Myeloid C/EBP? deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis.
ABSTRACT: CCAAT/enhancer binding protein ? (C/EBP?) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. Mice deficient for C/EBP? show protection against excitotoxic and ischemic CNS damage, but the involvement in this neuroprotective effect of the various C/EBP?-expressing cell types is not solved. Since C/EBP?-deficient microglia show attenuated neurotoxicity in culture, we hypothesized that specific C/EBP? deficiency in microglia could be neuroprotective in vivo. In this study, we have tested this hypothesis by generating mice with myeloid C/EBP? deficiency.Mice with myeloid C/EBP? deficiency were generated by crossing LysMCre and C/EBP?fl/fl mice. Primary microglial cultures from C/EBP?fl/fl and LysMCre-C/EBP?fl/fl mice were treated with lipopolysaccharide ± interferon ? (IFN?) for 6 h, and gene expression was analyzed by RNA sequencing. Gene expression and C/EBP? deletion were analyzed in vivo in microglia isolated from the brains of C/EBP?fl/fl and LysMCre-C/EBP?fl/fl mice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBP?fl/fl or control genotypes were subjected to experimental autoimmune encephalitis and analyzed for clinical signs for 52 days. One- or two-way ANOVA or Kruskal-Wallis with their appropriate post hoc tests were used.LysMCre-C/EBP?fl/fl mice showed an efficiency of C/EBP? deletion in microglia of 100 and 90% in vitro and in vivo, respectively. These mice were devoid of female infertility, perinatal mortality and reduced lifespan that are associated to full C/EBP? deficiency. Transcriptomic analysis of C/EBP?-deficient primary microglia revealed C/EBP?-dependent expression of 1068 genes, significantly enriched in inflammatory and innate immune responses GO terms. In vivo, microglial expression of the pro-inflammatory genes Cybb, Ptges, Il23a, Tnf and Csf3 induced by systemic lipopolysaccharide injection was also blunted by C/EBP? deletion. CNS expression of C/EBP? was upregulated in experimental autoimmune encephalitis and in multiple sclerosis samples. Finally, LysMCre-C/EBP?fl/fl mice showed robust attenuation of clinical signs in experimental autoimmune encephalitis.This study provides new data that support a central role for C/EBP? in the biology of activated microglia, and it offers proof of concept for the therapeutic potential of microglial C/EBP? inhibition in multiple sclerosis.
Project description:<h4>Background</h4>We generated a mouse model of primary microglial dysfunction by deleting two negative immune regulatory genes, Cx3cr1 and Socs3 (in LysM<sup>+</sup> cells). This study aimed to understand how primary microglial dysfunction impacts retinal neurons during aging.<h4>Methods</h4>The LysMCre-Socs3<sup>fl/fl</sup>Cx3cr1<sup>gfp/gfp</sup> double knockout (DKO), LysMCre-Socs3<sup>fl/fl</sup>, Cx3cr1<sup>gfp/gfp</sup> and Socs3<sup>fl/fl</sup> mice were maintained up to 12?months. Eyes were collected and processed for immunohistochemistry of IBA-1, cone arrestin, secretagogin, PKC? and GABA. Brain microglia from DKO and WT mice were stimulated with LPS?+?IFN-? or IL-4. The expression of TNF-?, IL-1?, IL-6, iNOS, IL-12p40, IL-23p19, CCL2, CCL5, CXCL2, IL-10, CD206 and Arg1 were examined by qRT-PCR and protein production was measured by Luminex assay. Retinal explants from C57BL/6?J mice were co-cultured with microglia from DKO or WT mice for 24?h, after which the number of cone arrestin<sup>+</sup> cells in retinal flatmount were quantified.<h4>Results</h4>In 3-5?month old mice, the number of microglia in retinal ganglion cell layer (GCL) and inner plexiform layer (IPL) were comparable in all strains of mice. The DKO mice had a significantly higher number of microglia in the outer plexiform layer (OPL) but significantly lower numbers of cone arrestin<sup>+</sup>, secretagogin<sup>+</sup> and GABA<sup>+</sup> cells compared to Socs3<sup>fl/fl</sup> and single KO mice. During aging, 57% of the DKO mice died before 12?months old. The 10-12?months old DKO mice had significantly higher numbers of microglia in GCL/IPL and OPL than age-matched Socs3<sup>fl/fl</sup> and single KO mice. The aged DKO mice developed retinal pigment epithelial (RPE) dysmorphology accompanied by subretinal microglial accumulation. The number of photoreceptors, bipolar cells (Secretagogin<sup>+</sup> or PKC?<sup>+</sup>) and GABA<sup>+</sup> amacrine cells was significantly lower in aged DKO mice compared to age-matched Socs3<sup>fl/fl</sup> and single KO mice. Microglia from DKO mice showed significantly higher levels of phagocytic activity and produced higher levels of TNF-?, IL-6, CCL2, CCL5, CXCL2 and CXCL10 compared to microglia from Socs3<sup>fl/fl</sup> mice. Co-culture of retinal explants with LPS?+?IFN-? or IL-4 pre-treated DKO microglia significantly reduced cone photoreceptor survival.<h4>Conclusions</h4>The LysMCre-Socs3<sup>fl/fl</sup>Cx3cr1<sup>gfp/gfp</sup> DKO mice displayed primary microglial dysfunction and developed age-related retinal microgliopathy characterized by aggragated microglial activation and multiple retinal neuronal and RPE degeneration.<h4>Trial registration</h4>Not applicable. The article does not contain any results from human participants.
Project description:CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. Mice deficient for C/EBPβ show protection against excitotoxic and ischemic CNS damage but the involvement of the various C/EBPβ expressing cell types in this neuroprotective effect is not solved. Since C/EBPβ-deficient microglia show attenuated neurotoxicity in culture we hypothesized that specific C/EBPβ deficiency in microglia could be neuroprotective in vivo. In this study we have tested this hypothesis by generating mice with myeloid C/EBPβ deficiency. Mice with myeloid C/EBPβ deficiency were generated by crossing LysMCre and C/EBPβfl/fl mice . Primary microglial cultures from C/EBPβfl/fl (named here as WT) and LysMCre-C/EBPβfl/fl (named here as KO) mice were treated with lipopolysaccharide ± interferon γ (IFNγ) for 6 h and gene expression was analyzed by RNA sequencing. LysMCre-C/EBPβfl/fl mice showed an efficiency of C/EBPβ deletion of 100% in cultured microglia. Transcriptomic analysis of C/EBPβ-deficient primary microglia revealed C/EBPβ-dependent expression of 1068 genes, significantly enriched in inflammatory and innate immune responses GO terms. This study provides new data that support a central role for C/EBPβ in the biology of activated microglia. Overall design: LysMCre-C/EBPβfl/fl genotype (12 samples): 4 samples treated with LPS, 4 with LPS +IFNg, and 4 vehicle. C/EBPβfl/fl genotype (9 samples): 3 samples treated with LPS, 3 with LPS +IFNg, and 3 vehicle. Design Case (Treatment LPS or LPS +INF) control (No treatment or vehicle) in LysMCre-C/EBPβfl/fl genotype and in C/EBPβfl/fl genotype
Project description:Among the pathogenic processes contributing to dopaminergic neuron (DN) death in Parkinson disease (PD), evidence points to non-cell-autonomous mechanisms, particularly chronic inflammation mounted by activated microglia. Yet little is known about endogenous regulatory processes that determine microglial actions in pathological states. We examined the role of glucocorticoid receptors (GRs), activated by glucocorticoids released in response to stress and known to regulate inflammation, in DN survival. Overall GR level was decreased in substantia nigra of PD patients and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. GR changes, specifically in the microglia after MPTP treatment, revealed a rapid augmentation in the number of microglia displaying nuclear localization of GR. Mice with selective inactivation of the GR gene in macrophages/microglia (GR(LysMCre)) but not in DNs (GR(DATCre)) showed increased loss of DNs after MPTP intoxication. This DN loss in GR(LysMCre) mice was not prevented by corticosterone treatment, in contrast to the protection observed in control littermates. Moreover, absence of microglial GRs augmented microglial reactivity and led to their persistent activation. Analysis of inflammatory genes revealed an up-regulation of Toll-like receptors (TLRs) by MPTP treatment, particularly TLR9, the level of which was high in postmortem parkinsonian brains. The regulatory control of GR was reflected by higher expression of proinflammatory genes (e.g., TNF-α) with a concomitant decrease in anti-inflammatory genes (e.g., IL-1R2) in GR(LysMCre) mice. Indeed, in GR(LysMCre) mice, alterations in phosphorylated NF-κB levels indicated its protracted activation. Together, our data indicate that GR is important in curtailing microglial reactivity, and its deregulation in PD could lead to sustained inflammation-mediated DN injury.
Project description:Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 has a crucial role in inhibiting STAT3 activation, cytokine signaling, and inflammatory gene expression in macrophages/microglia. To determine the role of SOCS3 in myeloid cells in neuroinflammation, mice with conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3(fl/fl)) were tested for experimental autoimmune encephalomyelitis (EAE). The myeloid-specific SOCS3-deficient mice are vulnerable to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with a severe, nonresolving atypical form of disease. In vivo, enhanced infiltration of inflammatory cells and demyelination is prominent in the cerebellum of myeloid-specific SOCS3-deficient mice, as is enhanced STAT3 signaling and expression of inflammatory cytokines/chemokines and an immune response dominated by Th1 and Th17 cells. In vitro, SOCS3-deficient macrophages exhibit heightened STAT3 activation and are polarized toward the classical M1 phenotype. SOCS3-deficient M1 macrophages provide the microenvironment to polarize Th1 and Th17 cells and induce neuronal death. Furthermore, adoptive transfer of M2 macrophages into myeloid SOCS3-deficient mice leads to delayed onset and reduced severity of atypical EAE by decreasing STAT3 activation, Th1/Th17 cells, and proinflammatory mediators in the cerebellum. These findings indicate that myeloid cell SOCS3 provides protection from EAE through deactivation of neuroinflammatory responses.
Project description:In multiple sclerosis plaques, oligodendroglial connexin (Cx) 47 constituting main gap junction channels with astroglial Cx43 is persistently lost. As mice with Cx47 single knockout exhibit no demyelination, the roles of Cx47 remain undefined. We aimed to clarify the effects of oligodendroglia-specific Cx47 inducible conditional knockout (icKO) on experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein peptide (MOG35-55) in PLP/CreERT;Cx47fl/fl mice at 14 d after tamoxifen injection. Cx47 icKO mice demonstrated exacerbation of acute and chronic relapsing EAE with more pronounced demyelination than Cx47 flox (fl)/fl littermates. CD3+ T cells more abundantly infiltrated the spinal cord in Cx47 icKO than in Cx47 fl/fl mice throughout the acute to chronic phases. CXCR3-CCR6+CD4+ and IL17+IFN?-CD4+ helper T (Th) 17 cells isolated from spinal cord and brain tissues were significantly increased in Cx47 icKO mice compared with Cx47 fl/fl mice, while MOG35-55-specific proliferation and proinflammatory cytokine production of splenocytes were unaltered. Microarray analysis of isolated microglia revealed stronger microglial activation toward proinflammatory and injury-response phenotypes with increased expressions of chemokines that can attract Th17 cells, including Ccl2, Ccl3, Ccl4, Ccl7, and Ccl8, in Cx47 icKO mice compared with Cx47 fl/fl mice. In Cx47 icKO mice, NOS2+ and MHC class II+ microglia were more enriched immunohistochemically, and A1-specific astroglial gene expressions and astroglia immunostained for C3, a representative A1 astrocyte marker, were significantly increased at the acute phase, compared with Cx47 fl/fl mice. These findings suggest that oligodendroglia-specific Cx47 ablation induces severe inflammation upon autoimmune demyelination, underscoring a critical role for Cx47 in regulating neuroinflammation.
Project description:The JAK/STAT pathway is critical for development, regulation, and termination of immune responses, and dysregulation of the JAK/STAT pathway, that is, hyperactivation, has pathological implications in autoimmune and neuroinflammatory diseases. Suppressor of cytokine signaling 3 (SOCS3) regulates STAT3 activation in response to cytokines that play important roles in the pathogenesis of neuroinflammatory diseases, including IL-6 and IL-23. We previously demonstrated that myeloid lineage-specific deletion of SOCS3 resulted in a severe, nonresolving atypical form of experimental autoimmune encephalomyelitis (EAE), characterized by lesions, inflammatory infiltrates, elevated STAT activation, and elevated cytokine and chemokine expression in the cerebellum. Clinically, these mice exhibit ataxia and tremors. In this study, we provide a detailed analysis of this model, demonstrating that the atypical EAE observed in LysMCre-SOCS3(fl/fl) mice is characterized by extensive neutrophil infiltration into the cerebellum and brainstem, increased inducible NO synthase levels in the cerebellum and brainstem, and prominent axonal damage. Importantly, infiltrating SOCS3-deficient neutrophils produce high levels of CXCL2, CCL2, CXCL10, NO, TNF-α, and IL-1β. Kinetic studies demonstrate that neutrophil infiltration into the cerebellum and brainstem of LysMCre-SOCS3(fl/fl) mice closely correlates with atypical EAE clinical symptoms. Ab-mediated depletion of neutrophils converts the atypical phenotype to the classical EAE phenotype and, in some cases, a mixed atypical/classical phenotype. Blocking CXCR2 signaling ameliorates atypical EAE development by reducing neutrophil infiltration into the cerebellum/brainstem. Thus, neutrophils lacking SOCS3 display elevated STAT3 activation and expression of proinflammatory mediators and play a critical role in the development of atypical EAE.
Project description:Neuron-microglia interactions have a crucial role in maintaining the neuroimmune system. The balance of neuroimmune system has emerged as an important process in the pathophysiology of depression. However, how neuron-microglia interactions contribute to major depressive disorders has been poorly understood. Herein, we demonstrated that microglia-derived synaptic changes induced antidepressive-like behavior by using microglia-specific signal transducer and activator of transcription 3 (STAT3) knockout (KO) (STAT3<sup>fl/fl</sup>;LysM-Cre<sup>+/-</sup>) mice. We found that microglia-specific STAT3 KO mice showed antidepressive-like behavior in the forced swim, tail suspension, sucrose preference, and open-field tests. Surprisingly, the secretion of macrophage colony-stimulating factor (M-CSF) was increased from neuronal cells in the brains of STAT3<sup>fl/fl</sup>;LysM-Cre<sup>+/-</sup> mice. Moreover, the phosphorylation of antidepressant-targeting mediators and brain-derived neurotrophic factor expression were increased in the brains of STAT3<sup>fl/fl</sup>;LysM-Cre<sup>+/-</sup> mice as well as in neuronal cells in response to M-CSF stimulation. Importantly, the miniature excitatory postsynaptic current frequency in the medial prefrontal cortex was increased in STAT3<sup>fl/fl</sup>;LysM-Cre<sup>+/-</sup> mice and in the M-CSF treatment group. Collectively, microglial STAT3 regulates depression-related behaviors via neuronal M-CSF-mediated synaptic activity, suggesting that inhibition of microglial STAT3 might be a new therapeutic strategy for depression.
Project description:Biallelic defects of the gene encoding for the intracellular enzyme 3â repair exonuclease (Trex)1 cause Aicardi-GoutiÃ¨res syndrome (AGS), a rare monogenic, lupus-like autoimmune disease, while heterozygous Trex1 loss of function alleles are associated with systemic lupus erythematosus (SLE). Trex1-/- mice develop lethal autoimmune multi-organ inflammation, which results from a chronic type I IFN response triggered by intracellular accumulation of a putative nucleic acid substrate of Trex1. This pathogenic nucleic acid is detected by the broadly, but not ubiquitously, expressed cytosolic DNA sensor cGAS, raising the question whether there are specific cell types that respond to Trex1 deficiency by production of IFN and induce autoimmunity. We generated mice with conditional knock out of the Trex1 gene and demonstrated that loss of Trex1 throughout the hematopoietic system and even selective loss in dendritic cells is sufficient to cause IFN release and autoimmunity. B cells showed no transcriptional response to Trex1 deficiency. Trex1-/- keratinocytes produced IFN but did not induce skin inflammation, whereas loss of Trex1 in cardiomyocytes triggered neither IFN response nor pathology. Trex1-deficient neurons and astrocytes did not release IFN in the CNS. In contrast, mice with selective inactivation of Trex1 in long-living CNS macrophages such as microglia locally produced IFN but did not reproduce the mild encephalitis seen in Trex1-/- mice. Collectively, individual cell types differentially respond to the loss of Trex1 and dendritic cells seem promising candidates for experiments addressing the molecular pathomechanism in Trex1 deficiency. We sorted CD19-positive B cells from spleens of Trex1fl/fl CD19-Cre+ and Trex1fl/fl CD19-Cre- mice and isolated total RNA for library construction to perform mRNA deep sequencing.
Project description:IL-6 is required for the response of mice against Listeria monocytogenes. Control of infection depends on classical IL-6 signaling via membrane IL-6R?, but IL-6 target cells and protective mechanisms remain unclear. We used mice with IL-6R?-deficiency in T cells (Il6rafl/fl×CD4cre) or myeloid cells (Il6rafl/fl×LysMcre) to define the role of these cells in IL-6-mediated protection. Abrogation of IL-6R? in T cells did not interfere with bacteria control and induction of TH1 and CD8+ T-cell responses. IL-6R?-deficiency in myeloid cells caused significant defects in listeria control. This defect was not associated with reduced recruitment of granulocytes and inflammatory monocytes, and both cell populations were activated and not impaired in cytokine production. However, IL-6R?-deficient inflammatory monocytes displayed diminished expression of IL-4R? and of CD38, a protein required for phagocytosis and innate control of listeria. In vitro studies revealed that IL-4 and IL-6 cooperated in induction of CD38. In listeria-infected mice, phagocytic activity of inflammatory monocytes correlated with CD38 expression levels on cells and inflammatory monocytes of Il6rafl/fl×LysMcre mice were significantly impaired in phagocytosis. In conclusion, we demonstrate that inhibition of classical IL-6 signaling in myeloid cells causes alterations in differentiation and function of these cells, which subsequently prevent effective control of L. monocytogenes.
Project description:Amyloid-? (A?) accumulation in the brain is a hallmark of Alzheimer's disease (AD) pathology. However, the molecular mechanism controlling microglial A? phagocytosis is poorly understood. Here we found that the E3 ubiquitin ligase Pellino 1 (Peli1) is induced in the microglia of AD-like five familial AD (5×FAD) mice, whose phagocytic efficiency for A? was then impaired, and therefore Peli1 depletion suppressed the A? deposition in the brains of 5×FAD mice. Mechanistic characterizations indicated that Peli1 directly targeted CCAAT/enhancer-binding protein (C/EBP)?, a major transcription factor responsible for the transcription of scavenger receptor CD36. Peli1 functioned as a direct E3 ubiquitin ligase of C/EBP? and mediated its ubiquitination-induced degradation. Consequently, loss of Peli1 increased the protein levels of C/EBP? and the expression of CD36 and thus, promoted the phagocytic ability in microglial cells. Together, our findings established Peli1 as a critical regulator of microglial phagocytosis and highlighted the therapeutic potential by targeting Peli1 for the treatment of microglia-mediated neurological diseases.