A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington's disease

Abstract

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by both neurological and systemic abnormalities. We examined the peripheral immune system and found widespread evidence of innate immune activation detectable in plasma throughout the course of HD. Interleukin 6 levels were increased in HD gene carriers with a mean of 16 years before the predicted onset of clinical symptoms. To our knowledge, this is the earliest plasma abnormality identified in HD. Monocytes from HD subjects expressed mutant huntingtin and were pathologically hyperactive in response to stimulation, suggesting that the mutant protein triggers a cell-autonomous immune activation. A similar pattern was seen in macrophages and microglia from HD mouse models, and the cerebrospinal fluid and striatum of HD patients exhibited abnormal immune activation, suggesting that immune dysfunction plays a role in brain pathology. Collectively, our data suggest parallel central nervous system and peripheral pathogenic pathways of immune activation in HD.

Figure 1.

Altered immune profile peripherally in HD. (A) Multiplex ELISA quantification of cytokine levels in plasma from HD patients (premanifest, early and moderate HD stages) compared with control subjects. Graphs show mean concentrations with standard error bars. Significant differences between individual groups are shown (ANOVA with post-hoc Tukey HSD test). (B) The overall trend for increasing levels of cytokines across all groups, analyzed using linear regression, was highly significant for IL-6 and IL-8 and significant for IL-4, IL-10, TNF-α, and IL-5. R-values (partial correlation coefficients) are corrected for age and sex. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 2.

Plasma Ig levels are unchanged in HD. Quantification of plasma IgG, IgA, and IgM by single radial immunodiffusion assays revealed no difference in Ig levels across disease stages, arguing against widespread activation of the adaptive immune system. Graphs show mean concentration standard error bars.

Figure 3.

Correlations between plasma cytokine levels and clinical severity scores in premanifest and manifest HD gene carriers. Levels of (A) IL-8 and (B) TNF-α correlated with worsening disease as demonstrated by increasing UHDRS chorea score and UHDRS total motor score and decreasing total functional capacity score.

Figure 4.

ROC curves demonstrating the ability of different combinations of plasma cytokine levels to discriminate between subject groups. In a ROC curve plot, the “true positive” diagnosis rate (sensitivity) is plotted against the “false positive” diagnosis rate (1-specificity) for a test with a binary outcome. The AUC summarizes the discrimination of the test, i.e., its ability to classify cases correctly. A perfect test would have an AUC of 1; a worthless test would have an AUC of 0.5. AUC values may be classified as follows: 0.9–1, excellent; 0.8–0.9, good; 0.7–0.8, fair; 0.6–0.7, poor; 0.5–0.6, fail (reference 36). For the present analysis, optimum combinations were identified by stepwise logistic regression analysis using a threshold of P = 0.05 for each cytokine removed from the model. A combination of IL-6, IL-10, and IL-5 best discriminated between controls and premanifest HD; a combination of IL-6, IL-8, and IL-10 best discriminated manifest from premanifest HD; and a combination of IL-6, IL-8, and IL-10 best discriminated controls from HD gene carriers (both premanifest and manifest).

Figure 5.

Human monocytes express WT and mutant huntingtin. (A) RT-QPCR studies of human monocytes obtained by flow cytometry demonstrated huntingtin expression in 100% of monocyte samples tested from controls (n = 2) and HD patients (n = 3). Expression ratios are relative to B2M. Graph shows mean expression ratios with standard error bars. (B) PCR amplification of CAG repeat tracts from huntingtin mRNA reveals that HD monocytes express both WT and mutant huntingtin, supporting the possibility of cell-autonomous dysfunction resulting in immune activation. WT and mutant CAG repeat lengths are shown. +, p4G6E4.0 plasmid expressing HTT exon 1 with 18 CAG repeats.

Figure 6.

HD monocytes, macrophages, and microglia are overactive when stimulated. (A) No IL-6 was detectable in the supernatant of monocytes from control (n = 9) or premanifest HD subjects (n = 8) in the unstimulated state or after priming with IFN-γ. Monocytes stimulated by the addition of both IFN-γ and 2 μg/ml LPS expressed IL-6, but expression levels were significantly higher from HD monocytes. (B) Alveolar macrophages from the YAC128 HD mouse model have similarly altered function when stimulated. YAC128 macrophages stimulated by the addition of both IFN-γ and 100 ng/ml LPS expressed significantly more IL-6. n = 3 WT and 4 YAC128. (C) Macrophages from YAC18 mice, which differ from YAC128 cells only in the number of CAG repeats, behaved no differently from WT cells (P = 0.231; n = 4 per genotype) in response to stimulation at the same LPS concentration, suggesting that the hyperactivity in the YAC128 is due to mutant huntingtin. (D) Microglia isolated from neonatal R6/2 mice are also hyperactive when stimulated by 10 ng/ml LPS (n = 4 per group). Graphs show mean concentrations with standard error bars. ND, not detected. Unpaired t tests: *, P < 0.05; **, P < 0.01.

Figure 7.

Altered expression of inflammatory transcripts in postmortem HD striatal tissue. Levels of IL-6, IL-8, and TNF-α RNA were significantly higher in the striatum of HD patients than in control striatum. Graphs show means with standard error bars. n = 6 controls and 17 HD patients (see Table S3). Unpaired t tests: *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Figure 8.

Correlations between matched CSF and plasma levels of IL-6 and IL-8. ELISA-quantified levels in CSF and matched plasma samples correlated strongly for both IL-6 and IL-8.

Figure 9.

Mouse models of HD recapitulate features of human immune dysfunction. Serum levels of cytokines, measured by multiplex ELISA, are elevated in both (A) R6/2 and (B) Hdh^Q150Q/Q150 knock-in mouse models (n = 9 per genotype) at end-stage. (C) In 12-mo YAC128 animals (equivalent to early human disease), serum IL-6 and mKC, a mouse functional homologue of IL-8, are significantly increased (n = 3 WT and 4 YAC128). Graphs show mean levels with standard error bars. Unpaired t tests: *, P < 0.05; **, P < 0.01.

Figure 10.

Immune activation, induced by mutant huntingtin, occurs both peripherally and centrally in HD. A cell-autonomous effect of the mutant protein may be responsible for the innate immune response. The NF-κB signaling pathway that triggers IL-6 release is known to be up-regulated by mutant huntingtin (reference 7), and microglia-derived toxicity can influence disease progression (references and 28). We find that the innate immune response detectable in plasma very early in the disease is strongly linked to disease progression and recapitulated in HD striatum, that human monocytes express mutant huntingtin, and that monocytes, macrophages, and microglia overexpress IL-6 when stimulated. Early innate immune activation could be a target in the development of disease-modifying therapies.