ myeloid dendritic cells. Results were shown as mean values ± SEM of 4 independent experiments. Non-parametric Two-way ANOVA corrected for multiple comparisons by controlling false discovery rate was used to calculate significant statistical differences. Star scale was assigned as follows: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

HLA-DR is basally expressed at high levels on both monocytes and mDC. Nevertheless, SARS-CoV-2 stimulation is able to induce it on both monocytes and mDC, further sustaining their activation status (Fig. 6 B). Although the differences were less pronounced of those observed for CD86, Tα1 treatment also dampens the induction of HLA-DR expression level mediated by SARS-CoV-2 in total as well as in the different monocyte subsets and in mDC (Fig. 6 B). In particular, a significant reduction was observed in inflammatory monocytes and mDC (Fig. 6 B).

3.5. Tα1 treatment impacts on the balance between pro- and anti-inflammatory cytokines released by PBMC in response to SARS-CoV-2 stimulation

Given the profound phenotypical changes mediated by Tα1 on innate immune cells during SARS-CoV-2 stimulation, we next evaluated whether this immune-modulation would also impact on the release of soluble mediators (Fig. 7 ). Stimulation of PBMC with SARS-CoV-2 strongly promoted the release of the pro-inflammatory cytokines TNF-α and IL-6 (Fig. 7 A) and of the neutrophil chemoattractant factor IL-8 (Fig. 7 B), thus supporting the existence of a hyperinflammatory status in presence of SARS-CoV-2. The release of these inflammatory factors from SARS-CoV-2 stimulated PBMC was significantly mitigated by Tα1 pre-treatment (Fig. 7 A and B). Most importantly, in this experimental setting we also found that the presence of Tα1 in SARS-CoV-2-stimulated PBMC cultures exerts its immune-modulatory/anti-inflammatory function by significantly up-regulating the anti-inflammatory cytokine IL-10 as compared to SARS-CoV-2 alone (Fig. 7 C).

Fig. 7.

Effect of Tα1 on innate cytokine profile in in vitro SARS-CoV-2-treated peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMC) were untreated (not stimulated, NS; grey color) or stimulated for 24 hours (h) with SARS-CoV-2 (CoV2) at 0.02 multiplicity of infection alone (green color) or in combination with a 3 h pre-treatment with 100 ng/mL of synthetic Thymosin α1 (Tα1) (purple color). PBMC stimulated with Tα1 alone (pink color) were included as experimental control. Results were shown as median values ± Interquartile range. Non-parametric Two-way ANOVA corrected for multiple comparisons with Tukey’s test to calculate significant statistical differences. Star scale was assigned as follows: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

Based on the well-recognized multiple therapeutic applications of Tα1 as enhancer of vaccine response, immunomodifier for the treatment of immunocompromised states and malignancies, homeostatic agent for controlling morbidity and mortality in sepsis and numerous infections [21], Tα1 was also employed in the treatment of COVID-19 [22].

In the attempt to further explore the immunomodulatory properties of Tα1 and its potential use in SARS-CoV-2 infection settings, in this study we interrogated a PBMC-based in vitro experimental model, whose capacity to mirror CP ex vivo immune cell profile and to respond to in vitro stimulation with SARS-CoV-2 was previously demonstrated [17]. Indeed, here we recapitulated in in vitro SARS-CoV-2-stimulated PBMC what we observed in ex vivo data collected in hospitalized patients with severe COVID-19 as well as in individuals with asymptomatic infection both showing a higher frequency of inflammatory and atypical monocytes and a more pronounced activation status in monocytes and mDC as compared to matched HD. Interestingly, the in vitro treatment of SARS-CoV-2-stimulated PBMC with Tα1 dampened the inflammatory state found in both monocytes and mDC. Consistently with these data, also the release of pro-inflammatory cytokines and chemokines was reduced, and the production of the anti-inflammatory IL-10 induced when Tα1 was added to SARS-CoV-2-treated cultures.

Over the years, many studies highlighted the significant role played by Tα1 treatment in activating and regulating phenotype and functions of various cell types of the innate and adaptive immunity in particular: by promoting the release of immunoregulatory cytokines from toll-like receptor stimulated mDC and pDC [23]; by increasing the number of activated T helper cells and controlling T cell differentiation and maturation by reducing immune cell apoptosis and by enhancing the antigen (Ag) presentation process [24]. In addition to the investigation in the area of infectious diseases and cancer, Tα1 has been also exploited in the context of autoimmunity and, in particular, in multiple sclerosis (MS) exerting an interesting and, until now, not-completely understood role as a homeostasis regulator, promoting in vitro the differentiation of IL-10 producing regulatory B cell subsets while dampening the high levels of IFN-γ and IL-17 production observed in MS patients’ cells [25], [26]. These diverse observations are consistent with the idea that Tα1 is able to establish a regulatory environment for the balance of inflammation and tolerance behaving as a context-dependent molecule exceptionally capable of multi-targeted interactions [27].

From an immunopathogenic point of view, COVID-19 can be considered as a multi-factorial disorder [3] whose outcome leads to damage caused by overactivation of innate and adaptive immunity, CD4⁺ and CD8⁺ lymphocyte depletion and functional exhaustion, uncontrolled cytokine cascade - better known as “cytokine storm” - mainly driven by IL-6 and other pro-inflammatory cytokines such as IL-1β, IL-8 and chemokines like CXCL-10 and CCL-2 [28]. All these clinical signs of the disease are more evident and frequent in fragile people as elderly population and immuno-compromised patients, who resulted to be more prone to progress into severe or critical COVID-19 disease given the over-induced pro-inflammatory response and injured immune functions [29], [30], [31]. Thus, the hypothesis that immunosenescence and age-related thymic dysfunction could play a relevant role in current COVID-19 disease scenario, has been formulated [30], [31]. In line with this view, Tα1 was repurposed as COVID-19 treatment alone or in combination with other immunoregulatory molecules mostly in early stage of COVID-19 pandemic when any vaccine or specific anti-viral drugs against COVID-19 were available [32].

The assumption that the age-related thymic involution contributes to immunosenescence and inflammaging [33] was also exploited to investigate whether aged thymic function participates in COVID-19 disease severity and, on the other hand, if its replenishment may improve protective immunity and efficient vaccination against viruses, including SARS-CoV-2, in the elderly. This hypothesis is currently under investigation in several phase II clinical trials, in which Tα1 is used to treat COVID-19 [11], [12], [22], [34], [35], [36], [37], [38], [39], [40]. A still debated picture on Tα1 efficiency in treating CP emerged from these clinical studies, since some data show that Tα1 administration reduces SARS-CoV-2 dependent mortality, while others suggested that Tα1 use had no impact on COVID-19 mortality rate [11], [12], [36], [37], [38], [39], [40]. Accordingly, meta-analyses conducted on the most relevant clinical studies evaluating the potential benefits of Tα1 in COVID-19 treatment, suggested neither significant advantages regarding patients’ mortality nor association between length of hospitalization and intensive care unit stay as well as invasive mechanical ventilation rate [41], [42]. However, several limitations of these studies should be considered before drawing the final conclusion, namely the heterogeneity in mean age and female/male ratio of the enrolled study cohorts, the differences in CP severity, the fact that most of the studies were limited to the Chinese population only and not lastly, the differences in the timing of Tα1 administration (Tα1 used at a later stage of COVID-19 disease was significantly associated with a poor prognosis than when its use started at an earlier stage).

Given the heterogenicity of clinical manifestations characterizing COVID-19 disease it was postulated and partly demonstrated that differences in innate immune response activation profoundly determine the fate of CP [43]. Accordingly, we and others previously demonstrated that the frequency as well as the absolute number of circulating pDC is reduced in CP with a magnitude dependent from the severity of the disease. In addition to alteration in the number of circulating pDC, functional differences were observed in pDC phenotype of CP which displayed a more inflammatory state [17], [19].

Moving from this evidence, here, we also analyzed in SARS-CoV-2 infected individuals with asymptomatic infection or hospitalized CP with severe disease, the frequency and blood cell count of monocyte and mDC compartment. We found that the absolute number of total monocytes and CD16^- monocyte subsets was significantly decreased while symptomatology and severity of disease increased, whereas CD16⁺ inflammatory and atypical monocyte subpopulations, those who mostly contribute to enhancement of inflammatory responses, was increased compared to matched HD, mirroring what was previously reported in other cohorts of CP [44], [45]. Similarly, CD11c⁺ mDC frequency and absolute numbers decreased significantly in CP-AS and were found further reduced in CP-H, in line with previous data showing a depletion of circulating mDC in mild and severe COVID-19 [46] that persisted also after months from recovery to infection [47]. Nonetheless, an inflammatory and activated phenotype was enhanced on CD16 expressing monocyte subsets as measured by the level of the costimulatory molecule CD86 and of the MHC class II HLA-DR. Very interestingly, then, in mDC, CD86 and HLA-DR expression was importantly increased mainly in asymptomatic subjects, showing an enhanced maturation status and Ag presentation capacity. These data indicate the capacity of mDC to respond to the viral insult by acquiring a mature phenotype propaedeutic to promote a proper anti-viral adaptive response in the asymptomatic subjects, while in severe COVID-19 this mDC-associated activation phenotype was only poorly induced likely contributing to diseases severity.

The tight regulation of monocyte immune-phenotype and the relative abundance of the different inflammatory subsets observed in CP and HD combined with serum levels of pro-inflammatory cytokines, inspired us to study in vitro the impact of Tα1 on the innate immune response during SARS-CoV-2 infection in human PBMC. This in vitro model was already employed in the past to study Tα1 action in other experimental settings, as that of MS [25], where 100 ng/ml of Tα1 was able to enhance the expansion of CD19⁺CD24⁺CD38^hi transitional-immature and CD24^low/-CD38^hi plasmablast-like regulatory B cell subsets, which likely inhibit both IFN-γ and IL-17 production by CD4 + cells. More recently, this human PBMC in vitro model has been exploited to study the SARS-CoV-2-elicited anti-viral and inflammatory responses [17]. Interestingly, here, we demonstrated that in in vitro SARS-CoV-2 stimulated PBMC, monocytes show a differentiation profile similar to that observed ex vivo in CP characterized by an increased percentage of CD16⁺ inflammatory and atypical monocytes. The expression of the co-stimulatory marker CD86 and of the MHC class II molecule HLA-DR was also enhanced in monocyte subsets, as well as in CD11c⁺ mDC. Accordingly, a consistent release of pro-inflammatory cytokines, such as IL-6 and TNF-α, and of the neutrophil chemoattractant IL-8 was observed in SARS-CoV-2-stimulated PBMC supernatants.

The presence of Tα1 in this context dampened the SARS-CoV-2-triggered inflammatory/activated status of monocytes and mDC, and mitigated the release of the pro-inflammatory mediators by reducing IL-6, TNF-α and IL-8 levels. In line with a containment of the inflammatory response mediated by Tα1 addition, a significant induction of the anti-inflammatory molecule IL-10 was found, further reinforcing the ability of this drug to tightly regulate its expression and establish a regulatory environment to balance inflammation and tolerance in different settings. Indeed, Tα1 treatment promotes IL-10 production in adaptive cell types by stimulating the differentiation of regulatory T and B cell subsets [13], [25], [48]. In this study, we extended this observation also to the innate immune cell subsets, namely monocytes and mDC, whose role in orchestrating the defense against infections including that of SARS-CoV-2, has been widely demonstrated [28], and highlight a Tα1-mediated induction of IL-10 in the first h after SARS-CoV-2 stimulation. Of note, these data well correlate with a study by Matteucci and colleagues describing that ex vivo Tα1 short treatment of blood cells from individuals with COVID-19 resulted in a significant decrease of IL-6, IL-1β, CCL2, TNF-α, and TRAF2 expression and an IL-10 up-regulation [49].

Moreover, our findings provide important cues on the biological and experimental versatility of the PBMC-based experimental setting that, by including the main immune cell subsets involved in the protection against pathogens, represents a robust in vitro tool to test novel immunopathological hypotheses. Most of the immune cells contained in PBMC possess indeed the ability to migrate in the site of infection/damage where they can influence function and phenotype of resident cells [50]. Here, we interrogate PBMC to understand whether Tα1-conditioned monocytes can exert an anti-inflammatory effect once recruited in the SARS-CoV-2 infected tissues, either represented by nasal or pulmonary epithelia. Nevertheless, the PMBC setting shows some limitations such as the sustained exposure - lasting up to 24 h - to a fixed Tα1 dose (100 ng/ml), that likely does not correspond to in vivo conditions. Indeed, Tα1 pharmacokinetics in healthy subjects has been evaluated in two studies showing that Tα1 concentration peaks in serum and plasma between 2 and 6 h and ranges around 100 ng/ml when 3.2 and 6.4 mg where administered subcutaneously, then returning to basal levels by 24 h [51], [52], [53]. In addition, our setting, being composed by only immune cells, lacks the contribution of other in vivo Tα1-cellular targets that, in turn, can further condition immune cell phenotype through bystander effects. Thus, it would be interesting to confirm our in vitro data by longitudinally studying ex vivo monocyte activation status in COVID-19 patients before and after Tα1 treatment to correlate the modulation of monocyte immune-phenotype with the therapeutic efficacy and the clinical outcome.

Having found that PBMC can sense the presence of infectious SARS-CoV-2 in an Angiotensin-converting enzyme 2-independent manner and, being at the same time not permissive to any SARS-CoV-2 variants isolated so far ([17] and our unpublished data), we can assume that Tα1, acting at different levels on immune cells, might be considered as a potential candidate for HDT. This is particularly important since HDT are generally not or poorly weakened by the genomic changes characterizing the current and emerging VOC. However, this hypothesis needs to be further verified in future studies aimed at testing Tα1 effects on the immune response induced by different VOC. Indeed, irrespective to the differential action that Tα1 may exert, these data might provide interesting cues on the dynamic interaction of the host immune system with new emerging VOC likely adapted to naturally acquired or vaccine-induced immunity as well as to mAbs-based targeted strategies. This piece of data could also shed light on novel therapeutic approaches for fragile persons carrying immune dysfunction or aged people, in which the potentiation of immune response is crucial to properly face infection-induced challenge - ranging from deaths, multiple re-infection alone or in combination with other respiratory viruses, to long COVID-19 sequelae - or to respond properly to the current and future vaccination plans.

Funding

This research was supported by EU funding within the NextGenerationEU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT).

CRediT authorship contribution statement

Daniela Ricci: Investigation, Formal analysis, Data curation, Visualization. Marilena Paola Etna: Investigation, Formal analysis, Data curation, Visualization, Writing – original draft, Writing – review & editing. Martina Severa: Formal analysis, Data curation, Writing – review & editing. Stefano Fiore: Investigation. Fabiana Rizzo: Investigation. Marco Iannetta: Resources. Massimo Andreoni: Resources. Stefano Balducci: Resources. Paola Stefanelli: Resources. Anna Teresa Palamara: Funding acquisition, Writing – review & editing. Eliana Marina Coccia: Conceptualization, Supervision, Funding acquisition, Writing – original draft, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article:

Data availability

No data was used for the research described in the article.