Dietary excess triggers accumulation of pro-inflammatory microglia in the mediobasal hypothalamus (MBH), but the components of this microgliosis and its metabolic consequences remain uncertain. Here, we show that microglial inflammatory signaling determines the immunologic response of the MBH to dietary excess and regulates hypothalamic control of energy homeostasis in mice. Either pharmacologically depleting microglia or selectively restraining microglial NF-κB-dependent signaling sharply reduced microgliosis, an effect that includes prevention of MBH entry by bone-marrow-derived myeloid cells, and greatly limited diet-induced hyperphagia and weight gain. Conversely, forcing microglial activation through cell-specific deletion of the negative NF-κB regulator A20 induced spontaneous MBH microgliosis and cellular infiltration, reduced energy expenditure, and increased both food intake and weight gain even in absence of a dietary challenge. Thus, microglial inflammatory activation, stimulated by dietary excess, orchestrates a multicellular hypothalamic response that mediates obesity susceptibility, providing a mechanistic rationale for non-neuronal approaches to treat metabolic diseases.

Here, we use multiple approaches to conditionally and specifically manipulate both the number and inflammatory activation state of resident microglia in mice. Attenuating microglial inflammatory capacity or depleting microglia altogether protects HFD-fed mice from hyperphagia and DIO and also mediates a striking reduction in microgliosis, in particular the elimination of MBH infiltration by bone-marrow-derived myeloid cells. By contrast, activating resident microglia is sufficient to trigger diet-independent weight gain and MBH recruitment of myeloid cells. Collectively, our results indicate that the inflammatory activation state of microglia controls the hypothalamic immune response to dietary excess and regulates the susceptibility to obesity.

The extent to which MBH microglial inflammatory activation regulates obesity pathogenesis also remains uncertain. On the whole, hypothalamic inflammation promotes overconsumption and weight gain in mice. For example, deleting or inhibiting the inflammatory master regulator NF-κB in neurons or astrocytes mitigated DIO while stimulating inflammation in the MBH impaired leptin and insulin signaling (). However, these studies have not addressed the potential role of microglia in this process. Indeed microglia can initiate and orchestrate processes that either exacerbate or protect against neurotoxicity, depending on the context (). We showed that microglia mediate neuronal stress and leptin resistance due to saturated fat ingestion () but have not yet explored their role in the more complex pathogenesis of obesity.

Diet-induced obesity (DIO) is associated with a form of low-grade inflammation involving macrophages and other immune cells in white adipose and other metabolic tissues and is implicated in the development of insulin resistance (). This process is paralleled by a more rapid response involving glial cell accumulation (gliosis) in the mediobasal hypothalamus (MBH), both in mice and humans (), and the inflammatory activation of MBH microglia is prominent in the gliosis induced by high-fat diet (HFD) or saturated fat consumption (). The porous blood-brain barrier (BBB) in the MBH may also allow infiltrating myeloid cells from the circulation to augment gliosis, as is seen in other CNS inflammatory conditions that alter BBB integrity (). However, because prior analyses of “microgliosis” in mice with DIO (e.g.,) used either common myeloid markers (e.g., Iba1, CD11b, Emr1) or methods that might damage the BBB (), the identity of these immune cells remains uncertain.

Clear public health concerns have spurred efforts to determine how to maintain CNS control over energy balance in the face of dietary excess. While most studies have focused on hypothalamic neurons (), comparatively few have investigated non-neuronal cells, which outnumber neurons in the brain. Here, we provide the first mechanistic evidence that microglia, the self-renewing population of CNS macrophages, orchestrate both the immunologic and physiologic responses of the hypothalamus to dietary excess and instruct the hypothalamic control of food intake, energy expenditure, and body weight.

Energy homeostasis depends on the integrated function of hypothalamic neurons that detect changes in nutrient availability through adiposity hormones, such as leptin, and coordinately control feeding behavior and metabolic rate (). However, the high prevalence of obesity indicates that environmental influences, such as dietary excess, can override this control system to promote weight gain.

Given that obesity induces inflammation in peripheral metabolic tissues and that the brain can modulate peripheral immunity through autonomic pathways, including some potentially involving hypothalamic neurons (), we tested whether microglial activation can produce peripheral inflammation. Comparing A20and control mice fed a CD revealed no differences in the mRNA levels of inflammatory genes in the WAT or livers ( Figure S7 C) or in the circulating levels of TNF and IL-6 ( Figure S7 D). These results both underscore cell-type specificity in the A20model and confirm that microglial inflammatory activation is not sufficient to directly drive peripheral tissue or systemic inflammation. Similarly, measures of peripheral tissue and systemic inflammation were not different between HFD-fed IKKβand control mice when assessed before their body weights diverged (data not shown).

Our finding that microglial inflammatory signaling regulates obesity susceptibility prompted us to test whether it may also regulate glucose homeostasis. We analyzed glucose tolerance in IKKβand A20mice ( Figures S7 A and S7B) at body weights equivalent to controls in order to avoid confounding by differential adiposity. Notably, the glucose tolerance of IKKβmice fed a HFD for 3 weeks and of A20mice fed a CD for 3 weeks was similar to that of corresponding diet-matched, tamoxifen-treated controls ( Figures S7 A and S7B).

The relative hyperphagia seen in A20mice prompted us to test their responsiveness to leptin, an important hormonal regulator of energy homeostasis. Just as chronic HFD consumption diminishes hypothalamic leptin signaling (), deleting microglial A20 significantly reduced leptin-induced STAT3 activation in MBH neurons, even in the absence of a dietary challenge ( Figures 7 F and 7G). This finding suggests that the inflammatory activation of microglia increases obesity susceptibility by reducing the sensitivity of MBH neurons to homeostatic signals such as leptin.

More impressively, the induction of microglial A20 deficiency in A20mice induced a spontaneous and rapid 4-fold increase in body weight gain despite the mice being maintained on a CD ( Figure 7 B). This effect occurred during the first week after tamoxifen treatment and lessened over subsequent weeks. To determine the physiological mechanism for this weight gain, we analyzed energy balance in individually housed A20and control mice using metabolic cages. Upon tamoxifen treatment, CD-fed A20mice also had a 33% increase in ad libitum food intake over 4 days ( Figure 7 C) and concomitant reductions in VO Figure S6 B), VCO Figure S6 C), and energy expenditure ( Figure 7 E) but no differences in respiratory exchange ratio (RER) ( Figure 7 D) or physical activity ( Figure S6 D) versus control mice. Corresponding with their lower metabolic rate, A20mice had reduced mRNA levels of several thermogenic genes (e.g., Ucp1, Cidea) in the brown adipose tissue (BAT), with a similar trend seen in the WAT ( Figure S6 E). These findings indicate that forcing microglial activation is sufficient to recapitulate key effects of HFD consumption on energy homeostasis.

The heightened inflammatory activation state of A20microglia prompted us to hypothesize that A20mice would have an intrinsic susceptibility to obesity. In testing this hypothesis, it was critical to avoid waiting for replacement of any circulating myeloid cells that had undergone tamoxifen-induced Cre-LoxP recombination. Therefore, we performed BMT on A20and appropriate control mice using WT (Cre) donors, again employing head/neck shielding to preserve BBB integrity during irradiation ( Figure 7 A). After allowing time for bone marrow reconstitution, treating this model with tamoxifen deleted Tnfaip3 exclusively in microglia (A20). Using ubiquitin-GFP mice as BMT donors to monitor peripheral myeloid cell infiltration into the MBH, we found that tamoxifen-treated A20mice showed robust accumulation of GFPcells in the MBH when fed only a CD ( Figure S6 A), indicating that microglial activation is sufficient on its own to recruit bone-marrow-derived myeloid cells into the MBH.

(A) Strategy to restrict A20 deletion to microglia by reconstituting irradiated and head-shielded CX3CR1 CreER/+ : Tnfaip3 F/F mice with WT bone marrow and then treating the resulting mice with tamoxifen following their recovery (A20 MGKO-BMT ).

Interestingly, some CD169cells recruited to the MBH of HFD-fed A20mice co-expressed Tmem119, confirming that these cells adopt features of resident microglia upon arrival in the niche ( Figures 6 G and 6H). Indeed, the robust MBH infiltration seen in HFD-fed A20mice may have induced the entering cells to undergo a more complete microglial conversion, as the analogous cells in WT mice altered their morphology and expressed both Iba1 and CX3CR1 but did not express Tmem119 ( Figures 3 E and 4 E–4G).

As predicted, A20mice fed a HFD had a phenotype opposite to that of IKKβmice, displaying marked increases over control mice in the number and size of microglia in the MBH, indicated by Iba1cells bearing morphological features of inflammatory activation ( Figures 6 C and 6D). Moreover, the diet-induced MBH microglial accumulation in A20mice included substantially more infiltrating CD169cells than in controls, a finding also opposite to the absence of these cells in IKKβmice ( Figures 6 E and 6F). Therefore, modulating the inflammatory activation potential of resident microglia in mice exerts reciprocal effects on microgliosis induced by HFD consumption in the MBH.

Based on the hypersensitivity of A20-deficient microglia to inflammatory stimuli, we analyzed MBH microgliosis in a cohort of A20and control mice fed a HFD beginning 4 weeks after tamoxifen administration. We first confirmed that, like IKKβmice, A20mice maintained microglial A20 deletion at this time point, whereas gene expression in peripheral leukocytes had returned to WT status ( Figure S5 B).

To delete A20 specifically in microglia, we bred CX3CR1mice with those expressing a conditional allele of A20 (Tnfaip3) (A20). Treating primary microglia from A20mice with 4-hydroxytamoxifen profoundly reduced Tnfaip3 mRNA levels ( Figure 6 A), resulting in increased basal and LPS-induced TNF and IL-6 secretion in vitro ( Figure 6 B).

(G) Co-localization of CD169 with the microglial marker Tmem119 (arrowheads) in the MBH of A20 MGKO mice fed a HFD for 4 weeks.

(E) Increased infiltration of CD169 + cells showing co-localization with the marker Iba1 (arrowheads) into the MBH of A20 MGKO fed a HFD for 4 weeks.

(C) Increased number and size of Iba1 + microglia in the MBH of A20 MGKO mice fed a HFD for 4 weeks.

(B) Increased TNF and IL-6 secretion by microglia isolated from Cx3cr1 CreER/+ : Tnfaip3 F/F (A20 MGKO ) mice, treated with 4-hydroxytamoxifen (5 μM, 48 hr), and then treated with LPS (100 ng/mL, 16 hr) (n = 5, ∗ p < 0.05 versus control).

We reasoned that if microglial inflammatory signaling mediates the impact of dietary excess on MBH neuronal function, then spontaneously activating microglia through genetic manipulation should be sufficient to drive both microgliosis and obesity in a diet-independent manner. To test this hypothesis, we targeted A20 (tumor necrosis factor alpha-induced protein 3 [TNFAIP3]), an anti-inflammatory molecule and primary negative regulator of NF-κB activity (). A20-deficient mice spontaneously develop neuroinflammation, which has been attributed to A20 deficiency in microglia (). Consistent with this, primary microglia isolated from A20-haplodeficient (Tnfaip3) mice had an exaggerated TNF and IL-6 secretory response to LPS and saturated fatty acid (palmitate) treatments ( Figure S5 A). These findings suggested that mice lacking A20 specifically in microglia might have a heightened vulnerability to HFD-induced microgliosis and metabolic dysfunction.

We also corroborated our findings with PLX5622 by examining monocyte markers in IKKβmice. As expected, IKKβmice fed a HFD recruited fewer monocyte-derived (CD169) cells into the MBH than did controls ( Figures 5 B and 5D). These models together indicate that resident microglial activation is required for diet-induced peripheral myeloid cell recruitment into the MBH.

We performed two flow cytometric analyses to confirm that PLX5622 treatment did not itself impact the pool of circulating myeloid cells available to infiltrate the MBH. First, we documented that PLX5622 treatment does not affect bone-marrow-derived (GFP) cell numbers in the circulation ( Figures S4 A and S4B). Next, we analyzed circulating CSF1R-expressing (GFP) cells in CSF1R-eGFP (“MacGreen”) mice fed a control or PLX5622-containing CD for 3 weeks ( Figures S4 C and S4D). Except for a slight (∼30%) reduction in non-classical (Ly6C) monocytes, PLX5622 treatment had no effect on the circulating GFPpool, including neutrophils and monocyte subsets ( Figures S4 C and S4D). These findings corroborate recent data indicating that PLX5622 treatment in mice did not lessen brain infiltration by circulating monocytes in response to systemic TNF treatment, indicating that it does not broadly deprive these cells of their chemotactic potential ().

Given that resident microglia were required for the development of DIO in mice, we hypothesized that these cells also control peripheral myeloid cell recruitment into the MBH. To test this, we again generated BMTmice and, after recovery from BMT, fed them a control or PLX5622-containing HFD. Remarkably, depleting resident microglia (Iba1/GFP) with PLX5622 abolished the presence of Iba1/GFPcells in the MBH of mice fed a HFD, indicating that resident microglia are required to recruit these peripheral myeloid cells into the MBH ( Figures 5 A and 5C ).

Our approach also revealed a population of CD68cells in the MBH that do not express Tmem119 or P2Y12 under basal CD-fed conditions, indicating that they are not typical resident microglia, and that are GFP, indicating that they are not recruited from the bone marrow ( Figures 4 and S3 ). Some of these singly CD68cells had morphologies characteristic of local perivascular and meningeal macrophages. The number of these cells increased modestly in response to HFD, suggesting that perivascular or meningeal macrophages also contribute to diet-induced microgliosis ( Figures 4 and S3 ). Alternatively, some of these cells may have been resident microglia that downregulated Tmem119 and P2Y12 expression in response to HFD ( Figures 4 and S3 ), consistent with the concomitant drop in the number of typical resident microglia (Tmem119/CD68or P2Y12/CD68) in the MBH.

To more specifically determine the cell types comprising HFD-induced microgliosis in BMTmice, we performed combined immunohistochemistry for GFP, the microglia-specific marker Tmem119, and the pan-myeloid marker CD68 ( Figures 4 E–4G). The GFP(bone-marrow-derived) cells in the MBH were always CD68, marking their myeloid origin, but never Tmem119, indicating that they are not typical resident microglia ( Figures 4 E–4G). These data together clearly indicate that HFD-induced microgliosis includes a prominent contribution from infiltrating bone-marrow-derived myeloid cells. We obtained essentially identical results when using P2Y12 to mark microglia ( Figures S3 C–S3E), strongly suggesting that the infiltrating cells express neither Tmem119 nor P2Y12 ( Figures 4 and S3 ).

Finally, to definitively identify bone-marrow-derived cells, we lethally irradiated WT mice while shielding their heads and necks with lead to preserve BBB integrity () and then performed bone marrow transplantation (BMT) with donor marrow from mice expressing ubiquitin-GFP (BMT) ( Figure 4 D). Following a 6-week recovery, we fed BMTand control mice a CD or HFD for 4 weeks and then scored for the presence of GFPcells in the MBH. Consistent with our prior approaches, the microgliosis induced in mice fed a HFD included a sharp increase in the number of GFP(bone-marrow-derived) cells ( Figures 4 E–4G and S3 B).

Second, we analyzed mice in which CX3CR1cells express GFP and CCR2cells (monocytes, but not resident microglia) express RFP (CX3CR1: CCR2) (). We saw that round, CCR2-expressing (RFP) cells reminiscent of monocytes appear in the MBH within a week of placing the mice on HFD ( Figure S3 A).

We used three separate strategies to define the contribution of infiltrating cells to hypothalamic microgliosis. First, we crossed CX3CR1mice with Rosa26-lox-stop-lox-tdTomato mice () and treated the appropriate progeny with tamoxifen to label all CX3CR1-expressing cells, including microglia. After waiting 4 weeks for peripheral immune cell replacement by unlabeled bone-marrow-derived precursors, the mice were fed a CD or HFD for an additional 4 weeks. In this model, all Iba1/tdTomatocells in the MBH reflect resident microglia, whereas Iba1/tdTomatocells reflect myeloid cells with a more rapid turnover rate that were not present in the MBH prior to tamoxifen administration ( Figure 4 A). This strategy revealed a cluster of Iba1/tdTomatocells in the MBH of HFD-fed mice, supporting the concept that microgliosis includes cells recruited from a non-microglial myeloid cell pool ( Figures 4 B and 4C).

(G) Quantification of the relative myeloid (CD68) composition of the MBH from sections in (E), showing a diet-induced emergence of infiltrating cells (GFP/Tmem119) cells, a decrease in the contribution of resident yolk sac-derive microglia (GFP/Tmem119), and an increase in the contribution of atypical microglia/hypothalamic macrophages (CD68cells negative for the other two markers). Values are mean ± SEM (n = 6–8,p < 0.05 versus CD; 3V, third ventricle; scale bar, 20 μm). See also Figure S3

(F) Data from singly and multiply stained MBH sections in (E), quantifying increased absolute numbers of total CD68 + cells, infiltrating myeloid cells and atypical myeloid cells, and a decreased number of resident microglia (Tmem119 + ) in response to a HFD ([+], positive staining by indicated antibody; [–], negative staining by indicated antibody; [blank], unstained for indicated antibody).

(E) Representative hypothalamic sections from BMT GFP mice fed a CD or HFD for 4 weeks and stained for markers of bone-marrow-derived infiltrating cells (GFP), resident microglia (Tmem119), and pan-myeloid identity (CD68), showing marked diet-induced myeloid cell infiltration (GFP + /CD68 + /Tmem119 − ) and an increased number of atypical myeloid cells (CD68 + /GFP − /Tmem119 − ) in the MBH (see insets).

(B) Representative Iba1 + /tdTomato − cells, indicating their blood-borne origin (arrowheads), at the edges of the MBH in mice generated as in (A).

(A) Scheme to distinguish between resident microglia and infiltrating cells using CX3CR1 CreER/+ : Rosa26-lox-stop-lox-tdTomato mice. 4 weeks after tamoxifen-induced Cre-LoxP recombination, microglia are tdTomato + while circulating monocytes are tdTomato − .

The MBH accumulation of Iba1- and CX3CR1-expressing cells lacking P2Y12 and Tmem119 in HFD-fed mice suggested the potential involvement of non-microglial myeloid cells in the gliosis response. Consistent with this hypothesis, HFD-fed mice had a significant rise in MBH cells expressing CD169 (Siglec1), which marks monocyte-derived cells, but not resident microglia () ( Figures 3 E and 3F). However, these CD169cells displayed a microglial morphology ( Figure 3 E, inset), suggesting that they may assume a hybrid, microglia-like state on arrival in the niche. Since BrdU incorporation showed no evidence for HFD-induced proliferation of resident MBH microglia (data not shown), we explored the possibility that these atypical cells might indeed be recruited from the circulation.

Using CX3CR1mice, we also compared the localization of cells expressing CX3CR1 in the MBH with that of cells expressing either P2Y12 or Tmem119. Mirroring the Iba1 data, this model revealed a cluster of CX3CR1-expressing (GFP) microglia induced by HFD specifically in the ME and ARC, most of which did not express P2Y12 or Tmem119 whether assessed after 4 or 8 weeks of HFD ( Figures 3 C and 3D). Indeed, typical double-positive resident microglia (CX3CR1/P2Y12or CX3CR1/Tmem119) were rarely seen in the cluster of microgliosis within the MBH of mice fed a HFD. Rather, their frequency increased progressively from the ME to the VMH, and they remained morphologically quiescent when compared to the cells in the ARC and ME ( Figures 3 C and 3D).

We first confirmed that Iba1cells with an activated morphology accumulate in response to HFD consumption (4 weeks) in a cluster within the median eminence (ME) and ARC, but not the ventromedial hypothalamus (VMH; Figures 3 A and 3B ). By contrast, the number of MBH cells staining for either P2Y12 or Tmem119 was reduced by HFD consumption ( Figures 3 A and 3B). Interestingly, both P2Y12and Tmem119cells were already relatively sparse as compared to Iba1cells in the ME and ARC of CD-fed mice, but this reciprocal relationship was much more pronounced on HFD ( Figures 3 A and 3B).

(C) Representative hypothalamic sections stained for P2Y12 or Tmem119 showing lack of co-localization with GFP (arrowheads) in CX3CR1-GFP mice following a 4-week HFD.

To investigate the cellular composition of HFD-induced MBH microgliosis, we devised a multi-parameter immunofluorescence approach to stain MBH sections. Since the common microglial markers Iba1 and CX3CR1 are also expressed in some monocytes (CX3CR1), tissue macrophages (Iba1 and CX3CR1), and potentially perivascular CNS macrophages (CX3CR1) (), we added two specific markers, P2Y12 and Tmem119, that are expressed exclusively by microglia (). Importantly, this approach also identifies the activation state of microglia, as microglial activation increases expression of Iba1 and reduces that of P2Y12 () but does not alter Tmem119 or CX3CR1 levels. Therefore, using these four markers in combination assesses both the role of inflammation in HFD-induced MBH microgliosis and the cell types comprising this response.

Next, we probed the impact of microglial IKKβ deficiency on the ability of chronic dietary excess to produce microgliosis in the MBH. Compared to control mice, IKKβmice fed a HFD had consistently less microglial activation and accumulation, assessed by immunofluorescence to quantify microglial size and number ( Figures 2 J and 2K). Together, these analyses indicate that microglial NF-κB-dependent signaling is necessary for HFD-induced hypothalamic microgliosis, hyperphagia, and DIO.

To investigate the metabolic impact of restraining microglial inflammatory activation, we studied IKKβand control mice fed either a CD or a HFD initiated 4 weeks after tamoxifen treatment. Remarkably, IKKβmice gained less weight than IKKβcontrols specifically when fed a HFD ( Figure 2 D), mirroring the effect of microglial depletion ( Figure 1 C). Similarly, microglial IKKβ deficiency did not affect consumption of a CD ( Figure 2 E) but, like PLX5622 treatment, reduced cumulative intake of the HFD ( Figure 2 F). As before, reduced body fat accounted for the relative decrease in body weight ( Figures 2 G and 2H). Both IKKβand control mice had similar rates of energy expenditure ( Figure 2 I), suggesting that reduced food intake is primarily responsible for the lean phenotype of IKKβmice. Finally, female IKKβmice were also resistant to DIO ( Figure S2 ).

Cre-mediated recombination is durable in microglia, which are long lived, but transient in peripheral CX3CR1cells, which are rapidly replaced by bone-marrow-derived precursors (). Thus, 4 weeks after tamoxifen treatment, Ikbkb mRNA levels remained reduced by ∼75% in flow-sorted microglia from IKKβmice ( Figures 2 A and 2B) but had returned to wild-type (WT) levels in peripheral CX3CR1monocytes ( Figure S2 A). Moreover, lipopolysaccharide (LPS) treatment dose dependently increased Tnfa (TNF) mRNA levels in microglia from IKKβcontrol mice, but not IKKβmice, indicating reduced microglial inflammatory capacity in IKKβmice ( Figure 2 C).

We sought to corroborate our findings from microglial depletion by using a genetic approach to specifically silence microglial NF-κB-mediated inflammatory signaling. Mice expressing tamoxifen-inducible Cre recombinase (CreER) in cells expressing CX3CR1 (CX3CR1mice) were thus crossed with mice harboring conditional alleles of Ikbkb, which encodes IKKβ, an essential cofactor for NF-κB activation (Ikbkb) (). Treating the progeny with tamoxifen generated IKKβmice, in which Ikbkb is deleted in microglia ( Figure 2 A) and a subset of circulating myeloid cells ( Figure S2 A) (), and IKKβcontrols.

(J) Reduced number and size of Iba1 + microglia in the hypothalami of IKKβ MGKO mice fed a HFD. Values are mean ± SEM (scale bar, 20 μm).

Depleting microglia significantly reduced weight gain in mice fed a HFD but not in mice fed a CD ( Figure 1 C). Similarly, PLX5622 treatment reduced total body fat (but not lean mass) ( Figure 1 D) and food intake only in HFD-fed mice ( Figures 1 E and 1F). Together, these findings indicate that microglia help sustain caloric intake in the context of a HFD, thus driving increased adiposity and body weight.

To investigate the overall contribution of microglia to energy balance and obesity susceptibility, we fed mice either a control HFD or one containing the CSF1R inhibitor PLX5622 to selectively deplete microglia (). Immunostaining for the microglial marker Iba1 revealed robust depletion of microglia in the MBH of PLX5622-treated mice, whether chronically fed a standard low-fat chow diet (CD) or a HFD ( Figures 1 A and 1B ). By contrast, PLX5622 treatment did not deplete macrophages from the white adipose tissue (WAT) of the same mice, indicating relative microglial specificity ( Figure S1 ).

(F) Reduced food intake in PLX5622-treated mice fed a HFD (cumulative intake normalized to intial body weight, n = 12, two-way RM-ANOVA,p < 0.05 orp < 0.01 versus HFD). Values are mean ± SEM (3V, third ventricle; scale bar, 20 μm). See also Figure S1

Discussion

Microglia rapidly accumulate in the MBH of rodents fed a HFD. However, it was previously unclear whether gliosis is a reactive consequence of weight gain or whether non-neuronal cells are instead instructive, regulating energy balance and contributing to obesity pathogenesis. Here, we provide the first definitive evidence that microglia, through their capacity for inflammatory signaling, are critical regulators of the susceptibility to DIO and an essential conduit linking dietary overconsumption to hypothalamic dysfunction.

We combine pharmacologic and genetic approaches to show that NF-κB-dependent microglial activation is critical for both the development of HFD-induced MBH microgliosis, including the ingress of bone-marrow-derived myeloid cells, and metabolic dysfunction. Restraining this inflammatory activation in mice, either by depleting microglia or by deleting microglial Ikbkb, reduces food intake, mitigates DIO, and prevents MBH infiltration. By contrast, genetically activating microglia is sufficient to induce diet-independent MBH microgliosis, myeloid cell infiltration, metabolic dysfunction, and obesity. Together, these findings identify microglia as central regulators of energy balance and suggest that manipulating their activation could be an effective therapeutic for metabolic pathologies including obesity.

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Whereas our findings point to a highly specialized interaction between microglia and neurons governing energy metabolism and associated behaviors, we found no weight-independent role for microglia in regulating glucose homeostasis or peripheral inflammation. These findings suggest that metabolic inflammation in peripheral tissues develops independently of the CNS, likely resulting from chronic nutrient overload and/or obesity itself. This finding may explain the slow progression of metabolic inflammation in peripheral tissues relative to the MBH and suggests the possibility of distinct sets of factors that mediate the “metabolic” activation of microglia versus peripheral macrophages.

How do activated microglia influence energy balance in the context of HFD feeding? Some insight may be gleaned from the features of A20MGKO-BMT mice. In particular, increased food intake in A20MGKO-BMT mice was associated with reduced leptin sensitivity by the MBH, and reduced whole-body energy expenditure was associated with a reduction in the mRNA levels of several thermogenic genes in the BAT, suggesting that microglial signaling may specifically interact with effector pathways of the sympathetic nervous system.

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Bischof F. The role of the ubiquitin-editing enzyme A20 in diseases of the central nervous system and other pathological processes. However, the specific factors mediating such microglia-neuronal cross-talk remain largely unknown. Candidate mechanisms include secreted cytokines (), cell-cell interactions (), modulation of tanycyte function (), and alterations to the integrity and selectivity of the BBB surrounding the MBH (). Additionally, A20 is involved in cell-type-specific processes beyond controlling NF-κB activity, including autophagy, aging, and cell death (). By further exploring A20 regulation of microglial function, we may uncover novel pathways and polarization states contributing to CNS metabolic regulation.

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Xu A.W. Modulation of AgRP-neuronal function by SOCS3 as an initiating event in diet-induced hypothalamic leptin resistance. The mouse models we generated also provide important new insights into the cellular components of diet-induced microgliosis within the MBH. First, we note that the cluster of cells involved in this response is confined to the ARC and ME, whereas surrounding areas are devoid of such microgliosis. This anatomical specificity is intriguing, given that this region of the MBH is postulated to lie outside the traditional BBB (), allowing cells within it to sense and respond to circulating factors, and for circulating cells to extravasate from the vasculature and join in the response.

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et al. Origin, fate and dynamics of macrophages at central nervous system interfaces. + cells in the MBH of HFD-fed mice had morphologies suggestive of a perivascular origin, akin to cells seen at the edges of the MBH in CD-fed mice. Future work using appropriate models should attempt to define the specific contribution of perivascular and meningeal macrophages to diet-induced MBH microgliosis and hypothalamic function. Second, HFD-induced microgliosis in the MBH largely involves cells lacking typical definitive microglial markers such as Tmem119 and P2Y12. Indeed, based on marker analysis, the number of resident microglia in the MBH decreased in mice fed a HFD. Coincidently, however, we saw a modest rise in the number of myeloid cells in the MBH expressing neither typical microglial markers nor markers indicative of a blood-borne origin. Though their identities remain uncertain, they may represent a non-microglial, yet long-lived, population of brain-resident myeloid cells. Recently, for example, perivascular and meningeal macrophages were shown to have surprisingly slow turnover rates (). Indeed, some of these “atypical” CD68cells in the MBH of HFD-fed mice had morphologies suggestive of a perivascular origin, akin to cells seen at the edges of the MBH in CD-fed mice. Future work using appropriate models should attempt to define the specific contribution of perivascular and meningeal macrophages to diet-induced MBH microgliosis and hypothalamic function.

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Julius D. The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Another possibility is that these cells are actually resident microglia that downregulate typical markers in response to inflammatory activation. P2Y12 expression, for example, declines with microglial activation (), and many of the cells accumulating in the MBH in response to dietary excess had morphological features (shortened branches, enlarged cell bodies), suggesting cellular activation. If this were the case, then HFD-induced MBH microgliosis shifts the polarization of resident microglia but does not induce proliferation.

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et al. Inhibiting microglia expansion prevents diet-induced hypothalamic and peripheral inflammation. Whereas this conclusion is supported by our negative BrdU data, a recent study showed increased BrdU incorporation in the hypothalami of mice fed a HFD (). However, whereas diet-induced microgliosis is tightly confined to the MBH, the other study described BrdU incorporation outside of this region. Also, as only Iba1 was used to mark myeloid cells, it is unclear what specific cell types were involved in that reported proliferative response. Therefore, whereas the hypothalamic response to HFD consumption may include myeloid proliferation, our data indicate that MBH-resident microglia are not involved. Rather, our findings prompt interest in determining the transcriptional signature of HFD-activated MBH microglia and whether it is different from that induced by other stimuli and from that of microglia in other brain regions.

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et al. Microglial repopulation model reveals a robust homeostatic process for replacing CNS myeloid cells. + myeloid cells infiltrating the MBH upon deleting microglial A20 coexpress Tmem119, normally a specific marker of resident microglia. This profound phenotypic conversion could have, in turn, acted to ameliorate the amplitude and kinetics of the inflammatory and metabolic responses initiated by tamoxifen treatment in A20MGKO-BMT mice, especially given that the infiltrating cells expressed A20. Indeed, future work should determine whether infiltrating cells amplify MBH inflammation initiated by microglia or whether they function to quell this inflammation and hasten a return to homeostasis. Upon entering the MBH, infiltrating myeloid cells display morphologies characteristic of resident microglia. The fate-switching capacity of peripheral monocytes was recently described (), and future studies should determine its full extent in the MBH. On the other hand, this hybrid identity complicates studies to determine the independent impact of microglia versus infiltrating myeloid cells on metabolic regulation. For example, some of the GFPmyeloid cells infiltrating the MBH upon deleting microglial A20 coexpress Tmem119, normally a specific marker of resident microglia. This profound phenotypic conversion could have, in turn, acted to ameliorate the amplitude and kinetics of the inflammatory and metabolic responses initiated by tamoxifen treatment in A20mice, especially given that the infiltrating cells expressed A20. Indeed, future work should determine whether infiltrating cells amplify MBH inflammation initiated by microglia or whether they function to quell this inflammation and hasten a return to homeostasis.

Our findings here begin to shed light on this question; it is remarkable that either treating mice with PLX5622 or deleting Ikbkb in microglia reduces DIO susceptibility in conjunction with abrogating peripheral cell infiltration into the MBH, whereas microglial A20 deficiency induces both metabolic dysfunction and the influx of peripheral myeloid cells into the MBH. This tight correlation between metabolic outcomes and cellular infiltration suggests that myeloid cells drawn to the MBH may indeed contribute to the pathogenesis of DIO, a hypothesis that awaits the molecular tools to independently manipulate different subtypes of myeloid cells.