Validation of CSF-1 receptor (CD115) staining for analysis of murine monocytes by flow cytometry.

CD115, the receptor for colony stimulating factor 1, is essential for survival and differentiation of monocytes and macrophages and is therefore frequently used to define monocyte subsets and their progenitors in immunological assays. However, CD115 surface expression and detection by flow cytometry is greatly influenced by cell isolation and processing methods, organ source, and disease context. In a systematic analysis of murine monocytes, we define experimental conditions that preserve or limit CD115 surface expression and staining by flow cytometry.

CSF-1 and Notch signaling cooperate in macrophage instruction and tissue repair during peripheral limb ischemia.

Ischemia causes an inflammatory response featuring monocyte-derived macrophages (MF) involved in angiogenesis and tissue repair. Angiogenesis and ischemic macrophage differentiation are regulated by Notch signaling Notch ligand Delta-like 1 (Dll1). Colony stimulating factor 1 (CSF-1) is an essential MF lineage factor, but its role in ischemic macrophage development and the interaction with Notch signaling is so far unclear.

Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures.

Tertiary lymphoid structures (TLS) are lymph node-like immune cell clusters that emerge during chronic inflammation in non-lymphoid organs like the kidney, but their origin remains not well understood. Here we show, using conditional deletion strategies of the canonical Notch signaling mediator Rbpj, that loss of endothelial Notch signaling in adult mice induces the spontaneous formation of bona fide TLS in the kidney, liver and lung, based on molecular, cellular and structural criteria. These TLS form in a stereotypical manner around parenchymal arteries, while secondary lymphoid structures remained largely unchanged.

Analysis of Monocyte Cell Fate by Adoptive Transfer in a Murine Model of TLR7-induced Systemic Inflammation.

Myeloid plasticity is a hallmark of the innate immune response to Toll-like receptor (TLR) activation. Here, we provide a protocol for monocyte cell fate tracking by adoptive transfer in the context of systemic inflammation induced by TLR7 activation, the principal innate immune receptor sensing viral RNA in mice. Defined monocyte subsets are isolated from the bone marrow of donor mice by cell sorting and adoptively transferred into the systemic circulation of congenic hosts, with or without concurrent activation of TLR7 via the topical application of the small molecule agonist, imiquimod, in a cream formulation that induces a systemic inflammatory response.

Notch and TLR signaling coordinate monocyte cell fate and inflammation.

Conventional Ly6C monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6C monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6C patrolling monocyte development from Ly6C monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6C monocyte development.

Retinal myeloid cells regulate tip cell selection and vascular branching morphogenesis via Notch ligand Delta-like 1.

During angiogenesis, single endothelial cells (EC) specialize into tip cells that guide vessel sprouting towards growth factor gradients and instruct the adjacent vessel stalk. The balance between tip and stalk cells is regulated by endothelial Notch signalling through the expression of Notch ligand Delta-like 4 (Dll4) in tip cells, which suppresses a tip cell fate in adjacent stalk cells. Here we show, using genetic reporter and conditional deletion strategies, that myeloid cells regulate tip cell numbers and Dll4 expression via the Notch ligand Dll1 during vascular development in the retina.

Multimodal and Multiscale Analysis Reveals Distinct Vascular, Metabolic and Inflammatory Components of the Tissue Response to Limb Ischemia.

Unlabelled: Ischemia triggers a complex tissue response involving vascular, metabolic and inflammatory changes.

Methods: We combined hybrid SPECT/CT or PET/CT nuclear imaging studies of perfusion, metabolism and inflammation with multicolor flow cytometry-based cell population analysis to comprehensively analyze the ischemic tissue response and to elucidate the cellular substrate of noninvasive molecular imaging techniques in a mouse model of hind limb ischemia.

Results: Comparative analysis of tissue perfusion with [Tc]-Sestamibi and arterial influx with [Tc]-labeled albumin microspheres by SPECT/CT revealed a distinct pattern of response to vascular occlusion: an early ischemic period of matched suppression of tissue perfusion and arterial influx, a subacute ischemic period of normalized arterial influx but impaired tissue perfusion, and a protracted post-ischemic period of hyperdynamic arterial and normalized tissue perfusion, indicating coordination of macrovascular and microvascular responses.

Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis.

In colorectal cancer patients, a high density of cytotoxic CD8 T cells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/β-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8 T cell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization.

The chemokine receptor CXCR1 coordinates monocyte recruitment and endothelial regeneration after arterial injury.

Regeneration of arterial endothelium after injury is critical for the maintenance of normal blood flow, cell trafficking, and vascular function. Using mouse models of carotid injury, we show that the transition from a static to a dynamic phase of endothelial regeneration is marked by a strong increase in endothelial proliferation, which is accompanied by induction of the chemokine CXCL1 in endothelial cells near the wound edge, leading to progressive recruitment of Ly6C monocytes expressing high levels of the cognate CXCR1 chemokine receptor. In -deficient mice recruitment of Ly6C monocytes, endothelial proliferation and regeneration of the endothelial monolayer after carotid injury are impaired, which is rescued by acute transfer of normal Ly6C monocytes.

Blood vessel control of macrophage maturation promotes arteriogenesis in ischemia.

Ischemia causes an inflammatory response that is intended to restore perfusion and homeostasis yet often aggravates damage. Here we show, using conditional genetic deletion strategies together with adoptive cell transfer experiments in a mouse model of hind limb ischemia, that blood vessels control macrophage differentiation and maturation from recruited monocytes via Notch signaling, which in turn promotes arteriogenesis and tissue repair. Macrophage maturation is controlled by Notch ligand Dll1 expressed in vascular endothelial cells of arteries and requires macrophage canonical Notch signaling via Rbpj, which simultaneously suppresses an inflammatory macrophage fate.

Blood flow controls bone vascular function and osteogenesis.

While blood vessels play important roles in bone homeostasis and repair, fundamental aspects of vascular function in the skeletal system remain poorly understood. Here we show that the long bone vasculature generates a peculiar flow pattern, which is important for proper angiogenesis. Intravital imaging reveals that vessel growth in murine long bone involves the extension and anastomotic fusion of endothelial buds.

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling.

A population of monocytes, known as Ly6C(lo) monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6C(hi) monocytes into Ly6C(lo) monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions.

Tumor-induced CD11b(+) Gr-1(+) myeloid-derived suppressor cells exacerbate immune-mediated hepatitis in mice in a CD40-dependent manner.

Immunosuppressive CD11b(+) Gr-1(+) myeloid-derived suppressor cells (MDSCs) accumulate in the livers of tumor-bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune-mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α-galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor-free mice.

Immunogenicity of necrotic cell death.

The mode of tumor cell death has significant effects on anti-tumor immunity. Although, previously it was thought that cell death is an inert effect, different investigators have clearly shown that dying tumors can attract, activate and mature professional antigen presenting cells and dendritic cells. In addition, others and we have shown that the type of tumor cell death not only controls the presence or absence of specific tumor antigens, but also can result in immunological responses ranging from immunosuppression to anti-tumor immunity.

IFN-γ regulates survival and function of tumor-induced CD11b+ Gr-1high myeloid derived suppressor cells by modulating the anti-apoptotic molecule Bcl2a1.

Myeloid derived suppressor cells (MDSCs) play a critical role in suppression of immune responses in cancer and inflammation. Here, we describe how regulation of Bcl2a1 by cytokines controls the suppressor function of CD11b(+) Gr-1(high) granulocytic MDSCs. Coculture of CD11b(+) Gr-1(high) granulocytic MDSCs with antigen-stimulated T cells and simultaneous blockade of IFN-γ by the use of anti-IFN-γ blocking antibody, IFN-γ(-/-) effector T cells, IFN-γR(-/-) MDSCs or STAT1(-/-) MDSCs led to upregulation of Bcl2a1 in CD11b(+) Gr-1(high) cells, improved survival, and enhanced their suppressor function.

Peptidases released by necrotic cells control CD8+ T cell cross-priming.

Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells.

Regulation of accumulation and function of myeloid derived suppressor cells in different murine models of hepatocellular carcinoma.

Background & Aims: Myeloid derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive activity. They accumulate in tumor-bearing mice and humans with different types of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the biology of MDSC in murine HCC models and to identify a model, which mimics the human disease.

Human Th17 cells in patients with cancer: Friends or foe?

The role of interleukin-17 (IL-17)-secreting CD4(+) T (Th17) cells in cancer is under intense investigation. We have demonstrated that CCR4(+)CCR6(+) Th17 cells not only are increased in the peripheral blood of patients affected by hepatocellular carcinoma, but also suppress CD8(+) T-cell functions in vitro. These results suggest that Th17 cells may exert immunosuppressive functions in hepatocellular carcinoma.

Primary sterile necrotic cells fail to cross-prime CD8(+) T cells.

Necrotic cells are known to activate the innate immune system and trigger inflammation by releasing damage associated molecular patterns (DAMPs). However, how necrotic cells influence the induction of antigen-specific CD8(+) T cell-mediated adaptive immune responses under sterile conditions, in the absence of pathogen associated molecular patterns (PAMPs), remains poorly understood. Here, we examined antigen-specific CD8(+) T-cell responses to primary sterile necrotic tumor cells both in vitro and in vivo.

Anti-Gr-1 antibody depletion fails to eliminate hepatic myeloid-derived suppressor cells in tumor-bearing mice.

Recent studies show that the liver is a preferred organ for the accumulation of MDSC. In this study, we examined the effect of systemic RB6-8C5 treatment on hepatic MDSC in tumor-bearing mice. EL4 tumor-bearing mice were injected i.

Comparative analysis of monocytic and granulocytic myeloid-derived suppressor cell subsets in patients with gastrointestinal malignancies.

Myeloid-derived suppressor cells (MDSC) are a heterogenous population of cells comprising myeloid progenitor cells and immature myeloid cells, which have the ability to suppress the effector immune response. In humans, MDSC have not been well characterized owing to the lack of specific markers, although it is possible to broadly classify the MDSC phenotypes described in the literature as being predominantly granulocytic (expressing markers such as CD15, CD66, CD33) or monocytic (expressing CD14). In this study, we set out to perform a direct comparative analysis across both granulocytic and monocytic MDSC subsets in terms of their frequency, absolute number, and function in the peripheral blood of patients with advanced GI cancer.

Human CCR4+ CCR6+ Th17 cells suppress autologous CD8+ T cell responses.

The role of Th17 cells in cancer patients remains unclear and controversial. In this study, we have analyzed the phenotype of in vitro primed Th17 cells and further characterized their function on the basis of CCR4 and CCR6 expression. We show a novel function for a subset of IL-17-secreting CD4(+) T cells, namely, CCR4(+)CCR6(+)Th17 cells.