JAK1/2 Regulates the Synergistic Effect Between Interferon γ and Lipopolysaccharide in Microglia

A research paper titled “JAK1/2 Regulates the Synergistic Effect Between Interferon γ and Lipopolysaccharide in Microglia” was published in the Journal of Neuroimmune Pharmacology in 2024. This article was completed by scholars Alexander P. Young and Eileen M. Denovan-Wright from the Department of Pharmacology at Dalhousie University. The study investigates the response mechanisms and signaling pathways of microglia when exposed to exogenous inflammatory stimuli such as lipopolysaccharide (LPS) and endogenous inflammatory mediators like interferon γ (IFNγ).

Research Background

Microglia, as resident immune cells in the brain, play a crucial role in regulating neuroinflammation. Although microglia can inhibit inflammation and promote tissue repair in the central nervous system, in some cases, they can be overactivated, leading to secondary neuronal damage and cognitive impairment. For example, in chronic neurodegenerative diseases such as Alzheimer’s disease (AD) and acute conditions like sepsis-associated encephalopathy, overactivation of microglia can exacerbate neuronal death.

Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria that can activate microglia through the TLR4 receptor. Interferon γ (IFNγ) is an endogenous pro-inflammatory cytokine that can synergize with LPS to enhance the pro-inflammatory phenotype of microglia. However, the mechanism of signal interaction between LPS and IFNγ at the microglial level is currently unclear, which poses challenges for drug development.

Research Objectives and Significance

This study aims to elucidate the mechanism of synergy between LPS and IFNγ by systematically inhibiting the downstream signaling pathways of TLR4 and IFNγ receptor (IFNGR), evaluate the differences in the effects of LPS from different bacterial species on microglia, and explore the therapeutic efficacy of blocking JAK1/2 signaling.

Research Methods and Procedures

Cell Culture and Treatment

The study used various cell models, including SIM-A9 cells for microglia, RAW 264.7 cells for macrophages, and STHDHQ7/Q7 neuronal cell line for experiments. Each cell type was maintained under specific culture conditions and treated with corresponding cytokines and inhibitors prior to experiments.

Synergistic Effect of LPS and IFNγ

The study measured NO release as a proxy for pro-inflammatory activity in microglia by gradually increasing the concentrations of IFNγ and LPS. Using the Griess reagent system to determine NO release, the study found that the combination of IFNγ and LPS could synergistically induce significant NO release in a concentration-dependent manner.

Signal Pathway Inhibition

By using specific signal pathway inhibitors such as Ruxolitinib for JAK1/2 and Tak-242 for TLR4, the study examined the effects of these inhibitors on NO release and mRNA expression when treating with IFNγ and LPS alone or in combination. Notably, Ruxolitinib was able to completely inhibit the pro-inflammatory response of microglia induced by IFNγ and LPS, demonstrating the critical role of JAK1/2 in this signaling process.

Impact of LPS from Different Bacterial Sources

To assess the differences in the effects of LPS from different sources, the study used LPS derived from Escherichia coli, Klebsiella pneumoniae, and Akkermansia muciniphila, finding that LPS from K. pneumoniae was more potent in activating microglia compared to LPS from other species.

Research Results and Analysis

Synergistic Effect of LPS and IFNγ

The study found that combined treatment with IFNγ and LPS could synergistically release NO. Isobologram and combination index analyses confirmed that their combination produced a significant enhancing effect. This suggests that microglia may be overactivated in various pathological states when circulating levels of LPS and IFNγ are elevated.

Effects of Signal Pathway Inhibition

By systematically inhibiting the downstream signaling pathways of TLR4 and IFNGR, the study showed that JAK1/2 plays a crucial regulatory role in the pro-inflammatory response induced by the combination of LPS and IFNγ. In particular, the use of the JAK1/2 inhibitor Ruxolitinib could completely block the pro-inflammatory response of microglia, indicating that JAK1/2 may be an important target for drug intervention in this process.

Differences in LPS from Various Bacterial Sources

The study compared the effects of LPS from different sources and found that LPS from K. pneumoniae strains had the strongest pro-inflammatory effect, while LPS from A. muciniphila was the least potent. This suggests that the source of LPS plays an important role in the activation of microglia, and LPS from different strains may affect TLR4 activation through different mechanisms.

Secondary Neuronal Damage

By modulating IFNγ and LPS in the established conditioned medium system, it was found that pre-treated microglial conditioned medium could cause varying degrees of damage to STHDHQ7/Q7 neuronal cells. Notably, Ruxolitinib in the conditioned medium significantly reduced neuronal death, further supporting the feasibility of JAK1/2 inhibition as a potential therapeutic strategy.

Conclusions and Value

This study reveals the specific mechanism of LPS and IFNγ synergistically activating microglia, particularly through the regulation of the JAK1/2 signaling pathway. The results suggest that in many pathological states, the synergistic effect of LPS and IFNγ may lead to overactivation of microglia, thereby exacerbating neuroinflammation. Therefore, inhibition targeting JAK1/2 may be an effective approach for treating related neuropathies. This study not only expands our understanding of microglial response mechanisms but also provides new ideas and directions for future drug development.

Research Highlights

1. Synergistic Effect: For the first time, the synergistic pro-inflammatory effect of LPS and IFNγ through the JAK1/2 signaling pathway in microglia was clearly demonstrated.
2. Inhibitor Efficacy: Ruxolitinib (JAK1/2 inhibitor) can completely block the pro-inflammatory response induced by the combination of LPS and IFNγ, showing significant potential for drug intervention.
3. Bacterial Species Differences: LPS from different bacterial species shows significant differences in activating microglia, providing new insights into understanding and preventing clinical bacterial infections.

By revealing more detailed signaling mechanisms of microglia under inflammatory conditions, this study lays an important foundation for future exploration of neuroinflammation intervention strategies.