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. 2019 Sep 2;38(17):e101064.
doi: 10.15252/embj.2018101064. Epub 2019 Jul 30.

Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome

Dario Tejera  1   2 , Dilek Mercan  1 , Juan M Sanchez-Caro  1 , Mor Hanan  3 , David Greenberg  3 , Hermona Soreq  3 , Eicke Latz  2   4   5 , Douglas Golenbock  4 , Michael T Heneka  1   2   4
Affiliations

Systemic inflammation impairs microglial Aβ clearance through NLRP3 inflammasome

Dario Tejera et al. EMBO J. .

Abstract

Alzheimer's disease is the most prevalent type of dementia and is caused by the deposition of extracellular amyloid-beta and abnormal tau phosphorylation. Neuroinflammation has emerged as an additional pathological component. Microglia, representing the brain's major innate immune cells, play an important role during Alzheimer's. Once activated, microglia show changes in their morphology, characterized by a retraction of cell processes. Systemic inflammation is known to increase the risk for cognitive decline in human neurogenerative diseases including Alzheimer's. Here, we assess for the first time microglial changes upon a peripheral immune challenge in the context of aging and Alzheimer's in vivo, using 2-photon laser scanning microscopy. Microglia were monitored at 2 and 10 days post-challenge by lipopolysaccharide. Microglia exhibited a reduction in the number of branches and the area covered at 2 days, a phenomenon that resolved at 10 days. Systemic inflammation reduced microglial clearance of amyloid-beta in APP/PS1 mice. NLRP3 inflammasome knockout blocked many of the observed microglial changes upon lipopolysaccharide, including alterations in microglial morphology and amyloid pathology. NLRP3 inhibition may thus represent a novel therapeutic target that may protect the brain from toxic peripheral inflammation during systemic infection.

Keywords: 2-photon; Alzheimer's; amyloid-beta; microglia; neuroinflammation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Systemic inflammation transiently affects microglia in an age‐dependent manner

  1. Representative 3D microglia reconstructions showing morphological changes upon LPS injection within the first 48 h. Scale bar: 10 μm.

  2. Morphological parameters quantification within 48 h after LPS injection (mean of 5–6 ± SEM; one‐way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001).

  3. Two‐photon microscopy experimental design.

  4. Representative 3D microglia reconstructions from 5‐ and 15‐month‐old mice showing microglia changes 2 and 10 days post‐LPS injection. Scale bar: 20 μm.

  5. Morphological parameters quantification for 5‐ and 15‐month‐old mice after LPS injection (mean of 5–6 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, #*P < 0.05, **P < 0.01, ***P < 0.001).

Figure EV1
Figure EV1. Transient increase in CD68 immunoreactivity upon LPS injection

  1. CD68 staining in cortex of 5 and 15 months old of wild‐type and Nlrp3 −/− mice. Scale bar: 20 μm.

  2. CD68 integrated density in wild‐type and Nlrp3 −/− mice (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, ***P < 0.001).

Figure 2
Figure 2. Nlpr3 knockout mice are refractory to peripheral immune challenge or age‐associated changes

  1. Schematic representation of two‐photon microscopy experimental design.

  2. Two‐photon representative images of wild‐type and Nlrp3 −/− mice (5 and 15 months old). Scale bar: 20 μm.

  3. Quantification of morphological parameters for wild‐type and Nlpr3 −/− mice after LPS injection (mean of 5‐6 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01).

Figure EV2
Figure EV2. LPS injection triggers a transient increase in pro‐inflammatory cytokines in both brain and periphery

  1. IL‐1β and TNF‐α ELISA measurement in brain lysates of wild‐type and Nlrp3 −/−. A significant NLRP3‐dependent increase of IL‐1β levels was observed 2 days after LPS injection and then a return to control levels. For TNF‐α, all groups showed a transient increase in its levels (mean of 6 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001).

  2. IL‐1β and TNF‐α ELISA measurement in liver lysates of wild‐type and Nlrp3 −/− mice. A transient increase in NLRP3‐dependent IL‐1β levels is observed after immune challenge. LPS injection triggered a transient increase in TNF‐α in all groups evaluated (mean of 6 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure EV3
Figure EV3. Astrocytes are transiently activated after peripheral immune challenge

  1. Representative cortical pictures of 5 and 15 months old wild‐type and Nlrp3 −/− stained with GFAP. A transient increase in GFAP immunoreactivity is observed 2 days after LPS injection in wild‐type but not in Nlrp3 −/− mice. Scale bar: 20 μm.

  2. GFAP integrated density in wild‐type and Nlrp3 −/− mice (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, ***P < 0.001).

Figure 3
Figure 3. Peripheral immune challenge affects amyloid deposition in APP/PS1 mice

  1. Representative cortical images of MXO4 staining for APP and APP/Nlrp3 −/− 15‐month‐old mice. Scale bar: 50 μm.

  2. Cortical amyloid plaque number and size quantification, and Amyloid‐beta1–40 and 1–42 ELISA quantification for APP and APP/Nlrp3 −/− mice (5‐ and 15‐month‐old) (mean of 8 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4
Figure 4. Microglia dynamics depend on distance to Aβ deposition in APP/PS1 mice

  1. Two‐photon images of microglia (eGFP) cells clustering around amyloid plaque for APP and APP/Nlrp3 −/− (5 and 15 months old). Scale bar: 20 μm.

  2. Quantification of morphological parameters in (A) (mean of 5–6 ± SEM).

  3. Flow cytometry plots from APP and APP/Nlrp3 −/− mice (15 months old), cells were gated on CD11b and MXO4 after microglia isolation.

  4. Relative Aβ microglia uptake quantification (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05).

Figure EV4
Figure EV4. Systemic inflammation alters beclin‐1 expression in an NLRP3‐dependent manner

  1. Western blot analysis of whole brain lysate from 15‐month‐old APP and APP/Nlrp3 −/− using beclin‐1 antibody.

  2. Quantification of beclin‐1 expression levels (mean of 2–5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05).

Figure 5
Figure 5. Microglia dynamics depend on distance to Aβ deposition in APP/PS1 mice

  1. Two‐photon microglia (eGFP) images from APP and APP/Nlrp3 −/− in areas free of plaque (5 and 15 months old mice). Scale bar: 20 μm.

  2. Quantification of APP and APP/Nlrp3 −/− morphological parameters for microglia in areas free of plaque (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01).

Figure 6
Figure 6. Peripheral myeloid cells infiltrate into APP/PS1 mice upon LPS injection
  1. A, B

    Iba‐1 (green), CD169 (red), and MXO4 staining in plaque‐associated areas (cortex and hippocampus) of 5 and 15 months old of APP and APP/Nlrp3 −/−. Note the colocalization between Iba‐1 and CD169 (white arrows) in APP 15‐month‐old mice 2 days post‐LPS injection. Scale bar: 20 μm.

Figure EV5
Figure EV5. Blood–brain barrier is compromised in old APP mice upon LPS injection

  1. Iba‐1 (green) and fibrinogen (red) staining in cortex of 5 and 15 months old of wild‐type and Nlrp3 −/− mice. Scale bar: 10 μm.

  2. Iba‐1 (green) and fibrinogen (red) staining in cortex of 5 and 15 months old of APP and APP/Nlrp3 −/− mice. Scale bar: 10 μm.

  3. Fibrinogen integrated density in wild‐type and Nlrp3 −/− mice (left panel) and APP and APP/Nlrp3 −/− mice (right panel) (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, ***P < 0.001).

Figure 7
Figure 7. Microglia proliferate in non‐APP mice upon peripheral immune challenge
  1. A, B

    Iba‐1, Ki67, and DAPI staining in the cortex and hippocampus of wild‐type and Nlrp3 −/− (5 and 15 months old). Microglia proliferate upon LPS injection (white arrows). Non‐microglia cells proliferation was observed as well (yellow arrows). Scale bar: 20 μm.

  2. C

    Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel) (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 8
Figure 8. Microglia proliferate in APP/PS1 mice upon peripheral immune challenge
  1. A, B

    Iba‐1, Ki67, and MXO4 staining in the cortex and hippocampus of APP and APP/Nlrp3 −/− (5 and 15 months old). Microglia proliferate upon LPS injection (white arrows). Non‐microglia cells proliferation was observed as well (yellow arrows). Scale bar: 20 μm.

  2. C

    Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel) (mean of 5 ± SEM; two‐way ANOVA followed by Tukey's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001).

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