Microglial TNFR2 signaling regulates the inflammatory response after CNS injury in a sex-specific fashion.

Microglia, the resident immune cells of the central nervous system (CNS), play a major role in damage progression and tissue remodeling after acute CNS injury, including ischemic stroke (IS) and spinal cord injury (SCI). Understanding the molecular mechanisms regulating microglial responses to injury may thus reveal novel therapeutic targets to promote CNS repair. Here, we investigated the role of microglial tumor necrosis factor receptor 2 (TNFR2), a transmembrane receptor previously associated with pro-survival and neuroprotective responses, in shaping the neuroinflammatory environment after CNS injury. By inducing experimental IS and SCI in Cx3cr1CreER:Tnfrsf1bfl/fl mice, selectively lacking TNFR2 in microglia, and corresponding Tnfrsf1bfl/fl littermate controls, we found that ablation of microglial TNFR2 significantly reduces lesion size and pro-inflammatory cytokine levels, and favors infiltration of leukocytes after injury. Interestingly, these effects were paralleled by opposite sex-specific modifications of microglial reactivity, which was found to be limited in female TNFR2-ablated mice compared to controls, whereas it was enhanced in males. In addition, we show that TNFR2 protein levels in the cerebrospinal fluid (CSF) of human subjects affected by IS and SCI, as well as healthy donors, significantly correlate with disease stage and severity, representing a valuable tool to monitor the inflammatory response after acute CNS injury. Hence, these results advance our understanding of the mechanisms regulating microglia reactivity after acute CNS injury, aiding the development of sex- and microglia-specific, personalized neuroregenerative strategies.

Peripherally derived myeloid cells induce disease-dependent phenotypic changes in microglia.

In central nervous system (CNS) injury and disease, peripherally derived myeloid cells infiltrate the CNS parenchyma and interact with resident cells, propagating the neuroinflammatory response. Because peripheral myeloid populations differ profoundly depending on the type and phase of injury, their crosstalk with CNS resident cells, particularly microglia, will lead to different functional outcomes. Thus, understanding how peripheral myeloid cells affect the phenotype and function of microglia in different disease conditions and phases may lead to a better understanding of disease-specific targetable pathways for neuroprotection and neurorepair. To this end, we set out to develop an in vitro system to investigate the communication between peripheral myeloid cells and microglia, with the goal of uncovering potential differences due to disease type and timing. We isolated peripheral myeloid cells from mice undergoing experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, or acute cerebral ischemia by permanent middle cerebral artery occlusion (pMCAO) at different times after disease and probed their ability to change the phenotype of primary microglia isolated from the brain of adult mice. We identified changes not only dependent on the disease model, but also on the timepoint after disease onset from which the myeloid cells were isolated. Peripheral myeloid cells from acute EAE induced morphological changes in microglia, followed by increases in expression of genes involved in inflammatory signaling. Conversely, it was the peripheral myeloid cells from the chronic phase of pMCAO that induced gene expression changes in genes involved in inflammatory signaling and phagocytosis, which was not followed by a change in morphology. This underscores the importance of understanding the role of infiltrating myeloid cells in different disease contexts and phases. Furthermore, we showed that our assay is a valuable tool for investigating myeloid cell interactions in a range of CNS neuroinflammatory conditions.

Systemic treatment with a selective TNFR2 agonist alters the central and peripheral immune responses and transiently improves functional outcome after experimental ischemic stroke.

Ischemic stroke often leaves survivors with permanent disabilities and therapies aimed at limiting detrimental inflammation and improving functional outcome are still needed. Tumor necrosis factor (TNF) levels increase rapidly after ischemic stroke, and while signaling through TNF receptor 1 (TNFR1) is primarily detrimental, TNFR2 signaling mainly has protective functions. We therefore investigated how systemic stimulation of TNFR2 with the TNFR2 agonist NewSTAR2 affects ischemic stroke in mice. We found that NewSTAR2 treatment induced changes in peripheral immune cell numbers and transiently affected microglial numbers and neuroinflammation. However, this was not sufficient to improve long-term functional outcome after stroke in mice.

Conditional Ablation of Myeloid TNF Improves Functional Outcome and Decreases Lesion Size after Spinal Cord Injury in Mice.

Spinal cord injury (SCI) is a devastating condition consisting of an instant primary mechanical injury followed by a secondary injury that progresses for weeks to months. The cytokine tumor necrosis factor (TNF) plays an important role in the pathophysiology of SCI. We investigated the effect of myeloid TNF ablation (peripheral myeloid cells (macrophages and neutrophils) and microglia) versus central myeloid TNF ablation (microglia) in a SCI contusion model. We show that TNF ablation in macrophages and neutrophils leads to reduced lesion volume and improved functional outcome after SCI. In contrast, TNF ablation in microglia only or TNF deficiency in all cells had no effect. TNF levels tended to be decreased 3 h post-SCI in mice with peripheral myeloid TNF ablation and was significantly decreased 3 days after SCI. Leukocyte and microglia populations and all other cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IFNγ) and chemokines (CCL2, CCL5, and CXCL1) investigated, in addition to TNFR1 and TNFR2, were comparable between genotypes. Analysis of post-SCI signaling cascades demonstrated that the MAPK kinase SAPK/JNK decreased and neuronal Bcl-XL levels increased post-SCI in mice with ablation of TNF in peripheral myeloid cells. These findings demonstrate that peripheral myeloid cell-derived TNF is pathogenic in SCI.