The Effect of SARS-CoV-2 on the Spleen and T Lymphocytes

Abstract

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has infected millions of individuals in the world. However, the long-term effect of SARS-CoV-2 on the organs of recovered patients remains unclear. This study is to evaluate the impact of SARS-CoV-2 on the spleen and T lymphocytes. Seventy-six patients recovered from COVID-19, including 66 cases of moderate pneumonia and 10 cases of severe pneumonia were enrolled in the observation group. The control group consisted of 55 age-matched healthy subjects. The thickness and length of spleen were measured by using B-ultrasound and the levels of T lymphocytes were detected by flow cytometry. Results showed that the mean length of spleen in the observation group was 89.57 ± 11.49 mm, which was significantly reduced compared with that in the control group (103.82 ± 11.29 mm, p < 0.001). The mean thicknesses of spleen between observation group and control group were 29.97 ± 4.04 mm and 32.45 ± 4.49 mm, respectively, and the difference was significant (p < 0.001). However, no significant difference was observed in the size of spleen between common pneumonia and severe pneumonia (p > 0.05). In addition, the decreased count of T lymphocyte was observed in part of recovered patients. The counts of T suppressor lymphocytes in patients with severe pneumonia were significantly decreased compared with those with moderate pneumonia (p = 0.005). Therefore, these data indicate that SARS-CoV-2 infection affects the size of spleen and T lymphocytes.

Introduction

Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was first reported in Wuhan, Hubei Province, China, in December 2019 (14,17,37). COVID-19 is generally susceptible to all populations and mainly spreads through respiratory droplets and close contact transmission (15,16,33). World Health Organization (WHO) declared that COVID-19 was a global public health emergency on February 2020 (30). Unfortunately, this infection is rapidly spreading worldwide, due to the absence of effective treatments and vaccines (6,36). COVID-19 is a systemic disease that can affect multiple organs, including the heart, liver, lung, kidney, spleen, and brain (1,3,4,21,25,32,34). Most patients with COVID-19 have a good prognosis, whereas a few patients discharged from hospital may be relapsed (7,19,29). Moreover, abnormal lung function was observed in patients after hospital discharge (2,22). However, the long-term effect of SARS-CoV-2 on the organs of recovered patients remains unknown.

Spleen is a secondary peripheral lymphoid organ, which plays a key role in immune response by regulating T and B cells (8,27). The size of spleen and the levels of T lymphocyte are susceptible to be influenced by infections (5,18). In this study, we are aimed to investigate the impact of SARS-CoV-2 on the spleen and T lymphocytes. Thus, the sizes of spleen were evaluated by using B-ultrasound in patients who were 1-month recovery from COVID-19. Meanwhile, the levels of T lymphocytes were detected in those discharged patients. Results showed that SARS-CoV-2 infection resulted in the significant decrease in the size of spleen compared with those healthy subjects. Moreover, results showed that SARS-CoV-2 infection resulted in the decrease of T lymphocyte.

Materials and Methods

Patient collection and classification

For this retrospective study, 76 cases of confirmed COVID-19 patients from January 1 to March 30, 2020 were enrolled in the observation group and 55 cases of age-matched healthy subjects were served as the control group. All subjects were collected from the Guangzhou Eighth People's Hospital. COVID-19 patients were confirmed by laboratory tests according to the WHO interim guidance, and discharged based on the 7th edition of the guidelines provided by the National Health Commission of China (28). COVID-19 patients were consisting of 66 moderate cases and 10 severe cases based on the diagnostic criteria recommended by the Chinese National Institute for Viral Disease Control and Prevention (28). Thirty-one males and 45 females ranged from 20 to 78 years were collected in the observation group, and the average age was 48.38 ± 14.70 years. COVID-19 patients met the discharge standards and returned to our hospital 1 month after discharge, and then follow-up data were collected. The inclusion criteria of patients in the observation group were (i) negative nucleic acid test; (ii) no respiratory symptoms and normal blood routine; (iii) no hepatitis B, liver cirrhosis, or blood system disease; and (iv) no history of schistosomiasis and tumor. Fifty-five cases of age-matched healthy people were served as the control group, including 34 males and 21 females. The age of healthy people ranged from 20 to 72 years, and the mean age of 47.98 ± 11.19 years. No difference was observed in sex and height between observation group and control group. The study was approved by the Guangzhou Eighth People's Hospital (no. 186).

B-ultrasound detection of spleen

The size of spleen was examined in all subjects by B-ultrasound. Patients was placed in the right or supine position the right or supine position, and the max thickness and length of spleen were measured. The normal range of thickness is 30–40 mm and normal length is 100–120 mm. The B-ultrasound examination machine was a GEVividE95 color ultrasonic diagnostic apparatus, and the probe frequency was 3.5 MHz.

Measurements of T lymphocyte

Two milliliters of peripheral blood samples were collected for laboratory tests by flow cytometry. According to the manufacturer's protocol, four-color fluorescently labeled monoclonal antibodies were added into the well-mixed anticoagulated blood and incubated at room temperature. Then, T lymphocytes subsets in blood specimen were analyzed by the Multiset automatic analysis software (BD).

Statistical analysis

All data were statistically analyzed using IBM^® SPSS^® 19.0 statistical software package, and the data were expressed as mean ± standard deviation. The comparison of two sample means was analyzed by the independent sample t test. The comparison of multiple sample means was analyzed by the chi-square test (χ ²). p < 0.05 indicated the difference was statistically significant.

Results

SARS-CoV-2 infection resulted in the decreased size of spleen in recovered patients

The size of spleen between observation group and control group is shown in Table 1. The length and thickness of spleen in the observation group were 89.57 ± 11.49 mm and 29.97 ± 4.04 mm, which were significantly reduced compared with that in control group (p < 0.05). Subsequently, the differences in the spleen of patients with different types of COVID-19 were further analyzed compared with normal healthy people. The results are shown in Table 2. No significant difference was observed in the size of spleen between moderate pneumonia and severe pneumonia groups. However, the thickness and length of spleen in patients with moderate and severe pneumonia were significantly reduced than those in healthy subjects (p < 0.05).

Table 1.

The Size of Spleen in Observation and Control Groups

Groups	n	Age (years)	Thickness (mm)	Length (mm)
Observation group	76	48.38 ± 14.70	29.97 ± 4.04^a	89.57 ± 11.49^b
Control group	55	47.98 ± 11.19	32.45 ± 4.49	103.82 ± 11.29

Compared with control group, p = 0.001.

Compared with control group, p < 0.001.

Table 2.

The Size of Spleen in Different Types of Subjects

Groups	n	Age (years)	Thickness (mm)	Length (mm)
Moderate pneumonia	66	47.38 ± 15.04	30.08 ± 4.00^a	89.41 ± 11.42^c
Severe pneumonia	10	55.00 ± 10.53	29.30 ± 4.45^b	90.60 ± 12.53^d
Healthy people	55	47.98 ± 11.19	32.45 ± 4.49	103.82 ± 11.29

Compared with healthy subjects, p = 0.003.

Compared with healthy subjects, p = 0.032.

Compared with healthy subjects, p < 0.001.

Compared with healthy subjects, p = 0.001.

SARS-CoV-2 infection affected the T lymphocytes

To investigate the effect of SARS-CoV-2 on the immune function in recovered patients, the levels of T lymphocytes were measured in peripheral blood by flow cytometry. Compared with the normal range, the decreased count of T lymphocytes was observed in part of patients (Table 3). The decreased count of T helper lymphocytes was shown in 28.9% of recovered patients and the reduced T suppressor lymphocytes was observed in 30.3% of cases. In addition, the counts of T suppressor lymphocytes in patients with severe pneumonia were significantly reduced than those with moderate pneumonia (p = 0.005, Table 3). Patients with severe pneumonia also presented more lower level of CD3⁺ CD45⁺ T lymphocytes compared with those with moderate pneumonia (p = 0.027, Table 3). However, no significant difference was observed in the count of CD45⁺ T lymphocytes, T helper lymphocytes and CD4⁺/CD8⁺ T lymphocytes in patients with moderate and severe pneumonia (p > 0.05).

Table 3.

The Level of Lymphocytes in Discharged Patients with COVID-19

Groups	n	Less than normal range	Normal range	Moderate pneumonia	Severe pneumonia	p
T lymphocytes (CD45⁺)	76	23 (30.3%)	1,488–4,483/μL	1,738 ± 501	1,548 ± 335	0.252
T helper lymphocytes (CD3⁺ CD4⁺)	76	22 (28.9%)	550–1,440/μL	661 ± 194	565 ± 128	0.135
T suppressor lymphocytes (CD3⁺ CD8⁺)	76	23 (30.3%)	320–1,250/μL	478 ± 253	358 ± 83	0.005
T lymphocytes (CD3⁺ CD45⁺)	76	0 (0%)	0.50–0.84%	0.69 ± 0.09%	0.62 ± 0.05%	0.027
T lymphocytes (CD4⁺/CD8⁺)	76	25 (32.3%)	1.4–2.0%	0.016 ± 0.006%	0.017 ± 0.005%	0.973

Discussion

Spleen is an important immune organ in human body, which contains a large number of lymphocytes and macrophages. Secondary splenomegaly is more common in patients with chronic hepatitis B, liver cirrhosis, thrombocytopenic purpura, and chronic myeloid leukemia (13,24,26,35). Hypersplenism is often accompanied by the decrease of blood cells and the enlargement of spleen (11). But the diminished spleen size is rarely reported, which might be associated with ischemic stroke and bacterial infection (9,12). Interestingly, a diminished spleen size and reduced spleen weights were reported in patients with COVID-19 (10,20,23). Meanwhile, the number of lymphocytes was significantly reduced and lymphoid follicles were atrophied, decreased, or absent in the spleen of COVID-19 patients (31). However, it is unclear whether the diminished spleen size would be back to normal in recovered patients with COVID-19.

In this study, the size of spleen was measured in 76 recovered patients with COVID-19 and 55 healthy people by B-ultrasound. Results showed that the thickness and length of spleen in patients with COVID-19 were significantly reduced compared with healthy people. Moreover, the thickness and length of spleen in patients with moderate and severe pneumonia were significantly decreased compared with healthy subjects. However, no significant difference was observed in the thickness and length of spleen between moderate pneumonia and severe pneumonia groups, which might be caused by the small sample size. These results indicated that the size of spleen was affected by SARS-CoV-2, which was consistent with current reports (10,20,23). The diminished spleen size in COVID-19 patients was connected with the reduction of lymphocytes and lymph follicles (14). In addition, Lin et al. reported that the exacerbated lymphopenia and decrease of CD8⁺ T cells were observed in discharged and hospitalized patients (14), which might change the body's immune status. In this study, the clinical manifestations and the blood test in recovered patients were as normal as healthy people based on the inclusion criteria. Results showed that the size of spleen was still smaller in recovered patients compared with age-matched healthy people, suggesting that the shrinkage of spleen might be the result of self-regulation and repair. Moreover, these data showed that the decreased amount of T lymphocytes was still observed in part of recovered patients. Compared with patients with moderate pneumonia, the levels of CD3⁺ CD45⁺ T lymphocytes and T suppressor lymphocytes were significantly decreased in those patients with severe pneumonia. These data indicated that the immune function in recovered patients was still influenced by SARS-CoV-2 and did not fully recover, which might be connected with the decreased size of spleen. However, whether the size of spleen and immune function would be fully recovered in a much longer time remains unknown. Thus, further investigations are needed in future. In addition, there are some limitations in this study. On the one hand, this study is limited by the small sample number. One the other hand, patients is 1-month recovery from COVID-19 and no multiple periodic observations was performed.

In conclusion, these data indicate that SARS-CoV-2 affects the size of spleen and T lymphocytes, which is correlated with immune function. The decreased size of spleen may be a risk factor for the recovery of immune function in recovered patients. B-ultrasound examination can help to assess the involvement and recovery of spleen after SARS-CoV-2 infection.

Footnotes

Acknowledgments

We thank all the participators who supported this study.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the Guangdong Medical Research Fund (no. A2019553) and National Natural Science Foundation of China (no. 81901464).

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