Sex Differences in Macrophage Responses to Obesity-Mediated Changes Determine Migratory and Inflammatory Traits.

The mechanisms whereby obesity differentially affects males and females are unclear. Because macrophages are functionally the most important cells in obesity-induced inflammation, we sought to determine reasons for male-specific propensity in macrophage migration. We previously determined that male mice fed a high-fat diet exhibit macrophage infiltration into the hypothalamus, whereas females were protected irrespective of ovarian estrogen, in this study, we show that males accumulate more macrophages in adipose tissues that are also more inflammatory. Using bone marrow cells or macrophages differentiated in vitro from male and female mice fed control or high-fat diet, we demonstrated that macrophages derived from male mice are intrinsically more migratory. We determined that males have higher levels of leptin in serum and adipose tissue. Serum CCL2 levels, however, are the same in males and females, although they are increased in obese mice compared with lean mice of both sexes. Leptin receptor and free fatty acid (FFA) receptor, GPR120, are upregulated only in macrophages derived from male mice when cultured in the presence of FFA to mimic hyperlipidemia of obesity. Unless previously stimulated with LPS, CCL2 did not cause migration of macrophages. Leptin, however, elicited migration of macrophages from both sexes. Macrophages from male mice maintained migratory capacity when cultured with FFA, whereas female macrophages failed to migrate. Therefore, both hyperlipidemia and hyperleptinemia contribute to male macrophage-specific migration because increased FFA induce leptin receptors, whereas higher leptin causes migration. Our results may explain sex differences in obesity-mediated disorders caused by macrophage infiltration.

Visceral adipose tissue imparts peripheral macrophage influx into the hypothalamus.

BACKGROUND: Obesity is characterized by a systemic inflammation and hypothalamic neuroinflammation. Systemic inflammation is caused by macrophages that infiltrate obese adipose tissues. We previously demonstrated that high-fat diet (HFD)-fed male mice exhibited peripheral macrophage infiltration into the hypothalamus, in addition to activation of resident microglia. Since this infiltration contributes to neuroinflammation and neuronal impairment, herein we characterize the phenotype and origin of these hypothalamic macrophages in HFD mice. METHODS: C57BL/6J mice were fed HFD (60% kcal from fat) or control diet with matching sucrose levels, for 12-16 weeks. Males and females were analyzed separately to determine sex-specific responses to HFD. Differences in hypothalamic gene expression in HFD-fed male and female mice, compared to their lean controls, in two different areas of the hypothalamus, were determined using the NanoString neuroinflammation panel. Phenotypic changes in macrophages that infiltrated the hypothalamus in HFD-fed mice were determined by analyzing cell surface markers using flow cytometry and compared to changes in macrophages from the adipose tissue and peritoneal cavity. Adipose tissue transplantation was performed to determine the source of hypothalamic macrophages. RESULTS: We determined that hypothalamic gene expression profiles demonstrate sex-specific and region-specific diet-induced changes. Sex-specific changes included larger changes in males, while region-specific changes included larger changes in the area surrounding the median eminence. Several genes were identified that may provide partial protection to female mice. We also identified diet-induced changes in macrophage migration into the hypothalamus, adipose tissue, and peritoneal cavity, specifically in males. Further, we determined that hypothalamus-infiltrating macrophages express pro-inflammatory markers and markers of metabolically activated macrophages that were identical to markers of adipose tissue macrophages in HFD-fed mice. Employing adipose tissue transplant, we demonstrate that hypothalamic macrophages can originate from the visceral adipose tissue. CONCLUSION: HFD-fed males experience higher neuroinflammation than females, likely because they accumulate more visceral fat, which provides a source of pro-inflammatory macrophages that migrate to other tissues, including the hypothalamus. Our findings may explain the male bias for neuroinflammation and the metabolic syndrome. Together, our results demonstrate a new connection between the adipose tissue and the hypothalamus in obesity that contributes to neuroinflammation and hypothalamic pathologies.

Leukemia Inhibitory Factor Represses GnRH Gene Expression via cFOS during Inflammation in Male Mice.

BACKGROUND: The mechanisms whereby neuroinflammation negatively affects neuronal function in the hypothalamus are not clear. Our previous study determined that obesity-mediated chronic inflammation elicits sex-specific impairment in reproductive function via reduction in spine density in gonadotropin-releasing hormone (GnRH) neurons. Neuroinflammation and subsequent decrease in GnRH neuron spine density was specific for male mice, while protection in females was independent of ovarian estrogens. METHODS: To examine if neuroinflammation-induced cytokines can directly regulate GnRH gene expression, herein we examined signaling pathways and mechanisms in males in vivo and in GnRH-expressing cell line, GT1-7. RESULTS: GnRH neurons express cytokine receptors, and chronic or acute neuroinflammation represses GnRH gene expression in vivo. Leukemia inhibitory factor (LIF) in particular represses GnRH expression in GT1-7 cells, while other cytokines do not. STAT3 and MAPK pathways are activated following LIF treatment, but only MAPK pathway, specifically p38α, is sufficient to repress the GnRH gene. LIF induces cFOS that represses the GnRH gene via the -1,793 site in the enhancer region. In vivo, following high-fat diet, cFOS is induced in GnRH neurons and neurons juxtaposed to the leaky blood brain barrier of the organum vasculosum of the lamina terminalis, but not in the neurons further away. CONCLUSION: Our results indicate that the increase in LIF due to neuroinflammation induces cFOS and represses the GnRH gene. Therefore, in addition to synaptic changes in GnRH neurons, neuroinflammatory cytokines directly regulate gene expression and reproductive function, and the specificity for neuronal targets may stem from the proximity to the fenestrated capillaries.

c-JUN Dimerization Protein 2 (JDP2) Is a Transcriptional Repressor of Follicle-stimulating Hormone ß (FSHß) and Is Required for Preventing Premature Reproductive Senescence in Female Mice.

Follicle-stimulating hormone (FSH) regulates follicular growth and stimulates estrogen synthesis in the ovaries. FSH is a heterodimer consisting of an α subunit, also present in luteinizing hormone, and a unique ß subunit, which is transcriptionally regulated by gonadotropin-releasing hormone 1 (GNRH). Because most FSH is constitutively secreted, tight transcriptional regulation is critical for maintaining FSH levels within a narrow physiological range. Previously, we reported that GNRH induces FSHß (Fshb) transcription via induction of the AP-1 transcription factor, a heterodimer of c-FOS and c-JUN. Herein, we identify c-JUN-dimerization protein 2 (JDP2) as a novel repressor of GNRH-mediated Fshb induction. JDP2 exhibited high basal expression and bound the Fshb promoter at an AP-1-binding site in a complex with c-JUN. GNRH treatment induced c-FOS to replace JDP2 as a c-JUN binding partner, forming transcriptionally active AP-1. Subsequently, rapid c-FOS degradation enabled reformation of the JDP2 complex. In vivo studies revealed that JDP2 null male mice have normal reproductive function, as expected from a negative regulator of the FSH hormone. Female JDP2 null mice, however, exhibited early puberty, observed as early vaginal opening, larger litters, and early reproductive senescence. JDP2 null females had increased levels of circulating FSH and higher expression of the Fshb subunit in the pituitary, resulting in elevated serum estrogen and higher numbers of large ovarian follicles. Disruption of JDP2 function therefore appears to cause early cessation of reproductive function, a condition that has been associated with elevated FSH in women.

Single-Cell Transcriptomics Identifies Pituitary Gland Changes in Diet-Induced Obesity in Male Mice.

Obesity is a chronic disease with increasing prevalence worldwide. Obesity leads to an increased risk of heart disease, stroke, and diabetes, as well as endocrine alterations, reproductive disorders, changes in basal metabolism, and stress hormone production, all of which are regulated by the pituitary. In this study, we performed single-cell RNA sequencing of pituitary glands from male mice fed control and high-fat diet (HFD) to determine obesity-mediated changes in pituitary cell populations and gene expression. We determined that HFD exposure is associated with dramatic changes in somatotrope and lactotrope populations, by increasing the proportion of somatotropes and decreasing the proportion of lactotropes. Fractions of other hormone-producing cell populations remained unaffected. Gene expression changes demonstrated that in HFD, somatotropes became more metabolically active, with increased expression of genes associated with cellular respiration, and downregulation of genes and pathways associated with cholesterol biosynthesis. Despite a lack of changes in gonadotrope fraction, genes important in the regulation of gonadotropin hormone production were significantly downregulated. Corticotropes and thyrotropes were the least affected in HFD, while melanotropes exhibited reduced proportion. Lastly, we determined that changes in plasticity and gene expression were associated with changes in hormone levels. Serum prolactin was decreased corresponding to reduced lactotrope fraction, while lower luteinizing hormone and follicle-stimulating hormone in the serum corresponded to a decrease in transcription and translation. Taken together, our study highlights diet-mediated changes in pituitary gland populations and gene expression that play a role in altered hormone levels in obesity.

Sexual dimorphism in obesity is governed by RELMα regulation of adipose macrophages and eosinophils.

Obesity incidence is increasing worldwide with the urgent need to identify new therapeutics. Sex differences in immune cell activation drive obesity-mediated pathologies where males are more susceptible to obesity co-morbidities and exacerbated inflammation. Here, we demonstrate that the macrophage-secreted protein RELMα critically protects females against high fat diet-induced obesity. Compared to male mice, RELMα levels were elevated in both control and high fat dietfed females and correlated with adipose macrophages and eosinophils. RELMα-deficient females gained more weight and had pro-inflammatory macrophage accumulation and eosinophil loss, while both RELMα treatment and eosinophil transfer rescued this phenotype. Single cell RNA-sequencing of the adipose stromal vascular fraction was performed and identified sex and RELMα-dependent changes. Genes involved in oxygen sensing and iron homeostasis, including hemoglobin and lncRNA Gm47283, correlated with increased obesity, while eosinophil chemotaxis and response to amyloid-beta were protective. Monocyte-to-macrophage transition was also dysregulated in RELMα-deficient animals. Collectively, these studies implicate a RELMα-macrophage-eosinophil axis in sex-specific protection against obesity and uncover new therapeutic targets for obesity.

Altered GnRH neuron and ovarian innervation characterize reproductive dysfunction linked to the Fragile X messenger ribonucleoprotein (Fmr1) gene mutation.

Introduction: Mutations in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene cause Fragile X Syndrome, the most common monogenic cause of intellectual disability. Mutations of FMR1 are also associated with reproductive disorders, such as early cessation of reproductive function in females. While progress has been made in understanding the mechanisms of mental impairment, the causes of reproductive disorders are not clear. FMR1-associated reproductive disorders were studied exclusively from the endocrine perspective, while the FMR1 role in neurons that control reproduction was not addressed. Results: Here, we demonstrate that similar to women with FMR1 mutations, female Fmr1 null mice stop reproducing early. However, young null females display larger litters, more corpora lutea in the ovaries, increased inhibin, progesterone, testosterone, and gonadotropin hormones in the circulation. Ovariectomy reveals both hypothalamic and ovarian contribution to elevated gonadotropins. Altered mRNA and protein levels of several synaptic molecules in the hypothalamus are identified, indicating reasons for hypothalamic dysregulation. Increased vascularization of corpora lutea, higher sympathetic innervation of growing follicles in the ovaries of Fmr1 nulls, and higher numbers of synaptic GABAA receptors in GnRH neurons, which are excitatory for GnRH neurons, contribute to increased FSH and LH, respectively. Unmodified and ovariectomized Fmr1 nulls have increased LH pulse frequency, suggesting that Fmr1 nulls exhibit hyperactive GnRH neurons, regardless of the ovarian feedback. Conclusion: These results reveal Fmr1 function in the regulation of GnRH neuron secretion, and point to the role of GnRH neurons, in addition to the ovarian innervation, in the etiology of Fmr1-mediated reproductive disorders.

Macrophage-Regulatory T Cell Interactions Promote Type 2 Immune Homeostasis Through Resistin-Like Molecule α.

RELMα is a small, secreted protein expressed by type 2 cytokine-activated "M2" macrophages in helminth infection and allergy. At steady state and in response to type 2 cytokines, RELMα is highly expressed by peritoneal macrophages, however, its function in the serosal cavity is unclear. In this study, we generated RELMα TdTomato (Td) reporter/knockout (RαTd) mice and investigated RELMα function in IL-4 complex (IL-4c)-induced peritoneal inflammation. We first validated the RELMαTd/Td transgenic mice and showed that IL-4c injection led to the significant expansion of large peritoneal macrophages that expressed Td but not RELMα protein, while RELMα+/+ mice expressed RELMα and not Td. Functionally, RELMαTd/Td mice had increased IL-4 induced peritoneal macrophage responses and splenomegaly compared to RELMα+/+ mice. Gene expression analysis indicated that RELMαTd/Td peritoneal macrophages were more proliferative and activated than RELMα+/+ macrophages, with increased genes associated with T cell responses, growth factor and cytokine signaling, but decreased genes associated with differentiation and maintenance of myeloid cells. We tested the hypothesis that RαTd/Td macrophages drive aberrant T cell activation using peritoneal macrophage and T cell co-culture. There were no differences in CD4+ T cell effector responses when co-cultured with RELMα+/+ or RELMαTd/Td macrophages, however, RELMαTd/Td macrophages were impaired in their ability to sustain proliferation of FoxP3+ regulatory T cells (Treg). Supportive of the in vitro results, immunofluorescent staining of the spleens revealed significantly decreased FoxP3+ cells in the RELMαTd/Td spleens compared to RELMα+/+ spleens. Taken together, these studies identify a new RELMα regulatory pathway whereby RELMα-expressing macrophages directly sustain Treg proliferation to limit type 2 inflammatory responses.

Obesity, Neuroinflammation, and Reproductive Function.

The increasing occurrence of obesity has become a significant public health concern. Individuals with obesity have higher prevalence of heart disease, stroke, osteoarthritis, diabetes, and reproductive disorders. Reproductive problems include menstrual irregularities, pregnancy complications, and infertility due to anovulation, in women, and lower testosterone and diminished sperm count, in men. In particular, women with obesity have reduced levels of both gonadotropin hormones, and, in obese men, lower testosterone is accompanied by diminished LH. Taken together, these findings indicate central dysregulation of the hypothalamic-pituitary-gonadal axis, specifically at the level of the GnRH neuron function, which is the final brain output for the regulation of reproduction. Obesity is a state of hyperinsulinemia, hyperlipidemia, hyperleptinemia, and chronic inflammation. Herein, we review recent advances in our understanding of how these metabolic and immune changes affect hypothalamic function and regulation of GnRH neurons. In the latter part, we focus on neuroinflammation as a major consequence of obesity and discuss findings that reveal that GnRH neurons are uniquely positioned to respond to inflammatory changes.

GnRH Receptor Expression and Reproductive Function Depend on JUN in GnRH Receptor‒Expressing Cells.

Gonadotropin-releasing hormone (GnRH) from the hypothalamus regulates synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gonadotropes. LH and FSH are heterodimers composed of a common α-subunit and unique ß-subunits, which provide biological specificity and are limiting components of mature hormone synthesis. Gonadotrope cells respond to GnRH via specific expression of the GnRH receptor (Gnrhr). GnRH induces the expression of gonadotropin genes and of the Gnrhr by activation of specific transcription factors. The JUN (c-Jun) transcription factor binds to AP-1 sites in the promoters of target genes and mediates induction of the FSHß gene and of the Gnrhr in gonadotrope-derived cell lines. To analyze the role of JUN in reproductive function in vivo, we generated a mouse model that lacks JUN specifically in GnRH receptor‒expressing cells (conditional JUN knockout; JUN-cKO). JUN-cKO mice displayed profound reproductive anomalies such as reduced LH levels resulting in lower gonadal steroid levels, longer estrous cycles in females, and diminished sperm numbers in males. Unexpectedly, FSH levels were unchanged in these animals, whereas Gnrhr expression in the pituitary was reduced. Steroidogenic enzyme expression was reduced in the gonads of JUN-cKO mice, likely as a consequence of reduced LH levels. GnRH receptor‒driven Cre activity was detected in the hypothalamus but not in the GnRH neuron. Female, but not male, JUN-cKO mice exhibited reduced GnRH expression. Taken together, our results demonstrate that GnRH receptor‒expression levels depend on JUN and are critical for reproductive function.

Diet-Induced Obesity Elicits Macrophage Infiltration and Reduction in Spine Density in the Hypothalami of Male but Not Female Mice.

Increasing prevalence in obesity has become a significant public concern. C57BL/6J mice are prone to diet-induced obesity (DIO) when fed high-fat diet (HFD), and develop chronic inflammation and metabolic syndrome, making them a good model to analyze mechanisms whereby obesity elicits pathologies. DIO mice demonstrated profound sex differences in response to HFD with respect to inflammation and hypothalamic function. First, we determined that males are prone to DIO, while females are resistant. Ovariectomized females, on the other hand, are susceptible to DIO, implying protection by ovarian hormones. Males, but not females, exhibit changes in hypothalamic neuropeptide expression. Surprisingly, ovariectomized females remain resistant to neuroendocrine changes, showing that ovarian hormones are not necessary for protection. Second, obese mice exhibit sex differences in DIO-induced inflammation. Microglial activation and peripheral macrophage infiltration is seen in the hypothalami of males, while females are protected from the increase in inflammatory cytokines and do not exhibit microglia morphology changes nor monocyte-derived macrophage infiltration, regardless of the presence of ovarian hormones. Strikingly, the anti-inflammatory cytokine IL-10 is increased in the hypothalami of females but not males. Third, this study posits a potential mechanism of obesity-induced impairment of hypothalamic function whereby obese males exhibit reduced levels of synaptic proteins in the hypothalamus and fewer spines in GnRH neurons, located in the areas exhibiting macrophage infiltration. Our studies suggest that inflammation-induced synaptic remodeling is potentially responsible for hypothalamic impairment that may contribute to diminished levels of gonadotropin hormones, testosterone, and sperm numbers, which we observe and corresponds to the observations in obese humans. Taken together, our data implicate neuro-immune mechanisms underlying sex-specific differences in obesity-induced impairment of the hypothalamic function with potential consequences for reproduction and fertility.