key: cord-0962690-uqosi0ck
authors: Wang, Xiaobing; Wei, Jia; Zhu, Ruiping; Chen, Liping; Ding, Feng; Zhou, Rui; Ge, Liuqing; Xiao, Jun; Zhao, Qiu
title: Contribution of CD4+ T cell mediated inflammation to diarrhoea in patients with COVID-19
date: 2022-04-06
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2022.04.006
sha: 4d46d9e682f0bbb2b90daed4e163a5c86908305e
doc_id: 962690
cord_uid: uqosi0ck

Objectives : This study aimed to explore the role of CD4+ T cells in the mechanisms of COVID-19 related diarrhoea. Methods : Lymphocyte subsets were analysed in COVID-19 patients, and the expression of angiotensin-converting enzyme 2 (ACE2), the transmembrane protease, serine 2 (TMPRSS2), and CD4+ T cell-related indicators in the colon were compared between patients with and without diarrhoea. Correlation analyses were performed for ACE2 and other indicators to identify the relationship between SARS-CoV-2 infection and CD4+ mediated inflammation. The expression and distribution of CD4+ T cell-associated chemokines and their receptors were detected to determine the possibility of migration of CD4+ T cells to inflammation sites. Results : The CD4+ T cell counts and percentages and CD4/CD8 ratio showed the most significant differences between the two groups. The diarrhoea group expressed higher levels of ACE2, Tbet, and TNFα at both the mRNA and protein levels, with no difference from the non-diarrhoea group for the percentage of ACE2+TNFα+ cells, indicating an indirect association between ACE2 and TNFα. The mRNA expression of CXCL10, CXCL11, and CXCR3, and the number of CD4+CXCR3+T cells was increased in the diarrhoea group. Conclusions : CD4+ T cell-mediated inflammation may contribute to COVID-19 related diarrhoea. CXCR3+ mediated migration of CD4+ T cells into the gut may perpetuate inflammation.

Coronavirus-2 (SARS-CoV-2) strain, has become a global pandemic that is a serious threat to human life and health. The most prominent manifestations of COVID-19 are located in the respiratory system, but an increasing number of extrapulmonary manifestations have also been reported (AlSamman et al., 2020; Gupta et al., 2020) . SARS-CoV-2 is also affect the gastrointestinal (GI) tract (Ma et al., 2020; Ziegler et al., 2020; Zou et al., 2020) , causing digestive symptoms, such as anorexia, diarrhoea, nausea, vomiting, abdominal pain, and abdominal discomfort (Cheung et al., 2020 , Elmunzer et al., 2021 . Among these GI presentations, diarrhoea is one of the most common symptoms (Cheung et al., 2020; Elmunzer et al., 2021) .

In the early phase of the COVID-19 epidemic, diarrhoea seemed to be underestimated, with a prevalence of less than 10% Guan et al., 2020; Huang et al., 2020; Wang et al., 2020; Xu et al., 2020) . As the epidemic progressed, the prevalence of diarrhoea in COVID-19 was observed to be more than 30% (Elmunzer et al., 2021; Guo et al., 2021) , and its importance has since been better described. Further, 6 diarrhoea may be associated with the severity of COVID-19, whereby patients with diarrhoea have a higher prevalence of required ventilator support, admission to the intensive care unit (ICU) (Wan et al., 2020) , and might be more prone to multiple organ damage than those without diarrhoea (Zhang et al., 2021) . In addition, based on the potential intestinal diarrhoeal infection and faecal-oral transmission of SARS-CoV-2, more attention should be paid to diarrhoea in the control and prevention of the COVID-19 epidemic, especially when collecting faecal samples or performing endoscopic examinations in these patients.

Although extensive studies have focused on diarrhoea in COVID-19 patients, most of the studies only analysed the correlations between diarrhoea and other clinical data.

Possible underlying causes of COVID-19 associated diarrhoea have been reported to include the direct cytopathic effects of SARS-CoV-2, robust systemic inflammation or so-called cytokine storm, gut microbiota dysbiosis, and antibiotic associated diarrhoea or adverse effects of antiviral agents such as hydroxy-chloroquine, remdesivir, and ritonavir-lopinavir (de Oliveira et al., 2021 , Guo et al., 2021 , Jin et al., 2021 , Perisetti et al., 2020 , Yan et al., 2021 . However, to date, no exact mechanism has been 7 proposed to explain the local inflammation in the intestine that leads to diarrhoea.

During clinical practice, we found that COVID-19 related diarrhoea was closely associated with lymphocyte subsets, specifically CD4+ T cells. Therefore, our aim was to explore the role of CD4+ T cells in the mechanism of diarrhoea-associated COVID-19.

We observed a difference in the lymphocyte count and percentage in COVID-19 patients with diarrhoea than those without. Retrospective analyses were performed to explore the involvement of lymphocytes in the process of COVID-19 associated diarrhoea.

Patients with a positive standard COVID-19-real-time polymerase chain reaction (PCR) and negative influenza virus-real time PCR test were included in the analysis.

Patients with mental illness, pregnancy, infectious disease, systemic disease, food allergy, a history of chronic diarrhoea such as inflammatory bowel disease (IBD), 8 celiac disease and bacillary dysentery before COVID-19 infection were excluded.

Further, patients with diarrhoea 15 days prior to COVID-19 infection and those who were deemed inappropriate by two independent doctors for any other conditions were also excluded. Based on these criteria, a total of 145 inpatients were included in this study.

Individual characteristics, symptoms on admission, chronic medical illnesses, laboratory results, disease severity, treatment during hospitalisation, and hospital stay were collected from patient records in this retrospective study. All data were crosschecked by two researchers (WJ and WX) and provided in table 1.

The retrospective analysis indicated that CD4+T cells might be associated with diarrhoea in COVID-19 patients. Therefore, the intestinal mucosa was collected for analysis from patients with COVID-19 with and without diarrhoea, patients with IBD, 9 and healthy controls.

All patients were recruited at the Digestive Endoscopy Centre of Zhongnan Hospital between January 2020 and December 2020, and their clinical characteristics are outlined in supplementary table S1. The diagnosis of COVID-19 was based on the WHO interim guidance.

Patients who had been diagnosed with COVID-19, with no prior history of diarrhoea (>15 days), that presented with diarrhoea (including that which persisted past viral infection) considered to be associated with COVID-19 (as opposed to other causes) were included in this part of the study.

The exclusion criteria were as follows:

Patients with incomplete clinical data, endoscopy, imaging and histological results, and follow-up information; taking drugs, radiotherapy, or chemotherapy before biopsy; with a history of chronic diarrhoea such as IBD or celiac disease before COVID-19 infection; and with mental illness, pregnancy, infectious disease, and food allergy were excluded from the study.

A total of 41 patients met these criteria and were included in the analysis and 10 underwent biopsy. Biopsies were collected using electronic colonoscopies, and the 41 biopsy samples were divided into five groups, namely ulcerative colitis (UC; n = 8), Crohn's disease (CD; n = 8), COVID-19 diarrhoea (n = 6), COVID-19 non-diarrhoea (n = 8), and healthy controls (HC; n = 11).

According to the guidelines, the human colon tissues were grinded, extracted by Trizol reagent (Invitrogen) and centrifuged to obtain total RNA. Reverse transcription of first-strand cDNA was performed according to the manufacturer"s instructions (Thermo Fisher Scientific, Waltham, MA, USA). Gel electrophoresis confirmed that the primers produced only one specific band. SYBR Green dye (CoWin BioSciences, Taizhou, Jiangsu, China) was used, and PCR was performed using a LightCycler ® 96 instrument (Roche Holding AG, Basel, Switzerland). Primers were obtained from TSINGKE Biosciences (Beijing, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the control. Primer sequences used are listed in supplementary table S2. Data were quantified using a LightCycler analyser and 11 normalised using the 2 -ΔΔCT method.

The human colon tissues were lysed with an ultrasonic crusher, and the cell lysate (which contained protease and phosphatase inhibitors; Servicebio Technology Co, Ltd, Wuhan, China) was used to split them. After centrifugation, the supernatant was collected and used as the protein extract. The protein was denatured by boiling in sodium dodecyl sulphate-polyacrylamide gel electrophoresis protein loading buffer.

An electrophoresis apparatus (Bio-Rad Laboratories, Hercules, CA, USA) was used to separate the proteins. The protein was transferred to a cellulose nitrate (PVDF) membrane (Millipore Corporation, Bedford, MA, USA) using a membrane transfer instrument (Bio-Rad). Membranes were blocked with 5% bovine serum albumin (BSA) at 20℃ for 2h and incubated with primary antibodies overnight at 4 °C. The membranes were incubated with secondary antibodies at 20 °C for 1 h. Finally, the membranes were treated with chemiluminescent detection reagents (Advansta Inc., San Jose, CA, USA) and imaged. The optical density of the protein was analysed 12 using the ImageJ software. All western blotting (WB) experiments were conducted in triplicate.

Fresh human biopsy samples were fixed with 4% paraformaldehyde and embedded in paraffin. Tissues were dewaxed and rehydrated with xylene, alcohol, and distilled water. After blocking the tissue with 5% BSA for 30 min, the sections were incubated with the primary antibodies overnight at 4 °C. After incubation and washing, the tissue was incubated with secondary antibodies at 20 °C for 1 h and kept away from light. After washing with phosphate-buffered saline (PBS), Vectashield with 4′,6-diamidino-2-phenylindole (DAPI; Baiqiandu Biotechnology Co, Wuhan, China) was used to cover the slides and the staining results were observed using a fluorescence microscope. A double-blinded method was used in this experiment. The details of all the antibodies are provided in supplementary table S3 and supplementary   table S4. 13

All data were analysed using GraphPad Prism (version 6.0; GraphPad Software, Inc., San Diego, CA). Categorical variables were described as counts and percentages and compared with the χ2 test. Continuous variables with normal distribution were described as the mean ± SD and compared using one-way ANOVA with a post hoc test (Bonferroni or Tokey). If not, continuous variables were presented as interquartile ranges (IQR) and compared using the Kruskal-Wallis test with the Nemenyi post hoc test. Spearman"s correlation coefficients were used to assess the correlation between the expression of angiotensin converting enzyme-2 (ACE2) and inflammatory cytokines in COVID-19 patients. A P-value <0.05 was considered statistically significant.

All steps of our study were in line with ethical requirements, and the study was approved by the Ethics Committee of the Zhongnan Hospital of Wuhan University (No. 2021052). Written informed consent was waived and oral consent was obtained 14 from all patients. The clinical courses were followed until 11 July 2021.

A total of 145 cases were included in the retrospective study, of which 21 were COVID-19 patients with diarrhoea and 124 were COVID-19 patients without diarrhoea. As shown in table 1, there was no significant difference between the two groups in terms of demographic features, concomitant symptoms, comorbidities, routine blood test results, blood inflammatory indicators, disease severity, treatment, and hospital stay.

Among the blood biochemistry indices, the diarrhoea group showed lower globulin levels than that of the non-diarrhoea group, whilst the other indices were comparable in both groups.

The lymphocyte subsets in the two groups showed significant differences. Compared with the non-diarrhoea group, the diarrhoea group had increased CD3+ and higher 15 CD4+ T cell counts and percentages. Additionally, CD8+ T cell counts were lower and the CD4/CD8 ratio was higher in the diarrhoea group than those of the non-diarrhoea group. Further, the diarrhoea group showed higher numbers of CD19+ B cells and lower numbers of CD16+CD56+ NK cells than those of the non-diarrhoea group.

Among these laboratory indicators, CD4+T cell counts, CD4+T cell percentage, and the CD4/CD8 ratio showed the most significant differences between the two groups.

Furthermore, the results for patients with common and serious illnesses were provided separately. The results showed that there was no significant difference in diarrhea between common patients and all patients (supplementary table S5 and supplementary   table S6 ). As there were only two patients with severe diarrhea, the number of cases was too small to represent the whole of severe diarrhea, which was considered to be of no statistical significance.

Using real-time PCR, we compared the relative mRNA expression of ACE2, transmembrane serine protease 2 (TMPRSS2), and CD4+T cell-related cytokines and transcription factors in the colonic mucosa of COVID-19 patients with and without diarrhoea, patients with IBD, and healthy controls.

As shown in Figure 1a , the relative level of ACE2 was significantly different between the diarrhoea and non-diarrhoea groups (P = 0.049). Compared to ACE2, there were no remarkable changes in TMPRSS2 expression between the two groups ( Figure 1b Moreover, the expression of tumour necrosing factor-alpha (TNFα; P = 0.031; Figure   1g ) and interleukin-10 (IL10; P = 0.018; Figure 1j ) in the diarrhoea group was higher than that in the non-diarrhoea group. These results suggest that diarrhoea in COVID-19 patients is related to ACE2 and the cytokines secreted by Th1, Th2, and Th17 cells at the mRNA level.

Inflammatory factors with significant differences at the mRNA level were selected for Western Blot testing. The results showed that ACE2 protein levels in the diarrhoea group were significantly higher than those in both the non-diarrhoea (P = 0.041) and HC groups (P = 0.047; Figure 2b ). When compared to that of both the non-diarrhoea (P = 0.049) and the HC group (P = 0.036), the protein expression of Tbet (Fig 2c) and

TNFα (P = 0.018; Figure 2f The results of the correlation analysis performed between ACE2 and CD4+ T cell-related cytokines suggested that ACE2 was moderately correlated with Th1-associated indicators, including Tbet and TNFα, at both mRNA and protein expression levels (r values ≥0.5; P <0.05; supplementary table S7; supplementary   table S8; supplementary table S9; supplementary table S10) . These results suggested a 18 close correlation between ACE2 and Th1 cells. To explore whether ACE2 was directly associated with CD4+ T cell-mediated inflammation, we performed double immunofluorescence of ACE2 and TNF-α in the colonic mucosa.

It was observed that ACE2 and TNFα were prominently expressed by lamina propria cells of colonic mucosa from COVID-19 patients with diarrhoea and IBD (Figure 3 ).

There were a significantly higher amount of ACE2 positive cells in the diarrhoea group than in both the non-diarrhoea (P = 0.034) and HC groups (P = 0.004). The number of TNF-α-positive cells in the diarrhoea group was also higher than that in both the non-diarrhoea (P = 0.012) and HC groups (P< 0.001). The number of ACE2+TNFα+ cells in the diarrhoea group was significantly higher than that in the HC group (P = 0.001). However, there were no significant differences in ACE2+TNFα cells were observed between the diarrhoeal and non-diarrheal groups (P = 0.090).

To assess the chemotactic activity of the CD4+T cell axis, the mRNA expression of chemokine receptors CXCR3, CCR5, CCR4, CCR6, and their ligands (CXCL9, CXCL10, CXCL11, CCL2, CCL3, CCL4, and CCL20) was determined with real-time PCR. Total mRNA was extracted from the colon tissues of the patients, and 21 cases were included in the analyses. 

Finally, we examined the expression and distribution of CXCR3 and CD4 by double IF analysis. Both CXCR3 and CD4 were distributed in the lamina propria of the colon ( Figure 5a ). CXCR3+ cells in the diarrhoea group showed a significant increase compared to those in both the non-diarrhoea (P = 0.027) and HC groups (P = 0.002).

The diarrhoea group also showed a higher amount of CD4+ T cells than that in the non-diarrhoea (P = 0.010) and HC groups (P = 0.001). Moreover, the number of CXCR3+CD4+ cells in the diarrhoea group was significantly higher than those in the non-diarrhoea (P = 0.017) and HC groups (P=0.002) (Fig 5(b) ).

Whilst diarrhoea may be common in COVID-19 patients, the exact pathogenesis is still unclear. Some studies have detected SARS-CoV-2 RNA in the faeces of COVID-19 patients using rapid RT-PCR (Holshue et al., 2020 , van Doorn et al., 2020 , and some positive faecal samples remained positive even after respiratory samples tested negative (van Doorn et al., 2020) , which indicates the possibility of delayed clearance of SARS-CoV-2 in the GI tract. Another study detected 4 SARS-CoV-2 RNA-positive faecal samples using electron microscopy, and a live virus was observed in two specimens from patients who did not have diarrhoea (Wang W. et al., 2020) . In addition, SARS-CoV-2 RNA can also be detected in biopsies of the oesophagus, stomach, duodenum, and rectum by endoscopy .

Further, positive viral nucleocapsid protein expression was identified by histological and immunofluorescent staining in the cytoplasm of gastric, duodenal, and rectal glandular epithelial cells , which enhances the evidence for GI infection of SARS-CoV-2. Furthermore, ACE2, which controls intestinal inflammation and diarrhoea , is a viral receptor for the entry of SARS-CoV-2 into human cells (Lan et al., 2020) . Different studies have found that ACE2 is highly expressed in digestive organs, except the stomach , Zou et al., 2020 , suggesting an increased susceptibility of digestive systems to SARS-CoV-2 infection and a potential risk of gastrointestinal symptoms such as diarrhoea after infection. Based on this evidence, we speculated that the direct cytopathic effects of SARS-CoV-2 might play an important role in the pathogenesis of COVID-19 associated diarrhoea.

Both systemic and local inflammation in the GI tract have been observed in COVID-19 patients, and many studies have found that hyperinflammation in peripheral blood is associated with disease severity and mortality (Carter et al., 2020, Gustine and Jones, 2021) . For local inflammation, numerous infiltrating lymphocytes 22 and plasma cells with interstitial oedema were observed in the lamina propria of the stomach, duodenum, and rectum biopsies obtained from patients with COVID-19 by endoscopy . To seek clues related to diarrhoea in COVID-19 patients, we performed a case-control analysis based on the symptom of "diarrhoea" in patients with COVID-19. Our study revealed that there were significant differences in lymphocyte subsets and globulin levels between COVID-19 patients with and without diarrhoea, with no significance for all other indicators, suggesting that immune factors might play a role in the occurrence of diarrhoea in COVID-19. In lymphocyte subset detection, patients with diarrhoea had different patterns of CD3+, CD4+, and CD8+ T cell percentages, CD19+ B cell counts, CD16+CD56+ NK cell percentages, and CD4/CD8 ratios. In view of the most significant differences observed in CD4+ count, percentage, and CD4/CD8 ratio between groups, and the change patterns of other subsets, we speculated that CD4+ T cells are a key factor associated with diarrhoea in patients with COVID-19.

To clarify the role of CD4+ T cell-mediated inflammation in COVID-19-associated diarrhoea, we collected colonic mucosa from COVID-19 patients with and without 23 diarrhoea, taking IBD patients as positive controls and healthy subjects as negative controls. CD4+ T cells are thought to play an important role in both innate and adaptive immune responses (Ruterbusch et al., 2020) , and have been identified as the main drivers of IBD and players perpetuating intestinal inflammation (Imam et al., 2018) .

ACE2 is a viral receptor for the entry of SARS-CoV-2 that is highly expressed in the intestine. Thus, the detection of ACE2 expression in the intestine reflects the invasion of SARS-CoV-2 into the intestine. TMPRSS2 is highly expressed in lung, heart, prostate, kidney, and intestinal tissues (Comperat et al., 2020; Hamming et al., 2004; Thunders and Delahunt, 2020) . SARS-CoV-2 uses ACE2 for entry and TMPRSS2 for S protein priming (Hoffmann et al. 2020) , and as a host cell factor, TMPRSS2 is essential for SARS-CoV-2 transmission (Hoffmann et al., 2020) . Therefore, TMPRSS2 is usually detected together with ACE2 as evidence of SARS-CoV-2 infection. CD4+ T cell-related factors to be detected include specific transcription factors and inflammatory effector cytokines that interact with ACE2 to facilitate viral entry and activation (Baughn et al., 2020) . The expression of inflammatory factors in 24 the intestinal tract reflects the occurrence of a CD4+ T cell-mediated immune response. Chemokines play an important role in the priming, differentiation, and regulation of specific effector T-cell subsets after acute pathogen challenge (Eberlein et al., 2020) . Thus, specific transcription factors of different CD4+ T cell subsets, such as Tbet of Th1, GATA3 of Th2, RORC of Th17, and FOXP3 of Tregs (Jin et al., 2018 , Ruterbusch et al., 2020 , and inflammatory effector cytokines such as TNFα, interferon (IFN) γ, IL10, IL17, IL25, and transforming growth factor (TGF) β were detected at both the mRNA and protein levels, with detection of ACE2 and TMPRSS2.

Our results revealed that COVID-19 patients with diarrhoea had a higher expression of ACE2, Tbet, and TNFα at both mRNA and protein levels than those patients without diarrhoea and healthy controls (Figure 1 S7; supplementary table S8; supplementary table S9; supplementary table S10).

Based on the above findings, we speculated that the close association between ACE2 and CD4+ T cells might be explained by two possibilities: 1) SARS-CoV-2 directly infects CD4+ T cells in the intestine, resulting in the occurrence of CD4+ T cell-mediated immune responses; or 2) SARS-CoV-2 infection occurs in the intestinal mucosa, leading to local intestinal inflammation and involvement of CD4+ T cells to perpetuate intestinal inflammation. Although ACE2 is rarely expressed in immune 26 cells (Berthelot et al., 2020) , a study has shown that immune cells, including CD4+ and CD8+ T cells, monocytes, and B cells, are susceptible to SARS-CoV-2 infection (Borsa and Mazet, 2020) . To exclude the direct association of ACE2 with CD4+ T cell-mediated inflammation, an immunofluorescence assay of dual labelling for ACE2 and TNFα was conducted. The results revealed that the percentages of ACE2+ cells and TNFα + cells in the diarrhoea group were higher than those in the non-diarrhoea group, although no statistical difference in the percentage of ACE2+TNFα + cells between the two groups was observed (Figure 3) , indicating an indirect association between ACE2 and TNFα secretion that usually originates from CD4+ T cells.

Chemokines play an important role in the priming, differentiation, and regulation of specific effector T-cell subsets after acute pathogen challenge (Eberlein et al., 2020) .

Thus, the expression of chemokines and their corresponding receptors was measured at the mRNA level, and it was found that COVID-19 patients with diarrhoea had higher expression levels of CXCL10, CXCL11, and CXCR3 than those without diarrhoea ( Figure 4) . Interestingly, CXCL10 and CXCL11 are both ligands for 27 CXCR3, which was found to be expressed on differentiated Th1 cells to modulate their migration into the gut in a murine colitis model (Wadwa et al., 2016) . In summary, our findings showed that both enhanced systemic and local inflammation in the GI tract mediated by CD4 + T cells were identified in COVID-19 patients with diarrhoea, and that the occurrence and persistence of inflammation in the gut might be closely associated with SARS-CoV-2 infection. CXCR3+-mediated migration of CD4+ T cells into the gut may contribute to perpetuating intestinal inflammation.

Future investigations may be performed to elucidate more detailed mechanisms of COVID-19 associated diarrhoea in animal models and in vitro to clarify the potential 28 therapeutic benefits of targeting CXCR3.

We thank all the patients included in this study. 

Xiaobing Wang and Jia Wei contributed equally to this paper.

All authors listed have approved the manuscript that is enclosed.

There are no conflicts of interest to be declared.

This study was reviewed and approved by ethical committee the ethics committee of Zhongnan Hospital of Wuhan University (No. 2021052) . This study was an observational study. Informed verbal consent was obtained from all patients in our study as written consent was waived by the ethics committee. The mRNA expression of CD4+T cells related cytokines(TNFα, IFNγ,IL25, IL10, IL17A and TGFβ).

"HC" represented Healthy control. (Kruskal-Wallis test with Nemenyi post hoc test; n=5-10 per group) Error bars denote the Mean ± SEM. ns = not significant, *p < 0.05, **p < 0.01, ***p<0.001 and ****p < 0.0001. 

Non-respiratory presentations of COVID-19, a clinical review

Targeting 30 TMPRSS2 in SARS-CoV-2 Infection

Lymphocyte Changes in Severe COVID-19: Delayed Over-Activation of STING?

Attacking the defence: SARS-CoV-2 can infect immune cells

Peripheral immunophenotypes in children with multisystem inflammatory syndrome associated with SARS-CoV-2 infection

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Gastrointestinal Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples From a

Systematic Review and Meta-analysis

The Genetic Complexity of Prostate Cancer

Microbiota Modulation of the Gut-Lung Axis in COVID-19

Digestive Manifestations in Patients Hospitalized With Coronavirus Disease

Clinical gastroenterology and hepatology : the official clinical practice journal of the

Clinical Characteristics of Coronavirus Disease 2019 in China

Potential intestinal infection and faecal-oral 32 transmission of SARS-CoV-2

Extrapulmonary manifestations of COVID-19

Immunopathology of Hyperinflammation in COVID-19

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

First Case of 2019 Novel Coronavirus in the United States

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Effector T Helper Cell Subsets in Inflammatory Bowel Diseases

Pathophysiological mechanisms underlying gastrointestinal symptoms in patients with COVID-19

Treg/Th17 imbalance in rheumatoid arthritis through miR-21

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Diarrhoea may be underestimated: a missing link in 2019 novel coronavirus

Gastrointestinal symptoms of 95 cases with SARS-CoV-2 infection

COVID-19 and the Digestive System

Putative Mechanisms of Diarrhea in COVID-19

Vivo CD4(+) T Cell Differentiation and Function: Revisiting the Th1/Th2 Paradigm

Gene of the month: TMPRSS2 (transmembrane serine protease 2)

Systematic review with meta-analysis: SARS-CoV-2 stool testing and the potential for faecal-oral transmission

downregulates CXCR3 expression on Th1 cells and interferes with their migration to intestinal inflammatory sites

Enteric involvement in hospitalised patients with COVID-19 outside Wuhan

Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China

Detection of SARS-CoV-2 in Different Types of Clinical Specimens

Evidence for Gastrointestinal Infection of SARS-CoV-2

Clinical findings in a group of patients infected with the 2019 novel coronavirus (SARS-Cov-2) outside of

China: retrospective case series

SARS-CoV-2-infected patients with diarrhea in relation to disease severity

Specific ACE2 expression in small intestinal enterocytes may cause gastrointestinal symptoms and injury after 2019-nCoV infection

Diarrhea and altered inflammatory cytokine pattern in severe coronavirus disease 2019: Impact on disease course and in-hospital mortality

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues

-e) Statistics of ACE2 and related transcription factors (Tbet, GATA3 and RORc) expression; (f, g) Statistics of CD4+T cells" cytokines expression (TNFα, IL10)

Representative fluorescence images (a) and quantification of optical density of ACE2 and TNFα (b). Panels from left to right: COVID-19-diarrhea

TNFα (green), and nucleus (DAPI; blue). (one-way ANOVA test with Bonferroni post hoc test

Quantitation of mRNA expression of CD4+T cells chemokines and receptors in COVID-19 patients with and without diarrhea

HC" represented Healthy control. (Kruskal-Wallis test with Nemenyi post hoc test; n=5-9 per group) Error bars denote the Mean ± SEM. ns = not significant

Figure 5. Distribution of CXCR3 and CD4 in colons of COVID-19 patients with and without diarrhea

Representative fluorescence images (a) and quantification of optical density of CXCR3 and CD4 (b)

CD4 (green), and nucleus (DAPI; blue). (one-way