ETV3 and ETV6 enable monocyte differentiation into dendritic cells by repressing macrophage fate commitment.

In inflamed tissues, monocytes differentiate into macrophages (mo-Macs) or dendritic cells (mo-DCs). In chronic nonresolving inflammation, mo-DCs are major drivers of pathogenic events. Manipulating monocyte differentiation would therefore be an attractive therapeutic strategy. However, how the balance of mo-DC versus mo-Mac fate commitment is regulated is not clear. In the present study, we show that the transcriptional repressors ETV3 and ETV6 control human monocyte differentiation into mo-DCs. ETV3 and ETV6 inhibit interferon (IFN)-stimulated genes; however, their action on monocyte differentiation is independent of IFN signaling. Instead, we find that ETV3 and ETV6 directly repress mo-Mac development by controlling MAFB expression. Mice deficient for Etv6 in monocytes have spontaneous expression of IFN-stimulated genes, confirming that Etv6 regulates IFN responses in vivo. Furthermore, these mice have impaired mo-DC differentiation during inflammation and reduced pathology in an experimental autoimmune encephalomyelitis model. These findings provide information about the molecular control of monocyte fate decision and identify ETV6 as a therapeutic target to redirect monocyte differentiation in inflammatory disorders.

Circadian Expression of Migratory Factors Establishes Lineage-Specific Signatures that Guide the Homing of Leukocyte Subsets to Tissues.

The number of leukocytes present in circulation varies throughout the day, reflecting bone marrow output and emigration from blood into tissues. Using an organism-wide circadian screening approach, we detected oscillations in pro-migratory factors that were distinct for specific vascular beds and individual leukocyte subsets. This rhythmic molecular signature governed time-of-day-dependent homing behavior of leukocyte subsets to specific organs. Ablation of BMAL1, a transcription factor central to circadian clock function, in endothelial cells or leukocyte subsets demonstrated that rhythmic recruitment is dependent on both microenvironmental and cell-autonomous oscillations. These oscillatory patterns defined leukocyte trafficking in both homeostasis and inflammation and determined detectable tumor burden in blood cancer models. Rhythms in the expression of pro-migratory factors and migration capacities were preserved in human primary leukocytes. The definition of spatial and temporal expression profiles of pro-migratory factors guiding leukocyte migration patterns to organs provides a resource for the further study of the impact of circadian rhythms in immunity.

Lymphocyte Circadian Clocks Control Lymph Node Trafficking and Adaptive Immune Responses.

Lymphocytes circulate through lymph nodes (LN) in search for antigen in what is believed to be a continuous process. Here, we show that lymphocyte migration through lymph nodes and lymph occurred in a non-continuous, circadian manner. Lymphocyte homing to lymph nodes peaked at night onset, with cells leaving the tissue during the day. This resulted in strong oscillations in lymphocyte cellularity in lymph nodes and efferent lymphatic fluid. Using lineage-specific genetic ablation of circadian clock function, we demonstrated this to be dependent on rhythmic expression of promigratory factors on lymphocytes. Dendritic cell numbers peaked in phase with lymphocytes, with diurnal oscillations being present in disease severity after immunization to induce experimental autoimmune encephalomyelitis (EAE). These rhythms were abolished by genetic disruption of T cell clocks, demonstrating a circadian regulation of lymphocyte migration through lymph nodes with time-of-day of immunization being critical for adaptive immune responses weeks later.

TLR or NOD receptor signaling skews monocyte fate decision via distinct mechanisms driven by mTOR and miR-155.

Monocytes are rapidly recruited to inflamed tissues where they differentiate into monocyte-derived macrophages (mo-mac) or dendritic cells (mo-DC). At infection sites, monocytes encounter a broad range of microbial motifs. How pathogen recognition impacts monocyte fate decision is unclear. Here, we show, using an in vitro model allowing the simultaneous differentiation of human mo-mac and mo-DC, that viruses promote mo-mac while Mycobacteria favor mo-DC differentiation. Mechanistically, we found that pathogen sensing through toll-like receptor (TLR) ligands increases mo-mac differentiation via mTORC1. By contrast, nucleotide-binding oligomerization domain (NOD) ligands favor mo-DC through the induction of TNF-α secretion and miR-155 expression. We confirmed these results in vivo, in mouse skin and by analyzing transcriptomic data from human individuals. Overall, our findings allow a better understanding of the molecular control of monocyte differentiation and of monocyte plasticity upon pathogen sensing.

Artery-Associated Sympathetic Innervation Drives Rhythmic Vascular Inflammation of Arteries and Veins.

BACKGROUND: The incidence of acute cardiovascular complications is highly time-of-day dependent. However, the mechanisms driving rhythmicity of ischemic vascular events are unknown. Although enhanced numbers of leukocytes have been linked to an increased risk of cardiovascular complications, the role that rhythmic leukocyte adhesion plays in different vascular beds has not been studied. METHODS: We evaluated leukocyte recruitment in vivo by using real-time multichannel fluorescence intravital microscopy of a tumor necrosis factor-α-induced acute inflammation model in both murine arterial and venous macrovasculature and microvasculature. These approaches were complemented with genetic, surgical, and pharmacological ablation of sympathetic nerves or adrenergic receptors to assess their relevance for rhythmic leukocyte adhesion. In addition, we genetically targeted the key circadian clock gene Bmal1 (also known as Arntl) in a lineage-specific manner to dissect the importance of oscillations in leukocytes and components of the vessel wall in this process. RESULTS: In vivo quantitative imaging analyses of acute inflammation revealed a 24-hour rhythm in leukocyte recruitment to arteries and veins of the mouse macrovasculature and microvasculature. Unexpectedly, although in arteries leukocyte adhesion was highest in the morning, it peaked at night in veins. This phase shift was governed by a rhythmic microenvironment and a vessel type-specific oscillatory pattern in the expression of promigratory molecules. Differences in cell adhesion molecules and leukocyte adhesion were ablated when disrupting sympathetic nerves, demonstrating their critical role in this process and the importance of ß2-adrenergic receptor signaling. Loss of the core clock gene Bmal1 in leukocytes, endothelial cells, or arterial mural cells affected the oscillations in a vessel type-specific manner. Rhythmicity in the intravascular reactivity of adherent leukocytes resulted in increased interactions with platelets in the morning in arteries and in veins at night with a higher predisposition to acute thrombosis at different times as a consequence. CONCLUSIONS: Together, our findings point to an important and previously unrecognized role of artery-associated sympathetic innervation in governing rhythmicity in vascular inflammation in both arteries and veins and its potential implications in the occurrence of time-of-day-dependent vessel type-specific thrombotic events.

The aryl hydrocarbon receptor shapes monocyte transcriptional responses to interleukin-4 by prolonging STAT6 binding to promoters.

Cytokines induce functional and metabolic adaptations in immune cells, typically through transcriptional responses that can be influenced by other extracellular signals and by intracellular factors. The binding of the cytokine interleukin-4 (IL-4) to its receptor induces the phosphorylation and activation of the transcription factor STAT6. The aryl hydrocarbon receptor (AhR), a transcription factor activated by various endogenous and microbe-derived metabolites, modulates the responses of immune cells to danger signals or inflammatory mediators such as cytokines. Here, we investigated cross-talk between the AhR and signaling stimulated by IL-4 in human and mouse monocytes. AhR activation was required for a subset of IL-4-induced transcriptional responses and inhibited the IL-4-induced metabolic switch to fatty acid ß-oxidation. The promoters of the genes that were induced by IL-4 in an AhR-dependent manner lacked canonical AhR binding sites, implying a nongenomic mechanism of AhR action. Mechanistically, AhR activation reduced the activity of SHP-1, a phosphatase that targets and inhibits STAT6, and prolonged STAT6 phosphorylation and binding to specific target loci, thus extending the duration of STAT6 activity. Our results identify AhR as a key player in the molecular control of responses to IL-4 in monocytes and suggest a nongenomic mechanism through which AhR ligands may influence the functional responses of cells to IL-4.

Homeostatic activation of aryl hydrocarbon receptor by dietary ligands dampens cutaneous allergic responses by controlling Langerhans cells migration.

Dietary compounds can affect the development of inflammatory responses at distant sites. However, the mechanisms involved remain incompletely understood. Here, we addressed the influence on allergic responses of dietary agonists of aryl hydrocarbon receptor (AhR). In cutaneous papain-induced allergy, we found that lack of dietary AhR ligands exacerbates allergic responses. This phenomenon was tissue-specific as airway allergy was unaffected by the diet. In addition, lack of dietary AhR ligands worsened asthma-like allergy in a model of 'atopic march.' Mice deprived of dietary AhR ligands displayed impaired Langerhans cell migration, leading to exaggerated T cell responses. Mechanistically, dietary AhR ligands regulated the inflammatory profile of epidermal cells, without affecting barrier function. In particular, we evidenced TGF-ß hyperproduction in the skin of mice deprived of dietary AhR ligands, explaining Langerhans cell retention. Our work identifies an essential role for homeostatic activation of AhR by dietary ligands in the dampening of cutaneous allergic responses and uncovers the importance of the gut-skin axis in the development of allergic diseases.

Influence of circadian clocks on adaptive immunity and vaccination responses.

The adaptive immune response is under circadian control, yet, why adaptive immune reactions continue to exhibit circadian changes over long periods of time is unknown. Using a combination of experimental and mathematical modeling approaches, we show here that dendritic cells migrate from the skin to the draining lymph node in a time-of-day-dependent manner, which provides an enhanced likelihood for functional interactions with T cells. Rhythmic expression of TNF in the draining lymph node enhances BMAL1-controlled ICAM-1 expression in high endothelial venules, resulting in lymphocyte infiltration and lymph node expansion. Lymph node cellularity continues to be different for weeks after the initial time-of-day-dependent challenge, which governs the immune response to vaccinations directed against Hepatitis A virus as well as SARS-CoV-2. In this work, we present a mechanistic understanding of the time-of-day dependent development and maintenance of an adaptive immune response, providing a strategy for using time-of-day to optimize vaccination regimes.

Isolation and Culturing of Primary Mouse and Human Macrophages.

Macrophages are mature immune cells involved in the defense of the organism. Since their discovery, the main function attributed to macrophages has been phagocytosis. However, in recent years, several new functions such as angiogenesis, tissue remodeling, clearance of apoptotic cells, pro- and anti-inflammatory properties and tumor growth have been attributed to macrophages. To perform such varied functions, macrophages acquire specific phenotypes in response to external signals. The possibility of replicating these phenotypes in vitro represents a cutting-edge tool to understand potential macrophage functions in vivo. This chapter outlines protocols used to isolate and culture murine bone marrow-derived and human monocyte-derived macrophages. Furthermore, macrophage polarization processes into different phenotypes, with special relevance to atherosclerosis, are indicated.

[Health risk behaviors by occupation in working population in Spain]. / Conductas de riesgo para la salud según la ocupación en población empleada en España.

OBJECTIVE: This study aims to analyze the variations in the prevalence of different health risk behaviors according to occupation in working population in Spain. METHOD: Cross-sectional study with data from the Spanish National Health Survey of 2017. The analysis includes adults between 18 and 65 years with employment at the time of the survey. Health risk behaviors are obesity, physical inactivity in free time, tobacco consumption and excessive alcohol consumption. The primary explanatory variable is the occupation, using the National Classification of Occupations of 2011. Sociodemographic characteristics are gender, age, country of birth and educational level. The prevalences (P) of risk behaviors have been calculated, as well as the odd and adjusted odds ratios (aOR). RESULTS: The highest figures of obesity are observed in operators of installations and machinery and assemblers (P: 20.0%; ORa: 1.26; A95%CI: 1.04-1.52). The higher level of physical inactivity during free time appears in elementary occupations (P: 83.4%; ORa: 1.70; A95%CI: 1.45-1.99). Tobacco consumption is higher in operators of installations and machinery and assemblies (P: 37.4%; ORa: 1.22; A95%CI: 1.05-1.43). Excessive alcohol consumption appears to a greater extent on skilled workers in the agricultural sector, livestock, forestry and fisheries (P: 3.9%; ORa: 1.51; A95%CI: 0.83-2.75). CONCLUSIONS: The results indicate a greater relationship between risk behaviors for health and manual or lower-skilled occupations.

Modulation of Immune Responses by Nutritional Ligands of Aryl Hydrocarbon Receptor.

Accumulating evidence indicates that nutrition can modulate the immune system through metabolites, either produced by host digestion or by microbiota metabolism. In this review, we focus on dietary metabolites that are agonists of the Aryl hydrocarbon Receptor (AhR). AhR is a ligand-activated transcription factor, initially characterized for its interaction with xenobiotic pollutants. Numerous studies have shown that AhR also recognizes indoles and tryptophan catabolites originating from dietary compounds and commensal bacteria. Here, we review recent work employing diet manipulation to address the impact of nutritional AhR agonists on immune responses, both locally in the intestine and at distant sites. In particular, we examine the physiological role of these metabolites in immune cell development and functions (including T lymphocytes, innate-like lymphoid cells, and mononuclear phagocytes) and their effect in inflammatory disorders.

Loss of direct adrenergic innervation after peripheral nerve injury causes lymph node expansion through IFN-γ.

Peripheral nerve injury can cause debilitating disease and immune cell-mediated destruction of the affected nerve. While the focus has been on the nerve-regenerative response, the effect of loss of innervation on lymph node function is unclear. Here, we show that the popliteal lymph node (popLN) receives direct neural input from the sciatic nerve and that sciatic denervation causes lymph node expansion. Loss of sympathetic, adrenergic tone induces the expression of IFN-γ in LN CD8 T cells, which is responsible for LN expansion. Surgery-induced IFN-γ expression and expansion can be rescued by ß2 adrenergic receptor agonists but not sensory nerve agonists. These data demonstrate the mechanisms governing the pro-inflammatory effect of loss of direct adrenergic input on lymph node function.

Differences in the Prevalence of Fruit and Vegetable Consumption in Spanish Workers.

The present study aims to examine the differences in daily fruit and vegetable consumption in the working population in Spain. A cross-sectional study was conducted, using data from the 2017 National Health Survey (n = 10,700 workers aged between 18 and 65 years). The daily consumption of fruit and vegetables was evaluated using two items included in a food frequency questionnaire. Occupations were classified into the 17 main groups of the National Classification of Occupations of 2011 (CNO-11). The prevalence (P) of daily fruit and vegetable consumption was calculated in relation to sociodemographic characteristics, health behaviors, work-related characteristics and occupations. Logistic regression analysis was performed to examine the association, with simple and adjusted Odds Ratio (aOR). The P of daily consumption of fruit and vegetables in workers was 60% for fruit and 40% for vegetables. After adjusting for sociodemographic characteristics and health behaviors, workers working night or rotating shifts had a lower consumption of fruits (aOR:0.9; p < 0.05), and those working on temporary contracts had a lower consumption of vegetables (aOR:0.8; p < 0.05). Engineers, scientists, health care workers and teachers had the highest fruit consumption (74.5%) and the highest vegetable consumption (55.1%). The lowest consumption of fruits was presented by the military (42.3%) and unskilled workers in the service sector (45.8%), and the lowest consumption of vegetables was presented by skilled construction workers (25.5%). These findings could aid in workplace health promotion and could be used in future studies to evaluate the impact of the activities adopted.

Isolation, Culture, and Polarization of Murine Bone Marrow-Derived and Peritoneal Macrophages.

Macrophages are the most specialized phagocytic cells, and acquire specific phenotypes and functions in response to a variety of external triggers. Culture of bone marrow-derived or peritoneal macrophages from mice represents an exceptionally powerful technique to investigate macrophage phenotypes and functions in response to specific stimuli, resembling as much as possible the conditions observed in various pathophysiological settings. This chapter outlines protocols used to isolate and culture murine bone marrow-derived and peritoneal macrophages. Furthermore, we describe how these macrophages can be "polarized" to obtain specific macrophage subsets with special relevance to atherosclerosis.

Circadian rhythms in leukocyte trafficking.

A broad range of immunological processes oscillates over the course of a day. Recent findings have identified a molecular basis for the circadian clock in the regulation of the immune system. These rhythms manifest themselves in oscillatory behavior of immune cells and proinflammatory mediators, which causes a time-dependent sensitivity in the reaction to pathogens. This rhythmicity impacts disease manifestations and severity and provides an option for therapy that incorporates chronopharmacological considerations. This review will focus on the current knowledge and relevance of rhythmic immune cell trafficking. It will provide an overview of the molecular clock machinery and its interrelations with leukocyte migration and the immune response.

In vivo inhibition of c-MYC in myeloid cells impairs tumor-associated macrophage maturation and pro-tumoral activities.

Although tumor-associated macrophages (TAMs) are involved in tumor growth and metastasis, the mechanisms controlling their pro-tumoral activities remain largely unknown. The transcription factor c-MYC has been recently shown to regulate in vitro human macrophage polarization and be expressed in macrophages infiltrating human tumors. In this study, we exploited the predominant expression of LysM in myeloid cells to generate c-Myc(fl/fl) LysM(cre/+) mice, which lack c-Myc in macrophages, to investigate the role of macrophage c-MYC expression in cancer. Under steady-state conditions, immune system parameters in c-Myc(fl/fl) LysM(cre/+) mice appeared normal, including the abundance of different subsets of bone marrow hematopoietic stem cells, precursors and circulating cells, macrophage density, and immune organ structure. In a model of melanoma, however, TAMs lacking c-Myc displayed a delay in maturation and showed an attenuation of pro-tumoral functions (e.g., reduced expression of VEGF, MMP9, and HIF1α) that was associated with impaired tissue remodeling and angiogenesis and limited tumor growth in c-Myc(fl/fl) LysM(cre/+) mice. Macrophage c-Myc deletion also diminished fibrosarcoma growth. These data identify c-Myc as a positive regulator of the pro-tumoral program of TAMs and suggest c-Myc inactivation as an attractive target for anti-cancer therapy.