Epigenetic and transcriptional control of the microglial inflammatory response: potential insights into neuro-inflammaging

Inflammation is an evolutionarily conserved host defense response during infection or injury that seeks to remove the causal agent that led to its initiation, repair the damaged tissue(s) and restore homeostasis. Thus, transient inflammation in response to an adequate threat with a quick return to a basal resting state is beneficial. However, when inflammation becomes inappropriately increased or prolonged it can have severe pathophysiological consequences. During aging, the immune system shifts to a proinflammatory state characterized by low-grade, chronic, sterile inflammation that has been termed ‘inflammaging’. The proinflammatory state largely results from chronic activation of the innate immune system and includes elevated circulating levels of inflammatory mediators including the immune cell signaling molecules (cytokines) Interleukin (IL)-1, IL-6, IL-8, IL-13, IL-18, tumor necrosis factor (TNFα) and antivirals (the type I interferons (IFN-I). Numerous factors are thought to contribute to inflammaging and amongst potential mechanisms high fat feeding/obesity and associated increases in gut permeability to bacterial endotoxins, as well as, cellular senescence have emerged as key contributors. Importantly, inflammaging is tightly correlated to global indicators of poor health status, multimorbidity, impairment in day-to-day living activities and is thought to underlie or accelerate most age-dependent chronic diseases (e.g. cardiovascular disease, diabetes, cancer, as well as, neurodegenerative conditions like Parkinson’s disease (PD) and Alzheimer’s disease (AD)).While mechanisms driving peripheral inflammaging are beginning to be understood, the etiology of neuro-inflammaging is a crucially unresolved issue. An important source of inflammation in the brain are the non-neuronal cell populations (glia) which provide structural, trophic, and other physiological support for neurons, and especially, the activity of microglia–the brain’s own resident innate immune cells. Microglia are essential for brain development, maintenance and protection throughout the life of an organism. As innate immune cells, however, they can also mount a full inflammatory response to infection or environmental challenge to restore brain health.Environmental factors, for example, changes in peripheral levels of fatty acids, bacterial endotoxins or proinflammatory mediators resulting from high fat feeding or gut permeabilization can cause microglia to undergo changes that signal activation. Most importantly, microglia lose their homeostatic function during aging, becoming less neuroprotective and increasingly neurotoxic. Microglia-mediated inflammation, for example, is strongly linked to age-induced cognitive impairment, is a common hallmark of both PD and AD, and is believed to be mechanistically important in driving pathogenesis. Thus, there is great interest in discovering factors that regulate age-related changes in microglial inflammatory function.Although inflammation is a complex and multicomponent response, a key point of its control occurs at the level of gene transcription and involves several classes of transcription factors, transcriptional co-regulators and chromatin modifications. A recent 2017 study by Soreq et al., identified a relatively unknown gene, PHD finger protein 15 (PHF15) as one of the top 25 differentially expressed genes in microglia during non-pathological aging in humans, with PHF15 levels increasing with age. Sequence and structural similarity to other members of the PHF family suggest that PHF15 might function as a putative chromatin-mediated gene regulator. I first sought to determine whether PHF15 could repress inflammatory function in microglia. If so, I wanted to investigate whether factors known to be causal in inflammaging (e.g. high fat feeding/obesity or cellular senescence) lead to age-dependent cognitive impairment via modulation of microglial PHF15. A major hallmark of senescent cells is the secretion of inflammatory mediators including various cytokines (lL-6, IL-1β, IL-8), chemoattractant cytokines (chemokines; for example C-X-C motif chemokine 10 (CXCL10), C-C motif chemokine ligand (CCL-) 5 (CCL5) and CCL20), antivirals (IFN-I), growth factors and extracellular matrix proteases termed the senescence-associated secretory phenotype (SASP). Secretion of the SASP is partially controlled by the Cyclic GMP-AMP (cGAMP) synthase (cGAS)-Stimulator of interferon genes (STING) (cGAS-STING) cytosolic DNA sensing pathway at the molecular level. Thus, peripheral changes induced by high fat feeding/obesity or the SASP could lead to increased neuroinflammation via inhibition of microglial PHF15.I show that Phf15 significantly represses proinflammatory gene expression in mouse microglia, modulating both the magnitude and duration of the inflammatory response. Importantly, Phf15 regulates both basal expression and signal-dependent upregulation of proinflammatory genes—which constitute different phases of the transcriptional inflammatory response and are controlled by distinct molecular mechanisms. Global transcriptional changes after Phf15 knockout in a microglial cell line further revealed that Phf15 may specifically regulate the antiviral response, as well as, proinflammatory factor production and secretion. Interestingly, loss of Phf15 resulted in increased IFN-I-dependent and inflammatory gene expression profiles that closely mimic transcriptional changes in aged microglia. Together, my data indicate that Phf15 is an important novel repressor of microglial inflammatory function which might counteract age-induced inflammation in the healthy, aging brain.Interestingly, I found decreased expression levels of Il-6, a key proinflammatory cytokine expressed by senescent cells in the hippocampus–an area which mediates various memory-related process–of aged (27-month old) STING-deficient mice. However, this decrease did not translate to improved working memory or differences in Phf15 mRNA expression in the brain, suggesting that expression of SASP-related inflammatory factors by the cGAS-STING pathway does not proceed via inhibition of Phf15. Similarly, prolonged treatment with a high fat diet/obesity did not affect working memory or levels of Phf15 in the mouse brain, suggesting that brain inflammation resulting from a HFD or obesity is likewise not a result of Phf15 downregulation.Overall, my results suggest that Phf15 could function as an immune regulatory checkpoint, restraining the transition from a homeostatic phenotype towards the chronic, proinflammatory, IFN-I responsive state seen in microglia in the aged brain. Further understanding of its exact mechanism of action could lend insight into possible future therapeutic intervention

The Role of RFamide-Related Peptide-3 in Age-Related Reproductive Decline in Female Rats.

Reproductive senescence, the point in time when females cease to show estrous cyclicity, is associated with endocrine changes in the hypothalamus, pituitary, and gonads. However, the mechanisms triggering this transition are not well understood. To gain a better understanding of the top-down control of the transition from reproductive competence to a state of reproductive senescence, we investigated middle-aged female rats exhibiting varying degrees of reproductive decline, including individuals with normal cycles, irregular cycles, and complete cessation of cycles. We identified hormonal changes in the brain that manifest before ovarian cycles exhibit any deterioration. We found that females exhibit an increase in RFamide-related peptide-3 (RFRP3) mRNA expression in the hypothalamus in middle age prior to changes in estrous cycle length. This increase is transient and followed by subsequent decreases in kisspeptin (KiSS1) and gonadotropin-releasing hormone (GnRH) mRNA expression. Expression of RFRP3 and its receptor also increased locally in the ovaries with advancing age. While it is well known that aging is associated with decreased GnRH release and downstream disruption of the hypothalamic-pituitary-gonadal (HPG) axis, herein, we provide evidence that reproductive senescence is likely triggered by alterations in a network of regulatory neuropeptides upstream of the GnRH system

Epigenetic and transcriptional control of the microglial inflammatory response: potential insights into neuro-inflammaging

Inflammation is an evolutionarily conserved host defense response during infection or injury that seeks to remove the causal agent that led to its initiation, repair the damaged tissue(s) and restore homeostasis. Thus, transient inflammation in response to an adequate threat with a quick return to a basal resting state is beneficial. However, when inflammation becomes inappropriately increased or prolonged it can have severe pathophysiological consequences. During aging, the immune system shifts to a proinflammatory state characterized by low-grade, chronic, sterile inflammation that has been termed ‘inflammaging’. The proinflammatory state largely results from chronic activation of the innate immune system and includes elevated circulating levels of inflammatory mediators including the immune cell signaling molecules (cytokines) Interleukin (IL)-1, IL-6, IL-8, IL-13, IL-18, tumor necrosis factor (TNFα) and antivirals (the type I interferons (IFN-I). Numerous factors are thought to contribute to inflammaging and amongst potential mechanisms high fat feeding/obesity and associated increases in gut permeability to bacterial endotoxins, as well as, cellular senescence have emerged as key contributors. Importantly, inflammaging is tightly correlated to global indicators of poor health status, multimorbidity, impairment in day-to-day living activities and is thought to underlie or accelerate most age-dependent chronic diseases (e.g. cardiovascular disease, diabetes, cancer, as well as, neurodegenerative conditions like Parkinson’s disease (PD) and Alzheimer’s disease (AD)).While mechanisms driving peripheral inflammaging are beginning to be understood, the etiology of neuro-inflammaging is a crucially unresolved issue. An important source of inflammation in the brain are the non-neuronal cell populations (glia) which provide structural, trophic, and other physiological support for neurons, and especially, the activity of microglia–the brain’s own resident innate immune cells. Microglia are essential for brain development, maintenance and protection throughout the life of an organism. As innate immune cells, however, they can also mount a full inflammatory response to infection or environmental challenge to restore brain health.Environmental factors, for example, changes in peripheral levels of fatty acids, bacterial endotoxins or proinflammatory mediators resulting from high fat feeding or gut permeabilization can cause microglia to undergo changes that signal activation. Most importantly, microglia lose their homeostatic function during aging, becoming less neuroprotective and increasingly neurotoxic. Microglia-mediated inflammation, for example, is strongly linked to age-induced cognitive impairment, is a common hallmark of both PD and AD, and is believed to be mechanistically important in driving pathogenesis. Thus, there is great interest in discovering factors that regulate age-related changes in microglial inflammatory function.Although inflammation is a complex and multicomponent response, a key point of its control occurs at the level of gene transcription and involves several classes of transcription factors, transcriptional co-regulators and chromatin modifications. A recent 2017 study by Soreq et al., identified a relatively unknown gene, PHD finger protein 15 (PHF15) as one of the top 25 differentially expressed genes in microglia during non-pathological aging in humans, with PHF15 levels increasing with age. Sequence and structural similarity to other members of the PHF family suggest that PHF15 might function as a putative chromatin-mediated gene regulator. I first sought to determine whether PHF15 could repress inflammatory function in microglia. If so, I wanted to investigate whether factors known to be causal in inflammaging (e.g. high fat feeding/obesity or cellular senescence) lead to age-dependent cognitive impairment via modulation of microglial PHF15. A major hallmark of senescent cells is the secretion of inflammatory mediators including various cytokines (lL-6, IL-1β, IL-8), chemoattractant cytokines (chemokines; for example C-X-C motif chemokine 10 (CXCL10), C-C motif chemokine ligand (CCL-) 5 (CCL5) and CCL20), antivirals (IFN-I), growth factors and extracellular matrix proteases termed the senescence-associated secretory phenotype (SASP). Secretion of the SASP is partially controlled by the Cyclic GMP-AMP (cGAMP) synthase (cGAS)-Stimulator of interferon genes (STING) (cGAS-STING) cytosolic DNA sensing pathway at the molecular level. Thus, peripheral changes induced by high fat feeding/obesity or the SASP could lead to increased neuroinflammation via inhibition of microglial PHF15.I show that Phf15 significantly represses proinflammatory gene expression in mouse microglia, modulating both the magnitude and duration of the inflammatory response. Importantly, Phf15 regulates both basal expression and signal-dependent upregulation of proinflammatory genes—which constitute different phases of the transcriptional inflammatory response and are controlled by distinct molecular mechanisms. Global transcriptional changes after Phf15 knockout in a microglial cell line further revealed that Phf15 may specifically regulate the antiviral response, as well as, proinflammatory factor production and secretion. Interestingly, loss of Phf15 resulted in increased IFN-I-dependent and inflammatory gene expression profiles that closely mimic transcriptional changes in aged microglia. Together, my data indicate that Phf15 is an important novel repressor of microglial inflammatory function which might counteract age-induced inflammation in the healthy, aging brain.Interestingly, I found decreased expression levels of Il-6, a key proinflammatory cytokine expressed by senescent cells in the hippocampus–an area which mediates various memory-related process–of aged (27-month old) STING-deficient mice. However, this decrease did not translate to improved working memory or differences in Phf15 mRNA expression in the brain, suggesting that expression of SASP-related inflammatory factors by the cGAS-STING pathway does not proceed via inhibition of Phf15. Similarly, prolonged treatment with a high fat diet/obesity did not affect working memory or levels of Phf15 in the mouse brain, suggesting that brain inflammation resulting from a HFD or obesity is likewise not a result of Phf15 downregulation.Overall, my results suggest that Phf15 could function as an immune regulatory checkpoint, restraining the transition from a homeostatic phenotype towards the chronic, proinflammatory, IFN-I responsive state seen in microglia in the aged brain. Further understanding of its exact mechanism of action could lend insight into possible future therapeutic intervention

The Role of RFamide-Related Peptide-3 in Age-Related Reproductive Decline in Female Rats.

Reproductive senescence, the point in time when females cease to show estrous cyclicity, is associated with endocrine changes in the hypothalamus, pituitary, and gonads. However, the mechanisms triggering this transition are not well understood. To gain a better understanding of the top-down control of the transition from reproductive competence to a state of reproductive senescence, we investigated middle-aged female rats exhibiting varying degrees of reproductive decline, including individuals with normal cycles, irregular cycles, and complete cessation of cycles. We identified hormonal changes in the brain that manifest before ovarian cycles exhibit any deterioration. We found that females exhibit an increase in RFamide-related peptide-3 (RFRP3) mRNA expression in the hypothalamus in middle age prior to changes in estrous cycle length. This increase is transient and followed by subsequent decreases in kisspeptin (KiSS1) and gonadotropin-releasing hormone (GnRH) mRNA expression. Expression of RFRP3 and its receptor also increased locally in the ovaries with advancing age. While it is well known that aging is associated with decreased GnRH release and downstream disruption of the hypothalamic-pituitary-gonadal (HPG) axis, herein, we provide evidence that reproductive senescence is likely triggered by alterations in a network of regulatory neuropeptides upstream of the GnRH system

Phf15 - a novel transcriptional repressor regulating inflammation in a mouse microglial cell line

Aim: Excessive microglial inflammation has emerged as a key player in mediating the effects of aging and neurodegeneration on brain dysfunction. Thus, there is great interest in discovering transcriptional repressors that can control this process. We aimed to examine whether Phf15 - one of the top differentially expressed genes in microglia during aging in humans - could regulate transcription of proinflammatory mediators in microglia.Methods: Real-time quantitative PCR was used to assess Phf15 mRNA expression in mouse brain during aging. Loss-of-function [short hairpin RNA (shRNA) -mediated knockdown (KD) and CRISPR/Cas9-mediated knockout (KO) of Phf15] and gain-of-function [retroviral overexpression (OE) of murine Phf15 cDNA] studies in a murine microglial cell line (SIM-A9) followed by immune activation with lipopolysaccharide were used to determine the effect of Phf15 on proinflammatory factor (Tnfα, IL-1β, and Nos2) mRNA expression. RNA sequencing was used to determine global transcriptional changes after Phf15 knockout under basal conditions and after lipopolysaccharide stimulation.Results:Phf15 expression increases in mouse brain during aging, similar to humans. KD, KO, and OE studies determined that Phf15 represses mRNA expression levels of proinflammatory mediators such as Tnfα, IL-1β, and Nos2. Global transcriptional changes after Phf15 KO showed that Phf15 specifically represses genes related to the antiviral (type I interferon) response and cytokine production in microglia.Conclusion: We provide the first evidence that Phf15 is an important transcriptional repressor of microglial inflammation, regulating the antiviral response and proinflammatory cytokine production. Importantly, Phf15 regulates both basal and signal-dependent activation and controls the magnitude and duration of the microglial inflammatory response

Knockdown of hypothalamic RFRP3 prevents chronic stress-induced infertility and embryo resorption.

Whereas it is well established that chronic stress induces female reproductive dysfunction, whether stress negatively impacts fertility and fecundity when applied prior to mating and pregnancy has not been explored. In this study, we show that stress that concludes 4 days prior to mating results in persistent and marked reproductive dysfunction, with fewer successful copulation events, fewer pregnancies in those that successfully mated, and increased embryo resorption. Chronic stress exposure led to elevated expression of the hypothalamic inhibitory peptide, RFamide-related peptide-3 (RFRP3), in regularly cycling females. Remarkably, genetic silencing of RFRP3 during stress using an inducible-targeted shRNA completely alleviates stress-induced infertility in female rats, resulting in mating and pregnancy success rates indistinguishable from non-stress controls. We show that chronic stress has long-term effects on pregnancy success, even post-stressor, that are mediated by RFRP3. This points to RFRP3 as a potential clinically relevant single target for stress-induced infertility

Gene Expression Signatures of Contact Lens-Induced Myopia in Guinea Pig Retinal Pigment Epithelium

PurposeTo identify key retinal pigment epithelium (RPE) genes linked to the induction of myopia in guinea pigs.MethodsTo induce myopia, two-week-old pigmented guinea pigs (New Zealand strain, n = 5) wore -10 diopter (D) rigid gas-permeable contact lenses (CLs), for one day; fellow eyes were left without CLs and served as controls. Spherical equivalent refractive errors (SE) and axial length (AL) were measured at baseline and one day after initiation of CL wear. RNA sequencing was applied to RPE collected from both treated and fellow (control) eyes after one day of CL-wear to identify related gene expression changes. Additional RPE-RNA samples from treated and fellow eyes were subjected to quantitative real-time PCR (qRT-PCR) analysis for validation purposes.ResultsThe CLs induced myopia. The change from baseline values in SE was significantly different (P = 0.016), whereas there was no significant difference in the change in AL (P = 0.10). RNA sequencing revealed significant interocular differences in the expression in RPE of 13 genes: eight genes were significantly upregulated in treated eyes relative to their fellows, and five genes, including bone morphogenetic protein 2 (Bmp2), were significantly downregulated. The latter result was also confirmed by qRT-PCR. Additional analysis of differentially expressed genes revealed significant enrichment for bone morphogenetic protein (BMP) and TGF-β signaling pathways.ConclusionsThe results of this RPE gene expression study provide further supporting evidence for an important role of BMP2 in eye growth regulation, here from a guinea pig myopia model