key: cord-0688843-bhjp6oyf
authors: Fricke-Galindo, Ingrid; Buendía-Roldán, Ivette; Ruiz, Andy; Palacios, Yadira; Pérez-Rubio, Gloria; Hernández-Zenteno, Rafael de Jesus; Reyes-Melendres, Felipe; Zazueta-Márquez, Armando; Alarcón-Dionet, Aimé; Guzmán-Vargas, Javier; Bravo-Gutiérrez, Omar Andrés; Quintero-Puerta, Teresa; Gutiérrez-Pérez, Ilse Adriana; Nava-Quiroz, Karol J; Bañuelos-Flores, José Luis; Mejía, Mayra; Rojas-Serrano, Jorge; Ramos-Martínez, Espiridión; Guzmán-Guzmán, Iris Paola; Chávez-Galán, Leslie; Falfán-Valencia, Ramcés
title: TNFRSF1B and TNF variants are associated with differences in soluble TNF receptors' levels in patients with severe COVID-19
date: 2022-03-16
journal: J Infect Dis
DOI: 10.1093/infdis/jiac101
sha: 1ce490006a89cfba2d3c1aa69f7c0dbc3b65dd54
doc_id: 688843
cord_uid: bhjp6oyf

BACKGROUND: The impact of genetic variants in the expression of TNF-α and its receptors in COVID-19 severity has not been previously explored. We evaluated the association of TNF (rs1800629 and rs361525), TNFRSF1A (rs767455 and rs1800693), and TNFRSF1B (rs1061622 and rs3397) variants with COVID-19 severity, assessed as invasive mechanical ventilation (IMV) requirement, and the plasma levels of soluble TNF-α, TNFR1, and TNFR2 in patients with severe COVID-19. METHODS: The genetic study included 1,353 patients. Taqman assays assessed the genetic variants. ELISA determined the soluble TNF, TNFR1, and TNFR2 in plasma samples from 334 patients. RESULTS: Patients carrying TT (TNFRSF1B rs3397) exhibited lower PaO(2)/FiO(2) levels than those with CT+CC genotypes. Differences in plasma levels of TNFR1 and TNFR2 were observed according to the genotype of TNFRSF1B rs1061622, TNF rs1800629, and rs361525. According to the studied genetic variants, there were no differences in the soluble TNF-α levels. Higher soluble TNFR1 and TNFR2 levels were detected in patients with COVID-19 requiring IMV. CONCLUSION: Genetic variants in TNF and TNFRSFB1 influence the plasma levels of soluble TNFR1 and TNFR2, implicated in the COVID-19 severity.

M a n u s c r i p t 5 

The coronavirus disease (COVID-19) presents a broad spectrum of clinical manifestations. Although most patients are expected to present a mild or moderate form of the disease, almost 15% progress to severe COVID-19 and 5% to a critical form characterized by acute respiratory distress syndrome (ARDS), septic shock, and/or multiple organ failure [1] . The most severe stage of COVID-19 is related to an extrapulmonary systemic hyper inflammation syndrome in which the enhancement in levels of cytokines promotes lung inflammation and ARDS development, multiple organ failure, and even death [2] [3] [4] .

Plasma cytokine levels have been evaluated in patients with COVID-19 and related to the worst disease outcome [5] . For instance, patients admitted to the intensive care unit (ICU) had higher plasma levels of IL-2, IL-7, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein 1-α (MIP1-α), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) [1] . Also, the TNF-α was found elevated in critical COVID-19 patients [6] and patients with ARDS and acute kidney injury [7] . In addition, a recent meta-analysis reported higher levels of soluble TNF-α (sTNF) in non-survivors compared to survivors of COVID-19 [8] .

Moreover, the TNF-α receptors (TNFR1 and TNFR2) play an essential role in cellular mechanisms mediated by this cytokine [9] , which could also be implicated in COVID-19 severity and mortality. In this sense, the serum levels of soluble TNFR1 (sTNFR1) have been reported significantly higher in ICU patients when compared to non-ICU patients with COVID-19 [10] . Recently, our research group reported an increase of sTNFR1 and ADAM17 related to severity and mortality of COVID-19, as well as higher levels of sTNFR1 and sTNFR2 in patients compared to healthy controls [11] .

Variants in the genes encoding TNF-α and its receptors have been widely studied and associated with autoimmune [12] [13] [14] , chronic [15] [16] [17] , and infectious diseases [12, 18] , as well as with cancer [19] .

TNF rs1800629 and rs361525 single-nucleotide polymorphisms are considered the most critical A c c e p t e d M a n u s c r i p t 6 variants in human disease susceptibility as these might influence the transcription of the cytokine gene [20] . The former has been associated with severe sepsis [21] , while the rs361525 was recently related to influenza A (H1N1) susceptibility [22] .

In COVID-19, only the TNF G-308 (rs1800629) promoter variant has been evaluated and associated with susceptibility and a more aggressive pattern of the disease [23] . Nevertheless, there is a lack of studies investigating other genetic variants in TNF, and its receptors' genes (TNFRSF1A and TNFRSF1B), on the course and outcome of the disease. We aimed to evaluate the association of single-nucleotide variants in TNF (rs1800629, rs361525), TNFRSF1A (rs767455, rs1800693), and TNFRSF1B (rs1061622, rs3397) with COVID-19 severity, and with sTNF, sTNFR1, and sTNFR2 plasma levels in severe COVID-19 patients. ancestry-informative markers [24] .

All the included patients were diagnosed with severe COVID-19 as they presented dyspnea, a respiratory rate of ≥30 breaths per minute, blood oxygen saturation ≤90%, and/or PaO 2 /FiO 2 ≤300 at the hospital admission [25] .

Blood samples for the genetic analysis and the proteins' determination were collected in tubes with EDTA. The sampling was performed during the patients' hospital stay. Available clinical data from A c c e p t e d M a n u s c r i p t 7 the electronic medical records were registered in the database and included for the statistical analyses (demographic data, co-morbidities, signs and symptoms, length of hospital stay, clinical outcome, and severity of COVID-19). The invasive mechanical ventilation (IMV) requirement (IMV and non-IMV) was considered a severity indicator and dependent variable for the association study.

All patients included in the study were genotyped. Genomic DNA was isolated from peripheral blood mononuclear cells using the commercial BDtract Genomic DNA isolation kit (Maxim Biotech, San

Francisco, CA, USA), and stored at 4°C until process. The TNF (rs1800629, rs361525), TNFRSF1A

(rs767455, rs1800693), TNFRSF1B (rs1061622, rs3397) variants were determined by TaqMan® SNP Genotyping Assays (C___7514879_10, C___2215707_10, C___2298465_20, C___2645714_10, C___8861232_20, C___8861228_20, respectively), according to the supplier instructions, in a 7300

Real-Time PCR System (Applied Biosystems/ThermoFisher Scientific Inc., Singapore).

Considering the TNF, TNFRSF1A, and TNFRSF1B genotypes, 334 patients with COVID-19 were included in a study to determine soluble TNF and TNF receptors (sTNF, sTNFR1, and sTNFR2) in Enzyme-Linked Immunosorbent Assay (ELISA) following the manufacturer's protocol. The absorbance was read at 450 nm. All samples were assessed in duplicate, reporting in pg/mL the mean value of the wells.

Categorical data are presented as frequencies and continuous values as mean ± standard deviation (SD) or median and interquartile range [IQR] . Normal distribution was assessed using the Kolmogorov-Smirnov Test. The differences in continuous variables among groups were evaluated using the Mann-Whitney U test and Fisher's exact test for categorical variables. Association studies and regression analysis were performed using PLINK v1.07 [26] . We employed Haploview [27] for the linkage disequilibrium analysis of the studied variants.

The differences in the plasma levels of sTNF, sTNFR1, and sTNFR2 among the different evaluated genotypes were assessed using the Kruskal-Wallis test; when a significant difference between groups was found, a pairwise comparison was performed using a Wilcoxon rank-sum test with continuity correction. The post-hoc power analysis for Wilcoxon-Mann-Whitney tests (two groups) was determined with G*Power v 3. 

The comparison of the clinical and demographical data among IMV and Non-IMV groups is shown in Dyspnea, fever, cough, myalgia, and arthralgia were the most common symptoms among patients included in the study. Dyspnea and cough were most frequent in the IMV group, while myalgia and anosmia were common among non-IMV patients ( Table 1 ).

The allele and genotype frequencies of the variants included in the study are shown in Supplementary We found a lack of association of the TNF, TNFRSF1A, and TNFRSF1B genetic variants with the IMV requirement (Supplementary Table 1 ). Only a marginal association was observed for the TNFRS1B rs1061622 (p=0.0702). Likewise, the genetic variants were not found as predictors variables of the IMV condition in the logistic regression model even when controlling for age and sex as covariates (Supplementary Table 2 ).

We also investigated the impact of TNF, TNFRSF1A, and TNFRSF1B genetic variants in the frequency of symptoms observed differently among IMV and Non-IMV groups ( Figure 3 and Supplementary Table 3) .

[ (Fig. 1a) . Likewise, we observed a trend of the sTNFR2 levels according to the same variant (p=0.06, Fig. 1b) . Meanwhile, the plasma levels of sTNF were similar among all rs1061622 genotypes (p>0.05) (Fig. 1c) .

The sTNFR2 plasma levels were different among the TNF rs1800629 and rs361525 genotypes, (p<0.05 in both cases) ( Fig. 2b and 3b) Meanwhile, the sTNF and sTNFR1 were not different among the TNF rs1800629 and rs361525 genotypes (Fig. 2a, 2c, 3a, and 3c) . Likewise, the plasma levels of sTNF, sTNFR1, and sTNFR2 were not significantly different among the genotypes of the remaining studied variants (TNFRSF1A rs767455, rs1800693, and TNFRSF1B rs3397) (Supplementary Figures 4-6) . In addition, we evaluated a correlation among the levels of sTNF, sTNFR1, sTNFR2, age, BMI, PaO 2 /FiO 2 , IMV days, and hospitalization stay. Only a significant correlation was observed among sTNFR1 and sTNFR2 levels (rho=0.59, p<0.0001, Spearman's rank correlation test, Supplementary   Figure7 ), suggesting that both receptors respond in parallel in the immune response.

We also studied if the increased levels of sTNF, sTNFR1, and sTNFR2 are implicated in patients with severe (Figure 4a and 4b) ; however, sTNF level did not show differences between IMV and Non-IMV (6 pg/mL [0.0-11 pg/mL] vs 6 pg/mL [0.0-9 pg/mL]) ( Figure 4c) . Interestingly, sTNFR1, and sTNFR2 levels were also observed differently when the severity of ARDS was considered (p<0.05 in all cases) (Figure 5a -5d).

Besides, we wanted to know if the sTNF, sTNFR1, and sTNFR2 plasma levels could vary in the stages described for COVID-19 and if this variability was related to the genetic variants. Therefore, considering that patients mainly presented severe COVID-19, we focused on the pulmonary condition, when patients are admitted to hospital and require IMV, and the hyperinflammation stage characterized by an extrapulmonary systemic hyperinflammation syndrome and poor prognosis [3] . sTNF and sTNFR2 plasma levels were similar in the two measurements. Meanwhile, an increase of sTNFR1 was observed in the Time 2 determination compared to the first one (Table 2) , suggesting some relevance of sTNFR1 in the hyperinflammatory phase. The variations of sTNF, sTNFR1, and sTNFR2 in the two stages of severe COVID-19 were independent of the genetic variants included in the study (Supplementary Figures 8-10 ). In addition, we wondered if this variation could impact the clinical outcome of patients, but we did not find any differences in the plasma levels of sTNF, sTNFR1, and sTNFR2 of survivors and non-survivors of COVID-19 (Supplementary Figure 11) .

Finally, the Δ value was assessed ( Table 2 ) and, in agreement with the last finding, the Δ value of sTNFR1 was the highest, and it was also not influenced by the TNF, TNFRSF1A, and TNFRSF1B

genetic variants included in the study (Supplementary Figures 12-14) .

The increase of sTNF, sTNFR1, and sTNFR2 levels has been previously linked with the COVID-19 severity and mortality. Herein, we report for the first time that genetic variants in the genes encoding these proteins (mainly TNF and TNFRSFB1) are associated with the plasma levels of the receptors sTNFR1 and sTNFR2 in patients with severe COVID-19. Although we were not able to find a difference with the studied phenotype (IMV and Non-IMV groups), we found a relation of the genetic variants with differences in the levels of the TNF receptors, and this could be linked to the severity of the disease since higher levels of these proteins were observed among patients with IMV and a severe ARDS.

Increased plasma levels of sTNFR1 were observed among patients with TT and GT genotypes of It has been reported that TNFR2 mediates the stimulatory activity of TNF on CD4 + Foxp3 + regulatory T cells (Tregs) and CD8 + Foxp3 + Tregs and is involved in the phenotypic stability, proliferation, activation, and suppressive activity of Tregs. This receptor can also be expressed on CD8 + effector T cells (Teffs), which delivers an activation signal and cytotoxic ability to CD8 + Teffs during the early immune response. An apoptosis signal terminates the immune response, which is uncontrolled during the cytokine release syndrome. Due to TNFR2 distribution and its pleiotropic effects, the receptor appears to be crucial for keeping the balance between Tregs and Teffs. It has been proposed as an efficient therapeutic target for impaired immune responses, such as cancer and autoimmune diseases [36] . In this sense, this study also confirms the previous hypothesis of targeting the TNF receptors for the COVID-19 treatment [11] .

A study from Egypt identified the TNF rs1800629 AA genotype associated with an aggressive COVID-19 pattern [23] . The severity of the disease was determined using several variables, including mechanical ventilation. Accordingly, we observed a higher frequency of AA and GA genotype among the IMV group when compared to Non-IMV; however, this was not statistically significant. Only marginal associations were observed for the genotype frequency of TNFRSF1B rs1061622.

A c c e p t e d M a n u s c r i p t 14 Meanwhile, the GA and AA genotypes of TNF rs361525 were associated with cough, a frequent symptom in COVID-19, particularly in the IMV group. This variant has been previously associated with increased local production and downstream inflammation in chronic obstructive pulmonary disease [37]; mainly, the A allele was related to increased transcriptional activation of the TNF promoter and susceptibility to several arthritic conditions [38] . We also evaluated the PaO 2 /FiO 2 values at the hospital admission as an indicator of COVID-19 severity, and these were different among the TNFRSF1B rs3397 genotypes. This variant has been related to an impaired TNFRSF1B M a n u s c r i p t 23 

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The authors acknowledge the support received from physicians and technicians from the clinical services at INER to confirm the study participants' diagnosis and clinical care.

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