Multiple sclerosis (MS) is a chronic neurological disease characterized by nerve damage and consequent impairments in vision, movement, balance and mental function. In MS, the immune system mistakenly starts attacking myelin, the protective sheath that surrounds axons (i.e., nerve fibers) in the brain, spinal cord and optic nerves.
Macrophages, immune cells responsible for detecting damaged cells, germs or other debris in the central nervous system (CNS) and eliminating them, have been found to play a key role in MS. These cells can adopt different functional states, which are associated with either inflammation or the repair of damage in the CNS.
Researchers at Ludwig-Maximilians-University Munich and Technical University Munich recently carried out a study aimed at further exploring the contribution of macrophages to the neuroinflammation observed in patients diagnosed with MS and some other neurological diseases.
Their paper, published in Nature Neuroscience, introduces a new approach to study immune cells and the signals driving their behavior in living organisms.
‘”Monocyte-derived macrophages play a dual role in neuroinflammatory conditions, contributing both to disease progression and to tissue repair,” Arek Kendirli, co-first author, told Medical Xpress.
“These opposing actions in experimental models of MS highlight the remarkable plasticity of macrophages. In our previous work, we showed that macrophages transition from an inducible nitric oxide synthase (iNOS)-positive, lesion-promoting phenotype to a lesion-resolving state characterized by arginase-1 (Arg1) expression. However, the molecular cues that govern these transitions in vivo—particularly within the inflamed central nervous system—remain poorly understood.”
The primary objective of the recent study by de la Rosa & Kendirli and their colleagues was to probe the molecular mechanisms that drive the behavior of macrophages in living organisms, examining several genes and signals simultaneously. To do this, they developed a new experimental approach that relies on a technique called CRISPR screening to switch off several genes in living mice and observe associated effects.
Most past studies applied CRISPR screening techniques in vitro, as opposed to living organisms. Studying the effects of knocking out specific genes in living animals, however, would more realistically capture the complexity of the CNS and the behavior of specific cell populations.
“We are interested in macrophages in the context of multiple sclerosis because they are the most abundant cells in active lesions and have this dual capacity to exert damage but also contribute to repair,” said Clara de la Rosa, co-first author of the paper.
“Therefore, there is a lot of untapped therapeutic potential in targeting this cell type to shift the balance of the disease from damaging to repair, but currently there are no therapies actively targeting it.”
A promising method to study mouse immune cells
Some of Kendirli and de la Rosa’s collaborators had previously created a type of immortalized mouse progenitor cell known as Hoxb8 cells. These cells can divide indefinitely in a laboratory setting without dying and can develop into two primary types of immune cells, myeloid cells (e.g., macrophages, monocytes and neutrophils) and lymphoid cells (e.g., T cells, B cells and natural killer cells).
As myeloid cells do not live very long, the researchers started exploring the possibility of transferring Hoxb8 cells that differentiated into myeloid cells into living mice. This would in turn allow them to model the full life cycle of myeloid cells in a living organism.
“The ability of Hoxb8 cells to be cultured long-term made them ideal for introducing CRISPR constructs,” explained Kendirli. “We transplanted these edited cells into mice with experimentally induced autoimmune encephalomyelitis (EAE) about a week before the peak of disease, enabling them to fully adopt the phenotype of endogenous macrophages.
“Using this approach, we screened more than 100 cytokine receptors and signaling components. We identified IFN-γ, TNF-α, GM-CSF, and TGF-β as key regulators of macrophage polarization in vivo, whereas IL-4, IL-10, and IL-13—well-known modulators in vitro—did not direct macrophage polarization in vivo in this MS model at the examined time point.”
CRISPR, the method employed by the authors, is a gene-editing technique that allows neuroscientists to target several genes at once. This ultimately reduces the number of experiments necessary to uncover molecular processes that drive the development and behavior of cells.
“As explained by Arek, we first had to adapt a method that worked to do CRISPR screens in macrophages in our disease model,” said de la Rosa.
“Once we established this method, we could screen for cytokine signaling genes and combine CRISPR with single-cell transcriptomics and intravital and fixed-tissue imaging to really delve deeper into the effects of the cytokines on macrophage functions.”
Further unraveling the underpinnings of neuroinflammation
Using their CRISPR screening-based method, the researchers were able to gather new insight into the molecular processes that contribute to the switching of macrophages to states associated with neuroinflammation. The team was able to identify several signaling proteins (i.e., cytokines) that control macrophage states in the brains of living organisms.
“Our method allows us to screen hundreds of genes within a week, rather than generating individual transgenic mouse lines for each target,” said Kendirli.
“By combining CRISPR screens with single-cell technologies, we can now map macrophage phenotypes at single-cell resolution. Through intravital imaging of edited macrophages within spinal cord lesions, we can then observe their behavior directly in living tissue.”
The researchers’ approach to introducing gene-edited immune precursor cells in healthy and genetically unmodified mice allows for greater experimental flexibility than other previously introduced experimental methods. In the future, the methodology could be used to explore further macrophage cell functions via biosensor-based readouts.
“In my opinion, the most important contribution of our study is methodological,” said de la Rosa. “Our methodology can accelerate the pace of discovery while reducing the work and number of experiments needed.
“Another contribution is the conceptual framework of studying candidate genes we think to be relevant to pathology in animal models in depth, to gather enough information that allows us to leverage data from human disease to try to determine whether the direction we take with the preclinical research will be relevant to people with MS.
“Since many preclinical findings do not translate well in clinical settings, I think it’s important to check for potential relevance during our basic research in disease models.”
The team’s future research plans
The experimental methods introduced by this team of researchers and the precursor cells they developed could soon be used to study target immune cell populations across different organs and in the context of various diseases.
Meanwhile, Kendirli, de la Rosa and their colleagues plan to use their method to perform additional CRISPR screenings, with the hope of uncovering genes that could be realistically targeted by pharmaceutical drugs and might reduce neuroinflammation.
“At the same time, we are working to better understand the dynamics of macrophage state transitions,” said Kendirli. “Despite advances in technology, many parameters—such as the cells’ precise spatial localization, time of arrival, and interactions with neighboring cells—still shape these transitions in ways we are only beginning to uncover.”
Past studies have consistently observed an abundance of macrophages in CNS lesions associated with MS, yet there are currently no treatments targeting these immune cells. The researchers thus plan to continue investigating the role of macrophages in MS, particularly the molecular processes that prompt them to shift between tissue-repairing and inflammation-related states.
“Now that we have a solid methodological pipeline, we want to delve deeper into aspects of macrophage biology that we think might be relevant for a pro-repair shift and that could also be lifestyle or drug-targetable,” added de la Rosa.
“For instance, are any known drug targets playing a role in macrophage regulation? Are there epigenetic changes sensitive to lifestyle and age shifting these cells from more to less pro-repair or pro-damage? There’s still a lot to explore.” https://medicalxpress.com/news/2026-01-insight-immune-inflammation-multiple-sclerosis.html
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