RNA-Seq of Huntington's disease patient myeloid cells reveals innate transcriptional dysregulation associated with proinflammatory pathway activation

Abstract

Innate immune activation beyond the central nervous system is emerging as a vital component of the pathogenesis of neurodegeneration. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The systemic innate immune system is thought to act as a modifier of disease progression; however, the molecular mechanisms remain only partially understood. Here we use RNA-sequencing to perform whole transcriptome analysis of primary monocytes from thirty manifest HD patients and thirty-three control subjects, cultured with and without a proinflammatory stimulus. In contrast with previous studies that have required stimulation to elicit phenotypic abnormalities, we demonstrate significant transcriptional differences in HD monocytes in their basal, unstimulated state. This includes previously undetected increased resting expression of genes encoding numerous proinflammatory cytokines, such as IL6 Further pathway analysis revealed widespread resting enrichment of proinflammatory functional gene sets, while upstream regulator analysis coupled with Western blotting suggests that abnormal basal activation of the NFĸB pathway plays a key role in mediating these transcriptional changes. That HD myeloid cells have a proinflammatory phenotype in the absence of stimulation is consistent with a priming effect of mutant huntingtin, whereby basal dysfunction leads to an exaggerated inflammatory response once a stimulus is encountered. These data advance our understanding of mutant huntingtin pathogenesis, establish resting myeloid cells as a key source of HD immune dysfunction, and further demonstrate the importance of systemic immunity in the potential treatment of HD and the wider study of neurodegeneration.

Figure 1.

Comparison of log2 fold changes in resting and stimulated HD and control monocytes. The log2 fold changes for the 130 differentially expressed genes in resting HD monocytes (FDR < 0.05) were compared between the unstimulated and stimulated datasets. The magnitude of the relative expression difference was greater in the unstimulated samples for 116 of these genes, suggesting that transcriptional differences between HD and control monocytes become less pronounced in response to stimulation. Genes which were only differentially expressed in the unstimulated samples are shown in blue, while the three genes which were differentially expressed in both datasets are shown in red. HTT is further shown in green, while the NFĸB transcription factor RELA (p65) is shown in purple (however neither of these genes were differentially expressed). The line x = y is also shown.

Figure 2.

Functional gene sets associated with innate immunity and inflammation are enriched in resting HD monocytes. GSEA identified biologically relevant gene sets that are enriched in resting HD monocytes. A network diagram of significant biological themes among the upregulated genes is shown, indicating number of genes (node size), statistical significance (darkest shading = lowest P-value) and gene content similarity via the Jaccard coefficient (edge thickness). Nodes with similar gene content cluster more closely due to an edge-weighted layout (modified for readability). An FDR cut-off of < 0.05 was used to determine gene set inclusion in the diagram, before the Jaccard coefficient was used to filter out gene sets with similar gene content. The diagram was rendered in Cytoscape 3.3.0.

Figure 3.

Specific intracellular signalling pathways are predicted to be abnormally activated in resting HD monocytes. IPA analysis of upstream regulators of gene transcription identified numerous signalling pathway components downstream of the TLR4 receptor which are predicted to be abnormally activated in resting HD monocytes. Upstream regulators with significantly inferred activation are shown in orange (darker colour = higher activation z-score). The activation z-score for each component is also shown (>2 or < −2 is considered to be significant).

Figure 4.

NFĸB but not ERK or p38 MAPK signalling is abnormally activated in resting HD myeloid cells. Monocyte-derived macrophages were isolated from HD and control peripheral blood samples and pulsed with LPS and IFN-γ for 15, 30, 60 and 120 min, or left unstimulated. Western blotting was then carried out on the unstimulated samples to quantify the basal levels of IĸBα, while additional Western blotting was carried out on all samples to quantify the levels of total and active, phosphorylated ERK and p38 MAPK. (A) Resting HD myeloid cells express significantly reduced IĸBα protein compared with control (control n = 7, HD n = 9). However, no significant differences were seen in (B) the ratio of phosphorylated to total enzyme, or (C) the ratio of total enzyme to β-actin for either ERK or p38 at any of the time points that were studied (n = 10). Error bars represent ± SEM, *P < 0.05.