Characterization of Gene Expression Phenotype in Amyotrophic Lateral Sclerosis Monocytes

Abstract

Importance: Amyotrophic lateral sclerosis (ALS) is a common adult-onset neurodegenerative disease characterized by selective loss of upper and lower motor neurons. Patients with ALS have persistent peripheral and central inflammatory responses including abnormally functioning T cells and activated microglia. However, much less is known about the inflammatory gene profile of circulating innate immune monocytes in these patients.

Objective: To characterize the transcriptomics of peripheral monocytes in patients with ALS.

Design, setting, and participants: Monocytes were isolated from peripheral blood of 43 patients with ALS and 22 healthy control individuals. Total RNA was extracted from the monocytes and subjected to deep RNA sequencing, and these results were validated by quantitative reverse transcription polymerase chain reaction.

Main outcomes and measures: The differential expressed gene signatures of these monocytes were identified using unbiased RNA sequencing strategy for gene expression profiling.

Results: The demographics between the patients with ALS (mean [SD] age, 58.8 [1.57] years; 55.8% were men and 44.2% were women; 90.7% were white, 4.65% were Hispanic, 2.33% were black, and 2.33% were Asian) and control individuals were similar (mean [SD] age, 57.6 [2.15] years; 50.0% were men and 50.0% were women; 90.9% were white, none were Hispanic, none were black, and 9.09% were Asian). RNA sequencing data from negative selected monocytes revealed 233 differential expressed genes in ALS monocytes compared with healthy control monocytes. Notably, ALS monocytes demonstrated a unique inflammation-related gene expression profile, the most prominent of which, including IL1B, IL8, FOSB, CXCL1, and CXCL2, were confirmed by quantitative reverse transcription polymerase chain reaction (IL8, mean [SE], 1.00 [0.18]; P = .002; FOSB, 1.00 [0.21]; P = .009; CXCL1, 1.00 [0.14]; P = .002; and CXCL2, 1.00 [0.11]; P = .01). Amyotrophic lateral sclerosis monocytes from rapidly progressing patients had more proinflammatory DEGs than monocytes from slowly progressing patients.

Conclusions and relevance: Our data indicate that ALS monocytes are skewed toward a proinflammatory state in the peripheral circulation and may play a role in ALS disease progression, especially in rapidly progressing patients. This increased inflammatory response of peripheral immune cells may provide a potential target for disease-modifying therapy in patients with ALS.

Figure 1.. Comparison of Differentially Expressed Genes (DEGs) in Monocytes of Patients With Amyotrophic Lateral Sclerosis (ALS) and Healthy Control Participants by Deep RNA Sequencing

Box and whisker plots (A) and heat map (B) illustrate top 10 upregulated DEGs that have the largest differences of medium expression between the ALS group (n = 23) and healthy control group (HC, n = 10). In box and whisker plots, upper ends of boxes represent the third quartiles of corresponding groups; lower ends of boxes represent the first quartiles of groups; middle lines indicate mediums; upper whiskers indicate the maximum; and lower whiskers indicate the minimum. Outlier data are shown as circles plotted beyond the whiskers.

Figure 2.. Expression of Inflammation-Related Differentially Expressed Genes Verified by Quantitative Reverse Transcription Polymerase Chain Reaction

Upregulation of IL1B (A), NLRP3 (B), IL8 (C), FOSB (D), CD83 (E), SOCS3 (F), CXCL1 (G), and CXCL2 (H) in monocytes of patients with amyotrophic lateral sclerosis (ALS) (n = 43) were verified by quantitative reverse transcription polymerase chain reaction. Arbitrary unit of each mRNA was normalized to β-actin and relative to the healthy control (HC) samples (n = 22). Error bars indicate the standard error. ^aP < .01 vs HC (IL1B, P < .001; NLRP3, P = .007; IL8, P = .002; FOSB, P = .009; and CXCL1, P = .002). ^bP < .05 vs HC (CD83, P = .02; SOCS3, P = .01; and CXCL2, P = .01).

Figure 3.. Heat Map of Differentially Expressed Genes Associated With Inflammatory Responses

More proinflammatory differentially expressed genes were upregulated in monocytes isolated from patients with rapidly progressing amyotrophic lateral sclerosis (ALS fast, n = 10) than from patients with slowly progressing ALS (ALS slow, n = 12) or control groups (HC, n = 10).