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In sepsis, a dysregulated immune response to infection can lead to immunosuppression and opportunistic infections. Understanding immune endotypes related to post-sepsis immunosuppression may lead to improved therapeutic approaches. NKT cells are master regulators of post-sepsis immunosuppression and can be leveraged to dissect endotypes in vivo. However, experimental studies can lack relevance to clinical disease. Here, we start with murine models and then apply cross-species comparative transcriptomics to better define clinical endotypes associated with post-sepsis immunosuppression and mortality. We use NKT-cell deficient (Cd1d-/-) to define the innate immune transcriptome regulated by NKT cells in post-endotoxemia immunosuppression. We then compare these murine findings to the transcriptomic analysis of peripheral blood mononuclear cells from septic shock patients at single-cell resolution. Three genes regulated by NKT cells in late endotoxemia–FPR1, FPR2, and MARCO--have increased expression across monocyte subpopulations in clinical septic shock. We previously emphasized the differences between the two monocyte states (MS1, MS3) expanded in sepsis; here, we identify a common signature shared by MS1 and MS3. In two independent clinical sepsis cohorts, FPR1 and FPR2 closely associate with Sepsis Response Signature 1 (SRS1), a previously defined transcriptional endotype predictive of immunosuppression and mortality. FPRs typically augment the antimicrobial response, but in sepsis these genes associate with immunosuppression in sepsis. (Note: to minimize revision mouse experiments, we are omitting mouse data on FPR1/2 blockade but could use for revisions). In summary, we use cross species transcriptomics to identify NKT cell-regulated genes that are enriched across the monocyte subpopulations of clinical septic shock and associate with an immunosuppressed endotype. This study illustrates how comparative transcriptomics can address the complementary limitations of experimental and clinical sepsis.

Background: Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterized by the loss of tolerance against self-antigens. Recent efforts have focused on the development of treatments towards the restoration of self–tolerance; in this context, tolerogenic dendritic cells (tolDCs) have become a target of interest due its ability to polarize the immune response towards an anti-inflammatory profile of the immune system. Although the genetic contribution to SLE has proven to be important there is still scarce information underlying the involved genes and mechanisms. Allele specific expression (ASE) is a gene regulation mechanism characterized by the overexpression of one allele, which can be associated with physiopathological processes and diseases. Objectives: The aim of this study is to characterize the ASE during tolerance induction on dendritic cells from Mexican women with SLE. Methods: Potential participants have been identified using the Mexican Registry of Lupus database (LupusRGMX). Blood and oral exudate samples were collected. From the blood sample peripheral blood mononuclear cells (PBMCs) were obtained using Lymphoprep gradient; from these PBMCs monocytes were isolated using a magnetic beads cell-enrichment kit. Cells were cultured in RPMI-1640 medium supplemented with GM-CSF and IL-4 for monocyte-derived dendritic cells (moDCs) and GM-CSF, IL-4 and IL-10 for tolerogenic dendritic cells (tolDCs). At 7th day cells were additionally stimulated with imiquimod (IMQ) in order to observe its response. Flow cytometry analysis was performed to evaluate cell isolation/differentiation. RNA-seq will be performed from cell cultures and differential gene expression will be determined. Genotype will be determined from oral exudate using the Infinium Global Diversity Array-8 and gene imputation will be performed. Finally, ASE dynamics will be determined. Results: Standardization of cultures and flow cytometry analysis was performed with samples from four healthy individuals. Higher percentage of cells expressing CD14 and CD11c were observed on both dendritic cell cultures when compared to monocytes. Similar pattern was observed for the percentage of cells expressing markers associated with antigen presentation (MHCII, CD40 and CD80) in both SLE (n=15) and healthy (n=5) groups. DNA has been isolated; whereas samples for RNA isolation have been obtained. Conclusion: Changes in the expression of surface markers involved in antigen presentation have been observed after the differentiation protocols. Further RNA-seq and genotyping analyses will allow us to identify ASE patterns associated with the tolerogenic potential of dendritic cells from Mexican women with SLE.