Tumor-infiltrating CD8+ T cells (TILs) show progressive loss of effector function and upregulation of inhibitory receptors. NR4A transcription factors have emerged as key regulators of this dysfunctional state. Here we developed degron-based systems enabling rapid degradation of endogenous NR4A proteins in both mouse and primary human T cells. We demonstrate that the continuous presence of each NR4A protein is required to maintain suppression of effector cytokines and expression of co-inhibitory receptors; degradation of individual NR4A proteins rapidly restored these functional features, with each NR4A protein exerting prominent effects on distinct as well as overlapping subsets of genes and surface markers associated with effector, memory and exhaustion programs. Transcriptional profiling of phenotypically defined populations revealed both shared and unique gene programs across NR4A family members. Through CRISPR-mediated endogenous gene editing in primary human CD8+ T cells, we show that targeted degradation of NR4A proteins with a small molecule degrader can maintain cytokine expression and suppress inhibitory receptor expression in cells subjected to chronic stimulation, providing a framework for a powerful strategy for therapeutic intervention.

In this issue of Cell Chemical Biology, Lane et al.1 introduce a transgene-based system to express fusion proteins that recruit transcription factors to E3 ligases. This approach expands the target repertoire for engineered cell therapies as exemplified by the targeting of SMAD proteins to overcome a TGFβ-mediated suppressive mechanism that weakens anti-tumor responses.

Persistent inflammation contributes to the development of cardiovascular disease (CVD) as an HIV-associated comorbidity. Innate immune cells such as monocytes are major drivers of inflammation in men and women with HIV. The study objectives are to examine the contribution of circulating non-classical monocytes (NCM, CD14dimCD16+) and intermediate monocytes (IM, CD14+CD16+) to the host response to long-term HIV infection and HIV-associated CVD. Women with and without chronic HIV infection (H) were studied. Subclinical CVD (C) was detected as plaques imaged by B-mode carotid artery ultrasound. The study included H-C-, H+C-, H-C+, and H+C+ participants (23 of each, matched on race/ethnicity, age and smoking status), selected from among enrollees in the Women's Interagency HIV Study. We assessed transcriptomic features associated with HIV or CVD alone or comorbid HIV/CVD comparing to healthy (H-C-) participants in IM and NCM isolated from peripheral blood mononuclear cells. IM gene expression was little affected by HIV alone or CVD alone. In IM, coexisting HIV and CVD produced a measurable gene transcription signature, which was abolished by lipid-lowering treatment. In NCM, versus non-HIV controls, women with HIV had altered gene expression, irrespective of whether or not they had comorbid CVD. The largest set of differentially expressed genes was found in NCM among women with both HIV and CVD. Genes upregulated in association with HIV included several potential targets of drug therapies, including LAG3 (CD223). In conclusion, circulating monocytes from patients with well controlled HIV infection demonstrate an extensive gene expression signature which may be consistent with the ability of these cells to serve as potential viral reservoirs. Gene transcriptional changes in HIV patients were further magnified in the presence of subclinical CVD.

Background Cryopreserved peripheral blood mononuclear cells (PBMCs) are frequently collected and provide disease- and treatment-relevant data in clinical studies. Here, we developed combined protein (40 antibodies) and transcript single-cell (sc)RNA sequencing (scRNA-seq) in PBMCs. Results Among 31 participants in the Women's Interagency HIV Study (WIHS), we sequenced 41,611 cells. Using Boolean gating followed by Seurat UMAPs (tool for visualizing high-dimensional data) and Louvain clustering, we identified 50 subsets among CD4+ T, CD8+ T, B, NK cells, and monocytes. This resolution was superior to flow cytometry, mass cytometry, or scRNA-seq without antibodies. Combined protein and transcript scRNA-seq allowed for the assessment of disease-related changes in transcriptomes and cell type proportions. As a proof-of-concept, we showed such differences between healthy and matched individuals living with HIV with and without cardiovascular disease. Conclusions In conclusion, combined protein and transcript scRNA sequencing is a suitable and powerful method for clinical investigations using PBMCs.

Cystic fibrosis (CF) is an inherited life-threatening disease accompanied by repeated lung infections and multiorgan inflammation that affects tens of thousands of people worldwide. The causative gene, cystic fibrosis transmembrane conductance regulator (CFTR), is mutated in CF patients. CFTR functions in epithelial cells have traditionally been thought to cause the disease symptoms. Recent work has shown an additional defect: monocytes from CF patients show a deficiency in integrin activation and adhesion. Because monocytes play critical roles in controlling infections, defective monocyte function may contribute to CF progression. In this study, we demonstrate that monocytes from CFTRΔF508 mice (CF mice) show defective adhesion under flow. Transplanting CF mice with wild-type (WT) bone marrow after sublethal irradiation replaced most (60-80%) CF monocytes with WT monocytes, significantly improved survival, and reduced inflammation. WT/CF mixed bone marrow chimeras directly demonstrated defective CF monocyte recruitment to the bronchoalveolar lavage and the intestinal lamina propria in vivo. WT mice reconstituted with CF bone marrow also show lethality, suggesting that the CF defect in monocytes is not only necessary but also sufficient to cause disease. We also show that monocyte-specific knockout of CFTR retards weight gains and exacerbates dextran sulfate sodium-induced colitis. Our findings show that providing WT monocytes by bone marrow transfer rescues mortality in CF mice, suggesting that similar approaches may mitigate disease in CF patients.

Double-positive CD4+CD8αβ+ (DP) cells are thought to reside as T cell progenitors exclusively within the thymus. We recently discovered an unexpected CD4+ and CD8αβ+ immune cell population in healthy and atherosclerotic mice by single-cell RNA sequencing. Transcriptomically, these cells resembled thymic DPs. Flow cytometry and three-dimensional whole-mount imaging confirmed DPs in thymus, mediastinal adipose tissue, and aortic adventitia, but nowhere else. Deep transcriptional profiling revealed differences between DP cells isolated from the three locations. All DPs were dependent on RAG2 expression and the presence of the thymus. Mediastinal adipose tissue DPs resided in close vicinity to invariant NKT cells, which they could activate in vitro. Thymus transplantation failed to reconstitute extrathymic DPs, and frequencies of extrathymic DPs were unaltered by pharmacologic inhibition of S1P1, suggesting that their migration may be locally confined. Our results define two new, transcriptionally distinct subsets of extrathymic DPs that may play a role in aortic vascular homeostasis.

AIMS:During virally suppressed chronic HIV infection, persistent inflammation contributes to the development of cardiovascular disease (CVD), a major comorbidity in people living with HIV (LWH). Classical blood monocytes (CMs) remain activated during antiretroviral therapy and are a major source of pro-inflammatory and pro-thrombotic factors that contribute to atherosclerotic plaque development and instability. METHODS AND RESULTS:Here, we identify transcriptomic changes in circulating CMs in peripheral blood mononuclear cell samples from participants of the Women's Interagency HIV Study, selected by HIV and subclinical CVD (sCVD) status. We flow-sorted CM from participants of the Women's Interagency HIV Study and deep-sequenced their mRNA (n = 92). CMs of HIV+ participants showed elevated interleukin (IL)-6, IL-1β, and IL-12β, overlapping with many transcripts identified in sCVD+ participants. In sCVD+ participants LWH, those reporting statin use showed reduced pro-inflammatory gene expression to a level comparable with healthy (HIV-sCVD-) participants. Statin non-users maintained an elevated inflammatory profile and increased cytokine production. CONCLUSION:Statin therapy has been associated with a lower risk of cardiac events, such as myocardial infarction in the general population, but not in those LWH. Our data suggest that women LWH may benefit from statin therapy even in the absence of overt CVD.

Background In transplant medicine, clinical decision making largely relies on histology of biopsy specimens. However, histology suffers from low specificity, sensitivity, and reproducibility, leading to suboptimal stratification of patients. We developed a histology-independent immune framework of kidney graft homeostasis and rejection. Methods We applied tailored RNA deconvolution for leukocyte enumeration and coregulated gene network analysis to published bulk human kidney transplant RNA transcriptomes as input for unsupervised, high-dimensional phenotype clustering. We used framework-based graft survival analysis to identify a biomarker that was subsequently characterized in independent transplant biopsy specimens. Results We found seven immune phenotypes that confirm known rejection types and uncovered novel signatures. The molecular phenotypes allow for improved graft survival analysis compared with histology, and identify a high-risk group in nonrejecting transplants. Two fibrosis-related phenotypes with distinct immune features emerged with reduced graft survival. We identified lysyl oxidase-like 2 (LOXL2)-expressing peritubular CD681 macrophages as a framework-derived biomarker of impaired allograft function. These cells precede graft fibrosis, as demonstrated in longitudinal biopsy specimens, and may be clinically useful as a biomarker for early fibrogenesis. Conclusions This study provides a comprehensive, data-driven atlas of human kidney transplant phenotypes and demonstrates its utility to identify novel clinical biomarkers.

Background: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4(+)T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B-100(apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (T(H)1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4(+)T cells with an atheroprotective, regulatory T cell (T-reg) phenotype in healthy individuals. Yet, the function of apoB-reactive T(regs)and their relationship with pathogenic T(H)1 cells remain unknown. Methods: To interrogate the function of autoreactive CD4(+)T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B978-993(apoB(+)) at the single-cell level. Results: We found that apoB(+)T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a T-reg-like transcriptome, although only 21% of all apoB(+)T cells expressed the T(reg)transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB(+)T cells formed several clusters with mixed T(H)signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of T(H)1, T helper cell type 2 (T(H)2), and T helper cell type 17 (T(H)17), and of follicular-helper T cells. ApoB(+)T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic T(H)1/T(H)17-like cells with proinflammatory properties and only a residual T(reg)transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed T(H)1/T(H)17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB(+)T(regs)in lineage tracing of hyperlipidemicApoe(-/-)mice. In adoptive transfer experiments, converting apoB(+)T(regs)failed to protect from atherosclerosis. Conclusions: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive T(regs)as a novel cellular target in atherosclerosis.

In this study, we demonstrate that correcting the monocyte adhesion defect in CFTRΔF508 mice (CF mice) by bone marrow transplantation significantly improved survival and reduced inflammation. Abstract Cystic fibrosis (CF) is an inherited life-threatening disease accompanied by repeated lung infections and multi-organ inflammation that affects tens of thousands of people worldwide. The causative gene, cystic fibrosis transmembrane conductance regulator (CFTR), is mutated in CF patients. Monocytes from CF patients show a deficiency in integrin activation and adhesion. Since monocytes play critical roles in controlling infections, defective monocyte function may contribute to CF progression. In this study, we demonstrate that monocytes from CFTRΔF508 mice (CF mice) show defective adhesion under flow. Transplanting CF mice with wild-type bone marrow after sublethal irradiation replaced most (60-80%) CF monocytes with wild-type monocytes, significantly improved survival, and reduced inflammation. Wild-type/CF mixed bone marrow chimeras directly demonstrated defective CF monocyte recruitment to the bronchoalveolar lavage and the intestinal lamina propria in vivo. Our findings show that providing wild-type monocytes by bone marrow transfer rescues gastrointestinal (GI) mortality in CF mice, suggesting that wild-type bone marrow stem cells might mitigate CF inflammation.

La Jolla Institute of Immunology, 9420 Athena Circle Drive La Jolla, CA 92037, USA; Eberhard Karls University, Tuebingen University Hospital, Department of Cardiology, OtfriedMueller-Strasse 10, 72076 Tuebingen, Germany; Department of Epidemiology and Population Health, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Belfer 13th floor, Bronx, NY 10461, USA; John H. Stroger, Jr. Hospital of Cook County, 1969 Ogden Ave, Chicago, IL 60612, USA; Department of Epidemiology, Johns Hopkins University, 265 Garland Hall, 3400 North Charles Street, Baltimore, MD 21218, USA; Department of Medicine, University of Alabama at Birmingham, 908 20th Street South, Birmingham, AL 35294, USA; Department of Medicine, SUNY-Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203, USA; Georgetown University Medical Center, Washington, DC, USA; Department of Preventive Medicine, University of Southern California, 2001 N Soto St, Los Angeles, CA 90033, USA; Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, 49 Jesse Hill Jr. Drive, Atlanta, GA 30303, USA; Department of Medicine, VAMC, Infectious Disease Section, 111W 4150 Clement St., San Francisco, CA 94121, USA; Department of Internal Medicine, Rush University Medical Center, 1735 West Harrison St, Chicago, IL 60612, USA and Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

AIMS:To test whether human immunodeficiency virus (HIV) infection and subclinical cardiovascular disease (sCVD) are associated with expression of CXCR4 and other surface markers on classical, intermediate, and non-classical monocytes in women. METHODS AND RESULTS:sCVD was defined as presence of atherosclerotic lesions in the carotid artery in 92 participants of the Women's Interagency HIV Study (WIHS). Participants were stratified into four sets (n = 23 each) by HIV and sCVD status (HIV-/sCVD-, HIV-/sCVD+, HIV+/sCVD-, and HIV+/sCVD+) matched by age, race/ethnicity, and smoking status. Three subsets of monocytes were determined from archived peripheral blood mononuclear cells. Flow cytometry was used to count and phenotype surface markers. We tested for differences by HIV and sCVD status accounting for multiple comparisons. We found no differences in monocyte subset size among the four groups. Expression of seven surface markers differed significantly across the three monocyte subsets. CXCR4 expression [median fluorescence intensity (MFI)] in non-classical monocytes was highest among HIV-/CVD- [628, interquartile range (IQR) (295-1389)], followed by HIV+/CVD- [486, IQR (248-699)], HIV-/CVD+ (398, IQR (89-901)), and lowest in HIV+/CVD+ women [226, IQR (73-519)), P = 0.006 in ANOVA. After accounting for multiple comparison (Tukey) the difference between HIV-/CVD- vs. HIV+/CVD+ remained significant with P = 0.005 (HIV-/CVD- vs. HIV+/CVD- P = 0.04, HIV-/CVD- vs. HIV-/CVD+ P = 0.06, HIV+/CVD+ vs. HIV+/CVD- P = 0.88, HIV+/CVD+ vs. HIV-/CVD+ P = 0.81, HIV+/CVD- vs. HIV-/CVD+, P = 0.99). All pairwise comparisons with HIV-/CVD- were individually significant (P = 0.050 vs. HIV-/CVD+, P = 0.028 vs. HIV+/CVD-, P = 0.009 vs. HIV+/CVD+). CXCR4 expression on non-classical monocytes was significantly higher in CVD- (501.5, IQR (249.5-887.3)) vs. CVD+ (297, IQR (81.75-626.8) individuals (P = 0.028, n = 46 per group). CXCR4 expression on non-classical monocytes significantly correlated with cardiovascular and HIV-related risk factors including systolic blood pressure, platelet and T cell counts along with duration of antiretroviral therapy (P < 0.05). In regression analyses, adjusted for education level, study site, and injection drug use, presence of HIV infection and sCVD remained significantly associated with lower CXCR4 expression on non-classical monocytes (P = 0.003), but did not differ in classical or intermediate monocytes. CONCLUSION:CXCR4 expression in non-classical monocytes was significantly lower among women with both HIV infection and sCVD, suggesting a potential atheroprotective role of CXCR4 in non-classical monocytes.

Rationale: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood. Objective: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis. Methods and Results: Using single-cell RNA-sequencing of aortic leukocytes from chow diet– and Western diet–fed Apoe−/− and Ldlr−/− mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe−/− and Ldlr−/− mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. Conclusions: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type–specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.

Objective: Atherosclerosis is a major cause of cardiovascular disease. Monocyte-endothelial cell interactions are partly mediated by expression of monocyte CX3CR1 and endothelial cell fractalkine (CX3CL1). Interrupting the interaction between this ligand-receptor pair should reduce monocyte binding to the endothelial wall and reduce atherosclerosis. We sought to reduce atherosclerosis by preventing monocyte-endothelial cell interactions through use of a long-acting CX3CR1 agonist. Methods: In this study, the chemokine domain of CX3CL1 was fused to the mouse Fc region to generate a long-acting soluble form of CX3CL1 suitable for chronic studies. CX3CL1-Fc or saline was injected twice a week (30 mg/kg) for 4 months into Ldlr knockout (KO) mice on an atherogenic western diet. Results: CX3CL1-Fc-treated Ldlr KO mice showed decreased en face aortic lesion surface area and reduced aortic root lesion size with decreased necrotic core area. Flow cytometry analyses of CX3CL1-Fc-treated aortic wall cell digests revealed a decrease in M1-like polarized macrophages and T cells. Moreover, CX3CL1-Fc administration reduced diet-induced atherosclerosis after switching from an atherogenic to a normal chow diet. In vitro monocyte adhesion studies revealed that CX3CL1-Fc treatment caused fewer monocytes to adhere to a human umbilical vein endothelial cell monolayer. Furthermore, a dorsal window chamber model demonstrated that CX3CL1-Fc treatment decreased in vivo leukocyte adhesion and rolling in live capillaries after short-term ischemia-reperfusion. Conclusion: These results indicate that CX3CL1-Fc can inhibit monocyte/endothelial cell adhesion as well as reduce atherosclerosis. (C) 2018 The Authors. Published by Elsevier GmbH.

Purpose of review The immune system plays a critical role in the development and modulation of atherosclerosis. New highparameter technologies, including mass cytometry (CyTOF) and single-cell RNA sequencing (scRNAseq), allow for an encompassing analysis of immune cells. Unexplored marker combinations and transcriptomes can define new immune cell subsets and suggest their functions. Here, we review recent advances describing the immune cells in the artery wall of mice with and without atherosclerosis. We compare technologies and discuss limitations and advantages. Recent findings Both CyTOF and scRNAseq on leukocytes from digested aortae show 10-30 immune cell subsets. Myeloid, T, B and natural killer cells were confirmed. Although cellular functions can be inferred from RNA-Seq data, some subsets cannot be identified based on current knowledge, suggesting they may be new cell types. CyTOF and scRNAseq each identified four B-cell subsets and three macrophage subsets in the atherosclerotic aorta. Limitations include cell death caused by enzymatic digestion and the limited depth of the scRNAseq transcriptomes. Summary High-parameter methods are powerful tools for uncovering leukocyte diversity. CyTOF is currently more powerful at discerning leukocyte subsets in the atherosclerotic aorta, whereas scRNAseq provides more insight into their likely functions.

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however, unipotent neutrophil progenitors are not well defined. Here, we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly, we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models, including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases, including cancer.

During virally-suppressed chronic human immunodeficiency virus (HIV) infection, persistent inflammation contributes to the development of cardiovascular disease, a major comorbidity in people living with HIV. Monocytes play a key role in atherosclerotic plaque development, inflammation, and stability, but their contribution to the CVD under viral suppression in people living with HIV remains unknown. Here, we investigated the transcriptomes of classical (CD14 ++ CD16 - ) blood monocytes from 92 women with and without chronic HIV infection and subclinical cardiovascular disease (sCVD), defined as the presence of focal carotid artery plaque, from the Women's Interagency HIV Study (WIHS). Differential gene expression, based on four two-way comparisons among participant groups (HIV-sCVD-, HIV+sCVD-, HIV-sCVD+, and HIV+sCVD+, 23 subjects each), identified large pro-inflammatory gene signatures for both sCVD and virally-suppressed HIV. These findings were further corroborated by Ingenuity Pathway Analysis. We found that classical monocytes persistently express common CVD-related markers of inflammation, including IL6, IL1β, and IL12B; overlapping with many transcripts identified in sCVD+ participants. In comorbid disease (HIV+sCVD+), those reporting statin use showed dramatically reduced pro-coagulant tissue factor (F3) and tissue factor pathway inhibitor (TFPI and TFPI2) gene expression to a level comparable with healthy participants. Cytokine expression profiles associated with the tissue factor pathway were also modified in participants on statins, suggesting that statins may benefit women with chronic HIV infection by limiting pro-coagulation and inflammation pathways in classical monocytes.

Neutrophils are innate immune cells greatly contributing to the host defense against bacterial and fungal infection and to various sterile inflammatory conditions. To reach their site of action, circulating neutrophils initially roll, then arrest on the activated vessel wall and then migrate out from the vessel lumen. During rolling at high shear stress, neutrophil microvilli anchored to the endothelium by selectin bonds are pulled out behind the cell into several micron long, thin membrane tubes called tethers. As the anchoring bonds break, the tethers turn into slings and may land in front of the rolling cell and provide adhesive substrate for rolling. Here, we show that both tethers and slings can detach and form elongated neutrophil derived particles, which we call shear‐derived particles (SDP).Cremaster intravital microscopy experiments of Ly6G‐AF647 injected male mice showed the presence of SDPs with or without intrascrotal pretreatment with tumor necrosis factor (TNF), suggesting that in mice the cremaster preparation induced trauma is sufficient to induce SDP formation. In 27 vessels of 8 TNF‐pretreated mice, 234 SDPs were observed with median length of 6.5 μm (range 1.9–112 μm). SDPs appeared when rolling neutrophils left tethers behind, but some were also generated from detached slings. While most SDP appeared arrested, 18% “sneaked” along the vessel wall with the blood flow at a median velocity of 1.13 μm/s (range 0.23–4.95 μm/s), which was significantly lower than the neutrophil rolling velocity of 3.47 μm/s (range 0.7–9.76 μm/s). Free‐floating SDPs were visible in the blood harvested from these mice.SDP production by tether and sling detachment was reproduced in flow chamber experiments using CD16‐AF647 labeled human neutrophils. 11.5 (range 0–42) SDPs per 0.1 μl were found in plasma of 20 healthy donors by a CD16 staining and a standardized confocal microscopy scanning protocol. In 24 septic donors, significantly more (median count 70.5, range 0–140) SDPs were found, which were also longer (median length 5.4 μm vs. 4 μm, range 1.7–23 μm and 1.7–12.8 μm). Image stream (Amnis) analysis confirmed the increase of SDP count in septic plasma and showed that the ratio between CD16+ SDPs and CD16+ vesicles is on the order of 1:1000. We speculate that some SDPs may fragment into spherical vesicles. All significances: p<0.0001 with Student's T‐test.We conclude that rolling or arrested neutrophils lose their tethers and slings. These form a new class of elongated microparticles in the blood, which are elevated in sepsis. Further work is required to better characterize the conditions when SDP form and to characterize the composition and function of these structures.Support or Funding InformationK.Ley: NIH grant HL 78784, A.Marki: AHA postdoctoral fellowship 17POST33410940This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rationale: The coincidence of inflammation and metabolic derangements in obese adipose tissue has sparked the concept of met-inflammation. Previous observations, however, suggest that inflammatory pathways may not ultimately cause dysmetabolism. Objective: We have revisited the relationship between inflammation and metabolism by testing the role of TRAF (tumor necrosis receptor–associated factor)-1, an inhibitory adapter of inflammatory signaling of TNF (tumor necrosis factor)-α, IL (interleukin)-1β, and TLRs (toll-like receptors). Methods and Results: Mice deficient for TRAF-1, which is expressed in obese adipocytes and adipose tissue lymphocytes, caused an expected hyperinflammatory phenotype in adipose tissue with enhanced adipokine and chemokine expression, increased leukocyte accumulation, and potentiated proinflammatory signaling in macrophages and adipocytes in a mouse model of diet-induced obesity. Unexpectedly, TRAF-1 −/− mice were protected from metabolic derangements and adipocyte growth, failed to gain weight, and showed improved insulin resistance—an effect caused by increased lipid breakdown in adipocytes and UCP (uncoupling protein)-1–enabled thermogenesis. TRAF-1–dependent catabolic and proinflammatory cues were synergistically driven by β3-adrenergic and inflammatory signaling and required the presence of both TRAF-1–deficient adipocytes and macrophages. In human obesity, TRAF-1–dependent genes were upregulated. Conclusions: Enhancing TRAF-1–dependent inflammatory pathways in a gain-of-function approach protected from metabolic derangements in diet-induced obesity. These findings identify TRAF-1 as a regulator of dysmetabolism in mice and humans and question the pathogenic role of chronic inflammation in metabolism.