ABSTRACT
Melanin-concentrating hormone (MCH) was initially identified in mammals as a hypothalamic neuropeptide regulating appetite and energy balance. However, the wide distribution of MCH receptors in peripheral tissues suggests additional functions for MCH which remain largely unknown. We have previously reported that mice lacking MCH develop attenuated intestinal inflammation when exposed to Clostridium difficile toxin A. To further characterize the role of MCH in host defense mechanisms against intestinal pathogens, Salmonella enterocolitis (using Salmonella enterica serovar Typhimurium) was induced in MCH-deficient mice and their wild-type littermates. In the absence of MCH, infected mice had increased mortality associated with higher bacterial loads in blood, liver, and spleen. Moreover, the knockout mice developed more-severe intestinal inflammation, based on epithelial damage, immune cell infiltrates, and local and systemic cytokine levels. Paradoxically, these enhanced inflammatory responses in the MCH knockout mice were associated with disproportionally lower levels of macrophages infiltrating the intestine. Hence, we investigated potential direct effects of MCH on monocyte/macrophage functions critical for defense against intestinal pathogens. Using RAW 264.7 mouse monocytic cells, which express endogenous MCH receptor, we found that treatment with MCH enhanced the phagocytic capacity of these cells. Taken together, these findings reveal a previously unappreciated role for MCH in host-bacterial interactions.
INTRODUCTION
Melanin-concentrating hormone (MCH) was initially described in 1983 by Kawauchi to be secreted from the salmon pituitary gland as a stress response to a predator (1). As its name indicates, MCH lightens fish skin color by segregating the pigment-carrying granules in melanocytes. It is highly conserved between species, as human and salmon MCH sequences are almost identical. MCH in mammals has been identified as one of the orexigenic neuropeptides in the hypothalamus, by demonstrating that intracerebroventricular injection of MCH stimulates feeding in rats (2). Subsequent studies revealed that MCH-deficient mice are leaner and resist diet-induced obesity (3–5). Despite the well-established role of MCH in energy balance, the distribution of MCH receptor 1 (MCHR1) not only in brain but also in peripheral tissues, including the gastrointestinal tract, indicates additional functions for MCH, which remain largely unexplored.
Our group has recently reported that mice deficient for MCH develop attenuated 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis (6), as well as attenuated Clostridium difficile toxin A-mediated enteritis. Moreover, in vitro studies revealed direct proinflammatory effects of MCH on colonic epithelial cells, resulting in upregulation of cytokine expression (6, 7). In mice, MCHR1 transcripts have been detected in spleen, thymus, lymph nodes, bone marrow, and blood, and fluorescence-activated cell sorter (FACS) analysis suggested that human CD3+ cells (T cells), CD19+ cells (B cells), and CD14+ cells (monocytes) express MCHR1 (8). Treatment of PBMCs with MCH resulted in upregulation of intracellular calcium and cyclic AMP (cAMP) levels (9). In other experiments, MCH inhibited the proliferation of in vitro-activated T cells and regulated interleukin-2 (IL-2) production (10). Furthermore, MCH has been shown to be induced by Th2-type cytokines in more than one cell type, suggesting its involvement in allergic diseases like asthma (11, 12). In rainbow trout, treatment with MCH was found to stimulate in vitro the phagocytic activity of leukocytes (13). However, the role of MCH in mammalian host responses against intestinal pathogens has not been investigated.
In the present study, we used a mouse model of Salmonella-induced enterocolitis to assess the impact of MCH deficiency on host-bacterial interactions. To our surprise, and in contrast to previously described proinflammatory effects of MCH in the intestine (6, 7), we observed increased inflammatory responses and mortality in MCH knockout (MCH-KO) mice infected with Salmonella, associated with impaired macrophage recruitment in the intestine and delayed bacterial clearance. Consistent with the in vivo findings, in vitro experiments using RAW 264.7 mouse monocytic cells revealed directs effects of MCH on monocyte migration and phagocytosis.
MATERIALS AND METHODS
Mice.MCH-KO mice were backcrossed onto the C57BL/6J genetic background for at least 11 generations. In this study, MCH-KO mice and their wild-type (WT) littermates were generated by heterozygous breeding and 5-month-old male mice were used. Mice were housed singly and maintained in a controlled environment with a 12-h light/dark cycle and ad libitum access to food and water. All studies were approved by the Institutional (MGH) Subcommittee on Research Animal Care.
Salmonella-induced enterocolitis.To induce enterocolitis, a streptomycin-resistant, invasion-competent strain of wild-type Salmonella enterica serovar Typhimurium (SL1344) was used. Mice were pretreated with 20 mg of streptomycin administered by gavage as previously described (14). Twenty-four hours later, mice received, by gavage, 8.8 × 107 CFU (5-day survival experiment, n = 16 per group), 4.4 × 107 CFU (24-h bacterial load experiment, n = 5 per group), or 1.35 × 107 CFU (48-h immune response experiment, n = 8 to 9 per group) of Salmonella. At 24 h postinfection, the bacterial load in blood, as well as in spleen and liver homogenates, was assessed based on the number of Salmonella colonies formed in LB agar plates supplemented with 50 μg/ml of streptomycin.
Histological and molecular assessment of inflammation.For the histological assessment of intestinal inflammation in mice infected with Salmonella, we followed the protocol described by Barthel et al. (14). In this model, mice pretreated with streptomycin and then administered Salmonella orally develop more pronounced inflammation in their cecum and a lesser degree of inflammation of their colon at 48 h postinfection, while the small intestine is not involved. Cecal inflammation was evaluated at the 48-h time point in hematoxylin-and-eosin (H&E)-stained histological sections based on the scoring system described in reference 14. Briefly, sections were scored (0 to 3) by a pathologist (R.N.) in a blinded manner for the following parameters: (i) submucosal edema, (ii) polymorphonuclear leukocyte (PMN) infiltration in the lamina propria, and (iii) epithelial integrity. The total score represents the sum of the above-described individual scores. In F4/80-stained (1:10,000; Serotec) cecal sections, the numbers of immunoreactive cells morphologically consistent with macrophages per high-power (×400) field were calculated (3 fields with the greatest density).
Frozen samples from cecal tissue were weighed and homogenized (50 mg tissue/ml) on ice in a phosphate-buffered saline (PBS) buffer containing 0.5% Triton X-100 and a protease inhibitor cocktail (Roche). Following two cycles of freeze-thaw, the tissue lysate was centrifuged to remove debris and insoluble material, and the supernatant was collected for cytokine measurements. Cecal levels of IL-1β, tumor necrosis factor alpha (TNF-α), CXCL1 (KC), IFN-γ, and serum levels of CXCL1 were determined by enzyme-linked immunosorbent assay (ELISA) (R&D Systems) and normalized by the total protein content (bicinchoninic acid [BCA] protein assay).
Thioglycolate-induced cell migration.WT and MCH-KO mice (n = 4 to 5 per group) were injected intraperitoneally with 1.5 ml of 3% Brewer's thioglycolate medium (Difco). Mice were sacrificed after 48 h, and the peritoneal exudate was collected by washing the peritoneal cavity with 10 ml of cold PBS. Cell differentials were determined by performing cytospins with Diff-Quick staining, and the number of macrophages, as well as the number of total cells per high-power field (4 fields from remote areas for each mouse), were counted.
Expression and regulation of MCHR1 in monocytic cells.RAW 264.7 murine monocytic-like cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% antibiotic/antimycotic and 10% fetal bovine serum (FBS). Peripheral blood from a healthy donor was kindly provided by the Boston Children's Hospital Blood Bank. Monocytes were separated using the EasySep (negative selection) human monocyte enrichment kit (StemCell Technologies) according to the manufacturer's instructions and cultured for 6 days in RPMI 1640 medium (supplemented with 10% FBS and 1% antibiotic/antimycotic).
For the detection of MCHR1 on monocytes, we used an antibody raised in rabbits, which we have previously functionally characterized by its ability to block MCH-mediated downregulation of intracellular cAMP levels (7). Cells grown on coverslips were fixed with 4% paraformaldehyde. After blocking (protein block; Dako), slides were incubated with a rabbit anti-mouse antibody against MCHR1 (1:200 dilution) at room temperature for 1 h, followed by incubation with a secondary Alexa Fluor 594 donkey anti-rabbit antibody (A-21207, 1:300 dilution; Molecular Probes) for 30 min at room temperature. Slides were mounted using Prolong Gold 4′,6-diamidino-2-phenylindole (DAPI) mounting solution (Invitrogen) and viewed under a Zeiss Axioimager M1 microscope. As a negative control, the primary antibody was omitted in the procedure.
RAW 264.7 cells were cultured in complete medium and treated with 100 ng/ml lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 (Sigma) or vehicle for 4 h. Each condition was tested in 4 independent cultures. MCHR1 mRNA expression in RAW cells was measured by real-time PCR as previously described (6, 7) using a predeveloped assay from Applied Biosystems. Results are normalized by the expression of TATA-binding protein (TBP) and presented as arbitrary units (AU; control = 100).
Phagocytosis assay.Cells were incubated overnight in serum-free medium, followed by incubation with medium containing 10% FBS plus various concentrations of MCH (Bachem) or LPS (100 ng/ml) for 2 h prior to the phagocytosis assay. To evaluate phagocytosis, cells were incubated with fluorescein-labeled E. coli bioparticles for 2 h using the Vybrant phagocytosis kit (Molecular Probes). Each experimental condition was run in 10 replicates, and the fluorescence retained intracellularly was measured at 480 nm excitation and 520 nm emission using a microplate reader (Versamax; Molecular Devices).
Cell migration assay.We used a transwell-based assay (Cytoselect 24-well cell migration colorimetric assay, 8-μm pore size; Cell Biolabs) to assess the migration of RAW 264.7 cells in response to MCH. Briefly, in a checkerboard design, cells that had been serum starved overnight were diluted at a concentration of 1 × 106 cells per ml of DMEM supplemented with 1 μM MCH or vehicle, and 300 μl of the cell suspension was placed inside each insert. To the lower well of the migration plate, we added 500 μl DMEM supplemented with 1% FBS, 1% FBS plus 1 μM MCH, or 10% FBS as a positive control. Cells were incubated at 37°C for 16 h. Each condition was run in four replicates.
Statistical analysis.For comparisons between WT and MCH-KO mice, as well as MCH versus vehicle treatments, we used a nonparametric test (Mann-Whitney). Differences in survival rates were evaluated by chi-square test. Results are expressed as mean ± standard error of the mean (SEM) unless otherwise indicated. Statistical analysis was performed using StatView (SAS Institute). Geometric means were calculated using an on-line calculator.
RESULTS
MCH-deficient mice are more susceptible to Salmonella infection.MCH-knockout mice and their wild-type littermates were infected with Salmonella, and bacterial load was measured in blood and various tissues after 24 h (Fig. 1A). We found that mice that were deficient in MCH had higher bacteremia (3,192 CFU/ml versus 165 CFU/ml, MCH-KO versus WT, respectively; P = 0.047), as well as higher bacterial load in the liver (99,544 CFU/mg tissue versus 3,246 CFU/mg tissue; P = 0.029) and the spleen (192,096 CFU/mg tissue versus 12,657 CFU/mg tissue; P = 0.0758). Moreover, and consistent with the increased bacterial burden, at 5 days postexposure to Salmonella, only 50% of the MCH-KO animals (8/16) had survived, compared to 94% of the wild-type controls (15/16; P = 0.015) (Fig. 1B).
Increased susceptibility to Salmonella infection in the absence of MCH. MCH knockout mice (MCH-KO) and their wild-type littermates (WT) were infected with Salmonella Typhimurium by gavage following a single dose of streptomycin. (A) At 24 h postinoculation, mice were sacrificed and bacterial loads in their blood and in spleen and liver homogenates were assessed by the number of bacterial colonies formed in streptomycin-agar plates. Results are presented on a logarithmic scale, and horizontal lines represent the geometric mean of each group. (B) The percentages of MCH-KO and WT mice surviving the Salmonella infection were evaluated at day 5 postinfection. *, P < 0.05, MCH-KO versus WT.
More-severe inflammation in MCH-deficient mice infected with Salmonella.We used a second cohort of mice (n = 8 or 9 per group) to characterize the inflammatory responses of the MCH-KO mice to Salmonella infection at the histological and molecular level. At 2 days postinfection, MCH-KO mice had lost more body weight than the WT group (3.126 ± 0.304 g versus 2.040 ± 0.388 g, respectively; P = 0.0417) (Fig. 2A), again an indication of more severe infection in this group and consistent with the bacterial load and survival results presented in Figure 1. Additional indications of more-severe inflammation in the MCH-KO mice include reduced cecal length (1.308 ± 0.068 cm versus 1.632 ± 0.108 cm, MCH-KO versus WT, respectively; P = 0.0275) (Fig. 2B) and cecal weight (0.264 ± 0.027 g versus 0.325 ± 0.006 g, MCH-KO versus WT, respectively; P = 0.0444) (Fig. 2C). Indeed, upon macroscopic examination, the cecum of MCH-KO mice appeared smaller and with increased thickness of the intestinal wall (Fig. 2D). At the microscopic level, we observed extended epithelial cell destruction and lack of regeneration in the MCH-KO mice, along with pronounced submucosal edema and substantial neutrophil infiltration in the lamina propria (Fig. 2E). Upon scoring of inflammation, MCH-KO mice had a score of 6.333 ± 0.972, while the WT mice had an average score of 3.444 ± 0.689 (P = 0.0275) (Fig. 2F).
More-severe inflammation in MCH-KO mice infected with Salmonella. MCH knockout mice (MCH-KO) and their wild-type littermates (WT) were infected with Salmonella Typhimurium and evaluated at 48 h postchallenge. (A) In response to Salmonella infection, MCH-KO mice lost significantly more weight than WT mice. (B and C) Cecal length (B) and weight (C) were reduced in MCH-KO mice infected with Salmonella as a result of inflammation. (D) Gross appearance of the cecum in WT and MCH-KO mice infected with Salmonella. WT mice receiving no treatment served as a control. (E) Representative H&E-stained sections of cecal tissue from Salmonella-infected WT and MCH-KO mice show more severe histopathology in the MCH-KO mice. (F) Histological scoring of cecal inflammation in MCH-KO and WT mice infected with Salmonella. Each sample was scored (0 to 3) for epithelial integrity, PMN infiltration, and submucosal edema, and the sum of these scores was calculated. *, P < 0.05, MCH-KO versus WT mice.
Impaired macrophage recruitment in MCH-deficient mice infected with Salmonella.Paradoxically, and despite the fact that MCH-KO mice developed more severe cecal inflammation after Salmonella exposure, evaluation of F4/80-stained cecal sections corresponding to 48 h postinfection (Fig. 3A) revealed that, while MCH-KO mice had higher numbers of immune cells in their submucosa (193 ± 36.1 cells versus 80.1 ± 7.8 cells per high-power field, MCH-KO versus WT, respectively; P = 0.0056), the percentage of macrophages among these cells was almost 2-fold lower in MCH-KO mice (13.7% ± 3.5% versus 27.7% ± 4.06%, MCH-KO versus WT, respectively; P = 0.0161) (Fig. 3B).
Impaired macrophage recruitment in MCH-deficient mice infected with Salmonella. MCH-KO mice and their WT littermates were infected with Salmonella and evaluated at 48 h postinoculation. (A) Representative images of F4/80-stained cecal sections (20× objective) are shown on the left. The areas marked with boxes are shown in a 10-fold magnification of the original picture on the right. (B) The total number of immune cells infiltrating the submucosa and the number of macrophages were quantified in 3 high-power fields of F4/80-stained cecal sections, and the percentage of macrophages among the infiltrating immune cells was calculated. *, P < 0.05, MCH-KO versus WT mice.
Increased intestinal and systemic cytokine levels in MCH-KO mice infected with Salmonella.At 48 h postinfection, MCH-KO mice, compared to their WT littermates, had elevated levels of cecal IFN-γ (939.04 ± 197 pg/mg tissue versus 321.78 ± 76.99 pg/mg tissue; P = 0.029) (Fig. 4A), CXCL1 (keratinocyte-derived chemokine) (355.29 ± 58.45 pg/mg tissue versus 160.31 ± 29.98 pg/mg tissue; P = 0.034) (Fig. 4B), TNF-α (163.75 ± 27.24 pg/mg tissue versus 83.95 ± 10.64 pg/mg tissue; P = 0.0343) (Fig. 4C), and IL-1β (484.35 ± 97.07 pg/mg tissue versus 227.4 ± 42.14 pg/mg tissue; P = 0.043) (Fig. 4D). Moreover, serum CXCL1 levels were also higher in the MCH-KO mice than in the WT (1,612.154 ± 211.164 pg/ml versus 910.142 ± 119.05 pg/ml, respectively; P = 0.0157) (Fig. 4E). These results are consistent with increased immune cell infiltration in the cecum of MCH-KO mice infected with Salmonella, as shown in Figure 3B. A separate cohort of MCH-KO and WT mice receiving no treatment was used to evaluate baseline levels of the above-named cytokines, which were found to be not statistically different between the two groups (Fig. 4). These findings are consistent with our previous report of similar mRNA expression levels of TNF-α, IFN-γ, and IL-1β in the ileum of MCH-KO and WT mice (7).
Local and systemic cytokine levels in MCH-KO mice infected with Salmonella. Streptomycin-pretreated MCH-KO mice and their WT littermates were infected with Salmonella, and cytokine levels in cecal lysates and serum were evaluated at 48 h postinfection. MCH-KO and WT mice receiving none of the above treatments served as baseline controls. Cecal levels of IFN-γ (A), CXCL1 (B), TNF-α (C), and IL-1β (D) and serum levels of CXCL1 (E) were determined by ELISA. *, P < 0.05, MCH-KO versus WT mice, both infected with Salmonella.
MCHR1 expression and regulation in RAW 264.7 cells.To start dissecting potential direct effects of MCH on monocyte function, we first examined whether MCHR1 is present in these cells. For this experiment, we used both human monocytes and RAW 264.7 mouse monocytic cells. We were able to detect MCHR1 immunoreactivity both in human (Fig. 5A, left) peripheral blood monocytes and in the mouse monocytic RAW 264.7 cell line (Fig. 5A, middle). We have previously demonstrated that treatment of human colonocytes with proinflammatory stimuli, such as IL-1β or C. difficile toxin A, could induce the MCHR1 mRNA levels (7, 15). Extending these observations, in the present study, we found significant upregulation of MCHR1 mRNA expression in RAW 264.7 cells in response to treatment with LPS (16,734 ± 2,872 AU versus 100 ± 17 AU, LPS versus vehicle; P = 0.0339) (Fig. 5B).
MCHR1 expression and regulation in monocytes. (A) MCHR1 immunoreactivity (red) in human and mouse monocytes. In the sections serving as negative control (neg. control), the primary anti-MCHR1 antibody was omitted. (B) RAW 264.7 mouse monocytic cells were treated with 100 ng/ml LPS or vehicle, and the mRNA expression of MCHR1 was measured by real-time PCR and expressed as arbitrary units (AU; vehicle treated = 100) normalized by the expression of a housekeeping gene (TBP). *, P < 0.05.
Treatment with MCH promotes phagocytosis by RAW 264.7 monocytes.One of the potential mechanisms that could explain the increased bacterial load in MCH-deficient mice could be defects in bacterial phagocytosis. RAW 264.7 cells were cultured in the presence of MCH or vehicle and subsequently tested for phagocytosis of fluorescein-labeled E. coli. Cells pretreated with MCH exhibited enhanced phagocytosis in a dose-dependent manner. Specifically, among the concentrations that we tested, 1 μM MCH produced the biggest effect on phagocytosis (100 ± 11 versus 233 ± 32 arbitrary phagocytosis units, vehicle versus 1 μM MCH; P = 0.0169) (Fig. 6), consistent with previous reports from groups other than ours of MCH being effective at the 1 μM range (16). A smaller but statistically significant effect was observed using 0.1 μM MCH (137 ± 9 arbitrary phagocytosis units compared to vehicle; P = 0.0022). As expected, fluorescent staining was restricted to the cytoplasm, as shown in Figure 6 at the top. In these experiments, treatment with LPS served as a positive control.
MCH enhances phagocytosis by RAW 264.7 monocytes. Raw 264.7 mouse monocytic cells were treated with various concentrations of MCH or LPS (as a positive control) for 2 h and subsequently incubated with fluorescein isothiocyanate (FITC)-labeled E. coli. (Top) Images under fluorescent microscopy demonstrating the presence of FITC-labeled E. coli bioparticles within the cytoplasm of RAW 264.7 cells in response to MCH or LPS treatment. (Bottom) Phagocytic activity of RAW 264.7 cells treated with MCH was assessed by measuring the fluorescent signal retained within the cells and is expressed as arbitrary units (AU) relative to the results of vehicle-treated cells (control = 100). *, P < 0.05, 1 μM MCH versus vehicle treatment; **, P < 0.005, 0.1 μM MCH versus vehicle.
MCH facilitates migration of monocytes.An alternative to defects in phagocytosis mechanism explaining more-severe inflammation in MCH-KO mice could be impairment in macrophage recruitment. This notion is also supported by data presented in Figure 3. We used two different approaches to test this hypothesis. Peritoneal macrophage recruitment in response to thioglycolate was assessed in MCH-KO mice and their WT littermates. We found an overall trend of reduced total cell content in the exudate of MCH-KO mice, though the differences did not reach statistical significance (57.2 ± 17.1 versus 105 ± 21.4 cells per high-power field, MCH-KO versus WT, respectively; P = 0.1322) (Fig. 7A). However, the absolute number of macrophages was significantly lower in the exudate of MCH-deficient mice (22.2 ± 4.3 versus 54.2 ± 10.5 macrophages per high-power field, MCH-KO versus WT, respectively; P = 0.031) (Fig. 7A), which is consistent with the lower numbers of infiltrating macrophages in the cecum of MCH-KO mice infected with Salmonella, as shown in Figure 3.
MCH promotes migration of monocytes. (A) MCH knockout mice and their wild-type littermates were injected with thioglycolate, and the numbers of total cells as well as macrophages in the peritoneal exudate were evaluated with Diff-Quick staining in cytospins (average of 4 high-power fields per sample). *, P < 0.05, MCH-KO versus WT mice. (B) RAW 264.7 cells treated with 1 μM MCH or vehicle were placed in the inner well of a transwell system, and migration of monocytes toward 1% FBS, 1% FBS plus 1 μM MCH, or 10% FBS was assessed by a colorimetric assay. Results are expressed relative to the migration of vehicle-treated cells (control = 100). *, P < 0.05, MCH versus vehicle-treated cells.
To further examine direct effects of MCH on monocytes, we used RAW 264.7 mouse monocytic cells in studies in vitro. The effect of MCH on RAW 264.7 monocyte migration was investigated in a transwell system. A checkerboard design of this experiment allowed us to test both random migration (chemokinesis) and migration toward a chemokine gradient (chemotaxis). We found that MCH increased primarily chemokinesis and to a lesser extent chemotaxis of RAW 264.7 cells (Fig. 7B). Specifically, when added to the insert well with the cells, MCH potentiated migration toward 1% FBS (164 ± 26 versus 100 ± 19, MCH versus vehicle; P = 0.09), 1% FBS plus MCH (193 ± 15 versus 126 ± 15, MCH versus vehicle; P = 0.019), or 10% FBS (224 ± 11 versus 163 ± 20, MCH versus vehicle; P = 0.037).
DISCUSSION
Host defense immune responses are evolutionarily conserved and critical for the organism's survival. In addition to mechanical barriers in the form of the skin and the epithelium lining surfaces exposed to pathogens, such as the gastrointestinal tract, cells of the myeloid lineage, such as tissue macrophages and dendritic cells, play a crucial role in sampling bacterial products through pathogen-associated molecular pattern recognition receptors, presenting bacterial antigens, accumulating to the sites of injury in response to proinflammatory signals, and eliminating bacteria by phagocytosis.
In the present study, infection with Salmonella revealed an impairment of MCH-deficient mice to fight a bacterial infection, suggesting that MCH is implicated in host defense against invasive intestinal pathogens. Furthermore, as a potential underlying mechanism of the in vivo findings, we identified direct effects of MCH on monocytes, resulting in enhanced migration and bacterial phagocytosis. However, the role of MCH on additional host defense mechanisms, for example, modulation of Salmonella invasion of epithelial cells, which also express MCHR1, cannot be excluded (6, 7).
There is a growing list of neuropeptides like MCH that modulate monocyte/macrophage functions (15, 17). For instance, substance P can elicit a variety of responses in monocytes, including differentiation (18), phagocytosis (15), production of proinflammatory cytokines (19), and chemotaxis (20). Furthermore, neuropeptide Y (NPY) regulates phagocytosis by neutrophils and monocytes at multiple levels (21). Despite the plethora of in vitro observations, little is known about the overall significance of neuropeptides in innate immune responses against pathogens such as Salmonella at the organism level. While we describe here that MCH-deficient mice developed a more-disseminated Salmonella infection which was associated with increased mortality under the conditions tested (Fig. 1), mice deficient for NPY or neurokinin-1 (the preferred substance P receptor) were found to be protected and to develop attenuated disease compared to their wild-type littermates (22, 23).
Equally important to MCH enhancement of phagocytosis by macrophages (Fig. 6), we provide both in vivo and in vitro evidence that MCH facilitates migration of monocytes (Fig. 4 and 5). In addition to monocytes, it has been shown in a scratch wound assay that MCH enhances migration of preadipocytes via actin polymerization and cytoskeleton reorganization (16), a mechanism that has been also described for the urotensin II-mediated monocyte chemotaxis (24). These observations suggest that, in addition to its immunomodulatory properties, MCH might also play a role in tissue repair processes. At this point, we can only speculate on the exact cellular pathways utilized by MCH to increase monocyte migration. For instance, in addition to actin filament modification, transactivation of chemokine receptors has been reported for substance P (20) and opiates (25).
In the inflamed intestine, there are several potential sources of MCH. We have previously demonstrated the presence of MCH and its receptor in the enteric neurons of the rat and guinea pig submucosal and myenteric plexus (6). In this context, MCH appears to be one of the important mediators of neuroimmune interactions, along with substance P, vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide, and somatostatin (26, 27). However, these neuropeptides are not released solely by neurons but also from immune cells, including T cells, mast cells, and monocytes, particularly in the presence of LPS or proinflammatory cytokines (28, 29). It is also plausible that, under certain conditions, MCH is produced by monocytes, macrophages, or dendritic cells in response to activation or exposure to inflammatory stimuli, as is the case for substance P, urotensin II, alpha-melanocyte-stimulating hormone (α-MSH), and calcitonin gene-related peptide (27, 29). However, this does not appear to be the case for the RAW 264.7 monocytes used in our in vitro experiments (data not shown).
In aggregate, our findings provide a link between MCH signaling via MCHR1 and macrophage function. Moreover, they suggest an integral role of MCH in host defense against infection with Gram-negative bacteria and, perhaps, additional types of invasive pathogens where phagocytosis is important for their elimination.
ACKNOWLEDGMENTS
We thank Eleftheria Maratos-Flier for providing us with the MCH-KO breeding pairs and Georgios Margonis for help with the thioglycolate experiment.
This study was supported by National Institutes of Health grants R01 DK080058 (E.K.) and R01 AI08900 (B.J.C.), a bridging grant from the American Gastroenterological Association (E.K.), and grant IBD-0165 from the Broad Medical Research Program of The Broad Foundation (E.K.).
The authors have nothing to disclose.
FOOTNOTES
- Received 28 May 2012.
- Returned for modification 22 June 2012.
- Accepted 15 October 2012.
- Accepted manuscript posted online 31 October 2012.
- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
