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

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.

Role of Mcpip1 in obesity-induced hepatic steatosis as determined by myeloid and liver-specific conditional knockouts

Monocyte chemoattractant protein-induced protein 1 (MCPIP1, alias Regnase1) is a negative regulator of inflammation, acting through cleavage of transcripts coding for proinflammatory cytokines and by inhibition of NFκB activity. Moreover, it was demonstrated, that MCPIP1 regulates lipid metabolism both in adipose tissue and hepatocytes. In this study, we investigated the effects of tissue-specific Mcpip1 deletion on the regulation of hepatic metabolism and development of non-alcoholic fatty liver disease (NAFLD).We used knock-in control Mcpip1fl/fl mice and animals with deletion of Mcpip1 in myeloid leukocytes (Mcpip1fl/flLysMCre) and in hepatocytes (Mcpip1fl/flAlbCre), which were fed chow or a high-fat diet (HFD) for 12 weeks. Mcpip1fl/flLysMCre mice were fed a chow diet were characterized by a significantly reduced hepatic expression of genes regulating lipid and glucose metabolism, which subsequently resulted in hypoglycemia and dyslipidemia. These animals also displayed systemic inflammation, demonstrated by increased concentrations of cytokines in the plasma. On the other hand, there were no significant changes in phenotype in Mcpip1fl/flAlbCre mice. Although we detected a reduced hepatic expression of genes regulating glucose metabolism and β-oxidation in these mice, they remained asymptomatic. Upon feeding them a HFD, Mcpip1fl/flLysMCre mice did not develop obesity, glucose intolerance, nor hepatic steatosis, but were characterized by hypoglycemia and dyslipidemia, along with proinflammatory phenotype with symptoms of cachexia. Mcpip1fl/flAlbCre animals, following a HFD, became hypercholesterolemic, but accumulated lipids in the liver at the same level as Mcpip1fl/fl mice, and no changes in the level of soluble factors tested in the plasma were detected.In conclusion, we have demonstrated that Mcpip1 protein plays an important role in the liver homeostasis. Depletion of Mcpip1 in myeloid leukocytes, followed by systemic inflammation, has a more pronounced effect on controlling liver metabolism and homeostasis than the depletion of Mcpip1 in hepatocytes.

Investigating the tumor-immune microenvironment in an autochthonous murine model of cholangiocarcinoma.

53 Background: Cholangiocarcinoma (CCA) is the most common liver malignancy within the biliary tree with increasing incidence and poor survival. Although surgical resection can be curative, prognosis remains abysmal with high rates of unresectability and recurrence, and new systemic therapies are needed. CCA tumors are characterized by an abundant inflammatory immune cell infiltrate, yet little is known about the immune dynamics underlying the disease. Here, we present a preclinical murine model of CCA for identifying potential targets susceptible to immune-based therapies. Methods: Mice with targeted activation of KrasG12D and loss of p53 (Kras-p53-/-) in the liver were exposed to 5-Diethoxycarbonyl-1.4-dihydrocollidine for 4 weeks to induce spontaneous CCA tumorigenesis. Immunohistochemistry staining was performed for mouse and human CCA. Immune compartments (bone marrow, spleen, peripheral blood, tumor draining and non-draining lymph nodes, and CCA tumor) of mice were processed for CyTOF analysis. In vitro assays of isolated myeloid leukocytes were performed to demonstrate function. Results: Tumor associated neutrophils (TANs) and tumor associated macrophages (TAMs) were present in high levels in human CCA tumors and the murine model. CyTOF analysis demonstrated mobilization of TANs, TAMs, and CCR2+ inflammatory monocytes into the tumor. The checkpoint marker PD-1 was upregulated on CD8+ and CD4+ T cells and associated with PD-L1+ TANs, TAMs, monocytes, and dendritic cells. The immunosuppressive tumor microenvironment was also characterized by TAMs expressing IL-10, TGFβ, and arginase 1; regulatory B cells expressing IL-10 and TGFβ; and Gata3+ TH2 cells. Isolated myeloid leukocytes suppressed T cell proliferation and induced invasive migration. Conclusions: Kras-p53-/- mice developed CCA tumors histologically similar to human disease: the data portray immunosuppressive cells in the tumor microenvironment, a possible obstacle for an effective anti-tumor response. Understanding the immune dynamics of immunosuppressive populations within the tumor microenvironment will be key to mount a potent therapeutic response for durable remission and improved survival.

A population of innate myelolymphoblastoid effector cell expanded by inactivation of mTOR complex 1 in mice

Adaptive autoimmunity is restrained by controlling population sizes and pathogenicity of harmful clones, while innate destruction is controlled at effector phase. We report here that deletion of Rptor in mouse hematopoietic stem/progenitor cells causes self-destructive innate immunity by massively increasing the population of previously uncharacterized innate myelolymphoblastoid effector cells (IMLECs). Mouse IMLECs are CD3-B220-NK1.1-Ter119- CD11clow/-CD115-F4/80low/-Gr-1- CD11b+, but surprisingly express high levels of PD-L1. Although they morphologically resemble lymphocytes and actively produce transcripts from Immunoglobulin loci, IMLECs have non-rearranged Ig loci, are phenotypically distinguishable from all known lymphocytes, and have a gene signature that bridges lymphoid and myeloid leukocytes. Rptor deletion unleashes differentiation of IMLECs from common myeloid progenitor cells by reducing expression of Myb. Importantly, IMLECs broadly overexpress pattern-recognition receptors and their expansion causes systemic inflammation in response to Toll-like receptor ligands in mice. Our data unveil a novel leukocyte population and an unrecognized role of Raptor/mTORC1 in innate immune tolerance.

Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin

ABSTRACT Pertussis (whooping cough), caused by Bordetella pertussis, is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species.