key: cord-0798960-rbubj3at
authors: Matteucci, Claudia; Minutolo, Antonella; Balestrieri, Emanuela; Petrone, Vita; Fanelli, Marialaura; Malagnino, Vincenzo; Ianetta, Marco; Giovinazzo, Alessandro; Barreca, Filippo; Di Cesare, Silvia; De Marco, Patrizia; Miele, Martino Tony; Toschi, Nicola; Mastino, Antonio; Sinibaldi Vallebona, Paola; Bernardini, Sergio; Rogliani, Paola; Sarmati, Loredana; Andreoni, Massimo; Grelli, Sandro; Garaci, Enrico
title: Thymosin alpha 1 mitigates cytokine storm in blood cells from COVID-19 patients
date: 2020-12-05
journal: Open Forum Infect Dis
DOI: 10.1093/ofid/ofaa588
sha: c83d6c800800aa46f8631e533d09f661c149b400
doc_id: 798960
cord_uid: rbubj3at

COVID-19 is characterized by immune-mediated lung injury and complex alterations of the immune system, such as lymphopenia and cytokine storm, that have been associated with adverse outcomes underlining a fundamental role of host response in SARS-CoV-2 infection and the pathogenesis of the disease. Thymosin alpha 1 (Tα1) is one of the molecules used in the management of COVID-19, since it is known to restore the homeostasis of the immune system during infections and cancer. Here we captured the interconnected biological processes regulated by Tα1 in CD8+ T cells under inflammatory conditions. Genes associated with cytokine signaling and production were found up-regulated in blood cells from COVID-19 patients and the ex-vivo treatment with Tα1 mitigated cytokines expression and inhibited lymphocytes activation in CD8+ T cell subset specifically, suggesting the potential role of Tα1 in modulating the immune response homeostasis and the cytokine storm in vivo.

A c c e p t e d M a n u s c r i p t 4 

Coronavirus disease caused by SARS-CoV-2 represents a potentially fatal disease of great global concern for public health. Immune system dysregulations, such as lymphopenia and cytokine storm, have been associated with the severity of the disease, suggesting a fundamental role of host response in the pathogenesis [1, 2] . SARS-CoV-2 uses the angiotensin-converting enzyme-2 (ACE-2) to infect target cells, and determines the activation of Toll-like receptors (TLRs), triggering the production of pro-inflammatory cytokines and chemokines from epithelial and immune effector cells [3] . Regardless of the activation of the cellular and humoral immune response, a number of individuals developed a persistent inflammation which can lead to a cytokine storm and diffuse organ involvement, mainly associated with the severe condition of COVID-19 patients such as acute respiratory distress syndrome (ARDS) [4] . Moreover, modification of total lymphocytes indicates a potential association between lymphocyte subsets alteration and viral pathogenic mechanism [5] . The function of NK and CD8+ T cells has been found affected during infection and the restoring after therapy highlights the association of functional exhaustion of cytotoxic lymphocytes with COVID-19 [6] . Most of the severe cases showed elevated levels of infection-related biomarkers and inflammatory cytokines [7] . Indeed, abnormal high plasma levels of innate or pro-inflammatory cytokines have been detected, and higher mortality was associated with elevated levels of interleukin (IL)-6 [8] . Unfortunately, only a small proportion of patients benefits from drugs currently used to manage COVID-19, such as antiviral drugs or drugs aimed at reducing inflammation or blocking a single cytokine. Thus, no univocal standard of care has been defined nowadays for COVID-19 patients, as well as no effective drugs have been identified to mitigate the cytokine storm. Thymosin alpha 1 (Tα1), one of the molecules that has been used in the management of COVID-19 [9] , is a thymic peptide endowed with the ability to restore the homeostasis of the immune system A c c e p t e d M a n u s c r i p t 5 [10, 11] . Tα1 has been chemically synthesized and used in diseases with hindered or impaired immune response, particularly infections and cancer [12, 13] . Tα1 has been used as immune enhancer in SARS patients demonstrating efficacy in controlling the spread of the disease [14, 15] . Recent data show Tα1 enhances the number of lymphocytes in COVID-19 patients with severe and critical disease, reverses T cell exhaustion, and induces immune reconstitution increasing thymus output [16] . Based on our previous study showing that Tα1 induces the release of antiviral soluble factors in CD8+ T cells stimulated by lipopolysaccharide (LPS) [17] , here the biological processes and networks modulated by Tα1 in that condition have been evaluated by an enrichment pathways analysis. The identified genes, including cytokines and immune regulatory factors, have been then analyzed in T1treated blood cells from COVID-19 patients, to investigate the ability of Tα1 to mitigate cytokine dysregulation in SARS-CoV-2 infection.

Fifteen SARS-CoV-2 positive individuals were enrolled in an open study by the Infectious 

Blood samples were diluted (1:2) in RPMI 1640 enriched with 2mM of L-glutamine, 100 U/mL of Penicillin, 0.1 mg/ml of Streptomycin, 10% fetal bovine serum. Blood samples were exposed for 8 hours at 37°C in 5%CO 2 to 50μg/ml Tα1 (SciClone, Pharmaceutical). After incubation, samples were recovered and analyzed by flow cytometry and Real time PCR.

Each culture condition was done in duplicate.

Blood samples were centrifuged and treated twice with red blood lysing buffer to remove red cells. After extraction, 100 ng of DNase treated RNA (Total RNA extraction kit blood, Grisp) was reverse transcribed into cDNA according to the manufacturer's protocol (ImProm-II™ Reverse Transcription System, Promega). The gene expression was assessed by Real-time PCR in the Bio-Rad instrument CFX96Real-Time System, using SYBR Green (SMOBIO) chemistry. Primer pairs sequences used in the Real time PCR analysis are listed in Table S2 [18]. software (Beckman Coulter). The gating strategy has been described in Figure S1 . Results were expressed as percentage of positive cells and Median Fluorescence Intensity (MFI).

For pathway and network analysis, the Metascape online tool (http://metascape.org) was used to identify the predominant biological processes and network that are regulated by Tα1 in CD8+ T cells stimulated with LPS. Briefly, after extracting the expression values from the gene expression data profile analyzed in a previous work [19] gene lists were divided into four groups (CD8 T cells, CD8 T cells + Tα1, CD8 T cells + LPS, CD8 T cells +LPS+Tα1).

Subsequently, enrichment analysis was performed using Metascape online tool using the gene lists to identify the significant biological processes and networks modulated by Tα1. The DAVID online tool (https://david.ncifcrf.gov/) was used to perform functional annotation clustering by reference database of human complex diseases and disorders Genetic Association Database (GAD_DISEASE) at p-value cut of point (p <0.050).

Statistical analysis of group-wise expression levels and response to treatment was performed through the nonparametric Kruskal-Wallis test in the case of independent samples, and through the Friedman test in the case of dependent samples. Pairwise associations between continuous variables was tested through the Spearman correlation coefficient was calculated.

In addition, to determine possible interactions between treatment effects and clinical disease score, all biomarkers were analyzed using multivariate linear mixed models with an unstructured estimate of the covariance matrix which modeled the presence of treatment as a repeated within-subject factor, as well as group and clinical state as between subject factor. In order to account for possible confounds due to inter-patient variability age and gender as A c c e p t e d M a n u s c r i p t 8 covariates of no interest. When a statistically significant (p<0.050) overall effect of time was found, pairwise comparisons between factor levels were performed and corrected for multiple comparisons using the Dunn-Šidák correction. Significant differences are shown as *p<0.050, **p<0.010 and *** p<0.001. Data analyses were performed using the SPSS statistical software system (version 23.0 for Windows, USA).

To identify the biological pathways on which the effects of T1 in vitro on peripheral blood cells from COVID-19 patients should be focused, an in silico enrichment analysis using the Metascape tool in cells treated with Tα1 and stimulated with LPS as pro-inflammatory condition was carried out. To this purpose, we took advantage of the results of our previous microarray-based study on genes modulation by T1 and/or LPS in CD8+ T cells from HDs [17] . Among the different pathways highlighted, several are related to cytokines network and regulation of immune response against pathogens ( Figure 1A and 2S) . Actually, most of the genes were involved in regulating the interaction between "cytokines and their receptors" (n =78), "cytokine signaling" (n=45) and "regulation of cytokines production" (n=32). Other genes regulated inflammatory processes mediated by NF-kB (n = 14), IL-17 (n = 17) and IL-12 (n = 12). To better understand the relationship between the various enriched biological pathways that were identified, Metascape network analysis for each treatment was performed ( Figure 1B) . The analysis enabled us to determine the interconnections between the enriched biological processes by collapsing multiple genes from a multi-network into a single node A c c e p t e d M a n u s c r i p t 9 intertwined by edges similarly score, according to their annotation. The single treatment with Tα1 resulted to affect genes involved in immune response, inflammation and response to infection pathways. LPS treatment, according to the Metascape analysis, affected several cytokines and inflammation related pathways, that were strongly regulated in co-treatment with Tα1. These networked cellular responses predominantly reflected that Tα1 differently regulates biological processes according to the pathophysiological condition analyzed. (Table   1 ). Moreover, the treatment with Tα1 in CD8+ T cells in the presence of LPS modulates many more and different biological processes than Tα1 or LPS alone ( Figure 2) . Notably, several pathways playing important roles in infection and inflammation were found negatively modulated (Toll-like receptors, IL-17 and IL-23, production of cytokines and chemokines, etc). In CD8+ T cells, it was found that LPS modulates 30 genes, Tα1 39 genes and combined treatment 41 genes (Table S3 and Figure 3S ). Only four genes were expressed and modulated in all three groups (AMHR2, CCL22, LTB4R2 and STAT4). Nine genes were modulated in the same manner both in Tα1 and in LPS+Tα1 (up-regulated:

CCL3, CCL4, CD3e; down-regulated IL6ST, IL7, NFATC2, TLR9, TRAF2 and TRAF3). Note that these genes play important roles in the regulation of inflammatory processes also in COVID-19 disease [7] . The analysis of genes exclusively modulated in the different treatments underlines that 24/30 genes are modulated exclusively upon LPS treatment, 24/39 genes exclusively in Tα1 treatment and 22/41 genes when in combination.

There is an increasing interest in the identification of genes network contributing to the etiology of complex diseases for drug-repositioning [19] . The DAVID online tool, to get a functional annotation clustering using reference database of human complex diseases and disorders, was A c c e p t e d M a n u s c r i p t 10 then applied to the list of total genes modulated by Tα1 in LPS stimulated CD8+ T cells utilized for the Metascape analysis. This tool allowed to point out the potential use of Tα1 treatment to modulate a span of gene sets dysregulated in several diseases. The wide range of disorders evidenced by the disease enrichment analysis is listed in Table 2 and the regulated genes in Table S4 . Of note, the top identified diseases are "respiratory diseases" such as respiratory syncytial virus infections, bronchiolitis and asthma, all disorder involved in SARS-CoV-2 infection, as well as rheumatoid arthritis, and diabetes type 2 that are also risk factors in COVID-19. 

Flow cytometry analysis confirmed a higher expression of IL-6 (p=0.045), CD38 (p=0.048) and HLA-DR (p=0.001) in CD8+ T cells from COVID-19 patients in respect to HDs (Figure   4 A-C) and in CD4+ T cells (IL-6 p=0.036, CD38 p=0.050) (Figure 4 D-F) . The treatment with Tα1 significantly attenuates the percentage of cells expressing IL-6 (p=0.050) and CD38 (p=0.044), as well as the relative MFI of IL-6 (p=0.050) in CD8+ T cells from COVID-19 patients (Figure 4A and 4B) . Conversely, the percentage of CD8+ T cells expressing CD38 increased in HDs after treatment ( Figure 4B) while no difference was observed in CD38 MIF. A significant decrease in the percentage of HLA-DR+CD8+ T cells (p=0.001) and in the HLA-DR MFI (p=0.001) was found after Tα1 treatment in COVID-19 blood cells, as well as in HDs ( Figure 4C) . In blood cells from COVID-19 patients the percentage of CD4+ T cells expressing IL-6 was higher than in those from HDs (p=0.047), and the IL-6 MFI was found down-modulated in both COVID-19 patients (p=0.039) and HDs (p=0.031) after Tα1 treatment ( Figure 4D) . Also, the percentage of CD4+ T cells expressing CD38, HLA-DR and the MIF of CD38 resulted higher in patients than HDs (Figure 4E and 4F) . Moreover, the treatment with Tα1 significantly decreased the percentage of CD38 (p=0.041) and HLA-DR MFI (p<0.001) in COVID-19 patients and in HDs (MFI CD38 p=0.048 and HLADR p=0.041), and also the percentage of CD4+ T cells expressing HLA-DR in COVID-19 patients was decreased (p<0.001) (Figure 4E and 4F) . All these statistically significant correlations were lost following in vitro treatment with T1 ( Figure 5 A-F) . Moreover, in the CD8+ T cell subset, the percentage of CD38, intracellular IL-6 and IFN after Tα1 treatment change upon the severity of the disease (Figure 5 G-I) .

In the last few months several studies pointed out COVID-19 as a disease distinguished by hyper-inflammation and immune-mediated lung injury. These features have been associated with adverse outcomes in patients and suggested the potential advantage of anti-inflammatory drugs [20] . Actually, COVID-19 resembles detrimental sepsis [21] , with complex alterations of the immune system ranging from inhibition to activation and exhaustion [22] . In this context, looking for effective and specific drugs to inhibit the SARS-CoV-2 infection, several molecules with inhibitory and immunomodulating activity have been studied [23,24].

Nevertheless, controversy in the scientific community concerning their effectiveness is still open and only part of the patients demonstrated to benefit from anti-inflammatory drugs such as glucocorticoids [25] .

Tα1 has been proposed for immunomodulation in COVID-19 [26, 27] . Indeed, Tα1 has been already used in China during the SARS-CoV-2 outbreak [16] for its known ability to restore homeostasis of the immune system during infections from different kinds of pathogens [11, 12] . Moreover, Tα1 has been already shown to reduced mortality and improved immune responses in patients with sepsis [28] . Herein, we demonstrate that Tα1 restored gene expression in CD8+ T cells under pro-inflammatory condition such as LPS stimulation.

A c c e p t e d M a n u s c r i p t 13 inflammatory response such as "IL-17 signaling pathways", cytokine and chemokine production and "TLRs cascade", but, conversely, upregulated "IL-10 signaling" and gene pathways related to "response to virus" and "aging". It has been demonstrated Tα1 interacts with TLRs signaling and activates intracellular signaling pathways such as NF-κB, p38 MAPK, and the MyD88-dependent [29, 30] . TLRs are the crucial sensors of bacterial and viral pathogen-associated molecular patterns [31] . Other than bacterial LPS, TLR4 can bind proteins of several viruses and, in silico, ex-vivo and in vitro analyses demonstrated TLR4 related pathways to be involved in recognizing molecular patterns from SARS-CoV-2 to induce inflammatory response similar to bacterial sepsis [32, 33] . Moreover, the TLR4 signaling pathway has been connected to inflammatory diseases seen as risk factors for COVID-19 [34] .

In the present study, the T1 related enrichment network analysis allowed the identification of cytokine signaling and production related genes, evocating the cytokine storm already described in COVID-19 [1, 35] , that we found indeed up-regulated in the blood cells from COVID-19 patients. We observed the over-expression of cytokines such as IL-6, TNF, LTA, IL-1 and IL-10, and chemokines such as CCL2, also known as MCP1, and CXCL6, in blood cells from COVID-19 patients when compared to HDs. Peculiarly, the ex-vivo treatment with T1 significantly down-regulates the transcriptional expression of IL-6, TNF and IL-1 in COVID-19 blood cells, while up-regulates the same cytokines in HDs, confirming the ability of Tα1 to regulate biological processes according to the cellular state of activation and to the physiopathological condition. Peculiarly, in LPS stimulated CD8+ T cells Tα1 inhibits the expression of important lymphocyte activation factors such as NFATC2 associated with the production of IL-6 [36] . It is important to underline that high levels of IL-6 and IL-10 have been already recognized as disease severity predictors in COVID-19 [37] . Interestingly, here we show that the T1-mediated inhibition of IL-6 is accompanied by the induction and A c c e p t e d M a n u s c r i p t 14 maintenance of high levels of IL-10, a cytokine well known as a master regulator of immune responses [38] , demonstrating that T1 differentially modulates functional genes to control immune response homeostasis. Also the pro-inflammatory cytokines IL-1 and TNF were found down-regulated by Tα1 in COVID-19 blood cells. TNF and IL-1b are important in acute inflammatory reactions, acting as an amplifier of inflammation, and anti-TNF or anti-IL-1 therapy have been evaluated in patients with COVID-19 [39, 40] . Interestingly, we found a strong down-regulation of the TNF receptor-associated factor TRAF2, but not of TRAF3, suggesting a fine regulation of the TNF signaling by T1 [30] .

Furthermore, several modulated chemokines are associated with pulmonary inflammatory processes. In LPS stimulated CD8+ T cells, Tα1 down-regulated LTB4R2 and CCL2 which play a pivotal role in the pathogenesis of airway inflammation [41] and ARDS [42] . In COVID-19 blood cells Tα1 down-modulated the transcriptional expression of CCL2 and slightly CXCL6, chemokines involved in fibrosis and leucocytes recruitment [41] [42] [43] .

The immunophenotyping analysis revealed a strong effect of T1 in T cell subsets. Recently, it has been demonstrated that Tα1 administration could increase lymphocytes count and be a potential approach to protect effector T cells during COVID-19 [44] . T1 significantly decreases the expression of the intracellular IL-6 and of the activation markers CD38 and HLA-DR in CD4+ T cells in both COVID-19 and HDs. Notably, a specific response to T1 appeared in CD8+ T cells. Indeed, the treatment with T1 determined a significant decrease of intracellular IL-6, as well as of CD38 and HLA-DR in CD8+ T cells from COVID-19 patients but not from HDs. Interestingly, the decrease of CD38 in CD8+ T cells has been found also significantly associated with clinical status of the patients. It is worth mentioning that CD38 is a crucial marker of inflammation in CD8+ T cells and is overexpressed during viral infections [45] . CD38 and HLADR have been found up-regulated in CD8+ subset in COVID-19 patients [46] , even if different expression has been found related to the stage of A c c e p t e d M a n u s c r i p t 15 the disease or drug treatment [47] . Interestingly, our study also evidenced specifically in CD8+ T cells, but not in CD4+ from COVID-19 patients, a significant positive correlation of CD38 detected by flow cytometry with the transcriptional expression of cytokines in blood, such as TNF, IL-10 and IFN, and a significant positive correlation of HLA-DR with the transcriptional expression in blood of TNF, IL-10 and IL-17RA. Noteworthy, the correlations were lost by in vitro T1 treatment.

By the functional annotation clustering of human complex diseases and disorders, we demonstrated that T1 is able to modulate genes related to respiratory diseases, such respiratory syncytial virus infections, bronchiolitis and asthma, and autoimmune diseases such rheumatoid arthritis and diabetes type 2, that are risk factors in COVID-19. Tα1 has already shown remarkable effects in the treatment of respiratory distress syndrome (ARDS) [48] . It was also demonstrated that CD8+ T cells are critical modulators of the rheumatoid arthritis disease, and LPS engagement of the functional TLR4 activates these lymphocytes contributing to the maintenance of chronic inflammatory processes in the disease [49] and furthermore suggesting a specific role of T1 in CD8+ T cells regulation. Currently, COVID-19 resulted much more severe in elderly patients, especially in those with different comorbidities [50] . Compared to children and adults, elderly patients can be affected by thymus involution and dysfunctions. Recently, it has been demonstrated the promotion of thymus output in parallel with the restoration of lymphocytopenia and acute exhaustion of T cells during administration of Tα1 in COVID-19 patients [16] . Moreover, Tα1 has been used also as an adjuvant in vaccinations, showing increase of antibody response in influenza vaccination in the elderly [51] , and increased expression of MHC class I surface molecules in cells of different origin including primary cultures of human macrophages essential for antigen presentation [52] . Networks layout of the clusters generated with the list of the genes regulated by LPS, Tα1 and LPS+ Tα1 in CD8+ T cells. Each circle node represents one enriched term, where its size is proportional to the number of input genes falling into that term, and its color representing its cluster identity (i.e. nodes of the same color belong to the same cluster). All similar terms with a Kappa similarity score >0.3 are connected by edges (the thicker the edge, the higher the similarity). One term from each cluster is selected to have its term description shown as label. Created by metascape [http://metascape.org]. 

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