Microglial production of interleukin-10, a cytokine that damps down the immune response, is dramatically elevated in presymptomatic mouse models of ALS caused by mutations in superoxide dismutase (SOD1), and blocking IL-10 hastens disease onset, while increasing its expression delays onset and prolongs survival. These findings, published in the January 20 Journal of Neuroscience, suggest that increasing IL-10 expression at the earliest stages disease could offer a potential therapeutic strategy in SOD1-ALS.
A prominent neuroinflammatory component, characterized by astrogliosis and microglial activation, is common to sporadic and familial ALS patients, as well as to SOD1 mouse models of the disease. In transgenic mice overexpressing human ALS-associated variants of SOD1, glial cells accelerate disease progression and directly contribute to motor neuron degeneration (Boillée et. al., 2006, Apr 2007 Alzforum news; Oct 2011 news). However, the role of glial cells at different stages of disease, and the mechanisms of toxicity are not as well understood.
To explore the role of microglia in early stages of SOD1-ALS, Jasna Kriz and colleagues at Laval University in Quebec, Canada, crossed SOD1 mice expressing the G93A or the G37R variants of SOD1 with a mouse model developed in the Kriz lab in which luciferase and green fluorescent protein expression are under control of a mouse toll-like receptor 2 (TLR2) promoter (Lalancette-Hébbert M et. al., 2009). TLR2 expression is minimal in quiescent microglia, but induced in response to infection or injury, Kriz said. Thus, greater light emission corresponds to activation of microglia.
In vivo biophotonic/bioluminescence imaging of double transgenic mice expressing SOD1-G93A revealed a marked reduction in TLR2 expression relative to wild-type controls. The most striking changes were apparent in the olfactory bulb (OB) and the brain from day 45 to day 90, before onset of clinical symptoms (weight loss and impaired hindlimb reflexes). In response to a challenge with lipopolysaccharide (LPS), a bacterial molecule commonly used to trigger inflammatory responses, the TLR2 response in the brain and spinal cord (SC) was significantly reduced, as was expression of TLR2 downstream targets, such as tumor necrosis factor (TNF)-a. The slower progressing SOD1-G37R mouse responded in a similar fashion. These results indicate that microglia in the presymptomatic phase in both SOD1 mouse models had adopted an anti-inflammatory phenotype, Kriz said.
First author Mathieu Gravel next examined the microglial activation profile in primary microglial cultures derived from WT or SOD1-G93A mice at day 60. SOD1 microglial cultures expressed consistently lower levels of TLR2. Cultures derived from SOD1 SC exhibited a 16-fold increase in IL-10 mRNA at baseline, and a 4-fold increase relative to WT following LPS challenge. The anti-inflammatory cytokine TGF-b was also elevated following immune activation. In contrast, production of pro-inflammatory cytokines (IL-1b, IL-6, TNFa) was only modestly different between microglial cultures from SOD1 and WT mice at rest, and only IL-1b expression was elevated in SC microglial cultures relative to controls following LPS stimulation. No increased IL-10 expression was seen in peripheral immune cells at baseline or after LPS challenge, indicating that the elevation of IL-10 is a property of resident microglia, rather than infiltrating macrophages.
The dramatic increase in IL-10 was surprising, Kriz said. “This is one of the most homeostatic cytokines, and plays a key role in modulation of inflammatory processes. The question for us was then whether this increase is helpful or not.”
To address that question, the team chronically infused an antibody that blocks the IL-10 receptor into the cerebral ventricles of SOD1-G93A mice beginning at day 60. The treatment precipitated disease onset 7 days earlier than untreated mice with no effect on survival. Since these results suggested that reducing IL-10 expression is detrimental to motor neuron survival in SOD1 mice, the researchers hypothesized that increasing IL-10 levels during the presymptomatic stages of disease may have neuroprotective effects. To this end, they selectively overexpressed IL-10 in microglia via a one-time injection of an adeno-associated viral (AAV) vector expressing IL-10 under control of the human CD11b promoter at 30-40 days. Onset was delayed by 9 days, and survival increased by 13 days, compared to untreated mice.
“Together, our results suggest that enhanced IL-10 levels together with downregulation of the TLR2 response represent an adaptive resident microglia-cell-specific mechanism that controls early disease propagation and onset,” the team concluded in the paper. “This is the first in vivo evidence showing that specifically targeting the microglial immune profile may have disease modifying effects in SOD1 mutant mice”, Kriz added.
The results of the study confirm the importance of microglia for understanding ALS and as targets for therapy, said Oleg Butovsky of Harvard Medical School, who was not involved in the study and has characterized microglial gene expression signatures in health and disease (Butovsky et. al., 2014., Feb 2015 news). But he raised a concern that the overall cytokine profile, including elevated IL-1b and TNFa in the SC, suggested at best a mixed, rather than definitively anti-inflammatory, phenotype. He noted that previous work has shown that microglia from SOD1-G93A mice undergo multiple and contradictory responses of several kinds during the presymptomatic phase, including proinflammatory changes (Chiu IM et al., 2013).
“It’s a complex, dynamic response,” Butovsky said, “and based only on IL-10, I don’t think we can conclude that this is an anti-inflammatory phenotype. However, I don’t think that reduces the interest of the paper, which shows that IL-10 overexpression [via AAV] suppresses inflammation.”
Much remains unknown about the IL-10 response observed in the mice, including what triggers it, when in the disease course the anti-inflammatory profile changes, and whether other mouse models—and critically, whether people who develop ALS—show the same changes.
Assuming the elevation of IL-10 proves relevant for human disease, Kriz said, “The question is, what is the temporal window of opportunity?” If increasing IL-10 levels, either with a drug or gene therapy, is helpful only presymptomatically, it may be difficult to develop, and perhaps as importantly, to test, such a therapy in people.
That point was emphasized by Jacob Ayers of the University of Florida, who was not involved in the study, but who in previous work showed that AAV delivery of IL-10 to newborn SOD1-G93A mice extended survival (Ayers, J et. al., 2015).
“I believe that when thinking about manipulation of IL-10 as a potential therapeutic strategy,” Ayers said, “we can look at the clinical studies of minocycline and other immunomodulatory therapies aimed at ALS, which resulted in their inability to significantly affect the disease course. These trials reveal that it is extremely difficult to affect the disease course when beginning treatment after symptoms have begun. Therefore, it may be difficult for any drug to have much of an effect on ALS disease pathogenesis until we can identify a reliable biomarker and start treating patients earlier.”
Gravel M, Béland LC, Soucy G, Abdelhamid E, Rahimian R, Gravel C, Kriz J. IL-10 Controls Early Microglial Phenotypes and Disease Onset in ALS Caused by Misfolded Superoxide Dismutase 1. J Neurosci. 2016 Jan 20;36(3):1031-48.[Pubmed]