A Novel Single Cell RNA-seq Analysis of Non-Myeloid Circulating Cells in Late Sepsis

Abstract

BackgroundWith the successful implementation of the Surviving Sepsis Campaign guidelines, post-sepsis in-hospital mortality to sepsis continues to decrease. Those who acutely survive surgical sepsis will either rapidly recover or develop a chronic critical illness (CCI). CCI is associated with adverse long-term outcomes and 1-year mortality. Although the pathobiology of CCI remains undefined, emerging evidence suggests a post-sepsis state of pathologic myeloid activation, inducing suboptimal lymphopoiesis and erythropoiesis, as well as downstream leukocyte dysfunction. Our goal was to use single-cell RNA sequencing (scRNA-seq) to perform a detailed transcriptomic analysis of lymphoid-derived leukocytes to better understand the pathology of late sepsis.MethodsA mixture of whole blood myeloid-enriched and Ficoll-enriched peripheral blood mononuclear cells from four late septic patients (post-sepsis day 14-21) and five healthy subjects underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq).ResultsWe identified unique transcriptomic patterns for multiple circulating immune cell subtypes, including B- and CD4+, CD8+, activated CD4+ and activated CD8+ T-lymphocytes, as well as natural killer (NK), NKT, and plasmacytoid dendritic cells in late sepsis patients. Analysis demonstrated that the circulating lymphoid cells maintained a transcriptome reflecting immunosuppression and low-grade inflammation. We also identified transcriptomic differences between patients with bacterial versus fungal sepsis, such as greater expression of cytotoxic genes among CD8+ T-lymphocytes in late bacterial sepsis.ConclusionCirculating non-myeloid cells display a unique transcriptomic pattern late after sepsis. Non-myeloid leukocytes in particular reveal a host endotype of inflammation, immunosuppression, and dysfunction, suggesting a role for precision medicine-guided immunomodulatory therapy.

Systemic inflammation impairs human myelopoiesis and interferon I responses

“…It has been reported that mice deficient for either IFNAR1 or IFNβ generate significantly less mature monocytes with reduced expression of ISG 29,30 , suggesting a potential causal link between diminished IFN-I response and impaired myelopoiesis. In accordance, we observed a similar significant loss of non-classical monocytes in late-stage sepsis patients 21 , further substantiating the clinical relevance of the LPS-SI model to other SI-related pathophysiological conditions such as sepsis. Most importantly, our functional experiments revealed that IFNβ reverses the immunosuppressed state of LPS-treated monocytes.…”

“…f , IFN-I activity over the inferred monocyte differentiation trajectory from HSCs to non-classical monocytes, colored based on time point. g , UMAP of blood monocytes from a late (>14 days) sepsis patient cohort (left panel 21 ), along with cell density calculated for each health status (healthy volunteer or sepsis patient, right panel). h , log2(relative abundance) of cell types compared between late sepsis patients and healthy controls.…”

“…6e-f). To investigate the similarity between sustained effects of LPS-SI and the late, hyporesponsive phase of sepsis, we compared our late BM data to those of a recently published dataset of blood samples from sepsis patients >14 days into the disease 21 . All patients in the cohort had prolonged ICU stays and unresolved organ dysfunction.…”

“… a , UMAP representation of blood monocytes obtained from late sepsis patients 21 , colored based on cell type. b , UMAP of late sepsis blood monocytes separated by health condition (healthy or late sepsis) and colored based on cell type.…”

Transcriptional switch of hepatocytes initiates macrophage recruitment and T-cell suppression in endotoxemia

“…S3E-G). 28,29 Co-expression of inhibitory receptors can substantially impair T lymphocyte function, such as the production of IL2, IL4, and TNF (Fig. 3I), and induce a reduction in cell number (Fig.…”