A myeloperoxidase enhancer drives myeloid cell-specific labeling in a transgenic frog line.

We have identified a myeloid cell-specific enhancer in the 5' flanking region of the Xenopus tropicalis myeloperoxidase gene. Transgenic reporter analysis using Xenopus laevis revealed that the expression of GFP was detected in the tail fin macrophages of a swimming tadpole, and the distributions of the GFP-positive and XL-2 (a pan-marker for leukocytes)-positive cells were mostly overlapping. The GFP-positive cells in the liver of the transgenic tadpole were localized in the same areas where the myeloid cells were present. Isolation of leukocytes from the peripheral blood cells followed by flow cytometric analysis revealed that the GFP-positive fraction was specifically enriched in neutrophils with lobulated nuclei. Furthermore, the macrophages purified from the peritoneal cavity were also GFP-positive. In summary, a transgenic frog line in which the myeloid cells are labeled with GFP provides a useful tool to elucidate the physiological role of myeloid cells of multiple origins in the embryo.

White adipose tissue-infiltrated CD11b+ myeloid cells are a source of S100A4, a new potential marker of hepatic damage.

CONTEXT: The endocrine and immunological properties of subcutaneous vs visceral adipose tissue (sWAT and vWAT, respectively) have turned a milestone in the study of metabolic diseases. The cytokine S100A4 is increased in obesity and has a role in adipose tissue dysfunction. However, the cellular source and its potential role in hepatic damage in obesity has not been elucidated. OBJECTIVE: We aim to study the regulation of S100A4 in immune cells present in sWAT and vWAT, as well as its potential role as a circulating marker of hepatic inflammation and steatosis. DESIGN: A cohort of 60 patients with obesity and distinct metabolic status was analyzed. CD11b+ myeloid cells and T cells were isolated from sWAT and vWAT by magnetic-activating cell sorting, and RNA was obtained. S100A4 gene expression was measured, and correlation analysis with clinical data was performed. Liver biopsies were obtained from 20 patients, and S100A4 circulating levels were measured to check the link with hepatic inflammation and steatosis. RESULTS: S100A4 gene expression was strongly upregulated in sWAT- vs vWAT-infiltrated CD11b+ cells, but this modulation was not observed in T cells. S100A4 mRNA levels from sWAT (and not from vWAT) CD11b+ cells positively correlated with glycemia, triglycerides, TNF-α gene expression and proliferation markers. Finally, circulating S100A4 directly correlated with liver steatosis and hepatic inflammatory markers. CONCLUSION: Our data suggest that sWAT-infiltrated CD11b+ cells could be a major source of S100A4 in obesity. Moreover, our correlations identify circulating S100A4 as a potential novel biomarker of hepatic damage and steatosis.

Bioluminescence for in vivo detection of cell-type-specific inflammation in a mouse model of uveitis.

This study reports the use of cell-type-specific in vivo bioluminescence to measure intraocular immune cell population dynamics during the course of inflammation in a mouse model of uveitis. Transgenic lines expressing luciferase in inflammatory cell subsets (myeloid cells, T cells, and B cells) were generated and ocular bioluminescence was measured serially for 35 days following uveitis induction. Ocular leukocyte populations were identified using flow cytometry and compared to the ocular bioluminescence profile. Acute inflammation is neutrophilic (75% of ocular CD45 + cells) which is reflected by a significant increase in ocular bioluminescence in one myeloid reporter line on day 2. By day 7, the ocular T cell population increases to 50% of CD45 + cells, leading to a significant increase in ocular bioluminescence in the T cell reporter line. While initially negligible (< 1% of CD45 + cells), the ocular B cell population increases to > 4% by day 35. This change is reflected by a significant increase in the ocular bioluminescence of the B cell reporter line starting on day 28. Our data demonstrates that cell-type-specific in vivo bioluminescence accurately detects changes in multiple intraocular immune cell populations over time in experimental uveitis. This assay could also be useful in other inflammatory disease models.

Bmal1 Deletion in Myeloid Cells Attenuates Atherosclerotic Lesion Development and Restrains Abdominal Aortic Aneurysm Formation in Hyperlipidemic Mice.

OBJECTIVE: Although the molecular components of circadian rhythms oscillate in discrete cellular components of the vasculature and many aspects of vascular function display diurnal variation, the cellular connections between the molecular clock and inflammatory cardiovascular diseases remain to be elucidated. Previously we have shown that pre- versus postnatal deletion of Bmal1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1), the nonredundant core clock gene has contrasting effects on atherogenesis. Here we investigated the effect of myeloid cell Bmal1 deletion on atherogenesis and abdominal aortic aneurysm formation in mice. Approach and Results: Mice lacking Bmal1 in myeloid cells were generated by crossing Bmal1 flox/flox mice with lysozyme 2 promoter-driven Cre recombinase mice on a hyperlipidemic low-density lipoprotein receptor-deficient background and were fed on a high-fat diet to induce atherosclerosis. Atherogenesis was restrained, concomitant with a reduction of aortic proinflammatory gene expression in myeloid cell Bmal1 knockout mice. Body weight, blood pressure, blood glucose, triglycerides, and cholesterol were unaltered. Similarly, myeloid cell depletion of Bmal1 also restrained Ang II (angiotensin II) induced formation of abdominal aortic aneurysm in hyperlipidemic mice. In vitro, RNA-Seq analysis demonstrated a proinflammatory response in cultured macrophages in which there was overexpression of Bmal1. CONCLUSIONS: Myeloid cell Bmal1 deletion retards atherogenesis and restrains the formation of abdominal aortic aneurysm and may represent a potential therapeutic target for inflammatory cardiovascular diseases.

Prognostic Accuracy of Soluble Triggering Receptor Expressed on Myeloid Cells (sTREM-1)-based Algorithms in Febrile Adults Presenting to Tanzanian Outpatient Clinics.

BACKGROUND: The inability to identify individuals with acute fever at risk of death is a barrier to effective triage and management of severe infections, especially in low-resource settings. Since endothelial and immune activation contribute to the pathogenesis of various distinct life-threatening infections, we hypothesized that measuring mediators of these pathways at clinical presentation would identify febrile adults at risk of death. METHODS: Plasma concentrations of markers of endothelial (angiopoetin-1/2, soluble fms-like tyrosine kinase-1, soluble vascular cell adhesion molecule-1, soluble intercellular adhesion molecule-1) and immune (soluble triggering receptor expressed on myeloid cells [sTREM-1], interleukin-6, interleukin-8, chitinase-3-like protein-1, soluble tumor necrosis factor receptor-1, procalcitonin [PCT], C-reactive protein [CRP]) activation pathways were determined in consecutive adults with acute fever (≥38°C) at presentation to outpatient clinics in Dar es Salaam, Tanzania. We evaluated the accuracy of these mediators in predicting all-cause mortality and examined whether markers could improve the prognostic accuracy of clinical scoring systems, including the quick sequential organ failure assessment (qSOFA) and Glasgow coma scale (GCS). RESULTS: Of 507 febrile adults, 32 died (6.3%) within 28 days of presentation. We found that sTREM-1 was the best prognostic marker for 28-day mortality (area under the receiver operating characteristic [AUROC] 0.87, 95% confidence interval [CI] 0.81-0.92) and was significantly better than CRP (P < .0001) and PCT (P = .0001). The prognostic accuracy of qSOFA and the GCS were significantly enhanced when sTREM-1 was added (0.80 [95% CI 0.76-0.83] to 0.91 [95% CI 0.88-0.94; P < .05] and 0.72 [95% CI 0.63-0.80] to 0.94 [95% CI 0.91-0.97; P < .05], respectively). CONCLUSIONS: Measuring sTREM-1 at clinical presentation can identify febrile individuals at risk of all-cause febrile mortality. Adding severity markers such as sTREM-1 to simple clinical scores could improve the recognition and triage of patients with life-threatening infections in resource-limited settings.

A novel imaging flow cytometry method for the detection of histone H4 acetylation in myeloid cells.

BACKGROUND: The histone deacetylase inhibitor (HDACI) valproic acid has been shown to inhibit the growth of multiple paediatric tumour types and is well tolerated in a childhood cancer setting. The current study was designed to develop a novel imaging flow cytometry method for the detection of histone H4 acetylation in white blood cells obtained from childhood cancer patients treated with valproic acid. MATERIALS AND METHODS: HL-60 cells and whole blood samples from healthy volunteers were incubated with valproic acid (0-8 mM) for 0-24 hours, with additional blood samples collected from ependymoma patients receiving valproic acid on the SIOP Ependymoma II clinical trial. An imaging flow cytometry method was developed using an ImageStream®χ flow cytometer, collecting 100 000 images per sample following excitation of PE tagged acH4 antibody and DAPI. RESULTS: The mean percentage of acH4-positive cells increased to a greater extent than increases in mean and median fluorescence intensity following incubation with valproic acid. Comparable results were observed for in vitro and ex vivo experiments, and the assay was shown to be appropriate for clinical sample analysis. Myeloid cells exhibited a smaller proportion of acH4-positive cells than the lymphoid population, but a greater fold increase above basal levels. CONCLUSIONS: The percentage of acH4-positive myeloid cells has the potential to be used as a robust pharmacodynamic biomarker for the measurement of acH4 for HDACIs. The developed assay is now being utilised in a clinical trial involving the treatment of childhood ependymoma patients with valproic acid.

Alterations in sialic-acid O-acetylation glycoforms during murine erythrocyte development.

We used Casd1-deficient mice to confirm that this enzyme is responsible for 9-O-acetylation of sialic acids in vivo. We observed a complete loss of 9-O-acetylation of sialic acid on the surface of myeloid, erythroid and CD4+ T cells in Casd1-deficient mice. Although 9-O-acetylation of sialic acids on multiple hematopoietic lineages was lost, there were no obvious defects in hematopoiesis. Interestingly, erythrocytes from Casd1-deficient mice also lost reactivity to TER-119, a rat monoclonal antibody that is widely used to mark the murine erythroid lineage. The sialic acid glyco-epitope recognized by TER-119 on erythrocytes was sensitive to the sialic acid O-acetyl esterase activity of the hemagglutinin-esterase from bovine coronavirus but not to the corresponding enzyme from the influenza C virus. During erythrocyte development, TER-119+ Ery-A and Ery-B cells could be stained by catalytically inactive bovine coronavirus hemagglutinin-esterase but not by the inactive influenza C hemagglutinin-esterase, while TER-119+ Ery-C cells and mature erythrocytes were recognized by both virolectins. Although the structure of the sialoglycoconjugate recognized by TER-119 was not chemically demonstrated, its selective binding to virolectins suggests that it may be comprised of a 7,9-di-O-acetyl form of sialic acid. As erythrocytes mature, the surfaces of Ery-C cells and mature erythrocytes also acquire an additional distinct CASD1-dependent 9-O-acetyl sialic acid moiety that can be recognized by virolectins from both influenza C and bovine coronavirus.

Analysis of cell proliferation and tissue remodelling uncovers a KLF4 activity score associated with poor prognosis in colorectal cancer.

BACKGROUND: Human cancers can be classified based on gene signatures quantifying the degree of cell proliferation and tissue remodelling (PR). However, the specific factors that drive the increased tissue remodelling in tumours are not fully understood. Here we address this question using colorectal cancer as a case study. METHODS: We reanalysed a reported cohort of colorectal cancer patients. The patients were stratified based on gene signatures of cell proliferation and tissue remodelling. Putative transcription factors activity was inferred using gene expression profiles and annotations of transcription factor targets as input. RESULTS: We demonstrate that the PR classification performs better than the currently adopted consensus molecular subtyping (CMS). Although CMS classification differentiates patients with a mesenchymal signature, it cannot distinguish the remaining patients based on survival. We demonstrate that the missing factor is cell proliferation, which is indicative of good prognosis. We also uncover a KLF4 transcription factor activity score associated with the tissue remodelling gene signature. We further show that the KLF4 activity score is significantly higher in colorectal tumours with predicted infiltration of cells from the myeloid lineage. CONCLUSION: The KLF4 activity score is associated with tissue remodelling, myeloid cell infiltration and poor prognosis in colorectal cancer.

Deleting MyD88 signaling in myeloid cells promotes development of adenocarcinomas of the colon.

Intestinal myeloid cells are not only essential for keeping local homeostasis, but also play an important role in regulating the occurrence of colitis and colitis-associated cancer (CAC). In these diseases, the manner in which the myeloid cells work and which molecular pathways influence them are still not fully understood. In our study, we discovered that MyD88 signaling in colonic myeloid cells participates in the development of CAC. Myeloid MyD88-deficient mice showed greater susceptibility to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CAC, as evidenced by the increase in the number and sizes of tumors. Myeloid MyD88 deletion markedly increased production of pro-inflammatory and pro-tumor cytokines; recruitment of more IL-1ß producing-neutrophils in colon from bone marrow; increased in epithelial cell apoptosis and decreased in epithelial cell proliferation; enhancement of colon mucosal expression of COX-2, p-STAT3, ß-catenin, and cyclinD1; induction of further DNA damage and ß-catenin mutation. To sum up, these results suggest that myeloid MyD88 signaling protects the intestine from tumorigenesis during the development of CAC.

Leukocytes as a reservoir of circulating oncogenic DNA and regulatory targets of tumor-derived extracellular vesicles.

Essentials Tumor-bearing mice were employed to follow oncogenic HRAS sequences in plasma, and blood cells. Cancer DNA accumulated in leukocytes above levels detected in exosomes, platelets and plasma. Extracellular vesicles and nucleosomes are required for uptake of tumor DNA by leukocytes. Uptake of tumor-derived extracellular vesicles by leukocytes triggers coagulant phenotype. SUMMARY: Background Tumor-derived extracellular vesicles (EVs) and free nucleosomes (NSs) carry into the circulation a wealth of cancer-specific, bioactive and poorly understood molecular cargoes, including genomic DNA (gDNA). Objective Here we investigated the distribution of extracellular oncogenic gDNA sequences (HRAS and HER2) in the circulation of tumor-bearing mice. Methods and Results Surprisingly, circulating leukocytes (WBCs), especially neutrophils, contained the highest levels of mutant gDNA, which exceeded the amount of this material recovered from soluble fractions of plasma, circulating EVs, platelets, red blood cells (RBCs) and peripheral organs, as quantified by digital droplet PCR (ddPCR). Tumor excision resulted in disappearance of the WBC-associated gDNA signal within 2-9 days, which is in line with the expected half-life of these cells. EVs and nucleosomes were essential for the uptake of tumor-derived extracellular DNA by neutrophil-like cells and impacted their phenotype. Indeed, the exposure of granulocytic HL-60 cells to EVs from HRAS-driven cancer cells resulted in a selective increase in tissue factor (TF) procoagulant activity and interleukin 8 (IL-8) production. The levels of circulating thrombin-antithrombin complexes (TAT) were markedly elevated in mice harboring HRAS-driven xenografts. Conclusions Myeloid cells may represent a hitherto unrecognized reservoir of cancer-derived, EV/NS-associated oncogenic gDNA in the circulation, and a possible novel platform for liquid biopsy in cancer. In addition, uptake of this material alters the phenotype of myeloid cells, induces procoagulant and proinflammatory activity and may contribute to systemic effects associated with cancer.

EGFR in Tumor-Associated Myeloid Cells Promotes Development of Colorectal Cancer in Mice and Associates With Outcomes of Patients.

BACKGROUND & AIMS: Inhibitors of the epidermal growth factor receptor (EGFR) are the first-line therapy for patients with metastatic colorectal tumors without RAS mutations. However, EGFR inhibitors are ineffective in these patients, and tumor level of EGFR does not associate with response to therapy. We screened human colorectal tumors for EGFR-positive myeloid cells and investigated their association with patient outcome. We also performed studies in mice to evaluate how EGFR expression in tumor cells and myeloid cells contributes to development of colitis-associated cancer and ApcMin-dependent intestinal tumorigenesis. METHODS: We performed immunohistochemical and immunofluorescent analyses of 116 colorectal tumor biopsies to determine levels of EGFR in tumor and stroma; we also collected information on tumor stage and patient features and outcomes. We used the Mann-Whitney U and Kruskal-Wallis tests to correlate tumor levels of EGFR with tumor stage, and the Kaplan-Meier method to estimate patients' median survival time. We performed experiments in mice lacking EGFR in intestinal epithelial cells (Villin-Cre; Egfrf/f and Villin-CreERT2; Egfrf/f mice) or myeloid cells (LysM-Cre; Egfrf/f mice) on a mixed background. These mice were bred with ApcMin/+ mice; colitis-associated cancer and colitis were induced by administration of dextran sodium sulfate (DSS), with or without azoxymethane (AOM), respectively. Villin-CreERT2 was activated in developed tumors by administration of tamoxifen to mice. Littermates that expressed full-length EGFR were used as controls. Intestinal tissues were collected; severity of colitis, numbers and size of tumors, and intestinal barrier integrity were assessed by histologic, immunohistochemical, quantitative reverse transcription polymerase chain reaction, and flow cytometry analyses. RESULTS: We detected EGFR in myeloid cells in the stroma of human colorectal tumors; myeloid cell expression of EGFR associated with tumor metastasis and shorter patient survival time. Mice with deletion of EGFR from myeloid cells formed significantly fewer and smaller tumors than the respective EGFR-expressing controls in an ApcMin/+ background as well as after administration of AOM and DSS. Deletion of EGFR from intestinal epithelial cells did not affect tumor growth. Furthermore, tamoxifen-induced deletion of EGFR from epithelial cells of established intestinal tumors in mice given AOM and DSS did not reduce tumor size. EGFR signaling in myeloid cells promoted activation of STAT3 and expression of survivin in intestinal tumor cells. Mice with deletion of EGFR from myeloid cells developed more severe colitis after DSS administration, characterized by increased intestinal inflammation and intestinal barrier disruption, than control mice or mice with deletion of EGFR from intestinal epithelial cells. EGFR-deficient myeloid cells in the colon of DSS-treated LysM-Cre; Egfrf/f mice had reduced expression of interleukin 6 (IL6), and epithelial STAT3 activation was reduced compared with controls. Administration of recombinant IL6 to LysM-Cre; Egfrf/f mice given DSS protected them from weight loss and restored epithelial proliferation and STAT3 activation, compared with administration of DSS alone to these mice. CONCLUSIONS: Increased expression of EGFR in myeloid cells from the colorectal tumor stroma associates with tumor progression and reduced survival time of patients with metastatic colorectal cancer. Deletion of EGFR from myeloid cells, but not intestinal epithelial cells, protects mice from colitis-induced intestinal cancer and ApcMin-dependent intestinal tumorigenesis. Myeloid cell expression of EGFR increases activation of STAT3 and expression of survivin in intestinal epithelial cells and expression of IL6 in colon tissues. These findings indicate that expression of EGFR by myeloid cells of the colorectal tumor stroma, rather than the cancer cells themselves, contributes to tumor development.

Myeloid cells are required for PD-1/PD-L1 checkpoint activation and the establishment of an immunosuppressive environment in pancreatic cancer.

BACKGROUND: Pancreatic cancer is characterised by the accumulation of a fibro-inflammatory stroma. Within this stromal reaction, myeloid cells are a predominant population. Distinct myeloid subsets have been correlated with tumour promotion and unmasking of anti-tumour immunity. OBJECTIVE: The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer and to understand the relationship between myeloid cells and T cell-mediated immunity within the pancreatic cancer microenvironment. METHODS: Primary mouse pancreatic cancer cells were transplanted into CD11b-diphtheria toxin receptor (DTR) mice. Alternatively, the iKras* mouse model of pancreatic cancer was crossed into CD11b-DTR mice. CD11b+ cells (mostly myeloid cell population) were depleted by diphtheria toxin treatment during tumour initiation or in established tumours. RESULTS: Depletion of myeloid cells prevented KrasG12D-driven pancreatic cancer initiation. In pre-established tumours, myeloid cell depletion arrested tumour growth and in some cases, induced tumour regressions that were dependent on CD8+ T cells. We found that myeloid cells inhibited CD8+ T-cell anti-tumour activity by inducing the expression of programmed cell death-ligand 1 (PD-L1) in tumour cells in an epidermal growth factor receptor (EGFR)/mitogen-activated protein kinases (MAPK)-dependent manner. CONCLUSION: Our results show that myeloid cells support immune evasion in pancreatic cancer through EGFR/MAPK-dependent regulation of PD-L1 expression on tumour cells. Derailing this crosstalk between myeloid cells and tumour cells is sufficient to restore anti-tumour immunity mediated by CD8+ T cells, a finding with implications for the design of immune therapies for pancreatic cancer.

Targeting distinct myeloid cell populations in vivo using polymers, liposomes and microbubbles.

Identifying intended or accidental cellular targets for drug delivery systems is highly relevant for evaluating therapeutic and toxic effects. However, limited knowledge exists on the distribution of nano- and micrometer-sized carrier systems at the cellular level in different organs. We hypothesized that clinically relevant carrier materials, differing in composition and size, are able to target distinct myeloid cell subsets that control inflammatory processes, such as macrophages, neutrophils, monocytes and dendritic cells. Therefore, we analyzed the biodistribution and in vivo cellular uptake of intravenously injected poly(N-(2-hydroxypropyl) methacrylamide) polymers, PEGylated liposomes and poly(butyl cyanoacrylate) microbubbles in mice, using whole-body imaging (computed tomography - fluorescence-mediated tomography), intra-organ imaging (intravital multi-photon microscopy) and cellular analysis (flow cytometry of blood, liver, spleen, lung and kidney). While the three carrier materials shared accumulation in tissue macrophages in liver and spleen, they notably differed in uptake by other myeloid subsets. Kupffer cells and splenic red pulp macrophages rapidly take up microbubbles. Liposomes efficiently reach dendritic cells in liver, lung and kidney. Polymers exhibit the longest circulation half-life and target endothelial cells in the liver, neutrophils and alveolar macrophages. The identification of such previously unrecognized target cell populations might open up new avenues for more efficient drug delivery.

Combination of Mass Cytometry and Imaging Analysis Reveals Origin, Location, and Functional Repopulation of Liver Myeloid Cells in Mice.

BACKGROUND & AIMS: Resident macrophages are derived from yolk sac precursors and seed the liver during embryogenesis. Native cells may be replaced by bone marrow precursors during extensive injuries, irradiation, and infections. We investigated the liver populations of myeloid immune cells and their location, as well as the dynamics of phagocyte repopulation after full depletion. The effects on liver function due to the substitution of original phagocytes by bone marrow-derived surrogates were also examined. METHODS: We collected and analyzed liver tissues from C57BL/6 (control), LysM-EGFP, B6 ACTb-EGFP, CCR2-/-, CD11c-EYFP, CD11c-EYFP-DTR, germ-free mice, CX3CR1gfp/gfp, CX3CR1gpf/wt, and CX3CR1-DTR-EYFP. Liver nonparenchymal cells were immunophenotyped using mass cytometry and gene expression analyses. Kupffer and dendritic cells were depleted from mice by administration of clodronate, and their location and phenotype were examined using intravital microscopy and time-of-flight mass cytometry. Mice were given acetaminophen gavage or intravenous injections of fluorescently labeled Escherichia coli, blood samples were collected and analyzed, and liver function was evaluated. We assessed cytokine profiles of liver tissues using a multiplexed array. RESULTS: Using mass cytometry and gene expression analyses, we identified 2 populations of hepatic macrophages and 2 populations of monocytes. We also identified 4 populations of dendritic cells and 1 population of basophils. After selective depletion of liver phagocytes, intravascular myeloid precursors began to differentiate into macrophages and dendritic cells; dendritic cells migrated out of sinusoids, after a delay, via the chemokine CX3CL1. The cell distribution returned to normal in 2 weeks, but the repopulated livers were unable to fully respond to drug-induced injury or clear bacteria for at least 1 month. This defect was associated with increased levels of inflammatory cytokines, and dexamethasone accelerated the repopulation of liver phagocytes. CONCLUSIONS: In studies of hepatic phagocyte depletion in mice, we found that myeloid precursors can differentiate into liver macrophages and dendritic cells, which each localize to distinct tissue compartments. During replenishment, macrophages acquire the ability to respond appropriately to hepatic injury and to remove bacteria from the blood stream.

CD38-Expressing Myeloid-Derived Suppressor Cells Promote Tumor Growth in a Murine Model of Esophageal Cancer.

Myeloid-derived suppressor cells (MDSC) are an immunosuppressive population of immature myeloid cells found in advanced-stage cancer patients and mouse tumor models. Production of inducible nitric oxide synthase (iNOS) and arginase, as well as other suppressive mechanisms, allows MDSCs to suppress T-cell-mediated tumor clearance and foster tumor progression. Using an unbiased global gene expression approach in conditional p120-catenin knockout mice (L2-cre;p120ctn(f/f)), a model of oral-esophageal cancer, we have identified CD38 as playing a vital role in MDSC biology, previously unknown. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation, and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in other mouse tumor models of esophageal carcinogenesis, and CD38(high) MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38(high) MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38(low) MDSCs, likely as a result of increased iNOS production. In addition, we have identified novel tumor-derived factors, specifically IL6, IGFBP3, and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38(+) MDSCs in peripheral blood of advanced-stage cancer patients and validated targeting CD38 in vivo as a novel approach to cancer therapy.

Effect of Cisplatin on the Frequency and Immuno-inhibitory Function of Myeloid-derived Suppressor Cells in A375 Melanoma Model.

BACKGROUND: To investigate the change of frequency and immuno-inhibitory function of myeloid-derived suppressor cells (MDSCs) after treatment of cisplatin (DDP) in A375 human melanoma model. MATERIALS AND METHODS: BALB/c nude mice were inoculated with A375 cells to establish the human melanoma model and randomly divided into control group given normal saline (NS) and experimental group treated with DDP (5 mg/ kg). The percentages of MDSCs in the tumor tissue and peripheral blood after DDP treatment were detected by flow cytometry. The proliferation and interferon-γ (IFN-γ) secretion of T cells co-cultured with MDSCs were analyzed through carboxyfluorescein succinimidyl ester (CFSE) labeling assay and enzyme-linked immunospot (ELISPOT) assay, respectively. RESULTS: In A375 human melanoma model, DDP treatment could significantly decrease the percentage of MDSCs in the tumor tissue, but exerted no effect on the level of MDSCs in peripheral blood. Moreover, DDP treatment could attenuate the immuno-inhibitory function of MDSCs. T cells co-cultured with DDP-treated MDSCs could dramatically elevate the proliferation and production of INF-γ. CONCLUSIONS: DDP can decrease the frequency and attenuate immuno-inhibitory function of MDSCs in A375 melanoma model, suggesting a potential strategy to augment the efficacy of combined immunotherapy.

Histologic and Immunohistochemical Features of the Skin Lesions in CANDLE Syndrome.

Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome is a newly characterized autoinflammatory disorder, caused by mutations in PSMB8. It is characterized by early-onset fevers, accompanied by a widespread, violaceous, and often annular cutaneous eruption. Although the exact pathogenesis of this syndrome is still obscure, it is postulated that the inflammatory disease manifestations stem from excess secretion of interferons. Based on preliminary blood cytokine and gene expression studies, the signature seems to come mostly from type I interferons, which are proposed to lead to the recruitment of immature myeloid cells into the dermis and subcutis. In this study, we systematically analyzed skin biopsies from 6 patients with CANDLE syndrome by routine histopathology and immunohistochemistry methods. Skin lesions showed the presence of extensive mixed dermal and subcutaneous inflammatory infiltrate, composed of mononuclear cells, atypical myeloid cells, neutrophils, eosinophils, and some mature lymphocytes. Positive LEDER and myeloperoxidase staining supported the presence of myeloid cells. Positive CD68/PMG1 and CD163 staining confirmed the existence of histiocytes and monocytic macrophages in the inflammatory infiltrate. CD123 staining was positive, demonstrating the presence of plasmacytoid dendritic cells. Uncovering the unique histopathological and immunohistochemical features of CANDLE syndrome provides tools for rapid and specific diagnosis of this disorder and further insight into the pathogenesis of this severe life-threatening condition.

Role of Myeloid-Derived Suppressor Cells in Glucocorticoid-Mediated Amelioration of FSGS.

The mechanism by which glucocorticoids alleviate renal inflammatory disorders remains incompletely understood. Here, we report that the efficacy of glucocorticoids in ameliorating FSGS depends on the capacity to expand myeloid-derived suppressor cells (MDSCs). After glucocorticoid treatment, the frequency of CD11b(+)HLA-DR(-)CD14(-)CD15(+) MDSCs in peripheral blood rapidly increased in patients with glucocorticoid-sensitive FSGS but remained unchanged in patients with glucocorticoid-resistant FSGS. The frequency of CD11b(+)Gr-1(+) MDSCs in mouse peripheral blood, bone marrow, spleen, kidney-draining lymph nodes (KDLNs), and kidney also increased after glucocorticoid treatment. The induced MDSCs from glucocorticoid-treated mice strongly suppressed T cells, dendritic cells, and macrophages but induced regulatory T cells in spleen, KDLNs, and kidney. Moreover, glucocorticoid treatment suppressed doxorubicin-induced T cell proliferation, dendritic cell and macrophage infiltration, and proinflammatory cytokine production, whereas this protective effect was largely abolished by depleting MDSCs using anti-Gr-1 antibody. Finally, the adoptive transfer of induced MDSCs into the doxorubicin-treated mice not only confirmed the protective role of MDSCs in doxorubicin-induced renal injury but also showed that the transferred MDSCs rapidly migrated into the lymphocyte-accumulating organs, such as the spleen and KDLNs, where they suppressed T cell proliferation. Taken together, these results demonstrate that glucocorticoid treatment ameliorates FSGS by expanding functional MDSCs and that this rapid elevation of MDSCs in peripheral blood may serve as an indicator for predicting the efficacy of glucocorticoid treatment.

Fractalkine-CX3CR1-dependent recruitment and retention of human CD1c+ myeloid dendritic cells by in vitro-activated proximal tubular epithelial cells.

Chemokines play pivotal roles in tissue recruitment and retention of leukocytes, with CX3CR1 recently identified as a chemokine receptor that selectively targets mouse kidney dendritic cells (DCs). We have previously demonstrated increased tubulointerstitial recruitment of human transforming growth factor-ß (TGF-ß)-producing DCs in renal fibrosis and chronic kidney disease (CKD). However, little is known about the mechanism of human DC recruitment and retention within the renal interstitium. We identified CD1c+ DCs as the predominant source of profibrotic TGF-ß and highest expressors of the fractalkine receptor CX3CR1 within the renal DC compartment. Immunohistochemical analysis of diseased human kidney biopsies showed colocalization of CD1c+ DCs with fractalkine-positive proximal tubular epithelial cells (PTECs). Human primary PTEC activation with interferon-γ and tumor necrosis factor-α induced both secreted and surface fractalkine expression. In line with this, we found fractalkine-dependent chemotaxis of CD1c+ DCs to supernatant from activated PTECs. Finally, in comparison with unactivated PTECs, we showed significantly increased adhesion of CD1c+ DCs to activated PTECs via a fractalkine-dependent mechanism. Thus, TGF-ß-producing CD1c+ DCs are recruited and retained in the renal tubulointerstitium by PTEC-derived fractalkine. These cells are then positioned to play a role in the development of fibrosis and progression of chronic kidney disease.