French researchers have shown that modifying peripheral macrophages could quell microglial activation in the central nervous system (CNS), slowing the rate of decline and extending survival in mouse models of amyotrophic lateral sclerosis (ALS).

The findings identify a new and more tractable target than attempts to suppress proinflammatory responses by directly targeting activated microglia.

"People are trying to modify microglia in the CNS. We thought it would be easier to target peripheral macrophages. To target inflammation in the CNS, you have to get into the CNS. But maybe you can just look at targeting the periphery," said Severine Boillee, group leader at the Paris Brain Institute and coauthor of the research, published in the October 19, 2020, online issue of Nature Neuroscience.

Boillee has spent 20 years investigating the pathological interaction between diseased motor neurons and their neighborhood microglia and macrophages in search of therapeutically promising pathways. She has used gene profiling of ALS mice, blood samples from patients and pluripotent stem cell-derived microglia and macrophages in a bid to uncover disease modifying targets at the periphery.

Recent studies of the genetic underpinnings of ALS have identified a number of genes encoding for proteins with roles in the immune system response to motor neuron damage, which when mutated lead to dysregulation of the immune response. Motor neuron degeneration is still held to be the initiating event, but as the resident macrophages of the CNS, the microglia reaction to motor neuron injury is seen as the number one driver of inflammation and disease progression. While the exact mechanisms are unclear, there is a feedback loop, with microglial activation increasing over the course of ALS.

ALS is mainly sporadic, but in the 10% of cases that are familial, more than 25 genes have been implicated, of which SOD1, which encodes superoxide dismutase is the second most frequent cause.

Removing mutant SOD1 slows disease progression in ALS mice, an effect that previously has been attributed to microglia alone. However, as Boillee pointed out, these experiments also involved manipulation of macrophages, meaning it is not possible to discern if there are contributions from the two cell types.

A foot in each world

In her work, she has studied spinal motor neurons, which are unusual in that their soma in the spinal cord are surrounded by microglia and their axons extend into the periphery and are surrounded by peripheral macrophages. That made it possible to differentiate the contributions of the two cell types and to characterize and assess in detail the disease modifying capacity of peripheral macrophages.

To do this, the researchers used busulfan to induce myeloablation, an approach that differs from previous work in which radiation was used, and which has the advantage that unlike radiation, it does not affect the CNS.

"We used busulfan because we knew irradiation would influence the CNS," Boillee said, noting that in addition, as an approved therapeutic, this increases the translational potential of the research.

Myeloablation of mutant SOD1 expressing macrophages in SOD1 mice and their replacement with normal controls did not promote neuroprotection. However, replacing them with macrophages engineered to express fewer neurotoxic reactive oxygen species slowed both peripheral and central pathology in ALS mice, but only during the early disease phase. There was no improvement in overall survival.

The researchers hypothesized that acting at disease onset, which would also be more clinically relevant, might have greater impact on survival. SOD1 mice grafted at disease onset reached the symptomatic stage later and had increased survival. That indicates the timing of replacement of peripheral macrophages is a crucial parameter, and it is possible to increase ALS mouse survival by modifying macrophages at the periphery.

"When anti-inflammatory drugs are used to target microglia in the CNS, they are administered systemically and don't make a distinction between the CNS and the periphery," Boillee said. "We have shown in the mouse model that the two cell types are very different, and remain different during the disease. So the first point to be aware of is that if any drug does not work it could be that it has an opposite or different effect [on the two cell types]."

The differences were highlighted in transcriptome analyses. On average there were 18,171 genes expressed in microglia and 14,080 in peripheral nerve macrophages. The two shared most of these genes, reflecting their common myeloid origins. But both cell types expressed genes the other did not.

Further investigation showed the survival-promoting effect of grafting modified macrophages was linked to a shift in inflammatory reactivity and to upregulation of 39 genes. The effect was to shift the response towards motor neuron support.

By separating out their effects and directly comparing peripheral macrophages to CNS microglia, the researchers have generated a longitudinal transcriptomic dataset of how macrophages in the two compartments of motor neurons react to neurodegeneration in ALS, and highlighted the differences in their response.

Taken overall, the research suggests that modifying peripheral macrophages can down regulate inflammation at the periphery, along nerves and in the CNS, and peripheral macrophages can influence disease progression and survival of ALS mice.

Bone marrow transplant is safe in ALS patients and the animal model proof of principal indicates that modulating macrophages at the periphery could be of therapeutic value in ALS, Boillee said. The same effect was seen using post mortem tissue from ALS patients, showing the findings are not just an artefact of SOD1-expressing mice, she noted.

Having established this proof of principle, Boillee is "really pushing" to find the best reactive oxygen species target and to modify the target. "What we chose [in the experiment] was based on the literature, now we have got more insight on how to target macrophages at the periphery," she said (Chiot, A. et al. Nat Neurosci 2020, Advanced publication).