Urocortin 2 Suppresses Host Resistance to Listeria monocytogenes Infection via Up-Regulation of Interleukin-10
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《内分泌学杂志》
Department of Bacteriology (H.S., A.N.) and Third Department of Internal Medicine (K.K., T.S.), Hirosaki University School of Medicine, Hirosaki 036-8562, Japan
Abstract
Previous studies have showed that corticotropin-releasing factor (CRF) modulates immune response during inflammation. We investigated the effect of CRF family peptides on host resistance to Listeria monocytogenes infection in mice. When mice were administered ip with CRF, urocortin (Ucn), or Ucn2 30 min prior a sublethal infection with L. monocytogenes, the numbers of bacteria in the organs of Ucn2-treated mice were dramatically increased, and most of these mice succumbed. However, host resistance to the infection was retained in CRF- or Ucn-treated mice. The suppressive effect of Ucn2 was dependent on CRF receptor type 2 because an antagonist to the receptor canceled the effect of Ucn2. IL-10 production was significantly increased, and interferon- and TNF production was decreased in the spleens of Ucn2-treated mice, compared with those in Ucn2-untreated control mice. The effect of Ucn2 was canceled by treatment with anti-IL-10 monoclonal antibody and in IL-10-deficient mice. The expression and activation of signal transducers and activators of transcription (STAT) 3 were up-regulated, and the expression and activation of STAT1 were down-regulated in the spleens from Ucn2-treated mice, compared with vehicle-treated mice. Moreover, suppression of TNF production and augmentation of IL-10 production and expression and activation of STAT3 by Ucn2 treatment were observed in heat-killed L. monocytogenes-stimulated macrophages. These results suggested that Ucn2 suppresses host resistance to L. monocytogenes infection via up-regulation of IL-10 production.
Introduction
CORTICOTROPIN-RELEASING FACTOR (CRF) is well known as a primary mediator of the mammalian stress response (1). CRF plays an important role at the initial step of neuroendocrine system, e.g. control behavior and autonomic adaptive changes through hypothalamo-pituitary-adrenal axis. Centrally produced CRF shows various effects including immunosuppression on the peripheral organs and tissues. CRF also activates the sympathetic nerve system. One of the effects of CRF to the central nervous system is the induction of anxiety and motor activity as well as the inhibition of food intake and sexual behavior. The effects of CRF are mediated by two types of receptors, CRF receptor (CRFR) type 1 and CRFR2 (2, 3, 4). CRFR1 is the main receptor of CRF; thus, it mediates the principal functions of CRF. CRF produced from hypothalamus acts through CRFR1 to stimulate the synthesis and release of ACTH by pituitary corticotropic cells (5, 6). ACTH stimulates the production of adrenocorticosteroids in adrenal glands (5). Several studies showed that CRF is present in the synovium of patients with rheumatoid arthritis (7), colonic mucosa of patients with ulcerative colitis (8), and inflammatory thyroid lesions (9). Peripherally produced CRF is also found in human placenta (10, 11), ovary (12), endometrium (13), and peripheral nerves (14). In human skin, peripheral CRF is produced on site, whereas peripheral CRF is produced from nerve endings in rodents, especially C57BL/6 mice (15).
Recent identification and characterization of a second mammalian member of the CRF family peptide, urocortin (Ucn), which possesses characteristics of an endogenous ligand for CRFR2 (16), was made. Ucn shares a considerable degree of homology with CRF. Ucn binds to CRFR1 and also binds to CRFR2 with high affinity (16). CRFR2 is abundant in the periphery, such as skeletal muscle, spleen, gastrointestinal tract, and heart (17). Similar to CRF, Ucn is detected in synovium of patients with rheumatoid arthritis (18), human placenta (19), fetal membranes (19), circulating leukocytes (20), and skin (21). These findings suggest that the peripheral presence of CRF and Ucn is involved in the modulation of local immune responses during inflammation.
Recently discovered CRF family peptide, Ucn2, can bind to only CRFR2 (22, 23). Ucn2 is a 38-amino acid peptide. Central administration of Ucn2 showed the reduction of food intake in the similar degree of CRF (23). Unlike CRF, Ucn2 treatment provoked no significant changes in gross motor activity (23). However, a peripheral effect of Ucn2 is still unclear.
There have been studies on the involvement of CRF family peptides in controlling noninfectious inflammation. CRF and Ucn suppress experimental autoimmune encephalomyelitis (24), and Ucn can inhibit production of TNF induced by stimulation with bacterial lipopolysaccharide (25). However, the effect of CRF family peptides on microbial infections is still unknown. Therefore, we investigated a role of CRF family peptides, CRF, Ucn, and Ucn2 in L. monocytogenes infection. Host resistance to infection with L. monocytogenes, an intracellular-growing bacterium, is controlled by cell-mediated immunity. Various cytokines are reportedly involved in the regulation of host resistance to L. monocytogenes. L. monocytogenes infection promotes the induction of a host T helper 1 response including interferon (IFN)-, which is critical in host resistance to L. monocytogenes (26, 27, 28). In contrast, IL-10 plays a regulatory role in L. monocytogenes infection including suppression of antilisterial resistance (29, 30, 31). In this study, we demonstrate that Ucn2 dramatically enhances the susceptibility to a sublethal infection with L. monocytogenes in mice and that IL-10 mediates the suppressive effect of Ucn2 on antilisterial resistance.
Materials and Methods
Animals
C57BL/6 mice and IL-10-deficient (–/–) mice on a C57BL/6 background were used in this study. C57BL/6 mice were purchased from CLEA Japan, Inc., Tokyo, Japan. IL-10–/– mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were used at 6–8 wk old. Animals were cared for under specific pathogen-free conditions in the Institute for Animal Experiment, Hirosaki University School of Medicine. All animal experiments in this paper were conducted in accordance with the Animal Research Ethics Committee, Hirosaki University School of Medicine and followed the Guidelines for Animal Experimentation, Hirosaki University.
Bacteria
L. monocytogenes 1b 1684 cells were prepared as described previously (32). The concentration of washed cells was adjusted spectrophotometrically at 550 nm, and cells were stocked at –80 C until use. Mice were infected iv with 0.2 ml of a solution containing 5 x 105 colony-forming units (CFU) of L. monocytogenes in PBS.
Preparation of CRF, Ucn1, Ucn2, and CRFR2 antagonist antisauvagine-30 (AS-30)
CRF and Ucn were purchased from Peptide Institute (Osaka, Japan). Mouse Ucn2 and AS-30 were synthesized by Asahi Techno Glass (Chiba, Japan). CRF, Ucn, and Ucn2 were given ip at a dose of 2.5 μg per 250 μl in PBS. Mice were injected ip with 2.5 μg per 250 μl of AS-30, a selective CRFR2 antagonist, in PBS 30 min before the injection of Ucn2 or PBS.
Determination of numbers of viable L. monocytogenes in the organs
The spleens and livers of infected animals were homogenized in PBS or 1% 3-[(cholamidopropyl)dimethylammonio]1-propanesulfate (Wako Pure Chemical Industries Ltd., Osaka, Japan). The numbers of viable bacteria in the organs of infected animals were counted by plating serial 10-fold dilutions of organ homogenates on tryptic soy agar (BD Diagnosis Systems, Sparks, MD). Colonies were routinely counted 24 h later.
Antibody
A hybridoma cell line secreting monoclonal antibody (mAb) against mouse IL-10 (JES5–2A5, rat IgG1) was injected into pristane-primed CD-1 / mice. The mAb found in the ascite fluid was partially purified by (NH4)2SO4 precipitation (32). The mice were given a single iv injection with anti-IL-10 mAb 24 h before infection. All in vivo effects of mAbs described were verified by use of reagents tested by the Limulus amoebocyte lysate assay to contain less than 0.1 ng per injected dose.
Cytokine assays
The liver and spleen homogenates for cytokine assays were prepared as follows. The organs were aseptically removed from the mice and suspended in RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) containing 1% 3-[(cholamidopropyl)dimethylammonio]1-propanesulfate, and 10% (wt/vol) homogenates were prepared with a Dounce grinder. They were clarified by centrifuging at 2000 x g for 20 min. The organ extracts were stored at –80 C until the cytokine assays were performed (33). Titers of IFN, TNF, and IL-10 in organ homogenates and culture supernatants were determined by double sandwich ELISA as described previously (34).
Western blotting
Spleens from mice infected with L. monocytogenes were homogenized in lysis buffer (0.05 M Tris-HCl, 2% sodium dodecyl sulfate, 6% 2-mercaptoethanol, 10% glycerol). The protein concentration of each sample was determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Each amount of protein was developed by SDS-PAGE using 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to Immobilon-P transfer membrane (Millipore Corp., Bedford, MA). After blocking, the membrane was incubated with primary antibodies specific to signal transducers and activators of transcription (STAT)1, phosphorylated (p) STAT1, STAT3, and pSTAT3. Primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). After washing, the membrane was incubated with horseradish peroxidase-conjugated antirabbit IgG. Immunoreactive bands were visualized using 3',3'-diaminobenzidine (Wako) or enhanced chemiluminescence Western blotting analysis system (Amersham Biosciences, Buckinghamshire, UK). Density of detected band was quantified using Scion Image (Scion Corp., Frederick, MD).
Real-time quantitative RT-PCR
Total RNA was isolated from pieces of spleens and livers (0.05 g each) by a guanidium thiocyanate-phenol-chloroform single-step method (35). First-strand cDNAs were synthesized by reverse transcription of 1 μg total RNA using random primers (Takara, Shiga, Japan) and reverse transcriptase Moloney murine leukemia virus (Invitrogen Corp., Carlsbad, CA). The following primers were used: STAT1, forward, 5'-GCCCGACCCTATTACAAAAA-3', and reverse, 5'-CTGCCAACTCAACACCTCTG-3'; STAT3, forward, 5'-CAAAACCCTCAAGAGCCAAGGAGAC-3', and reverse, 5'-GCCGGTGCTGCACGATAGGG-3'; glyceraldehydes-3-phosphate dehydrogenase (GAPDH), forward, 5'-TGAAGGTCGGTGTGAACGGATTTGG-3', and reverse, 5'-ACGACATACTCAGCACCAGCATCAC-3'. SYBR Green Supermix (Bio-Rad) was used as a PCR solution. PCR was run following protocol: initial activation of Taq DNA polymerase at 95 C for 5 min, 30 sec at 95 C for denaturing, 30 sec at 55 C for annealing, and 30 sec at 72 C for elongation, and 40 PCR cycles were performed. All experiments were run in duplicate and nontemplate controls and dissociation curves were used to detect primer-dimer conformation and nonspecific amplication. The threshold cycle (CT) of each target product was determined and set in relation to the amplification plot of GAPDH. The detection threshold is set to the log linear range of the amplification curve and kept constant (0.05) for all data analysis. Difference in CT values (CT) of two genes was used to calculate the relative expression: relative expression = 2–(CT of STATs – CT of GADPH) = 2–CT.
Cell culture
Murine macrophage cell line RAW264.7 was purchased from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan) and was cultured in DMEM (Nissui, Tokyo, Japan) supplemented with 10% of fetal bovine serum (JRH Biosciences, Lenexa, KS) and 3% L-glutamine (Wako). Cells at 2 x106/ml were stimulated with heat-killed L. monocytogenes (HKLM) at 2 x107/ml in the presence or absence of Ucn2 for 48 h. Culture supernatants and cells were collected for further analyses.
Statistical evaluation of the data
Data were expressed as mean ± SD in bacterial numbers, cytokine titers, and relative expression of STATs. Six to nine samples were used in each figure. One-way ANOVA was performed to determine the significance of the differences of bacterial counts, cytokine titers, and relative expression of STATs in the organs between the control and experimental groups, followed by Scheffé’s F post hoc test. A 2 test was carried out to determine the significance of the difference of survival rate among CRF-, Ucn-, or Ucn2-treated and vehicle-treated group.
Results
Ucn2 enhanced the susceptibility to a sublethal infection with L. monocytogenes
We investigated the effect of administration of CRF family peptides on the susceptibility to a sublethal infection with L. monocytogenes. Mice were administered ip with 2.5 μg CRF, Ucn1, or Ucn2 in PBS or PBS only 30 min before iv infection with 5 x105 CFU of L. monocytogenes. The numbers of viable bacteria in the spleens and livers of mice were counted 5 d after infection (Fig. 1A), and the survival rate of each group was also determined (Fig. 1B). The bacterial numbers in the organs of mice treated with Ucn2 showed significant increment, compared with those of PBS-injected mice (Fig. 1A; P < 0.01). In contrast, the treatment with CRF or Ucn1 showed no significant change in bacterial numbers (Fig. 1A). The survival rate of Ucn2-treated mice during L. monocytogenes infection was dramatically decreased, compared with PBS-injected mice (Fig. 1B; P < 0.05). Again, no change was shown in survival rates when mice were treated with CRF or Ucn1 before infection (Fig. 1B). These results showed that the treatment with Ucn2 dramatically enhanced the susceptibility to a sublethal infection with L. monocytogenes. Next, we assessed the kinetics of L. monocytogenes in the organs of mice treated with Ucn2 or PBS. The numbers of bacteria were continuously increased during 5 d after infection in the spleens and livers of Ucn2-treated mice, whereas the bacterial numbers were increased up to 3 d after infection and then decreased in the organs of PBS-injected mice (Fig. 1C). These results suggested that the treatment with Ucn2 suppressed host resistance to L. monocytogenes infection.
The effect of Ucn2 on L. monocytogenes infection was dose dependent and mediated via CRFR2
We investigated the dose dependency of the effect of Ucn2 on host resistance to L. monocytogenes infection. Mice were administered ip with various doses of Ucn2 30 min before infection. The numbers of viable bacteria in the organs of mice were counted 5 d after infection (Fig. 2). As the dose of Ucn2 was decreased, the numbers of bacteria in the organs of mice were decreased. The effect of Ucn2 treatment was canceled by the pretreatment of CRFR2 antagonist, AS-30. These results showed that the suppressive effect of Ucn2 on host resistance to L. monocytogenes infection was dose dependent and mediated by CRFR2.
Ucn2 up-regulated IL-10 production and down-regulated IFN and TNF production in the spleens of mice infected with a sublethal dose of L. monocytogenes
IFN and TNF play protective roles, and IL-10 plays a detrimental role in host resistance to L. monocytogenes infection (26, 28, 29, 30, 31). Therefore, we assessed the production of cytokines in Ucn2-treated mice during L. monocytogenes infection. Mice were administered ip with 2.5 μg of Ucn2 30 min before infection. The spleens were obtained from the infected mice at 1, 2, 3, or 5 d after infection, and the titers of cytokines in organ homogenates were determined (Fig. 3). The IFN and TNF titers in the spleens from Ucn2-treated mice were significantly lower than those in PBS-treated mice (Fig. 3, A and B; P < 0.01). In contrast, the IL-10 titers in the spleens from Ucn2-treated mice were significantly higher than those in the control group (Fig. 3C; P < 0.01). These results suggested that Ucn2 induced the up-regulation of IL-10 and the down-regulation of IFN and TNF during L. monocytogenes infection in vivo.
Absence of IL-10 canceled the effect of Ucn2 on a sublethal infection with L. monocytogenes
To investigate whether the suppressive effect of Ucn2 is mediated by IL-10, C57BL/6 mice and IL-10–/– mice were injected ip with 2.5 μg of Ucn2 30 min before L. monocytogenes infection. The numbers of viable bacteria in the organs of mice were counted 5 d later. The Ucn2 treatment showed no effect on the bacterial numbers when administered into IL-10–/– mice (Fig. 4A). Next, we confirmed the effect of Ucn2 in mice that endogenous IL-10 had been neutralized by antimouse IL-10 mAb. Mice were injected ip with various doses of antimouse IL-10 mAb or isotype-matched IgG 24 h before infection and the bacterial numbers were counted 5 d later. As expected, anti-IL-10 mAb canceled the suppressive effect of Ucn2 when mice had received 100 or 1000 μg of anti-IL-10 mAb (Fig. 4B). These results suggested that IL-10 played a critical role in the suppression of host resistance to L. monocytogenes infection by Ucn2.
STAT3 expression was up-regulated and STAT1 expression was down-regulated in the spleens of Ucn-2 treated mice
STATs are signal transducers of various cytokines. IFN activates STAT1 (36) and IL-10 activates STAT3 (37). Therefore, we investigated the effect of Ucn2 on the expression and activation of STAT1 and STAT3 during a sublethal infection with L. monocytogenes. We prepared mRNAs and proteins from the spleens of Ucn2-treated mice 24 and 48 h after infection. Real-time quantitative RT-PCR showed that STAT1 expression was up-regulated in the spleens from PBS-treated mice 24 h after infection, whereas STAT1 expression was not up-regulated in the spleens from Ucn2-treated mice (Fig. 5A). In contrast, STAT3 expression was up-regulated in the spleens from Ucn2-treated mice 24 h after infection, whereas the spleens from PBS-treated mice showed no change in the expression of STAT3 (Fig. 5B). Next, we confirmed the expressions of nonphosphorylated STATs and pSTATs by Western blotting. The expressions of STAT1 (Fig. 5, C and D) and pSTAT1 (Fig. 5, C and E) were up-regulated in the spleens from PBS-treated mice 48 h after infection, whereas the up-regulation of neither STAT1 (Fig. 5, C and D) nor pSTAT1 (Fig. 5, C and D) expression was observed in the spleens from Ucn2-treated mice. Conversely, the expressions of STAT3 (Fig. 5, C and F) and pSTAT3 (Fig. 5, C and G) were up-regulated in the spleens from Ucn2-treated mice, compared with PBS-treated mice 48 h after infection. These results suggested that Ucn2 up-regulated STAT3 and down-regulated STAT1 during L. monocytogenes infection.
Ucn2 up-regulated IL-10 production and down-regulated TNF production in macrophages stimulated with HKLM
Next, we investigated the effect of Ucn2 on cytokine responses by stimulating with HKLM for 48 h in murine macrophage cell line RAW264.7. Cytokine titers in the culture supernatants were determined by ELISA (Fig. 6, A and B). Treatment of Ucn2 significantly decreased the TNF titers (Fig. 4A) and increased the IL-10 titers (Fig. 6B) in a dose-dependent manner. IFN was not detected in any group of culture supernatants (Fig. 6B) (data not shown). To investigate the activation of STAT3 by the increased IL-10, pSTAT3 was detected in Ucn2-treated RAW264.7 cells by Western blotting. Ucn2 treatment up-regulated the expression of pSTAT3 dose dependently (Fig. 6C). These results suggested that Ucn2 treatment induced up-regulation of IL-10 and down-regulation of TNF in vitro.
Discussion
CRF secreted from hypothalamus stimulates ACTH production from pituitary gland, and ACTH promotes the production of glucocorticoids from adrenal gland. Glucocorticoids are known to be potent antiinflammatory agents (38) and modulate the production of inflammatory factors such as cytokines (39). Although it has not yet been established whether physiologic adrenal glucocorticoid secretion modulates the immune responses to specific antigens, central CRF and its family peptides may exhibit the effect on inflammatory responses mediated by the hypothalamo-pituitary-adrenal axis. A previous report showed that the peripheral secretion of CRF and Ucn is involved in the modulation of the peripheral immune responses by acting at specific receptors on multiple populations of immune cells to produce a wide range of effects (40). In this study, although the doses of CRF, Ucn, and Ucn2 that we used herein were supraphysiological, only Ucn2 showed the significant suppressive effect on host resistance to a sublethal infection with L. monocytogenes (Fig. 1). The suppressive effect of Ucn2 on host resistance to L. monocytogenes infection was dose dependent (Fig. 2). Administration of Ucn2 during 2 h before to 1 h after infection exhibited the suppressive effect on host resistance against L. monocytogenes infection, and the effect was maximum when Ucn2 was administered 30 min prior infection (data not shown). The suppressive effect was reversed by pretreatment with AS-30, an antagonist of CRFR2 (Fig. 2). CRFR2 reportedly distributes in the rat spleen and thymus (41). Although CRFR2 is expressed in skin and spleen in mouse (42), cells that express CRFR2 have not been specified yet. It is possible that CRFR2 expressed on immune cells mediates the suppressive effect of Ucn2 on host resistance to L. monocytogenes infection. Ucn binds to both CRFR1 and CRFR2. However, our present results revealed that Ucn showed no significant effect on host resistance against L. monocytogenes infection (Fig. 1). Although it is now impossible to explain why Ucn2 but not Ucn suppresses host resistance to L. monocytogenes infection despite the fact that both Ucn and Ucn2 bind CRFR2 as a common receptor, Ucn and Ucn2 may drive different immunological pathways in listerial infection.
L. monocytogenes infection induces T helper 1 response in the host (27), and IFN plays a critical role in host resistance to L. monocytogenes infection (26, 28). IFN produced by natural killer cells can activate macrophages (43). TNF is also essential for primary host defense against infection with L. monocytogenes (44, 45, 46, 47). TNF can activate resident macrophages (45), and production of reactive oxygen and reactive nitrogen intermediates by activated macrophage is important for bactericidal activity during L. monocytogenes infection (48). Our results showed that the treatment with Ucn2 treatment down-regulated IFN and TNF production (Fig. 3, A and B). Treatment with neither CRF nor Ucn showed a significant effect on IFN and TNF production during L. monocytogenes infection (data not shown). These results suggested that the diminished up-regulation of IFN and TNF is involved in impaired host resistance to L. monocytogenes infection in Ucn2-treated mice. TNF production from macrophage in response to HKLM stimulation was suppressed by treatment with Ucn2 in vitro (Fig. 6A), presuming that Ucn2 might suppress activation of macrophages. Activated macrophages exhibit enhanced synthesis of nitric oxide that is involved in bactericidal activity (49). Therefore, we assessed the expression of inducible nitric oxide synthase (iNOS) in spleens of Ucn2-treated mice during listerial infection. However, there was no significant difference in the expression of iNOS between Ucn2-treated and untreated group (date not shown). In vitro study using murine macrophage cell line also showed no significant difference in the expression of iNOS between Ucn2-treated and untreated group (data not shown). This result suggested that Ucn2 might not affect on iNOS activation in macrophages.
In a signaling pathway, IFN activates STAT-1 (36). Herein STAT-1 expression was down-regulated in the Ucn2-treated mice (Fig. 5, A, C, D, and E), compared with the PBS-treated mice in both mRNA and protein levels, suggesting that down-regulation of IFN production might be involved in the suppressive effect on host resistance to L. monocytogenes infection.
IL-10 is known to mediate antiinflammatory responses. IL-10 inhibits the production of proinflammatory cytokines such as IL-1, IL-6, and TNF in cutaneous inflammation (50), and IL-10 down-regulates IL-12 production from dendritic cells (51). Studies on experimental inflammation models have shown that IL-10 suppresses the production of TNF and IL-6 from macrophages, synoviocytes, and T cells (52). Moreover, IL-10 seems to mediate CD25+CD4+ T cell-mediated immunosuppression in autoimmune or inflammatory disease (53, 54). IL-10 plays a detrimental role in host resistance to L. monocytogenes infection (29, 30, 31, 55). In this study, IL-10 production induced by L. monocytogenes infection was up-regulated by Ucn2 treatment in vivo (Fig. 3C). Neither CRF nor Ucn showed a significant effect on IL-10 production (data not shown). Macrophages can produce IL-10 in response to bacterial infections (56, 57). In vitro study showed that IL-10 production from macrophages was also up-regulated by Ucn2 treatment (Fig. 6B). Although turnover of Ucn2 is very short, the up-regulation of IL-10 in the early phase of infection might cause the sustained suppression of host resistance against L. monocytogenes infection. Ucn2 revealed no suppressive effect on host resistance to L. monocytogenes infection when IL-10–/– mice were used (Fig. 4A). Moreover, the depletion of endogenous IL-10 by injection of anti-IL-10 mAb also canceled the suppressive effect of Ucn2 on host resistance to L. monocytogenes infection (Fig. 4B). These results suggested that the effect of Ucn2 is mediated by IL-10, not by a direct effect of Ucn2. In a signaling pathway, IL-10 activates STAT3 (37). In this study, STAT3 expression was up-regulated in Ucn2-treated mice (Fig. 5, B, C, and F). pSTAT-3 was also up-regulated in Ucn2-treated mice, compared with PBS-treated mice (Fig. 5, C and G). pSTAT3 expression was also up-regulated in Ucn2-treated macrophages in vitro (Fig. 6C). These results suggested that the suppressive effect of Ucn2 on host resistance to L. monocytogenes might be mainly mediated by IL-10 and sustained up-regulation of IL-10 production could suppress host resistance against L. monocytogenes infection.
Finally, our present results demonstrated that CRF family peptide Ucn2 converts a sublethal infection with L. monocytogenes to the lethal infection in mice through up-regulation of IL-10. Our study supported that the existence of close interaction between endocrine system and immune system during bacterial infection.
Footnotes
This work was supported in part by a grant-in-aid for general scientific research (13670261 and 16590352) provided by the Japanese Ministry of Education, Science, Sports, and Culture.
Abbreviations: AS-30, Antisauvagine-30; CFU, colony-forming unit; CRF, corticotropin-releasing factor; CRFR, CRF receptor; CT, threshold cycle; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; HKLM, heat-killed L. monocytogenes; IFN, interferon; iNOS, inducible nitric oxide synthase; mAb, monoclonal antibody; p, phosphorylated; STAT, signal transducers and activators of transcription; Ucn, urocortin.
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Abstract
Previous studies have showed that corticotropin-releasing factor (CRF) modulates immune response during inflammation. We investigated the effect of CRF family peptides on host resistance to Listeria monocytogenes infection in mice. When mice were administered ip with CRF, urocortin (Ucn), or Ucn2 30 min prior a sublethal infection with L. monocytogenes, the numbers of bacteria in the organs of Ucn2-treated mice were dramatically increased, and most of these mice succumbed. However, host resistance to the infection was retained in CRF- or Ucn-treated mice. The suppressive effect of Ucn2 was dependent on CRF receptor type 2 because an antagonist to the receptor canceled the effect of Ucn2. IL-10 production was significantly increased, and interferon- and TNF production was decreased in the spleens of Ucn2-treated mice, compared with those in Ucn2-untreated control mice. The effect of Ucn2 was canceled by treatment with anti-IL-10 monoclonal antibody and in IL-10-deficient mice. The expression and activation of signal transducers and activators of transcription (STAT) 3 were up-regulated, and the expression and activation of STAT1 were down-regulated in the spleens from Ucn2-treated mice, compared with vehicle-treated mice. Moreover, suppression of TNF production and augmentation of IL-10 production and expression and activation of STAT3 by Ucn2 treatment were observed in heat-killed L. monocytogenes-stimulated macrophages. These results suggested that Ucn2 suppresses host resistance to L. monocytogenes infection via up-regulation of IL-10 production.
Introduction
CORTICOTROPIN-RELEASING FACTOR (CRF) is well known as a primary mediator of the mammalian stress response (1). CRF plays an important role at the initial step of neuroendocrine system, e.g. control behavior and autonomic adaptive changes through hypothalamo-pituitary-adrenal axis. Centrally produced CRF shows various effects including immunosuppression on the peripheral organs and tissues. CRF also activates the sympathetic nerve system. One of the effects of CRF to the central nervous system is the induction of anxiety and motor activity as well as the inhibition of food intake and sexual behavior. The effects of CRF are mediated by two types of receptors, CRF receptor (CRFR) type 1 and CRFR2 (2, 3, 4). CRFR1 is the main receptor of CRF; thus, it mediates the principal functions of CRF. CRF produced from hypothalamus acts through CRFR1 to stimulate the synthesis and release of ACTH by pituitary corticotropic cells (5, 6). ACTH stimulates the production of adrenocorticosteroids in adrenal glands (5). Several studies showed that CRF is present in the synovium of patients with rheumatoid arthritis (7), colonic mucosa of patients with ulcerative colitis (8), and inflammatory thyroid lesions (9). Peripherally produced CRF is also found in human placenta (10, 11), ovary (12), endometrium (13), and peripheral nerves (14). In human skin, peripheral CRF is produced on site, whereas peripheral CRF is produced from nerve endings in rodents, especially C57BL/6 mice (15).
Recent identification and characterization of a second mammalian member of the CRF family peptide, urocortin (Ucn), which possesses characteristics of an endogenous ligand for CRFR2 (16), was made. Ucn shares a considerable degree of homology with CRF. Ucn binds to CRFR1 and also binds to CRFR2 with high affinity (16). CRFR2 is abundant in the periphery, such as skeletal muscle, spleen, gastrointestinal tract, and heart (17). Similar to CRF, Ucn is detected in synovium of patients with rheumatoid arthritis (18), human placenta (19), fetal membranes (19), circulating leukocytes (20), and skin (21). These findings suggest that the peripheral presence of CRF and Ucn is involved in the modulation of local immune responses during inflammation.
Recently discovered CRF family peptide, Ucn2, can bind to only CRFR2 (22, 23). Ucn2 is a 38-amino acid peptide. Central administration of Ucn2 showed the reduction of food intake in the similar degree of CRF (23). Unlike CRF, Ucn2 treatment provoked no significant changes in gross motor activity (23). However, a peripheral effect of Ucn2 is still unclear.
There have been studies on the involvement of CRF family peptides in controlling noninfectious inflammation. CRF and Ucn suppress experimental autoimmune encephalomyelitis (24), and Ucn can inhibit production of TNF induced by stimulation with bacterial lipopolysaccharide (25). However, the effect of CRF family peptides on microbial infections is still unknown. Therefore, we investigated a role of CRF family peptides, CRF, Ucn, and Ucn2 in L. monocytogenes infection. Host resistance to infection with L. monocytogenes, an intracellular-growing bacterium, is controlled by cell-mediated immunity. Various cytokines are reportedly involved in the regulation of host resistance to L. monocytogenes. L. monocytogenes infection promotes the induction of a host T helper 1 response including interferon (IFN)-, which is critical in host resistance to L. monocytogenes (26, 27, 28). In contrast, IL-10 plays a regulatory role in L. monocytogenes infection including suppression of antilisterial resistance (29, 30, 31). In this study, we demonstrate that Ucn2 dramatically enhances the susceptibility to a sublethal infection with L. monocytogenes in mice and that IL-10 mediates the suppressive effect of Ucn2 on antilisterial resistance.
Materials and Methods
Animals
C57BL/6 mice and IL-10-deficient (–/–) mice on a C57BL/6 background were used in this study. C57BL/6 mice were purchased from CLEA Japan, Inc., Tokyo, Japan. IL-10–/– mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were used at 6–8 wk old. Animals were cared for under specific pathogen-free conditions in the Institute for Animal Experiment, Hirosaki University School of Medicine. All animal experiments in this paper were conducted in accordance with the Animal Research Ethics Committee, Hirosaki University School of Medicine and followed the Guidelines for Animal Experimentation, Hirosaki University.
Bacteria
L. monocytogenes 1b 1684 cells were prepared as described previously (32). The concentration of washed cells was adjusted spectrophotometrically at 550 nm, and cells were stocked at –80 C until use. Mice were infected iv with 0.2 ml of a solution containing 5 x 105 colony-forming units (CFU) of L. monocytogenes in PBS.
Preparation of CRF, Ucn1, Ucn2, and CRFR2 antagonist antisauvagine-30 (AS-30)
CRF and Ucn were purchased from Peptide Institute (Osaka, Japan). Mouse Ucn2 and AS-30 were synthesized by Asahi Techno Glass (Chiba, Japan). CRF, Ucn, and Ucn2 were given ip at a dose of 2.5 μg per 250 μl in PBS. Mice were injected ip with 2.5 μg per 250 μl of AS-30, a selective CRFR2 antagonist, in PBS 30 min before the injection of Ucn2 or PBS.
Determination of numbers of viable L. monocytogenes in the organs
The spleens and livers of infected animals were homogenized in PBS or 1% 3-[(cholamidopropyl)dimethylammonio]1-propanesulfate (Wako Pure Chemical Industries Ltd., Osaka, Japan). The numbers of viable bacteria in the organs of infected animals were counted by plating serial 10-fold dilutions of organ homogenates on tryptic soy agar (BD Diagnosis Systems, Sparks, MD). Colonies were routinely counted 24 h later.
Antibody
A hybridoma cell line secreting monoclonal antibody (mAb) against mouse IL-10 (JES5–2A5, rat IgG1) was injected into pristane-primed CD-1 / mice. The mAb found in the ascite fluid was partially purified by (NH4)2SO4 precipitation (32). The mice were given a single iv injection with anti-IL-10 mAb 24 h before infection. All in vivo effects of mAbs described were verified by use of reagents tested by the Limulus amoebocyte lysate assay to contain less than 0.1 ng per injected dose.
Cytokine assays
The liver and spleen homogenates for cytokine assays were prepared as follows. The organs were aseptically removed from the mice and suspended in RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) containing 1% 3-[(cholamidopropyl)dimethylammonio]1-propanesulfate, and 10% (wt/vol) homogenates were prepared with a Dounce grinder. They were clarified by centrifuging at 2000 x g for 20 min. The organ extracts were stored at –80 C until the cytokine assays were performed (33). Titers of IFN, TNF, and IL-10 in organ homogenates and culture supernatants were determined by double sandwich ELISA as described previously (34).
Western blotting
Spleens from mice infected with L. monocytogenes were homogenized in lysis buffer (0.05 M Tris-HCl, 2% sodium dodecyl sulfate, 6% 2-mercaptoethanol, 10% glycerol). The protein concentration of each sample was determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Each amount of protein was developed by SDS-PAGE using 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to Immobilon-P transfer membrane (Millipore Corp., Bedford, MA). After blocking, the membrane was incubated with primary antibodies specific to signal transducers and activators of transcription (STAT)1, phosphorylated (p) STAT1, STAT3, and pSTAT3. Primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). After washing, the membrane was incubated with horseradish peroxidase-conjugated antirabbit IgG. Immunoreactive bands were visualized using 3',3'-diaminobenzidine (Wako) or enhanced chemiluminescence Western blotting analysis system (Amersham Biosciences, Buckinghamshire, UK). Density of detected band was quantified using Scion Image (Scion Corp., Frederick, MD).
Real-time quantitative RT-PCR
Total RNA was isolated from pieces of spleens and livers (0.05 g each) by a guanidium thiocyanate-phenol-chloroform single-step method (35). First-strand cDNAs were synthesized by reverse transcription of 1 μg total RNA using random primers (Takara, Shiga, Japan) and reverse transcriptase Moloney murine leukemia virus (Invitrogen Corp., Carlsbad, CA). The following primers were used: STAT1, forward, 5'-GCCCGACCCTATTACAAAAA-3', and reverse, 5'-CTGCCAACTCAACACCTCTG-3'; STAT3, forward, 5'-CAAAACCCTCAAGAGCCAAGGAGAC-3', and reverse, 5'-GCCGGTGCTGCACGATAGGG-3'; glyceraldehydes-3-phosphate dehydrogenase (GAPDH), forward, 5'-TGAAGGTCGGTGTGAACGGATTTGG-3', and reverse, 5'-ACGACATACTCAGCACCAGCATCAC-3'. SYBR Green Supermix (Bio-Rad) was used as a PCR solution. PCR was run following protocol: initial activation of Taq DNA polymerase at 95 C for 5 min, 30 sec at 95 C for denaturing, 30 sec at 55 C for annealing, and 30 sec at 72 C for elongation, and 40 PCR cycles were performed. All experiments were run in duplicate and nontemplate controls and dissociation curves were used to detect primer-dimer conformation and nonspecific amplication. The threshold cycle (CT) of each target product was determined and set in relation to the amplification plot of GAPDH. The detection threshold is set to the log linear range of the amplification curve and kept constant (0.05) for all data analysis. Difference in CT values (CT) of two genes was used to calculate the relative expression: relative expression = 2–(CT of STATs – CT of GADPH) = 2–CT.
Cell culture
Murine macrophage cell line RAW264.7 was purchased from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan) and was cultured in DMEM (Nissui, Tokyo, Japan) supplemented with 10% of fetal bovine serum (JRH Biosciences, Lenexa, KS) and 3% L-glutamine (Wako). Cells at 2 x106/ml were stimulated with heat-killed L. monocytogenes (HKLM) at 2 x107/ml in the presence or absence of Ucn2 for 48 h. Culture supernatants and cells were collected for further analyses.
Statistical evaluation of the data
Data were expressed as mean ± SD in bacterial numbers, cytokine titers, and relative expression of STATs. Six to nine samples were used in each figure. One-way ANOVA was performed to determine the significance of the differences of bacterial counts, cytokine titers, and relative expression of STATs in the organs between the control and experimental groups, followed by Scheffé’s F post hoc test. A 2 test was carried out to determine the significance of the difference of survival rate among CRF-, Ucn-, or Ucn2-treated and vehicle-treated group.
Results
Ucn2 enhanced the susceptibility to a sublethal infection with L. monocytogenes
We investigated the effect of administration of CRF family peptides on the susceptibility to a sublethal infection with L. monocytogenes. Mice were administered ip with 2.5 μg CRF, Ucn1, or Ucn2 in PBS or PBS only 30 min before iv infection with 5 x105 CFU of L. monocytogenes. The numbers of viable bacteria in the spleens and livers of mice were counted 5 d after infection (Fig. 1A), and the survival rate of each group was also determined (Fig. 1B). The bacterial numbers in the organs of mice treated with Ucn2 showed significant increment, compared with those of PBS-injected mice (Fig. 1A; P < 0.01). In contrast, the treatment with CRF or Ucn1 showed no significant change in bacterial numbers (Fig. 1A). The survival rate of Ucn2-treated mice during L. monocytogenes infection was dramatically decreased, compared with PBS-injected mice (Fig. 1B; P < 0.05). Again, no change was shown in survival rates when mice were treated with CRF or Ucn1 before infection (Fig. 1B). These results showed that the treatment with Ucn2 dramatically enhanced the susceptibility to a sublethal infection with L. monocytogenes. Next, we assessed the kinetics of L. monocytogenes in the organs of mice treated with Ucn2 or PBS. The numbers of bacteria were continuously increased during 5 d after infection in the spleens and livers of Ucn2-treated mice, whereas the bacterial numbers were increased up to 3 d after infection and then decreased in the organs of PBS-injected mice (Fig. 1C). These results suggested that the treatment with Ucn2 suppressed host resistance to L. monocytogenes infection.
The effect of Ucn2 on L. monocytogenes infection was dose dependent and mediated via CRFR2
We investigated the dose dependency of the effect of Ucn2 on host resistance to L. monocytogenes infection. Mice were administered ip with various doses of Ucn2 30 min before infection. The numbers of viable bacteria in the organs of mice were counted 5 d after infection (Fig. 2). As the dose of Ucn2 was decreased, the numbers of bacteria in the organs of mice were decreased. The effect of Ucn2 treatment was canceled by the pretreatment of CRFR2 antagonist, AS-30. These results showed that the suppressive effect of Ucn2 on host resistance to L. monocytogenes infection was dose dependent and mediated by CRFR2.
Ucn2 up-regulated IL-10 production and down-regulated IFN and TNF production in the spleens of mice infected with a sublethal dose of L. monocytogenes
IFN and TNF play protective roles, and IL-10 plays a detrimental role in host resistance to L. monocytogenes infection (26, 28, 29, 30, 31). Therefore, we assessed the production of cytokines in Ucn2-treated mice during L. monocytogenes infection. Mice were administered ip with 2.5 μg of Ucn2 30 min before infection. The spleens were obtained from the infected mice at 1, 2, 3, or 5 d after infection, and the titers of cytokines in organ homogenates were determined (Fig. 3). The IFN and TNF titers in the spleens from Ucn2-treated mice were significantly lower than those in PBS-treated mice (Fig. 3, A and B; P < 0.01). In contrast, the IL-10 titers in the spleens from Ucn2-treated mice were significantly higher than those in the control group (Fig. 3C; P < 0.01). These results suggested that Ucn2 induced the up-regulation of IL-10 and the down-regulation of IFN and TNF during L. monocytogenes infection in vivo.
Absence of IL-10 canceled the effect of Ucn2 on a sublethal infection with L. monocytogenes
To investigate whether the suppressive effect of Ucn2 is mediated by IL-10, C57BL/6 mice and IL-10–/– mice were injected ip with 2.5 μg of Ucn2 30 min before L. monocytogenes infection. The numbers of viable bacteria in the organs of mice were counted 5 d later. The Ucn2 treatment showed no effect on the bacterial numbers when administered into IL-10–/– mice (Fig. 4A). Next, we confirmed the effect of Ucn2 in mice that endogenous IL-10 had been neutralized by antimouse IL-10 mAb. Mice were injected ip with various doses of antimouse IL-10 mAb or isotype-matched IgG 24 h before infection and the bacterial numbers were counted 5 d later. As expected, anti-IL-10 mAb canceled the suppressive effect of Ucn2 when mice had received 100 or 1000 μg of anti-IL-10 mAb (Fig. 4B). These results suggested that IL-10 played a critical role in the suppression of host resistance to L. monocytogenes infection by Ucn2.
STAT3 expression was up-regulated and STAT1 expression was down-regulated in the spleens of Ucn-2 treated mice
STATs are signal transducers of various cytokines. IFN activates STAT1 (36) and IL-10 activates STAT3 (37). Therefore, we investigated the effect of Ucn2 on the expression and activation of STAT1 and STAT3 during a sublethal infection with L. monocytogenes. We prepared mRNAs and proteins from the spleens of Ucn2-treated mice 24 and 48 h after infection. Real-time quantitative RT-PCR showed that STAT1 expression was up-regulated in the spleens from PBS-treated mice 24 h after infection, whereas STAT1 expression was not up-regulated in the spleens from Ucn2-treated mice (Fig. 5A). In contrast, STAT3 expression was up-regulated in the spleens from Ucn2-treated mice 24 h after infection, whereas the spleens from PBS-treated mice showed no change in the expression of STAT3 (Fig. 5B). Next, we confirmed the expressions of nonphosphorylated STATs and pSTATs by Western blotting. The expressions of STAT1 (Fig. 5, C and D) and pSTAT1 (Fig. 5, C and E) were up-regulated in the spleens from PBS-treated mice 48 h after infection, whereas the up-regulation of neither STAT1 (Fig. 5, C and D) nor pSTAT1 (Fig. 5, C and D) expression was observed in the spleens from Ucn2-treated mice. Conversely, the expressions of STAT3 (Fig. 5, C and F) and pSTAT3 (Fig. 5, C and G) were up-regulated in the spleens from Ucn2-treated mice, compared with PBS-treated mice 48 h after infection. These results suggested that Ucn2 up-regulated STAT3 and down-regulated STAT1 during L. monocytogenes infection.
Ucn2 up-regulated IL-10 production and down-regulated TNF production in macrophages stimulated with HKLM
Next, we investigated the effect of Ucn2 on cytokine responses by stimulating with HKLM for 48 h in murine macrophage cell line RAW264.7. Cytokine titers in the culture supernatants were determined by ELISA (Fig. 6, A and B). Treatment of Ucn2 significantly decreased the TNF titers (Fig. 4A) and increased the IL-10 titers (Fig. 6B) in a dose-dependent manner. IFN was not detected in any group of culture supernatants (Fig. 6B) (data not shown). To investigate the activation of STAT3 by the increased IL-10, pSTAT3 was detected in Ucn2-treated RAW264.7 cells by Western blotting. Ucn2 treatment up-regulated the expression of pSTAT3 dose dependently (Fig. 6C). These results suggested that Ucn2 treatment induced up-regulation of IL-10 and down-regulation of TNF in vitro.
Discussion
CRF secreted from hypothalamus stimulates ACTH production from pituitary gland, and ACTH promotes the production of glucocorticoids from adrenal gland. Glucocorticoids are known to be potent antiinflammatory agents (38) and modulate the production of inflammatory factors such as cytokines (39). Although it has not yet been established whether physiologic adrenal glucocorticoid secretion modulates the immune responses to specific antigens, central CRF and its family peptides may exhibit the effect on inflammatory responses mediated by the hypothalamo-pituitary-adrenal axis. A previous report showed that the peripheral secretion of CRF and Ucn is involved in the modulation of the peripheral immune responses by acting at specific receptors on multiple populations of immune cells to produce a wide range of effects (40). In this study, although the doses of CRF, Ucn, and Ucn2 that we used herein were supraphysiological, only Ucn2 showed the significant suppressive effect on host resistance to a sublethal infection with L. monocytogenes (Fig. 1). The suppressive effect of Ucn2 on host resistance to L. monocytogenes infection was dose dependent (Fig. 2). Administration of Ucn2 during 2 h before to 1 h after infection exhibited the suppressive effect on host resistance against L. monocytogenes infection, and the effect was maximum when Ucn2 was administered 30 min prior infection (data not shown). The suppressive effect was reversed by pretreatment with AS-30, an antagonist of CRFR2 (Fig. 2). CRFR2 reportedly distributes in the rat spleen and thymus (41). Although CRFR2 is expressed in skin and spleen in mouse (42), cells that express CRFR2 have not been specified yet. It is possible that CRFR2 expressed on immune cells mediates the suppressive effect of Ucn2 on host resistance to L. monocytogenes infection. Ucn binds to both CRFR1 and CRFR2. However, our present results revealed that Ucn showed no significant effect on host resistance against L. monocytogenes infection (Fig. 1). Although it is now impossible to explain why Ucn2 but not Ucn suppresses host resistance to L. monocytogenes infection despite the fact that both Ucn and Ucn2 bind CRFR2 as a common receptor, Ucn and Ucn2 may drive different immunological pathways in listerial infection.
L. monocytogenes infection induces T helper 1 response in the host (27), and IFN plays a critical role in host resistance to L. monocytogenes infection (26, 28). IFN produced by natural killer cells can activate macrophages (43). TNF is also essential for primary host defense against infection with L. monocytogenes (44, 45, 46, 47). TNF can activate resident macrophages (45), and production of reactive oxygen and reactive nitrogen intermediates by activated macrophage is important for bactericidal activity during L. monocytogenes infection (48). Our results showed that the treatment with Ucn2 treatment down-regulated IFN and TNF production (Fig. 3, A and B). Treatment with neither CRF nor Ucn showed a significant effect on IFN and TNF production during L. monocytogenes infection (data not shown). These results suggested that the diminished up-regulation of IFN and TNF is involved in impaired host resistance to L. monocytogenes infection in Ucn2-treated mice. TNF production from macrophage in response to HKLM stimulation was suppressed by treatment with Ucn2 in vitro (Fig. 6A), presuming that Ucn2 might suppress activation of macrophages. Activated macrophages exhibit enhanced synthesis of nitric oxide that is involved in bactericidal activity (49). Therefore, we assessed the expression of inducible nitric oxide synthase (iNOS) in spleens of Ucn2-treated mice during listerial infection. However, there was no significant difference in the expression of iNOS between Ucn2-treated and untreated group (date not shown). In vitro study using murine macrophage cell line also showed no significant difference in the expression of iNOS between Ucn2-treated and untreated group (data not shown). This result suggested that Ucn2 might not affect on iNOS activation in macrophages.
In a signaling pathway, IFN activates STAT-1 (36). Herein STAT-1 expression was down-regulated in the Ucn2-treated mice (Fig. 5, A, C, D, and E), compared with the PBS-treated mice in both mRNA and protein levels, suggesting that down-regulation of IFN production might be involved in the suppressive effect on host resistance to L. monocytogenes infection.
IL-10 is known to mediate antiinflammatory responses. IL-10 inhibits the production of proinflammatory cytokines such as IL-1, IL-6, and TNF in cutaneous inflammation (50), and IL-10 down-regulates IL-12 production from dendritic cells (51). Studies on experimental inflammation models have shown that IL-10 suppresses the production of TNF and IL-6 from macrophages, synoviocytes, and T cells (52). Moreover, IL-10 seems to mediate CD25+CD4+ T cell-mediated immunosuppression in autoimmune or inflammatory disease (53, 54). IL-10 plays a detrimental role in host resistance to L. monocytogenes infection (29, 30, 31, 55). In this study, IL-10 production induced by L. monocytogenes infection was up-regulated by Ucn2 treatment in vivo (Fig. 3C). Neither CRF nor Ucn showed a significant effect on IL-10 production (data not shown). Macrophages can produce IL-10 in response to bacterial infections (56, 57). In vitro study showed that IL-10 production from macrophages was also up-regulated by Ucn2 treatment (Fig. 6B). Although turnover of Ucn2 is very short, the up-regulation of IL-10 in the early phase of infection might cause the sustained suppression of host resistance against L. monocytogenes infection. Ucn2 revealed no suppressive effect on host resistance to L. monocytogenes infection when IL-10–/– mice were used (Fig. 4A). Moreover, the depletion of endogenous IL-10 by injection of anti-IL-10 mAb also canceled the suppressive effect of Ucn2 on host resistance to L. monocytogenes infection (Fig. 4B). These results suggested that the effect of Ucn2 is mediated by IL-10, not by a direct effect of Ucn2. In a signaling pathway, IL-10 activates STAT3 (37). In this study, STAT3 expression was up-regulated in Ucn2-treated mice (Fig. 5, B, C, and F). pSTAT-3 was also up-regulated in Ucn2-treated mice, compared with PBS-treated mice (Fig. 5, C and G). pSTAT3 expression was also up-regulated in Ucn2-treated macrophages in vitro (Fig. 6C). These results suggested that the suppressive effect of Ucn2 on host resistance to L. monocytogenes might be mainly mediated by IL-10 and sustained up-regulation of IL-10 production could suppress host resistance against L. monocytogenes infection.
Finally, our present results demonstrated that CRF family peptide Ucn2 converts a sublethal infection with L. monocytogenes to the lethal infection in mice through up-regulation of IL-10. Our study supported that the existence of close interaction between endocrine system and immune system during bacterial infection.
Footnotes
This work was supported in part by a grant-in-aid for general scientific research (13670261 and 16590352) provided by the Japanese Ministry of Education, Science, Sports, and Culture.
Abbreviations: AS-30, Antisauvagine-30; CFU, colony-forming unit; CRF, corticotropin-releasing factor; CRFR, CRF receptor; CT, threshold cycle; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; HKLM, heat-killed L. monocytogenes; IFN, interferon; iNOS, inducible nitric oxide synthase; mAb, monoclonal antibody; p, phosphorylated; STAT, signal transducers and activators of transcription; Ucn, urocortin.
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