IL-17A produced by POMC neurons regulates diet-induced obesity

Rosa Gallo; Ana Teijeiro; Mariana Angulo-Aguado; Nabil Djouder

doi:10.1016/j.isci.2024.110259

. 2024 Jun 12;27(7):110259. doi: 10.1016/j.isci.2024.110259

IL-17A produced by POMC neurons regulates diet-induced obesity

Rosa Gallo ^1,², Ana Teijeiro ^1,², Mariana Angulo-Aguado ^1,², Nabil Djouder ^1,^3,^∗

PMCID: PMC11255842 PMID: 39027371

Summary

Overeating leads to obesity, a low-grade inflammatory condition involving interleukin-17A (IL-17A). While pro-opiomelanocortin (POMC) neurons regulate feeding, their connection with IL-17A is not well understood. To impair IL-17A signaling in POMC neurons, IL-17A receptor (Il17ra) was deleted by crossing IL17ra-flox and Pomc-Cre mice. Despite effective deletion, these mice showed no differences in body weight or adiposity compared to control mice, challenging the idea that IL-17A induces obesity through POMC neuron regulation. However, both groups exhibited reduced weight gain and adiposity upon high-fat diet compared to mice carrying only the floxed alleles, suggesting independent effects of Pomc-Cre transgene on body weight. Further analysis reveals that POMC neurons express IL-17A, and reduction in number of POMC neurons in Pomc-Cre mice could be linked to decreased IL-17A expression, which correlates with reduced adipocyte gene expression associated with obesity. Our data underscore an unexpected crosstalk between IL-17A-producing POMC neurons and the endocrine system in obesity regulation.

Subject areas: Genetics, Molecular biology, Neuroscience, Immunology

Graphical abstract

Highlights

•
Pomc-Cre mouse causes unintended weight loss
•
POMC neurons produce IL-17A upon high-fat diet
•
Pomc-Cre mouse has reduced levels of POMC neurons and IL-17A affecting body weight

Genetics; Molecular biology; Neuroscience; Immunology

Introduction

Nutrient overload can lead to obesity, a low-grade inflammatory disease.¹ In mammals, feeding behavior is regulated by neurons that express pro-opiomelanocortin (POMC) or agouti-related protein (AgRP) in the arcuate nucleus (ARC) region of the hypothalamus. Whereas AgRP neurons enhance food intake,² POMC neurons play a critical role in the regulation of body weight and energy balance through the production of alpha-melanocyte-stimulating hormone (α-MSH) that can act as an appetite suppressant by binding to and activating the melanocortin receptors in the brain.³^,⁴^,⁵ In obese individuals, there is often a reduction in POMC expression, leading to decreased α-MSH production and increased appetite.⁶^,⁷ Additionally, genetic mutations in the POMC gene have been linked to early-onset obesity in humans.⁸^,⁹ Leptin, a hormone produced by adipocytes and increased in obese individuals also contributes to the activation of POMC neurons.¹⁰ Therefore, dysregulation of POMC neurons plays a critical role in the onset of obesity by increasing food intake.

The pro-inflammatory cytokine interleukin-17A (IL-17A) is produced by innate and adaptive lymphocytes, plays fundamental functions in tissue physiology and influences a wide range of biological processes beyond host defense, such as neural repair or adipocyte metabolism.¹^,¹¹ Not only have increases in circulating IL-17A levels been observed after feeding in mice and humans,¹¹^,¹² it also exerts a direct influence on adipocytes, triggering metabolic reprogramming that fosters diet-induced obesity (DIO) and contributes to the development of metabolic syndrome, without affecting food intake.¹ Interestingly, systemic administration of IL-17A causes a temporal increase in both POMC and AgRP expression.¹¹^,¹² Accordingly, hypothalamic astrocytes and microglia express IL-17RA,¹¹^,¹³ suggesting a broader role of IL-17A in hypothalamus. Additionally, IL-17A has been reported to play a role in the regeneration of sensory neurons.¹⁴ While a distinct role of IL-17A in adipocyte reprogramming has been proposed to be crucial for obesity, these findings suggest that IL-17A may promote obesity through the modulation of POMC neurons, linking brain to adipocyte biology.

The Cre-lox system has proven to be a valuable tool in studying the role of arcuate neurons in the regulation of feeding and energy expenditure in mice. The Pomc-Cre mouse that expresses the Cre recombinase enzyme under the control of the Pomc promoter has been widely used in research to study the function of POMC neurons in vivo.¹⁵^,¹⁶^,¹⁷^,¹⁸ By using the Pomc-Cre mouse line in combination with mice that carry loxP-flanked genes, researchers can selectively delete or modify genes specifically in POMC neurons, allowing them to investigate the role of these genes in physiology. For instance, the Pomc-Cre has been used to study the function of melanocortin pathway in the regulation of body weight.¹⁶ It has also been utilized to understand how melanocortin neurons integrate endocrine signals to protect female mice against DIO.¹⁸ Additionally, deleting the regulatory molecule miR-29a in the Pomc-Cre mouse has been shown to attenuate obesity in adult mice fed with high-fat diet (HFD).¹⁷ Therefore, studies using Pomc-Cre mouse models have provided valuable insights into the functions of POMC neurons in the regulation of appetite and energy balance and have helped to identify potential therapeutic targets for obesity and related metabolic disorders.

Here, we sought to investigate the role of IL-17A in modulating POMC neurons and its effect on obesity. To this end, we deleted IL-17A receptor (IL-17RA) specifically in POMC neurons and analyzed the effects on body weight, comparing the results with the Pomc-Cre line mouse. Our study demonstrates off-target effects induced by the Pomc-Cre transgene, which may contribute to physiological and metabolic phenotypes that are inherent to the Cre-lox system rather than the result of the deletion of the genes of interest in the POMC neurons. Precisely, we demonstrate that POMC neurons express IL-17A during DIO and this could be implicated in the off-target effects elicited by the Pomc-Cre line.

Results

Deletion of IL-17RA in POMC neurons does not affect mouse body weight

To elucidate the function of IL-17A signaling in POMC neurons, IL-17RA was deleted in these neurons by crossing IL17ra-flox mice¹⁹ (hereafter Il17ra^(flox-flox) mice) with Pomc-Cre mice¹⁵ (hereafter Il17ra^(+/+)Pomc mice) in order to produce Il17ra^(Δ/Δ)Pomc mice. Adult mice were fed for 2 months with HFD (Figure 1A). Co-immunofluorescence (CIF) and bioinformatics analysis from published datasets²^,²⁰ confirmed that IL-17RA is abundantly expressed in POMC neurons in the ARC region of the hypothalamus, and this expression is efficiently abolished when IL-17RA is genetically depleted (Figures 1B, S1A, and S1B). Notably, Il17ra^(Δ/Δ)Pomc mice with ablation of Il17ra in POMC neurons had similar body weight (Figures 1C–1F) and adiposity (Figures 1G–1I) to Il17ra^(+/+)Pomc littermate controls, suggesting that IL-17A does not act on POMC neurons to induce obesity. However, upon HFD treatment, both cohorts of mice expressing the Pomc-Cre mouse gained less weight and had less adiposity than Il17ra^(flox-flox) littermates (Figures 1G–1I). Notably, Il17ra^(Δ/Δ)Pomc mice and Il17ra^(+/+)Pomc control littermates fed with normal diet (ND) showed similar body weight and adiposity (Figures 1C–1F). These data strongly points out that the Pomc-Cre strain has an effect on body weight following HFD that is independent on the absence of Il17ra in POMC neurons. Therefore, IL-17A regulates adipocyte biology independently of POMC neurons or food intake to promote obesity, as previously shown.¹

Deletion of SOCS3 in POMC neurons does not affect mouse body weight

To confirm the off target-effects of the Pomc-Cre mouse, we sought investigating the role of the suppressor of cytokine signaling 3 (Socs3) in modulating obesity via regulation of POMC neurons. SOCS3 acts as a negative regulator of cytokine signaling, inhibiting the activation of JAK/STAT signaling pathway, resulting in gene expression changes.²¹^,²²^,²³ Mice with neural cell-specific Socs3 deletion display increased insulin sensitivity and are protected against DIO,²⁴ whereas overexpression of Socs3 has the opposite effect.²⁵ Notably, Wang et al. and Kievit et al. published important findings indicating that the obesogenic effects of Socs3 and associated metabolic dysfunctions are mediated through the regulation of the POMC neurons in the hypothalamus.²⁶^,²⁷ Depletion of SOCS3 specifically in murine POMC neurons protects against metabolic dysfunctions and DIO.²⁴^,²⁶^,²⁷ However, noteworthy authors did not use Pomc-Cre littermate controls to compare the obtained data, which is especially important because during development, POMC is transiently and broadly expressed in various neurons of the central nervous system that differ from POMC neurons in adulthood.²⁸ Therefore, to check whether Pomc-Cre transgene is indeed generating effects on body weight loss, as demonstrated for IL-17RA deletion, we conducted a crossbreeding experiment between Pomc-Cre¹⁵ mice (hereafter Socs3^+/+(Pomc) mice) with Socs3-flox²⁹ (hereafter Socs3^(flox-flox) mice) mice, which resulted in the generation of Socs3^(Δ/Δ)Pomc mice with specific deletion of Socs3 in POMC neurons (Figure 2A). IF analysis indicated that Socs3 was efficiently deleted in POMC neurons (Figures S2A and S2B). However, in contrast to the findings reported by Wang et al. and Kievit et al.,²⁶^,²⁷ our results indicate that Socs3^(Δ/Δ)Pomc mice fed with HFD had similar body weights (Figures 2B–2E) and adiposity (Figures 2F–2H) to those of the Socs3^(+/+)Pomc wild-type littermates. Importantly, Socs3^(flox-flox) mice upon HFD gained more weight (Figures 2B–2E) and had more adiposity than the littermates that had the Pomc-Cre line (Socs3^(+/+)Pomc) (Figures 2F–2H). Notably, Socs3^(flox-flox) mice and Socs3^(+/+)Pomc wild-type littermates (Pomc-Cre line) had similar body weight without any effects on adiposity upon ND (Figures 2B–2H). These results confirm that increased body weight observed upon HFD is primarily caused by the transgenic Pomc-Cre gene rather than the ablation of Socs3, as also shown for IL-17RA depletion.

POMC neurons express IL-17A upon HFD

Next, we investigated the mechanisms through which the transgenic Pomc-Cre mouse model gained less body weight when fed with HFD. Notably, there was an evident decrease in the number of neurons expressing POMC in the ARC region of the hypothalamus, in Pomc-Cre mice compared to the wild-type littermate control (Figures 3A and 3B). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) confirmed a decrease in Pomc levels without any significant effects on Socs3 mRNA levels in the hypothalamus of the Pomc-Cre mice (Figures 3C and 3D), suggesting the decrease in POMC neurons. Remarkably, it became evident that Pomc-Cre mice also exhibited reduced IL-17A levels (Figure 3E), suggesting that the insertion of the transgene could affect IL-17A expression. Accordingly, bioinformatics analysis conducted on various publicly available datasets highlighted that POMC neurons expressed IL17a and Rorc, a well-known transcription factor associated with IL-17A (Figure 3F).¹ Co-IF confirmed that IL-17A is highly expressed in POMC neurons, and its expression is significantly reduced in the ARC region of the hypothalamus of the Pomc-Cre mice compared to the wild-type littermates (Figures 3A and 3G).

Considering the pivotal role of IL-17A in mediating obesity through adipocyte reprogramming and phosphorylation of PPARγ, the observed phenotype in Pomc-Cre mice could potentially find its explanation in the diminished POMC neurons and reduced IL-17A levels. As a result of this, the white adipose tissue of Pomc-Cre mice had decreased expression of phosphorylated PPARγ-targeted genes associated with obesity (Figure 3H). Hence, POMC neurons could secrete IL-17A, which may assume a pivotal role in adipocyte biology, as previously shown,¹ operating independently of its impact on POMC neurons. Any genetic disruptions resulting in reduction of IL-17A levels could potentially result in bight loss, irrespective of food intake.

Discussion

This study explores the interplay between IL-17A signaling, POMC neurons, and obesity. The anorexigenic axis mediated by POMC neurons plays a crucial role in regulating food intake and energy expenditure, and its dysregulation can lead to obesity and metabolic disorders. Here we demonstrate that IL-17RA deletion in POMC neurons using the Pomc-Cre transgenic mouse model does not affect body weight. The mechanistic insights into how the transgenic Pomc-Cre mouse could result in reduced body weight still require detailed resolution. However, our data demonstrate that POMC neurons unexpectedly could express IL-17A, the levels of which are diminished in the Pomc-Cre mice. There is a speculative possibility that this decrease might arise from the reduced number of POMC neurons with a reduction in POMC expression, potentially stemming from unknown mechanisms in response to variable integration of transgenic Pomc-Cre copies into the mouse genome.

The diminished count of POMC neurons might unlikely be the direct cause of reduced body weight gain. Accordingly, alterations in POMC neurons carrying the Cre would more likely result in increased food intake and obesity rather than satiety. However, the reduction in POMC neurons in the ARC of the hypothalamus leads to subsequent decreases in IL-17A levels, as POMC neurons can be a source of IL-17A production. The reduction in IL-17A signaling might thus contribute to body weight loss, as IL-17A is known to promote obesity through metabolic reprogramming of adipocytes,¹ rather than by regulating food intake via actions on POMC neurons. It is thus likely that the levels of IL-17A are systemically decreased in Pomc-Cre mice fed with HFD.

In agreement with this, our work provides evidence indicating that IL-17A signaling does not exert an impact on POMC neurons to modulate the obese state in mice. IL-17A could be therefore secreted by POMC neurons upon overnutrition affecting white adipose tissue biology. This highlights a potential unexpected crosstalk between the IL-17A-producing POMC neurons and the endocrine system to regulate obesity.

The overlooked possibility of non-specific Cre-related targets and the disparities observed in our study compared to previous findings could be also attributed to other factors.²⁶^,²⁷ One potential factor is the variation in the composition of the HFD used, which might influence the outcome of the studies. Similar to our study, Wang et al. used a diet with 45% fat, whereas Kievit et al. treated their mice with a diet containing a higher proportion of fat (58%).²⁶^,²⁷ Additionally, differences in the microbiota among the various mouse strains might play a role in influencing the outcomes. Another factor that should be taken into account is the variation in body weight between male and female mice, which can also impact the experimental results. To ensure accurate and reliable results, it is crucial to carefully address these potential confounding variables in future research. Properly controlling for diet composition, considering the impact of microbiota differences, and accounting for sex-related variations will strengthen the validity of our conclusions and enhance the translational potential of our findings.

Despite the extensive utilization of the transgenic Pomc-Cre mouse model¹⁵ in exploring the in vivo functions of POMC neurons,¹⁶^,¹⁷^,¹⁸^,²⁶^,²⁷ our study emphasizes that the mere ectopic expression of Pomc-Cre, irrespective of the presence of the floxed allele, induces alterations in the body weight of mice when fed an HFD. Notably, Pomc-eGFP reporter expression does not mimic the expression of tdTomato observed in the Pomc-Cre; ROSA-tdTomato mouse model, arguing that other cells than POMC neurons might be targeted by Pomc-Cre during embryogenesis.²⁸ Furthermore, it is reportedly showed that a subpopulation of cells expresses Pomc-Cre only during development but not in the adults.³⁰ Hence, it is crucial to validate the data obtained from the Pomc-Cre mouse model¹⁵ by employing alternative Pomc-Cre mice that undergo recombination at a distinct stage of embryonic development, as indicated by previous studies.³¹ Nevertheless, tracking POMC neurons across both developmental and adult stages could provide additional insights into this characteristic. On the other hand, the use of the Cre-lox system to delete genes of interest temporally could certainly be useful. In fact, using Tg(Pomc-Cre/ERT2) to delete serotonin receptor 2C in POMC neurons shows no significant effects on body weight when compared to their littermate controls.³² This tamoxifen-induced CreERT2 system mainly promotes the expression of Cre-ERT2 in adult POMC neurons, bypassing any issues encountered during embryogenesis.¹⁰^,³³ However, it is important to acknowledge that tamoxifen treatments might potentially influence adult neurogenesis. Another alternative would be the employment of the CRISPR-mediated gRNA to generate a knock-in mouse model. However, it is essential to consider that this method may also give rise to considerable off-target effects.

In sum, our work uncovers notable constraints in the transgenic Pomc-Cre mouse model, resulting in diminished IL-17A levels within POMC neurons and subsequently causing decreased body weight in response to DIO. Although the Cre-lox system is a valuable tool, it is not without its potential drawbacks. Due to the stochastic nature of DNA recombination, the efficacy of the system may be limited, and the expression of the Cre recombinase enzyme may cause inefficient or non-specific deletion of the gene of interest,³⁴^,³⁵ resulting in off-target effects that may yield unexpected results. Therefore, it is critical to use appropriate controls such as Cre-littermate controls (mice expressing the Cre enzyme) when employing the Cre-lox system to distinguish phenotypes triggered by the modulation of the gene of interest’s expression from phenotypes linked to the Cre’s insertion into the genome.

Limitations of the study

This study primarily focuses on investigating the off-target effects of the Pomc-Cre mouse model.¹⁵ Our data suggest that POMC neurons can secrete IL-17A in response to HFD, and the reduction of POMC neurons in the Pomc-Cre mouse may contribute to lower body weight. Genetic ablation of IL-17A or RORγt in POMC neurons could serve as valuable models to demonstrate that IL-17A is secreted by these neurons and to elucidate the function of POMC IL-17A⁺ neurons in the context of the Pomc-Cre mouse model and in obesity. Moreover, the precise molecular mechanisms driving this phenomenon require further exploration, and it remains unclear whether systemic levels of IL-17A are decreased. We hypothesize that IL-17A’s potential action may involve autocrine signaling within white adipose tissue, given that compromising IL-17A signals in this tissue has been shown to reduce body weight gain.¹ Additionally, it would be of great interest to monitor the function and activity of POMC neurons in the Pomc-Cre mouse through electrophysiological analysis. It is also important to acknowledge that our study is limited to one specific Pomc-Cre mouse model. To gain a comprehensive understanding of potential off-target effects in metabolic studies, it would be valuable to investigate other Pomc-Cre lines, including the Pomc-creERT2 mouse model.³¹^,³² This will help determine if the observed effects are specific to the Pomc-Cre mouse or if they are consistent across various Pomc-Cre models. Furthermore, it is worth noting that our study does not exclude the possibility that the reduction of POMC neurons we observed could be due to aberrations during embryonic development. Therefore, a thorough tracking of POMC neurons throughout both the developmental and adult stages could provide valuable insights into this aspect.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Anti-goat Alexa Fluor 488	Invitrogen	Cat# A11055; RRID: AB_2534102
Anti-streptavidin Alexa Fluor 555	AAT Bioquest	Cat#16989; RRID: N/A
Anti-POMC	Sigma Aldrich	Cat# SAB2500826; RRID: AB_10605136
Anti-SOCS3	Proteintech	Cat# 66797-1-Ig; RRID: AB_2882141
Anti-IL17RA	Santa Cruz	Cat# sc-376374; RRID: AB_10991331
Anti-IL-17A	Invitrogen	Cat# PA5-79470; RRID: AB_2746586

Chemicals, peptides, and recombinant proteins

DAPI	Sigma	D9542
Mowiol 4-88	Sigma	81381
BSA	Merck	A7906
Chow diet	Teklad	2918
HFD 45%	Brogaarden	D12451
TRIzol	Sigma	15596026

Critical commercial assays

Vectastain ABC HRP Kit (Peroxidase, Mouse IgG)	Vector Laboratories	PK-4002
Avidin-biotin blocking kit	Palex	416430
M-MLV Reverse Transcriptase GoTaq SYBR Green Master Mix	Thermofisher Promega	28025013 A6002
Mouse: Pomc-Cre	Balthasar et al.,¹⁵	MGI: 3640616
Mouse: Il17ra^(flox-flox)	El Malki et al.,¹⁹	MGI:5498882
Mouse: Socs3^(flox-flox)	Yasukawa et al.,²⁹	MGI:3051525

Oligonucleotides

Primers for Genotyping Primers for RT-qPCR	This Paper Table S1	N/A

Software and algorithms

GraphPad Prism 9.4.0	GraphPad Software	https://www.graphpad.com/scientific-software/prism/
Illustrator 26.3.1	Adobe	https://.adobe.com/products/illustrator.html
Image J 53c software	NIH	https://imagej.nih.gov/ij/
Photoshop 23.4.0 R Statistical Package V4.0.3 and R Studio	Adobe	https://adobe.com/products/photoshop.html https://www.r-studio.com/

Other

Fluorescence confocal microscopy	Leica	TCS SP5 MP

Open in a new tab

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Nabil Djouder (ndjouder@cnio.es). The sharing of materials described in this work could be subject to standard material transfer agreements.

Materials availability

This study did not generate unique reagents.

Data and code availability

•
This study did not generate any datasets. All data reported in this paper will be shared by the lead contact upon request.
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

Animal models

Il17ra^(Δ/Δ)Pomc and Socs3^(Δ/Δ)Pomc mice were generated in our lab by crossing Pomc-Cre¹⁵ (MGI: 3640616) mice with Il17ra^{(flox-flox) 19} (MGI:5498882) and Socs3^{(flox-flox) 29} (MGI:3051525) mice, respectively. More precisely, matings were conducted between Pomc-Cre mice heterozygous for the Cre allele and heterozygous for the floxed allele (+/flox). Littermate controls from the same cohort and strain of mice were always used in this study.

Only 8 week-old males were used for the study. All experiments were approved by the CNIO-ISCIII (Instituto de Salud Carlos III) Ethics Committee, Institutional Animal Care and Use Committee (IACUC), the Ethical Committee for animal experimentation (CEIyBA) (PROEX 172/18; PROEX 173.3/22) and Community of Madrid (CAM) and performed in accordance with the guidelines for ethical conduct in the care and use of animals as stated in the international guiding principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences and as described in the ARRIVE guidelines.³⁶

Method details

Mouse conditions and housing

All mice have been housed at the animal facility of the Spanish National research Centre (CNIO) in pathogen-free conditions, with a 12-h light–dark cycle between 8:00 and 20:00 in a temperature-controlled room (23 ± 1°C) following the recommendations of the EUMORPHIA Consortium for animal housing, unless stated otherwise. All mice have been backcrossed to C57BL/6 for at least seven generations.

Mouse diets

Mice were fed either a ND (18% fat, 58% carbohydrates and 24% proteins) (Harlan Laboratories, 2018S) or a HFD (45% fat, 35% carbohydrates and 20% proteins) (Research Diets, D12451). Food and water were provided ad libitum.

Mouse genotyping

The genotyping was performed as previously reported¹^,³⁷^,³⁸^,³⁹ using the following couples of primers:

Cre-Fw: 5’-CCATCTGCCACCAGCCAG-3’

Cre-Rv: 5’-TCGCCATCTTCCAGCAGG-3’

Cre control Fw: 5’- ACTGGGATCTTCGAACTCTTTGGAC-3’

Cre control Rv: 5’- GATGTTGGGGCACTGCTCATTCACC-3’

Socs3 Fw: 5’-GCGGGCAGGGGAAGAGACTGTCTGGGGTTG-3’

Socs3 Rv1: 5’-GGCGCACGGAGCCAGCGTGGATCTGCG-3’

Socs3 Rv2: 5’-AGTCCGCTTGTCAAAGGTATTGTCCCAC-3’

Il17ra Fw: 5’- GGCAGCCTTTGGGATCCCAAC-3’

Il17ra Rv: 5’-CTACTCTTCTCACCAGCGCGC-3’

Body weight

Mouse body weight has been measured weekly beginning at 8 weeks by using an analytical balance as previously described.¹

Tissue preparation

Freshly collected tissues were fixed in 10% buffered formalin solution overnight and embedded in paraffin and processed as previously reported.¹^,³⁷^,³⁸^,⁴⁰ Mice were euthanized using CO₂ chambers. Tissues were quickly removed from mice and weighted using an analytical balance. For molecular analysis, the tissues were washed in PBS and frozen at -80.

Immunofluorescence

Avidin-biotin immunofluorescence technique has been performed as previously described.⁴¹ Briefly, tissue sections of 3 μm were deparaffinized, rehydrated and antigen retrieved by using 1 M sodium citrate buffer (pH 6.5). After blocking endogenous peroxidase using 3% H₂O₂ for 10 min, and permeabilized in 0.5% Triton X-100 in PBS for 1 hour, sections were blocked for 1 hr at room temperature with avidin-biotin blocking kit diluted in 5% BSA dissolved in PBS 1X. Furthermore, sections were incubated with primary antibodies (anti-POMC, anti-IL17R, anti-SOCS3 and anti-IL17A) overnight at 4°C using the following concentrations. Then, the sections were incubated with biotinylated secondary antibodies (1:200) diluted in PBS/ 5% BSA solution for 1h at RT, followed by the incubation with streptavidin-HRP (1:200) diluted in PBS/ 5% BSA solution for 30 min at RT.

Eventually, the sections were incubated with secondary antibodies anti-goat AF488 (1:500) and anti-streptavidin AF555 (1:500) and DAPI for 1 hour at RT. The pictures were acquired with confocal microscope and quantified with Image J software.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from frozen tissue, using TRIzol as previously reported⁴² or extracted from paraffin-embedded tissue as previously described.⁴³ The cDNA was synthesized with M-MLV reverse transcriptase and GoTaq Real-Time was used to carry out RT-qPCRs.⁴⁴ Ct values of each gene were normalized to Ct value of 18s and 2^-ΔΔCt was calculated. Finally, the fold changes of each gene were obtained normalizing the value of different groups to the control samples of the experiment. RT-qPCR primers are listed in Table S1.

Bioinformatics analysis

Publicly available RNA sequencing technology single-cell sequencing and trap-sequencing datasets from neurons https://cellxgene.cziscience.com/ and precisely POMC-expressing neurons GSE153753, GSE181539 ²^,²⁰ were used to analyze Il17a, Il17ra, Pomc, and Rorc genetic expression profiles. The average of the normalized count values (base mean) from the different datasets were used to represent the genetic expression.

Quantification and statistical analysis

Image analysis and quantifications

To quantify the immunofluorescence staining, 5 images per slide from each mouse were obtained. Single or double positive cells located anatomically near the ventral part of the third ventricle, specifically within the arcuate nucleus (ARC), were counted and normalized to the total number of DAPI-positive cells in the same ARC region, as indicated above. Data are represented as percentages and were calculated using the formula: (number of DAPI-positive cells in the arcuate nucleus / 100) x number of positive cells, as previously reported.²^,⁴⁵ All quantifications were performed manually in a blind manner. Each dot in the graphs represents data from an individual mouse.

Statistical analysis

Statistical analyses were performed using GraphPad PrismV8.3.0 software. Statistical significance (p value) (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001) between the means of two groups was determined using unpaired two-tailed Student’s t test or two-way ANOVA. Results are expressed as the mean value ± s.e.m. n represents the number of mice used in each experiment.

Acknowledgments

We are very grateful to Jesus Gómez Alonso from the Confocal Microscopy Unit at CNIO for helping to process confocal pictures and images. We thank Eduardo Caleiras, pathologist from the Histopathology Unit at CNIO for assisting us with the histological and IF analysis. We thank all mouse providers. We are also thankful to the CNIO Mouse Genome Editing Core Unit and Animal Facility for the mouse re-derivation and maintenance, respectively. This work was funded by grants to N.D. supported by the State Research Agency (MICIU/AEI/10.13039/501100011033) from the Spanish Ministry of Science, Innovation and Universities (MICIU) (RTI2018-094834-B-I00 and PID2021-122695OB-I00), also including the iDIFFER network of Excellence (RED2022-134792-T), and cofunded by European Regional Development Fund (ERDF), by the Asociación Española Contra el Cáncer (AECC) (PRYGN211184DJOU), and by the BBVA Foundation grants for biomedicine (EIC21-1-243). This work was developed at the CNIO funded by the Health Institute Carlos III (ISCIII).

Author contributions

R.G., A.T., M.A.A., and N.D. designed the experiments. R.G. and A.T. performed most of the experiments and statistical analyses. M.A.A. assisted in IF workflow and analysis, performed the bioinformatics analysis and RT-qPCR. N.D. conceived the project and study and secured funding. N.D. wrote the manuscript with R.G. All authors reviewed and contributed to the editing of the manuscript.

Declaration of interests

The authors declare no competing interests.

Published: June 12, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110259.

Supplemental information

Document S1. Figures S1 and S2 and Table S1

mmc1.pdf^{(894KB, pdf)}

References

1.Teijeiro A., Garrido A., Ferre A., Perna C., Djouder N. Inhibition of the IL-17A axis in adipocytes suppresses diet-induced obesity and metabolic disorders in mice. Nat. Metab. 2021;3:496–512. doi: 10.1038/s42255-021-00371-1. [DOI] [PubMed] [Google Scholar]
2.Biglari N., Gaziano I., Schumacher J., Radermacher J., Paeger L., Klemm P., Chen W., Corneliussen S., Wunderlich C.M., Sue M., et al. Functionally distinct POMC-expressing neuron subpopulations in hypothalamus revealed by intersectional targeting. Nat. Neurosci. 2021;24:913–929. doi: 10.1038/s41593-021-00854-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Krashes M.J., Lowell B.B., Garfield A.S. Melanocortin-4 receptor–regulated energy homeostasis. Nat. Neurosci. 2016;19:206–219. doi: 10.1038/nn.4202. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Quarta C., Claret M., Zeltser L.M., Williams K.W., Yeo G.S.H., Tschöp M.H., Diano S., Brüning J.C., Cota D. POMC neuronal heterogeneity in energy balance and beyond: an integrated view. Nat. Metab. 2021;3:299–308. doi: 10.1038/s42255-021-00345-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Huszar D., Lynch C.A., Fairchild-Huntress V., Dunmore J.H., Fang Q., Berkemeier L.R., Gu W., Kesterson R.A., Boston B.A., Cone R.D., et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell. 1997;88:131–141. doi: 10.1016/s0092-8674(00)81865-6. [DOI] [PubMed] [Google Scholar]
6.Cui J., Ding Y., Chen S., Zhu X., Wu Y., Zhang M., Zhao Y., Li T.R.R., Sun L.V., Zhao S., et al. Disruption of Gpr45 causes reduced hypothalamic POMC expression and obesity. J. Clin. Invest. 2016;126:3192–3206. doi: 10.1172/jci85676. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Souza G.F.P., Solon C., Nascimento L.F., De-Lima-Junior J.C., Nogueira G., Moura R., Rocha G.Z., Fioravante M., Bobbo V., Morari J., et al. Defective regulation of POMC precedes hypothalamic inflammation in diet-induced obesity. Sci. Rep. 2016;6 doi: 10.1038/srep29290. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Farooqi I.S., Drop S., Clements A., Keogh J.M., Biernacka J., Lowenbein S., Challis B.G., O’Rahilly S. Heterozygosity for a POMC-Null Mutation and Increased Obesity Risk in Humans. Diabetes. 2006;55:2549–2553. doi: 10.2337/db06-0214. [DOI] [PubMed] [Google Scholar]
9.Krude H., Biebermann H., Luck W., Horn R., Brabant G., Grüters A. Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans. Nat. Genet. 1998;19:155–157. doi: 10.1038/509. [DOI] [PubMed] [Google Scholar]
10.Caron A., Dungan Lemko H.M., Castorena C.M., Fujikawa T., Lee S., Lord C.C., Ahmed N., Lee C.E., Holland W.L., Liu C., Elmquist J.K. POMC neurons expressing leptin receptors coordinate metabolic responses to fasting via suppression of leptin levels. Elife. 2018;7 doi: 10.7554/eLife.33710. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Douglas A., Stevens B., Lynch L. Interleukin-17 as a key player in neuroimmunometabolism. Nat. Metab. 2023;5:1088–1100. doi: 10.1038/s42255-023-00846-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Nogueira G., Solon C., Carraro R.S., Engel D.F., Ramalho A.F., Sidarta-Oliveira D., Gaspar R.S., Bombassaro B., Vasques A.C., Geloneze B., et al. Interleukin-17 acts in the hypothalamus reducing food intake. Brain Behav. Immun. 2020;87:272–285. doi: 10.1016/j.bbi.2019.12.012. [DOI] [PubMed] [Google Scholar]
13.Chen J., Zhong H., Yu H., Sun J., Shen B., Xu X., Huang S., Huang P., Zhong Y. Interleukin-17A modulates retinal inflammation by regulating microglial activation via the p38 MAPK pathway in experimental glaucoma neuropathy. FASEB J. 2023;37 doi: 10.1096/fj.202202056RR. [DOI] [PubMed] [Google Scholar]
14.Enamorado M., Kulalert W., Han S.J., Rao I., Delaleu J., Link V.M., Yong D., Smelkinson M., Gil L., Nakajima S., et al. Immunity to the microbiota promotes sensory neuron regeneration. Cell. 2023;186:607–620.e17. doi: 10.1016/j.cell.2022.12.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Balthasar N., Coppari R., McMinn J., Liu S.M., Lee C.E., Tang V., Kenny C.D., McGovern R.A., Chua S.C., Jr., Elmquist J.K., Lowell B.B. Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis. Neuron. 2004;42:983–991. doi: 10.1016/j.neuron.2004.06.004. [DOI] [PubMed] [Google Scholar]
16.Li H., Xu Y., Jiang Y., Jiang Z., Otiz-Guzman J., Morrill J.C., Cai J., Mao Z., Xu Y., Arenkiel B.R., et al. The melanocortin action is biased toward protection from weight loss in mice. Nat. Commun. 2023;14:2200. doi: 10.1038/s41467-023-37912-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Ma Y., Murgia N., Liu Y., Li Z., Sirakawin C., Konovalov R., Kovzel N., Xu Y., Kang X., Tiwari A., et al. Neuronal miR-29a protects from obesity in adult mice. Mol. Metab. 2022;61 doi: 10.1016/j.molmet.2022.101507. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.González-García I., García-Clavé E., Cebrian-Serrano A., Le Thuc O., Contreras R.E., Xu Y., Gruber T., Schriever S.C., Legutko B., Lintelmann J., et al. Estradiol regulates leptin sensitivity to control feeding via hypothalamic Cited1. Cell Metab. 2023;35:438–455.e7. doi: 10.1016/j.cmet.2023.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.El Malki K., Karbach S.H., Huppert J., Zayoud M., Reissig S., Schüler R., Nikolaev A., Karram K., Münzel T., Kuhlmann C.R.W., et al. An alternative pathway of imiquimod-induced psoriasis-like skin inflammation in the absence of interleukin-17 receptor a signaling. J. Invest. Dermatol. 2013;133:441–451. doi: 10.1038/jid.2012.318. [DOI] [PubMed] [Google Scholar]
20.Yu H., Rubinstein M., Low M.J. Developmental single-cell transcriptomics of hypothalamic POMC neurons reveal the genetic trajectories of multiple neuropeptidergic phenotypes. Elife. 2022;11 doi: 10.7554/eLife.72883. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Shuai K., Liu B. Regulation of JAK–STAT signalling in the immune system. Nat. Rev. Immunol. 2003;3:900–911. doi: 10.1038/nri1226. [DOI] [PubMed] [Google Scholar]
22.Carow B., Rottenberg M.E. SOCS3, a Major Regulator of Infection and Inflammation. Front. Immunol. 2014;5 doi: 10.3389/fimmu.2014.00058. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Abdelaal M., le Roux C.W., Docherty N.G. Morbidity and mortality associated with obesity. Ann. Transl. Med. 2017;5:161. doi: 10.21037/atm.2017.03.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Mori H., Hanada R., Hanada T., Aki D., Mashima R., Nishinakamura H., Torisu T., Chien K.R., Yasukawa H., Yoshimura A. Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity. Nat. Med. 2004;10:739–743. doi: 10.1038/nm1071. [DOI] [PubMed] [Google Scholar]
25.Reed A.S., Unger E.K., Olofsson L.E., Piper M.L., Myers M.G., Jr., Xu A.W. Functional Role of Suppressor of Cytokine Signaling 3 Upregulation in Hypothalamic Leptin Resistance and Long-Term Energy Homeostasis. Diabetes. 2010;59:894–906. doi: 10.2337/db09-1024. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Wang Z., do Carmo J.M., da Silva A.A., Bailey K.C., Aberdein N., Moak S.P., Hall J.E. Role of SOCS3 in POMC neurons in metabolic and cardiovascular regulation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2019;316:R338–R351. doi: 10.1152/ajpregu.00163.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Kievit P., Howard J.K., Badman M.K., Balthasar N., Coppari R., Mori H., Lee C.E., Elmquist J.K., Yoshimura A., Flier J.S. Enhanced leptin sensitivity and improved glucose homeostasis in mice lacking suppressor of cytokine signaling-3 in POMC-expressing cells. Cell Metab. 2006;4:123–132. doi: 10.1016/j.cmet.2006.06.010. [DOI] [PubMed] [Google Scholar]
28.Padilla S.L., Reef D., Zeltser L.M. Defining POMC neurons using transgenic reagents: impact of transient Pomc expression in diverse immature neuronal populations. Endocrinology. 2012;153:1219–1231. doi: 10.1210/en.2011-1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Yasukawa H., Ohishi M., Mori H., Murakami M., Chinen T., Aki D., Hanada T., Takeda K., Akira S., Hoshijima M., et al. IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat. Immunol. 2003;4:551–556. doi: 10.1038/ni938. [DOI] [PubMed] [Google Scholar]
30.Bouret S.G., Bates S.H., Chen S., Myers M.G., Jr., Simerly R.B. Distinct roles for specific leptin receptor signals in the development of hypothalamic feeding circuits. J. Neurosci. 2012;32:1244–1252. doi: 10.1523/jneurosci.2277-11.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Xu A.W., Kaelin C.B., Takeda K., Akira S., Schwartz M.W., Barsh G.S. PI3K integrates the action of insulin and leptin on hypothalamic neurons. J. Clin. Invest. 2005;115:951–958. doi: 10.1172/jci24301. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Berglund E.D., Liu C., Sohn J.W., Liu T., Kim M.H., Lee C.E., Vianna C.R., Williams K.W., Xu Y., Elmquist J.K. Serotonin 2C receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis. J. Clin. Invest. 2013;123:5061–5070. doi: 10.1172/jci70338. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Lee C.H., Song D.K., Park C.B., Choi J., Kang G.M., Shin S.H., Kwon I., Park S., Kim S., Kim J.Y., et al. Primary cilia mediate early life programming of adiposity through lysosomal regulation in the developing mouse hypothalamus. Nat. Commun. 2020;11:5772. doi: 10.1038/s41467-020-19638-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Ryding A.D., Sharp M.G., Mullins J.J. Conditional transgenic technologies. J. Endocrinol. 2001;171:1–14. doi: 10.1677/joe.0.1710001. [DOI] [PubMed] [Google Scholar]
35.Shao X., Somlo S., Igarashi P. Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract. J. Am. Soc. Nephrol. 2002;13:1837–1846. doi: 10.1097/01.asn.0000016444.90348.50. [DOI] [PubMed] [Google Scholar]
36.Kilkenny C., Browne W.J., Cuthill I.C., Emerson M., Altman D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010;8 doi: 10.1371/journal.pbio.1000412. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Garrido A., Kim E., Teijeiro A., Sánchez Sánchez P., Gallo R., Nair A., Matamala Montoya M., Perna C., Vicent G.P., Muñoz J., et al. Histone acetylation of bile acid transporter genes plays a critical role in cirrhosis. J. Hepatol. 2022;76:850–861. doi: 10.1016/j.jhep.2021.12.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Chaves-Perez A., Santos-de-Frutos K., de la Rosa S., Herranz-Montoya I., Perna C., Djouder N. Transit-amplifying cells control R-spondins in the mouse crypt to modulate intestinal stem cell proliferation. J. Exp. Med. 2022;219 doi: 10.1084/jem.20212405. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Gomes A.L., Teijeiro A., Burén S., Tummala K.S., Yilmaz M., Waisman A., Theurillat J.P., Perna C., Djouder N. Metabolic Inflammation-Associated IL-17A Causes Non-alcoholic Steatohepatitis and Hepatocellular Carcinoma. Cancer Cell. 2016;30:161–175. doi: 10.1016/j.ccell.2016.05.020. [DOI] [PubMed] [Google Scholar]
40.Bosso G., Cipressa F., Moroni M.L., Pennisi R., Albanesi J., Brandi V., Cugusi S., Renda F., Ciapponi L., Polticelli F., et al. NBS1 interacts with HP1 to ensure genome integrity. Cell Death Dis. 2019;10:951. doi: 10.1038/s41419-019-2185-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Berman J.W., Basch R.S. Amplification of the biotin-avidin immunmofluorescence technique. J. Immunol. Methods. 1980;36:335–338. doi: 10.1016/0022-1759(80)90138-6. [DOI] [PubMed] [Google Scholar]
42.Bosso G., Cipressa F., Tullo L., Cenci G. Co-amplification of CBX3 with EGFR or RAC1 in human cancers corroborated by a conserved genetic interaction among the genes. Cell Death Discov. 2023;9:317. doi: 10.1038/s41420-023-01598-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Kumar K., Oli A., Hallikeri K., Shilpasree A.S., Goni M. An optimized protocol for total RNA isolation from archived formalin-fixed paraffin-embedded tissues to identify the long non-coding RNA in oral squamous cell carcinomas. MethodsX. 2022;9 doi: 10.1016/j.mex.2021.101602. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Piñeiro-Hermida S., Bosso G., Sánchez-Vázquez R., Martínez P., Blasco M.A. Telomerase deficiency and dysfunctional telomeres in the lung tumor microenvironment impair tumor progression in NSCLC mouse models and patient-derived xenografts. Cell Death Differ. 2023;30:1585–1600. doi: 10.1038/s41418-023-01149-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Zhan C., Zhou J., Feng Q., Zhang J.E., Lin S., Bao J., Wu P., Luo M. Acute and long-term suppression of feeding behavior by POMC neurons in the brainstem and hypothalamus, respectively. J. Neurosci. 2013;33:3624–3632. doi: 10.1523/jneurosci.2742-12.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1 and S2 and Table S1

mmc1.pdf^{(894KB, pdf)}

Data Availability Statement

•
This study did not generate any datasets. All data reported in this paper will be shared by the lead contact upon request.
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

[bib1] 1.Teijeiro A., Garrido A., Ferre A., Perna C., Djouder N. Inhibition of the IL-17A axis in adipocytes suppresses diet-induced obesity and metabolic disorders in mice. Nat. Metab. 2021;3:496–512. doi: 10.1038/s42255-021-00371-1. [DOI] [PubMed] [Google Scholar]

[bib2] 2.Biglari N., Gaziano I., Schumacher J., Radermacher J., Paeger L., Klemm P., Chen W., Corneliussen S., Wunderlich C.M., Sue M., et al. Functionally distinct POMC-expressing neuron subpopulations in hypothalamus revealed by intersectional targeting. Nat. Neurosci. 2021;24:913–929. doi: 10.1038/s41593-021-00854-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib3] 3.Krashes M.J., Lowell B.B., Garfield A.S. Melanocortin-4 receptor–regulated energy homeostasis. Nat. Neurosci. 2016;19:206–219. doi: 10.1038/nn.4202. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4.Quarta C., Claret M., Zeltser L.M., Williams K.W., Yeo G.S.H., Tschöp M.H., Diano S., Brüning J.C., Cota D. POMC neuronal heterogeneity in energy balance and beyond: an integrated view. Nat. Metab. 2021;3:299–308. doi: 10.1038/s42255-021-00345-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5.Huszar D., Lynch C.A., Fairchild-Huntress V., Dunmore J.H., Fang Q., Berkemeier L.R., Gu W., Kesterson R.A., Boston B.A., Cone R.D., et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell. 1997;88:131–141. doi: 10.1016/s0092-8674(00)81865-6. [DOI] [PubMed] [Google Scholar]

[bib6] 6.Cui J., Ding Y., Chen S., Zhu X., Wu Y., Zhang M., Zhao Y., Li T.R.R., Sun L.V., Zhao S., et al. Disruption of Gpr45 causes reduced hypothalamic POMC expression and obesity. J. Clin. Invest. 2016;126:3192–3206. doi: 10.1172/jci85676. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib7] 7.Souza G.F.P., Solon C., Nascimento L.F., De-Lima-Junior J.C., Nogueira G., Moura R., Rocha G.Z., Fioravante M., Bobbo V., Morari J., et al. Defective regulation of POMC precedes hypothalamic inflammation in diet-induced obesity. Sci. Rep. 2016;6 doi: 10.1038/srep29290. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Farooqi I.S., Drop S., Clements A., Keogh J.M., Biernacka J., Lowenbein S., Challis B.G., O’Rahilly S. Heterozygosity for a POMC-Null Mutation and Increased Obesity Risk in Humans. Diabetes. 2006;55:2549–2553. doi: 10.2337/db06-0214. [DOI] [PubMed] [Google Scholar]

[bib9] 9.Krude H., Biebermann H., Luck W., Horn R., Brabant G., Grüters A. Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans. Nat. Genet. 1998;19:155–157. doi: 10.1038/509. [DOI] [PubMed] [Google Scholar]

[bib10] 10.Caron A., Dungan Lemko H.M., Castorena C.M., Fujikawa T., Lee S., Lord C.C., Ahmed N., Lee C.E., Holland W.L., Liu C., Elmquist J.K. POMC neurons expressing leptin receptors coordinate metabolic responses to fasting via suppression of leptin levels. Elife. 2018;7 doi: 10.7554/eLife.33710. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib11] 11.Douglas A., Stevens B., Lynch L. Interleukin-17 as a key player in neuroimmunometabolism. Nat. Metab. 2023;5:1088–1100. doi: 10.1038/s42255-023-00846-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12.Nogueira G., Solon C., Carraro R.S., Engel D.F., Ramalho A.F., Sidarta-Oliveira D., Gaspar R.S., Bombassaro B., Vasques A.C., Geloneze B., et al. Interleukin-17 acts in the hypothalamus reducing food intake. Brain Behav. Immun. 2020;87:272–285. doi: 10.1016/j.bbi.2019.12.012. [DOI] [PubMed] [Google Scholar]

[bib13] 13.Chen J., Zhong H., Yu H., Sun J., Shen B., Xu X., Huang S., Huang P., Zhong Y. Interleukin-17A modulates retinal inflammation by regulating microglial activation via the p38 MAPK pathway in experimental glaucoma neuropathy. FASEB J. 2023;37 doi: 10.1096/fj.202202056RR. [DOI] [PubMed] [Google Scholar]

[bib14] 14.Enamorado M., Kulalert W., Han S.J., Rao I., Delaleu J., Link V.M., Yong D., Smelkinson M., Gil L., Nakajima S., et al. Immunity to the microbiota promotes sensory neuron regeneration. Cell. 2023;186:607–620.e17. doi: 10.1016/j.cell.2022.12.037. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib15] 15.Balthasar N., Coppari R., McMinn J., Liu S.M., Lee C.E., Tang V., Kenny C.D., McGovern R.A., Chua S.C., Jr., Elmquist J.K., Lowell B.B. Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis. Neuron. 2004;42:983–991. doi: 10.1016/j.neuron.2004.06.004. [DOI] [PubMed] [Google Scholar]

[bib16] 16.Li H., Xu Y., Jiang Y., Jiang Z., Otiz-Guzman J., Morrill J.C., Cai J., Mao Z., Xu Y., Arenkiel B.R., et al. The melanocortin action is biased toward protection from weight loss in mice. Nat. Commun. 2023;14:2200. doi: 10.1038/s41467-023-37912-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib17] 17.Ma Y., Murgia N., Liu Y., Li Z., Sirakawin C., Konovalov R., Kovzel N., Xu Y., Kang X., Tiwari A., et al. Neuronal miR-29a protects from obesity in adult mice. Mol. Metab. 2022;61 doi: 10.1016/j.molmet.2022.101507. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18.González-García I., García-Clavé E., Cebrian-Serrano A., Le Thuc O., Contreras R.E., Xu Y., Gruber T., Schriever S.C., Legutko B., Lintelmann J., et al. Estradiol regulates leptin sensitivity to control feeding via hypothalamic Cited1. Cell Metab. 2023;35:438–455.e7. doi: 10.1016/j.cmet.2023.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib19] 19.El Malki K., Karbach S.H., Huppert J., Zayoud M., Reissig S., Schüler R., Nikolaev A., Karram K., Münzel T., Kuhlmann C.R.W., et al. An alternative pathway of imiquimod-induced psoriasis-like skin inflammation in the absence of interleukin-17 receptor a signaling. J. Invest. Dermatol. 2013;133:441–451. doi: 10.1038/jid.2012.318. [DOI] [PubMed] [Google Scholar]

[bib20] 20.Yu H., Rubinstein M., Low M.J. Developmental single-cell transcriptomics of hypothalamic POMC neurons reveal the genetic trajectories of multiple neuropeptidergic phenotypes. Elife. 2022;11 doi: 10.7554/eLife.72883. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] 21.Shuai K., Liu B. Regulation of JAK–STAT signalling in the immune system. Nat. Rev. Immunol. 2003;3:900–911. doi: 10.1038/nri1226. [DOI] [PubMed] [Google Scholar]

[bib22] 22.Carow B., Rottenberg M.E. SOCS3, a Major Regulator of Infection and Inflammation. Front. Immunol. 2014;5 doi: 10.3389/fimmu.2014.00058. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] 23.Abdelaal M., le Roux C.W., Docherty N.G. Morbidity and mortality associated with obesity. Ann. Transl. Med. 2017;5:161. doi: 10.21037/atm.2017.03.107. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib24] 24.Mori H., Hanada R., Hanada T., Aki D., Mashima R., Nishinakamura H., Torisu T., Chien K.R., Yasukawa H., Yoshimura A. Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity. Nat. Med. 2004;10:739–743. doi: 10.1038/nm1071. [DOI] [PubMed] [Google Scholar]

[bib25] 25.Reed A.S., Unger E.K., Olofsson L.E., Piper M.L., Myers M.G., Jr., Xu A.W. Functional Role of Suppressor of Cytokine Signaling 3 Upregulation in Hypothalamic Leptin Resistance and Long-Term Energy Homeostasis. Diabetes. 2010;59:894–906. doi: 10.2337/db09-1024. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib26] 26.Wang Z., do Carmo J.M., da Silva A.A., Bailey K.C., Aberdein N., Moak S.P., Hall J.E. Role of SOCS3 in POMC neurons in metabolic and cardiovascular regulation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2019;316:R338–R351. doi: 10.1152/ajpregu.00163.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib27] 27.Kievit P., Howard J.K., Badman M.K., Balthasar N., Coppari R., Mori H., Lee C.E., Elmquist J.K., Yoshimura A., Flier J.S. Enhanced leptin sensitivity and improved glucose homeostasis in mice lacking suppressor of cytokine signaling-3 in POMC-expressing cells. Cell Metab. 2006;4:123–132. doi: 10.1016/j.cmet.2006.06.010. [DOI] [PubMed] [Google Scholar]

[bib28] 28.Padilla S.L., Reef D., Zeltser L.M. Defining POMC neurons using transgenic reagents: impact of transient Pomc expression in diverse immature neuronal populations. Endocrinology. 2012;153:1219–1231. doi: 10.1210/en.2011-1665. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib29] 29.Yasukawa H., Ohishi M., Mori H., Murakami M., Chinen T., Aki D., Hanada T., Takeda K., Akira S., Hoshijima M., et al. IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat. Immunol. 2003;4:551–556. doi: 10.1038/ni938. [DOI] [PubMed] [Google Scholar]

[bib30] 30.Bouret S.G., Bates S.H., Chen S., Myers M.G., Jr., Simerly R.B. Distinct roles for specific leptin receptor signals in the development of hypothalamic feeding circuits. J. Neurosci. 2012;32:1244–1252. doi: 10.1523/jneurosci.2277-11.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib31] 31.Xu A.W., Kaelin C.B., Takeda K., Akira S., Schwartz M.W., Barsh G.S. PI3K integrates the action of insulin and leptin on hypothalamic neurons. J. Clin. Invest. 2005;115:951–958. doi: 10.1172/jci24301. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib32] 32.Berglund E.D., Liu C., Sohn J.W., Liu T., Kim M.H., Lee C.E., Vianna C.R., Williams K.W., Xu Y., Elmquist J.K. Serotonin 2C receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis. J. Clin. Invest. 2013;123:5061–5070. doi: 10.1172/jci70338. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib33] 33.Lee C.H., Song D.K., Park C.B., Choi J., Kang G.M., Shin S.H., Kwon I., Park S., Kim S., Kim J.Y., et al. Primary cilia mediate early life programming of adiposity through lysosomal regulation in the developing mouse hypothalamus. Nat. Commun. 2020;11:5772. doi: 10.1038/s41467-020-19638-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib34] 34.Ryding A.D., Sharp M.G., Mullins J.J. Conditional transgenic technologies. J. Endocrinol. 2001;171:1–14. doi: 10.1677/joe.0.1710001. [DOI] [PubMed] [Google Scholar]

[bib35] 35.Shao X., Somlo S., Igarashi P. Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract. J. Am. Soc. Nephrol. 2002;13:1837–1846. doi: 10.1097/01.asn.0000016444.90348.50. [DOI] [PubMed] [Google Scholar]

[bib36] 36.Kilkenny C., Browne W.J., Cuthill I.C., Emerson M., Altman D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010;8 doi: 10.1371/journal.pbio.1000412. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib37] 37.Garrido A., Kim E., Teijeiro A., Sánchez Sánchez P., Gallo R., Nair A., Matamala Montoya M., Perna C., Vicent G.P., Muñoz J., et al. Histone acetylation of bile acid transporter genes plays a critical role in cirrhosis. J. Hepatol. 2022;76:850–861. doi: 10.1016/j.jhep.2021.12.019. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib38] 38.Chaves-Perez A., Santos-de-Frutos K., de la Rosa S., Herranz-Montoya I., Perna C., Djouder N. Transit-amplifying cells control R-spondins in the mouse crypt to modulate intestinal stem cell proliferation. J. Exp. Med. 2022;219 doi: 10.1084/jem.20212405. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib39] 39.Gomes A.L., Teijeiro A., Burén S., Tummala K.S., Yilmaz M., Waisman A., Theurillat J.P., Perna C., Djouder N. Metabolic Inflammation-Associated IL-17A Causes Non-alcoholic Steatohepatitis and Hepatocellular Carcinoma. Cancer Cell. 2016;30:161–175. doi: 10.1016/j.ccell.2016.05.020. [DOI] [PubMed] [Google Scholar]

[bib40] 40.Bosso G., Cipressa F., Moroni M.L., Pennisi R., Albanesi J., Brandi V., Cugusi S., Renda F., Ciapponi L., Polticelli F., et al. NBS1 interacts with HP1 to ensure genome integrity. Cell Death Dis. 2019;10:951. doi: 10.1038/s41419-019-2185-x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib41] 41.Berman J.W., Basch R.S. Amplification of the biotin-avidin immunmofluorescence technique. J. Immunol. Methods. 1980;36:335–338. doi: 10.1016/0022-1759(80)90138-6. [DOI] [PubMed] [Google Scholar]

[bib42] 42.Bosso G., Cipressa F., Tullo L., Cenci G. Co-amplification of CBX3 with EGFR or RAC1 in human cancers corroborated by a conserved genetic interaction among the genes. Cell Death Discov. 2023;9:317. doi: 10.1038/s41420-023-01598-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib43] 43.Kumar K., Oli A., Hallikeri K., Shilpasree A.S., Goni M. An optimized protocol for total RNA isolation from archived formalin-fixed paraffin-embedded tissues to identify the long non-coding RNA in oral squamous cell carcinomas. MethodsX. 2022;9 doi: 10.1016/j.mex.2021.101602. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib44] 44.Piñeiro-Hermida S., Bosso G., Sánchez-Vázquez R., Martínez P., Blasco M.A. Telomerase deficiency and dysfunctional telomeres in the lung tumor microenvironment impair tumor progression in NSCLC mouse models and patient-derived xenografts. Cell Death Differ. 2023;30:1585–1600. doi: 10.1038/s41418-023-01149-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib45] 45.Zhan C., Zhou J., Feng Q., Zhang J.E., Lin S., Bao J., Wu P., Luo M. Acute and long-term suppression of feeding behavior by POMC neurons in the brainstem and hypothalamus, respectively. J. Neurosci. 2013;33:3624–3632. doi: 10.1523/jneurosci.2742-12.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

IL-17A produced by POMC neurons regulates diet-induced obesity

Rosa Gallo

Ana Teijeiro

Mariana Angulo-Aguado

Nabil Djouder

Summary

Graphical abstract

Highlights

Introduction

Results

Deletion of IL-17RA in POMC neurons does not affect mouse body weight

Figure 1.

Deletion of SOCS3 in POMC neurons does not affect mouse body weight

Figure 2.

POMC neurons express IL-17A upon HFD

Figure 3.

Discussion

Limitations of the study

STAR★Methods

Key resources table

Resource availability

Lead contact

Materials availability

Data and code availability

Experimental model and study participant details

Animal models

Method details

Mouse conditions and housing

Mouse diets

Mouse genotyping

Body weight

Tissue preparation

Immunofluorescence

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Bioinformatics analysis

Quantification and statistical analysis

Image analysis and quantifications

Statistical analysis

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases