TLR4 regulates pulmonary vascular homeostasis and remodeling via redox signaling.

Pulmonary arterial hypertension (PAH) contributes to morbidity and mortality of patients with lung and heart diseases. We demonstrated that hypoxia induced PAH and increased pulmonary arterial wall thickness in wild-type mice. Mice deficient in toll-like receptor 4 (TLR4-/-) spontaneously developed PAH, which was not further enhanced by hypoxia. Echocardiography determined right ventricular hypertrophy and decreased pulmonary arterial acceleration time were associated with the development of PAH in TLR4(-/-) mice. In pulmonary arterial smooth muscle cells (PASMC), hypoxia decreased TLR4 expression and induced reactive oxygen species (ROS) and Nox1/Nox4. Inhibition of NADPH oxidase decreased hypoxia-induced proliferation of wild-type PASMC. PASMC derived from TLR4(-/-) mice exhibited increased ROS and Nox4/Nox1 expression. Our studies demonstrate an important role of TLR4 in maintaining normal pulmonary vasculature and in hypoxia-induced PAH. Inhibition of TLR4, by genetic ablation or hypoxia, increases the expression of Nox1/Nox4 and induces PASMC proliferation and vascular remodeling. These results support a novel function of TLR4 in regulating the development of PAH and reveal a new regulatory axis contributing to TLR4 deficiency-induced vascular hypertrophy and remodeling.

TLR2-mediated expansion of MDSCs is dependent on the source of tumor exosomes.

Exosomes released from tumor cells having been shown to induce interleukin-6 release from myeloid-derived suppressor cells in a Toll-like receptor 2/Stat3-dependent manner. In this study, we show that exosomes released from tumor cells re-isolated from syngeneic mice are capable of inducing interleukin-6 in a Toll-like receptor 2-independent manner, whereas the data generated from exosomes of tumor cells having undergone numerous in vitro passages induce interleukin-6 in a Toll-like receptor 2-dependent manner. This discrepancy may be due to the source of tumor cells used to generate the exosomes for this study. These results suggest that exosomes released from tumor cells that are not within a tumor microenvironment may not realistically represent the role of tumor exosomes in vivo. This is an important consideration since frequently passing tumor cells in vivo is an accepted practice for studying tumor exosome-mediated inflammatory responses.

Contribution of MyD88 to the tumor exosome-mediated induction of myeloid derived suppressor cells.

In this study we observed that mice pretreated with tumor exosomes had a significant acceleration of tumor metastasis in the lung. Tumor metastasis correlated significantly with an increase in recruitment of more Myeloid-derived suppressor cells (MDSCs) in the lung of C57BL/6j (B6) mice pretreated with tumor exosomes. These effects were blunted when MyD88 knockout (KO) mice were pretreated with tumor exosomes. MDSCs induced by tumor exosomes and isolated from wild-type B6 mice also more potently inhibited T cell activation and induction of interleukin-6 and tumor necrosis factor-alpha than MDSCs isolated from the lung of MyD88 KO mice. In vitro, addition of tumor exosomes to bone marrow-derived CD11b(+)Gr-1(+) cells isolated from wild-type B6 mice resulted in more cytokine production, including tumor necrosis factor-alpha, interleukin-6, and the chemokine CCL2, than CD11b(+)Gr-1(+) cells isolated from MyD88 KO mice. Moreover, lower levels of CCL2 were observed in the lungs in MyD88 KO mice pretreated with tumor exosomes than that in wild-type mice. Together these data demonstrate a pivotal role for MyD88 in tumor exosome-mediated expansion of MDSCs and tumor metastasis.

Adipose tissue exosome-like vesicles mediate activation of macrophage-induced insulin resistance.

OBJECTIVE: We sought to determine whether exosome-like vesicles (ELVs) released from adipose tissue play a role in activation of macrophages and subsequent development of insulin resistance in a mouse model. RESEARCH DESIGN AND METHODS: ELVs released from adipose tissue were purified by sucrose gradient centrifugation and labeled with green fluorescent dye and then intravenously injected into B6 ob/ob mice (obese model) or B6 mice fed a high-fat diet. The effects of injected ELVs on the activation of macrophages were determined through analysis of activation markers by fluorescence-activated cell sorter and induction of inflammatory cytokines using an ELISA. Glucose tolerance and insulin tolerance were also evaluated. Similarly, B6 mice with different gene knockouts including TLR2, TLR4, MyD88, and Toll-interleukin-1 receptor (TIR) domain-containing adaptor protein inducing interferon-beta (TRIF) were also used for testing their responses to the injected ELVs. RESULTS: ELVs are taken up by peripheral blood monocytes, which then differentiate into activated macrophages with increased secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Injection of obELVs into wild-type C57BL/6 mice results in the development of insulin resistance. When the obELVs were intravenously injected into TLR4 knockout B6 mice, the levels of glucose intolerance and insulin resistance were much lower. RBP4 is enriched in the obELVs. Bone marrow-derived macrophages preincubated with recombinant RBP4 led to attenuation of obELV-mediated induction of IL-6 and TNF-alpha. CONCLUSIONS: ELVs released by adipose tissue can act as a mode of communication between adipose tissues and macrophages. The obELV-mediated induction of TNF-alpha and IL-6 in macrophages and insulin resistance requires the TLR4/TRIF pathway.

Immature myeloid cells induced by a high-fat diet contribute to liver inflammation.

UNLABELLED: Chronic inflammation plays a critical role in promoting obesity-related disorders, such as fatty liver disease. The inflammatory cells that mediate these effects remain unknown. This study investigated the accumulation of immature myeloid cells in the liver and their role in liver inflammation. We found that the accumulation of immature myeloid cells, i.e., CD11b(+)Ly6C(hi)Ly6G(-) cells, in the liver of B6 mice fed a high-fat diet contribute to liver inflammation. Adoptive transfer of CD11b(+)Ly6C(hi)Ly6G(-) cells isolated from the liver of obese B6 mice, but not from lean B6 mice, resulted in liver damage that was evident by an increase in the activity of liver transferases in serum. CD11b(+)Ly6C(hi)Ly6G(-) cells isolated from the liver of obese mice are more easily activated by way of Toll-like receptor (TLR) stimulation resulting in interleukin 12 and other inflammatory cytokine expression in an MyD88-dependent fashion. TLR7-activated CD11b(+)Ly6C(hi)Ly6G(-) cells also enhance liver natural killer T cell (NKT) death in an Fas-dependent manner. Experiments using mice depleted of Gr-1(+) immature myeloid cells demonstrated the important role of CD11b(+)Ly6C(hi)Ly6G(-) in liver inflammation. Repeated injection of exosome-like particles causes CD11b(+) cell activation and subsequent homing to and accumulation of the cells in the liver. CONCLUSION: Consumption of a high-fat diet by B6 mice triggers an accumulation of immature myeloid cells in the liver. The immature myeloid cells release proinflammatory cytokines and induce NKT cell apoptosis. Activation-induced NKT apoptosis further promotes excessive production of Th-1 cytokines. This diet-induced accumulation of immature myeloid cells may contribute to obesity-related liver disease.

Phospholipase Cgamma2 mediates RANKL-stimulated lymph node organogenesis and osteoclastogenesis.

Phospholipase Cgamma2 (PLCgamma2) is an important signaling effector of multiple receptors in the immune system. Here we show that PLCgamma2-deficient mice displayed impaired lymph node organogenesis but normal splenic structure and Peyer's patches. Receptor activator of NF-kappaB ligand (RANKL) is a tumor necrosis factor family cytokine and is essential for lymph node organogenesis. Importantly, PLCgamma2 deficiency severely impaired RANKL signaling, resulting in marked reduction of RANKL-induced activation of MAPKs, p38 and JNK, but not ERK. The lack of PLCgamma2 markedly diminished RANKL-induced activation of NF-kappaB, AP-1, and NFATc1. Moreover, PLCgamma2 deficiency impaired RANKL-mediated biological function, leading to failure of the PLCgamma2-deficient bone marrow macrophage precursors to differentiate into osteoclasts after RANKL stimulation. Re-introduction of PLCgamma2 but not PLCgamma1 restores RANKL-mediated osteoclast differentiation of PLCgamma2-deficient bone marrow-derived monocyte/macrophage. Taken together, PLCgamma2 is essential for RANK signaling, and its deficiency leads to defective lymph node organogenesis and osteoclast differentiation.