Systemic inflammatory response and neuromuscular involvement in amyotrophic lateral sclerosis

Abstract

Objective: To evaluate the combined blood expression of neuromuscular and inflammatory biomarkers as predictors of disease progression and prognosis in amyotrophic lateral sclerosis (ALS).

Methods: Logistic regression adjusted for markers of the systemic inflammatory state and principal component analysis were carried out on plasma levels of creatine kinase (CK), ferritin, and 11 cytokines measured in 95 patients with ALS and 88 healthy controls. Levels of circulating biomarkers were used to study survival by Cox regression analysis and correlated with disease progression and neurofilament light chain (NfL) levels available from a previous study. Cytokines expression was also tested in blood samples longitudinally collected for up to 4 years from 59 patients with ALS.

Results: Significantly higher levels of CK, ferritin, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, IL-2, IL-8, IL-12p70, IL-4, IL-5, IL-10, and IL-13 and lower levels of interferon (IFN)-γ were found in plasma samples from patients with ALS compared to controls. IL-6, TNF-α, and IFN-γ were the most highly regulated markers when all explanatory variables were jointly analyzed. High ferritin and IL-2 levels were predictors of poor survival. IL-5 levels were positively correlated with CK, as was TNF-α with NfL. IL-6 was strongly associated with CRP levels and was the only marker showing increasing expression towards end-stage disease in the longitudinal analysis.

Conclusions: Neuromuscular pathology in ALS involves the systemic regulation of inflammatory markers mostly active on T-cell immune responses. Disease stratification based on the prognostic value of circulating inflammatory markers could improve clinical trials design in ALS.

Figure 1.. Principal component analysis (PCA) of patients with amyotrophic lateral sclerosis (ALS) vs controls

PCA analysis shows a good separation between ALS and controls of the expression levels of the 11 cytokines under investigation, more significant when sex-specific groups are considered. (A) ALS (green squares) and controls (yellow squares), (B) male only ALS (green squares) and controls (yellow squares), and (C) female only ALS (green squares) and controls (yellow squares).

Figure 2.. Odds ratios (ORs) from the multivariate logistic regression analysis and hazard ratios (HRs) from the Cox regression analysis

(A) OR (filled circle) and relative 95% confidence interval (CI) (whiskers) of plasma markers in amyotrophic lateral sclerosis (ALS) and controls from mutually adjusted multivariate logistic regression models; dashed line represents null hypothesis (no difference between ALS and controls). (B) HR (filled circles) and relative 95% CI (whiskers) of plasma markers estimating risk of death among patients with ALS from mutually adjusted multivariate Cox regression analysis. The dashed line represents null hypothesis (no association with survival). p < 0.5, 0.5 < p < 0.1, and p > 0.1 are shown in blue, mulberry, and black, respectively. CRP = C-reactive protein; CVD = cardiovascular disease; IFN = interferon; IL = interleukin; TNF = tumor necrosis factor.

Figure 3.. Longitudinal expression levels of interleukin (IL)–6 for patients with amyotrophic lateral sclerosis (ALS)

The scatterplots show IL-6 plasma levels (black dots) obtained at each follow-up time point from the 59 patients with ALS included in the longitudinal cohort. Median and quartile ranges at each visit are presented with red bars. Patients were subgrouped according to (A.a, A.b) progression rate calculated at baseline (with a cutoff value of 0.5); (B.a, B.b) sex; (C.a, C.b) site of disease onset; (D.a, D.b) ALS Functional Rating Scale–Revised (ALSFRS-R) score at V1 (cutoff value of 40); and (E.a, E.b) whether they were on riluzole or not. *p Value for Kruskal-Wallis test examining difference between all visits; **p value adjusted for multiple comparison between V1 and V6; ***p value adjusted for multiple comparison between V2 (D only) and V6.