Protocol for making an animal model of "blindsight" in macaque monkeys.

Patients with damage to the primary visual cortex (V1) can respond correctly to visual stimuli in their lesion-affected visual field above the chance level, an ability named blindsight. Here, we present a protocol for making an animal model of blindsight in macaque monkeys. We describe the steps to perform pre-lesion training of monkeys on a visual task, followed by lesion surgery, post-lesion training, and evaluation of blindsight. This animal model can be used to investigate the source of visual awareness. For complete details on the use and execution of this protocol, please refer to Yoshida et al. (2008)1 and Takakuwa et al. (2021).2.

IL-23 production in human macrophages is regulated negatively by tumor necrosis factor α-induced protein 3 and positively by specificity protein 1 after stimulation of the toll-like receptor 7/8 signaling pathway.

The IL-23/IL-17 axis plays an important role in the development of autoimmune diseases, but the mechanism regulating IL-23 production is mainly unknown. We investigated how TNFAIP3 and Sp1 affect IL-23 production by human macrophages after exposure to resiquimod, a TLR7/8 agonist. IL-23 production was significantly upregulated by resiquimod but only slightly by LPS (a TLR4 agonist). Interestingly, IL-23 levels were significantly attenuated after sequential stimulation with LPS and resiquimod, but IL-12p40 and IL-18 levels were not. TLR4-related factors induced by LPS may regulate IL-23 expression via TLR7/8 signaling. LPS significantly enhanced TNFAIP3 and IRAK-M levels but reduced Sp1 levels. After exposure to resiquimod, RNA interference of TNFAIP3 upregulated IL-23 significantly more than siRNA transfection of IRAK-M did. In contrast, knockdown of Sp1 by RNA interference significantly attenuated IL-23 production. Transfection with siRNA for TNFAIP3 enhanced IL-23 expression significantly. After stimulation with resiquimod, GW7647-an agonist for PPARα (an inducer of NADHP oxidase)-and siRNA for UCP2 (a negative regulator of mitochondrial ROS generation) enhanced TNFAIP3 and reduced IL-23. siRNA for p22phox and gp91phox slightly increased Sp1 levels. However, after exposure to resiquimod siRNA-mediated knockout of DUOX1/2 significantly enhanced Sp1 and IL-23 levels, and decreased TNFα-dependent COX-2 expression. Concomitantly, TNFAIP3 levels was attenuated by DUOX1/2 siRNA. TNFAIP3 and Sp1 levels are reciprocally regulated through ROS generation. In conclusion, after stimulation of the TLR7/8 signaling pathway IL-23 production in human macrophages is regulated negatively by TNFAIP3.

TRIM28/TIF1ß and Fli-1 negatively regulate peroxynitrite generation via DUOX2 to decrease the shedding of membrane-bound fractalkine in human macrophages after exposure to substance P.

The chemokine fractalkine is synthesized as a membrane-bound protein, but studies have shown that serum levels of soluble fractalkine are elevated in inflammatory and autoimmune diseases. Patients with autoimmune diseases also have increased serum levels of neuropeptide substance P (SP). The shedding activity of the ADAM family is induced by peroxynitrite, but that of SP is unclear. Treatment of human macrophages with SP upregulated levels of membrane-bound fractalkine. Interestingly, small interfering RNA (siRNA) for DUOX2 further increased membrane-bound fractalkine but decreased soluble fractalkine compared with cells treated with SP alone. SP induced nitric oxide 2/inducible nitric oxide synthase (NOS2/iNOS) mRNA and increased levels of nitrotyrosine, a biomarker of peroxynitrite, whereas transfection with DUOX2 siRNA blunted upregulation of nitrotyrosine. Most importantly, N(ω)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) decreased protein levels of nitrotyrosine and concomitantly increased expression of membrane-bound fractalkine after exposure to SP. As for the signaling pathway of TGFß1 (an inhibitor of iNOS mRNA expression), silencing of RNA for TAK-1 upregulated membrane-bound fractalkine, but silencing of RNA for the Smad family did not. Interfering RNA of transcription factor specificity protein 1 (Sp1) upregulated protein levels of TGFß1/LAP. Most importantly, double transfection with siRNA for Sp1 and TRIM28/TIF1ßor Fli-1 led to a significant increase in TGFß1/LAP levels and a corresponding reduction of NOS2/iNOS, which inhibited the shedding of membrane-bound fractalkine. In conclusion, TRIM28/TIF1ß and Fli-1 negatively regulate TGFß1 expression to upregulate the generation of peroxynitrite, leading to increased shedding of membrane-bound fractalkine induced by SP.

Di-(2-Ethylhexyl) Phthalate Promotes Release of Tissue Factor-Bearing Microparticles From Macrophages via the TGFß1/Smad/PAI-1 Signaling Pathway.

BACKGROUND: Plasminogen activator inhibitor type 1 promotes formation of endothelial microparticles with procoagulant activity. However, it remains unclear whether di-(2-ethylhexyl) phthalate, a peroxisome proliferator-activated receptor α agonist, influences microparticle formation. MATERIALS AND METHODS: The effect of di-(2-ethylhexyl) phthalate on release of tissue factor-bearing microparticles was investigated using human M1 macrophages. RESULTS: Exposure of M1 macrophages to di-(2-ethylhexyl) phthalate significantly upregulated expression of plasminogen activator inhibitor type 1, whereas incubation of macrophages with small interfering RNA for peroxisome proliferator-activated receptor α attenuated it. Di-(2-ethylhexyl) phthalate significantly increased the tissue factor protein level in culture supernatants of M1 macrophages, but not M2 macrophages. After purification of proteins by centrifugal filtration, western blotting detected 2 high molecular weight bands of tissue factor-bearing microparticles in culture supernatants of M1 macrophages. The upper band showed binding to factor VIIa and tissue factor pathway inhibitor, unlike the lower band. This suggested heterogeneity of the procoagulant activity of tissue factor-bearing microparticles, presumably dependent upon encryption/decryption of tissue factor. Phosphatidylserine contributes to tissue factor decryption, and western blotting revealed that the density of phosphatidylserine was reduced in the upper tissue factor band compared with the lower band. Di-(2-ethylhexyl) phthalate also upregulated transforming growth factor-ß1 protein production by M1 macrophages. Moreover, silencing of Smad2, Smad3 or Smad4 attenuated plasminogen activator inhibitor type 1 expression and tissue factor-release from macrophages after di-(2-ethylhexyl) phthalate stimulation. CONCLUSIONS: Di-(2-ethylhexyl) phthalate promotes formation of tissue factor-bearing microparticles in human M1 macrophages via the transforming growth factor-ß1/Smad/ plasminogen activator inhibitor type 1 signaling pathway.

Transcription factor specificity protein 1 modulates TGFß1/Smad signaling to negatively regulate SIGIRR expression by human M1 macrophages stimulated with substance P.

The stimuli inducing expression of single immunoglobulin IL-1-related receptor (SIGIRR) and the relevant regulatory mechanisms are not well defined. Transforming growth factor ß1 (TGFß1) delays internalization of neurokinin-1 receptor (NK1R) and subsequently enhances cellular signaling. This study investigated the effect of TGFß1 on SIGIRR protein production by human M1 macrophages in response to stimulation with substance P (SP). SP caused upregulation of SIGIRR expression in a concentration-dependent manner, whereas aprepitant (an NK1R inhibitor) blunted this response. Silencing p38γMAPK or TAK-1 partially attenuated the response to SP stimulation, while TGFß1/2/3 siRNA dramatically diminished it. SP induced much greater SIGIRR protein production than either lipopolysaccharide (a TLR4 agonist) or resiquimod (a TLR7/8 agonist). Unexpectedly, silencing of transcription factor specificity protein 1 (Sp1) led to significant upregulation of SIGIRR expression after SP stimulation, while KLF2 siRNA only partially enhanced it and Fli-1 siRNA reduced it. SP also upregulated TGFß1 expression, along with a corresponding increase of SIGIRR protein, whereas silencing TGFß1/2/3 blunted these responses. Sp1 siRNA or mithramycin (a gene-selective Sp1 inhibitor) significantly enhanced the expression of TGFß1 and SIGIRR by macrophages after SP stimulation. Importantly, this effect of Sp1 siRNA on TGFß1 and SIGIRR was blunted by siRNA for Smad2, Smad3, or Smad4, but not by TAK-1 siRNA. Next, we investigated the influence of transcription factor cross-talk on SIGIRR expression in response to SP. Co-transfection of macrophages with Sp1 siRNA and C/EBPß or TIF1ß siRNA attenuated the upregulation of SIGIRR by SP, while a combination of Sp1 siRNA and Fli-1 siRNA dramatically diminished it. In conclusion, TGFß1 may be an intermediary between SP/NK1R activation and SIGIRR expression in Sp1 siRNA-transfected macrophages. In addition, Sp1 modulates TGFß1/Smad signaling and negatively regulates SIGIRR protein production by macrophages after SP stimulation.