The NLRP3 and NLRP1 inflammasomes are activated in Alzheimer's disease

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.

Fig. 1

Messenger RNA expression levels of genes within the inflammasome pathway. Expression of 84 genes involved in the inflammasome pathway assessed by real-time quantitative RT-PCR-array in monocytes of individuals with a diagnosis of either severe Alzheimer’s disease (AD), moderate Alzheimer’s disease (MILD) or Mild Cognitive Impairment (MCI) and of age- and sex-matched Healthy Controls (HC). Heat maps of Log₂ Fold changes are presented. Results obtained upon stimulating cells with LPS or Aβ₄₂ alone, or upon LPS-priming followed by Aβ₄₂ stimulation are presented. Fold-changes >10 in the expression of LPS + Aβ₄₂ stimulated genes of importance are summarized in the Table

Fig. 2

mRNA expression by Real-Time PCR. Single Real-Time PCR results obtained in LPS and Aβ₄₂ -stimulated monocytes of individuals with a diagnosis of either severe Alzheimer’s disease (AD), moderate Alzheimer’s disease (MILD) or Mild Cognitive Impairment (MCI) and of age- and sex-matched Healthy Controls (HC). a NLRP1, NLRP3, caspase 8, PYCARD, caspase 1 and caspase 5 are shown in panel a; IL-1β and IL-18 in panel b and IL-33 and IL-37 in panel c. The results are shown as fold-change expression from the un-stimulated samples. Gene expression was calculated relative to GAPDH housekeeping gene. Summary results are shown in the bar graphs. The boxes stretch from the 25 to the 75 percentile; the line across the boxes indicates the median values; the lines stretching from the boxes indicate extreme values. Outside values are displayed as separate points. Statistical significance is shown *(p <0.05), **(p <0.01)

Fig. 3

NLRP3s expression in Monocyte by Western blot: NLRP3s protein expression assessed by western blotting in monocyte of individuals with a diagnosis of either severe Alzheimer’s disease (AD), moderate Alzheimer’s disease (MILD) or Mild Cognitive Impairment (MCI) and of age- and sex-matched Healthy Controls (HC). The same protein concentration of whole-cells lysates was loaded into the gel, as confirmed by actin. Representative results obtained in un-stimulated or in LPS-primed and Aβ₄₂-stimulated monocytes are presented in the upper panel. Quantitative evaluation (arbitrary unit, AU) of NLRP3s expression obtained comparing band density (normalized to actin) in un-stimulated or in LPS-primed Aβ42-stimulated monocytes is shown in the lower panel. (N = 5, mean ± s.e.m., T test, *p <0.05). (N = 5/group, mean ± S.E.M and statistical significance are shown. *(p <0.05)

Fig. 4

FACS analyses: co-expression of inflammasome proteins in CD14+ cells. Co-expression of inflammasome proteins in LPS-primed and Aβ₄₂-stimulated CD14+ of individuals with a diagnosis of either severe Alzheimer’s disease (AD), moderate Alzheimer’s disease (MILD) or Mild Cognitive Impairment (MCI) and of age- and sex-matched Healthy Controls (HC). Two-hundred-thousand cells were acquired and gated on CD14 expression and side scatter properties. CD14+ cells co-expressing NLRP3-associated inflammasome components are shown in panel a; CD14+ cells co-expressing NLRP1-associated inflammasome components are shown in panel b. Summary results are shown in the bar graphs. The boxes stretch from the 25° to the 75°percentile; the line across the boxes indicates the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown *(p <0.05), **(p <0.01)

Fig. 5

Confocal microscopy: co-localization of inflammasome proteins in CD14+ cells. Representative confocal fluorescence images (63× magnification) of experiments (N = 5) showing the effect of LPS-priming and Aβ₄₂-stimulation on NLRP3/PYCARD (a) and NLRP1/caspase 1 (b) co-localization in monocytes of individuals with a diagnosis of either severe Alzheimer’s disease (AD), moderate Alzheimer’s disease (MILD) or Mild Cognitive Impairment (MCI) and of age- and sex-matched Healthy Controls (HC). Summary results of NLRP3/PYCARD and NLRP1/caspase-1 PCC (Pearson co-localization efficiency) are presented in (c) and (d), respectively. The boxes stretch from the 25 to the 75 percentile; the line across the boxes indicates the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown *(p <0.05), **(p <0.01)

Fig. 6

IL-1β, IL-18, IL-33 and IL-37 production. IL-1β, (panel a), IL-18 (panel b), IL-33 (panel c) and IL-37 (panel d). Interleukin-1β and IL-18 production was assessed by multiplex ELISA in supernatants. CD14+/IL-1β+, CD14+/IL-33 and CD14+/IL-37+ cells were analyzed by flow-cytometry. In both cases, LPS-primed and Aβ₄₂-stimulated monocytes of individuals with a diagnosis of either severe Alzheimer’s disease (AD), moderate Alzheimer’s disease (MILD) or Mild Cognitive Impairment (MCI) and of age- and sex-matched Healthy Controls (HC) were analyzed. Summary results are shown in the bar graphs. The boxes stretch from the 25° to the 75°percentile; the line across the boxes indicates the median values; the lines stretching from the boxes indicate extreme values. Statistical significance is shown *(p <0.05), **(p <0.01)