TPL2 kinase activity regulates microglial inflammatory responses and promotes neurodegeneration in tauopathy mice
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eLife assessment
In this study, the authors provide important findings supporting a key role for TLP2 as a regulator of neurotoxic and pro-inflammatory cytokine and chemokine release following acute and chronic neuroinflammation. They provide convincing data supporting that the abrogation of TPL2 kinase activity ameliorates disease pathogenesis in a mouse model of tauopathy. This manuscript will be of broad interest to readers in the fields of neuroimmunology and neurodegenerative disease who are interested in the pathogenic effects of innate immune signaling pathways in disease.
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Abstract
Tumor progression locus 2 (TPL2) (MAP3K8) is a central signaling node in the inflammatory response of peripheral immune cells. We find that TPL2 kinase activity modulates microglial cytokine release and is required for microglia-mediated neuron death in vitro. In acute in vivo neuroinflammation settings, TPL2 kinase activity regulates microglia activation states and brain cytokine levels. In a tauopathy model of chronic neurodegeneration, loss of TPL2 kinase activity reduces neuroinflammation and rescues synapse loss, brain volume loss, and behavioral deficits. Single-cell RNA sequencing analysis indicates that protection in the tauopathy model was associated with reductions in activated microglia subpopulations as well as infiltrating peripheral immune cells. Overall, using various models, we find that TPL2 kinase activity can promote multiple harmful consequences of microglial activation in the brain including cytokine release, iNOS (inducible nitric oxide synthase) induction, astrocyte activation, and immune cell infiltration. Consequently, inhibiting TPL2 kinase activity could represent a potential therapeutic strategy in neurodegenerative conditions.
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Author Response
Reviewer #1 (Public Review):
Wang, Y. et al. investigated the role of TPL2 signaling in acute and chronic neuroinflammatory conditions using small molecule inhibitors and a TPL2 kinase-dead mutant mouse line. They find that TPL2 is upregulated by various brain-resident cells, including microglia, astrocytes, and endothelial cells, during neurodegenerative disease progression and following peripheral LPS injection. They show that upon pharmacological and genetic inhibition during acute LPS stimulation, pro-inflammatory cytokine concentration, microgliosis, and neuronal loss can be reversed. In chronic neuroinflammation, as seen in a tauopathy mouse model, the loss of TPL2 rescues reactive gliosis, immune cell infiltration, neurodegeneration, and cognitive health. Interestingly, TPL2 loss of function was not …
Author Response
Reviewer #1 (Public Review):
Wang, Y. et al. investigated the role of TPL2 signaling in acute and chronic neuroinflammatory conditions using small molecule inhibitors and a TPL2 kinase-dead mutant mouse line. They find that TPL2 is upregulated by various brain-resident cells, including microglia, astrocytes, and endothelial cells, during neurodegenerative disease progression and following peripheral LPS injection. They show that upon pharmacological and genetic inhibition during acute LPS stimulation, pro-inflammatory cytokine concentration, microgliosis, and neuronal loss can be reversed. In chronic neuroinflammation, as seen in a tauopathy mouse model, the loss of TPL2 rescues reactive gliosis, immune cell infiltration, neurodegeneration, and cognitive health. Interestingly, TPL2 loss of function was not significantly beneficial in models of nerve injury and stroke. By analyzing their multiple sequencing datasets and those of other research teams, the authors find that TPL2 aids to upregulate transcripts for the DAM signature, immediate early genes, and astrocyte reactivity. These data build together to further emphasize the intricacy and importance of the immune component in neurodegeneration and other neuroinflammatory conditions.
The conclusions of this paper are mostly well supported by their data, but further confirmation of sequencing results and microglia intrinsic mechanisms need to be expanded.
- In the discussion section, it will be important to highlight that TPL2 could also be directly contributing to tauopathy disease progression through its actions in brain-resident endothelial cells. They spend a lot of time characterizing the effects of TPL2 on in vitro microglial responses and do not adequately discuss the potential that their disease phenotypes in the tauopathy model have more to do with TPL2's ability to regulate BBB permeability or facets of endothelial biology. It will be important to highlight that there are various discrete cellular mechanisms (e.g. functions for TPL2 in microglia, endothelial cells, astrocytes, peripheral immune cells, etc.) that could be underlying the disease readouts seen in their global TPL2 kinase-dead mice. They should discuss this in the context of previous literature demonstrating roles for TPL2 in other non-microglial cell types (e.g. Nanou et al PMID: 34038728).
Thank you for this comment. We agree that while TPL2 is most highly expressed in microglia in the brain, TPL2 expression in endothelial cells and other cell types could potentially contribute to the disease. We have added discussion of this to the manuscript including discussion of the Nanou et al paper which raises the possibility that the TPL2-dependent infiltration of peripheral immune cells in TauP301S mice could be due to regulation of the BBB by TPL2 activity in endothelial cells. We also discuss potential roles for TPL2 in the various other cell types. In addition, we have now added characterization of cell-autonomous TPL2-dependent phenotypes in cultured astrocytes and have provided additional analysis of TPL2-dependent changes in endothelial cells in the scRNAseq experiment in TauP301S mice.
- Hippocampal single-cell RNA sequencing led the authors to report that TLP2KD in the PS19 model of tauopathy reduced the number of T-cell and dendritic cell (DC) infiltrates into the brain. The authors should corroborate this finding with immunohistochemistry or flow cytometry to confirm the presence of changing CD4+, CD8+, and DC populations. Most notably, it is critical for them to enumerate the cell numbers in an effort to validate that there are indeed empirical, and not just proportional, reductions in these cell populations.
Thank you for the suggestion. We have performed immunohistochemistry to examine T cells in fixed brain tissue sections. We have included the data for T cell staining in Figure 5-figure supplement 2. We focused the IHC analysis on staining for CD8+ T cells based on the substantially greater abundance of CD8+ T cells compared to CD4+ T cells or DC in the single cell data (Figure 5C, Figure 5-figure supplement 5) and the availability of an antibody that worked well in our hands. These results corroborate the single cell data by empirically showing significantly increased numbers of T cells in TauP301S mice and significantly reduced numbers in the TauP301S x TPL2KD mice (Figure 5-figure supplement 2).
- The authors concluded from Figure 3 that TPL2 plays a key role in in vivo microglia and astrocyte activation. Adding in an in vitro study, like those done in Figures 1, 2, and S4, that looks at a cell-autonomous role for TPL2 in astrocyte reactivity would strengthen this claim and rule out a microglial-independent pathway of TPL2 inflammation.
Thank you for the suggestion. To investigate the potential cell-autonomous role of TPL2 in astrocytes, we cultured primary mouse astrocyte and stimulated astrocytes with either LPS or cytokines, in the absence or presence of TPL2 inhibitor and measured stimulation induced changes in cytokine release and gene expression. Data are included in Figure 3-figure supplement 1 and the results are discussed in the manuscript. In contrast to the broader TPL2-dependence of cytokine release by cultured microglia only a much more restricted set of cytokines exhibited TPL2-dependence in cultured astrocytes. Furthermore, RT-qPCR analysis of TPL2-dependent activated astrocyte genes identified in the LPS in vivo study found much less TPL2-dependent activation in cultured astrocytes. We discuss that these results suggest that the TPL2-dependent astrocyte activation observed in vivo was probably largely contributed to indirectly by the function of TPL2 in microglia, but there was also potentially some contribution of cell-autonomous function of TPL2 in astrocytes.
- Although the TPL2KD mouse line is a valuable tool to impair TPL2's function while retaining its expression, the researchers failed to comment on the potential effects a global mutation in TPL2 could have in their model systems. Peripheral immunological challenges, like their IP injections of LPS, could behave differently and affect the nervous system in a microglia-independent pathway if monocyte/macrophage signaling is also impaired.
We agree that during peripheral immunological challenges TPL2 could affect the nervous system in a microglia-independent manner. We have added this point to the discussion.
- Oligodendrocytes and OPCs have comparable numbers of DEGs to astrocytes (Figure S11a). What is changing within their transcriptional profile?
In this manuscript we focused on TPL2-dependent DEGs in the Tauopathy model, which were all in microglia. We agree the TPL2-independent changes in the TauP301S mice in other cell types are also interesting. This data set has been uploaded to public data repository (GSE180041) and analysis of the changes in oligodendrocytes has been performed from this data set, as well as other disease models, in a recent publication: “Disease-associated oligodendrocyte responses across neurodegenerative diseases” (PMID: 36001972).
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eLife assessment
In this study, the authors provide important findings supporting a key role for TLP2 as a regulator of neurotoxic and pro-inflammatory cytokine and chemokine release following acute and chronic neuroinflammation. They provide convincing data supporting that the abrogation of TPL2 kinase activity ameliorates disease pathogenesis in a mouse model of tauopathy. This manuscript will be of broad interest to readers in the fields of neuroimmunology and neurodegenerative disease who are interested in the pathogenic effects of innate immune signaling pathways in disease.
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Reviewer #1 (Public Review):
Wang, Y. et al. investigated the role of TPL2 signaling in acute and chronic neuroinflammatory conditions using small molecule inhibitors and a TPL2 kinase-dead mutant mouse line. They find that TPL2 is upregulated by various brain-resident cells, including microglia, astrocytes, and endothelial cells, during neurodegenerative disease progression and following peripheral LPS injection. They show that upon pharmacological and genetic inhibition during acute LPS stimulation, pro-inflammatory cytokine concentration, microgliosis, and neuronal loss can be reversed. In chronic neuroinflammation, as seen in a tauopathy mouse model, the loss of TPL2 rescues reactive gliosis, immune cell infiltration, neurodegeneration, and cognitive health. Interestingly, TPL2 loss of function was not significantly beneficial in …
Reviewer #1 (Public Review):
Wang, Y. et al. investigated the role of TPL2 signaling in acute and chronic neuroinflammatory conditions using small molecule inhibitors and a TPL2 kinase-dead mutant mouse line. They find that TPL2 is upregulated by various brain-resident cells, including microglia, astrocytes, and endothelial cells, during neurodegenerative disease progression and following peripheral LPS injection. They show that upon pharmacological and genetic inhibition during acute LPS stimulation, pro-inflammatory cytokine concentration, microgliosis, and neuronal loss can be reversed. In chronic neuroinflammation, as seen in a tauopathy mouse model, the loss of TPL2 rescues reactive gliosis, immune cell infiltration, neurodegeneration, and cognitive health. Interestingly, TPL2 loss of function was not significantly beneficial in models of nerve injury and stroke. By analyzing their multiple sequencing datasets and those of other research teams, the authors find that TPL2 aids to upregulate transcripts for the DAM signature, immediate early genes, and astrocyte reactivity. These data build together to further emphasize the intricacy and importance of the immune component in neurodegeneration and other neuroinflammatory conditions.
The conclusions of this paper are mostly well supported by their data, but further confirmation of sequencing results and microglia intrinsic mechanisms need to be expanded.
1. In the discussion section, it will be important to highlight that TPL2 could also be directly contributing to tauopathy disease progression through its actions in brain-resident endothelial cells. They spend a lot of time characterizing the effects of TPL2 on in vitro microglial responses and do not adequately discuss the potential that their disease phenotypes in the tauopathy model have more to do with TPL2's ability to regulate BBB permeability or facets of endothelial biology. It will be important to highlight that there are various discrete cellular mechanisms (e.g. functions for TPL2 in microglia, endothelial cells, astrocytes, peripheral immune cells, etc.) that could be underlying the disease readouts seen in their global TPL2 kinase-dead mice. They should discuss this in the context of previous literature demonstrating roles for TPL2 in other non-microglial cell types (e.g. Nanou et al PMID: 34038728).
2. Hippocampal single-cell RNA sequencing led the authors to report that TLP2KD in the PS19 model of tauopathy reduced the number of T-cell and dendritic cell (DC) infiltrates into the brain. The authors should corroborate this finding with immunohistochemistry or flow cytometry to confirm the presence of changing CD4+, CD8+, and DC populations. Most notably, it is critical for them to enumerate the cell numbers in an effort to validate that there are indeed empirical, and not just proportional, reductions in these cell populations.
3. The authors concluded from Figure 3 that TPL2 plays a key role in in vivo microglia and astrocyte activation. Adding in an in vitro study, like those done in Figures 1, 2, and S4, that looks at a cell-autonomous role for TPL2 in astrocyte reactivity would strengthen this claim and rule out a microglial-independent pathway of TPL2 inflammation.
4. Although the TPL2KD mouse line is a valuable tool to impair TPL2's function while retaining its expression, the researchers failed to comment on the potential effects a global mutation in TPL2 could have in their model systems. Peripheral immunological challenges, like their IP injections of LPS, could behave differently and affect the nervous system in a microglia-independent pathway if monocyte/macrophage signaling is also impaired.
5. Oligodendrocytes and OPCs have comparable numbers of DEGs to astrocytes (Figure S11a). What is changing within their transcriptional profile? -
Reviewer #2 (Public Review):
The authors used both pharmacological inhibition and genetic TPL2 kinase dead (KD) mice to test the hypothesis, that inhibition of TPL2 attenuates the microglia inflammatory response to stimuli such as LPS and in the context of chronic (tau mouse model) and acute (optic nerve crush/stroke) neurodegenerative models. The use of TPL2 kinase dead mice rather than KO mice is elegant and important because of the non-enzymatic role of TPL2 in stabilization of its interacting partner ABIN-2. The authors convincingly demonstrated that pharmacological and genetic inhibition of TLP2 in primary microglia reduced the production of pro-inflammatory cytokines, chemokines, and iNOS and consequently reduced neuronal cell death in neuronal-microglial cocultures. Genetic inhibition of TLP2 reduced partially the inflammatory …
Reviewer #2 (Public Review):
The authors used both pharmacological inhibition and genetic TPL2 kinase dead (KD) mice to test the hypothesis, that inhibition of TPL2 attenuates the microglia inflammatory response to stimuli such as LPS and in the context of chronic (tau mouse model) and acute (optic nerve crush/stroke) neurodegenerative models. The use of TPL2 kinase dead mice rather than KO mice is elegant and important because of the non-enzymatic role of TPL2 in stabilization of its interacting partner ABIN-2. The authors convincingly demonstrated that pharmacological and genetic inhibition of TLP2 in primary microglia reduced the production of pro-inflammatory cytokines, chemokines, and iNOS and consequently reduced neuronal cell death in neuronal-microglial cocultures. Genetic inhibition of TLP2 reduced partially the inflammatory response of microglia in the PS31 tau model. Furthermore, the authors observed reduced infiltration of T cells and dendritic cells. Notably, TLP2 inhibition rescued behavioral deficits in PS31 mice. Overall, these studies support the possibility that inhibition of TPL2 kinase may have translational potential in prevention or treatment of tau-driven neurodegeneration. One aspect of the study that merits further investigation is the alteration in the population structure of myeloid cells in the brain under the various conditions that were evaluated using single cell RNA seq. Very high representation of microglia was observed in TauP301S mice at nine months of age. These findings could reflect bias in recovery of cells for the single cell RNA sequencing assay and independent validation of microglia cellularity by immunohistochemistry would address this question.
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