key: cord-288044-ver1nrsz
authors: Sierra, Beatriz; Pérez, Ana B; Aguirre, Eglis; Bracho, Claudia; Valdés, Odalys; Jimenez, Narciso; Baldoquin, Waldemar; Gonzalez, Guelsys; Ortega, Lilia M; Montalvo, Maria C; Resik, Sonia; Alvarez, Delmis; Guzmán, Maria G
title: Association of early nasopharyngeal immune markers with COVID-19 clinical outcome: predictive value of CCL2/MCP-1
date: 2020-09-03
journal: Open Forum Infect Dis
DOI: 10.1093/ofid/ofaa407
sha: 
doc_id: 288044
cord_uid: ver1nrsz

Early recognition of severe forms of COVID-19 is essential for an opportune and effective intervention, reducing life risk complications. An altered inflammatory immune response seems to be associated to COVID-19´s pathogenesis and progression to severity. Here we demonstrated the utility of the early nasopharyngeal swab sample for detection of the early expression of immune markers and the potential value of CCL2/MCP-1 in predicting disease outcome

A c c e p t e d M a n u s c r i p t In view of this we considered the possible utility of early nasopharyngeal swabs samples to detect the presence of messenger RNA of proinflammatory and regulatory mediators already associated to COVID-19 disease, like TNF, CCL2/MCP-1, CCL3/MIP-1, and TGF and IL-10, respectively. We hypothesized that the activity of this set of cytokines/chemokines could trigger/suppress the severe outcome of infection, resulting in confirmed COVID-19 patients with asymptomatic infections or different clinical evolutions.

We found significant differences between the groups with favorable and unfavorable clinical evolution, suggesting the value of the early detection of TNFα, CCL2/MCP-1, CCL3/MIP-1 A c c e p t e d M a n u s c r i p t alpha mRNA in nasopharyngeal swab samples, and the predictive value of CCL2/MCP-1 for the COVID-19 clinical outcome.

Nasopharyngeal samples were collected separately with sterile polyester tipped swabs (Puritan Medical Products Co., LLC, Guilford, ME, USA) following the PAHO/WHO A c c e p t e d M a n u s c r i p t Symptomatic cases were classified according to the number of symptoms, and the presence of symptoms previously associated to severity in Cuban patients (distress respiratory, dyspnea and asthenia). Patients with 1 or 2 symptoms, excluding those associated to severity, were classified as Mild Disease (eleven patients). Patients with 5 or more symptoms, including asthenia and dyspnea, were classified as Very Symptomatic (nine patients); Those patients with respiratory distress (respiratory frequency>30/min, O 2 Saturation index <93 %, PaO 2 /FiO 2 Ratio <300) and intensive care requirement, but progressing to recovering phase, were classified as Severe (three patients), and those with respiratory distress and intensive care requirement, that unfortunately died, as Fatal (three patients). None of the asymptomatic or mild disease cases showed levels of C-reactive protein over 20 mg/l, levels of ferritin over 400 μg/L, or lymphopenia (lymphocyte count below 1.0× 10 9 /l). In order to identify biomarkers which discriminate between a favorable and unfavorable clinical evolution, we joined asymptomatic subjects and patients with mild disease in the category of "favorable evolution", and all those patients who developed a more severe picture (Very symptomatic, complicated and fatal cases) in the category "unfavorable evolution" ( Table 1 ).

All swab samples from symptomatic patients were collected between the first and fifth day after onset of symptoms for COVID-19. Samples from 16 healthy individuals confirmed as negative for SARS-CoV-2 were used as controls. The information about age, sex, days from symptoms onset to sample collection and co-morbidities of the studied subjects are shown in table 1.

The cDNA was synthesized from mRNA with poly(dT) primers and Superscript II reverse Experiments were conducted in triplicate. Sequence of primers used in this work is described in supplementary data.

Distribution of data was tested using Kolmogorov-Smirnov test (data not normally distributed 

The objective of this study was to explore the early expression of TNF-alpha, CCL2/MCP-1, CCL3/MIP-1 alpha, IL-10, and TGFβ, previously associated with the uncontrolled cytokines response in the ARDS pathogenesis produced by human pathogenic coronaviruses, thereby taking advantage of the nasopharyngeal swab sample, mandatory for diagnosis of COVID-19 diagnosis in Cuban suspected cases.

To determine the possible association of early gene expression of immune mediators in nasopharyngeal swab sample with the clinical evolution, we studied patients with a mild or severe COVID-19 clinical picture, and asymptomatic subjects. SARS-CoV-2 negative individuals were included as controls. Figure 1a shows the comparison of TNF alpha expression among negative controls, patients with favorable and unfavorable evolution. We did not find significant differences between the controls and patients with favorable evolution The group of unfavorable evolution showed a significantly higher TNF alpha expression in comparison to the patients with favorable evolution (p0,001), and with negative controls (p0,001).

A significantly higher expression however of CCL2 was observed in cases with unfavorable outcome compared to cases with favorable evolution (p0,001) or to negative controls (p0,001) (figure 1b). Similar results were obtained for CCL3: also here a significantly A c c e p t e d M a n u s c r i p t higher expression in cases with unfavorable outcome was observed, compared to cases with favorable evolution (p0,001) or to controls (p0,001) (figure 1c).

The analysis of TGFβ expression among the three groups is shown in figure 1d . Individuals grouped as favorable outcome showed a significantly higher expression compared to controls (p0,05) . Patients with unfavorable outcome showed higher expression than controls (p0,001), and cases with favorable outcome (p0,01).In contrast, we did not find statistical relevant differences in IL-10 expression between individuals with favorable and unfavorable evolution or with negative controls (supplementary data)

We also analyzed if there were significant differences in the gene expression of each cytokine according to the time from symptom onset to sample collection. We found a significantly higher expression only for TGF beta in the period from 0 to 3 days, compared to the period from 4 to 5 days (p=0.005). We checked then which group of cases, according to clinical evolution, was supporting this difference in the in the period from 0 to 3 days, and we found significantly higher expression in cases with unfavorable evolution compare those with favorable evolution (p=0,033).

An association of TNFα with marked asthenia (p=0,0004), and with the dyspnea presence (p=0,006) in the unfavorable evolution group was found. Dyspnea has been reported to be more frequent in COVID-19 severe cases, and indeed, in some studies, it was included as a marker of severe disease (15, 16) . A similar association with these clinical symptoms was observed for CCL2/MCP-1 and TGFβ (asthenia: p=0,003 and p=0,006 resp.; dyspnea:

p=0,043 and p=0,006 resp.) while CCL3/MIP-1 alpha was shown to be associated only with asthenia (p=0,004).

The univariate logistic regression models showed that the coefficients of three of the five cytokines evaluated were statistically associated with the unfavorable outcome (CCL2/MCP-A c c e p t e d M a n u s c r i p t 1, TGFβ and TNFα). Nevertheless, only CCL2/MCP-1 was significant in the multivariate analysis-beta_(Estimate = 2.93, p= 0.024).

Early recognition of severe forms of COVID-19 is crucial for an opportune and effective early intervention that reduces life risk complications.

A deregulated antiviral immune response, resulting in the release of large amounts of proinflammatory cytokines and subsequent uncontrolled local or systemic inflammation, has been recognized as causal for severity and lethality in pathogenic human coronavirus infections, including COVID-19 (3) (4).

As a consequence, several inflammatory markers, in particular cytokines, have been associated to severity and prognosis of COVID-19 (6) . However, most of these were detected in peripheral blood, and after the fifth day of onset of symptoms, which is relatively late (7).

To our knowledge, this is the first study evaluating mRNA expression of cytokines/chemokines, already linked to COVID-19 pathogenesis, in swab samples from the upper airway at the early start of the symptoms (first 5 days). Although the sample size was rather limited (16 healthy controls, 23 patients with favorable evolution and 15 patients with unfavorable evolution), clearly statistical differences were observed.

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

Concurring with recent reports in Chinese COVID-19 patients, we found significantly higher expression of TNF, CCL2/MCP-1 and CCL3/MIP-1 in COVID-19 cases with an unfavorable evolution compared to those with a favorable evolution (1) (8).

These markers have a high amplifying potential, quickly enhancing the inflammatory cytokine/chemokine responses in the upper airway, probably predicting later pathologic events in the lower airway associated to ARDS (9) (10) (11) .

The higher levels of TGFβ in cases with severest outcome could be in apparent contradiction with the regulatory and anti-inflammatory role attributed to this cytokine (12) . However, the TGFβ function seems to be highly dependent on location and context and could play a major role under inflammatory conditions (13) , in fact, it has been also associated to hypoxia and lung damage (14) . The analysis of IL-10 gene expression did not showed statistical relevant differences among the groups.

Cytokines are pleiotropic and interact as a network, in which the analysis of a single cytokine may not provide trustable information about serial immune events. Considering this, univariate logistic statistical analysis was conducted to detect differences in concentrations of each measured cytokines, and to detect the confounders in patients with favorable versus patients with unfavorable evolution. Also, in order to control the effect confounders like age, sex and the presence of comorbidities, a multivariate logistic regression analysis was used.

The univariate logistic regression model confirmed the association of TNF, CCL2/MCP-1

and TGFβ with the COVID-19 outcome. However, after discard the "noise" introduced by confounder variables, with a multivariate logistic regression model, only CCL2/MCP-1 was significant which suggested a predictive value for this marker.

CCL2/MCP-1 regulates the migration and infiltration of monocytes, memory T lymphocytes, and natural killer (NK) cells, favoring inflammatory process in tissues, including lung (2) .

A c c e p t e d M a n u s c r i p t M a n u s c r i p t M a n u s c r i p t A c c e p t e d M a n u s c r i p t Tables. Table 1 : Characterization of the studied subjects. A c c e p t e d M a n u s c r i p t Figure 1 

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Initial viral load and the outcomes of SARS

Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology

COVID-19: consider cytokine storm syndromes and immunosuppression

World Health Organization. Laboratory testing strategy recommendations for COVID-19: interim guidance

Association of inflammatory markers with the severity of COVID-19: A meta-analysis

Mild versus severe COVID-19: Laboratory markers

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

Monocyte chemoattractant protein-1 (MCP-1): an overview

TNF-alpha signalling and inflammation: interactions between old acquaintances

Airway Epithelial Derived Cytokines and Chemokines and Their Role in the Immune Response to Respiratory Syncytial Virus Infection

Anti-and Pro-inflammatory Roles of TGF-β, IL-10, and IL-22 in Immunity and Autoimmunity

Hypoxia down-regulates expression of secretory leukocyte protease inhibitor in bronchial epithelial cells via TGF-beta1

Human Fibrotic Diseases: Current Challenges in Fibrosis Research

Plasma IP-10 and MCP-3 levels are highly associated with disease severity and predict the progression of COVID-19

IP-10 and MCP-1 as Biomarkers Predicting Disease Severity of COVID-19 (6/2/2020)

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