Increased ratio of circulating neutrophils to monocytes in amyotrophic lateral sclerosis

Abstract

Objective: To elucidate amyotrophic lateral sclerosis (ALS) biomarkers and potential mechanisms of disease, we measured immune cell populations in whole blood from a large cohort of patients with ALS.

Methods: Leukocytes were isolated from the blood of 44 control patients and 90 patients with ALS. The percentages and total numbers of each cell population were analyzed using flow cytometry and matched with patient ALS Functional Rating Scale-Revised (ALSFRS-R) score to correlate leukocyte metrics with disease progression.

Results: We show a significant increase in the percentage of neutrophils and a significant decrease in the percentage of CD4 T cells and CD16(-) monocytes in the blood of patients with ALS compared to controls; however, only CD16(-) monocyte levels correlated with disease progression. We also examined the monocyte surface expression of CCRL2 and CCR3; CD16(-) monocytes displayed decreased percentages and total numbers expressing CCR3, but these numbers did not correlate with ALSFRS-R score. We found that combining multiple disease metrics yielded the most accurate predictor of disease progression: the ratio of neutrophils to CD16(-) monocytes (N:M ratio) is significantly increased in patients with ALS and better correlates with disease progression than any other single metric.

Conclusions: These observations implicate neutrophils and monocytes as important factors in late disease progression.

Figure 1.. Immune cell populations are altered in the peripheral blood of patients with ALS

(A) The total number of leukocytes and (B) the percent and total number of specific leukocyte populations are shown. Filled black circles represent individual healthy human control patients, and empty circles represent patients with ALS. Average and SEM are displayed. Asterisks above bars indicate significance (% of CD45⁺ cells: *p < 0.01, ***p < 0.0001; # of leukocytes: *p < 0.008). ALS = amyotrophic lateral sclerosis.

Figure 2.. Percentage of CD16⁻ monocytes in the blood is reduced during disease progression

The percentage and total number of leukocytes in the blood of patients with amyotrophic lateral sclerosis are correlated with their ALSFRS-R score at the time of blood draw. Data are displayed with best-fit line and 95% confidence intervals. R² value and p value are also shown. ALSFRS-R scores are displayed from high (A) to low (B) to illustrate disease progression. ALSFRS-R = Amyotrophic Lateral Sclerosis Functional Rating Scale–Revised.

Figure 3.. Percentage and total number of CD16⁻ monocytes expressing CCR3 is reduced in patients with ALS

The percentage and total number of CD16⁺ and CD16⁻ monocytes expressing CCRL2 (A) and CCR3 (B) in the peripheral blood are shown. Filled black circles represent individual healthy human control patients, and empty circles represent patients with ALS. Average and SEM are displayed. Asterisks above bars indicate significance (*p < 0.025). ALS = amyotrophic lateral sclerosis.

Figure 4.. The N:M ratio is significantly increased in patients with ALS and correlates with disease progression

(A) The N:M ratio in healthy control patients (filled black circles) and patients with ALS (empty circles) is shown. Average and SEM are displayed. Asterisks above bars indicate significance (***p < 0.001). (B) The N:M ratio in the blood of patients with ALS is correlated with the ALSFRS-R score at the time of blood draw. Data are displayed with best-fit line and 95% confidence intervals. R² value and p value are also shown. ALSFRS-R scores are displayed from high (left) to low (right) to illustrate disease progression. ALS = amyotrophic lateral sclerosis; ALSFRS-R = ALS Functional Rating Scale–Revised; N:M ratio = neutrophil to CD16⁻ monocyte ratio.