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. 2014 Mar;137(Pt 3):819-33.
doi: 10.1093/brain/awt355. Epub 2014 Jan 22.

HTT-lowering Reverses Huntington's Disease Immune Dysfunction Caused by NFκB Pathway Dysregulation

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Free PMC article

HTT-lowering Reverses Huntington's Disease Immune Dysfunction Caused by NFκB Pathway Dysregulation

Ulrike Träger et al. Brain. .
Free PMC article

Abstract

Huntington's disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington's disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington's disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function-the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington's disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington's disease.

Keywords: Huntington’s disease; gene lowering; immunology; myeloid cells.

Figures

Figure 1
Figure 1
Pro-inflammatory cytokine production by monocytes and macrophages is elevated in patients with Huntington’s disease. Innate immune regulators such as IL-6, IL-8 and TNFα were elevated in Huntington’s disease patients (A) blood monocytes and (B) macrophages collected from two independent patient cohorts, stimulated in vitro with 10 ng/ml IFNγ and 2 µg/ml lipopolysaccharide for 24 h. Data show mean concentrations ± SEM, n = individual biological repeats, ANOVA with post hoc Tukey HSD test. *P < 0.05; **P < 0.01.
Figure 2
Figure 2
Glucan encapsulated small interfering RNA particles (GeRPs) can effectively knock-down total HTT in primary human immune cells. (A) GeRPs deliver small interfering RNA (siRNA) efficiently when phagocytosed by myeloid cells, as shown in primary human monocytes after 12 h incubation in culture (GeRPs = green; DAPI = blue). (B) Ninety per cent of macrophages take up GeRPs when incubated at 1:10 cell: particle ratio for 12 h as quantified by flow cytometry. Data shown as mean [n = 2 for controls and n = 3 for Huntington’s disease (HD)] ± SEM. (C) Total HTT RNA measured by quantitative PCR and protein levels measured by TR-FRET were reduced by 70% and 50%, respectively, in macrophages treated for 3 days with GeRPs containing anti-HTT small interfering RNA. Data shown as mean HTT levels (each combining two independent experiments, n = individual biological repeats) ± SEM. Data are normalized to the scrambled small interfering RNA treated condition for each genotype.
Figure 3
Figure 3
Knock-down of total HTT reverses the hyper-reactive cytokine production by Huntington’s disease macrophages. Huntington’s disease (HD) and control macrophages were treated with either anti-HTT or scrambled small interfering RNA (siRNA) for 3 days, before the cells were stimulated with 10 ng/ml IFNγ and 2 µg/ml lipopolysaccharide for 24 h. Measuring cytokine production with multiplex ELISA assays showed that lowering HTT levels reduces IL-6, IL-8 and TNFα levels after stimulation. Data shown as mean concentrations (n = 9 for controls and n = 8 for Huntington’s disease, combined from three independent experiments, n = individual biological repeats) ± SEM, two-way ANOVA with Bonferroni post-tests. *P < 0.05; **P < 0.01, ***P < 0.001.
Figure 4
Figure 4
Expression of mutant HTT induces elevated cytokine production. U937 cells were lentivirally-transduced with mutant (m)HTT exon 1 containing either 29, 71 or 129 glutamine (Q) repeats or an empty vector. (A) Expression of mutant HTT protein post-transduction was confirmed, with increased levels of soluble HTT in all three cell lines expressing exogenous HTT. (B) Innate immune regulators were elevated in PMA-differentiated mutant HTT expressing U937 cells stimulated for 24 h with 10 ng/ml IFNγ and 2 µg/ml lipopolysaccharide (LPS). Data shown as mean concentrations (n = 3 technical repeats for all conditions) ± SEM, two-way ANOVA with Bonferroni post-tests, *P < 0.05; **P < 0.01, ***P < 0.001. The experiment was repeated three times independently and showed similar results.
Figure 5
Figure 5
HTT interacts directly with the NFκB pathway, which is dysregulated in Huntington’s disease (HD). (A) HTT interacts directly with IKK, as shown by proximity ligation assays. Monocyte-derived macrophages were differentiated on glass cover-slips and stained for HTT and IKKγ or IKKα/β before antibodies binding in close proximity were visualized using proximity ligation assay (PLA) probes as red spots, shown here. Cells stained with a single primary antibody did not result in red spots. (B) Quantification of the number of spots per cell using the Volocity software shows increased binding between IKKγ and HTT in Huntington’s disease compared with control cells (P = 0.06). Binding of HTT to the α and β subunit of IKK showed a similar, but smaller trend (P = 0.1). Two-tailed unpaired t-test used for statistical analysis. (C) In control cells lipopolysaccharide (LPS)-induced degradation of IκB occurred within 15 min of stimulation and recovered within 2 h, whereas Huntington’s disease monocytes demonstrated a more rapid loss of IκB and no recovery of the protein. Shown is an example blot of samples from one control subject and one patient with Huntington’s disease. (D) Translocation of the NFκB transcription factor RelA to the nucleus after lipopolysaccharide stimulation was measured using imaging flow cytometry; example images are shown here. In untranslocated cells the green RelA staining surrounds the nuclear DAPI staining, whereas in cells demonstrating translocation of RelA the colours merge. (E) Increased RelA translocation into the nucleus following lipopolysaccharide stimulation was observed in Huntington’s disease monocytes (n = 7) compared to controls (n = 8). n = individual biological repeats. Data shown as mean concentrations ± SEM, two-way ANOVA with Bonferroni post-tests, *P < 0.05; **P < 0.01. All experiments were repeated at least twice with the same results.
Figure 6
Figure 6
Lowering total HTT levels reverses transcriptional changes found in Huntington’s disease monocytes. Huntington’s disease and control monocytes were incubated with either scrambled or anti-HTT small interfering RNA (siRNA) containing GeRPs for 3 days before RNA isolation. Using quantitative PCR, efficient HTT knock-down was demonstrated as well as lowering of key NFκB pathway molecules IRAK1, CD40 and JUN in (A) Huntington’s disease patient cells but not (B) controls. Data shown as relative gene expression (n = 10 individual biological repeats for controls and Huntington’s disease) ± SEM, paired t-test. *P < 0.05; **P < 0.01.
Figure 7
Figure 7
Mechanism of immune dysfunction in Huntington’s disease. (A) In normal wild-type HTT expressing myeloid cells, lipopolysaccharide binds the TLR4 receptor activating the NFκB pathway triggering production of pro-inflammatory cytokines such as IL-6 and TNFα. (B) Mutant (m)HTT interferes with the NFκB pathway by two distinct mechanisms. The mutant protein binds IKKγ to directly cause increased IκB degradation and NFκB transcription factor translocation, allowing increased transcription of target genes such as IL-6 and TNFα. Moreover, mutant HTT causes transcriptional changes leading to increased expression of key molecules within the signalling cascade likely to increase signalling transduction rate.

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