Our results show that, in a cross-sectional study, 64% of sALS and fALS subjects have strongly increased serum concentrations of the cytokine IL-17A, compared to normal subjects, and the concentrations of IL-17A fluctuate, which could result in false-negative results in some subjects. The spinal cord of deceased sALS patients show a milieu in which polarization of CD3 cells to IL-17A-producing cells can develop in response to products of macrophages, T cells and mast cells; including IL-1β, TNF-α, IL-6, IL-23, and probably eicosanoids (Figure 2 and 6). The cytokine IL-17A is pathogenic in inflammatory and autoimmune diseases such as multiple sclerosis , psoriasis, inflammatory bowel disease, systemic lupus erythematosus, and rheumatoid arthritis . CD8 cells in gray matter might have a role in tissue destruction by cytotoxic cytokines, the cytotoxic molecules granzyme B, and nitric oxide (NO), resembling the role of CD8 cells in multiple sclerosis . Although IL-17A is expressed on CD4 cells in the animal model of multiple sclerosis, experimental allergic encephalitis , IL-17A is expressed also on other cells, such as macrophages in asthma , CD8 cells in Behcet disease and psoriasis , and mast cells in rheumatoid arthritis synovium.
The ALS spinal cord is infiltrated by IL-17A-positive T cells and IL-17A-positive mast cells in gray matter and by TNF-α-positive macrophages/microglia in gray and white matter (Figure. 2, 3, 4). Macrophages/microglia were found to co-localize with neurons, reminiscent of previously demonstrated large phagocytic cells surrounding atrophic neurons . Although the identification of these cells as macrophages or microglia is not possible since both are CD68-positive, blood-derived macrophages may penetrate into the ALS spinal cord, as suggested by their presence around the vessels with disrupted ZO-1 junctions , and into Alzheimer disease brain, as shown by their invasion across brain endothelial cells with disrupted ZO-1 junction .
To clarify the induction of IL-17A in ALS patients, we focused attention on IL-1β, IL-6 , and IL-23, which are known to induce IL-17A and can be produced by macrophages and/or dendritic cells (Figure 6). The development of IL-17A-producing TH17 cells is initiated by transforming growth factor-β and IL-6, which induce phosphor-STAT-3 and the transcription factor RORγt, and is stabilized and expanded by the cytokines IL-21 and IL-23 [35, 36]. Human TH17 cell differentiation requires IL-6, IL-1β and IL-21 or IL-23 . In human studies, transforming growth factor-β has not been found to be essential . In a recent mouse study, TH17 cells, which were induced by IL-1β, IL-6 and IL-23, were more pathogenic than those induced in presence of transforming growth factor-β . The cytokines and chemokines required for TH17 polarization, IL-1α, IL-6, and CCL20, and matrix metalloproteinase 1, were transcriptionally stimulated more in ALS patients than in controls (Figure 8).
The presence of IL-17A in mast cells in the spinal cord of patients with ALS and Alzheimer disease (Figure 3) has not been previously reported. Mast cells together with macrophages produce eicosanoids , which are important in polarization of the TH17 subset . Mast cells are emerging as master regulators with bi-functional role in both innate and adaptive immunity . In the setting of autoimmunity, mast cells have a role in the initiation of the pathological immune response in experimental allergic encephalomyelitis through modulation of regulatory T cells into pathogenic Th17 cells . Mast cells foster inflammation through the production of IL-6 and the shift of regulatory T cells to TH17 cells .
Fibrillar and APO forms of wild type SOD-1, but not the AI form, induced the key cytokines, IL-1β and IL-6, indicating their crucial role in inflammation of sALS patients (Figure 6). These autoantigens are likely present in the inclusions with non-native/misfolded forms of SOD-1, which are present in sporadic ALS spinal cords , and might be released from live or dying neurons [45, 46] and be presented to autoimmune T cells by macrophages and dendritic cells.
Whole-genome expression analysis revealed that stimulation by SOD-1 increased in mononuclear cells of both patients and controls the transcription of cytokines, chemokines and matrix metallopeptidases. In patients' cells, however, the pro-inflammatory cytokines IL-1α and IL-6 were enhanced more and the anti-inflammatory cytokine IL-10 was enhanced less than in controls' cells (Figure 8). The chemokines expressed at a high level even before stimulation include CCL2 (MCP-1), CXCL1 (GROα), and CXCL3 (GROγ). The chemokine CCL20 (MIP-3α), a chemoattractant for CCR6, the marker of Th17 cells , was increased by SOD-1 stimulation more in patients' than in controls' cells. Matrix metallopeptidase 1, an effector of tissue remodeling , and tissue factor pathway inhibitor 2 were strongly stimulated in patients' cells, suggesting global pathology in ALS . In agreement with the constitutive production of IL-17A in PBMC's, no increase in the transcription of IL-17A upon 18-hr SOD-1 stimulation was observed.