Neutrophil extracellular trap-enriched supernatants carry microRNAs able to modulate TNF-α production by macrophages.

Neutrophil extracellular traps (NETs) emerge from the cell as a DNA scaffold associated with cytoplasmic and granular proteins, able to immobilize and kill pathogens. This association occurs following nuclear and granular membrane disintegration, allowing contact with the decondensed chromatin. Thus, it is reasonable to speculate that the DNA can also mix with miRNAs and carry them in NETs. Here, we report for the first time the presence of the miRNA carriers associated with NETs and miRNAs present in NET-enriched supernatants (NET-miRs), thus adding a novel class of molecules and new proteins that can be released and transported in the NET platform. We observed that the majority of NET-miRs were common to all four stimuli used (PMA, interleukin-8, amyloid fibrils and Leishmania), and that miRNA-142-3p carried by NETs down-modulates protein kinase Cα and regulates TNF-α production in macrophages upon NET interaction with these cells. Our findings unveil a novel role for NETs in the cell communication processes, allowing the conveyance of miRNA from neutrophils to neighboring cells.

Neuroendocrine Control of Macrophage Development and Function.

Macrophages carry out numerous physiological activities that are essential for both systemic and local homeostasis, as well as innate and adaptive immune responses. Their biology is intricately regulated by hormones, neuropeptides, and neurotransmitters, establishing distinct neuroendocrine axes. The control is pleiotropic, including maturation of bone marrow-derived myeloid precursors, cell differentiation into functional subpopulations, cytotoxic activity, phagocytosis, production of inflammatory mediators, antigen presentation, and activation of effector lymphocytes. Additionally, neuroendocrine components modulate macrophage ability to influence tumor growth and to prevent the spreading of infective agents. Interestingly, macrophage-derived factors enhance glucocorticoid production through the stimulation of the hypothalamic-pituitary-adrenal axis. These bidirectional effects highlight a tightly controlled balance between neuroendocrine stimuli and macrophage function in the development of innate and adaptive immune responses. Herein, we discuss how components of neuroendocrine axes impact on macrophage development and function and may ultimately influence inflammation, tissue repair, infection, or cancer progression. The knowledge of the crosstalk between macrophages and endocrine or brain-derived components may contribute to improve and create new approaches with clinical relevance in homeostatic or pathological conditions.

Protective effect of gedunin on TLR-mediated inflammation by modulation of inflammasome activation and cytokine production: Evidence of a multitarget compound.

Activation of toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) triggers an innate immune response, via cytokine production and inflammasome activation. Herein, we have investigated the modulatory effect of the natural limonoid gedunin on TLR activation in vitro and in vivo. Intraperitoneal (i.p.) pre- and post-treatments of C57BL/6 mouse with gedunin impaired the influx of mononuclear cells, eosinophils and neutrophils, as well as the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and nitric oxide (NO), triggered by lipopolysaccharide (LPS) in mouse pleura. Accordingly, in vitro post-treatment of immortalized murine macrophages with gedunin also impaired LPS-induced production of such mediators. Gedunin diminished LPS-induced expression of the nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) on pleural leukocytes in vivo and in immortalized macrophages in vitro. In line with this, gedunin inhibited LPS-induced caspase-1 activation and the production of IL-1ß in vivo and in vitro. In addition, gedunin treatment triggered the generation of the anti-inflammatory factors IL-10 and heme oxigenase-1 (HO-1) at resting conditions or upon stimulation. We also demonstrate that gedunin effect is not restricted to TLR4-mediated response, since this compound diminished TNF-α, IL-6, NO, NLRP3 and IL-1ß, as well as enhanced IL-10 and HO-1, by macrophages stimulated with the TLR2 and TLR3 agonists, palmitoyl-3-Cys-Ser-(Lys)4 (PAM3) and polyriboinosinic:polyribocytidylic acid (POLY I:C), in vitro. In silico modeling studies revealed that gedunin efficiently docked into caspase-1, TLR2, TLR3 and to the myeloid differentiation protein-2 (MD-2) component of TLR4. Overall, our data demonstrate that gedunin modulates TLR4, TLR3 and TLR2-mediated responses and reveal new molecular targets for this compound.

HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR.

HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.