“…Stimulated myeloid cells normally contain only one or two ASCcontaining inflammasomes (Baroja-Mazo et al, 2014;Franklin et al, 2014) (see also Fig 3C) that are also significantly larger than most of the NLRP3 signals seen in these cells (see Fig EV4C and D). Murine NLRP3 has a calculated molecular weight of 113 kDa and the pyrin domain has to exist at least as a trimer to trigger IL-1beta production, which is apparently independent of the formation of ASCcontaining inflammasome (Susjan et al, 2017). Thus, it seems plausible that the observed smaller NLRP3 aggregates present NLRP3 trimers or higher oligomeric forms as suggested for NLRP2 (Faustin et al, 2007) or NLRC4 (Hu et al, 2015;Zhang et al, 2015).…”
Section: Discussionmentioning
confidence: 87%
“…Non treated microglia showed, in correlation to their weak band in the Western blot, a diffuse cytoplasmic staining with a few small NLRP3 puncta ( Fig 4A). Stimulation of microglia with LPS/ATP resulted in the emergence of more and larger NLRP3 puncta/specs of different sizes ( Fig 4A, arrows), indicating formation of NLRP3 oligomers or aggregates (Susjan et al, 2017) (see also Discussion). Quantification of NLRP3 aggregates in Becn1 +/À and wild-type microglia showed a higher number of NLRP3 aggregates in Becn1 +/À microglia ( Fig EV4A).…”
Section: Nlrp3 Is a Substrate For Autophagymentioning
Alzheimer's disease is characterized not only by extracellular amyloid plaques and neurofibrillary tangles, but also by microglia‐mediated neuroinflammation. Recently, autophagy has been linked to the regulation of the inflammatory response. Thus, we investigated how an impairment of autophagy mediated by BECN1/Beclin1 reduction, as described in Alzheimer's disease patients, would influence cytokine production of microglia. Acutely stimulated microglia from Becn1+/− mice exhibited increased expression of IL‐1beta and IL‐18 compared to wild‐type microglia. Becn1+/−APPPS1 mice also contained enhanced IL‐1beta levels. The investigation of the IL‐1beta/IL‐18 processing pathway showed an elevated number of cells with inflammasomes and increased levels of NLRP3 and cleaved CASP1/Caspase1 in Becn1+/− microglia. Super‐resolation microscopy revealed a very close association of NLRP3 aggregates and LC3‐positive vesicles. Interestingly, CALCOCO2 colocalized with NLRP3 and its downregulation increased IL‐1beta release. These data support the notion that selective autophagy can impact microglia activation by modulating IL‐1beta and IL‐18 production via NLRP3 degradation and thus present a mechanism how impaired autophagy could contribute to neuroinflammation in Alzheimer's disease.
“…Stimulated myeloid cells normally contain only one or two ASCcontaining inflammasomes (Baroja-Mazo et al, 2014;Franklin et al, 2014) (see also Fig 3C) that are also significantly larger than most of the NLRP3 signals seen in these cells (see Fig EV4C and D). Murine NLRP3 has a calculated molecular weight of 113 kDa and the pyrin domain has to exist at least as a trimer to trigger IL-1beta production, which is apparently independent of the formation of ASCcontaining inflammasome (Susjan et al, 2017). Thus, it seems plausible that the observed smaller NLRP3 aggregates present NLRP3 trimers or higher oligomeric forms as suggested for NLRP2 (Faustin et al, 2007) or NLRC4 (Hu et al, 2015;Zhang et al, 2015).…”
Section: Discussionmentioning
confidence: 87%
“…Non treated microglia showed, in correlation to their weak band in the Western blot, a diffuse cytoplasmic staining with a few small NLRP3 puncta ( Fig 4A). Stimulation of microglia with LPS/ATP resulted in the emergence of more and larger NLRP3 puncta/specs of different sizes ( Fig 4A, arrows), indicating formation of NLRP3 oligomers or aggregates (Susjan et al, 2017) (see also Discussion). Quantification of NLRP3 aggregates in Becn1 +/À and wild-type microglia showed a higher number of NLRP3 aggregates in Becn1 +/À microglia ( Fig EV4A).…”
Section: Nlrp3 Is a Substrate For Autophagymentioning
Alzheimer's disease is characterized not only by extracellular amyloid plaques and neurofibrillary tangles, but also by microglia‐mediated neuroinflammation. Recently, autophagy has been linked to the regulation of the inflammatory response. Thus, we investigated how an impairment of autophagy mediated by BECN1/Beclin1 reduction, as described in Alzheimer's disease patients, would influence cytokine production of microglia. Acutely stimulated microglia from Becn1+/− mice exhibited increased expression of IL‐1beta and IL‐18 compared to wild‐type microglia. Becn1+/−APPPS1 mice also contained enhanced IL‐1beta levels. The investigation of the IL‐1beta/IL‐18 processing pathway showed an elevated number of cells with inflammasomes and increased levels of NLRP3 and cleaved CASP1/Caspase1 in Becn1+/− microglia. Super‐resolation microscopy revealed a very close association of NLRP3 aggregates and LC3‐positive vesicles. Interestingly, CALCOCO2 colocalized with NLRP3 and its downregulation increased IL‐1beta release. These data support the notion that selective autophagy can impact microglia activation by modulating IL‐1beta and IL‐18 production via NLRP3 degradation and thus present a mechanism how impaired autophagy could contribute to neuroinflammation in Alzheimer's disease.
“…This design appropriately reproduces NLRP3 inflammasome activation; wild-type NLRP3 is activated only in the presence of inflammasome trigger nigericin, while CAPS-associated NLRP3 variant R258W is constitutively active (Supplementary Fig. 2c ) 36 . Fig.…”
NLRP3 is a cytosolic sensor triggered by different pathogen- and self-derived signals that plays a central role in a variety of pathological conditions, including sterile inflammation. The leucine-rich repeat domain is present in several innate immune receptors, where it is frequently responsible for sensing danger signals and regulation of activation. Here we show by reconstitution of truncated and chimeric variants into Nlrp3−/− macrophages that the leucine-rich repeat domain is dispensable for activation and self-regulation of NLRP3 by several different triggers. The pyrin domain on the other hand is required to maintain NLRP3 in the inactive conformation. A fully responsive minimal NLRP3 truncation variant reconstitutes peritonitis in Nlrp3−/− mice. We demonstrate that in contrast to pathogen-activated NLRC4, the constitutively active NLRP3 molecule cannot engage wild-type NLRP3 molecules in a self-catalytic oligomerization. This lack of signal amplification is likely a protective mechanism to decrease sensitivity to endogenous triggers to impede autoinflammation.
“… 21 Although the thioredoxin interacting protein (Txnip) was identified as a redox sensitive ligand of NLRP3, 46 not enough evidence suggested that the signaling pathway leading to NLRP3 activation requires Txnip. 47 …”
Diabetic retinopathy (DR) is a well-known microvascular complication related to inflammation. Mcc950 is a potent and specific inhibitor of the NLRP3 inflammasome but its influence on DR has not been studied. Thus, we evaluated the anti-inflammatory effects of Mcc950 on high-glucose-induced human retinal endothelial cells (HRECs) and the potential underlying mechanism. In surgical excised proliferative membranes from DR patients, high expression of NLRP3, caspase 1 and IL-1β was observed and co-localization of NLRP3 and IL-1β occurred in CD31+ labeled HRECs. Moreover, in high-glucose-stimulated HRECs, increased production of the NLRP3 inflammasome activation and severe apoptosis were rescued with Mcc950 treatment. Additionally, the inhibitory effect of Mcc950 was mimicked through downregulation of NEK7 by siRNA in high-glucose-induced HRECs and Mcc950 treatment remarkably inhibited Nek7 and NLRP3 interactions by co-immunoprecipitation, suggesting that Mcc950 may be a potentially protective agent against inflammation, likely via downregulation of the Nek7-NLRP3 pathway. In conclusion, Mcc950 inhibited HREC dysfunction under high-glucose conditions and this research may offer insight for future pharmaceutical approaches for treating DR.
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