The involvement of NOTCH signaling in macrophage activation by Toll receptors has been clearly established, but the factors and pathways controlling NOTCH signaling during this process have not been completely delineated yet. We have characterized the role of TSPAN33, a tetraspanin implicated in a disintegrin and metalloproteinase (ADAM) 10 maturation, during macrophage proinflammatory activation. Tspan33 expression increases in response to TLR signaling, including responses triggered by TLR4, TLR3, and TLR2 activation, and it is enhanced by IFN-γ. In this study, we report that induction of Tspan33 expression by TLR and IFN-γ is largely dependent on NOTCH signaling, as its expression is clearly diminished in macrophages lacking Notch1 and Notch2 expression, but it is enhanced after overexpression of a constitutively active intracellular domain of NOTCH1. TSPAN33 is the member of the TspanC8 tetraspanin subgroup more intensely induced during macrophage activation, and its overexpression increases ADAM10, but not ADAM17, maturation. TSPAN33 favors NOTCH processing at the membrane by modulating ADAM10 and/or Presenilin1 activity, thus increasing NOTCH signaling in activated macrophages. Moreover, TSPAN33 modulates TLR-induced proinflammatory gene expression, at least in part, by increasing NF-κB–dependent transcriptional activity. Our results suggest that TSPAN33 represents a new control element in the development of inflammation by macrophages that could constitute a potential therapeutic target.

Plant exosomes exhibit high stability and easy absorption, and have emerged as promising bioactive tools due to their potential health benefits and biomedical applications. Saffron tepals contain abundant metabolites with potential therapeutic properties and were used for exosome extraction by ultracentrifugation and gradient purification. The exosomes showed an average particle size of 151.5 ± 79.6 nm and exhibited a spherical morphology. Five well-conserved miRNAs—miR157, miR166, miR168, miR396, and miR398—were identified in the exosomes, which are involved in the coordination of growth and physiological plant responses with endogenous and environmental abiotic and biotic signals, and their potential targets in mammals are upregulated in specific cancer types and associated with inflammation. Proteome analysis revealed an enrichment of proteasome proteins, ribosomal proteins, and proteins involved in the cytoskeleton, transport across the membrane (ABC transporters), and vesicle trafficking (RAB GTPases, TM9SF and Coatomer subunits). Metabolite analyses showed mainly anthocyanins. The exosomes have selective stimulatory activity on macrophages, increasing the expression of surface molecules (CD80 and CD86), and cytokines (IL-1β, IL-6, and TNF-α), but not the levels of IL-10. Overall, these results indicated that saffron flowers are an effective and abundant source of exosomes as new nanomedicines for human health.

Inhibition of Notch signalling in T cells attenuates the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Growing evidence indicates that myeloid cells are also key players in autoimmune processes. Thus, the present study evaluates the role of the Notch1 receptor in myeloid cells on the progression of myelin oligodendrocyte glycoprotein (MOG)_35‐55‐induced EAE, using mice with a myeloid‐specific deletion of the Notch1 gene (MyeNotch1KO). We found that EAE progression was less severe in the absence of Notch1 in myeloid cells. Thus, histopathological analysis revealed reduced pathology in the spinal cord of MyeNotch1KO mice, with decreased microglia/astrocyte activation, demyelination and infiltration of CD4⁺ T cells. Moreover, these mice showed lower Th1 and Th17 cell infiltration and expression of IFN‐γ and IL‐17 mRNA in the spinal cord. Accordingly, splenocytes from MyeNotch1KO mice reactivated in vitro presented reduced Th1 and Th17 activation, and lower expression of IL‐12, IL‐23, TNF‐α, IL‐6, and CD86. Moreover, reactivated wild‐type splenocytes showed increased Notch1 expression, arguing for a specific involvement of this receptor in autoimmune T cell activation in secondary lymphoid tissues. In summary, our results reveal a key role of the Notch1 receptor in myeloid cells for the initiation and progression of EAE.

The mechanisms by which tumor-associated macrophages, key components of the glioblastoma (GBM) microenvironment, impair chemotherapy efficacy remain poorly understood. Resistance to temozolomide (TMZ), the standard chemotherapeutic agent for GBM, is associated with poor prognosis due to efficient DNA repair mechanisms. While low expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) has been linked to improved TMZ response, our previous findings suggest that N-methylpurine-DNA glycosylase (MPG) may also contribute to chemoresistance in GBM. Here, we report for the first time that conditioned medium from pro-inflammatory macrophages (CM-M1) enhances TMZ cytotoxicity by suppressing STAT3 phosphorylation, resulting in decreased MGMT and MPG expression in GBM cells. Proteomic profiling of CM-M1 revealed a unique, cytokine-rich secretome that may promote STAT1 activation, thereby inhibiting pSTAT3 and reducing DNA repair enzymes levels. Clinically, elevated MGMT and MPG protein levels were associated with increased pSTAT3 in our GBM patient cohort, and analysis of the TCGA database further showed that their combined overexpression correlates with significantly reduced progression-free survival. Gene silencing experiments confirmed the contribution of both enzymes to TMZ resistance, with dual knockdown producing a synergistic sensitizing effect. These findings uncover a novel mechanism of macrophage secretome–mediated chemoresistance and support the development of M1-based strategies to improve TMZ efficacy in GBM.

Transient receptor potential canonical (TRPC) channels are calcium channels with diverse expression profiles and physiological implications in the retina. Neurons and glial cells of rat retinas with photoreceptor degeneration caused by retinitis pigmentosa (RP) exhibit basal calcium levels that are above those detected in healthy retinas. Inner retinal cells are the last to degenerate and are responsible for maintaining the activity of the visual cortex even after complete loss of photoreceptors. We considered the possibility that TRPC1 and TRPC5 channels might be associated with both the high calcium levels and the delay in inner retinal degeneration. TRPC1 is known to mediate protective effects in neurodegenerative processes, while TRPC5 promotes cell death. In order to comprehend the implications of these channels in RP, the co-localization and subsequent physical interaction between TRPC1 and TRPC5 in healthy retina (Sprague-Dawley rats) and degenerating (P23H-1, a model of RP) retina were detected by immunofluorescence and proximity ligation assays. There was an overlapping signal in the innermost retina of all animals where TRPC1 and TRPC5 physically interacted. This interaction increased significantly as photoreceptor loss progressed. Both channels function as TRPC1/5 heteromers in the healthy and damaged retina, with a marked function of TRPC1 in response to retinal degenerative mechanisms. Furthermore, our findings support that TRPC5 channels also function in partnership with STIM1 in Müller and retinal ganglion cells. These results suggest that increase in TRPC1/5 heteromers may contribute to slowing of the degeneration of the inner retina during the outer retinal degeneration.

Transient receptor potential canonical (TRPC) channels are calcium channels with diverse expression profiles and physiological implications in the retina. Neurons and glial cells of rat retinas with photoreceptor degeneration caused by retinitis pigmentosa (RP) exhibit basal calcium levels that are above those detected in healthy retinas. Inner retinal cells are the last to degenerate and are responsible for maintaining the activity of the visual cortex, even after complete loss of photoreceptors. We considered the possibility that TRPC1 and TRPC5 channels might be associated with both the high calcium levels and the delay in inner retinal degeneration. TRPC1 is known to mediate protective effects in neurodegenerative processes while TRPC5 promotes cell death. In order to comprehend the implications of these channels in RP, the co-localization and subsequent physical interaction between TRPC1 and TRPC5 in healthy retina (Sprague-Dawley rats) and degenerating (P23H-1, a model of RP) retina were detected by immunofluorescence and proximity ligation assays. There was an overlapping signal in the innermost retina of all animals where TRPC1 and TRPC5 physically interacted. This interaction increased significantly as photoreceptor loss progressed. Both channels function as TRPC1/5 heteromers in the healthy and damaged retina, with a marked function of TRPC1 in response to retinal degenerative mechanisms. Furthermore, our findings support that TRPC5 channels also function in partnership with STIM1 in Müller and retinal ganglion cells. These results suggest that an increase in TRPC1/5 heteromers may contribute to the slowing of the degeneration of the inner retina during the outer retinal degeneration.