Inflammatory signature in amyotrophic lateral sclerosis predicting disease progression

Abstract

Experimental studies identified a role of neuroinflammation in the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the role of inflammatory molecules as diagnostic and prognostic biomarkers in patients with ALS is unclear. In this cross-sectional study, the cerebrospinal fluid (CSF) levels of a set of inflammatory cytokines and chemokines were analyzed in 56 newly diagnosed ALS patients and in 47 age- and sex-matched control patients without inflammatory or degenerative neurological disorders. The molecules analyzed included: interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-17, granulocyte colony stimulating factor (GCSF), macrophage inflammatory protein (MIP)-1a, MIP-1b, tumor necrosis factors (TNF), eotaxin. Principal component analysis (PCA) was used to explore possible associations between CSF molecules and ALS diagnosis. In addition, we analyzed the association between CSF cytokine profiles and clinical characteristics, including the disease progression rate score, and peripheral inflammation assessed using the Neutrophil-to-lymphocyte ratio (NLR). PCA identified six principal components (PCs) explaining 70.67% of the total variance in the CSF cytokine set. The principal component (PC1) explained 26.8% of variance and showed a positive load with CSF levels of IL-9, IL-4, GCSF, IL-7, IL-17, IL-13, IL-6, IL-1β, TNF, and IL-2. Logistic regression showed a significant association between PC1 and ALS diagnosis. In addition, in ALS patients, the same component was significantly associated with higher disease progression rate score and positively correlated with NLR. CSF inflammatory activation in present in ALS at the time of diagnosis and may characterize patients at higher risk for disease progression.

Keywords: Amyotrophic lateral sclerosis (ALS); Cerebrospinal fluid (CSF); Cytokines; Disease progression; Neuroinflammation; Neutrophil-to-lymphocytes ratio (NLR).

Figure 1

Association of individual CSF cytokines with the first 6 PCs. (A) Principal component analysis (PCA) results: load of individual cytokines with the first 6 PCs, significant associations (cut-off = 0.4) are shown (red = positive; blue = negative). (B) Logistic regression: associations between diagnosis group (ALS vs controls) and the first 6 PCs, OR and 95% CI are shown, *p < 0.05. ALS amyotrophic lateral sclerosis, CI confidence interval, CSF cerebrospinal fluid, GCSF granulocyte colony-stimulating factor, IFN interferon, IL interleukin, MIP macrophage inflammatory protein, OR odds ratio, PC principal component, TNF tumor necrosis factor.

Figure 2

CSF cytokines in ALS and control patients. Boxplot showing the CSF levels of inflammatory cytokines in ALS and controls. The circles represent outlier patients. The star marks the extreme values. Mann–Whitney p and B–H corrected p are shown. ALS amyotrophic lateral sclerosis, B–H Benjamini–Hockberg, CSF cerebrospinal fluid, GCSF granulocyte colony-stimulating factor, IL interleukin.

Figure 3

Correlations between CSF cytokines and ALSFRS-R total and bulbar scores. Cytokine concentrations are expressed in pg/ml. Spearman’s Rho, p, and B-H corrected p are shown. ALSFRS-R ALS Functional Rating Scale-Revised, CSF cerebrospinal fluid, B–H Benjamini–Hockberg, CSF cerebrospinal fluid, GCSF granulocyte colony-stimulating factor, IFN interferon, IL interleukin, MIP macrophage inflammatory protein, TNF tumor necrosis factor.

Figure 4

PCA components and Disease progression rate. Logistic regression: associations between ALS disease progression rate group (low vs medium/high) and the first 6 PCs, OR and 95%CI are shown, **p < 0.05. ALS amyotrophic lateral sclerosis, CI confidence interval, GCSF granulocyte colony-stimulating factor, OR Odds ratio, PC principal component.

Figure 5

CSF cytokines and disease progression rate. Boxplot showing the CSF levels of inflammatory cytokines in ALS and controls. The circles represent outlier patients. The star marks the extreme values. Mann–Whitney p and B–H corrected p are shown. ALS amyotrophic lateral sclerosis, B–H Benjamini–Hockberg, CSF cerebrospinal fluid, GCSF granulocyte colony-stimulating factor, IL interleukin, MIP macrophage inflammatory protein.