Exposure Of Bone Marrow-derived Macrophages Research Articles

Interleukin-23 receptor expressing γδ T cells locally promote early atherosclerotic lesion formation and plaque necrosis in mice.

Atherosclerosis is a chronic inflammatory disease of the vessel wall controlled by local and systemic immune responses. The role of interleukin-23 receptor (IL-23R), expressed in adaptive immune cells (mainly T-helper 17 cells) and γδ T cells, in atherosclerosis is only incompletely understood. Here, we investigated the vascular cell types expressing IL-23R and addressed the function of IL-23R and γδ T cells in atherosclerosis. IL-23R+ cells were frequently found in the aortic root in contrast to the aorta in low-density lipoprotein receptor deficient IL-23R reporter mice (Ldlr-/-Il23rgfp/+), and mostly identified as γδ T cells that express IL-17 and GM-CSF. scRNA-seq confirmed γδ T cells as the main cell type expressing Il23r and Il17a in the aorta. Ldlr-/-Il23rgfp/gfp mice deficient in IL-23R showed a loss of IL-23R+ cells in the vasculature, and had reduced atherosclerotic lesion formation in the aortic root compared to Ldlr-/- controls after 6 weeks of high-fat diet feeding. In contrast, Ldlr-/-Tcrδ-/- mice lacking all γδ T cells displayed unaltered early atherosclerotic lesion formation compared to Ldlr-/- mice. In both HFD-fed Ldlr-/-Il23rgfp/gfp and Ldlr-/-Tcrδ-/- mice a reduction in the plaque necrotic core area was noted as well as an expansion of splenic regulatory T cells. In vitro, exposure of bone marrow-derived macrophages to both IL-17A and GM-CSF induced cell necrosis, and necroptotic RIP3K and MLKL expression, as well as inflammatory mediators. IL-23R+ γδ T cells are predominantly found in the aortic root rather than the aorta and promote early atherosclerotic lesion formation, plaque necrosis, and inflammation at this site. Targeting IL-23R may thus be explored as a therapeutic approach to mitigate atherosclerotic lesion development.

Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related steatohepatitis

ObjectiveBone marrow-derived myeloid cells accumulate in the liver as monocytes and macrophages during the progression of obesity-related non-alcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH). Myeloid cells comprise heterogeneous subsets,...

Analysis of the Trichuris suis excretory/secretory proteins as a function of life cycle stage and their immunomodulatory properties

Parasitic worms have a remarkable ability to modulate host immune responses through several mechanisms including excreted/secreted proteins (ESP), yet the exact nature of these proteins and their targets often remains elusive. Here, we performed mass spectrometry analyses of ESP (TsESP) from larval and adult stages of the pig whipworm Trichuris suis (Ts) and identified ~350 proteins. Transcriptomic analyses revealed large subsets of differentially expressed genes in the various life cycle stages of the parasite. Exposure of bone marrow-derived macrophages and dendritic cells to TsESP markedly diminished secretion of the pro-inflammatory cytokines TNFα and IL-12p70. Conversely, TsESP exposure strongly induced release of the anti-inflammatory cytokine IL-10, and also induced high levels of nitric oxide (NO) and upregulated arginase activity in macrophages. Interestingly, TsESP failed to directly induce CD4+ CD25+ FoxP3+ regulatory T cells (Treg cells), while OVA-pulsed TsESP-treated dendritic cells suppressed antigen-specific OT-II CD4+ T cell proliferation. Fractionation of TsESP identified a subset of proteins that promoted anti-inflammatory functions, an activity that was recapitulated using recombinant T. suis triosephosphate isomerase (TPI) and nucleoside diphosphate kinase (NDK). Our study helps illuminate the intricate balance that is characteristic of parasite-host interactions at the immunological interface, and further establishes the principle that specific parasite-derived proteins can modulate immune cell functions.

Galatrox is a C-type lectin in Bothrops atrox snake venom that selectively binds LacNAc-terminated glycans and can induce acute inflammation.

Previous studies indicate that snake venom contains glycan-binding proteins (GBPs), although the binding specificity and biological activities of many of these GBPs is unclear. Here we report our studies on the glycan binding specificity and activities of galatrox, a Bothrops atrox snake venom-derived GBP. Glycan microarray analysis indicates that galatrox binds most strongly to glycans expressing N-acetyllactosamine (LacNAc), with a significant preference for Galβ1-4GlcNAcβ over Galβ1-3GlcNAcβ compounds. Galatrox also bound immobilized laminin, a LacNAc-dense extracellular matrix component, suggesting that this GBP can bind LacNAc-bearing glycoproteins. As several endogenous mammalian GBPs utilize a similar binding LacNAc binding preference to regulate neutrophil and monocyte activity, we hypothesized that galatrox may mediate B. atrox toxicity through regulation of leukocyte activity. Indeed, galatrox bound neutrophils and promoted leukocyte chemotaxis in a carbohydrate-dependent manner. Similarly, galatrox administration into the mouse peritoneal cavity induced significant neutrophil migration and the release of pro-inflammatory cytokines IL-1α and IL-6. Exposure of bone marrow-derived macrophages to galatrox induced generation of pro-inflammatory mediators IL-6, TNF-α, and keratinocyte-derived chemokine. This signaling by galatrox was mediated via its carbohydrate recognition domain by activation of the TLR4-mediated MyD88-dependent signaling pathway. These results indicate that galatrox has pro-inflammatory activity through its interaction with LacNAc-bearing glycans on neutrophils, macrophages and extracellular matrix proteins and induce the release of pro-inflammatory mediators.

TLR4 is necessary for hyaluronan-mediated airway hyperresponsiveness after ozone inhalation.

Ozone is a common environmental air pollutant that contributes to hospitalizations for respiratory illness. The mechanisms, which regulate ozone-induced airway hyperresponsiveness, remain poorly understood. We have previously reported that toll-like receptor 4 (TLR4)-deficient animals are protected against ozone-induced airway hyperresponsiveness (AHR) and that hyaluronan (HA) mediates ozone-induced AHR. However, the relation between TLR4 and hyaluronan in the airway response to ozone remains unexplored. We hypothesized that HA acts as an endogenous TLR4 ligand for the development of AHR after ozone-induced environmental airway injury. TLR4-deficient and wild-type C57BL/6 mice were exposed to either inhaled ozone or intratracheal HA and the inflammatory and AHR response was measured. TLR4-deficient mice have similar levels of cellular inflammation, lung injury, and soluble HA levels as those of C57BL/6 mice after inhaled ozone exposure. However, TLR4-deficient mice are partially protected from AHR after ozone exposure as well as after direct intratracheal instillation of endotoxin-free low molecular weight HA. Similar patterns of TLR4-dependent cytokines were observed in the bronchial alveolar lavage fluid after exposure to either ozone or HA. Exposure to ozone increased immunohistological staining of TLR4 on lung macrophages. Furthermore, in vitro HA exposure of bone marrow-derived macrophages induced NF-kappaB and production of a similar pattern of proinflammatory cytokines in a manner dependent on TLR4. Our observations support the observation that extracellular matrix HA contributes to ozone-induced airways disease. Furthermore, our results support that TLR4 contributes to the biological response to HA by mediating both the production of proinflammatory cytokines and the development of ozone-induced AHR.

Immunological Consequences of Apoptotic Cell Phagocytosis

Cells undergo apoptosis in development, tissue homeostasis, and disease and are subsequently cleared by professional and nonprofessional phagocytes. There is now overwhelming evidence that phagocyte function is profoundly altered following apoptotic cell uptake, with consequences for the ensuing innate and adaptive immune response. Pathogens and tumors exploit the changes in macrophage function following apoptotic cell uptake. Here, we will outline the consequences of apoptotic cell phagocytosis and illustrate how apoptotic cells could be used to manipulate the immune response for therapeutic gain.

TLR2 but not TLR4 Signalling is Critically Involved in the Inhibition of IFN‐γ‐induced Killing of Mycobacteria by Murine Macrophages

Gamma-interferon (IFN-gamma) plays a determinant role in activating macrophages that are critical to control Mycobacterium tuberculosis infection. However, M. tuberculosis can escape killing by attenuating the response of macrophages to IFN-gamma by blocking the transcription of a subset of IFN-gamma inducible genes. This inhibition occurs after signalling through Toll-like receptor 2 (TLR2). While most studies have investigated the inhibition of IFN-gamma responsive genes after TLR2 signalling, the present study focuses on the functional implications of inhibition of IFN-gamma signalling in macrophages with regard to mycobacteria killing. Here, we provide evidence that exposure of the murine macrophage cell line J774 to the TLR2 ligands; 19-kDa or zymosan, but not the TLR4 ligand LPS, inhibits IFN-gamma-induced killing of Mycobacterium bovis Bacillus Calmette-Guérin (BCG). Moreover, exposure of bone marrow-derived macrophages (BMM) from TLR4-deficient and wild-type (WT), but not from TLR2-deficient mice to 19-kDa lipoprotein (19-kDa) or zymosan, results in an impairment of IFN-gamma-mediated killing. We demonstrate that 19-kDa and zymosan inhibit the ability of IFN-gamma to activate murine macrophages to kill BCG without inhibiting nitric oxide (NO) or tumour necrosis factor (TNF) production. Finally, we demonstrate that the inhibitory effect of 19-kDa on IFN-gamma signalling is overcome with increasing amounts of IFN-gamma indicating that the refractoriness could be reversed at optimal IFN-gamma concentrations. The critical role of TLR2 but not TLR4 signalling in the inhibition of IFN-gamma promoted killing of mycobacteria is discussed.

Phagocytosis enhances murine macrophage activation by interferon‐γ and tumor necrosis factor‐α

Previously, we reported that exposure of bone marrow-derived macrophages (M phi) to a phagocytic stimulus in the simultaneous presence of interferon-gamma (IFN-gamma) induced these cells to generate nitrite (NO2-). This effect was achieved using both living (i.e. promastigotes of the protozoan parasite Leishmania enriettii) and inert (latex beads) particles. When the phagocytic stimulus was Leishmania, enhanced intracellular killing accompanied elevated NO2- secretion. As shown here, the capacity of phagocytosis to elicit NO2- production by IFN-gamma-treated M phi was inhibited by antibody to murine recombinant tumor necrosis factor-alpha (rTNF-alpha), suggesting that phagocytosis enabled IFN-gamma to activate M phi via the induction of TNF-alpha as an autocrine second signal. M phi NO2- production in response to rIFN-gamma and either exogenous TNF-alpha or Leishmania was strongly enhanced by prostaglandin E2, consistent with such a mechanism. However, addition of either Leishmania promastigotes or latex beads to M phi cultures simultaneously exposed to both IFN-gamma and exogenous murine or human rTNF-alpha further potentiated activation as measured by NO2- release. Furthermore, anti-TNF antibody failed to inhibit M phi responses to rIFN-gamma and bacterial lipopolysaccharide (LPS) in the presence or absence of Leishmania; also exogenous rTNF-alpha did not significantly affect NO2- production by IFN-gamma/LPS cultures despite a strong enhancement by Leishmania. These results suggest that phagocytosis enhances M phi responses by a process more complex than the sole induction of TNF-alpha. Phagocytosis also increased M phi NO2- production elicited by IFN-gamma plus TNF-alpha in L-arginine-deficient media. These results indicate that phagocytosis may be an important mechanism of up-regulating M phi microbicidal activity, and could be particularly relevant upon arginine depletion which occurs during an inflammatory response.