key: cord-0692219-44adrtg3
authors: Wang, Teng; Zhang, Xianglong; Liu, Zhanguo; Yao, Tong; Zheng, Dongying; Gan, Jianwei; Yu, Shuang; Li, Lin; Chen, Peng; Sun, Jian
title: Single-cell RNA sequencing reveals the sustained immune cell dysfunction in the pathogenesis of sepsis secondary to bacterial pneumonia
date: 2021-03-07
journal: Genomics
DOI: 10.1016/j.ygeno.2021.01.026
sha: 6a7179886718468e1b2f72ad0ba44806637dbeac
doc_id: 692219
cord_uid: 44adrtg3

Sepsis is a leading cause of mortality in intensive care unit worldwide, it's accompanied by immune cell dysfunction induced by multiple factors. However, little is known about the specific alterations in immune cells in the dynamic pathogenesis of sepsis secondary to bacterial pneumonia. Here, we used single cell RNA sequencing (scRNA-seq) to profile peripheral blood mononuclear cells (PBMCs) in a healthy control and two patients with sepsis secondary to bacterial pneumonia, including acute, stable and recovery stage. We analyzed the quantity and function of immune cells. During disease course, interferon gamma response was upregulated; T/NK cell subtypes presented activation and exhaustion properties, which might be driven by monocytes through IL-1β signaling pathways; The proportion of plasma cells was increased, which might be driven by NK cells through IFN signaling pathways; Additionally, interferon gamma response was upregulated in sepsis secondary to pneumonia induced by SARS-COV-2 compared with that induced by influenza virus and bacteria.

IFNGR1, IFITM2, IFITM3, IFI6, ISG15 and ISG20 were increased in disease progression ( Fig.2D) , suggesting sustained effects of interferon response in the disease course. In addition, TNFA signaling via NFκB was also upregulated in the acute and recovery stages compared to healthy controls ( Fig.2A, C) . Patients in recovery stage also showed upregulated TNFA signaling via NFκB compared with stable stage. Genes involved in this pathway such as NFκBIA, JUNB, TNFAIP3, CCL4 and SOCS3 were highly expressed in disease stages compared with healthy controls (Fig.2D ). To explore whether these pathway alterations among disease course and healthy controls were driven by certain cell subtypes, we further expanded pathway enrichment analysis in all immune cell subpopulations. Surprisingly, interferon gamma response was upregulated in all disease stages in T/NK cells (Fig.2E, FigS.1D All of these data indicated the global alterations of signaling pathways in sepsis secondary to pneumonia was possibly associated with T/NK cells and myeloid cells.

Next, we explored the dynamically transcriptional alterations in gene expression during disease progression. Eight clusters with different time-dependent expression patterns were found, the biological functions of genes in these clusters were also assessed (Fig.2G ). Cluster1 comprised 2,989 genes with declined expression along disease course. The function of genes in cluster1 were enriched in autophagy (Fig.2G ). Previous studies demonstrated that the repair of autophagy lead to the dysfunction of proximal tubular in sepsis [16] , indicating the dynamic gene expression in cluster1 may be associated with kidney injury in sepsis. Cluster 6 contained 2,283 genes with sustained increased expression during disease stages. These genes were enriched in regulation of type I interferon production (Fig.2G) , which was consistent with the sustained upregulation of the interferon response genes in disease stages (Fig.2D ).

including PRDM1 and LAG3 were upregulated during disease progression in all T and NK cell subtype expect for CD4 + CD28 + T cells (Fig.3D ).

We next investigated the immunological changes implicated in T and NK cell subtypes during the progression of sepsis secondary to bacterial pneumonia. For the perspective of cell composition, T and NK cells displayed a divergent landscape between disease conditions and healthy controls.

CD4 + Tn cells and CD8 + Tn cells were attenuated in sepsis patients, indicating the immunosuppression status of these cell subpopulations. Both of these two cell types showed a similar decreasing trend along disease progress, with initially declined in the acute stage, slightly increased in the stable stage and further decreased in the recovery stage (Fig.3E ). By contrast, the proportion of CD8 + Te cells and CD8 + Tem cells were risen in disease course, which agreed with their function in lymphocyte activation, indicating enhanced immune effector effects of them.

Similar ascension trend was observed in CD8 + Te cells and CD8 + Tem cells along disease stages, which increased in the acute stage, slightly dropped in the stable stage, and increased continually in the recovery stage (Fig.3E) . Additionally, the proportion of NK cells was increased in disease process compared to healthy controls. While the proportion of NK cells were comparable between healthy controls and the acute stage, it was gradually increased in the following two disease stages ( Fig.3E ).

To further explore the cellular and functional alterations in NK cells. We performed re-cluster again and divided NK cells into two clusters: NK1 and NK2 (Fig.3F ). NK1 highly expressed As myeloid cells showed a continuously increasing trend across three disease stages compared with healthy controls, we next investigated whether this phenotypic remodeling was existed with more meticulous scenario. Thus, we gained insights into the compositional alterations during disease progression. Unexpectedly, the proportion of myeloid cell subtypes was comparable in the acute and stable stages compared with healthy controls. A newly discovered cell type of monocytes named CD24 + Mono, was specifically emerged in the recovery stage, which functioned in regulated exocytosis, response to bacterium and defense response to other organisms ( Next, we sought to investigate the potential mechanisms underlying the proliferation of plasma cells in the acute and stable stage. Intriguingly, NK cells were observed to be enriched in B cell proliferation and leukocyte activation involved in immune response, as well as positive regulation of apoptotic process (Fig.3C) . Therefore, we tried to explore the potential regulation network between NK cells and plasma cells to dissect the effects of NK cells on plasma cells proliferation.

In the acute and stable stages, 

signaling pathways T cells presented both immunological activation and suppression status, varies subpopulations of monocytes were observed to regulate lymphocyte activation and involved in apoptosis pathways.

Additionally, it was shown that T cell function was suppressed in a monocyte dependent fashion in sepsis patients [18] . We next aimed to explore whether intracellular interaction exists between monocytes and T cells. Thus, we constructed a putative cellular interaction network between monocytes and T cells in different disease stages compared with healthy controls to investigate whether monocytes impact the activation and suppression of T cells.

For the purpose of exploring the roles of monocytes on T cell activation, we selected subsets of monocytes including CD14 + Mono2, CD16 + Mono, CD24 + Mono, HLA-DR + Mono and LDHB + Mono as sender cells because they were highly associated with lymphocyte activation (Fig.4C ).

Journal Pre-proof CD8 + Te cells and CD8 + Tem cells were chosen as receiver cells due to their sustained increasing proportion across disease stages versus healthy controls. NicheNet [19] was employed to predict interactions between selected monocytes and T cells based on differential expressed genes in CD8 + Te cells and CD8 + Tem cells upon disease induction. For CD8 + Te cells, ligand -target interactions were centered on the recovery stage, whereas were sparse in the acute stages (Fig.6A,   FigS .5A). Concurrently, the ligand-target interactions between selected monocytes and CD8 + Tem cells were also enriched in the recovery stage (Fig.6B) . Thus, we focused on the ligand-mediated intracellular interactions mainly in the recovery stage. Interestingly, among the top predicted ligands, we found IL1B was expressed by selected monocytes in regulation of both CD8 + Te cells and CD8 + Tem cells. Moreover, S100A9, JUN and S100A8 were the common target genes in CD8 + Te cells and CD8 + Tem cells potentially regulated by IL1B (Fig.6A, B) . Further analysis of inferring the potential signaling pathways between IL1B and its target genes discovered some transcriptional regulators including SPl1, RELA, FOS, EP300, STAT3, IRAK1 and MYC (Fig.6C ).

To investigate the regulatory relationship and potential mechanisms in monocytes on T cells suppression, we selected CD14 + Mono2 and CD16 + Mono as sender cells, due to their function in positive regulation of cell death, apoptotic signaling pathway, negative regulation of cell proliferation and negative regulation of immune system process (Fig.4C) . Additionally, CD4 + Tn cells and CD8 + Tn cells were served as the receiver cells due to their continuously declined proportion across disease course (Fig.3E ).

For CD4 + Tn cells, ligand-target interactions were concentrated in the acute and stable stages, but were not predicted in the recovery stage for no differential expression genes were found between recovery stage and healthy controls (Fig.6D, FigS.5B ). Further analysis on CD8 + Tn cells showed that ligand-target interactions were enriched in all disease stages, suggesting the selected monocytes may exert their regulatory functions on CD8 + Tn cells during disease progression (Fig.6E, FigS.5C, D) . To dissect the common mechanisms of regulatory relationships underlying the suppression of CD4 + Tn cells and CD8 + Tn cells induced by monocytes, we looked for the shared ligands and their target genes. Surprisingly, IL1B was also expressed by selected monocytes in regulating both CD4 + Tn cells and CD8 + Tn cells (Fig.6D, E) . Concurrently, PRDM1

and ITGB1 were among the commonly predicted target genes in CD4 + Tn cells and CD8 + Tn cells driven by IL1B (Fig.6D, E) . Of particular interest, PRDM1 was found to be upregulated in T cell subtypes across disease stages, supporting that monocytes may promote the exhaustion of both CD4 + Tn cells and CD8 + Tn cells with the target of PRDM1. Furthermore, some transcription regulators such as SPl1, IRAK1, STAT3, RELA, SMAD3 and PRTN3 were observed to be suggested that the interferon gamma response may be aberrant with highest degree in SARS-COV-2 induced sepsis, secondly in influenza virus induced sepsis and last in bacterial sepsis.

Next, we attempted to explore the cellular immunological difference behind viral sepsis and bacterial sepsis. For the purpose of an unbiased comparison, the cell subtypes of these downloaded scRNA-seq datasets were annotated according to the acute stage of sepsis secondary to bacterial pneumonia in our study with SciBet [22] . As a result, all of the cell subpopulations were annotated in these downloaded scRNA-seq datasets (Fig.7B, C) . The proportion of T/NK cells was lower in both SARS-COV-2 induced sepsis and influenza virus induced sepsis compared with bacterial sepsis (Fig.7B) . GO analysis showed that defense response to other organism; response to bacterium and apoptotic signaling pathway were enriched when differentially expressed genes Furthermore, interferon-gamma-mediated signaling pathway and type I interferon signaling pathway were upregulated when differentially expressed genes were compared with influenza virus induced sepsis and bacterial sepsis (TableS4). We then focused on the immunological difference underlying cell subpopulations with a finer resolution. For innate immune response, the percentage of CD14 + Mono1 and NK cells especially NK1 cells was highest in SARS-COV-2 induced sepsis, secondly in bacterial sepsis, and last in influenza virus induced sepsis (Fig.7C) .

Moreover, the proportion of CD16 + Mono was higher in influenza virus induced sepsis compared with SARS-COV-2 induced sepsis and bacterial sepsis (Fig.7C ). However, with regard to adaptive immune response, the proportion of CD4 + Tn cells was highest in bacterial sepsis, secondly in SARS-COV-2 induced sepsis and last in influenza virus induced sepsis, whereas the percentage of Bm cells was highest in influenza virus induced sepsis, secondly in SARS-COV-2 induced sepsis J o u r n a l P r e -p r o o f Journal Pre-proof and last bacterial sepsis (Fig.7C) . Additionally, the proportion of CD4 + Tpm cells, CD8 + Tn cells, and CD8 + Tem cells were highest in bacterial sepsis, secondly in influenza virus induced sepsis and last in SARS-COV-2 induced sepsis (Fig.7C) . Conversely, the percentage of Bn cells were highest in SARS-COV-2 induced sepsis, secondly in influenza virus induced sepsis and last in bacterial sepsis (Fig.7C) . Furthermore, the proportion of CD8 + Te cells was lower in SARS-COV-2 induced sepsis compared with bacterial sepsis and influenza virus induced sepsis (Fig.7C) . [23, 24] . Thus, we (Fig.7E ).

It is established that sepsis altered both the innate and adaptive immune response for a long period after clinical "recovery" [5] , which prompted us to explore the detailed alterations of immune response and discover potential therapeutic treatment. However, global picture of immune cell dysfunction cannot be obtained with regard to all subpopulations using conventional bulk RNA sequencing (bulk RNA-seq). The emerging of scRNA-seq technology helps us to understand the cellular and molecular features with higher resolution and accuracy in sepsis-induced immune dysregulation [14, 15, 25] . However, considering the highly heterogeneous properties of sepsis regarding primary cause and infecting pathogens, their immune response may be divergent. A study has shown that intra-abdominal sepsis and pneumonia-derived sepsis presented different immune response, manifested by different number of immune cell subpopulations [7] . Thus, it is 

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The proportion immune cell subpopulations were changed during disease progression of sepsis

Interferon gamma response was upregulated in all PBMCs especially in T/NK cells

T/NK cell subtypes presented activation and exhaustion properties, which might be driven by monocytes through IL-1β signaling pathways

The proportion of plasma cells was increased, which might be induced by NK cells through IFN signaling pathways

interferon gamma response was the common upregulated pathway in sepsis secondary to pneumonia induced by SARS-COV-2, influenza virus and bacteria

The authors declare that they have no conflict of interest.