key: cord-0886547-edd9188e
authors: Özgeriş, Fatma B.; Koçak, Ömer F.; Kurt, Nezahat; Parlak, Emine; Yüce, Neslihan; Keleş, Mevlüt S.
title: High Serum Progranulin Levels in COVID-19 Patients: A Pilot Study
date: 2022-03-11
journal: Biochemistry (Mosc)
DOI: 10.1134/s0006297922030026
sha: 0cb956917edea375f97e13fbb8e1cda21c1e6a70
doc_id: 886547
cord_uid: edd9188e

In this study, we aimed to determine whether the progranulin level in serum predicts the course and severity of the disease in COVID-19 (+) patients and whether it can be used as a biomarker in these patients. Therefore, we sampled 61 people infected with COVID-19, and the cases were divided into the following groups: asymptomatic, noncomplicated, moderate, and severe. Concentrations of progranulin, TNF-α, IL-6 from in serum obtained from all participants were measured using commercially available ELISA kits, as well as WBC, PLT, NE, LY, ALT, AST, Hb, PCT, and CRP were examined with clinical analyzer. All measurements obtained for the patient samples were compared with those of 20 healthy individuals. The serum progranulin concentration was statistically higher in the COVID-19 (+) patient group than in the control group of healthy individuals [112.6 ± 54.8, 0.0 (0.0-54.2 pg/ml, respectively p = 0.000)]. ROC analysis was performed to evaluate the progranulin potential as a biomarker for COVID-19 (+) patients. A larger AUC (0.931 ± 0.08) value and a more significant p-value for progranulin than for CRP (p = 0.000) was detected. As a result, we believe that progranulin reaches high levels in the COVID-19 disease and may be a determinant in diagnosis and prognosis, and may be a better biomarker than CRP.

Coronavirus disease of 2019 (COVID 19) , caused by the severe acute respiratory syndrome coronavirus 2 (SARS CoV 2), is associated with substantial pulmonary disorders, and extrapulmonary manifestations, including thromboembolism, bleeding, myocardial injury, renal failure, gastrointestinal symptoms [1] . The spectrum of clinical presentations ranges from asymptomatic, mild courses, or severe and life threatening diseases requiring intensive care treatment [2] . As the COVID 19 epidemic intensifies, there is an urgent need to identify clinical and laboratory predictors for progression towards severe and fatal forms of this disease.

Interleukin 6 (IL 6) and tumor necrosis factor α (TNF α) are important members of the cytokine network with both anti inflammatory and pro inflammatory BIOCHEMISTRY (Moscow) Vol. 87 No. 3 2022 effects. Dysregulated cytokine production promotes devel opment of autoimmune disorders or simply persistence of infection, leading to the maintenance of a chronic patho logical inflammatory state [3] . IL 6 is a proinflammatory cytokine that stimulates acute phase responses, hematopoiesis, and specific immune reactions. Recently, it has been reported that IL 6 is responsible for high mor tality rate in COVID 19 [4] . Among the many cytokines studied, TNF α was noted to be particularly important as it is located upstream of the inflammation cascade and has many complex functions in the immune system [5] .

Progranulin (PGRN), also known as the granulin epithelin precursor, proepithelin [6] , acrogranin [7] , and epithelial transforming growth factor, is a glycoprotein composed of tandem repeats of 12 cysteine modules, called the granulin or epithelial domain. PGRN, a pleiotropic growth factor, is known to play an important role in maintaining and regulating homeostatic dynamics of normal tissue development, proliferation, regenera tion, and host defense response, and therefore has been widely studied in infectious diseases, wound healing, tumorigenesis, neuroproliferative and degenerative dis eases [8] . Interactions between PGRN and inflammation are complex. PGRN binds receptors for TNF α and inhibits downstream TNF α signaling. PGRN blocks the ability of TNF α to stimulate, among other effects, neu trophil respiratory burst. PGRN is therefore considered as anti inflammatory factor [9] .

C reactive protein (CRP) is one of the nonspecific acute phase response proteins synthesized in liver with participation of the IL 6 pathway. Its increased level serves as a biomarker of inflammation, infection, and tis sue damage [10, 11] . Serum CRP concentrations have been found to rise in COVID 19 (+) patients in the stud ies [12 14] . COVID 19 is a new infectious disease for which cur rent laboratory markers are not available to assess severi ty of the disease. For this reason, objective of this study was to investigate whether the progranulin levels predict the course and severity of the disease in the COVID 19 (+) patients and whether it can be used as a biomarker in these cases.

Patients and healthy people were selected from the patients who visited the Department of Infectious Diseases, Atatürk University Research Hospital. The groups consisted of COVID 19 (+) patients and control group with similar demographic characteris tics. COVID 19 infection was diagnosed by the real time polymerase chain reaction (RT PCR) test through exam ination of nasopharyngeal and oropharyngeal samples taken from the patients. Healthy volunteers with demo graphic characteristics similar to the patients, with nega tive for RT PCR test and without any complaints were included in the control group. All samples were taken after approval of the study by the clinical research ethics com mittee (No: B.30.2.ATA.0.01.00/411). All COVID 19 cases were managed according to the national guidelines. COVID 19 patients were divided into the following groups: asymptomatic (AG, no symptom, no pulmonary involvement in chest imaging studies), noncomplicated (NCG, patients with symptoms such as fever, muscle, joint pain, cough and sore throat, and no respiratory dis tress -respiratory rate per minute <24, SpO 2 > 93% in room air, and normal chest X ray and/or lung tomogra phy), moderate (MG, patients with symptoms such as fever, muscle, joint pain, cough and sore throat, respira tory rate <30/min, SpO 2 > 90 in room air, and mild to moderate pneumonia signs on chest X ray or tomogra phy), and severe (SG, patients with symptoms such as fever, muscle, joint pain, cough and sore throat, with tachypnea (≥30/min), SpO 2 level below ≤90% in room air, and bilateral diffuse pneumonia signs on chest X ray or tomography). On the first day of their admission, blood was taken from the patients in the MG and SG groups. These were also patients who took various antiviral drugs (Tamiflu, Plaquanil, Favipavir) and did not have chronic diseases (such as COPD, tuberculosis, or AIDS). Patients using immunosuppressive drugs or receiving chemother apy were not included in the study. Healthy volunteers were adults over 18 years of age who were similar in age to the patient group, had normal physical examination find ings and normal results of routine tests, were not preg nant, and did not have chronic diseases (such as COPD, tuberculosis, or AIDS). All of COVID 19 (+) patients in the severe group were patients treated in the intensive care unit (ICU) and nine patients were ventilated. At the beginning of the study, blood was taken from 95 people and they were included in the study. However, consider ing the inclusion/exclusion criteria, four people younger than 20 years old, eight people whose serum samples were hemolyzed, and two people who had COVID 19 (-) test in the PCR retest were excluded from the study, and the study was carried out with total of 81 people.

Examination of biochemical and clinical profile. Blood samples were taken from the patients admitted to our hospital. While some of the blood samples were sent to the hospital laboratory for routine analysis, another portion was sent to the Atatürk University, Faculty of Medicine, Medical Biochemistry Department Research laboratory for testing Il 6, TNF α, and PGRN. Serum Il 6, TNF α, and PGRN concentrations in the samples from each patient were recorded, along with the informa tion on their age, gender, comorbid characteristics, and biochemical parameters (ALT, AST, WBC, NE, LY, PLT, Hb, PCT, and CRP).

Serum IL 6, TNF α α, and PGRN analysis. Serum TNF α concentrations were measured using a human specific sandwich enzyme linked immunosorbent assay BIOCHEMISTRY (Moscow) Vol. 87 No. 3 2022 (Hu TNF α ELISA kit; Cat. No. KE00154, AuthentiKine, USA). Serum IL 6 concentrations were measured using a human specific sandwich enzyme linked immunosorbent assay (Hu IL 6 ELISA kit; Cat. No. KE00139, AuthentiKine). Serum PGRN con centrations were measured using a human specific sand wich enzyme linked immunosorbent assay (Hu PGRN ELISA kit; Cat. No. MBS163502, MyBioSource, Belgium). IL 6, TNF α, and PGRN analyses were per formed using a microplate reader (XS Powerwave, BioTEK, USA).

Statistical analysis. IBM SPSS 20.0 software pack age was applied for data analysis in the study. Data were presented as mean, standard deviation, minimum, maxi mum, percentage, and number. The Kolmogorov-Smirnov test was used to determine whether the continu ous variables were normally distributed. In comparisons between the two independent groups, Independent Samples t test was used for the data with normal distribu tion, and Mann-Whitney U test was used in other cases. In the comparison of continuous variables with more than two independent groups, the ANOVA test was used when normal distribution condition was met, and the Kruskal-Wallis test was used, when it was not. Pearson correlation analysis was used for the data with normal dis tribution, and Spearman correlation analysis was used for other data.

Receiver operating characteristic (ROC) analysis test was employed to determine whether the continuous vari able could be used in the diagnosis and to determine the cut off value, positive predictive value (PPV), negative predictive value, and area under the curve (AUC). Statistical significance level for all data was set at p < 0.05.

Demographic characteristics and radiological images of the COVID 19 (+) patient group and the healthy con trol group are given in Table 1 . There was no statistical dif ference between the mean age of the combined patient group and the control group (p = 0.997). 49.3% of the 81 participants were female, 50.7% were male.

While progranulin was detected in only 4 of 20 sub jects in the control group, it was not detected in only 6 of 61 subjects in the patient group. Since absorbance of the samples where progranulin levels could not be detected was the same as the blank, concentration of progranulin in these samples was evaluated as 0.00 mg/liter. Serum progranulin concentration was statistically higher in the COVID 19 (+) patient group than in the healthy control group (p = 0.000). There was no statistical difference between the progranulin levels in the COVID 19 (+) sub groups (p = 0.078). Boxblot graph of both COVID 19 (+) patient subgroups and healthy control group are given in Fig. 1 . There was a significant difference in the TNF α and IL 6 levels between the patient and the control groups or the patient subgroups (p = 0.000). No differ ence in the biochemical data was observed between the COVID 19 (+) subgroups in ALT, AST, WBC, NE, LY, PLT, Procal, and Hb values. There was a significant decrease in the NE, LY, and PLT values and a significant increase in the Hb values in the combined patient group compared to the healthy group. On the other hand, the CRP concentration was significantly different both between the patient and control group and between the COVID 19 (+) subgroups. Data on biochemical parame ters of all groups are presented in Table 2 .

No significant correlation was observed between the levels of progranulin and other parameters in the COVID 19 (+) patient group. In the COVID 19 (+) sub groups, positive correlation was observed with CRP (r = 0.474, p = 0.017) in the MG group of patients, and negative significant correlation with PLT in the severe group of patients (r = -0.578, p = 0.038) (Fig. 2) . On the other hand, when all participants were considered, signif icant positive correlation was observed between the pro granulin and the TNF α and IL 6 levels (r = 0.260, p = 0.018, r = 0.461, p = 0.000, respectively) (Fig. 3) .

ROC analysis was performed to evaluate potential of progranulin as a biomarker for COVID 19 (+) patients. It detected a larger AUC value and a more significant p value for progranulin than for CRP. The cut off value was 38 pg/ml for progranulin and 6 mg/ml for CRP, and positive predictive value (PPV) and negative predictive value (NPV) were calculated. The determined parameters of ROC analysis are presented in Table 3 and Fig. 4 .

The course of COVID 19 disease, which causes sig nificant health problem all over the world, varies from per son to person, and, therefore, knowing the parameters to monitor the course severity and follow up of the disease will make an important contribution to pathophysiology. Quantitative parameters showing the stage of development of the COVID 19 disease are extremely important for classifying patients according to the disease severity and applying effective treatment quickly. This study looked at the serum concentrations of Il 6, TNF α, and PGRN as well as biochemical parameters in COVID 19 patients of varying severity, compared them to the parameters in healthy people, and suggested new diagnostic targets by evaluating these potentially important data. IL 6 is rapidly produced through transcriptional and post transcriptional mechanisms as an immune response to infection and tissue injury. Dysregulated expression of IL 6 exerts a pathological effect on chronic inflammation and autoimmunity [15] . It is commonly accepted that IL 6 plays a pivotal role in the cytokine storm induced by SARS CoV 2 as a multifunctional mediator of inflam mation and participates in the interstitial pneumonia and ARDS observed in the severe cases of COVID 19 [16] . In the acute phase of inflammation and infection, IL 6 is produced by immune cells, especially T helper 17 (TH17) cells in the COVID 19 patients [17] . In addition, the IL 6 production is also induced by TNF α as a pyro genic cytokine released from immune cells in response to chronic inflammatory and autoimmune diseases, suggest ing that IL 6 is a downstream effector of TNF α [18] . Using meta analysis of cumulative data from 24 studies, Wu et al. [19] investigated the role of inflammatory cytokines, including IL 6, IL 10, and TNF α, in the severity and mortality of COVID 19. They reported that IL 6 levels were significantly increased in the COVID 19 patients. In another study conducted in individuals with COVID 19 disease, it was reported that plasma IL 6 lev els increased with the increase in severity of the dis ease [20] . In our study, there was a statistically significant difference in the TNF and IL 6 levels between the patient and control groups, as well as between the patient subgroups (p = 0.000). COVID 19 patients showed higher plasma proin flammatory cytokine levels including chemokines such as IFN γ, IL 1β, IP 10, and MCP 1, while higher concen trations of TNF α in the severe cases requiring admission to intensive care units were found [21] . In the study, serum cytokine and chemokine levels were investigated in asymptomatic, symptomatic, and convalescent COVID 19 cases and healthy controls, and the levels of TNF α were found to be higher in the symptomatic group as compared to the healthy controls [22] .

Eight further meta analysis studies by Wu et al. [19] investigating TNF α levels in the circulation of the severe (n = 725) and non severe COVID 19 (n = 587) patients showed no statistically significant difference between the groups. In our study, we could not find a sig PGRN is a pleiotropic growth factor and immune regulatory molecule found in epithelium, bone marrow, and variety of immune cells such as T cells, dendritic cells, monocytes, and macrophages [23] . By binding to the membrane bound receptors TNF R1, TNF R2, or Toll like receptor 9, PGRN performs variety of functions in the innate and adaptive immune systems. In the adaptive immune system, PGRN is crucial for T cell regulation as it promotes differentiation of CD4 + T cells into the Foxp regulatory T cells (Tregs) and protects Tregs from negative regulation by TNF α [24, 25] . PGRN prevents regulatory NK cell cytotoxicity against antiviral T cells and inhibits lipopolysaccharide mediated IL 6 and TNF α secretion from macrophages [9] . In our study, PGRN was found to be at the significantly higher levels in the COVID 19 pos itive patients compared to the control group. When we looked at the severity of the disease, we did not observe any statistical difference between the PGRN levels (p = 0.078). Serum protein expression in the COVID 19 patients was examined in the literature, and there was no difference in IL 6 levels in the SARS CoV 2 positive patients compared to the patients with similar symptoms and disease severity but negative for SARS CoV 2, where as a specific increase of the PGRN level has been shown [26] . Yao et al. [27] found that serum VCAM 1 and PGRN levels were higher in the patients with COVID 19 than in the healthy controls in their study of PRGN and VCAM 1 levels in the patients with COVID 19.

In the study of PGRN levels in sepsis, community acquired bacterial pneumonia (CAP), and COVID 19, researchers discovered that the COVID 19 patients with confirmed pneumonia had significantly higher levels of PGRN than the non COVID 19 CAP patients. They emphasized that for the COVID 19 patients with con firmed pneumonia, progranulin is even better biomarker than procalcitonin and CRP [28] .

Studies have shown that CRP levels reach high val ues at the onset of COVID 19 disease and are determina tive in diagnosis and prognosis [13, 14] . CRP is a bio marker used not only in the diagnosis of the disease, but also in determining its severity, evaluating treatment response, and predicting mortality [29] . In the multicen ter retrospective study conducted by Feng et al. [12] it was reported that the CRP level increased as the clinical pic ture worsened and reached a significant difference in the critically ill patients in 476 cases. As a result of our ROC analysis evaluating potential of PGRN as a biomarker for the COVID 19 (+) patients, we determined that it has a larger AUC value and a more significant p value than CRP. Thus, we believe that PGRN may be a good bio marker for COVID 19 disease.

CONCLUSIONS COVID 19 is recognized as a global health threat, so it is essential that clinicians have access to reliable rapid pathogen testing and applicable differential diagnosis based on clinical identification during their first contact with the suspected patients. In comparison with the TNF α and IL 6 levels, the serum level of PGRN was almost never detected in healthy people, while it was found to be very high in the COVID 19 (+) patients. It has been observed that it is more sensitive than CRP in distinguishing patients with COVID 19 from the healthy people. Furthermore, low PCT levels in the COVID 19 patients suggest that PGRN is increased due to the viral origin of the infection. We believe that progranulin levels reach high values in the COVID 19 disease and may be a determinant in diagnosis and prognosis, and may be a better biomarker than CRP.

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The authors thank all the medical care workers who participated in collecting samples.Ethics declarations. Authors declare no conflicts of interest. The study was approved by the Clinical Researches of Ethical Committee of the Atatürk University (No: B.30.2.ATA.0.01.00/411). All COVID 19 cases were managed with guidance by the national guideline.