An extensive phenotypic characterization of the hTNFα transgenic mice
- Michael D Hayward†1Email author,
- Beverly K Jones†1,
- Arman Saparov1,
- Heather S Hain1,
- Anne-Cecile Trillat1,
- Michelle M Bunzel1,
- Aaron Corona1,
- Bifang Li-Wang1,
- Bryan Strenkowski1,
- Caroline Giordano1,
- Hai Shen1,
- Emily Arcamone1,
- Jeffrey Weidlick1,
- Maria Vilensky1,
- Marina Tugusheva1,
- Roland H Felkner1,
- William Campbell1,
- Yu Rao1,
- David S Grass1 and
- Olesia Buiakova1
© Hayward et al; licensee BioMed Central Ltd. 2007
Received: 02 February 2007
Accepted: 10 December 2007
Published: 10 December 2007
Tumor necrosis factor alpha (TNFα) is implicated in a wide variety of pathological and physiological processes, including chronic inflammatory conditions, coronary artery disease, diabetes, obesity, and cachexia. Transgenic mice expressing human TNFα (hTNFα) have previously been described as a model for progressive rheumatoid arthritis. In this report, we describe extensive characterization of an hTNFα transgenic mouse line.
In addition to arthritis, these hTNFα transgenic mice demonstrated major alterations in body composition, metabolic rate, leptin levels, response to a high-fat diet, bone mineral density and content, impaired fertility and male sexual function. Many phenotypes displayed an earlier onset and a higher degree of severity in males, pointing towards a significant degree of sexual dimorphism in response to deregulated expression of TNFα.
These results highlight the potential usefulness of this transgenic model as a resource for studying the progressive effects of constitutively expressed low levels of circulating TNFα, a condition mimicking that observed in a number of human pathological conditions.
Increased production of the proinflammatory cytokine TNFα has been implicated in a number of human diseases involving inflammation such as autoimmune disorders, vascular disease and a number of cancers . Most notably, aberrant expression of the TNFα gene is associated with rheumatoid arthritis (RA) in both humans and animal models [2–5]. In addition to the inflammatory component of RA, rheumatoid cachexia is characterized by reduced body weight, increased metabolic rate and restricted motor activity and also correlates with increased circulating TNFα levels . Interestingly, TNFα is not only expressed in macrophages, lymphocytes, neutrophils, endothelial cells, keratinocytes and fibroblasts  but is also expressed in adipose tissue and is elevated in a number of experimental obesity models [8–11] and obese humans [12, 13]. The pleiotropic actions of this cytokine have stimulated interest in animal models involving genetic manipulation of TNFα itself and components of its cellular signaling pathway.
Inactivation of the endogenous TNFα gene and its two cognate receptors have demonstrated its role in inflammation as well as obesity [14, 15]. Transgenic mice expressing the TNFα gene under the control of various regulatory sequences have been used as models for RA as well as in vivo models to study wasting, ischemia, and lymphoid abnormalities [5, 16–18]. Transgenic mice expressing a non-cleavable membrane bound TNFα have reduced adipose mass  and cardiac-specific overexpression of TNFα in mice results in cardiomyopathy . The pleiotropic effects observed in genetic modifications of this cytokine in vivo therefore confirm the observations in humans and support the use of mutant mice as models for human diseases where a TNFα role has been described. A comprehensive phenotypic characterization of deregulated expression of TNFα would thus help inform investigators in their decision to use this specific animal model.
Our objective in this series of experiments was to determine the effects of deregulated hTNFα expression at low circulating levels on a broad range of murine physiological and behavioral measures. To achieve this goal, we comprehensively screened the hTNFα mice using sequential bioassays encompassing multiple physiological systems on the same groups of mice. A comprehensive phenotypic characterization of genetically-modified animals is limited by the time and expense in the selective breeding required to generate subjects for a large number of assays. To address this issue we have designed a serial phenotyping procedure used here that requires significantly fewer animals. By using such an approach, we were able to conduct a large number of tests in a relatively short period of time and used significantly fewer subjects than would be used in a study limited to experimentally naïve subjects. We have confidence that the results are reliable because we validated the order of these tests in wild-type animals by comparing data obtained by the serial protocol to data obtained in experimentally naïve subjects of comparable ages in pilot studies not included in this study. Because of the serial nature of our experimental approach, the results from the transgenic mice are relevant only to the age at which the specific assay was conducted and results could differ at different ages (e.g., due to the progressive-age related- deterioration of their health).
We found that in addition to serving as an improved model for progressive RA, these hTNFα transgenic mice exhibited numerous phenotypes including decreased activity, alterations in soft tissue and bone composition, impaired physical and metabolic responses to a high fat diet (HFD), increased food intake, markedly decreased leptin levels, significantly enhanced energy expenditure, impaired fertility and erectile dysfunction. The results from this study demonstrate that this transgenic line is a valuable model for the study of TNFα role in disease progression in multiple therapeutic areas including metabolism, obesity, bone homeostasis and male sexual health.
hTNFα transgenic (TG) and age- and sex-matched C57BL6/N wildtype (WT) mice were obtained from Taconic Farms (Hudson, NY, ). The hTNFα TG mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene, including the entire coding region and promoter, fused to the human β-globin 3' untranslated region (UTR) that replaces the endogenous 3'UTR of the human TNFα gene . This TG line was produced by pronuclear microinjection of B6SJL(F2) hybrid zygotes. The animals have been backcrossed for over 10 generations onto the C57BL6/N genetic background.
Mice were singly housed in a temperature- and humidity-controlled barrier facility. The mice were maintained on a 12 hr reverse dark/light cycle (lights go off at 11 AM). Animals had free access to food and water unless indicated otherwise in protocols for specific bioassays. All experiments were approved by the Xenogen Biosciences Institutional Animal Care and Use Committee.
List of bioassays used for characterization in the initial screen and result summary
Cardiovascular, renal and bladder function
Bladder function (F)
Blood pressure/heart rate (M)
Urine chemistry panel (F)
CNS and Behavior
Irwin test (M,F)
Automated blood chemistry panel (M)
Body weight measurements (M,F)
Immunology and Inflammation
FACS analysis (M)
LPS challenge (M)
Monocyte infiltration (F)
Pulmonary inflammation (F)
Wound healing (F)
Metabolism and Body Composition
Excised bone DEXA (F)
Food intake (M,F)
Food intake on HFD (M)
Metabolic response to HFD (M)
Physical response to HFD (M)
Selected muscle weight (F)
Formalin pain assay (M)
Hot-plate assay (F)
Histopathology of reproductive system (M)
Induced erection test (M)
Male contact sexual behavior (M)
Male fertility (M)
Age of mice and order of experiments
Body Weight (RC)
Body Weight (RC)
Body Weight (RC)
Male Sexual Behavior
Body Weight (RC)
Male Sexual Behavior
Body Weight (RC)
Male Sexual Behavior Insulin
Body Weight (RC)
Body Weight (RC)
DEXA(pre-HFD & Low Ca)
Body Weight (RC)
Body Weight (RC)
Body Weight (HFD)
Body Weight (RC)
Body Weight (HFD)
Body Weight (RC)
Body Weight (HFD)
Body Weight (RC)
Body Weight (HFD)
MicroCT OGTT (post-HFD)
Body Weight (RC)
Insulin DEXA(post-Low Ca)
Body Weight (HFD)
DEXA (post HFD)
Body Weight (RC)
Body Weight (HFD)
Insulin Tolerance Test
Lipopolysaccharide (LPS)-induced Inflammation
The mice were injected intraperitoneally (i.p.) with either 1 or 10 μg lipopolysaccharide (LPS, Sigma-Aldrich, Inc., St. Louis, MO) made up in 2 or 20 mg D-galactosamine (Sigma-Aldrich, Inc), respectively. Mice from the negative control group were injected with a phosphate buffered saline (PBS) vehicle alone. All mice were anesthetized with isofluorane 1.5 hours after injection, and blood was obtained from a retro-orbital vein. Serum was prepared by spinning clotted blood for 20 min at 4°C at 1200 × g. Human and mouse TNFα levels in the serum of the mice were detected by ELISA according to the manufacturer's protocol with a lower limit of detection in the mice assay of 23.4 pg/ml and 0.5 pg/ml in the human form (R&D Systems, Minneapolis, MN).
Irwin Observational Battery
Phenotypes in the Irwin observational panel
Body weight (g)
31.5 ± 18.8
20.3 ± 0.9
25.5 ± 1.7*
22.2 ± 0.9
Body length (mm)
92.8 ± 0.9
86.6 ± 0.5
89.4 ± 1.0*
87.8 ± 1.1
Grip strength (score 0–4)
2.4 ± 0.2
1.0 ± 0
0.8 ± 0.2**
1.6 ± 0.2
Wire maneuver (score 0–4)
1.4 ± 0.2
2.8 ± 0.5
4.0 ± 0***
2.0 ± 0.6
Tail elevation (score 0–2)
2.0 ± 0
1.4 ± 0.2
1.2 ± 0.2*
1.2 ± 0.2
Appropriate statistical tests were used to compare the TG mice to their WT counterparts (Fisher's exact t, Mann-Whitney U, unpaired t-tests).
Food Intake on Regular Chow
The mice were singly housed in clean cages with one sheet of Iso-pad bedding (Harlan-Teklad, Indianapolis, IN) and a weigh boat containing dustless precision pellets (Bio-Serv, Frenchtown, NJ). They were habituated to this environment for 72 hours. At the end of the habituation period, the mice were placed in new cages containing Iso-pad bedding and 20 food pellets. The weights of both the food and the mice were recorded. The experimental phase lasted 72 hours at which point the remaining food was recovered and weighed. Food intake was expressed as mg food consumed/g body weight/24 hours. An unpaired t-test was used to analyze differences between genotypes.
Blood was collected via retro-orbital bleeds from fasted or freely fed mice. Serum was prepared by spinning clotted blood for 20 min at 4°C at 1200 × g and frozen at -20°C. The leptin ELISA was performed according to the manufacture's protocol (R&D Systems). An unpaired t-test was used to analyze differences due to genotype.
Responses to HFD
The HFD challenge used the Western high fat/high carbohydrate diet (42.7 kcal % carbohydrate, 40.3 kcal % fat, 17 kcal % protein, D12079B, Research Diets, Inc., New Brunswick, NJ). The body weights of the mice were measured weekly and the following metabolic, anatomical and behavioral assays were conducted prior to and at the end of the HFD challenge.
Whole Body Dual Energy X-ray Analysis (DEXA)
An unpaired t-test was used to analyze differences between genotypes for each measure. At the end of the HFD period final body weights were taken and a post-HFD DEXA scan was also obtained.
Food Intake on HFD
During week 4 of the HFD food intake measurements were performed while mice were singly housed in clean cages on standard corn cob bedding and weighed at the start of the experimental period, which lasted 72 hours. At the beginning of the assay, mice were given approximately 15 grams of pre-weighed high-fat food pellets. At the end of the experiment the remaining food was recovered and weighed. Food intake was expressed as the mg food consumed/g body weight/24 hours. An unpaired t-test was used to analyze differences due to genotype.
During week 4 of the HFD the sizes of regional fat depots were determined by microCT analysis. Images of anesthetized mice were obtained using a commercially available microCT system (MicroCAT®, ImTek Inc. Oak Ridge, TN) with a high-resolution CCD/phosphor screen detector. Images were acquired with the X-ray biased at 40 kVp and 400 μA using a 0.5 mm aluminum filter. Each scan consisted of 196 individual projections with an exposure time of 320 ms/projection. Image reconstruction, whereby the 196 projections acquired in the scan were manipulated to produce two-dimensional cross sectional images of the mouse, was performed using the MicroCAT® Reconstruction, Visualization, and Analysis Software (ImTek Inc., Oak Ridge, TN). Slices were selected spanning the region between vertebral landmarks S4-S3 and L1–T13 using a 90° scout radiograph projection. Two-dimensional slices were reconstructed using a Shepp-Logan filtered back projection algorithm on a 256 × 256-pixel grid where the pixel size is 260 × 260 μm and the slice thickness is 1.6 mm. After reconstruction, the 2D-transaxial slice images were exported in *.bmp format for image analysis. Image analysis and volumetric fat calculations for renal/retroperitoneal, mesenteric, epididymal and inguinal depots were accomplished in 3D-Doctor (Able Software Corp., Lexington, MA). An unpaired t-test was used to analyze differences between mice of different genotypes for each parameter.
Metabolic Responses to HFD
Mice fed the HFD for five weeks and mice maintained on regular low fat chow (rodent Purina diet #5053, Fisher Feeds, Bound Brook, NJ) were fasted overnight and then bled from the retro-orbital sinuses. The samples were left to coagulate and then centrifuged at 4°C to isolate the serum. The serum was immediately frozen and stored at -80°C. The frozen serum samples collected from all of the mice before and at the end of the HFD were defrosted and analyzed in parallel for serum insulin by ELISA (UltraSensitive Mouse Insulin ELISA; ALPCO, Salem, NH) according to the manufacturer recommendations. To assess glucose tolerance, mice maintained on regular chow or the same mice after feeding on the HFD for 5 weeks were fasted for 16 – 20 hrs prior to the oral glucose tolerance test (OGTT). The animals were weighed immediately before the test and then the baseline serum glucose level was measured in a drop of blood obtained from a cut at the tip of the tail. The glucose levels were measured by a Glucometer Elite (Bayer, Leverkusen, Germany). The mice then received 1 g/kg body weight of 100 mg/ml glucose solution (Sigma, St. Louis, MO) in sterile water delivered by oral gavage. At 30, 60 and 120 min after the gavage, glucose concentration was measured as described above. The insulin tolerance test (ITT) was performed on ad libitum fed males during the 7th week of the HFD with human regular insulin at a dose of 75 U/kg injected i.p. Body weights, OGTT and ITT results were analyzed by two-way ANOVA. All other endpoints were also analyzed using an unpaired t-test to identify differences due to genotype.
Comprehensive Laboratory Animal Monitoring System
Mice fed the HFD for six weeks and mice maintained on the regular chow diet were assessed for 48 hours in the Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments, Columbus, Ohio). Measurements included oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory exchange ratio (RER, calculated), calculated heat production, total horizontal activity, ambulatory activity, non-ambulatory horizontal activity, vertical (rearing) activity and licking frequency. Results were averaged by photoperiod and analyzed by two-way ANOVA. For the heat production, oxygen consumption and carbon dioxide production endpoints, weight change was used as a covariate in the ANCOVA.
Low Calcium Dietary Challenge
Following a baseline DEXA scan of bone composition, the mice were maintained on diet # D02040301 from Research Diets (New Brunswick, NJ) for a period of four weeks. This diet contains 0.01% calcium. After four weeks on the diet a second DEXA scan was performed. The percent change from baseline was calculated for each endpoint and an unpaired t-test was used to analyze differences between mice of different genotypes for each time point.
On the first day of testing, CD1 females were introduced to the experimental males before the beginning of the dark phase of the light/dark cycle and left with males for one week. Vaginal mating plugs were checked daily at the end of the light phase. On day 8 the pairs were separated. Females were individually housed and allowed to deliver litters. The number of litters and litter size were determined for males of each genotype.
Male Contact Sexual Behavior
This assay was performed on the same male mice used in the Male Fertility assay. Prior to testing, the males were trained twice per week for two consecutive weeks. 8–10 Week old ovariectomized ICR female mice (Taconic Farms, Hudson, NY) implanted with a 2.5 mg 17-β-estradiol pellet (Innovative Research of America, Sarasota, FL) and injected with progesterone (Sigma-Aldrich, Inc) (500 μg in 0.2 ml of sesame oil 5 hours prior to the start of the experiment) were introduced to the cages of experimental males 1 hr after the onset of the dark phase for 4 hrs. After each training session females were observed for copulative plugs. Thirty minutes prior to the testing session the males were evaluated in the induced erection test as described below. Then the ovariectomized females supplemented with estrogen and progesterone were introduced to the male cages. The cages were placed in testing video cabinets (Noldus Information Technology, Wageningen, Netherlands) and sessions were digitally recorded for 45 min (Numeriscope™, Viewpoint, Champagne, France). The testing sessions were analyzed by a trained operator blind to the genotype using Observer software (Noldus Information Technology). The total number of mounts, intromissions and ejaculations were determined for every male. Latencies to the first mount and to the first intromission (from the beginning of the experiment) and latency to ejaculation from the first mount were also determined.
Induced Erection Test
The testing was performed during the dark phase of the light cycle under red light. The male mice were held tightly in supine position. The penile sheath was retracted and gentle pressure was applied to the mouse abdomen for 10–15 s. The occurrence of penile erection was recorded. Erections were scored as present (1) or absent (0). Between group differences were analyzed by Chi-square test.
Histolopathological Evaluation of the Reproductive Tissues
The reproductive tissues from two TG and two WT male mice were fixed in 4% formalin and sent without genotype identification to the Pathology Associates, a Charles River Company (Wilmington, MA) for histopathological evaluation. Testes were trimmed, embedded in glycol methacrylate (GMA), and stained with PAS/hematoxylin. The epididymides, prostates, seminal vesicles and penises were trimmed, embedded in paraffin, sectioned at approximately 5 microns, and stained with hematoxylin and eosin. Tissues were examined microscopically by a certified veterinary pathologist.
Excised Bone DEXA Analysis
The animals were euthanized and the right femurs and the L1 – L6 region of the spine were excised from the bodies and scanned on PIXImus2 as described in the manufacturer's manual. The region of interest was adjusted to fit the excised bone only. For both excised femur and L1 – L6 spine, the following measurements were made: bone mineral density, bone mineral content, bone area, bone length and width. An unpaired t-test was used to analyze differences between mice of different genotypes for each measure.
Statistical analysis was performed for each assay as indicated. Two-factor ANOVAs (with or without repeated measures) were performed using the SPSS statistical package (SPSS Inc., Chicago, IL). T-tests (paired or unpaired) were performed using Graphpad Prism (San Diego, CA) or Microsoft Excel. The data were assessed for outliers defined as two or more standard deviations from the mean and values exceeding these parameters were excluded.
Characterization of Transgene Expression
Irwin Observational Panel
The Irwin Observational Panel was performed on mice at 15 weeks of age. This test encompasses basic observations in four categories: physical characteristics, autonomic responses, behavioral responses, and neurological responses . The male TG mice differed significantly from WT mice in 5 out of 45 observations (Table 3). Assessment of physical characteristics found that body weight and body length were decreased in male hTNFα mice. Results in three tests designed to measure neurological responses related to muscle tone differed between the male TG and WT mice: hTNFα TG males exhibited reduced grip strength, were unable to grasp a wire in the wire maneuver test and displayed less elevated tail positions than the WT males (Table 3). Interestingly, the female TG mice at this age did not differ from their WT counterparts in any observation (Table 3). The sexual dimorphism seen in the Irwin test suggests that despite possessing similar levels of circulating hTNFα, the female TNFα mice had a milder phenotype than age-matched males, which is consistent with our observation that they tend to manifest arthritic symptoms later than the male TG animals (data not shown).
Energy Homeostasis on Regular Chow
Food Intake, Body Weight and Soft Tissue Composition
Soft tissue composition and body weight
22.99 ± 0.30 (n = 7)
27.34 ± 0.59 (n = 8)
31.70 ± 0.67 (n = 8)
21.96 ± 0.51 (n = 5)
24.66 ± 0.33**(n = 7)
24.87 ± 0.49*** (n = 5)
Total tissue mass
22.18 ± 0.57
32.00 ± 1.22
40.46 ± 1.48
20.92 ± 0.69
26.09 ± 0.53**
26.63 ± 0.97***
2.47 ± 0.22
6.37 ± 0.8
15.33 ± 1.05
1.97 ± 0.12
3.20 ± 0.26**
5.45 ± 0.68***
19.71 ± 0.36
25.63 ± 0.59
25.13 ± 0.47
18.95 ± 0.57
22.89 ± 0.35**
21.81 ± 0.35***
11.15 ± 0.72
19.53 ± 1.67
37.57 ± 1.37
9.39 ± 0.28
12.20 ± 0.82**
20.08 ± 1.91***
88.85 ± 0.72
80.47 ± 1.67
62.43 ± 1.37
90.61 ± 0.28
87.80 ± 0.82**
79.92 ± 1.91***
24.16 ± 1.02
32.19 ± 1.15
40.17 ± 1.46
22.01 ± 0.75
26.46 ± 0.50**
26.72 ± 0.91***
Leptin levels were measured in serum from 25 week-old fasted and 26 week old ad libitum fed males maintained on regular chow. Both fasted and fed blood leptin levels were significantly lower in TG males compared to WT animals (WT fasted: 2.663 ± 0.590 ng/ml [n = 7] vs. TG fasted: 0.019 ± 0.012 ng/ml [n = 7], P = 0.004; WT fed: 5.997 ± 0.758 ng/ml [n = 7] vs. TG fed: 1.015 ± 0.270 ng/ml [n = 6], P = 0.0004). Leptin levels usually correlate closely with the amount of white adipose tissue. However, in the hTNFα TG males leptin levels appeared disproportionately low as they were undetectable in fasted mice while in the fed state were reduced six-fold, but fat mass in the TG was reduced only two-fold. Twenty six week old TG females also showed a marked decrease in leptin levels compared to the WT controls; leptin levels in fasted WT females were 1.356 ± 0.385 ng/ml [n = 6] but were 0.144 ± 0.089 ng/ml [n = 6] in TG females (P = 0.028). These data demonstrate a striking dissociation between fat mass and leptin secretion since TG females had unchanged fat mass compared to WT but exhibited an almost ten-fold reduction in circulating leptin levels. Additionally, the food-intake measurements in mice fed regular diet did not detect any difference between genotypes despite significantly lower leptin levels. As leptin was measured nine to ten weeks after the food intake experiment we cannot rule out the possibility that the reduced serum leptin levels were a manifestation of a progressive decrease in body weight of the TG mice.
Energy Homeostasis on HFD
Food intake, weight gain and soft tissue composition
Regional Adipose Distribution
OGTT, Insulin Levels and Insulin Sensitivity
Fasting insulin levels were determined in serum samples collected pre and post HFD challenge at 20 and 27 weeks of age, respectively. There were no detectable differences in fasting insulin levels between the two groups prior to the HFD challenge but following the diet WT insulin levels significantly increased while there was no significant change in insulin levels in the TG males (Figure 5C). A direct measurement of insulin tolerance was performed after 7 weeks on the HFD when the mice were 29 weeks old. The WT mice failed to show a significant decrease in circulating glucose levels at all time points tested in response to an i.p. injection of insulin, demonstrating diet-induced insulin-resistance (Figure 5D). In contrast, the TG males were extremely sensitive to insulin with a pronounced reduction in glucose levels at all time points evaluated up to two hours following insulin treatment, resulting in both a main effect of genotype (F1,10 = 10.48, P = 0.009) and a significant interaction between genotype and time (F3,30 = 13.72, P < 0.0001) in the ANOVA analysis (Figure 5D). These results confirmed that the TG mice had higher insulin sensitivity compared to the WT controls following a HFD challenge.
Metabolic Rate and Physical Activity
Metabolic measures and activity measured in the CLAMS
WT (n = 8)
TG (n = 6)
TG Percent of WT
WT (n = 7)
TG (n = 5)
TG Percent of WT
Oxygen consumption ml/kg/hr
2989 ± 93
3823 ± 110
2770 ± 106
3454 ± 197
Carbon dioxide production ml/kg/hr
2336 ± 114
2932 ± 124
2510 ± 160
2985 ± 220
Heat production Kcal/g
0.014 ± 0.001
0.018 ± 0.001
0.014 ± 0.001
0.017 ± 0.001
Respiratory exchange ratio
0.789 ± 0.015
0.764 ± 0.016
0.902 ± 0.036
0.856 ± 0.025
Ambulatory activity beam breaks/hr
1220 ± 65
458 ± 102
1839 ± 283
448 ± 72
Vertical activity beam breaks/hr
2124 ± 91
468 ± 133
2550 ± 289
603 ± 166
Bone Density and Response to a Low Calcium Dietary Challenge
Male sexual behavior and fertility
Number of hTNFα and WT mice performing male sexual behaviors
Induced erection (1)
Induced erection (2)
Penile erection was tested in two separate experiments comparing TG and WT males in the induced erection test. In the first experiment significantly fewer TG males displayed an erection compared to WTs (Chi-square, P = 0.005). In the second study there was still a difference but it did not reach statistical significance (Chi-square, P = 0.06, Table 6).
A significantly smaller number of the TG males sired pregnancies after one week with CD1 females (Table 6). All pregnant females gave birth to live litters. There was no effect of the transgene on the litter size detected at this age (12.75 ± 0.9, n = 4, TG; 12.67 ± 0.4, n = 9, WT). The analysis of hematoxylin/eosin stained sections of the prostate, epididymis, seminal vesicles and penises, in addition to examination of PAS/hematoxylin stained sections of testes from two TG and two WT mice, did not reveal any differences due to the presence of the transgene (data not shown).
The TG mice reported here develop progressive arthritis  similar to previously described transgenic lines generated with this construct  but do so at a later age, consistent with the lower levels of circulating TNFα than in the lines reported earlier [18, 23] and more closely resembling the progression of RA in humans. We generated several lines of transgenic mice with this construct and although all of the lines had an arthritis phenotype we chose this line for a more thorough characterization because of its slower onset of the arthritis phenotype. In the generation of transgenic mice the integration site can be a factor in observed phenotypes because there is no control over the copy-number of transgenes that integrate and possible direct or indirect influences from the integration (unintended mutations of other genes or cis-genetic effects influencing expression). Thus, although the expression level in this line has utility as a model for human RA progression, a direct comparison between multiple lines with similar, low levels of expression is required to formally prove that the phenotypes observed here are due to the deregulated TNFα expression. Nonetheless, in addition to RA found in this line as well as previously published lines, the reduced body weight, increased metabolic rate and restricted motor activity correlate well with similar findings described in humans with rheumatoid cachexia . Bone loss, which has also been previously described  was documented in the line described here. Lastly, the male TG mice have some sexual behavior dysfunctions that could be attributable to moderate disease progression of RA but the low scores for induced erection tests suggest that the sexual behavior phenotypes may be due to multiple causes. Our approach of investigating the pleiotropic actions of TNFα by phenotyping the same mice in multiple in vivo assays confirms the results of many studies conducted on different lines of transgenic mice. Further, our results are consistent with an interpretation that many of the effects of deregulated TNFα expression are not directly a result of the development of RA.
Mice expressing the hTNFα transgene driven by its endogenous promoter but containing the β-globin 3' flanking region in place of hTNFα 3'sequences have previously been described as a model for progressive arthritis [5, 16]. The replacement of the endogenous 3' UTR of the hTNFα gene with β-globin 3' sequences renders the gene non-inducible in macrophages and also stabilizes the mRNA in hematopoetic and stromal cells, leading to constitutive expression of the transgene [16, 24]. Consistent with these studies, we demonstrated here that LPS treatment did not induce expression of the transgene but inducibility of the endogenous gene remained intact, as expected. Several lines have been generated using this modified construct  and common observations include progressive arthritis and a wasting phenotype. In the most extensively studied line (TG197) both phenotypes are manifest by 4 to 6 weeks of age and early mortality is common . This rapid onset of arthritis and wasting may be attributable to the relatively high circulating concentrations of hTNFα in this line [18, 23]. The hTNFα transgenic line studied here was generated using the same construct as line TG197 but expresses significantly lower levels of circulating hTNFα and has a later onset of arthritis in addition to longer life spans, which more closely models the disease progression in humans. Therefore, this mouse line may provide a more appropriate model for the pathophysiological effect of increased levels of TNFα. One caveat that should be pointed out in this model is that our transgene is likely expressed principally in stromal cells, since it was previously shown that deletion of the 3'-region of the human TNFα gene resulted in the loss of macrophage-specific TNFα expression . This pattern of expression may not be consistent with all clinical pathologies associated with increased TNFα levels.
Humans and mice both possess two receptors for TNFα, TNFR1A (p55) and TNFR1B (p75) . The generation of knockout mice for both TNFR1A and TNFR1B, have identified specific roles for these two receptors. For example, TNFR1B is required for TNF-induced T-cell toxicity and tissue necrosis  but is not required for TNFα-mediated lipolysis or inhibition of insulin-stimulated glucose transport . TNFR1A is thought to be the primary signaling receptor on most cell types involved in resistance to bacterial infections and the lethal response to endotoxins . TNFR1A also inhibits the differentiation of adipocytes, negatively affects lipid accumulation and insulin sensitivity in mature adipocytes , and regulates leptin production . Additionally, TNFR1A mediates negative effects on bone density , primarily by increasing the number of osteoclast progenitors and stimulating their differentiation and activity, but also by suppressing the differentiation of osteoblasts. A species specificity was demonstrated since human TNFα binds with high affinity only to murine TNFR1A  thus, phenotypes identified in this study should mainly reflect processes mediated by TNFR1A.
The hTNFα TG mice in this study demonstrated sexual dimorphisms in a number of physiological measures despite similar levels of circulating hTNFα. The female TG mice appeared less affected by the expression of the transgene than the males. For example, significant differences between the sexes were observed in the Irwin test, where females were not phenotypically distinct from the WT controls but age-matched males suffered reduced grip strength and wire maneuver, likely as a result of the advanced arthritis in the males (Table 3). TG males also displayed a more pronounced decrease in body weight and soft tissue composition than did the females (Figure 2; Table 4). While the basis for the sexual dimorphisms in this TG line was not investigated, previous studies have demonstrated that estrogen exerts a protective effect against chronic exposure to hTNFα . Significantly fewer TNFR1A receptors in heart muscle of female mice have also been reported although the liver and peripheral white blood cells display equivalent numbers of TNFR1A receptors . It is unknown whether the density of this receptor in females is also lower in other tissue types. By 14 weeks of age many male TG mice had already developed paw swelling and eritema consistent with moderate arthritis, which is likely responsible for the observed phenotypes in the neurological tests in Irwin that were specific to the males (see  under "phenotype" for supplemental data). There were no visible signs of arthritis in age-matched females.
The dramatic decrease in BMD and BMC in the TG mice of both sexes (see Results), and the accelerated bone loss observed on a low calcium diet in the TG females (Figure 6) are consistent with the established role of TNFα in bone resorption. A significant loss of BMD is also characteristic of advanced RA [29, 31–33].
Numerous studies have investigated the ability of TNFα to modulate leptin expression and secretion. Acute administration of LPS or relatively high levels of TNFα increase leptin in both humans and mice [34–36]. However, low levels of leptin have been measured in patients with both chronic inflammatory conditions and cachexia [37–40], raising the possibility that repression of leptin reflects biological compensation in response to these conditions. In addition to leptin's well established roles in inhibiting food intake and increasing energy expenditure there is also a growing body of evidence indicating that leptin can regulate bone density, potentially through a central hypothalamic response [41–43]. Thus, the results from our comprehensive screen could be interpreted as a down-regulation of leptin in an effort to reduce metabolic rate, bone loss, and/or depletion of adipose stores. Alternatively, suppression of circulating leptin may result from chronic stimulation of receptor signaling pathways shared with TNFα receptors.
Contradictory results describing effects of TNFα on glucose tolerance and insulin sensitivity have been previously discussed [44, 45]. In the TG mice described here low circulating levels of TNFα did not significantly affect glucose disposal in mice fed regular chow. However, the TG mice did not develop impairments in insulin sensitivity and glucose tolerance like the WT mice did when fed the HFD for 7 weeks. These findings are likely the result of the blunted weight response of the TG mice to the HFD as well as their maintenance of normal insulin levels.
The reduced body weights, altered soft tissue composition, and increased metabolic rate in the male TG mice presented here are hallmarks of the cachectic state in humans. Cachexia caused by arthritis, cancer and AIDS have all been correlated with elevated TNFα levels . Common observations include catabolic effects on body mass with lean tissue, particularly skeletal muscle, more affected than adipose stores [46–48]. An increased resting metabolic rate is a frequent finding, and anorexia is found in many but not all cases. In mice, a lethal wasting phenotype has been reported in animals expressing hTNFα under the control of T-cell specific regulatory sequences  and decreased body weight was observed in previously reported lines harboring the transgenic construct used in this study . In the phenotypic analysis of the hTNFα TG mice presented here, the metabolic rate was significantly elevated regardless of diet and both adipose tissue and lean mass were significantly decreased. However, our results indicate that in this model, fat mass is affected more than lean mass. In addition, the TG mice did not display anorexic behavior since they consumed comparable amounts of regular chow and were even hyperphagic on the high-fat diet. However, the profoundly decreased physical activity of the TG mice is consistent with "sickness behavior" [49, 50]. Despite the obvious alterations in metabolic rate, body composition, and body weight, these TG mice do not demonstrate excessively early mortality. Male TG mice were maintained for over a year without obvious differences in survival compared to WT mice (unpublished observation). These data suggest that the cachexia seen in RA may not be directly caused by TNFα but the cytokine may be a contributing factor and is responsible for many of the other metabolic and physiological symptoms observed in rheumatoid cachexia as well as in our mouse model.
Although some of the phenotypes in male sexual behavior in the hTNFα TG mice observed in this study were likely due to arthritic-like lesions of the limbs and the compromised health status of the animals we also showed that the erectile function of the TG mice was decreased, which is likely an additional factor contributing to the impairment in male sexual behaviors. It has been proposed that iNOS-mediated chronic NO overproduction in the penile tissue could be a factor in declining erectile function associated with aging and in Peyronie's disease in humans and rat models [51, 52]. A number of studies suggest that TNFα activates iNOS mediated NO production in endothelial and smooth muscle cells of blood vessels [53, 54]. Sustained up-regulation of iNOS may result in chronic overproduction of NO in the penile tissue of the hTNFα TG mice and be eventually responsible for the impaired erectile function of the mice observed in this study. The reduced sexual activity of the TG mice likely contributed to the decreased number of pregnancies sired by the TG males compared to the WT controls observed in this study. In addition, chronic exposure to the circulating hTNFα might also affect the germ cell development and sperm maturation in the TG males, as multiple effects of TNFα on the biology of the testicular Sertoli cells were reported in the literature [55–58].
Our results have confirmed many of the previously demonstrated traits associated with constitutive expression of TNFα. In addition, this study revealed several novel physiological effects of TNFα such as the effects of TNFα on induced erection and a sexual dimorphism of many of the observed phenotypes. This study demonstrates a number of divergent phenotypes in the same TG line and even the same cohort of subjects, illustrating the pleiotropic role of TNFα and the cross-physiological effects of cytokines such as TNFα, which regulate or modulate immunological, metabolic and nervous system processes.
In summary, our phenotypic analysis of the hTNFα TG line demonstrates the utility of this mouse line as a model for multiple human diseases in which chronically elevated TNFα levels are present including its established use in studies of RA. Chronic low levels of circulating hTNFα resulted in defects in bone homeostasis, a hypermetabolic state characterized by low body weight and hyperphagia but coincident with low motor activity, depletion of adipose tissue and lean mass, lack of a weight increase with a HFD challenge, inappropriately low leptin levels and impaired male sexual health. In addition, these studies also revealed that a significant level of sexual dimorphism exists with respect to TNFα 's role in the progression and severity of several biologically relevant phenotypes.
We thank Dr. Sylvie Ramboz for her comments and suggestions.
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