Analysis of IL-4/STAT6 Signaling in Macrophages.

Activation of signal transducer and activator of transcription 6 (STAT6) is a key signaling pathway in macrophage function, and is required for the so-called alternative (M2) activation of macrophages. Interleukin (IL)-4 and IL-13 are important M2 polarizing cytokines that act through STAT6 by inducing its phosphorylation and promoting transcription of STAT6-responsive genes. Inactivation of STAT6 signaling in macrophages has not been fully explored; however, a recent model suggests that inactivation of STAT6 signaling can occur via ubiquitination and proteasomal degradation. In this chapter, we describe a combination of techniques that can be used to study the activation/inactivation of STAT6 signaling in macrophages.

Isolation and Characterization of Adipose Tissue Macrophages.

This chapter describes a technique that can be used to isolate adipose tissue macrophages (ATMs) from the visceral white adipose tissue. Nevertheless, this technique can also be used to isolate ATMs from subcutaneous white adipose tissue and brown adipose tissue from mouse, human subcutaneous fat depot, and also from the fat body of the toad Xenopus. We detail the flow-cytometric gating strategy that has been developed to identify ATM population, and we describe the isolation of RNA from this population and its use for gene expression profiling. Finally, we describe in vitro culture of ATMs for downstream applications.

The environmental obesogen bisphenol A increases macrophage self-renewal.

Self-renewal of macrophages is important for the healthy development and replenishment of tissue-resident macrophage pools. How this mechanism is controlled by endocrine signals is still largely unexplored. Here, we show that the endocrine disruptor bisphenol A (BPA) increases macrophage self-renewal. This effect was associated with phosphorylation of extracellular signal-regulated kinase (ERK) and a slight increase in the expression of liver X receptor alpha (LXRα). We found that LXRα inhibition induced, while LXRα activation impeded, macrophage self-renewal. LXRα signaling hence may protect from excessive macrophage expansion. Self-renewing macrophages, however, had negligible LXRα expression when compared with quiescent macrophages. Accordingly, tissue-resident macrophage pools, which are dominated by quiescent macrophages, were rich in LXRα-expressing macrophages. Overall, we show that BPA increases macrophage self-renewal and that this effect, at least in part, can be inhibited by increasing LXRα expression. Since BPA is accumulated in the adipose tissue, it has the potential to increase self-renewal of adipose tissue macrophages, leading to a condition that might negatively impact adipose tissue health.

Neuropeptide FF increases M2 activation and self-renewal of adipose tissue macrophages.

The quantity and activation state of adipose tissue macrophages (ATMs) impact the development of obesity-induced metabolic diseases. Appetite-controlling hormones play key roles in obesity; however, our understanding of their effects on ATMs is limited. Here, we have shown that human and mouse ATMs express NPFFR2, a receptor for the appetite-reducing neuropeptide FF (NPFF), and that NPFFR2 expression is upregulated by IL-4, an M2-polarizing cytokine. Plasma levels of NPFF decreased in obese patients and high-fat diet-fed mice and increased following caloric restriction. NPFF promoted M2 activation and increased the proliferation of murine and human ATMs. Both M2 activation and increased ATM proliferation were abolished in NPFFR2-deficient ATMs. Mechanistically, the effects of NPFF involved the suppression of E3 ubiquitin ligase RNF128 expression, resulting in enhanced stability of phosphorylated STAT6 and increased transcription of the M2 macrophage-associated genes IL-4 receptor α (Il4ra), arginase 1 (Arg1), IL-10 (Il10), and alkylglycerol monooxygenase (Agmo). NPFF induced ATM proliferation concomitantly with the increase in N-Myc downstream-regulated gene 2 (Ndrg2) expression and suppressed the transcription of Ifi200 cell-cycle inhibitor family members and MAF bZIP transcription factor B (Mafb), a negative regulator of macrophage proliferation. NPFF thus plays an important role in supporting healthy adipose tissue via the maintenance of metabolically beneficial ATMs.

Adipose tissue macrophages develop from bone marrow-independent progenitors in Xenopus laevis and mouse.

ATMs have a metabolic impact in mammals as they contribute to metabolically harmful AT inflammation. The control of the ATM number may have therapeutic potential; however, information on ATM ontogeny is scarce. Whereas it is thought that ATMs develop from circulating monocytes, various tissue-resident Mϕs are capable of self-renewal and develop from BM-independent progenitors without a monocyte intermediate. Here, we show that amphibian AT contains self-renewing ATMs that populate the AT before the establishment of BM hematopoiesis. Xenopus ATMs develop from progenitors of aVBI. In the mouse, a significant amount of ATM develops from the yolk sac, the mammalian equivalent of aVBI. In summary, this study provides evidence for a prenatal origin of ATMs and shows that the study of amphibian ATMs can enhance the understanding of the role of the prenatal environment in ATM development.

Adipose tissue macrophages in non-rodent mammals: a comparative study.

The stromal vascular fraction (SVF) of adipose tissue in rodents and primates contains mesenchymal stem cells and immune cells. SVF cells have complex metabolic, immune and endocrine functions with biomedical impact. However, in other mammals, the amount of data on SVF stem cells is negligible and whether the SVF hosts immune cells is unknown. In this study, we show that the SVF is rich in immune cells, with a dominance of adipose tissue macrophages (ATMs) in cattle (Bos primigenius taurus), domestic goat (Capra aegagrus hircus), domestic sheep (Ovis aries), domestic cat (Felis catus) and domestic dog (Canis familiaris). ATMs of these species are granulated lysosome-rich cells with lamellipodial protrusions and express the lysosome markers acid phosphatase 5 (ACP-5) and Mac-3/Lamp-2. Using ACP-5 and Mac-3/Lamp-2 as markers, we additionally detected ATMs in other species, such as the domestic horse (Equus ferus caballus), wild boar (Sus scrofa) and red fox (Vulpes vulpes). Feline and canine ATMs also express the murine macrophage marker F4/80 antigen. In the lean condition, the alternative macrophage activation marker CD206 is expressed by feline and canine ATMs and arginase-1 by feline ATMs. Obesity is associated with interleukin-6 and interferon gamma expression and with overt tyrosine nitration in both feline and canine ATMs. This resembles the obesity-induced phenotype switch of murine and human ATMs. Thus, we show, for the first time, that the presence of ATMs is a general trait of mammals. The interaction between the adipose cells and SVF immune cells might be evolutionarily conserved among mammals.

Three-dimensional tumor spheroids for in vitro analysis of bacteria as gene delivery vectors in tumor therapy.

BACKGROUND: Several studies in animal models demonstrated that obligate and facultative anaerobic bacteria of the genera Bifidobacterium, Salmonella, or Clostridium specifically colonize solid tumors. Consequently, these and other bacteria are discussed as live vectors to deliver therapeutic genes to inhibit tumor growth. Therapeutic approaches for cancer treatment using anaerobic bacteria have been investigated in different mouse models. In the present study, solid three-dimensional (3D) multicellular tumor spheroids (MCTS) of the colorectal adenocarcinoma cell line HT-29 were generated and tested for their potential to study prodrug-converting enzyme therapies using bacterial vectors in vitro. RESULTS: HT-29 MCTS resembled solid tumors displaying all relevant features with an outer zone of proliferating cells and hypoxic and apoptotic regions in the core. Upon incubation with HT-29 MCTS, Bifidobacterium bifidum S17 and Salmonella typhimurium YB1 selectively localized, survived and replicated in hypoxic areas inside MCTS. Furthermore, spores of the obligate anaerobe Clostridium sporogenes germinated in these hypoxic areas. To further evaluate the potential of MCTS to investigate therapeutic approaches using bacteria as gene delivery vectors, recombinant bifidobacteria expressing prodrug-converting enzymes were used. Expression of a secreted cytosine deaminase in combination with 5-fluorocytosine had no effect on growth of MCTS due to an intrinsic resistance of HT-29 cells to 5-fluorouracil, i.e. the converted drug. However, a combination of the prodrug CB1954 and a strain expressing a secreted chromate reductase effectively inhibited MCTS growth. CONCLUSIONS: Collectively, the presented results indicate that MCTS are a suitable and reliable model to investigate live bacteria as gene delivery vectors for cancer therapy in vitro.