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"Time sweet time": circadian characterization of galectin-1 null mice
Journal of Circadian Rhythms volume 8, Article number: 4 (2010)
Recent evidence suggests a two-way interaction between the immune and circadian systems. Circadian control of immune factors, as well as the effect of immunological variables on circadian rhythms, might be key elements in both physiological and pathological responses to the environment. Among these relevant factors, galectin-1 is a member of a family of evolutionarily-conserved glycan-binding proteins with both extracellular and intracellular effects, playing important roles in immune cell processes and inflammatory responses. Many of these actions have been studied through the use of mice with a null mutation in the galectin-1 (Lgals1) gene. To further analyze the role of endogenous galectin-1 in vivo, we aimed to characterize the circadian behavior of galectin-1 null (Lgals1 -/-) mice.
We analyzed wheel-running activity in light-dark conditions, constant darkness, phase responses to light pulses (LP) at circadian time 15, and reentrainment to 6 hour shifts in light-dark schedule in wild-type (WT) and Lgals1 -/- mice.
We found significant differences in free-running period, which was longer in mutant than in WT mice (24.02 vs 23.57 h, p < 0.005), phase delays in response to LP (2.92 vs 1.90 circadian h, p < 0.05), and also in alpha (14.88 vs. 12.35 circadian h, p < 0.05).
Given the effect of a null mutation on circadian period and entrainment, we indicate that galectin-1 could be involved in the regulation of murine circadian rhythmicity. This is the first study implicating galectin-1 in the mammalian circadian system.
Circadian systems represent an endogenous mechanism adapted to cycling environmental conditions. In mammals, the central circadian clock is located in the suprachiasmatic nuclei (SCN), guiding circadian-regulated biological variables such as the sleep-wake cycle, hormonal secretions and locomotor activity . Another physiological process that exhibits circadian fluctuations, with obvious implications in disease progression and outcome, is the regulation of immune function.
The link between the circadian and the immune systems has been extensively investigated [2–4]. Circadian rhythms within the immune system were described in several tissues and cellular populations . In humans, the number of lymphocytes and granulocytes peaks during the night, whereas monocytes and neutrophil levels fall during the day . Major humoral immune responses undergo circadian changes, and rhythms in plasmatic levels of pro-inflammatory cytokines, as well as peptide hormones produced and secreted by immune cells, were also reported [3, 5–7].
In addition, circadian outputs might affect the central clock through a feedback mechanism that fine-tunes the system. The influence of the immune over the circadian system has been studied in recent years, and there are evidences of the effects of cytokines or chemokines in the central nervous system (CNS) [8–13]. A growing number of recent evidences point to the existence of a bidirectional interaction between the immune and circadian systems which might be essential in the study of disease progression and treatment. Recent work in our laboratory has shown that acute doses of bacterial LPS induce phase changes in mice [4, 14], probably mediated through NF-κB activation . Moreover, cytokines activate clock genes in CNS glial cells, which could release factors to synchronize CNS neurons .
Galectins, a family of evolutionarily-conserved β-galactoside-binding proteins, can control a variety of biological processes including the regulation of neuroimmunoendocrine networks [17, 18]. Galectin-1 (Gal1), a homodimeric protein of this family, can regulate immune cell homeostasis by modulating cytokine synthesis, T cell survival and dendritic cell physiology (reviewed in ). Blockade of Gal1 expression within tumor tissue results in heightened T-cell mediated tumor rejection and increased survival of antitumor Th1 cells . Recent evidence from our laboratory provided rational explanations for these selective immunosuppressive effects demonstrating the ability of Gal1 to preferentially regulate the survival of particularly glycosylated T-cell subsets  and induce the amplification of tolerogenic signals delivered from dendritic cells to T cells in a paracrine circuit involving IL-27 and IL-10 . Although the major role for Gal1 has been studied in the context of immune regulation, tumor progression or inflammation [18, 22–25], recent evidence suggests that it might also play an important role in nerve regeneration , muscle innervation  and neurogenesis [28, 29]. In fact, Gal1 is expressed in both motor and sensory neurons  and modifies the expression of neuronal NMDA glutamate receptors . Galectins have been associated with several biological processes in physiological as well as in pathological conditions (reviewed in ); yet the direct involvement of galectins in animal behavior is largely unexplored.
Based on the evidence that several cytokines and immune factors are known to affect circadian rhythms[13, 14, 32–34], we wished to explore whether the individual functions of Gal1 displayed on different biological settings, may influence or compromise behavioral parameters. The aim of this study is to establish a role for Gal1 in the mammalian circadian system, by characterizing the circadian activity behavior of the Gal1 null mutant (Lgals1 -/-) .
Lgals1 -/- mice (C57BL/6J) were generated by F.P. at the Jacques Monod Institute (Paris), and were bred at the Animal facilities of the Faculty of Exact and Natural Sciences (University of Buenos Aires) and the Institute of Biology and Experimental Medicine (CONICET). Male mutants (n = 5) and male littermate wild-type mice (n = 4) were kept and manipulated according to the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by our institutional research committee. At 90 days of age, mice were placed in individual cages provided with a 17-cm running wheel, under a 12:12 light-dark schedule (LD), with water and food ad libitum. Mice were kept in constant darkness (DD) in order to characterize the endogenous circadian period.
Wheel-running locomotor activity recording
Wheel-running activity was constantly monitored by a digital system (Archron, Argentina) which recorded the number of wheel revolutions every 5 minutes. Activity phase onset was taken as circadian time 12 (CT12). Light pulses (> 300 lux) were administered at CT 15 for the analysis of phase delays.
For reentrainment experiments, animals were initially maintained under a 12:12-h light-dark cycle (LD) for at least 10 days. Then, mice were subjected to an abrupt 6-h delay or advance in the phase of the LD cycle by shortening (for advances) or lengthening (for delays) the night previous to the shift. Time for reentrainment to the new LD cycle was defined as the time it took for each animal to adjust its activity onset with the new cycle according to the phase angle of the onset on the original LD scheme.
All data presented is expressed as mean ± SEM. Activity data was analyzed using the tools included in El Temps software (Antoni Díez Noguera, University of Barcelona). Free-running activity periods were determined by Chi-square periodograms. To analyze alpha (the time devoted to nocturnal locomotion) and phase angle (the difference between activity onset and the time of lights off), mean waveforms were obtained from the activity recordings in the free-running interval and the "activity" period (alpha) was defined as the area of the curve on top of the median of activity. The area under the curve was calculated to analyze amplitude and activity concentration; amplitude was defined as the percent of total activity represented in the activity period, and concentration of activity was calculated as the ratio of this percent to alpha. In order to achieve a correct analysis of activity patterns, each individual waveform was built from relative activity data, obtained by determining the ratio of absolute activity counts to average counts in the analyzed period for each animal; this is later referred to as "Relative activity". Activity onset on each individual day was defined as the first 5-min bin that contained at least 80 wheel revolutions, followed by another bin of at least 80 wheel revolutions within 40 min. Phase shifts were calculated by measuring the phase difference between eye-fitted regression lines through 4-6 consecutive activity onset times immediately prior to the light pulse, and at least 10 consecutive activity onset times after the light pulse (excluding the 5 cycles immediately after the pulse) .
In classical behavioral tests, both wild-type and Lgals1 -/- mice exhibited normal entrainment to LD cycles, with a small but significant difference in phase angle: WT animals tended to start their locomotor activity before lights off while mutants presented their activity onset after ZT 12 (p < 0.05, Student's t test, Table 1). Upon transfer to DD conditions, the endogenous period of wheel-running activity was determined. Significant differences were found between circadian period for both groups; for WT mice, mean period was 23.57 ± 0.10 h, while for Lgals1 -/- mice was 24.02 ± 0.03 h (p = 0.002, Student's t test).
The length of the activity interval (i.e., alpha) was found to be significantly different between mutants and WT mice. Alpha for mutants was 14.88 ± 0.88 circadian hours while it was 12.35 ± 0.38 circadian hours for WT mice (p < 0.05, Student's t test). Amplitude was found to be similar in both groups, as activity intervals represented 97% of total activity. In consequence, the amount of activity as related to the total time spent in the activity interval was significantly higher in WT animals as compared to mutants. All data is summarized in Table 1, and representative actograms of both groups are shown in Fig. 1. A tendency towards a bimodal pattern in locomotor activity can be seen in mutant mice, although a three-block analysis of alpha revealed no significant differences between groups (Fig. 2).
Phase delays in response to light pulses at CT15 were significantly larger for mutant mice (2.92 ± 0.26 circadian h) than for WT animals (1.90 ± 0.34 circadian h) (p < 0.05, Student's t test). Data is summarized in Table 1 and representative actograms are shown in Fig. 1.
No significant differences were found between the groups for reentrainment to a phase delay or phase advance in the LD cycle. After a 6-h advance of the LD cycle, WT animals took 8.7 ± 0.3 days and mutants needed 8.2 ± 0.4 days for reentrainment. After 6-h delays the corresponding figures were 2.0 ± 0.6 and 2.4 ± 0.4 days, respectively. Low-amplitude phase advances were found for both WT and mutant mice when subjected to light stimulation at CT21, without any significant difference between them (data not shown).
According to our results, a deficit in Gal1 expression induces subtle, albeit significant, changes in the circadian behavior of mice. Mutant animals exhibited a longer circadian period and, as expected after this result, a larger phase delay response to light pulses (according to the phase response curve of mice; larger phase delays would allow animals with a longer endogenous period to entrain to a 24-h LD cycle). However, the similarities between reentrainment rate between mutant and WT animals suggest that Gal1 might act on a non-parametric photic mechanism (i.e., the response to light pulses) rather than a parametric effect, such as a complete photoperiod synchronization.
In addition, we have preliminary evidence for Gal-1 expression in the suprachiasmatic region of control mice, which was absent in knockout animals (see additional File 1: Immunohistochemical analysis of Gal-1 in the suprachiasmatic area). Indeed, the expression of Gal1 in the suprachiasmatic nuclei (SCN) region of the CNS would suggest that this factor might be implicated in both the generation and the entrainment of circadian rhythms. It is tempting to speculate that the lack of Gal1 might affect the normal development of SCN neurons, as has been found for other CNS areas such as the olfactory bulb [37–39]. It is possible that small structural differences along neural developmental may elicit changes in the adult circadian behavior and responses to light.
Moreover, Gal1 expression in glial cells [28, 29, 40, 41] provides an additional substrate for immune-related effects on circadian responses, since glial cells have been implicated in the generation and modulation of circadian rhythms [16, 42]. In particular, astrocytic-derived Gal1 has also been reported to modulate glutamate neurotoxicity by altering the expression of NMDA receptors , which are key actors in the signal transduction pathway for circadian responses to light . Moreover, changes in these receptors, present in the retinorecipient portion of the SCN and responsive to photic stimulation, might explain at least in part the variation in light-induced phase shifts in the mutants.
The differences reported for circadian period and light-induced phase delays in Gal1 mutants should also be taken into account when employing this model in immune or behavioral research. Further research of daily or circadian fluctuations in specific immune variables should be performed in order to establish the possible implications of Gal1 in the mammalian circadian system. To illustrate this concept, Gal1-mediated tolerogenic mechanisms, including promotion of T cell apoptosis, modulation of Th2 cytokine balance or the control of dendritic cell physiology may be selectively influenced by the circadian system. This implies caution in the monitoring of these immunoregulatory effects which may substantially fluctuate during the light-dark cycle. In conclusion, we provide the first evidence of alterations in the behavior of mice devoid of Gal1, which prompts further investigation of the relevance of this endogenous protein in physiopathological processes.
Dunlap JC, Loros JJ, DeCoursey PJ: Chronobiology: Biological Timekeeping. First edition. Sunderland, MA, USA: Sinauer Associates, Inc. Publishers; 2004.
Halberg F, Johnson EA, Brown BW, Bittner JJ: Susceptibility rhythm to E. coli endotoxin and bioassay. Proc Soc Exp Biol Med 1960, 103:142–144.
Coogan AN, Wyse CA: Neuroimmunology of the circadian clock. Brain Res 2008, 1232:104–112.
Marpegan L, Leone MJ, Katz ME, Sobrero PM, Bekinschtein TA, Golombek DA: Diurnal rhythm in endotoxin-induced mortality in mice: correlation with proinflammatory factors and lack of circadian variation. Chronobiol Int 2009, 26:1–13.
Leone MJ, Marpegan L, Chiesa JJ, Golombek DA: A time to kill and a time to heal. Pathophysiological interactions between the circadian and the immune systems. Physiol Minirev 2007, 2:60–69.
Haus E, Smolensky MH: Biologic rhythms in the immune system. Chronobiol Int 1999, 16:581–622.
Petrovsky N, Harrison LC: Diurnal rhythmicity of human cytokine production: a dynamic disequilibrium in T helper cell type 1/T helper cell type 2 balance? J Immunol 1997, 158:5163–5168.
Bauhofer A, Witte K, Celik I, Pummer S, Lemmer B, Lorenz W: Sickness behaviour, an animal equivalent to human quality of life, is improved in septic rats by G-CSF and antibiotic prophylaxis. Langenbecks Arch Surg 2001, 386:132–140.
Bauhofer A, Witte K, Lemmer B, Middeke M, Lorenz W, Celik I: Quality of life in animals as a new outcome for surgical research: G-CSF as a quality of life improving factor. Eur Surg Res 2002, 34:22–29.
Ebong S, Call D, Nemzek J, Bolgos G, Newcomb D, Remick D: Immunopathologic alterations in murine models of sepsis of increasing severity. Infect Immun 1999, 67:6603–6610.
Ebong SJ, Call DR, Bolgos G, Newcomb DE, Granger JI, O'Reilly M, Remick DG: Immunopathologic responses to non-lethal sepsis. Shock 1999, 12:118–126.
Motzkus D, Albrecht U, Maronde E: The human PER1 gene is inducible by interleukin-6. J Mol Neurosci 2002, 18:105–109.
Nava F, Carta G, Haynes LW: Lipopolysaccharide increases arginine-vasopressin release from rat suprachiasmatic nucleus slice cultures. Neurosci Lett 2000, 288:228–230.
Marpegan L, Bekinschtein TA, Costas MA, Golombek DA: Circadian responses to endotoxin treatment in mice. J Neuroimmunol 2005, 160:102–109.
Marpegan L, Bekinschtein TA, Freudenthal R, Rubio MF, Ferreyra GA, Romano A, Golombek DA: Participation of transcription factors from the Rel/NF-kappa B family in the circadian system in hamsters. Neurosci Lett 2004, 358:9–12.
Leone MJ, Marpegan L, Bekinschtein TA, Costas MA, Golombek DA: Suprachiasmatic astrocytes as an interface for immune-circadian signalling. J Neurosci Res 2006, 84:1521–1527.
van Kooyk Y, Rabinovich GA: Protein-glycan interactions in the control of innate and adaptive immune responses. Nat Immunol 2008, 9:593–601.
Yang RY, Rabinovich GA, Liu FT: Galectins: structure, function and therapeutic potential. Expert Rev Mol Med 2008, 10:e17.
Rabinovich GA, Toscano MA: Turning 'sweet' on immunity: galectin-glycan interactions in immune tolerance and inflammation. Nat Rev Immunol 2009, 9:338–352.
Rubinstein N, Alvarez M, Zwirner NW, Toscano MA, Ilarregui JM, Bravo A, Mordoh J, Fainboim L, Podhajcer OL, Rabinovich GA: Targeted inhibition of galectin-1 gene expression in tumor cells results in heightened T cell-mediated rejection; A potential mechanism of tumor-immune privilege. Cancer Cell 2004, 5:241–251.
Toscano MA, Bianco GA, Ilarregui JM, Croci DO, Correale J, Hernandez JD, Zwirner NW, Poirier F, Riley EM, Baum LG, Rabinovich GA: Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death. Nat Immunol 2007, 8:825–834.
Ilarregui JM, Croci DO, Bianco GA, Toscano MA, Salatino M, Vermeulen ME, Geffner JR, Rabinovich GA: Tolerogenic signals delivered by dendritic cells to T cells through a galectin-1-driven immunoregulatory circuit involving interleukin 27 and interleukin 10. Nat Immunol 2009, 10:981–991.
Camby I, Le Mercier M, Lefranc F, Kiss R: Galectin-1: a small protein with major functions. Glycobiology 2006, 16:137R-157R.
Liu FT: Regulatory roles of galectins in the immune response. Int Arch Allergy Immunol 2005, 136:385–400.
Liu FT, Rabinovich GA: Galectins as modulators of tumour progression. Nat Rev Cancer 2005, 5:29–41.
Plachta N, Annaheim C, Bissiere S, Lin S, Ruegg M, Hoving S, Muller D, Poirier F, Bibel M, Barde YA: Identification of a lectin causing the degeneration of neuronal processes using engineered embryonic stem cells. Nat Neurosci 2007, 10:712–719.
Svensson A, Tagerud S: Galectin-1 expression in innervated and denervated skeletal muscle. Cell Mol Biol Lett 2009, 14:128–138.
Ishibashi S, Kuroiwa T, Sakaguchi M, Sun L, Kadoya T, Okano H, Mizusawa H: Galectin-1 regulates neurogenesis in the subventricular zone and promotes functional recovery after stroke. Exp Neurol 2007, 207:302–313.
Sakaguchi M, Shingo T, Shimazaki T, Okano HJ, Shiwa M, Ishibashi S, Oguro H, Ninomiya M, Kadoya T, Horie H, et al.: A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells. Proc Natl Acad Sci USA 2006, 103:7112–7117.
Gaudet AD, Steeves JD, Tetzlaff W, Ramer MS: Expression and functions of galectin-1 in sensory and motoneurons. Curr Drug Targets 2005, 6:419–425.
Lekishvili T, Hesketh S, Brazier MW, Brown DR: Mouse galectin-1 inhibits the toxicity of glutamate by modifying NR1 NMDA receptor expression. Eur J Neurosci 2006, 24:3017–3025.
Kwak Y, Lundkvist GB, Brask J, Davidson A, Menaker M, Kristensson K, Block GD: Interferon-gamma alters electrical activity and clock gene expression in suprachiasmatic nucleus neurons. J Biol Rhythms 2008, 23:150–159.
Cavadini G, Petrzilka S, Kohler P, Jud C, Tobler I, Birchler T, Fontana A: TNF-alpha suppresses the expression of clock genes by interfering with E-box-mediated transcription. Proc Natl Acad Sci USA 2007, 104:12843–12848.
Bentivoglio M, Grassi-Zucconi G, Peng ZC, Bassetti A, Edlund C, Kristensson K: Trypanosomes cause dysregulation of c-fos expression in the rat suprachiasmatic nucleus. Neuroreport 1994, 5:712–714.
Poirier F, Robertson EJ: Normal development of mice carrying a null mutation in the gene encoding the L14 S-type lectin. Development 1993, 119:1229–1236.
Edelstein K, de la Iglesia HO, Schwartz WJ, Mrosovsky N: Behavioral arousal blocks light-induced phase advances in locomotor rhythmicity but not light-induced Per1 and Fos expression in the hamster suprachiasmatic nucleus. Neuroscience 2003, 118:253–261.
Crandall JE, Dibble C, Butler D, Pays L, Ahmad N, Kostek C, Puschel AW, Schwarting GA: Patterning of olfactory sensory connections is mediated by extracellular matrix proteins in the nerve layer of the olfactory bulb. J Neurobiol 2000, 45:195–206.
Puche AC, Poirier F, Hair M, Bartlett PF, Key B: Role of galectin-1 in the developing mouse olfactory system. Dev Biol 1996, 179:274–287.
St John JA, Key B: Expression of galectin-1 in the olfactory nerve pathway of rat. Brain Res Dev Brain Res 1999, 117:171–178.
Wei Q, Eviatar-Ribak T, Miskimins WK, Miskimins R: Galectin-4 is involved in p27-mediated activation of the myelin basic protein promoter. J Neurochem 2007, 101:1214–1223.
Kajitani K, Nomaru H, Ifuku M, Yutsudo N, Dan Y, Miura T, Tsuchimoto D, Sakumi K, Kadoya T, Horie H, et al.: Galectin-1 promotes basal and kainate-induced proliferation of neural progenitors in the dentate gyrus of adult mouse hippocampus. Cell Death Differ 2009, 16:417–427.
Prolo LM, Takahashi JS, Herzog ED: Circadian rhythm generation and entrainment in astrocytes. J Neurosci 2005, 25:404–408.
Golombek DA, Rosenstein RE: The physiology of circadian entrainment. Physiol Rev 2009, in press.
Blois SM, Ilarregui JM, Tometten M, Garcia M, Orsal AS, Cordo-Russo R, Toscano MA, Bianco GA, Kobelt P, Handjiski B, et al.: A pivotal role for galectin-1 in fetomaternal tolerance. Nat Med 2007, 13:1450–1457.
We thank the staff of the Animal Facilities of the Faculty of Exact and Natural Sciences (University of Buenos Aires), the National University of Quilmes and the Institute of Biology and Experimental Medicine. This work was supported by grants from the Argentinean National Agency for Promotion of Science and Technology (PICT 2006-541 to D.A.G. and PICT 2006-603 to G.A.R.), the National University of Quilmes (DAG) and Fundación Sales (G.A.R.). L.C. is a fellow from CONICET and D.O.C. is a postgraduate fellow from ANPCyT.
The authors declare that they have no competing interests.
LPC performed the behavioral experiments and analysis; DOC performed the immunohistochemistry; FP generated and provided the mutant mice; LPC, GAR and DAG designed the experiments, analyzed the results and wrote the manuscript. All authors approved the final version of the manuscript.
Electronic supplementary material
Additional file 1: Immunohistochemical analysis of Galectin-1 in the suprachiasmatic area. Brains from 3 WT or 2 Gal-1 deficient (Lgals1 -/-) mice housed under 12:12 LD were perfused at zeitgeber time 6 with paraformaldehyde 4% and stored in PBS/sucrose until use. Sections (40-50 μm) were cut in vibratome and incubated in PBS 10% normal goat serum for 1 h at room temperature. Samples were then incubated with anti-Gal1 rabbit polyclonal IgG (1:200 dilution) used as described [22, 44] or control pre-immune rabbit IgG (same dilution), in PBS containing 2% goat serum overnight at 4°C. Subsequently, samples were rinsed in PBS for 30 min and then incubated with FITC-conjugated goat anti-rabbit IgG (BD Bioscience 1:200) for 2 h at room temperature. Following extensive washing, samples were mounted in anti-fading solution on glass slides and analyzed on a Nikon E800 scanning laser confocal microscope. As a control, primary antibody was omitted in some sections and processed as described above. Cellular nuclei were stained with propidium iodine. To determine whether Gal1 plays a role in the regulation of mice circadian behavior, we evaluated expression of this glycan-binding protein in the suprachiasmatic region of the brain. In the pictures, propidium iodine-stained nuclei are shown in red and Gal-1 expression is stained in green. A widespread and specific immunoreactivity of Gal-1 was observed (A), in contrast to what has been reported for Lgals1 -/- mice . In addition, strong gal-1 expression was found in the olfactory bulb of wild-type mice (data not shown). (B) As a control, Lgals1 -/- showed no expression of galectin-1. (PDF 1 MB)
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Casiraghi, L.P., Croci, D.O., Poirier, F. et al. "Time sweet time": circadian characterization of galectin-1 null mice. J Circad Rhythms 8, 4 (2010). https://doi.org/10.1186/1740-3391-8-4