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Nature sub issue: after rehabilitation, still immune deficiency. What exactly did COVID-19 do to B cells?!

  At the end of 2019, a New Coronavirus (SARS-CoV-2) was identified as the causative agent of severe acute respiratory tract infection in COVID-19. The clinical manifestations of sars-cov-2 infected patients vary widely, from asymptomatic to life-threatening severe, including acute respiratory distress syndrome (ARDS). The so-called & ldquo; Cytokine storm & rdquo;, That is, the uncontrollable activation of inflammatory response significantly promotes the occurrence of ARDS. Similar to other diseases that trigger cytokine storms, inhibiting inflammatory immune response may improve the outcome of severe sars-cov-2 infection.

  The current evidence shows that compared with healthy individuals, the serum metabolic components of covid-19 patients have changed significantly, which is related to the severity of the disease. The metabolic environment, such as the change of glucose concentration, the acquisition of short chain fatty acids and oxygen supply, affects the survival, activation and differentiation of B cells. Therefore, the specific metabolic environment in patients with covid-19 may change B cell biology, and B cells also play a variety of roles in the progression of covid-19 disease.

  The purpose of this study was to evaluate the signal characteristics of B cells in patients with covid-19, and to determine how changes in the abundance of specific metabolites affect B cell receptor signals by analyzing serum metabolic components obtained from patients with covid-19.

  1. Sars-cov-2 infection changes the immunophenotype and B cell function of rehabilitated patients. Specific B cell subsets play a key role in antiviral humoral immunity. In order to study whether sars-cov-2 infection affects B cell immunophenotype, we first detected naive B cells (CD27 - IgD +), atypical (CD27 - IgD -), switched (CD27 + IgD -) and non switched (CD27 + IgD +) memory B cells, transition B cells (CD38 + cd24hi) and plasmacytes (PBC, CD38 + CD24 & minus;). In the resting phase, the healthy control group and rehabilitation patients had similar B cell populations, but the frequency of PBC increased significantly compared with the healthy control group. To detect the response of B cells to BCR dependent signals, we used f (AB & rsquo;) 2 anti human Ig (M + G) antibody stimulated B cells for 24 hours. However, the number of atypical memory B cells increased compared with the resting state (Fig. 1a-e). Compared with the resting state, the frequency of stimulated non switched memory B cells in patients decreased, but there was no change in the healthy control group (Fig. 1a, c). Interestingly, the expression of CD19, a key regulator of BCR signaling pathway, decreased significantly in total B cells and almost all B cell subsets in rehabilitated patients compared with healthy controls (Fig. 1F). However, there was no change in CD19 mRNA expression in B cells of rehabilitated patients compared with healthy controls (Fig. 1g). Immunofluorescence experiment showed that the expression of CD19 in spleen of sars-cov-2 infected mice was significantly lower than that of uninfected (simulated) mice (Fig. 1H, I). These results suggest that sars-cov-2 infection may change the immunophenotype and function of B cells by inhibiting the expression of CD19.

  2. Sars-cov-2 infection changes BCR signal and B cell metabolism in rehabilitated patients. CD19 is a key regulator of BCR signal transduction, B cell development and humoral immune response. Compared with the healthy control group, the levels of total CD19, phosphorylated CD19 and phosphorylated Btk in B cells of patients were significantly reduced (Fig. 2a). CD19 is an important activator of PI3K signaling pathway in anti Ig (M + G) - stimulated cells. When detecting the activation of PI3K and its downstream signals by anti Ig (M + G) - stimulated B cells, the phosphorylated PI3K, Akt, FoxO1, S6 and mTOR in patient B cells were significantly lower than those in healthy control group (Fig. 2b). Because PI3K Signal is the main regulator and redox signal of B cell metabolism, we speculate that sars-cov-2 infection changes B cell metabolism. Abnormal production of reactive oxygen species (ROS) can lead to impaired survival of normal and germinal center B cells, and mitochondrial ROS inhibits humoral immune response. Compared with untreated patient B cells, the levels of CD19 and PBTK on antioxidant treated patient B cells were significantly increased (Fig. 2C). To evaluate the redox status of B cells in patients with covid-19, we used f (AB & rsquo;) 2 anti human Ig (M + G) stimulated B cells of patients and healthy controls, and the total ROS production was measured. It was found that patients' B cells produced more ROS than healthy controls (Fig. 2D). Mitochondrial complex III is necessary for hypoxia induced ROS production and cellular oxygen perception. Mitochondrial swelling in patient B cells increased compared with healthy controls (Fig. 2e). Endoplasmic reticulum (ER) homeostasis is maintained by regulating mitochondrial calcium level and shaping B cell metabolism and metabolic stress response. Compared with the healthy control group, the MFI of ER and Mito on B cells in patients increased significantly at 0 and 5 min, but decreased at 30 min (Fig. 2f-i). In addition, compared with the healthy control group, the co localization between BCR and ER or Mito decreased significantly during the whole process of B cell activation (Fig. 2j-k). We also found that the co localization of ER and Mito on B cells in patients was significantly reduced at 0, 5 and 30 minutes compared with healthy controls (Fig. 2L). These results suggest that sars-cov-2 infection may change the metabolic activity of B cells and increase the production of ROS, thus reducing the expression of CD19 and BCR dependent signal transduction.

  3. SARS-CoV-2 infection novel coronavirus pneumonia and B L cells, which had changed the serum metabolites and transcriptome, and the levels of L arginine, l glutamic acid, l isoleucine, l cystine and L - cysteine in serum of patients with pneumonia recovered significantly. Compared with the healthy control group, the levels of serum glutamate and cysteine in the rehabilitated covid-19 patients were significantly lower. By detecting the difference of B cell transcriptome between the rehabilitated covid-19 patients and the healthy control group, it was found that compared with the healthy control group, the expression of 48 genes decreased significantly, and the expression of 10 genes increased significantly (Fig. 3f). KEGG functional enrichment analysis found that 20 pathways were enriched in differentiation genes / metabolic genes, most of which were metabolic signaling pathways, especially amino acid synthesis and metabolism (Fig. 3G). Finally, using KEGG to identify the metabolic pathways of genes and metabolites, it was found that there were significant differences in 30 metabolic pathways between healthy control group and rehabilitated patients (Fig. 3H). In the transcriptome, longevity regulatory pathways - multispecies, valine, leucine and isoleucine biosynthesis, neomycin, kanamycin and gentamicin biosynthesis, carbon metabolism and estrogen signaling pathways are highly enriched (Fig. 3H). In the metabolome, purine metabolism and carbon metabolism were highly enriched (Fig. 3H). The correlation analysis of genes and metabolites detected in each difference group showed that the Pearson correlation coefficient was > 0.8. These results suggest that sars-cov-2 may change B cell metabolism through these pathways. In conclusion, sars-cov-2 infection may affect BCR signaling pathway by changing the metabolome and transcriptome spectrum of B cells.

  4. Sars-cov-2 infection changes the BCR signal pathway by affecting the early activation of B cells. In terms of B cell diffusion, compared with the original B cells and memory B cells in the healthy control group, the contact area of the patient's initial B cells decreased at 3 minutes of activation, and the contact area of the patient's memory B cells decreased significantly at 5 minutes of activation (Fig. 4a-c). For the total BCR signal, the MFI of Py in patients with uninfected B cells and memory B cells decreased significantly at 3 and 5 minutes compared with uninfected B cells and memory B cells in the healthy control group (FIG. 4A, B, d). Compared with naive B cells and memory B cells in the healthy control group, the MFI of BCRs decreased significantly at 3 and 5 minutes (Fig. 5a-c). In addition, compared with uninfected B cells and memory B cells in the healthy control group, the MFI of pShip decreased significantly 5 minutes after activation of uninfected B cells and memory B cells (Fig. 5a, B, d). For the activation of CD19, compared with uninfected B cells and memory B cells in the healthy control group, the MFI of pcd19 in uninfected B cells and memory B cells in patients decreased significantly at 3 and 5 minutes (Fig. 6a-c). These results suggest that sars-cov-2 infection alters the BCR signaling pathway by inhibiting the early activation of naive B cells and memory B cells.

  In this study, through transcriptome and metabolic studies, it was found that the gene expression related to metabolism and metabolites was maladjusted, and the ROS of B cells in rehabilitated patients increased, indicating that the metabolism of B cells in rehabilitated patients increased. Interestingly, the use of antioxidants can partially save the expression of CD19 in rehabilitated patients. In addition, we found that the expression of CD19 in the spleen of sars-cov-2 infected mice was also reduced, or it can be considered that the expression of CD19 may also be reduced in infected patients. It is worth paying attention to detect the expression of CD19 and the metabolic state of B cells in patients with mild and severe infection. In addition, the correlation between CD19 expression and different metabolic states of B cells is worth studying. More exciting, correcting the increased ROS in B cells saved the expression of CD19 and BCR signal, which may correct the immune status of rehabilitated patients. It is unclear whether rehabilitated patients with reduced CD19 expression are more likely to re infect sars-cov-2. If this is true, metabolic regulators such as NAC have the potential to prevent secondary infection in rehabilitated patients. Although the expression of CD19 was found to be decreased, there was no change in the level of CD19 mRNA in B cells of rehabilitated patients, suggesting that sars-cov-2 infection may reduce the level of CD19 expression in the post-translational period. We also detected several molecules in the BCR signaling pathway from RNA SEQ, but found no differences in CD19 mRNA and molecules in the BCR signaling pathway between healthy controls and patients. This may be the reason why the BCR signal pathway does not appear in the KEGG enriched signal pathway. Bacterial and fungal infections are common complications in patients with viral pneumonia and lead to increased mortality. Analyzing the signal characteristics of B cells in patients with covid-19 is helpful to evaluate whether covid-19 infection will make patients more susceptible to other diseases, and better understand how metabolic abnormalities in patients' serum affect B cell signals, which may inspire the development of new treatment strategies for specific metabolic pathways. Different compounds that alter cell metabolism and redox status are currently being tested in preclinical and clinical studies for the treatment of autoimmune diseases, and may also be used to inhibit the hyperactive immune response during viral infection. Source: Jing et al. Sars-cov-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism Signal Transduction and Targeted Therapy (2021) 6:345 ; https://doi.org/10.1038/s41392-021-00749-3


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