Immuno-in ammatory Characters in Low Severity COVID-19 Patients with Digestive Symptoms


 Background: Coronavirus disease 2019 (COVID-19) is an emerged infection raised widely concerns for pneumonia and respiratory manifestations. It is noteworthy that digestive symptoms are frequently observed in COVID-19 patients. We sought to describe the immune-inflammatory characteristics of COVID-19 patients with digestive symptoms and mild disease severity.Methods: We designated enrolled mild patients into three subtypes depending on the patients with or without digestive symptoms, including Digestive only (digestive symptoms only), Respiratory only (respiratory symptoms only) and Digestive+ Respiratory (Both digestive and respiratory symptoms). Patient discharge was based on negative results of rRT-PCR testing for SARS-CoV-2 from at least two sequential respiratory tract specimens collected ≥24 hours apart. The multiorgan function, immune-inflammatory characteristics were analyzed among three groups.Results: Mild liver damage and the activation of immuno-inflammatory system are the most abnormalities in mild patients but no significant differences were found (p＞0.05). Compared with the Respiratory only group, patients with digestive symptoms were more likely to have slightly higher and later peak values of inflammatory cytokines during the subsequent course of disease(P＜0.05). Additionally, we also found that there was a significant correlation between IL-2 and TNF level in the Digestive only cases (P＜0.05). Conclusions: Mild patients only/accompanied with digestive symptoms are a special subtype of COVID-19. Patients in this group were more likely to have slightly higher and later peak values of inflammatory cytokines during the subsequent course of disease. The prevention and clinical management of this type should be taken into consideration.


Introduction
The Coronavirus disease 2019 (COVID-19) has spread throughout worldwide now and has been declared an international public health emergency by the World Health Organization (WHO) [1,2]. Respiratory symptoms including cough, shortness of breath, dyspnea etc. are considered as common clinical manifestations of COVID-19 [3]. However, Series of recent researches have reported digestive symptoms, such as nausea, vomit and diarrhea were also as accompanied or only symptoms of the patients [4,5].
The virus target cell receptor, angiotensin converting enzyme 2 (ACE2), was highly expressed in gastrointestinal organs such as small intestine and duodenum, providing the foundation of gastrointestinal infection [6]. On the other hand, the viral nucleic acid could also be detected in the feces.
23.29% of patients had positive stools after negative respiratory samples, indicating "cured" patients may still result in the damage [7,8]. The digestive involvement of 2019-nCoV should be recognized.
Actually, although the virus is sweeping the worldwide, 80.9-86.0% of patients were with mild severity [9].
More importantly, mild patients facilitate the rapid dissemination of 2019-nCoV and are the main cause of the spread infection [10]. Therefore, understanding the characters of this group could help for treating and controlling the dissemination at early stage. Our previous study has shown that patients with digestive symptoms are more likely to have a longer delay before viral clearance compared to patients with only respiratory symptoms [11,12]. However, the underlying reasons remain largely unknown.
In highly pathogenic human coronaviruses (hCoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and middle east respiratory syndrome CoV (MERS-CoV) infection, cytokine storm and immunosuppression led to deteriorate clinical results [13]. Similarly, it is con rmed that severe COVID-19 cases had markedly higher levels of in ammatory cytokines and lower absolute number of T lymphocytes [14]. Further research showed SARS-CoV-2 virus infection led to lymphopenia and cytokine storm which correlated with disease severity [15]. Nevertheless, the in ammatory and immune characters in patients with digestive symptoms and mild severity is largely unknown. On this account, the present study is aim to illustrate the immuno-in ammatory characteristics and their longitudinally variations in mild patients.

Patient diagnosis and Groups
The study was done at Union Hospital, Tongji Medical College (Wuhan, China), which was a designated hospital to management of patients with COVID-19. We enrolled 206 mild patients during February 13th to February 29th, 2020, through laboratory-con rmed by real-time reverse-transcriptase polymerase-chainreaction (RT-PCR) assay for nasal and pharyngeal swab specimens [16], which was conducted in accordance with the protocol established by the World Health Organization [17]. Mild patients were de ned as patients without dyspnea, without clinical evidence of respiratory distress, and able to maintain blood oxygen saturation above 93% in resting condition [3,11]. Based on whether having digestive (nausea, vomit, diarrhea and anorexia,) and/or respiratory symptoms (cough/expectoration, chest tightness, pharyngalgia, shortness of breath), patients were divided into three groups, named "Digestive only," "Respiratory only" and "Digestive + Respiratory".

Data Collection
In order to obtain more accurate gastrointestinal symptoms of patients, we conducted a telephone survey and face to face to obtain more detailed information. We also interviewed patients who had only respiratory symptoms to make sure they did not have gastrointestinal symptoms. The epidemiological, clinical manifestation, laboratory, and outcome data was extracted from medical and nursing records.
Laboratory assessments consisted of complete blood count, stool routine, blood chemistry (including electrolytes, liver and renal function, lactate dehydrogenase and creatine kinase, etc.), coagulation test, in ammatory biomarkers (including C-reactive protein, erythrocyte sedimentation rate (ESR),   procalcitonin, IL-2, IL-4, IL-6, IL-10, TNF, etc.), immunity index (including percentage of NK cells, B /CD3 +   /CD4 + /CD8 + lymphocytes, C3, C4, IGE, IGG, IGM,     Mild liver damage and activation of the immunoin ammatory system are the most common manifestations in mild patients on admission Mild liver injury was the most common observed in mild patients on admission. In the increased ALT (30.6%) and AST (15.0%) level, the mean value was 36.4 U/L and 31.9 U/L, respectively. Most (17.5%) of the abnormal ALT and AST are 40 ~ 80U/L, and those ≥ 80U/L are less than 5%. Abnormal LDH was also common accounted for 32.0%. It seems that the proportion of abnormal liver function in the Respiratory only group was slightly higher than that of the other two groups but not statistically signi cant (Table 2, p = 0.186). The function of bile secretion was also slightly declined with increased levels of TBil and DBil, and increased activities of ALP and GGT. The levels of TP and albumin were slightly decrease which implied hepatic synthetic function was acceptable. Other abnormal creatinine, electrolyte disturbance, troponin, platelets, coagulation indexes and D-dimer were not common. Overall, hepatobiliary system is more vulnerable to damage while renal and cardiac disfunctions were rare in mild patients.

Dynamic Longitudinal Changes Of Immune Systems In Mild Patients
We then analyzed the dynamic changes of white blood cells (WBCs), neutrophils, lymphocytes and different lymphocyte subsets in the peripheral blood of these mild patients. No obvious differences of WBC and neutrophil counts between three groups was found during the course of disease ( Fig. 1A and B). The lymphocyte counts in Digestive only groups were higher than Respiratory only group at 4-6 days but without a statistical signi cance (1.46 vs. 1.10, p = 0.153) and became close during the following period of disease progression (Fig. 1C). As for lymphocyte subsets, CD3 + and CD8 + T cell counts of patients  Fig. 1D-F). No signi cant differences in B cell and NK cell counts were observed during the whole course ( Fig. 1G and H). The dynamic pro le of immunoglobulin and complement levels were also analyzed among the three groups but no signi cant differences were found (Fig. 2).

Dynamic Longitudinal Changes Of In ammatory Cytokines In Mild Patients
We further analyzed the dynamic changes of peripheral IL-2, IL-4, IL-6, IL-10 and TNF-α levels. As is shown in Fig. 3, IL-6 presented a uctuation during day 19 to 39, especially in patients with only respiratory symptoms. It was shown a peak at 31-33 days (Fig. 3A). However, compared with the moderate trend of

Discussion
In the present study, we compared organ function, in ammation and immune characteristics in subgroups of patients with mild disease severity divided by whether presenting with digestive symptoms or not. We found that mild liver disorder and activation of the immuno-in ammatory system are the most commonly abnormality in mild patients. Additionally, compared with the Respiratory only group, in ammatory markers showed slightly higher and later peak values during the subsequent course of disease in the Digestive group.
Liver disorder is the most commonly abnormality in mild patients in our study. Recently, the lancet also published an article which reported 43 of the 99 cases (43.4%) of COVID-19 had different degrees of liver function injury [9]. However, those are the characteristics of liver function in critically ill patients. Our study founded that hepatobiliary system is more vulnerable to damage even the patients are relatively mild.
Our results further showed that levels of some in ammatory cytokines such as IL-2, IL-4 and IL-10 in patients with digestive symptoms were slightly higher compared to those with respiratory symptoms and had later peak values during the courses. These might be the reason that patients have a longer delay before viral clearance and more likely to be associated with poorer prognosis compared to patients with respiratory symptoms [18].
Compared with the Respiratory Only group, there was a signi cant correlation between IL-2 and TNF level in the Digestive Only group. IL-2 has an important role in inducing and enhancing cytotoxic activity. Early studies presented a model of T cell differentiation characterized by a switch from ''antigen mode'', where antigen driven IL-2 expression is abundant, to ''in ammation mode'', where responses to antigen signals are attenuated, IL-2 gene transcription is de cient, and responses to cytokine signals may predominate [19]. And TNF regulates the switch from ''antigen mode'' to ''in ammation mode'' during terminal T cell differentiation [20]. Therefore, these seem to suggest that the appearance of different symptoms may be the comprehensive manifestation of different action mechanisms and different action results. It provides a new way for us to further study the pathogenesis and treatment of the disease. The mechanism of gastrointestinal symptoms and its signi cance still need to be further studied.
Our study has some notable limitations. First, it would be better to include as many patients as possible in Wuhan, in other cities in China, and even in other countries to get a more comprehensive understanding of 2019 nCoV with digestive system. However, the data in this study permit an early assessment of the in ammatory immune characteristics of gastrointestinal ndings with COVID-19. Second, no data were included on critically ill patients. However, the aim to study immune in ammatory characteristics in mild cases might be our advantage. Third, there were not many obvious differences in the longitudinal analysis were found in immuno-in ammatory characteristics. Further study focused on these aspects are needed.
In conclusion, patients only/with gastrointestinal symptoms are a special group of COVID-19. Patients in this group were more likely to have slightly higher and later peak values of in ammatory cytokines during the subsequent course of disease. The prevention and clinical management of this type should be taken into consideration.

Conclusions
In conclusion, mild patients with digestive symptoms are a special subtype of COVID-19. This kind of patients were more likely to have slightly higher and later peak values of in ammatory cytokines during the subsequent course of disease.

Author contribution
A. Duan and S. Zhang collected medical records data, analyzed the data, drafted the manuscript and contributed equally to this paper. Wang J helped for data statistics. W. Qian supported data entry and sorting; Hou X contributed revisions of the manuscript for important intellectual content. C. Han designed, supervised the study and revised the manuscript as the corresponding author.
This study was approved by the Medical Ethical Review Committee, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, China ([2020] No.0033). Written informed consent was waived due to the rapid emergence of this infectious disease and their information had been anonymized and de-identi ed.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.