2.2 Single cell atlas and molecular changes of alveolar target cells
As the lungs is the main target organ of SARS-CoV-2 infection, we further explored lung cell compositions and changes in COVID19 patients and healthy people by using cell dimensionality reduction clustering and Uniform Manifold Approximation and Projection (UMAP) analysis . We herein identified a variety of lung epithelial cells including type I alveolar cells (AT1) (AGER+), type II alveolar cells (AT2) (SFTPC+), club cells (SCGB3A2+), ciliated cells (TPPP3+), basal cells (PTTG1+) in the lungs (Fig. 2A ~ 2B). Of note, the types of immune cells in the lungs mainly include common neutrophils (Neu) (S100A8+), monocyte (MS4A6A+), T cells (CD3D+), natural killer cells (NK) (GNLY+), plasma cells (MZB1+), mast cells (TPSB2+) and macrophages (Macro) (CD68+) (Fig. 2A ~ 2B). Besides, endothelial cells (End) (CLDN5+) and fibroblasts (Fib) (DCN+) were also found in the lungs (Fig. 2A ~ 2B). Surprisingly, we discovered a cluster of MKI67 progenitor cells (MKI67+) with highly expressed MKI67 (Fig. 2A ~ 2B). Remarkably, the ratio of multiple cell types in the lungs is changed after infection, in particular the proportion of immune cells, including Neu, T, NK and Mono cells, is obviously increased (Fig. 2C), implying that the invasion of SARS-CoV-2 might lead to the enrichment of certain immune cells to the lungs. Additionally, the ratio of both Fib and club cells is respectively significantly increased (Fig. 2C), and their increases were usually related to lung fibrosis and increased lung mucus [15, 16]. In turn, we found that the ratio of AT1 and AT2 cells is drastically decreased (Fig. 2C), in particular many genes that maintain the integrity and homeostasis of alveolar cells, such as NKX2-1 as a key transcription factor, SFTP family members, IRX2, NPNT, SDC4, SHROOM3, TGFBR2 and TMED2, are significantly down-regulated after infection (Fig. 2D ~ E) [17–20]. Whilst it was clear from the results of hematoxylin-eosin staining (HE) that the alveolar cells of the left lower lung of patient 1 and the right lower lung of patient 2 are serious damage (Fig. 2F ~ G). Taken together, these above results seem to indicate that SARS-CoV-2 infection might destroy the integrity and homeostasis of alveolar cells.
In order to explain the above issue, we further performed a functional enrichment analysis on all differentially expressed genes in AT1 and AT2 cells after infection. We found that these differentially expressed genes are significantly enriched in energy electron chains, microtubule and vascular bundle assembly, response to virus, virus life cycle, interferon response and neutrophil chemotaxis (Fig. 2H). Usually, both AT1 and AT2 as epithelial cells do not have the ability to kill viruses [21, 22], but AT1 and AT2 cells can release a large number of molecules to induce immune and antiviral signal response after SARS-CoV-2 infection based on the enrichment analysis (Fig. 2H). Herein, we used the cell communication analysis to further illustrate this finding. Our results demonstrated that the number of cell communications in the entire lung are significantly reduced after infection (COVID-19: 3,275 vs Health: 6,573) (Fig S1A ~ S1B), which may be caused by the death of a large number of lung cells after infection. Interestingly, the cell types with the highest communication frequency among AT2/AT1 target cells are mainly MKI67+, End and basal cells in the normal group, but the most frequent cell type of communication are mainly Macro, Neu and Mono cells in the infection group (Fig. 2I ~ 2J and Fig S1C ~ S1D). Especially, we find that many receptor and ligand genes altered significantly of AT1 and AT2 cells are obviously enriched in myeloid cell migration, granulocyte migration, monocyte migration and chemotaxis (Fig. 2K). Taken together, these results seemed to imply that these significantly changed receptor and ligand genes play a crucial role in inducing AT2 and AT1 cells to communicate with innate immune cells in the invasion of SARS-CoV-2.
2.3 Dysregulated ligands and receptors cause the disorder of inflammatory genes and immune cell migration
Why can SARS-CoV-2 infection obviously increase the cell communication frequency among AT1 and AT2 cells and immune cells? We herein speculated that this increased communication frequency might be responsible for recruiting immune cells to kill SARS-CoV-2. Remarkably, our above results have demonstrated significant increased myeloid cells and T cells in the lungs after SARS-CoV-2 infection (Fig. 2C), which is consistent with previous studies [7, 23, 24]. However, as the first site of SARS-CoV-2 infection, how the AT1 and AT2 cells after infection induce the recruitment of myeloid cells and T cells, which is still unclear. Interestingly, we here found that a group of ligand and receptor genes, such as ANXA1_FPR1, CD74_APP, CD74_COPA, CXCL1_CXCR1, CXCL2_CXCR2, might participate in the recruitment of myeloid cells, and mediate the cell communication between AT1/AT2 cells and myeloid cells (Neu, Mono, Macro) after SARS-CoV-2 infection through in-depth cell communication analysis (Fig. 3A). Of note, previous studies have indicated that the CXCLs_CXCRs family as known chemokines can induce the recruitment of Neu and Mono , and the ANXA1_FPR1 is related to the increase of myeloid cells of COVID-19 patients , as well as both CD74_COPA and CD74_APP serve as inflammatory recruitment signals, in particular the COPA gene is also involved in the occurrence of autoimmune interstitial lung, arthritis, kidney disease and lipopneumonia [26, 27]. These results implied that the dysregulations of some certain receptor and ligand genes might cause the disorder of their downstream genes associated with inflammation in myeloid cells after SARS-CoV-2 infection.
We thus further explored the expressions of inflammation-related genes. Surprisingly, we found that the myeloid cells recruited by both AT1 and AT2 cells have higher inflammatory activity accompanied by significantly increased inflammatory gene set scores in Neu and Mono/Macro cells (Fig. 3B ~ 3C). Especially, many inflammation-related genes, such as C5AR1, CD55, CSF3R, CXCL8, HIF1A, NAMPT and NFKB1A, are significantly higher expressed in the Neu cells (Fig. 3D), as well as C5AR1, CD14, CSF3R, CXCL8, HIF1A, IFNAR1, NAMPT and NFKB1A are obvious higher expressed in the Mono/Macro cells of the lungs after SARS-CoV-2 infection (Fig. 3E). Previous reports have shown that both the ligand ANXA1 and receptor FPRs can served as an upstream signal to promote the up-regulation of granulocyte macrophage colony stimulating factor (GM-CSF) and cause the maturation and migration of neutrophil cells and macrophages . In particular CSF3R as an inflammation marker can act as a receptor for GM-CSF , as well as the FPR-1 and its ligand are also necessary for effectively recruiting neutrophils to the damaged lung tissue and the reduction of neutrophils are protected from pulmonary fibrosis . Additionally, C5AR1 can act as an upstream signal of Toll-like pathways (NFKB1, TLR1, TLR2, etc.) [31–33], which can further activate the downstream inflammatory cytokines, such as TNFRA family members (TNFAIP6, TNFRSF1B, TNFSF10) and IL family members (IL18R1, IL1B, IL7R) [34, 35]. And that epithelial cells can release CXCLs to induce neutrophil infiltration and up-regulate the expression of the inflammatory gene NAMPT . These studies indicate that the dysregulations of receptor and ligand genes may result in the disorders of inflammatory genes. Together, our study seemed to suggest that the inflammatory storm of different myeloid cells might be driven by the dysregulations of certain ligand and receptor genes after SARS-CoV-2 infection.
Herein, we also explored how the AT1 and AT2 cells recruit T cells into the lungs to kill SARS-CoV-2 after infection. We firstly found that there is almost no cell communication between AT1 and AT2 cells and T cells after infection (Fig. 3F), implying that the recruitment of T cells in the lung might be not directly related to AT1 and AT2 cells. Secondly, we found that T cells could communicate with myeloid cells in the lung, in particular their communication can be mainly mediated by some ligand and receptor genes such as ANXA1_FPR1, C5AR1_RPS19 and CCL5_CCR1 (Fig. 3F). Interestingly, studies have demonstrated that the combination of C5AR1 and RPS19 is one of the main reasons for the activation of the coagulation complement system [37, 38], and C5AR1 monoclonal antibody is also considered to be a effective drug for the treatment of coagulopathy caused by SARS-CoV-2 [13, 39], as well as the combination of CCL5 and CCR1 can induce the recruitment of T cells to inflammation and viral sites to inhibit the virus [40–42]. Taken together, these results seemed to indicate that myeloid cells have obvious duality in the lungs after SARS-CoV-2 infection. On the one hand, these myeloid cells induced by infected AT1/AT2 through inflammatory ligands and receptors may be the source of the lung inflammation storm . On the other hand, these myeloid cells of the lungs can further induce T lymphocytes to migrate into the lungs and kill SARS-CoV-2 (Fig. 3G).
2.4 Lung inflammation drives the increase of fibrosis and mucus accumulation
In this study, the HE staining shows a significant fibrosis increase in the lung tissues of the two dead COVID-19 patients (Fig. 4A ~ 4B), and the apoptosis score of fibroblasts is also significantly reduced compared with the control (Fig. 4C), which implies that severe lung inflammation might lead to the increase of lung fibrosis in the two COVID-19 patients. Remarkably, under certain physiological and pathological conditions, epithelial cells can undergo phenotypic transformation and become myofibroblasts that can produce extracellular matrix, which is called epithelial-mesenchymal transition (EMT) . In particular, the type II EMT can induce fibroblasts and other related cells for tissue repair after trauma and inflammatory injury, and the characteristic of tissue fibrosis is endless wound repair caused by continuous inflammation [45, 46]. Therefore, we here further investigate gene expressions in fibroblasts of the lungs. Our results demonstrated that differentially expressed genes in fibroblasts are significantly enriched in the biological processes of extracellular matrix decomposition, cell adhesion, and extracellular matrix assembly (Fig. 4D), indicating that the cytoskeleton and cell morphology had been altered after SARS-CoV-2 infection. Interestingly, we also found that collagen-related proteins (e.g. COL1A1, COL1A2, COL3A1), laminin proteins (e.g. LAMA2, LAMB1), inflammatory marker proteins (e.g. S100A8, S100A9), the matrix metalloproteinase MMP2, fibronectin FN1 and the key regulator ZEB1 of EMT were significantly upregulated in the fibroblasts of COVID-19 patients (Fig. 4E). Of note, these above genes are all related to the increase of EMT-mediated fibrosis [45–47]. As expected, fibrosis in COVID-19 patients can indeed be driven by EMT, with a significantly increased EMT score (Fig. 4F) and the decrease of MAL2, CLND4 and TJP1 of epithelial cell as well as the increase of VIM and CDH2 of mesenchymal cell in AT1/AT2 cells (Fig S2). Especially, previous studies have demonstrated that the activation of certain receptors and ligands (e.g. CD74_APP and CD74_COPA) signals affects downstream gene expressions and promotes inflammation and lung diseases [48–50], and the activation of COL1A2_a2b1, COL3A1_a2b1 and FN1_a3b1 signals drives the process of EMT, interstitial pneumonia and pulmonary fibrosis [10, 51, 52]. Together, our work suggested that some receptors and ligands, such as CD74_APP, CD74_COPA, COL1A2_a2b1, COL3A1_a2b1 and FN1_a3b1 not only mediate the cell communication between AT2 cells and fibroblasts but also facilitate the increase of lung fibrosis EMT-mediated by the lung inflammation (Fig. 4G).
In addition to pulmonary fibrosis, whether can the patient's respiratory failure be caused by lung obstruction? Interestingly, the HE staining revealed significant mucus increase in the lungs of these two COVID-19 patients (Fig. 4H ~ I), and the entire alveolar cell space filled by these thick mucus might cause dyspnea or failure in the patients. Remarkably, this phenotype from the HE staining was consistent with the increase of the proportion of club cells, a main cell type that produces mucus, in the two COVID19 patients (Fig. 2C). Particularly, we found that some mucus components (e.g. MUC5B and MUC4) and genes related to mucus viscosity (e.g. TGM2 and TFF3) were significantly up-regulated in the two COVID19 patients (Fig. 4J), and the mucus secretion score of club cells was accordingly increased significantly in COVID19 patients (Fig. 4K). These results indicated that SARS-CoV-2 infection improves the ratio of club cells and mucus production. Herein, we further performed functional and transcription factor enrichment analysis to explore the reason that drives the increase of club cell-based mucus secretion. Our results demonstrated that differentially expressed genes in club cells were mainly enriched in these processes such as hypoxia, C-type lectin, virus life cycle and interferon response (Fig. 4L). Especially, HSF1, IRF8, IRF3, IRF2 and IRF1 are the top 5 enriched transcription factors (Fig. 4M). Of note, previous studies have shown that HSF1, as a stress-induced and DNA-binding transcription factor, could participate in regulating HIV-1 transcriptional activation and latent escape [53, 54], and the IRF family members as interferon regulatory factors could play vital regulatory roles in the upstream of antiviral immunity and inflammation [55, 56]. Additionally, our results demonstrated that many disordered genes in ciliated cells for cleaning up mucus, were mainly enriched in these biological processes such as protein translation, transcription initiation, microtubule movement, cilia assembly and cilia structure (Fig. 4O), implying that the structure and movement of the cilia might be affected. Among them, some cilia production-related regulators (e.g. FOXJ1, RFX2, and RFX3) and genes related to cilia structure, cilia movement and microtubule bundle movement (e.g. DRC1, TEKT1, ARMC4, LRRC6, RSPH9) are significantly disordered after infection (Fig. 4P) [57–62]. Especially, ATP synthesis-related genes (e.g. ATP5F1A, ATP5F1B, ATP5F1C, ATP5F1D, ATP5F1E, ATP5PO, ATP5PF) are significantly increased in COVID-19 patients (Fig. 4P). Remarkably, under the stimulation with high levels of extracellular ATP, the frequency of cilia beating will be destroyed and the secretion of airway mucin can increase thousands of times [57, 63]. These results indicated that SARS-CoV-2 may severely affect the functional homeostasis of ciliated cells, thereby resulting in the impaired function for cleaning mucus. Taken together, our study revealed that the dysregulation of receptors and ligands causes lung inflammation and results in the expression dysregulation of many mucus secretion- and cleaning-related genes to further induce the increase of mucus production in club cells and the functional defect of cleaning mucus, thereby causing COVID-19 patient's respiratory failure.
2.5 Single cell transcriptome and communication analyses in the blood uncover the roles of receptors and ligands in immune responses and cell migration
Both integrating signals from different pathways and cell migrations are indispensable for diverse physiological processes such as cell survival, cell development and immune response as well as wound healing [64–66]. Especially, as a multifunctional cell type, immune cells can not only act as sensors to receive external signals and instruct cell migration as well as connect to distant tissues and organs for combating pathogens or viruses together, but also serve as regulators to control immune response and tolerance as well as homeostasis [64–66]. This implies that the pulmonary microenvironment and immune response disturbances caused by SARS-CoV-2 infection may be also associated with the disorder of other tissues and organs. To answer this issue, we herein further detected the single-cell atlas of blood from a few days before two COVID-19 patients died.
Based on the single-cell atlas of the blood of COVID-19 patients, we identified multiple kinds of myeloid cells including Neu (CSF3R+), CD14 monocyte (CD14+), CD16 monocyte (FCGR3A+), dendritic cells (DC) (CD1C+), plasmacytoid dendritic cells (pDC) (CLEC4C+) and mast cells (CPA3+) (Fig. 5A ~ 5B), and numerous immune cells including B cell (MS4A1+), plasma cell (MZB1+), CD4 T (CD3D+, IL7R+), CD8 T (CD3D+, CD8A+) and NK (Fig. 5A ~ 5B), as well as other cells such as granulocyte-macrophage progenitor (GMP) (ELANE+), megakaryocytes (Meg) (PF4+) and erythroid cells (Ery) (HBA1+) (Fig. 5A ~ 5B). Of note, the significantly increased cell type in the blood of COVID-19 patients is Neu cells (Fig. 5C), but the significantly decreased cell types are T cells and NK cells (Fig. 5C), which are agreement with the multiple blood test results from the two COVID-19 patients (Fig. 5D ~ 5E). After admission, the ratio of their Neu cells was significantly higher than the normal value (Fig. 5D), but the lymphocyte ratio was significantly lower (Fig. 5E). Remarkably, compared with the blood, the lymphocyte cells (e.g. T cells and NK cells) is obviously increased in the lungs after SARS-CoV-2 infection (Fig. 2C). The reason might be that SARS-CoV-2 infection prompts immune cells of the blood to migrate to the lungs and causes the enrichment of many certain immune cells in the lungs. Moreover, we found that the two COVID-19 patient's monocytes presented an abnormally disordered state (Fig. 5F), in particular both Neu and Mono cells in the blood of COVID-19 patients demonstrated a higher inflammatory state with a significantly increased inflammatory score (Fig. 5G ~ 5H). Besides, we also found that multiple ligand and receptor genes as well as inflammation-related genes, such as C5AR1, CD55, CSF3R, CXCL8, FPR1, TLR1, HIF1A, IFNAR1A, IRF1 and NFKB1A, are also significantly up-regulated in the immune cells of the blood of COVID-19 patients (Fig. 5I), which are very similar to the lungs (Fig. 3D ~ 3E). These results indicated that the dysfunctions of some receptor and ligand as well as inflammation-related genes caused an excessive inflammation in the blood of patients after SARS-CoV-2 infection, and facilitated immune cells of the blood to migrate to the lungs.
Remarkably, ACE2 and TMPRSS2 have been demonstrated to be extremely important for SARS-CoV-2 to enter cells . To our surprise, we could hardly detect the expressions of ACE2 and TMPRSS2 in the blood of COVID-19 patients (Fig S3A ~ S3B). So why and how do immune responses and inflammation in the blood of COVID-19 patients be caused without the expressions of ACE2 and TMPRSS2? In this work, we further explored the issue. Interestingly, we found the highly expressed genes C5AR1 and FPR1 in myeloid cells of the patient blood (Fig. 5I), which have been proved to are cell communication molecules that can transmit inflammatory signals [37, 38, 68, 69]. The previous study urges us to investigate further which signal molecules could cause inflammation of the blood. We thus detailedly analyzed these significantly changed ligands and receptors in the blood after SARS-CoV-2 infection. As shown in Fig S3C, these significantly altered ligand and receptor genes in the blood are mainly enriched in the migration, proliferation and cell adhesion functions of leukocytes and myeloid cells, suggesting that these ligand and receptor genes could be responsible for inflammation and cell migrations. Especially, we found that some significantly changed ligand and receptor genes in the blood such as ALOX5_ALOX5AP, ANXA1_FPR1, ANXA1_FPR2, C5AR1_RPS19, CCL5_CCR1 and CD74_MIF (Fig. 5J), which are similar to that in lung (Fig. 3). Of note, many significantly changed ligand and receptor genes in blood have been demonstrated to be inflammation and cell migration. For example, arachidonate 5-lipoxygenase (ALOX5) and arachidonic acid 5-lipoxygenase activating protein (ALOX5AP) are key enzymes and membrane proteins for the synthesis of pro-inflammatory products leukotrienes that have been proven to be related to the respiratory system and the heart . Patients with sickle cell disease have increased inflammation and circulating leukotrienes and a high incidence of hyperresponsiveness, which is related to the increase in ALOX5AP induced by the up-regulated placental growth factor PlGF . In addition, ALOX5AP is highly upregulated in circulating monocytes in patients with severe sepsis and can predict the clinical prognosis of patients . Besides, CXCLs released by astrocytes and neurons could induce inflammatory neutrophils to enter the blood-brain barrier to increase encephalitis after herpes simplex virus infection . The lungs could recruit a large number of inflammatory neutrophils in the blood transfusion-related Balb/c mouse acute lung injury model, as well as combinating the selectin ligand PSGL-1 and CXCR2 could promote the vasculature migration of neutrophils and combine with activated platelets to cause pathogenic inflammation . Especially lack of PSGL-1 receptor or inhibition of PSGL-1 can prevent liver damage during endotoxemia . Additionally, studies have revealed that the up-regulated C5AR1 could also lead to excessive inflammation and coagulation complement system disorders , as well as the CD74 could be involved in MIF-mediated inflammation via interacting with MIF on the membrane surface of alveolar macrophages to activate the p44/p43 MAPK signaling pathway and induce the accumulation of neutrophils in the alveolar cavity .
Taken together, our study seemed to imply that certain receptor and ligand genes in the blood of patients can act as sensors and cell communication molecules to respond to inflammatory signals from the lung, subsequently induce immune responses and promote immune cells (e.g. T cells) to migrate to the lungs for fighting on SARS-CoV-2 together. On the one hand, thereby their dysregulations further lead to an increase in inflammatory cells and a decrease in immune cells (e.g. lymphocytes) in the blood. On the other hand, they can serve as regulators and inflammation-related genes to participate in the regulations of immune response, tolerance and homeostasis, thus their dysfunctions result in an excessive inflammation in the blood of COVID-19 patients.
2.6 The decrease of lymphocytes in bone marrow is responsible for immune clearance failure in the blood and lungs
Our above works have demonstrated that SARS-CoV-2 infection caused the decrease of lymphocytes (especially T cells) in the blood, which is agreement with previous studies [77, 78]. In fact, studies have also revealed a significant reduction of lymphocytes of patient's blood after coronaviruses SARS-CoV or MERS-CoV infections [79, 80]. On the contrary, lymphocytes are significantly increased in the blood after other virus infections (e.g. Influenza virus, Epstein-Barr virus, Cytomegalovirus, Measles virus) [81–83]. The reasons for the decrease of lymphocytes in the blood after SARS-CoV-2 infection have be suggested to be the circulating migration to the peripheral target organs for destroying SARS-CoV-2 or be due to apoptosis and autophagy lead to lymphocyte failure . Another reason is thought to be impairment of the bone marrow's function to produce lymphocytes, thereby promoting the decrease of lymphocytes in the blood of patients after SARS-COV-2 infection . To further reveal whether the bone marrow is involved in response to SARS-CoV-2 infection and is responsible for the decline of lymphocytes, we here systematically analyzed the single cell data of the bone marrow from the two COVID-19 patients.
Herein, we identified common progenitor cells and stem cells in bone marrow including erythroid progenitor cells (Eryp) (GYPA+), Ery (HBA1+), GMP (CTSG+), hematopoietic stem cells (HSC) (MYB+), mesenchymal stem cells (MSC) (CD34+) and so on, and many myeloid cells such as Neu (CSF3R+), monocyte-derived dendritic cells (MDCs) (CST3+), Mono (LYZ+), pDC (CLEC4C+) and so on, as well as T cells (CD3D+), NK cells (GNLY+), B cells (CD79A+), plasma cells (MZB1+) and other immune cells (Fig. 6A ~ 6B). Furthermore, we identified Meg (PLEK+) and a small number of End cells (PDK4+) (Fig. 6A ~ 6B). Of note, among all these cell types, the increased proportion of Neu, GMP and Plasma cells is highest after SARS-CoV-2 infection (Fig. 6C). Remarkably, the proportion of Neu in bone marrow of COVID-19 patients is also increased significantly (Fig. 6C), which is similar with the blood and lungs (Fig. 5C and Fig. 2C), whilst the proportion of T cells and Mono in the bone marrow of COVID-19 patients was significantly reduced (Fig. 6C), which is also similar with the blood (Fig. 5C). These results indicate that the bone marrow does respond to SARS-CoV-2 infection.
We then evaluated the inflammatory scores of Neu and Mono cells in the bone marrow, finding that the inflammatory scores COVID-19 patients are significantly higher than those of normal people (Fig. 6D ~ 6E). Particularly, inflammation genes such as C5AR1, CD55, CSF3R, CXCL8, FPR1, KLF6 and NFKB1A were significantly up-regulated in Neu and Mono cells of the bone marrow of COVID-19 patients (Fig. 6F). It is worth noting that the inflammatory scores in the bone marrow area are not as strong as those in the lungs and blood because the number of inflammatory genes in the bone marrow are obvious lower that the lungs and blood in the COVID-19 patients (Fig. 3, Fig. 5 and Fig. 6). We further found that the bone marrow of COVID-19 patients almost detected the expressions of ACE2 and TMPRSS2 (Fig S4A ~ S4B), suggesting that the inflammatory response of the bone marrow myeloid cells might be caused by signal transmission between distinct cells or tissues, rather than SARS-CoV-2 directly infect the bone marrow . Interestingly, we performed the functional enrichment analysis of ligands and receptors and found that many signaling molecules of the bone marrow cells are widely enriched in the leukocyte and myeloid cell migration, activation, proliferation and chemotaxis (Fig S4C). Therefore, we next focused on the significantly changed ligands and receptors in the bone marrow of COVID-19 patients. We surprisingly found that ALOX5_ALOX5AP, ANXA1_FPR1, CCL5_RPS19, CCL5_CCR1, CD74_COPA, etc. (Fig. 6G) also exist in both the lung and blood (Fig. 5 and Fig. 3), implying that the excessive inflammatory response of myeloid cells in three different tissues and the recruitment of T cells are mediated by a set of similar signaling molecules.
Interestingly, many studies have urged that immune cells from bone marrow and lymph can migrate to peripheral organs to perform a variety of specific functions. For example, the large number of neutrophils could be quickly infiltrated the heart to promote tissue healing and damage in the mouse model of cardiac infarction . Remarkably, the up-regulated expressed CXCL9, CXL10 and CXCL1 can respectively function as CXCR3 ligands on T cells to ensure CD8 + T cells migration and location to the specifically peripheral organs or tissues to further kill virus-containing cells . Especially, CXCR3 can act as a lymph node memory molecule to induce migration of CD8 + T cells to the infected peripheral tissues during the secondary viral infection , as well as the up-regulation expression of CXCR3 allows T cells with circulating effectors to home to inflamed lymph nodes . Additionally, the high expressions of CXCL10 and CCL2 were observed in SARS-CoV infected patients, especially their up-regulation can inhibit the development of hematopoietic precursor cells and cause lymphopenia in patients infected with SASR-CoV [88–91]. Of note, our present results revealed that the multiple members of CXCLs and CXCRs families are also obviously up-regulated in the lungs, blood and bone marrow of COVID-19 patients. Together, our study seemed to indicate that after the lungs was infected by SARS-CoV-2, ligands and receptors of the blood and bone marrow tissues quickly respond to the inflammatory signaling from the lung and promote the activation of immune cells and migrate them to the lungs for fighting SARS-CoV-2. However, the abnormal expressions of many ligands and receptors further disrupt the normal movement and circulation of T cells among the lungs, blood and bone marrow tissues, thereby causing the increase of T cells and inflammatory storm in the lung, while the decrease of T cells in the blood and bone marrow of COVID-19 patients.
In this work, we next further explored the effects of apoptosis, autophagy and cell proliferation on changes of T cells in the bone marrow, blood and lungs of COVID-19 patients. We firstly demonstrated that after SARS-CoV-2 infection, the apoptosis scores of T cells were significantly increased in the bone marrow, blood and lungs of COVID-19 patients (Fig. 6H). In particular, T cells in the lungs have the highest apoptosis score (Fig. 6H), suggesting that the high viral load might exacerbate the apoptosis of T cells in the lungs. Secondly, we found that the proliferation scores of bone marrow and lungs significant increase after SARS-CoV-2 infection, but the blood is obviously decreased (Fig. 6I). Remarkably, the lung cell proliferation score and apoptosis score are significant increasing at the same time, implying that the high virus environment in the lungs not only induces the continuous proliferation of T cells, but continuously promotes the apoptosis T cells, which seems to be caused by continuous struggle between T cells and SARS-CoV-2 in the lungs. Thirdly, we discovered that the autophagy score of bone marrow is significantly increased after SARS-CoV-2 infection and is also the highest in COVID-19 patients, but the autophagy scores of both blood and lungs are significantly reduced (Fig. 6J). Of note, we observed changes in the order of T cell depletion in different tissues in the normal and COVID-19 groups suggesting SARS-CoV-2 infection reshaped the distribution pattern of T cell apoptosis, proliferation and autophagy in different tissues. For example, T cells in the bone marrow had the lowest apoptosis score in the healthy group, but the blood T cells in the infected group (Fig. 6H). Overall, our work revealed that the depletion patterns of T cells from three different tissues are different, i.e. the lungs are mainly through the pattern of increasing apoptosis, the blood is mainly through the patterns of increasing apoptosis and decreasing proliferation, as well as the bone marrow is mainly through the pattern of increasing apoptosis and autophagy.