TLR2 deficient mice develop mature-onset obesity

Our initial serendipitous observation, that aged TLR2-deficient (TLR2D) mice had an increased body weight relative to age-matched wild type (WT) mice, led us to consider TLR2 as a possible regulator of metabolic functions. We therefore first monitored the weight gain and metabolic state of WT vs. TLR2D mice of different ages. The body weight of TLR2D mice began to deviate from that of WT at approximately 4 months of age. By 12 months of age, the TLR2D mice weighed 30% more, on average, than their WT counterparts ( Fig. 1A,B ) and exhibited a marked increase in fat mass, as measured by both body composition MRI ( Fig. 1C ) and T 2 -weighted MRI images ( Fig. 1D ).

Figure 1 TLR2 deficient mice develop mature-onset obesity. (A) Photograph of 12 month old WT and TLR2D mice. (B–G) Comparison between middle-aged TLR2D and WT mice for weight (B; ANOVA, F = 28.568, P = 0.001), total fat mass (C; Student's t-test; *P < 0.05), representative T 2 -weighted axial images (D), blood cholesterol and triglyceride levels (E; for each; Student's t-test *P < 0.05), glucose (F; Repeated ANOVA, F = 2.779, P = 0.0286) and insulin tolerance tests (G; Repeated ANOVA, F between groups = 4.068, P = 0.0061). (H–J) Respiratory exchange ratio (H; RER; Repeated ANOVA, F F between groups = 2.923, P = 0.0032), daily food intake in [g] or corrected to body weight [g food /g body weight 0.75] (I; ANOVA, F = 20.697, P = 0.001; J; Student's t-test *P = 0.012) and metabolic parameters (K; Student's t-test; *P < 0.05) of TLR2D and WT mice as measured in metabolic cages. n = 10 for all tested groups. Asterisks denote significant differences relative to control. Full size image

The increased weight gain observed in TLR2D mice prompted us to test whether this phenomenon was accompanied by metabolic alterations. We found elevated plasma levels of both cholesterol and triglycerides in TLR2D mice compared to the WT animals ( Fig. 1E ). Furthermore, the TLR2D mice showed decreased ability to handle a glucose load (assessed by glucose-tolerance test; GTT; Fig. 1F ) and reduced insulin sensitivity (determined by insulin-tolerance test; ITT; Fig. 1G ) as compared to their WT controls. Indirect calorimetry revealed that the respiratory exchange ratio (RER) of 12-month old TLR2D mice was higher throughout the day cycle, both in the dark (active) phase and the light phase, suggesting that TLR2D mice oxidized more carbohydrates and less fat as an energy source ( Fig. 1H ). Furthermore, while food consumption was similar in the two strains at 3 months of age, at 4 and 12 months, TLR2D mice consumed 17% and 23% more food than WT controls, respectively ( Fig. 1I,J ). Nevertheless, water intake, heat production, dark cycle locomotion, VO 2 (a measurement of oxygen consumption) and VCO 2 (a measurement of carbon dioxide production) did not differ between strains ( Fig. 1K ). Together, these findings suggest that deficiency in TLR2 led to metabolic alterations that contributed to the development of mature onset obesity.

TLR2 deficiency in adulthood exacerbates obesity induced by high fat diet

Once we established that elderly TLR2D mice are more prone to develop obesity than their WT counterparts, we next tested the ability of adult TLR2D mice to cope with homeostatic challenge, such as high-fat diet (HFD). To this end, we maintained 3-month old TLR2D and WT mice, slightly before the emergence of mature onset obesity (3–4 months of age; Fig. 1B ), on a HFD (45% kcal from fat) for 50 days. During their time on HFD, the response of TLR2D mice deviated from that of the WT mice, showing a greater rate of weight gain ( Fig. 2A ). Though TLR2D mice maintained on regular diet also exhibited increased body weight compared to their age matched WT controls, those maintained on HFD displayed even more pronounced weight gain compared to their HFD-maintained WT counterparts, suggesting that TLR2-deficiency resulted in a higher susceptibility to diet-induced obesity (DIO) ( Fig. 2B ). Furthermore, on HFD, TLR2D mice had higher food intake (expressed in grams, corrected for body weight, or not) compared to WT ( Fig. 2C ), their total fat mass was greater ( Fig. 2D ) and their percent lean mass of total body weight was lower, as measured by whole body composition MRI ( Fig. 2E ). Notably, comparison of age matched controls maintained on regular diet versus the HFD fed mice revealed that TLR2D mice maintained with HFD increased their total fat by 4.1 ± 0.05 fold, whereas the WT animals fed with HFD increased body fat by 3.1 ± 0.3 fold relative to those kept on regular chow ( Fig. 2D ). Moreover, more pronounce differences between TLR2D and their WT control in the % lean mass were noticed when the mice were kept on HFD ( Fig. 2E ). Taken together, these results suggest that in addition to its role in restricting age-induced obesity, TLR2 expression provides physiological protection at early adulthood against diet-induced obesity.

Figure 2 TLR2 protects against high-fat diet-induced obesity at adulthood. (A) TLR2D and WT mice (3 month old) maintained on HFD were monitored for weight change during 8 weeks on HFD (Repeated ANOVA; left F = 10.9, P = < 0.0001; right F = 6.7, P = <0.0001). (B) Weight and weight gain in [g] by TLR2D and WT mice, maintained either on regular chow or on HFD (ANOVA; left P strain = 0.001, P chow < 0.0001; right P strain < 0.0001, P chow < 0.0001). (C) Daily food intake of TLR2D and WT mice maintained on HFD (; Student's t-test; *P < 0.05). (D,E) Total fat mass (D; ANOVA, P < 0.0001) and percent lean mass (E; ANOVA, P < 0.0001) of TLR2D and WT mice, maintained either on regular chow or on HFD as measured by body composition MRI [gm]. Asterisks denote significant differences relative to control. Full size image

The TLR2- mediated anorexic function extends beyond the hematopoietic system

The TLRs are a family of receptors mainly known for their immunological roles. Therefore, their contribution to the development of obesity was mainly linked to their ability to induce inflammation in peripheral metabolic organs5,6,7,8,9,10,11,12,13,27. As novel non-immune functions have recently been attributed to TLRs14,15,17,18,19,22, we aimed here to determine whether TLR2-mediated regulation of age-related obesity extends beyond their role associated with hematopoietic cells. To test this, we created bone marrow chimeras, in which following total body irradiation, the bone marrow (BM) of the TLR2D mice was replaced with wild type BM cells that have normal TLR2 expression (termed [wt>TLR2D]). As a control for this manipulation, we similarly manipulated age matched WT mice and replaced their BM with the same WT BM cells used for the TLR2 chimera preparation (these control chimeras are termed [wt>wt]). The identical wild type (TLR2-positive) BM cells used for the reconstitution of both chimera types allowed the attribution of any difference(s), if observed, to TLR2 deficiency by the non-hematopoietic, radio-resistant cells. We first verified that due to the manipulation, the [wt>TLR2D] chimeric mice gained TLR2 expression in their hematopoietic cells within the periphery, as demonstrated by the expression of TLR2 on myeloid cells (CD11b+) present in hematopoietic (blood, spleen, lymph-nodes, bone marrow) and metabolic organs (fat, liver and pancreas) (representative data from fat tissues is shown in Fig. 3A ). In all tested organs, TLR2 expression levels on myeloid cells were comparable between TLR2D recipient mice that received BM from WT donors ([wt>TLR2D] chimeras) and WT mice that received WT BM ([wt>wt] chimeras) ( Fig. 3A ). Although displaying similar peripheral hematopoietic expression of TLR2, with maturity, the TLR2D mice that received the WT BM ([wt>TLR2D] chimeric) showed increased weight gain, similarly to the non-chimeric TLR2D mice, relative to their [wt>wt] control chimeras ( Fig. 3B–D ). Moreover, similar to the results observed in the whole-body TLR2D mice ( Fig. 1F ), [wt>TLR2D] chimeras demonstrated a robust decrease in glucose tolerance compared with the [wt>wt] chimeric controls ( Fig. 3E ). Our results thus demonstrated that the TLR2-mediated protective role in preventing age-related obesity extends beyond hematopoietic contribution and is mediated, at least in part, via TLR2 expression by non-hematopoietic, radio-resistant cells.

Figure 3 TLR2 mediated protection against age-related obesity extends beyond its expression by cells of the hematopoietic lineage. (A–D) [wt>wt] and [wt>TLR2D] chimeras were analyzed for TLR2 expression by flow cytometry (A; Representative analysis of fat tissue isolated from TLR2D as well as from [wt>wt] and [wt>TLR2D] chimeras; histograms were pre-gated on myeloid cells (CD11b+); percentages of TLR2+ out of pre-gated cells are indicated), for weight gain (B,C,D) and for glucose sensitivity (E) (B-Repeated ANOVA, P between groups < 0.0001; C- Student's t-test, ***P < 0.0001; E- Repeated ANOVA, P between groups = 0.0052). n = 11−14 mice per group. Full size image

TLR2 is an intrinsic metabolic regulator in hypothalamic neurons

Food intake, energy balance and body weight are regulated in the CNS by metabolic neurons of the hypothalamus, which receive input from the periphery regarding the energetic state of the body and relay this information to higher brain areas28. The hypothalamic arcuate nucleus (ARC) has a key role in regulating feeding behavior and energy expenditure. The ARC contains two major neuronal populations with opposing effects on energy balance. One population co-expresses the orexigenic peptides neuropeptide Y (NPY) and agouti-related protein (AGRP) and the other population expresses pro-opiomelanocortin (POMC) which is cleaved to produce the anorectic peptide α-melanocyte stimulating hormone (α-MSH) and co-express cocaine-and-amphetamine-regulating transcript (CART).

The observed involvement of TLR2 in the regulation of food intake ( Fig. 1I ), together with the finding of a non-hematopoietic associated role of TLR2 in the prevention of age-induced obesity ( Fig. 3 ), encouraged us to test whether such protection is orchestrated by TLR2 activity in the metabolic region of the CNS. To address this question, we first analyzed the ARC, of middle-aged TLR2D mice and their age matched controls for the levels of α-MSH, one of the most potent anorectic peptides implicated in the central control of food intake28. Coronal brain slices, which included the ARC, were chosen according to the Allen Brain Atlas, as detailed in the Methods. All slices included the hippocampal dentate gyrus and the third ventricle at the level of hypothalamus, as a mean to verify regional location; the adjacent Median Eminence (ME) is indicated. In accordance with their increased appetite ( Fig. 1I ), reduced levels of hypothalamic α-MSH immunoreactive fibers within the ARC were found in TLR2D mice ( Fig. 4A,B ). These results could be either an outcome of the obesity developed in the TLR2D mice, or a direct effect of TLR2 deficiency within the hypothalamus, indicating a role of TLR2 in the central regulation of food intake. Considering the latter option, we tested TLR2 expression in the brain of middle-aged wild type mice. TLR2 was found to be restricted to the hypothalamus, localized exclusively in the ARC ( Fig. 5A ). Within this metabolic nucleus, TLR2 expression was not detected in glial cells, including astrocytes (GFAP+) and microglia (evaluated in Cx 3 cr1GFP/+ mouse, in which microglia express GFP) ( Fig. 5B,C ). Consistent with the observed TLR2-dependent alteration in α-MSH ( Fig. 4 ), a cleaved product of POMC, TLR2 expression was co-localized with POMC+ metabolic neurons (Identified in either reporter mice carrying RFP under the control of Pomc promoter, or by co-immunostaining; Fig. 5D,E ), known to respond to leptin by signaling satiety to higher brain areas28. About 54% out of the POMC positive cells were TLR2+. We could not detect co-localization of AGRP with TLR2. TLR2 co-localization with POMC neurons further confirmed localization to the ARC.

Figure 4 TLR2 deficient mice display reduced levels of hypothalamic α-MSH. (A,B) α-MSH expression, assessed by immunostaining, in the ARC of middle-aged TLR2D and WT mice (n = 3 mice per group; 3 depths). 3V- third ventricle; ME- median eminence. Student's t-test *P = 0.05. Scale bars indicate 20 μm. Full size image

Figure 5 TLR2 is an intrinsic metabolic regulator acting in hypothalamic neurons. (A) Representative pictures for TLR2 staining in various coronal brain sections of middle-aged mice showing localization of this receptor to the arcuate nucleus (ARC). (B,C) Co-staining of TLR2 with astrocytes (GFAP+; B) or microglia (Cx 3 cr1GFP/+; C) in the ARC of WT mice. (D, E) Co-localization of TLR2 to POMC neurons; staining for TLR2 in brain sections of mature PomcRFP mice (D) or co-staining with POMC (E) in WT mice. (F) Staining and quantification of TLR2 and TLR4 in the hypothalamus of adult and middle-aged WT mice (n = 3 − 4 mice per group; 3 depths). (G) Staining and quantification of TLR2 in ARC of mice fed with HFD or with regular chow (n = 3 − 4 mice per group; 3 depths; Student's t-test ***P < 0.001). 3V- third ventricle; Arc- arcuate nucleus; ME- median eminence. Scale bars indicate 20 μm. Full size image

The levels of arcuate TLR2 expression were found to be correlated with the age of the mice; TLR2 was induced as the mice matured ( Fig. 5F ). This age-related expression might explain the age-dependent development of obesity in TLR2-deficient animals and further support the notion that TLR2 is expressed during aging as an intrinsic central mechanism aimed at fighting age-related obesity. Of note, the expression of hypothalamic TLR4, the main TLR family member implicated in pathological inflammatory processes that underlie the development of obesity6,8,10,11, remained unchanged with age ( Fig. 5F ). Similarly to the age-related expression of TLR2, we further observed elevated expression of TLR2 in the ARC of mice housed on HFD relative to age-matched mice which have been fed with regular chow ( Fig. 5G ).

To further establish the role of TLR2 in regulating metabolic signals within hypothalamic neurons, we utilized the murine N42 hypothalamic neuronal cell line, previously shown to be suitable for in-vitro studies of hypothalamic metabolic pathways29. We found that N42 cells express TLR2 ( Fig. 6A ) and respond to their well known pharmacological activators (the lipopeptide, Pam3CysSK 4 (P3C) and peptidoglycan (PG)), as manifested by increased c-Fos expression, an established indicator of recent neuronal activity ( Fig. 6B,C ). To evaluate whether such TLR2 activation by the hypothalamic neurons would lead to metabolic consequences, we tested the effect of the TLR2 ligands on the expression of resistin (rstn) and fasting-induced adipose factor (fiaf; also known as angiopoietin-like 4), adipokines that have been previously shown to be regulated in this cell line and expressed in the brain, where they modulate hypothalamic signaling pathways and control energy homeostasis30. Administration of the TLR2 ligands, resulted in reduced mRNA transcript levels of resistin ( Fig. 6D ), a phenomenon previously linked to an increase in POMC and α-MSH and known to induce an anorectic response mediated at the hypothalamus30. The TLR2-mediated reduction in resistin was correlated with increased mRNA transcript levels of fiaf ( Fig. 6D ), in line with the reported inhibitory effect of resistin on fiaf30, which has been implicated in the control of body composition and glucose metabolism31,32,33. Addition of TLR2-neutralizing antibody diminished the ligand-induced effects on both resistin and fiaf expression, further supporting the TLR2-mediated metabolic effect ( Fig. 6E,F ). These data further support the possibility that neuronal-associated TLR2 plays a role as a metabolic regulator within the hypothalamus.