The Signicance of KL-6 as Prognosis Monitoring Biomarker in Patients With Severe COVID-19 From Stabilized Stage Toward Convalescence

Background: This study aims to identify some biomarkers for monitoring the recovery of lung injury in severe COVID-19 patients from stabilized stage toward convalescence. Methods: We enrolled participants who diagnosed with severe COVID-19 (n = 28) and health volunteers (n = 25) from Taikang Tongji (Wuhan) Hospital. The patients were in a stabilized stage and had a course of 48.1±12.8 days. We followed these patients for 90 days. The blood routine, cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17A, TNF-α, IFN-α, IFN-γ), type II alveolar epithelium injury indicators (Surfactant protein A (SP-A), Krebs von den Lungen-6 (KL-6)) and chest CT were tested on the 1, 30, 60, and 90 days after enrollment. Results: In stabilized stage, the parameters of blood routine and some cytokines (IL-1β, IL-2, IL-4, IL-12p70, TNF-α) had bounced back to normal (p>0.05). Some cytokines (IL-5, IL-6, IL-10, IL-17A, IFN-α, IFN-γ) and type II alveolar epithelium injury indicators (SP-A and KL-6) were still higher than normal (p<0.05). During the stabilized stage to convalescence, in spite of the variation of monocyte count, monocyte/lymphocyte ratio, IL-5, IL-10, IL-12p70, IL-17A, IFN-γ, IFN-α, SP-A and KL-6 were downward trend (p<0.05), only KL-6 level (p<0.05) could simultaneously reect the lung injury volume which be measured by CT. Conclusions: Our preliminary data indicated that KL-6 could be an effective prognostic biomarker for monitoring the recovery of lung function in patients with severe COVID-19 from stabilized stage toward Lungen-6; phosphatidylcholine.

Conclusions: Our preliminary data indicated that KL-6 could be an effective prognostic biomarker for monitoring the recovery of lung function in patients with severe COVID-19 from stabilized stage toward convalescence.

Background
Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) caused the corona virus disease 2019 (COVID- 19) and has become a public health emergency of international concern. Previous studies on the emergence of COVID-19 and its clinical features suggested that older age, high BMI index, male gender, neutrophil-to-lymphocyte ratio, decreased lymphocyte and some indicators (such as D-dimer, IL-6 and Creactive protein (CRP) ) elevated at initial stage or progressive stage might be the risk factors for developing into severe type of COVID-19 [1,2]. However, in patients with severe COVID-19 from stabilize to convalescent stage, there has been no literature report on which indicators maybe related to lung injury repair.
As known that type alveolar epithelial cells are the key cells for maintaining lung structure and function [3], meanwhile its expressed angiotensin-converting enzyme 2 (ACE2) which is the main receptor of SARS-COV-2 virus [4]. Therefore, COVID-19 lung injury is mainly caused by damage to type II alveolar epithelium. Alveolar type II alveolar epithelium mainly uses pulmonary surfactant to maintain the normal structure and function of the lung [5]. Among them, the most studied pulmonary surfactants are surfactant protein A (SP-A) [6][7][8] and Krebs von den Lungen-6 (KL-6) [9,10]. There is evidence that SP-A secreted by type II alveolar cells increases during infection [11]. These protein are responsible for recognizing and conditioning microorganisms and presenting them to immune cells, such as alveolar macrophages, to enhance microbial and virus clearance [12]. KL-6 is a high-molecular-weight glycoprotein expressed on the extracellular surface of type II alveolar cells and bronchial epithelial cells [13]. KL-6 is elevated in serum of patients with interstitial lung diseases (ILDs), such as idiopathic pulmonary brosis and hypersensitivity pneumonitis [14,15]. It is mainly produced by damaged or regenerating alveolar type II alveolar cells [16]. This preliminary study analyzed the variation of serum concentrations of type II alveolar epithelial injury indicators (SP-A and KL-6) in severe COVID19 patients from stabilize to convalescent stage, and to verify their potential as the monitoring indicators for COVID-19 lung injury rehabilitation.

Participants
In this study, we enrolled participants (n = 28) who diagnosed with severe COVID-19 and health volunteers (n = 25) from Taikang Tongji (Wuhan) Hospital. The patients with severe COVID-19 had a course of 48.1±12.8 days and were in a stabilized stage when enrolled. We followed these patients for 90 days (day 1, 30, 60 and 90 after enrollment). All participants gave written informed consent and the study was approved by the Ethics Review Board at Tongji University. All the patients had given their informed consent.

Blood routine tests
Blood was collected via venipuncture of an antecubital vein into one 5 mL K 2 EDTA (BD Vacutainer) tube and measured immediately by measured by commercially available assay kits according to the manufacturers' protocols (XN2000, Sysmex, Japan).

Serum collection
Whole blood (5 mL) was collected from patients and healthy controls at enrollment (day 1), and on 30, 60 and 90 days. The sample was centrifuged at 3,000 r/min for 10 min and the serum was sub-packed and stored at -80 °C.
Then 1ml PBS was added for washing, and 200g centrifuged for 5 minutes to remove supernatant, and 100ul of PBS was added for blending. Finally, the sample was detected by BD FACS Calibur (Becton, Dickinson and Company, New Jersey, USA) ELISA assay SP-A levels were measured using a commercial ELISA kit (OKAN05694, Aviva Systems Biology, San Diego, USA) as previously described [17].
CT image acquisition and imaging data analysis Chest CT images were obtained by using ONE scanners: SIEMENS SOMATOM go.Top&ALL. A tube voltage of 130kV and automatic tube current modulation (100-400mA) were used. Images were reconstructed with a slice thickness of 1.0mm and an interval of 1.0mm, respectively. Using the pneumonia post-processing software developed by uAI Discover-2019 nCoV (Shanghai United Imaging Intelligence Healthcare Co.,Ltd.) and making quantitative analysis. This software provides automatic segmentation and quantitative analysis of lung lobes. Automatically segment the lung and extract the lung infection area from the thin chest CT image. The baseline and follow-up CT focus volumes of each patient were recorded and compared by two doctors with more than 5 years of experience in chest imaging diagnosis.

Statistical analysis
A p-value less than 0.05 was considered statistically signi cant. Statistical analysis and graphic representation of the data was performed with SPSS 22.0 and GraphPad Prism 8.0 software.
The blood indexes variation of severe COVID-19 patients from stabilized stage toward convalescence From the above results, we know that during the stabilized stage of severe COVID-19 patients, the parameters of blood routine and some cytokines (IL-1β, IL-2, IL-4, IL-12p70 and TNF-α) returned to normal, but some cytokines (IL-5, IL-6, IL-10, IL-17A, IFN-γ and IFN-α) and type II alveolar epithelial injury indicators (SP-A and KL-6) were still higher than healthy control group. Therefore, we continuously monitored the blood indexes of severe COVID-19 patients from stabilized stage to convalescence (day 1, 30, 60 and 90 of experiment enrollment). The variation of WBC count (F=0.789, p=0.453) (Figure 2A The COVID-19 patients group were divided into lung injury range>1000 mm 3 group (n=10) and lung injury range<1000 mm 3 group (n=18) based on the CT results of day 1 enrollment. Following, we continuously monitored the blood indexes from stabilized stage to convalescence (day 1, 30, 60 and 90 of experiment enrollment). Our data showed that the WBC count (F=0.301, p=0.589) ( Figure 3A Figure 3Q) and SP-A (F=0.005, p=0.942) ( Figure 3R) were no statistically signi cant between two groups. Meanwhile, we found that the serum KL-6 (F=5.824, p=0.023) ( Figure 3S) was statistically signi cant between the two groups, and KL-6 was higher in lung injury range>1000 mm 3 group than lung injury range<1000 mm 3 group. Above data implied that KL-6 maybe serve as a good indicator for monitoring lung injury repair in severe COVID-19 patient from stabilized stage toward convalescence.
The clinical characteristics of a severe COVID-19 patient case from stabilized stage toward convalescence We selected a 57-year-old female patient con rmed severe COVID-19. Figures 4A to 4D showed the CT imaging results of this patient at different time points (day 1, 30, 60 and 90 of experiment enrollment). Figure 4E was the variation trend of CT results, it showed that the lung injury range decreased along time. Figure 4F and 4G respectively showed the variation of serum SP-A and KL-6 levels at the time points corresponding to the CT results. Combining the results of CT and serology, we found that as the extent of lung injury shrinks, the levels of SP-A and KL-6 gradually decrease. In addition, the variation of parameters of blood routine and cytokines did not match with the CT results, and the data not show.

Discussion
The COVID-19 virus has been shown to invade human alveolar type II alveolar epithelium through mainly ACE2 and induce the lung injury [18]. It is reported that alveolar type II alveolar epithelium are the key cells for maintaining the normal structure and function of the lung, and it is mainly through pulmonary surfactant (SP-A, KL-6 and so on) [3,5]. Both SP-A and KL-6 are produced by type II alveolar epithelial cells, which proliferate following lung injury. Therefore, them have been extensively studied as the biomarkers of lung injury [19].
Most of the SP-A found in the alveolar space is bound to dipalmitoyl phosphatidylcholine (DPPC), which is the main component of pulmonary surfactant. Meanwhile, SP-A could interact with alveolar macrophages (AM), and regulates many of its functions under infection and oxidative stress condition for participating in congenital lung defense and non-antibody-mediated immune response [20]. KL-6 is a high molecular weight human transmembrane mucin 1 and promotes the migration and proliferation of lung broblasts [21]. The serum level of KL-6 was capable of discriminating between ILD and other common lung diseases. A cutoff of 500 U/ml was established to distinguish patients with ILD from healthy controls and those with other lung disorders. Therefore, KL-6 may be an effective biomarker for ILD diagnosis and monitoring [22]. It is also reported that the serum concentrations of KL-6 may be a prognostic biomarker of COVID-19 severity in the stage of disease initiation and progression [9]. However, it is not clear whether SP-A and KL-6 could be used as indicators for monitoring lung injury repair in patients with severe COVID patients from stabilize to convalescent stage.
In stabilized stage, the parameters of blood routine and some cytokines (IL-1β, IL-2, IL-4, IL-12p70, TNF-α) had bounced back to normal. Some cytokines (IL-5, IL-6, IL-10, IL-17A, IFN-α, IFN-γ) and type II alveolar epithelium injury indicators (SP-A and KL-6) were still higher than normal. This suggests that some of the indicators that are meaningful for the initial and progression stages of COVID-19 may not be appropriate for disease surveillance in stabilized stage.
Following, we continuously monitored the blood indexes of severe COVID-19 patients from stabilized stage to convalescence (day 1, 30, 60 and 90 of experiment enrollment). Despite the variation of monocyte count, monocyte/lymphocyte ratio, IL-5, IL-10, IL-12p70, IL-17A, IFN-γ, IFN-α, SP-A and KL-6 were downward trend, only KL-6 level could simultaneously re ect the lung injury volume. Thus, our data implied that KL-6 maybe serve as a good indicator for monitoring lung injury repair in severe COVID-19 patient from stabilized stage toward convalescence, especially for some primary medical settings where CT imaging is not available.
Meanwhile, we selected a 57-year-old female patient with severe COVID-19 pneumonia, and we dynamically followed up the CT imaging changes in the lung of this patient, and also detected SP-A, KL-6, blood routine and cytokines. We found that the patient had gone from the stable stage of the disease to the stage of rehabilitation, among the measured hematological indicators, only the changes of SP-A and KL-6 were corresponding to the changes of pulmonary CT, suggesting that SP-A and KL-6 could well imply the outcome of the disease. At the same time, we found that the KL-6 level of the patient had basically returned to normal when the endpoint of observation was reached, and there was no statistical difference compared with the normal group, but SP-A was still signi cantly higher than normal. Whether this indicates persistent lung injury or a normal pattern of lung injury recovery in patient with severe COVID-19 pneumonia, it requires more clinical research.

Conclusion
In conclusion, our preliminary data indicate that serum type II alveolar epithelium injury indicators, especially KL-6 could be useful markers for monitoring and evaluating the recovery of lung injury in severe COVID-19 patients from stabilized stage toward convalescence. This hypothesis will need to be further tested in the future.