In silico identification of potential miRNAs -mRNA inflammatory networks implicated in the pathogenesis of COVID-19

COVID-19 has been found to affect the expression profile of several mRNAs and miRNAs, leading to dysregulation of a number of signaling pathways, particularly those related to inflammatory responses. In the current study, a systematic biology procedure was used for the analysis of high-throughput expression data from blood specimens of COVID-19 and healthy individuals. Differentially expressed miRNAs in blood specimens of COVID-19 vs. healthy specimens were then identified to construct and analyze miRNA-mRNA networks and predict key miRNAs and genes in inflammatory pathways. Our results showed that 171 miRNAs were expressed as outliers in box plot and located in the critical areas according to our statistical analysis. Among them, 8 miRNAs, namely miR-1275, miR-4429, miR-4489, miR-6721-5p, miR-5010-5p, miR-7110-5p, miR-6804-5p and miR-6881-3p were found to affect expression of key genes in NF-KB, JAK/STAT and MAPK signaling pathways implicated in COVID-19 pathogenesis. In addition, our results predicted that 25 genes involved in above-mentioned inflammatory pathways were targeted not only by these 8 miRNAs but also by other obtained miRNAs (163 miRNAs). The results of the current in silico study represent candidate targets for further studies in COVID-19.Copyright © 2023 Elsevier B.V.

ENDOTHELIAL INFLAMMATION AND DYSFUNCTION INDUCED BY SARS-COV-2 SPIKE PROTEIN 1 INVOLVE ADAM17 AND INTERFERON ACTIVATION PATHWAYS

Objective: COVID19 is associated with vascular inflammation. IFN-alpha (IFNa) and IFN-lambda3 (IFNl3) are potent cytokines produced in viral infections. Their effects involve interferon-stimulated genes (ISGs) and may influence expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. We hypothesized that S1P-induced immune/inflammatory responses in endothelial cells (EC) are mediated via IFN-activated pathways Design and methods: Human ECs were stimulated with S1P (1 mg/mL), IFNa (100ng/mL) or IFNl3 (100IU/mL). Because ACE2, ADAM17 and TMPRSS2 are important for SARS-CoV-2 infection, we used inhibitors of ADAM17 (marimastat, 3.8 nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50 mM). Gene and protein expression was investigated by real-time PCR and immunoblotting, respectively. Vascular function was assessed in mesenteric arteries from wild-type (WT) normotensive and hypertensive (LinA3) mice and in ISG15-deficient (ISG15KO) mice. Results: S1P increased expression of IFNa (3-fold), IFNl3 (4-fold) and ISGs (2-fold) in EC (p < 0.05). EC responses to IFNa (ISG15: 16-fold) were greater than to IFNl3 (ISG15: 1.7-fold) (p < 0.05). S1P increased gene expression of IL-6 (1.3-fold), TNFa (6.2-fold) and IL-1b (3.3-fold), effects that were amplified by IFNs. Only the ADAM17 inhibitor marimastat inhibited S1P effects. IFNa and IFNl3 increase protein expression of ADAM17 (27%) and TMPRSS2 (38%). No changes were observed on ACE2 expression. This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). EC production of IL-6 was increased by IFNa (1,230pg/mL) and IFNl3 (1,124pg/mL) vs control (591pg/mL). Nitric oxide generation and eNOS phosphorylation (Ser1177) were reduced by IFNa (40%) and IFNl3 (40%). Vascular functional responses demonstrated that endothelium-dependent vasorelaxation (% Emax) in vessels from WT-mice stimulated with IFNa (67%) and IFNl3 (71%) were reduced vs control (82%) (p < 0.05). Responses were not altered in vessels from ISG15KO mice. Increased contraction was observed only in vessels from hypertensive mice treated with IFNa (9.1 ± 0.5mN vs control: 7.3 ± 0.3mN) (p < 0.05). Conclusions: In ECs, S1P, IFNa and IFNl3 increased ISG15 and IL-6 by mechanisms dependent on ADAM17. IFNs amplifies endothelial cell inflammatory responses and induced vascular dysfunction through ISG15-dependent mechanisms, with augmented effects in hypertension. Our findings demonstrate that S1P induces immune/inflammatory responses that may be important in endotheliitis associated with COVID-19. This may be especially important in the presence of cardiovascular risk factors, including hypertension.

IMMUNE DYSREGULATION INDUCED BY SPIKE PROTEIN 1 OF SARS-COV-2 INCREASES ENDOTHELIAL CELL DYSFUNCTION VIA TYPE I AND TYPE III INTERFERON ACTIVATION PATHWAYS

Objective: COVID19-associated immunopathology is associated with increased production of interferon (IFN)-alpha (IFNα) and lambda3 (IFNL3). Effects of IFNs are mediated by interferon-stimulated genes (ISGs) and influence expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. Increasing evidence indicates vascular inflammation in cardiovascular sequelae of COVID19. We hypothesized that S1P-induced immune/inflammatory responses in endothelial cells (EC) are mediated via IFNα and IFNL3. Design and method: Human ECs were stimulated with S1P (1 μg/mL), IFNα (100ng/mL) or IFNL3 (100IU/mL). Because ACE2, metalloproteinase domain-17 (ADAM17) and type-II transmembrane serine protease (TMPRSS2) are important for SARS-CoV-2 infection, cells were treated with inhibitors of ADAM17 (marimastat, 3.8 nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50 μM). Gene and protein expression was investigated by real-time PCR immunoblotting, respectively. Vascular function was assessed in mesenteric arteries from wild-type (WT) normotensive and hypertensive mice and in ISG15-deficient (ISG15KO) mice. Results: EC stimulated with S1P increased expression of IFNα (3-fold), IFNL3 (4-fold) and ISG (2-fold)(p < 0.05). EC exhibited higher responses to IFNα (ISG15: 16-fold) than to IFNL3 (ISG15: 1.7-fold)(p < 0.05). S1P increased gene expression of IL-6 (1.3-fold), TNFα (6.2-fold) and IL-1β (3.3-fold), effects that were maximized by IFNs. Only marimastat inhibited S1P effects. IL-6 was increased by IFNα (1,230pg/mL) and IFNL3 (1,124pg/mL) vs control (591pg/ mL). This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). Nitric oxide production and eNOS phosphorylation (Ser1177) were reduced by IFNα and (40%) and IFNL3 (40%). Reduced endothelium relaxation maximal response (%Emax) was observed in vessels from WTmice stimulated with IFNα (67%) and IFNL3 (71%) vs control (82%)(p < 0.05) but not in vessels from ISG15KO mice. Increased contraction was observed only in vessels from hypertensive mice treated with IFNα (9.1 ± 0.5mN vs control: 7.3 ± 0.3mN, p < 0.05). Conclusions: In ECs, S1P, IFNα and IFNL3 increased ISG15 and IL-6, processes that involve ADAM17. Inflammation induced by S1P was amplified by IFNs. IFNs induce vascular dysfunction through ISG15-dependent mechanisms, with augmented effects in hypertension. Our findings demonstrate that S1P induces immune/inflammatory responses that may be important in endotheliitis associated with COVID-19. This is especially important in the presence of cardiovascular risk factors, including hypertension.

BIOMARKERS EVOLUTION IN PATIENTS WITH SARSCOV- 2 PNEUMONIA TREATED WITH BARICITINIB

Background and importance A randomised clinical trial has demonstrated that baricitinib reduces the mortality of patients with SARS-CoV-2 that require hospitalisation. However, the evolution of biomarkers that predict the patients' outcome is not well described. Aim and objectives To analyse the evolution of biomarkers in hospitalised adults with SARS-CoV-2 pneumonia treated with baricitinib. Material and methods We conducted a retrospective observational study in a tertiary university hospital (760 beds). We included 31 patients positive for SARS-CoV-2 between January and February 2021. All received baricitinib 4 mg daily for ≥5 days (2 mg daily if glomerular filtration <60 mL/ min). We evaluated five biomarkers: lymphocytes, C-reactive protein (CRP), ferritin, lactate dehydrogenase (LDH) and Ddimer. The results were obtained on the day of admission (D +0) and on days 2 (D+2), 5 (D+5), 7 (D+7) and 10 (D +10) after starting baricitinib. A pharmacist was involved in the multidisciplinary team taking part in COVID-19 protocol drafting, validation of treatments, dose adjustments, interactions, and monitoring of adverse effects. The REDCap database was used for data collection and the G-STAT-2.0.1 for statistical analysis (paired t-test/Holm-Bonferroni correction). Results A total of 31 patients were included: 6 women and 25 men. Median age (IQR) was 64 (55;75) years. Main comorbidities were dyslipidaemia (39%), hypertension (35%), pulmonary disease (29%), diabetes (16%) and cardiopathy (16%). During admission, 15 (48%) received corticosteroids and 18 (58%) remdesivir, 7 (23%) needed high-flow oxygen, 5 (16%) required intensive care unit (ICU) admission and 2 (6%) died. Baseline biomarkers, as median (IQR), were: CRP 8.2 (5;11) mg/dL, ferritin 402 (176;794) ng/mL, LDH 280 (237;340) U/L, lymphocytes 0.6 (0.4;0.9) 109/L and D-dimer 500 (300;700) ng/mL. The change in the biomarkers is shown in Figure 1. There was a decrease in CRP which was statistically significant from D+5 (p=0.0144) onwards and an increase in lymphocyte count significant from D+2 (p=0.0148) onwards. LDH, ferritin and D-dimer did not significantly improve. No patient had thromboembolic complications or other adverse reactions associated with treatment. Conclusion and relevance Patients with severe SARS-CoV-2 pneumonia treated with baricitinib showed a significant increase in lymphocyte counts as well as a significant decrease in CRP shortly after baricitinib treatment. This fact, together with the low mortality, and good tolerance supports the use of baricitinib for patients with COVID-19 pneumonia.

DIFFERENTIALLY EXPRESSED IMMUNOLOGICAL GENES in MILD and SEVERE CASES of COVID-19

Background: Coronavirus disease 2019 (COVID19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has varied clinical presentations from mild subclinical to severe disease with high mortality. Our aim was to determine whether examining immune-related gene expression early in infection could predict progression to severe disease. Methods: In subjects of the All Ireland Infectious Diseases Cohort study, we analysed expression of 579 genes with the NanoString nCounter Immunology panel in peripheral blood mononuclear cells in those with confirmed SARS-CoV-2 infection collected within 5 days of symptom onset and matched SARS-CoV-2 negative controls with respiratory infection. Subsequent maximum COVID19 disease severity was classified as mild or severe. Read counts were normalized using panel housekeeping genes. Expression changes in severity groups were estimated against control baseline. Results: Between April and July of 2020, we recruited 120 subjects, 62 with COVID19 and 58 controls, with average age 59 y.o. (IQR 34-88), 66% males and 69% Caucasian ethnicity. Maximal disease severity was used to separate COVID19 cases into mild (n=31) and severe (n=31). We identified 20 significantly deregulated genes between those with COVID19 and controls (;log2 fold;>0.5, p<0.05, Benjamin-Yekutieli p-adjustment). Function of 12 of these genes related to cytokine signaling, 9 upregulated genes to type I interferon signaling (MX1, IRF7, IFITM1, IFI35, STAT2, IRF4, PML, BST2, STAT1), while 7 downregulated genes mapped to innate immune function (IRF7, ICAM2, SERPING1, IFI16, BST2, FCER1A, PTK2). Expression in the severe group showed downregulation of FCER1A (innate immunity regulation), IL1B and TNF (inflammatory cytokines), and PTGS2 (inflammatory mediator) and greater upregulation of TNFSF4 (cytokine signaling) and PTK2 (innate immunity). Mild cases presented higher upregulation of IFIT2 (type I interferon signaling). Conclusion: Observed early downregulation of regulators and mediators of inflammation in those who developed severe COVID19, suggested dysregulation of inflammation. Specifically, IFIT2 upregulation in mild cases and FCER1A downregulation in severe cases, points to early differences in host responses centered on deregulation of the interferon and inflammation responses. Whether these patterns reflect delayed interferon involvement in pathways to control the infection and contribute to pathological inflammation and cytokine storms observed in severe COVID19 requires further research.

Whole exome sequencing of MIS-C patients: Analysis of genes involved in inborn errors of type I IFN immunity, hemophagocytic lymphohistiocytosis (HLH), Kawasaki disease (KD) and TLR7 gene

Introduction: After spring 2020, a series of reports from Europe and USA described clusters of children, presenting life-threatening multisystem inflammatory syndrome in children (MIS-C), associated with antecedent exposure to SARS-CoV-2 (1). In patients with life threatening COVID-19 3.5% were found to have inborn errors in type I IFN signalling pathway (2). A case series of 4 young patients with severe COVID-19 reported rare loss-of-function variants in the TLR7 gene associated with impaired type I IFN responses (3). Clinically, MIS-C shares features with secondary hemophagocytic lymphohistiocytosis (HLH) and Kawasaki disease (KD), which were also associated with possible infectious trigger and might share a common genetic cause (4). Objectives: We analysed whether MIS-C patients have an underlying presence of genetic variants in exomes associated with inborn errors of type I IFN immunity, HLH, KD and presence of variants in TLR7 gene. Methods: Blood was drawn from 17 MIS-C patients upon submission into the hospital, DNA from peripheral blood was isolated and whole exome sequencing was performed. Variants in the following genes were investigated: type I IFN immunity (TLR3, UNC93B1, TRAF3, TBK1, IRF3/9, IRF7, IFNAR1/2, STAT1/2, IKBKG, TRIF), HLH (AP3B1, CD27, FADD, FAS, FASLG, HPLH1, ITK, LYST, MAGT1, MYO5A, NLRC4, PRF1, RAB27A, RECQL4, SH2D1A, STX11, STXBP2, UNC13D, XIAP, TNFRSF9, CDC42), KD (ITPKC, CD40, FCGR2A, BLK, CASP3, TRX-CAT1-7, PGBD1, LTA, TSBP1, HLA-DQB1/2, HLA-DOB, IGHV1-69) and TLR7 genes. Analysis was focused on rare (GnomAD<0.01) exonic or splicing variants. Results: No common genetic denominators were found in analysed genes. Five rare variants were observed in four patients (4/17). According to ACMG classification variants of uncertain significance (VUS) were found in LYST (2), IKBKG (1), IRF3 (1) and NLRC4 (1) in heterozygous genotype. No clinical evidence was found in ClinVar database for any of the variants, except for one variant in LYST (c.3931A>G:p.M1311V) with uncertain significance for Chédiak-Higashi syndrome and medium prediction scores. Variants in LYST (c.5990C>G:p.A1997G), NLRC4 (c.772T>C:p.C258R) and IRF3 (c.325G>C: p.G109R) have high CADD, Mutation Taster, Polyphen and SIFT prediction scores. And IKBKG (c.325C>G:p.L109V) variant had medium prediction scores. Conclusion: Our findings suggest that MIS-C patients do not share a rare loss-of-function variant in type I IFN immunity genes, TLR7 gene or genes associated either with HLH or KD. Despite numerous clinical, immunological and genetic research of the MIS-C patients, the syndromes pathogenesis and etiologic cause remain elusive.