Characteristics of inflammatory factors and lymphocyte subsets in patients with severe COVID‐19

Abstract To investigate the inflammatory factors and lymphocyte subsets which play an important role in the course of severe coronavirus disease 2019 (COVID‐19). A total of 27 patients with severe COVID‐19 who were admitted to Tongji Hospital in Wuhan from 1 to 21 February 2020 were recruited to the study. The characteristics of interleukin‐1β (IL‐1β), IL‐2 receptor (IL‐2R), IL‐6, IL‐8, IL‐10, tumor necrosis factor‐α (TNF)‐α, C‐reactive protein (CRP), serum ferritin and procalcitonin (PCT), and lymphocyte subsets of these patients were retrospectively compared before and after treatment. Before treatment, there was no significant difference in most inflammatory factors (IL‐1β, IL‐2R, IL‐6, IL‐8, IL‐10, CRP, and serum ferritin) between male and female patients. Levels of IL‐2R, IL‐6, TNF‐α, and CRP decreased significantly after treatment, followed by IL‐8, IL‐10, and PCT. Serum ferritin was increased in all patients before treatment but did not decrease significantly after treatment. IL‐1β was normal in most patients before treatment. Lymphopenia was common among these patients with severe COVID‐19. Analysis of lymphocyte subsets showed that CD4+ and particularly CD8+ T lymphocytes increased significantly after treatment. However, B lymphocytes and natural killer cells showed no significant changes after treatment. A pro‐inflammatory response and decreased level of T lymphocytes were associated with severe COVID‐19.

of proinflammatory cytokines and chemokines by immune effector cells. [7][8][9][10] A cytokine storm triggers a violent attack by the immune system on the body, causing ARDS and multiple organ failure, and finally, leading to death. However, the main cytokines and chemokines involved in SARS-CoV and MERS-CoV infections differ. 7,9 In COVID- 19, it has been reported that patients being treated on intensive care units (ICU's) have higher plasma levels of interleukin-2 (IL-2), IL-7, IL-10, granulocyte-colony stimulating factor, interferon γ-inducible protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein-1A, and tumor necrosis factor-α (TNF-α) compared to non-ICU patients. 2 Regarding other inflammatory factors, elevated IL-6, serum ferritin, and C-reactive protein (CRP) have been most commonly reported in patients with  A cytokine storm is also associated with apoptosis of lymphocytes, leading to severe and transient lymphopenia. [12][13][14][15] Lymphopenia is a common feature in patients with COVID-19 and might be a critical factor associated with disease severity and mortality. 2,11,16 One of the most recent reports has shown that the number of CD4 + and CD8 + T cells in the peripheral blood of a SARS-CoV-2-infected patient is significantly reduced, whereas the status of CD4 + and CD8 + T cells are excessive activation. 4 With increasing evidence on the key pathophysiological role of inflammatory factors in patients with COVID-19, immunomodulatory agents including corticosteroids, tocilizumab, and lucitanib have been considered for use in clinics. However, more laboratory and clinical evidence for their use are needed.
In this study, the characteristics of several inflammatory factors (IL-1β, IL-2 receptor (IL-2R), IL-6, IL-8, IL-10, TNF-α, CRP, serum ferritin, and procalcitonin [PCT]) and lymphocyte subsets of 27 patients with severe COVID-19 patients were examined. We aimed to find appropriate targets for early intervention in patients with severe COVID-19 by comparing relevant laboratory indicators before and after treatment. We found that levels of IL-2R, IL-6, TNF-α, and CRP decreased significantly after corticosteroid therapy, followed by IL-8, IL-10, and PCT. Analysis of lymphocyte subsets showed that CD4 + and particularly CD8 + T lymphocytes increased significantly after treatment. However, B lymphocytes and natural killer (NK) cells showed no significant change after treatment. to attenuate lung inflammation and was given for no more than 1 week.

| Patients and samples
Blood laboratory tests and chest computed tomography were performed every 5 to 7 days. All 27 patients experienced improvements in their condition and as of 12 March 2020, eight male patients and nine female patients had been discharged from hospital. Blood samples of the patients were collected before treatment and after the condition improved significantly (ie, body temperature is normal for more than 3 days, respiratory symptoms improve obviously, and pulmonary imaging shows obvious absorption of inflammation).

| Ethics approval
This study received ethical approval from the Medical Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All participants gave written informed consent, and the study was carried out in accordance with the Declaration of Helsinki.

| Statistical analysis
All statistical analyses were performed using SPSS 22.0 (SPSS Inc, Chicago, IL). Data are expressed as mean ± standard deviation and were compared using the independent-samples t test. A P < .05 was considered statistically significant.

| Lymphocyte subsets and most inflammatory factors were not significantly different between male and female patients
The median age of male and female patients was 61 and 60 years, respectively. Before treatment, lymphocyte subsets and inflammatory factors were evaluated (Table 1). Lymphopenia was seen in 71.4% <5 pg/mL) was elevated only in three female patients, with a maximum of 12.2 pg/mL. There was no significant difference in IL-1β between male and female patients.

| Most inflammatory factors decreased significantly after treatment
Inflammatory factors (IL-1β, IL-2R, IL-6, IL-8, IL-10, TNF-α, CRP, serum ferritin, and PCT) of the 27 patients were compared before and after treatment (Figure 1). After treatment, the respiratory symptoms of all patients were significantly relieved and most of the inflammatory factors were decreased from their pretreatment levels.
CRP, IL-6, TNF-α, and IL-2R were significantly decreased after treatment, followed by IL-8, IL-10, and PCT. IL-8 and IL-10 showed a pretreatment increase in fewer than 50% of the patients. Although PCT was elevated in 63% (17/27) of patients, the maximum level was only 0.41 ng/mL. Levels of IL-1β and serum ferritin did not change significantly after treatment. In fact, as described above, IL-1β levels were only elevated slightly in just three female patients. Serum ferritin, however, was elevated in all patients and did not decrease significantly after treatment. It is likely that this inflammatory factor decreased slower than the others.

| DISCUSSION
The COVID-19 outbreak is a major challenge for clinicians. The disease pathogenesis remains to be fully characterized, and no phar- CRP plays an important role in innate immunity as an early defense mechanism against infections. Another inflammatory plasma marker that is extensively used in clinical practice is the ferritin. Unlike many bacterial infections, viral infections are commonly characterized by increased circulating ferritin concentrations. [29][30][31][32][33] In our study, CRP and serum ferritin were increased above normal levels in all patients with severe COVID-19, but only CRP decreased significantly after treatment. This is probably because serum ferritin levels decreased at a slower rate, but confirmation of this is required. PCT is known for its sensitivity to bacterial infections. 34 In our investigation, the PCT level was only slightly increased in patients with severe COVID-19, with the highest level less than 0.5 ng/mL, which does not support bacterial infection. PCT has been investigated for its ability to predict the development of inflammation.
However, the clinical effectiveness of this parameter is controversial. 35,36 In our study, PCT levels decreased significantly after treatment.
In our study, CD4 + and particularly CD8 + T lymphocyte subtypes were reduced in patients with severe COVID-19, which is consistent with the general characteristics of viral pneumonia 37 and reflects the deficiency of the adaptive immune response. Previous research on viral infections has indicated that adaptive T cells, especially CD8 + T cells, provide broader and more lasting cross-reactive cellular immunity with fewer limitations of strain-specific restriction. 38 Histological examination of the lungs of patients who have died of COVID-19 has revealed interstitial mononuclear inflammatory infiltrates, dominated by lymphocytes. 4 This finding has been correlated with lower CD4 + and CD8 + T cell counts in the peripheral blood samples of patients with severe COVID-19. In general, a significantly negative correlation has been reported between the number and function of both CD4 + and CD8 + T cells. 39 We found that proinflammatory factors were increased significantly in patients with severe COVID-19, which may be related to the decrease in T lymphocytes. After treatment, the number of T lymphocytes recovered alongside the decrease in proinflammatory factors.
The dynamic changes of lymphocytes function in this process need further study.
It had been reported that COVID-19 mostly affected men because immune genes are more expressed on the X chromosome. Although studies have provided evidence that cytokine storms and immunopathology can occur during pathogenic human coronaviruses infections, 5,6,8,9,11,20, Some limitations of this study should also be acknowledged. This was a retrospective, single-center, observational study, and unavoidable biases occurred when including participants. Furthermore, the sample size was very small. Despite these limitations, the study reflects the "real life" clinical situation.
The SARS-CoV-2 infection affect primarily T lymphocyte, particularly CD8 + T cells. The lymphocytes function in patients with severe COVID-19 need to be further clarified.

ACKNOWLEDGMENTS
The authors would like to thank the patients who participated in this study, and the staff at Tongji Hospital for their assistance with study enrollment, data collection, and sample examination.

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

AUTHOR CONTRIBUTIONS
MN researched literature and wrote the first draft of the manuscript. F-BT and D-DX collected the patients' clinical data.
BY designed the investigation, and reviewed and modified the manuscript. All authors have read and approved the final version of the manuscript.

DATA AVAILABILITY STATEMENT
The data used during the current report are available from the corresponding author on reasonable request.

ETHICS STATEMENT
This study received ethical approval from the Medical Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Written informed consent for publication of these clinical details was obtained from each patient. Copies of the consent form are available for review by the editor of this journal.