The cytokine IL-6 has been widely studied in a wide range of neurodegenerative disorders, including ALS. However, there is still the need to elucidate the exact role of IL-6 during the disease in different tissues. In this work we have analyzed the IL-6 protein expression in two of the most damaged tissues in the SOD1G93A mouse model, the spinal cord and the skeletal muscle, at different stages of the disease to assess its evolution over time. In addition, we have measured IL-6 gene and protein expression levels in blood from ALS patients to study whether or not IL-6 could be considered a marker of the peripheral inflammatory response in ALS.
Our results showed lower IL-6 protein levels in spinal cord at the pre-symptomatic and terminal stages in SOD1G93A mice. On the one hand, this finding supports the early anti-inflammatory or neuroprotective compensatory response found in central nervous system (CNS) [9]. On the other hand, the decreased expression found at the final stage is not in accordance with a unique previous study reporting increased IL-6 levels in the spinal cord of SOD1G93A mice [10]. An explanation for the lower levels of IL-6 found may be that the progressive change to a pro-inflammatory status occurring in later stages of ALS could be governed through other pro-inflammatory cytokines, such as IL-1β and tumor necrosis factor (TNF)-α [11], having IL-6 a less relevant effect. On the other hand, the predominant inflammatory status in the terminal stage in ALS could be explained by higher IL-6 mRNA expression, which could not be translated to protein due to the aberrant RNA processing occurring in ALS, as previously observed in the SOD1G93A mouse model [12]. Nevertheless, very little has been found in the literature about the evolution of this cytokine in the spinal cord in ALS over time and further research could help to better understand the role of this mediator in the spinal cord.
Regarding the skeletal muscle, we found increasing levels of IL-6 from the symptomatic stage to the terminal stage in EDL, but not in SOL. Unlike SOL, EDL is mostly composed of fast-twitch and glycolytic fibers. Previous studies have shown that IL-6 is a myokine whose expression is affected by fiber type [13, 14]. For instance, under no disease condition, IL-6 release was only inducible from SOL but not EDL muscle, leading to higher IL-6 protein levels in slow oxidative muscle fibers [13]. However, fast glycolytic fibers (EDL) are more vulnerable than slow-twitch oxidative fibers (SOL) under a variety of atrophic conditions [15], such as those occurring in ALS. In addition, in ALS, activated inflammation and abnormal glial responses occur in the limb muscle [16]. Therefore, our finding showing an unusual increase of IL-6 in EDL could indicate that the inherent pathological processes of ALS are responsible for this stimulation. In addition, IL-6 can perform as an anti-inflammatory myokine [17], suggesting that increased IL-6 release could represent a neuroprotective reaction against the excitotoxicity damage [18], as observed in an ALS animal model [19]. In this sense, the progressive muscle atrophy may be accompanied by increasing IL-6 levels in EDL reflecting the damage suffered by this type of muscles. Interestingly, inactivation of STAT3-IL6 signaling in fibro-adipogenic progenitors, activated in response to muscle injury, effectively countered muscle atrophy and fibrosis in mouse models of acute denervation and SODG93A mice [20]; this suggests that IL-6 could be a potential target to be considered in ALS therapies. Accordingly, previous studies have shown that neuroprotective molecules such as the fragment C of tetanus toxin may directly act on modulation of IL-6, improving the disease condition in the SOD1G93A mouse model [21].
In relation to circulating IL-6, we analyzed both gene and protein expression of IL-6 in blood samples from patients. We found significant lower levels of IL-6 mRNA in ALS patients compared to the levels obtained in healthy subjects. This result is in agreement with a previous study where they found a downregulation of Il6 gene expression in the peripheral blood of sporadic ALS patients [22]. Similarly, at protein level we found that the absence of IL-6 in blood was significantly associated with the ALS patients group. Despite this association, the no detection of IL-6 could be caused by the very low concentration of this protein found in blood due to the method used to collect the blood samples, PAX tubes, which could be interfering in the integrity of this cytokine. Therefore, the method employed to extract protein from the whole blood may imply a limitation on this study. Although PAX tubes are proper and reliable for mRNA analysis, other methods should be considered for protein study. In addition, the few mRNA transcripts found in blood of ALS patients could be missed in the translation to protein due to a dysfunction in the RNA processing, which is similar to our findings related to the spinal cord of the mouse model. A great variability of protein levels of this cytokine in blood has been found in the literature. Although many studies reported that higher levels were associated with ALS and correlated with faster progression of the disease course [5, 19, 23–26], others found no significant differences [4, 27, 28] or even decreasing IL-6 levels in ALS patients over time [29]. These controversial results found in blood suggest that this inflammatory mediator could be susceptible to variations due to processes not dependent on ALS, e.g. the gender and obesity [30]. In addition, a recent study reported blood IL-6 protein levels increased with aging in both ALS patients and healthy controls [4]. In the same study, they also found an association of higher IL-6 levels with respiratory dysfunction, proposing that this cytokine could be explored as a marker of respiratory failure [4]. In view of our results, another point to consider when analyzing IL-6 protein levels in blood would be the methodology followed from obtaining the whole blood from patients to the ELISA, as it may have a great influence on the results. Therefore, the instability of circulating IL-6 could become a barrier for its consideration by itself as a specific biomarker of ALS.
In our study, the most relevant finding is a consistent increasing protein IL-6 level in EDL in the SOD1G93A mouse model over time, which could correlate with the atrophy progression, and could be an indicator of an anti-inflammatory response to damage by exacerbating the inflammatory status in the muscle. In contrast, we found lower protein IL-6 expression in the spinal cord at pre-symptomatic and terminal stages as well as lower mRNA IL-6 expression in blood of ALS patients, suggesting that IL-6 might not be the main pro-inflammatory mediator in these tissues. On the other hand, given the controversial results found in blood in the literature, we suggest that tissues damaged directly by the disease, such as the skeletal muscle predominantly composed of fast glycolytic muscle fibers, could provide a closer insight of the mechanisms involved in ALS. Accordingly, future studies focused on the study of the role of IL-6 in the skeletal muscle of ALS patients by muscle biopsies could be significance. Although further research is needed to shed light on this, IL-6 could be a potential indicator of damage and the imbalanced inflammatory status of fast glycolytic muscle fibers in ALS.