In vitro modelling the neuroimmune system

The molecular mechanisms associated with neuroinflammatory processes are highly interesting prospects for pharmacological intervention in a broad range of diseases in the central and peripheral nervous systems. We support our clients’ drug discovery programs in this area by developing advanced in vitro models which capture various aspects of acute and chronic neuroinflammation.

Cell-based assays for neuroinflammation research

Neuroinflammation refers to the brain’s immune response to injury, infection, or other harmful stimuli, which causes activation of glial cells, such as microglia and astrocytes, and subsequent release of inflammatory mediators. These processes may have important therapeutic implications and our goal is to establish in vitro disease models that accurately mimics various aspects of acute and chronic neuroinflammation.

Mounting evidence suggests that inflammatory responses in the CNS play a key role in the development of a broad range of diseases¹^,²^, ³. However, while the nervous and immune systems employ overlapping mechanisms and shared mediators that promote crosstalk between the two systems, emerging evidence suggests that inflammatory reactions can also be neuroprotective⁴. Consequently, the dual role of CNS inflammation likely has important therapeutic implications.

Inflammatory molecules released during neuroinflammation, such as cytokines, chemokines, complement factors and reactive oxygen species, can directly damage neurons and disrupt neuronal function⁵. Additionally, disease-associated microglia are known to exhibit dysfunction in their ability to accurately perform synaptic pruning, and can also mediate transfer of toxic disease associated peptides and proteins between neurons, leading to progression of neuronal cell death⁶.

Understanding the relationship between neuroinflammation and disease development is crucial for developing effective therapies in multiple therapeutic areas. For example, therapeutic strategies targeting neuroinflammation are being investigated as potential treatments to mitigate neuronal damage and improve outcomes in neurodegenerative diseases such as Alzheimer’s disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS).

Jonathan Levenson
CSO at FireCyte Therapeutics &
Scientific Advisor at Cellectricon

Our collaboration with Cellectricon enabled us to establish advanced in vitro platforms amenable for neuroinflammatory compound screening

Working in a true collaborative fashion, our teams together efficiently explored different in vitro concepts, always with the focus on identifying a concept that allows for scale up to robust high-precision assays amenable for screening. In this way, Cellectricon helped us to identify a platform that recapitulates neuroinflammation systems biology in vitro and progress our work to develop new treatments for progressive neurodegenerative diseases of the eye.

Our approach to modeling neuroinflammation

Neuron-glia interactions and neuroinflammation may be dysfunctional in neurodevelopmental and neurodegenerative disorders. However, relevant in vitro models for investigating such interactions in a drug discovery context are lacking. Using our long-standing experience from developing standardized, advanced in vitro cultures, we are therefore establishing both chimeric and fully humanized models for the NI service module.

Our chimeric model is based on our rodent cortical cultures where neurons become synaptically connected with time in culture. These cultures contain both neurons and astrocytes, whereas the endogenous microglia do not survive the baseline culture conditions. We take advantage of this and add back iPSC-derived microglia of human origin along with factors promoting their survival. With the resulting model, we can study the effect of manipulating human targets in a fully integrated model where we quantify both morphological aspects, such as morphology of astrocytes and microglia, and functional aspects, such as neuronal function and cytokine release from the microglia, and how these are affected by inflammatory insults⁷. Another advantage is that the microglia can also be of patient origin.

While the chimeric culture system is a way of rapidly and reproducibly generating synaptically active tri-cultures, they do not fully represent the human biology. To complement this, we are also developing fully humanized tri-culture systems based on hiPSC-derived neurons, astrocytes, and microglia. This culture system is useful for applications where the target of interest is located downstream from and/or in other cell types than the microglia, for example in the complement system⁸.

High content imaging and functional readouts

We have developed a range of assays that enable quantification of endpoints relevant to neuronal and microglial structure and function in the presence of neuroinflammation. For example, exposure to inflammatory mediators causes a robust increase in pro-inflammatory cytokine secretion as measured using cytokine profiling on the Luminex platform.

High content imaging

Confocal and super-resolution high content imaging and analysis platforms are used for assessment of effects of neuroinflammation on cellular morphology, for example the morphology of astrocytes and microglia.

Functional assays

Our proprietary optical electrophysiology platform and multielectrode array technology are applied to assess effects on neuronal excitability and synaptic function.

Cytokine and protein profiling

Large-scale profiling of cytokines and proteins relevant for neuroinflammation and neurodegeneration are assessed using e.g. the Luminex platform.

Gene expression analysis

Effects of on gene expression following, for example, neuronflammatory insults can be assessed by qPCR, Quantigene or by full transcriptome analysis.

Johan Pihl
CSO

In collaboration with some of our most valued clients, we have developed the neuroinflammation service module

“We have developed functional and end-point assays for studying neuroinflammation in vitro. By utilizing advanced technologies, such as high content imaging and optical electrophysiology, the assays in the NI module can answer important questions on, for example, the connection between inflammatory insults, cytokine release from microglia, and how this leads to altered neuronal function.”

Case studies

All assays in the neuroinflammation service module are designed for high-capacity testing, which makes them suitable for early discovery activities such as Target Discovery and screening of test compounds as well as mechanism of action studies of more advanced molecules.

NEW - Download Case Study (PDF)

References

1. Patani R, Hardingham GE, Liddelow SA. Functional roles of reactive astrocytes in neuroinflammation and neurodegeneration. Nat Rev Neurol. 19:395–409 (2023).

2. Tansey MG, Wallings RL, Houser MC, Herrick MK, Keating CE, Joers V. Inflammation and immune dysfunction in Parkinson disease. Nat Rev Immunol. 22:657–673 (2022).

3. Wang C, Zong S, Cui X, Wang X, Wu S, Wang L, Liu Y, Lu Z. The effects of microglia-associated neuroinflammation on Alzheimer’s disease. Front Immunol. 14:1117172 (2023).

4. Gao C, Jiang J, Tan Y, et al. Microglia in neurodegenerative diseases: mechanism and potential therapeutic targets. Sig Transduct Target Ther. 8:359 (2023).

5. Zipp F, Bittner S, Schafer DP. Cytokines as emerging regulators of central nervous system synapses. Immunity. 9:914-925 (2023).

6. Zhang W, Dan X, Qinwen M, Haibin X. Role of Neuroinflammation in Neurodegeneration Development. Signal Transduction and Targeted Therapy. 8:267 (2023).

7. C. NODIN1, J. PIHL1, B. MA2, M. KARLSSON1, J. LEVENSON2;
1 Cellectricon AB, Mölndal, Sweden; 2 FireCyte Therapeutics, Inc., Beverly, MA. Characterization of a Systems Biology Platform Incorporating Human Microglia with Rodent Neurons and Astrocytes. Program No. PSTR448.04. 2023 Neuroscience Meeting Planner. Washington, D.C.: Society for Neuroscience, 2023. Online.

8. Schartz, ND., Tenner, AJ. The Good, the Bad, and the Opportunities of the Complement System in Neurodegenerative Disease. Journal of Neuroinflammation. 17:354 (2020).