CYLD inhibits NF-κB activation, which is a key pathway in regulating microglia during inflammation and in various CNS diseases [29]. However, the specific mechanisms by which NF-κB is regulated in microglia and the potential pathways targeted by CYLD beyond NF-κB remain incompletely understood. Given the detrimental effects of A20 deletion in microglia [20, 21], an enzyme that functions similar to CYLD, we hypothesized that CYLD overexpression in microglia could have the opposite effect and might protect mice against CNS inflammation. Contrary to our expectations, overexpression of CYLD in microglia did not affect microglial gene expression or function in LPS-induced neuroinflammation and did not influence disease severity in the EAE model. Additionally, in the steady state, CYLD overexpression had no effect on immune cell infiltration into the CNS or on microglia numbers in different brain regions. Thus, we conclude that constitutive overexpression of CYLD in microglia cannot serve as a strategy to protect mice from neuroinflammation.
Our most compelling evidence for this conclusion comes from the model of LPS-induced neuroinflammation. LPS is the most common stimulus for microglia activation in vivo and in vitro [30] and acts by activating the NF-κB pathway in microglia via the activation of TLR4. We induced the overexpression of CYLD in microglia by tamoxifen injection and analyzed the microglial response to LPS 12 weeks later. To our surprise, no differences between R26-Cyld-OECx3 mice and R26RFP/WT littermate controls could be observed on the protein and gene expression levels, despite clear expression changes between naïve and LPS-treated mice, which confirmed that our analysis approach in general worked. This was further corroborated in the EAE model, where CYLD overexpression in microglia did not influence disease severity or immune cell infiltration into the CNS.
In contrast, deletion of the deubiquitinating enzyme A20 in microglia significantly enhanced microglia activation and neuroinflammation. Mice with A20 deletion were more susceptible to LPS and EAE and exhibited a hyperactive microglial phenotype with increased proinflammatory cytokine production [20]. One possible explanation for our observation that microglial CYLD overexpression had no effect, while microglial A20 deletion was detrimental, is that even though CYLD and A20 share a number of substrates, they also have distinct substrate specificities and A20 has non-catalytic functions that CYLD lacks [22]. Furthermore, while mice with full deletion of A20 suffer from multiorgan inflammation and die at a young age [31], the loss of CYLD is not problematic [25] indicating that A20 is more critical in preventing excessive immune responses. This could be due to the fact that A20 is upregulated in a NF-κB-dependent manner which serves as a negative feedback mechanism to terminate NF-κB signaling [32]. Another factor could be that CYLD is expressed at much lower levels in microglia compared to A20 [33], potentially limiting its role in regulating microglia function under physiological conditions. However, even if CYLD is physiologically not required for microglia function, we hypothesized that its overexpression could nonetheless protect against neuroinflammation by restraining NF-κB signaling. One hypothesis for the lack of this protective effect is that the artificially expressed CYLD could be functionally inactivated, possibly through phosphorylation or cleavage [22]. In conclusion, we are convinced that CYLD overexpression in microglia is not effective to protect mice from neuroinflammation. However, we cannot rule out the possibility that endogenous CYLD in microglia could play a role in the regulation of neuroinflammation. Further studies with conditional CYLD deletion in microglia are needed to explore this possibility.
Also in the steady state, CYLD overexpression in microglia did not affect microglia numbers across different brain regions or the infiltration of peripheral immune cells into the CNS. In contrast, mice with A20 deletion in microglia showed increased microglia numbers [20, 21] and spontaneous infiltration of CD8+ T cells in the CNS [21], again suggesting that A20 is more critical for the regulation of microglia physiology. We observed a slight change in microglia morphology in the cortex of R26-Cyld-OECx3 mice twelve weeks post-tamoxifen injection, but this was not seen at two weeks or six months, questioning the significance of this finding. While no studies with a microglia-specific Cyld deletion have been conducted so far, global Cyld−/− mice displayed altered microglial numbers and morphology, along with elevated levels of the proinflammatory cytokines IL-1β and TNF-α in the dorsal striatum, indicating a more activated microglia phenotype [14, 19]. This may result from the loss of CYLD in the microglia themselves, which might remove a regulatory brake on microglia proliferation. The additional overexpression of CYLD in our model does not necessarily need to have the opposite effect, it rather seems likely that it would not have any additional benefit in this context. Furthermore, CYLD is crucial for maintaining neuronal activity, synaptic transmission and normal behavior of mice [14–17]. The loss of CYLD in microglia could contribute to these neurological phenotypes in Cyld−/− mice, for example due to alterations in synaptic pruning conducted by the microglia. Nevertheless, it seems more likely that the absence of CYLD in neurons is the primary cause for these phenotypes, since CYLD highly accumulates in the postsynaptic density in a neuronal activity-dependent manner [34] and therefore can directly regulate synaptic signaling pathways.
In conclusion, overexpression of CYLD in microglia does not protect mice against neuroinflammation and thus is not a viable therapeutic strategy. Further studies using conditional CYLD knock-out mice are necessary to fully elucidate CYLD’s role in microglial regulation. Our novel Rosa26-Cyld-tdTomato mice provide a valuable tool for exploring CYLD’s function across various cell types, potentially advancing our understanding of inflammatory disorders and cancer.