Saresella et al. Molecular Neurodegeneration (2016) 11:23 DOI 10.1186/s13024-016-0088-1 RESEARCHARTICLE Open Access The NLRP3 and NLRP1 inflammasomes are activated in Alzheimer’s disease Marina Saresella1*†, Francesca La Rosa1†, Federica Piancone1, Martina Zoppis1, Ivana Marventano1, Elena Calabrese1, Veronica Rainone2, Raffaello Nemni1,3, Roberta Mancuso1 and Mario Clerici1,3 Abstract Background: Interleukin-1 beta (IL-1β) and its key regulator, the inflammasome, are suspected to play a role in the neuroinflammation observed in Alzheimer’s disease (AD); no conclusive data are nevertheless available in AD patients. Results: mRNA for inflammasome components (NLRP1, NLRP3, PYCARD, caspase 1, 5 and 8) and downstream effectors (IL-1β, IL-18) was up-regulated in severe and MILD AD. Monocytes co-expressing NLRP3 with caspase 1 or caspase 8 were significantly increased in severe AD alone, whereas those co-expressing NLRP1 and NLRP3 with PYCARD were augmented in both severe and MILD AD. Activation of the NLRP1 and NLRP3 inflammasomes in AD was confirmed by confocal microscopy proteins co-localization and by the significantly higher amounts of the pro-inflammatory cytokines IL-1β and IL-18 being produced by monocytes. In MCI, the expression of NLRP3, but not the one of PYCARD or caspase 1 was increased, indicating that functional inflammasomes are not assembled in these individuals: this was confirmed by lack of co-localization and of proinflammatory cytokines production. Conclusions: The activation of at least two different inflammasome complexes explains AD-associated neuroinflammation. Strategies targeting inflammasome activation could be useful in the therapy of AD. Keywords: Alzheimer’s disease, Neuroinflammation, Inflammasome, Beta amyloid, Mild cognitive impairment Background The inflammasomes are multiprotein complexes mainly Alzheimer’s disease (AD) is a devastating neurodegener- expressed in myeloid cells that are required for the activa- ative condition characterized by neuronal cell death and tion of caspase 1 protease and the downstream secretion of progressive dementia. It is widely accepted that the two of its substrates, the proinflammatory cytokines IL-1β extracellular accumulation of amyloid-β (Aβ) in senile and IL-18 [2]. Inflammasome-dependent innate immune plaques and the formation of neurofibrillary tangles responses are initiated by Nod-like receptors (NLRs), cyto- (NFT) inside neurons as a result of the abnormal phos- plasmic pattern recognition receptors that detect invading phorylation of the microtubules-associated tau protein pathogens. NLRs are activated by bacterial, fungal, or viral are the main event in the pathogenesis of AD, but the molecules that contain pathogen-associated molecular cellular events leading to plaque-induced neuronal dys- patterns (PAMPs) or by non-microbial danger signals function are less clear [1]. Inflammatory mediators play (DAMPs) released by damaged cells [3, 4]. NLR activation an essential role in the neuroinflammation observed in leads to their oligomerization to form multiprotein inflam- AD. In particular interleukin IL-1β is increased in this masome complexes that serve as platforms for the recruit- condition, and the activation of its key regulator, the ment, cleavage, and activation of inflammatory caspases. inflammasome, is suspected to be involved in the patho- Four inflammasome complexes (NLRP1, NLRP3, IPAF, genesis of the disease. and AIM2) have been identified. These complexes contain either a specific NLR family protein or AIM2, as well as the PYCARD and/or Cardinal adaptor proteins and pro- caspases-1, 5 and 8 [5, 6]. NLRP3 is the best-characterized * Correspondence: [email protected] inflammasome; its formation requires multiple steps. In a †Equal contributors 1Don C. Gnocchi Foundation, IRCCS, Piazza Morandi, 3, 20121 Milan, Italy priming step, transcriptionally active signalling receptors Full list of author information is available at the end of the article induce the NF-kB-dependent induction of NLRP3 itself as © 2016 Saresella et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Saresella et al. Molecular Neurodegeneration (2016) 11:23 Page 2 of 14 well as that of the caspase 1 substrates of the pro-IL-1β (nFold >10) compared to HC, with no detectable differ- family [7, 8]. The NLRP3 is, at this stage, in a signalling in- ences being observed between individuals with a diag- competent conformation; this is modified upon a second nosis of AD or MCI; and 2) PYCARD, caspase 1 and signal, that will induce the assembly of a multimolecular caspase 5 expression was increased in AD (nFold ≥10) complex with PYCARD and caspase 1. Multiple signals, but not in MCI individuals. Only minor differences which are potentially provided in combination, thus trigger were seen between patients and controls in the expres- the formation of an active inflammasome, which, in turn, sion of these genes in monocytes-stimulated with either will stimulate the cleavage and the release of bioactive cy- LPS or Aβ42 alone; both stimuli induced IL-1β and IL-6 tokines including IL-1β and IL-18 [8–18]. Although these up-regulation in HC (nFold >10) (Fig. 1). cytokines have a beneficial role in promoting inflammation RT-PCR performed on each individual specimen was and eliminating infectious pathogens, mutations that result used next to verify the expression of NLRP1, NLRP3, in constitutive inflammasome activation and overproduc- PYCARD, caspase 1, caspase 5 and caspase 8 in LPS and tion of IL-1β and IL-18 have been linked to inflammatory Aβ42−stimulated monocytes of patients and HC. Results and autoimmune disorders [19–21]. of mRNA expression confirmed that: 1) NLRP1, NLRP3 The NLRP3 inflammasome complex is suspected to play and caspase 8 are significantly increased in AD and MCI a role in AD, as its activation in the microglia by Aβ trig- individuals compared to HC (p <0.05), with the highest gers neuroinflammation [14, 22, 23]. Notably, NLRP3 values observed in severe AD, and 2) PYCARD, caspase inflammasome deficiency favours the differentiation of 1 and caspase 5 are significantly increased in AD com- microglia cells to an M2 (anti-inflammatory) phenotype pared to MCI and HC (p <0.05) (Fig. 2a). and results in a decreased deposition of amyloid-β in the APP/PSI model of AD; these results reinforce the sugges- Up-regulation of inflammasome-related cytokines in tion that the NLRP3 inflammasome is involved in the LPS-primed and Aβ42−stimulated- monocytes of AD patients pathogenesis of the disease [24]. No conclusive data are Stimulation of monocytes with either LPS or Aβ42-alone nevertheless available in patients. To better investigate the resulted in the moderate increase of IL-1β and IL-6 ex- involvement of the inflammasome in AD we performed pression; notably LPS or Aβ42- alone were not sufficient in-depth analyses of the expression of genes and proteins to trigger inflammasome assembly, as no differences were that are part of the inflammasome complex in individuals detect in these conditions either in NLRP3 or IL-18 with a diagnosis of either AD or mild cognitive impair- mRNA (Fig. 1). Once cells were activated with LPS and ment (MCI). Results indicated that two different inflam- Aβ42, nevertheless, both IL-1β (nFold >10) and IL-18 masomes, NLRP3 and NLRP1, are activated in AD but (nFold ≥10) mRNA was significantly increased in severe not in individuals with a diangnosis of mild cognitive im- and MILD AD compared to MCI and HC (Fig. 1). Results pairment (MCI). obtained by single-PCR confirmed that in these experi- mental conditions IL-1β and IL-18 mRNA (p <0.05 vs. all Results other groups) is significantly increased in severe and Up-regulation of inflammasome genes in LPS-primed and MILD AD, with the highest values in severe AD (Fig. 2b). Aβ42-stimulated-monocytes of AD patients IL-33 and IL-37, two relatively novel cytokines that are mRNA expression of 84 genes involved in the assembly, members of the IL-1 family, were analyzed as well in the the activation, and the down-stream signalling of inflam- study. We found that IL-33 gene expression was greatly mosomes was quantified by qPCR in all patients and con- increased in MCI (nFold >10) compared to AD and HC; trols. Data are expressed as the fold change (nFold) no significant differences, on the other hand, were ob- between the results obtained in unstimulated cells and served in IL-37 gene expression among groups analyzed those obtained upon stimulations of cells with either LPS, (Fig. 1). Single RT-PCR confirmed these results (Fig. 2c). Aβ42 or LPS and Aβ42. In this initial set of experiments, pools of cells from donors with the same clinical diagnosis NLRP3 protein is significantly augmented in LPS-primed (MCI; MILD AD; severe AD) as well as from HC were and Aβ42−stimulated- monocytes of AD and MCI patients created. Data obtained in LPS-primed and Aβ42−stimu- The expression of NLRP3 protein, the best-characterized lated-monocytes, showed the presence of a significant up- protein within the inflammasome complex, was investi- regulation involving genes, that codify for the proteins that gated next by Western Blot analyses in LPS-primed and form the inflammasome in MILD and severe AD, as well Aβ42-stimulated-monocytes of all patients and controls. as in MCI. In cells of MCI individuals, though, not all the Different NLRP3 isoforms are known: the long form genes that are necessary for the assembly of a fully func- (118 KDa) is prevalent in cell lines (THP1 or Jurkat) tional inflammasome complex were up-regulated.