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Sepsis describes a complex clinical syndrome that results from an infection, setting off a cascade of systemic inflammatory responses that can lead to multiple organ failure and death. Leptin is a 16 kDa adipokine that, among its multiple known effects, is involved in regulating immune function. Here we demonstrate that leptin deficiency in ob/ob mice leads to higher mortality and more severe organ damage in a standard model of sepsis in mice (cecal ligation and puncture, CLP). Moreover, systemic leptin replacement improved the immune response to CLP. Based on the molecular mechanisms of leptin regulation of energy metabolism and reproductive function, we hypothesized that leptin acts in the central nervous system (CNS) to efficiently coordinate peripheral immune defense in sepsis. We now report that leptin signaling in the brain increases survival during sepsis in leptin-deficient as well as in wild-type mice and that endogenous CNS leptin action is required for an adequate systemic immune response. These findings reveal the existence of a relevant neuroendocrine control of systemic immune defense, and suggest a possible therapeutic potential for leptin analogues in infectious disease.
Scattered evidence from clinical experience suggests that the CNS may play a functionally relevant role in maintaining an efficiently coordinated immune system. For example, brain-injured patients admitted to intensive care units have an elevated risk to acquire infections (Dziedzic et al., 2004), and infections are a leading cause of mortality in patients with acute CNS trauma (Meisel et al., 2005). Increased susceptibility to infectious agents is believed to be partly due to transient immunodepression triggered by brain damage (Prass et al., 2003). This concept is further supported by observations suggesting that stroke-induced immunodepression increases the susceptibility to infection, and infection in turn is the most relevant complication in stroke patients (Dirnagl et al., 2007). A series of elegant studies by Tracey and colleagues indicates that the autonomic nervous system, especially the vagus nerve and the cholinergic antiinflammatory pathway, robustly regulates cytokine-mediated damage in local and systemic infectious disease (Czura and Tracey, 2005; Huston et al., 2007; Tracey, 2007). Interestingly, Tracey and colleagues also report that the systemic inflammatory response during endotoxemia is controlled by central muscarinic cholinergic pathways (Pavlov et al., 2006; Pavlov and Tracey, 2006). However, the molecular underpinnings of an obviously fine-balanced interplay between the central nervous and the immune system remain largely unknown.
Leptin is a 16kDa adipocyte derived circulating cytokine (Zhang et al., 1994) known to achieve the vast majority of its biological effects by acting in the CNS (Zhang et al., 1994; Campfield et al., 1995; Halaas et al., 1995; Pelleymounter et al., 1995; Friedman, 2002). Some of the well studied biological roles of leptin are its CNS-mediated participation in the control of food intake, body weight (Campfield et al., 1995; Halaas et al., 1995; Pelleymounter et al., 1995), and reproductive function (Barash et al., 1996; Chehab et al., 1996; Chehab et al., 1997; Bluher and Mantzoros, 2007). However, a considerable body of data also suggests that leptin may be involved in the regulation of immune responses in both rodents and humans (Flier, 1998; Lord et al., 1998; Fantuzzi and Faggioni, 2000; Farooqi et al., 2002; De Rosa et al., 2006; Tilg and Moschen, 2006; Lago et al., 2008). Leptin can functionally activate human monocytes in vitro by inducing the production of cytokines such as TNF-α and IL-6 (Santos-Alvarez et al., 1999). Lord et al. first reported that leptin has a specific effect on T-lymphocyte responses by differentially regulating the proliferation of naive and memory T cells. Leptin increased Th1 and suppressed Th2 cytokine production (Lord et al., 1998). The possibility that these potent effects of leptin on the immune system is a result of actions in the CNS rather than directly on immune cells has been suggested (Flier, 1998; Lord et al., 1998), but not yet rigorously tested.
Human congenital leptin deficiency is rare with less than 20 patients reported worldwide to date (Ozata et al., 1999; Farooqi et al., 2002). The phenotype is characterised by severe early onset obesity, hypogonadatropic hypogonadism and an increased frequency of infections. T-cell number and function is impaired in patients with congenital leptin deficiency with a decrease in CD4+ T-cells, reduced T-cell proliferation to a number of antigens and a predominant Th2 cytokine response (Farooqi et al., 2002). These findings are consistent with the T cell responses seen in leptin deficient ob/ob mice and are normalised when therapeutic doses of recombinant human leptin are administered to these patients (Farooqi et al., 2002).
Sepsis, which is defined as a systemic inflammatory response syndrome (SIRS) that occurs during infection (Santos-Alvarez et al., 1999), is associated with several clinical conditions and high mortality rates (Takahashi et al., 2004). This definition of SIRS, however, does not encompass fully the immunologic derangements that accompany the septic process. As sepsis progresses, immunodepression can become severe, leaving an already vulnerable patient ill-equipped to fight off the primary or new secondary infections (Hotchkiss and Karl, 2003). With no convincingly effective specific therapies available so far, the predominantly supportive treatment does not reduce the death rate of sepsis significantly (Zanotti and Kumar, 2002).
Here we demonstrate, using a well-established murine model of sepsis, that leptin action in the CNS optimizes the immune response and increases survival in normal as well as in leptin-deficient mice. We also demonstrate that leptin-deficient ob/ob mice are highly susceptible to sepsis and have more severe organ damage than controls. These studies, using pharmacological as well as genetic approaches, suggest that leptin-initiated neuroendocrine pathways play an important role in the functional coordination of the systemic immune response.
Male C57BL/6J wild type (WT, 6–10 weeks-old) and leptin-deficient C57BL/6J-ob/ob (ob/ob) as well as leptin receptor deficient mice C57BL/6J-db/db (db/db) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained under pathogen-free conditions. The mice had 12 backcrosses on the C57BL/6J strain. All mice were kept in standard environmental conditions (lights on from 7am to 7pm) and were fed with a commercial pelleted diet and water ad libitum.
Male C57BL/6J wild type mice between 8–10 weeks of age (Jackson Laboratories) were fed with high fat diet (58 kcal% fat w/sucrose; purchased from Surwit Diet, Research Diets, NJ, USA).
Neuron-specific enolase (NSE)- cre and Lepr(flox/flox) mice were generated as reported previously (Kowalski et al., 2001; Chua et al., 2004; de Luca et al., 2005) and were fed with a commercial pelleted diet and water ad libitum.
All experiments involving animals were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati.
Male mice between 8 to 10 weeks of age (21 to 29 grams (WT) and 49 to 57 grams (ob/ob)) were utilized. Polymicrobial sepsis was induced similarly as described (Murphey et al., 2004). Briefly, the cecal ligation and puncture (CLP) operations were always performed between 8 am and 1 pm. Mice were anesthetized by 2.5% isoflurane in oxygen via facemask. The skin was shaved and disinfected. After a 1-cm laparatomy (midline incision through the linea alba while avoiding injury to the abdominal vasculature), the latter 80% of the cecum was ligated with a 3-0 silk tie (Ethicon, Inc., Somerville, NJ, USA) and punctured once on the anti-mesenterial side with a 23-gauge needle. A small amount of the bowel contents was extruded through the puncture hole in order to assure a full thickness perforation. Care was taken not to obstruct the bowel, and this was tested after the animals' death. The cecum was replaced in its original location and the midline incision closed by two-layer suture with 4-0 silk (Schein, Inc., Melville, NY, USA). The animals were resuscitated with one milliliter of sterile saline subcutaneously and kept on a heating blanket with additional oxygen supply for 1 hour. Sham-treated controls underwent the same surgical procedures (i.e., laparotomy and resuscitation), but the cecum was neither ligated nor punctured.
Recombinant murine leptin (Henry et al., 1999) was replaced intraperitoneally in ob/ob and WT mice (1 μg/g of body weight) at 9a.m. and 6 p.m. prior to and following CLP for a total of four injections prior to blood, peritoneal lavage and organ harvest. This leptin replacement protocol has been shown to achieve circulating leptin levels (Lord et al., 1998) and was chosen to incorporate the range of serum levels measured in humans (Considine et al., 1996).
For experiments involving chronic intracerebroventricular (icv) leptin administration, 8–10 week-old male WT and ob/ob mice were anesthetized with 2.5% isoflurane in oxygen for icv-minipump implantation. A cannula was positioned in the right lateral cerebral ventricle (coordinates: anteroposterior = −0.07 mm to bregma; lateral = −0.12 mm to bregma and dorsoventral = −0.2 mm to the cranial calota) fixed to the skull with cyanocrylate and connected via a polyethylene catheter to a subcutaneous (sc) osmotic mini-pump (Alzet Osmotic Pumps; Durect Corp., Cupertino, CA) delivering leptin or vehicle (isotonic saline) (Nogueiras et al., 2007). Following surgery, the animals received a single dose of 0.28 mg/kg buprenorphine (Buprenex; Reckitt Benckiser Healthcare). Leptin was infused at a concentration of 1 μg/day for 14 days. CLP was induced after a 3-day surgery recovery. For experiments involving peripheral leptin administration (1 μg/day), 8–10 week-old male ob/ob mice were randomized and anesthetized with 2.5% isoflurane in oxygen. Osmotic minipumps were implanted subcutaneously in the upper back, delivering leptin or vehicle at a rate of 0.2 μl/h for 14 days. Following surgery, the animals received a single dose of 0.28 mg/kg buprenorphine.
For all experiments the osmotic minipumps were filled the evening before surgery and primed in a water bath overnight at 37 °C.
For intracerebroventricular leptin administration, 8-month-old, male C57BL/6 or ob/ob mice purchased from The Jackson Laboratory were housed in individual cages under conditions of controlled temperature (23°C) and illumination (12 h light/dark cycle). They were allowed ad libitum access to water and pelleted low-fat laboratory chow. Food intake and body weight were measured one day prior to surgery and daily during the experimental phase in all groups. Blood-plasma was also collected in all groups at these time points. Mice were anesthetized via inhalation of isofluorane and maintained in the anesthetic plane with constant administration throughout the procedure. Brain infusion cannulae were stereotaxically placed with their tips in the lateral cerebral ventricle using the following coordinates: 0.7 mm posterior to bregma, 1.2 mm lateral to the midsagittal suture, and to a depth of 2.0 mm, with bregma and lambda at the same vertical dimension. Cannulae were fixed to these coordinates using veterinary-grade glue. A catheter tube (ALZET Brain Infusion Kit 3; Alzet Osmotic Pumps; DURECT) was connected from the brain infusion cannula to an osmotic minipump flow moderator (model 1003D; Alzet). A subcutaneous pocket on the dorsal surface was created using blunt dissection, and the osmotic minipump was inserted. The incision was closed with veterinary-grade glue, and mice were kept warm until fully recovered. Mice were then infused with either saline, or Leptin (1.0 μg/d, Provider) for 7 d. After surgery, all mice received a single subcutaneous dose of 0.28 mg/kg buprenorphin (Buprenex; Reckitt Benckiser Healthcare). At the termination of all of the experiments, the animals were euthanized by decapitation, trunk blood was collected, and several tissues were removed and stored at −80°C for later analysis. Corticosterone Analysis: Corticosterone levels were measured in plasma from all groups. Daily measurements were obtained from tail bleedings collected in EDTA coated capillary tubes (Sarstedt AG & Co., Nümbrecht, Germany). Final sample made from trunk blood obtained at time of euthanization in the presence of EDTA. Samples were centrifuged at 4500 rcf for 15 minutes at 4 °C, and plasma was transferred to fresh tube. Samples were diluted 1:200 and analyzed via radiolabeled immunoassay (MP Biomedicals, Solon, OH).
Cells were resuspended in fluorescent-activated cell sorting (FACS) buffer (PBS with 1% bovine albumin and 0.1% azide). Nonspecific binding to cells was controlled by adding 5% rat serum (Invitrogen, Carlsbad, CA) and 1 μg/tube of Fc Block (BD Pharmingen, Franklin Lakes, NY) to the FACS buffer. Cells were stained in a five-color configuration using Pacific Blue-, FITC-, phycoerythrin (PE)-, APC-, or Alexa Fluor 700-labeled antibodies.
Myeloid cells were surface-stained with the following antibodies: Alexa Fluor 700-labeled CD11b (clone M1/70.15, BD Pharmingen, Franklin Lakes, NY); Alexa 488-labeled anti neutrophil (clone: 7/4, Serotec); and APC-labeled Gr-1 (clone: RB6-8C5, BD Pharmingen, Franklin Lakes, NY). For ph-p38 staining, surface stained samples were washed twice with PBS and fixed in 2% paraformaldehyde for 5 min at 37°C. Samples were then washed twice with FACS buffer. The cell pellet was cooled for 1 min on ice, then 1 ml of 95% Methanol was added to each sample while vortexing. Samples were kept on ice for additional 30 min and incubated overnight at −20°C. Then samples were washed with FACS buffer and stained with ph-p38 for 20 min at 4°C. Samples were washed thoroughly again with FACS buffer and run on a Becton Dickinson LSR.
Bacterial counts were performed on aseptically harvested blood by cardiac puncture and peritoneal fluid. Peritoneal fluid was harvested from mice by peritoneal lavage after aseptic preparation of the abdominal wall followed by injection of 9 ml of sterile saline into the peritoneal cavity and aspiration of peritoneal fluid. Samples were serially diluted in sterile saline and cultured on tryptic soy agar pour plates. Plates were incubated at 37°C for 24 hours and colony counts were performed.
The peritoneal fluid was harvested 24 h after CLP and mixed with fluorescently labeled (Alexa Fluor 488) E. coli and opsonizing reagent (Molecular Probes, Carlsbad, CA). This suspension was incubated at 37 °C for 1 h. Following the incubation period, the excess bacteria were removed by washing three times with FACS-buffer. Furthermore, to quench the fluorescence of adherent bacteria, trypan blue was added after the first acquisition and 1 min before the second acquisition. Quenching with trypan blue reduced the FITC fluorescence of adherent bacteria by excitation energy transfer (Szollosi et al., 1984; Hed, 1986).
Core temperature was measured rectally 24 h before and after CLP using a Fisher thermometer (Fisher Scientific, Pittsburgh, PA).
Levels of aspartate transaminase (AST), alanine transaminase (ALT) and blood urea nitrogen (BUN) were measured in serum using a human assay system.
All samples were well mixed with equal volume of Laemmli-buffer (0.125 M Tris-Cl −40% sodium dodecyl sulfate −20% glycerol, with 5% β-mercaptoethanol). Mixed samples were denaturated at 60°C for 20 min. Protein extracts and standard markers (BioRad, Hercules, CA) were resolved on a NuPage 12% SDS-polyarcylamide gel electrophoresis gel (Invitrogen, Hercules, CA) with 200 V for 40 minutes and transferred electrophoretically onto a nitrocellulose membrane (Millipore, Bedford, MA) under electrical conditions of 75 V per membrane for 90 minutes. The membranes were blocked with PBS containing 5% nonfat milk powder for 60 minutes and incubated with monoclonal primary antibody at 4°C overnight. P38, ph-p38 and actin protein were detected using a monoclonal antibody (Cell signaling technology, INC., Danvers, MA, USA and Sigma Chemical Co., St. Louis, MO, USA). After washing in T-PBS, membranes were incubated in secondary FITC-antibody for 120 minutes and washed thoroughly again in T-PBS. Membranes were then incubated in tertiary anti-FITC antibody for 60 minutes and again washed with T-PBS. Membranes were then placed in enhanced chemiluminescence Western blotting detection reagent (ECF from GE Healthcare, USA) for 10 minutes. The density of the bands was quantified by using a computerized imaging system (Alpha EaseSC).
Peritoneal fluid was harvested from mice by peritoneal lavage after aseptic preparation of the abdominal wall followed by injection of 9 ml of sterile saline into the peritoneal cavity and aspiration of peritoneal fluid. Serum was collected by cardiac puncture. IL-6, KC, MIP-2, MCP-1, G-CSF, IL-10 levels in the peritoneal fluid and serum were analyzed using ELISA according to the manufacturer's protocol (IL-6, IL-10 purchased from BioSource Inc., Camarillo, CA; MCP-1, MIP-2 purchased from PeproTech Inc., Rocky Hill, NJ; KC, G-CSF purchased from R&D Systems, Minneapolis, MN).
Plasma insulin and resistin levels were measured by RIA as described previously (Lopez et al., 2006; Nogueiras et al., 2007) using reagents provided in commercial kits (LINCO Research,Inc., St. Charles, MO).
Kidney tissues were fixed in 10% formalin and then embedded in paraffin for light microscopy. Sections were stained with hematoxylin and eosin (H&E) for histological examination.
Quantitative data are presented as mean ± standard error of the mean (S.E.M). Statistical comparisons were performed using Kaplan Meier LogRank (survival) and Student's t-test (2 groups). StatView (SAS Institute) and GraphPad Prism 3.0 were utilized for statistical analyses. A value of p ≤ 0.05 was considered statistically significant.
To date studies in humans have provided evidence for the effects of leptin on T-cell mediated immune responses. However, in addition, it is notable that of our patients with congenital leptin deficiency (n=12), four have died of sepsis. Although the precise details of the infectious episode in each case are unknown, and there may be additional factors that contribute to the untimely death of these patients, in all of the cases a failure to respond to conventional antimicrobial therapy was reported. We have similarly seen an increased incidence of sepsis and sepsis-related mortality in patients with homozygous loss of function mutations in the leptin receptor (5 of the 16 patients have died). However, homozygous mutations in the Melanocortin 4 receptor, a downstream target of leptin action in the brain, are not associated with increased sepsis and sepsis related mortality despite a comparable degree of obesity (no deaths amongst 16 patients).
To explore whether leptin controls systemic immune defense mechanisms in a functionally relevant manner, we initially asked whether leptin deficiency affects survival in sepsis by using an established murine model of sepsis (cecal ligation and puncture, CLP). CLP is a clinically relevant and acceptable model of acute septic peritonitis that has been used to study the systemic response to infection (Wichterman et al., 1980). CLP closely resembles the clinical situation of bowel perforation, inducing peritonitis due to mixed intestinal flora.
The survival of leptin-deficient (ob/ob) mice and WT control mice was assessed over a 10-day period following CLP. Ob/ob mice had a higher mortality than wild type mice (p = 0.0127, Fig. 1a). Indeed, all ob/ob mice died within 72 hours in response to CLP, while 30% of the WT control mice survived that period. Consistent with that observation, the mean survival time was decreased in ob/ob mice (44.4 ± 3.8 h, n = 20, p=0.025) compared to WT mice (66.1 ± 9.0 h, n = 17). To determine whether increased mortality after CLP in ob/ob mice was secondary to their higher body fat mass, we also assessed survival after CLP-induced sepsis in body weight-matched diet-induced obese (DIO) C57/BL6 mice maintained on a high-fat diet. However, despite a comparably increased body weight as ob/ob mice, the DIO mice had a survival of 55% within a 10-day period following CLP. Their survival rate was significantly higher than that of both lean WT mice and ob/ob mice (p = 0.002 and 0.028; Fig. 1a), implying that if anything, the obesity of ob/ob mice might have been anticipated to help protect them from septic mortality.
Obesity is linked to insulin resistance, and elevated plasma insulin and resistin levels are important surrogate hallmarks of insulin resistance (Emilsson et al., 1997; Rajala et al., 2004). As expected, we found higher circulating insulin and resistin levels in ob/ob mice (P<0.01), but there were no differences in pH, bicarbonate (HCO3−), base deficit (BE), hematocrit (Hct) or hemoglobin (Hb) between ob/ob and WT mice 24 hours after CLP (data not shown). Circulating interleukin 6 (IL-6) levels are predictive of mortality outcome in human sepsis as well as in the CLP mouse model (Remick et al., 2002). Consistent with our observations on survival, IL-6 was significantly (p = 0.0005) higher in ob/ob mice as compared to the WT or the DIO mouse group (Fig. 1b). None of the sham control mice showed increased cytokine levels (data not shown). These data suggest that endogenous leptin may protect against sepsis by modulating proinflammatory agents. Another independent predictor of mortality in sepsis is hypothermia (Pittet et al., 1996). However, ob/ob mice showed a similar drop in core temperature following CLP as WT mice suggesting that decreased survival in leptin-deficiency is unlikely to be a consequence of altered thermogenesis (data not shown). Since the measured systemic levels of the proinflammatory agent IL-6 suggested that leptin may directly promote immune defense against bacteria, we next determined whether ob/ob mice have impaired bacterial clearance. Colony counts of viable bacteria present in blood 24 h after CLP (Fig. 1c) were more than 2 orders of magnitude greater in ob/ob mice than in WT mice, while no bacteremia was observed in sham-operated mice (data not shown). Moreover, the number of ob/ob mice that developed bacteremia 24 hours after CLP was increased (93%) relative to WT controls (54%). These findings are consistent with the decreased survival of ob/ob mice in sepsis and suggest that endogenous leptin signaling modulates systemic inflammatory processes as an essential component of the physiological defense against bacteremia.
To determine if the observed systemic involvement of leptin in the control of immune defense is paralleled by local impact at the primary site of infection, bacterial colony counts were made in peritoneal lavage 24 h after CLP. ob/ob mice had more than two orders of magnitude more colonies than WT controls (Fig. 2a). No bacterial load was detected in sham-operated mice (data not shown). Because neutrophils play a key role in eliminating bacteria (Lee et al., 2003), we next determined whether enhanced mortality and impaired bacterial clearance in leptin deficiency may be due to defective neutrophil phagocytosis. Peritoneal neutrophil phagocytosis of opsonized E. coli was significantly (p=0.021) reduced in ob/ob mice as compared to WT controls (Fig. 2b–c). However, there were no differences in numbers of neutrophils at the site of infection in ob/ob mice as compared to WT controls 24 h after CLP (3.23 ± 03 vs. 3.02 ± 0.7, n = 7/group). These data imply that although there is no reduction in the number of neutrophils, leptin deficiency may result in loss of neutrophil function at the site of infection. In order to determine the molecular underpinnings of the decreased neutrophil function, we examined key signal transduction pathways involved in neutrophil function. p38 MAP kinase, an essential component of neutrophil function (Zu et al., 1998; van den Blink et al., 2004), was significantly reduced in peritoneal neutrophils; i.e., ob/ob mice had a significant reduction of phosphorylated p38 MAP kinase 24 h after CLP as compared to septic WT mice as indicated by fluorescence-activated cell sorting (FACS) (Fig. 2d, n = 5/group, >1000 cells interrogated/group). To corroborate this important finding with a second method, we quantified neutrophil p38 MAP kinase phosphorylation after CLP using western blot analysis. Consistent with the observations from FACS analysis, septic obob mice had reduced phosphorylated p38 MAP kinase, with no change of total p38 MAP kinase, compared to WT controls (Fig. 2e).
Collectively, these data indicate that leptin deficiency contributes substantially to mortality and compromises systemic and local immune responding following CLP-induced sepsis. Neutrophil function, but not neutrophil number, is also diminished in the absence of leptin, and this is reflected by reduced phosphorylation of p38 MAP kinase.
The enhanced mortality and insufficient immune response of ob/ob-mice following CLP could be a consequence of irreversible developmental deficiencies due to a lack of leptin during critical periods or alternatively could result from the absence of leptin signaling in sepsis. We therefore asked whether intraperitoneal (i.p.) administration of leptin has a beneficial influence on the immune response in sepsis. Leptin was replaced i.p. in ob/ob and WT mice (1 μg/g of body weight) at 9:00 AM and 6:00 PM prior to and following CLP for a total of four injections prior to blood, peritoneal lavage and organ harvest. This leptin replacement protocol has been shown to achieve circulating leptin levels (Lord et al., 1998) in the range of serum levels measured in humans (Considine et al., 1996). Replacement of leptin completely normalized IL-6 levels in serum and peritoneal lavage of septic ob/ob mice relative to untreated septic ob/ob and WT mice (Fig. 3a). Leptin administration in sepsis even produced a tendency to decrease IL-6 levels in WT mice, as compared to untreated WT mice (Fig. 3a). The anti-inflammatory cytokine IL-10 is an inhibitor of T cell-, monocyte-, and macrophage function. It limits and ultimately terminates inflammatory responses (Moore et al., 2001) and enhances the function of natural killer cells (Mocellin et al., 2003). IL-10 levels however did not differ between septic ob/ob and WT mice with or without leptin administration (Fig. 3b), suggesting that leptin regulates some but not all inflammatory processes.
To determine whether modulation of inflammatory processes by leptin also affects sepsis-induced tissue damage, we measured blood urea nitrogen (BUN) concentration, a surrogate parameter of kidney damage, 24 h after CLP. ob/ob mice had significantly higher BUN levels than the WT group (p=0.001, Fig. 3c), and the number of ob/ob mice that developed a BUN level higher than 75 mg/dl (as a critical sign of acute renal failure) was also increased (10 out of 13; 77%) relative to WT controls (4 out of 14; 29%). Leptin administration normalized mean BUN levels and reduced the number of mice with acute renal failure (2 out of 8; 25%) in both, ob/ob and WT mice.
We corroborated these indirect findings with histopathological examination of the kidney in ob/ob and WT mice 24 h after CLP (Fig. 3d). After CLP, ob/ob mice exhibited enhanced swelling of the cortical tubules, diffuse vacuolar degeneration and focal tubular necrosis, as well as a slightly increased cast formation compared to WT mice. Treatment with leptin decreased cast formation of the cortical tubules and lowered vacuolar degeneration as well as focal necrosis, without much impact on tubular swelling (Fig. 3d). In leptin-treated WT mice the diffuse degeneration and focal necrosis of the tubular epithelium (proximal as well as distal tubules) appeared to be less extensive, while tubular swelling and degeneration was still present (Fig. 3d). Overall, these results indicate that leptin protects against cellular kidney damage in sepsis, at least to some extent.
To examine the level of liver damage after CLP, we measured alanine aminotransferase (ALT), a serum marker of liver cell damage (Amacher, 2002; Ozer et al., 2008), 24 h after CLP. Ob/ob mice had significantly higher ALT levels than the WT group reflecting more liver damage (P<0.05, data not shown). We assessed liver histology in the same mice and found that hydropic degeneration and focal necrosis of hepatocytes was more severe in ob/ob mice than WT controls (data not shown).
Beneficial effects of leptin administration in CLP-induced sepsis could theoretically be a secondary consequence of the hormone's well known catabolic action including reduced body weight. In none of these acute experiments, however, did body weights differ between acutely leptin-treated mice and control mice at any time point (data not shown). In summary, these findings indicate that leptin action appears to reduce the IL-6 response, seems to decrease hepatic markers of liver damage and may reduce renal damage in sepsis.
Bacterial colony counts were significantly (p=0.03) lower in leptin-treated than untreated ob/ob mice 24 h after CLP (Fig. 4a). Leptin treatment of WT mice also decreased bacterial counts as compared to saline-injected WT controls. In control experiments, no bacteremia was observed in sham mice (data not shown). To determine whether the reduced bacterial burden after leptin administration was associated with improved phagocytosis, we compared peritoneal neutrophil phagocytosis of opsonized E. coli in vitro. As depicted in Fig. 4b, phagocytosis of E.coli in sepsis was increased in ob/ob mice with leptin replacement (21%) compared to levels in untreated ob/ob mice. Septic WT mice had only a slight increase in phagocytosis (10%) following leptin administration. The enhanced phagocytosis observed following leptin replacement in ob/ob mice suggests that endogenous leptin controls neutrophil clearing of bacteria.
To further dissect the molecular underpinnings of this observation we screened plasma and peritoneal lavage fluid for monocytes chemotactic protein-1 (MCP-1), keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2) and granulocyte colony-stimulating factor (G-CSF). These chemokines are pivotal in the recruitment and activation of neutrophils and monocytes (Kurihara et al., 1997; Czuprynski et al., 1998). The expression of MIP-2 and KC (both powerful chemoattractants for, and activators of, neutrophils (Chensue, 2001), MCP-1 (monocyte activation and recruitement to the site of infection (Adams and Lloyd, 1997; Lu et al., 1998)) and G-CSF (stimulation of development of progenitors to neutrophils and enhancement of neutrophilic functional activity and differentiation (Nagata et al., 1986; Kamezaki et al., 2005) in serum and peritoneal lavage were determined 24 h after CLP. Ob/ob mice had significantly higher levels of each of these chemokines in both serum and peritoneal lavage relative to septic WT controls (Fig. 4c–f). Administration of leptin to ob/ob mice reduced chemokine levels nearly to the levels seen in WT serum and peritoneal lavage. Leptin administration in WT mice resulted in no change or a slight decrease of chemokine levels relative to the untreated WT group. Interestingly, elevated MCP-1 levels in ob/ob mice was not associated with increased numbers of monocytes at the site of infection. In both WT and ob/ob mice, leptin administration did not change monocyte counts 24 h after CLP (data not shown). Taken together, these results suggest that leptin potently regulates neutrophil function via modulation of several specific chemokines.
The experiments on loss-of-function and gain-of-function described above indicate that endogenous leptin controls systemic inflammation and immune defense to the extent that leptin signaling promotes survival and prevents organ damage in sepsis. However, the organ specificity of essential leptin receptor activation remains unclear. Numerous studies on the role of leptin in metabolism and reproductive biology have shown that action of leptin in the CNS is sufficient to fulfill almost all of its physiological functions. To determine whether leptin's role in the control of immune defense follows a similar paradigm, we implanted icv-minipumps containing leptin (1 μg/24hours) or vehicle (saline, 1 μg/24hours) into ob/ob mice prior to CLP. Leptin replacement into the CNS of ob/ob-mice increased survival rates significantly (p = 0.027) relative to vehicle-infused ob/ob controls (Fig. 5a). Leptin administration into the CNS also significantly decreased serum IL-6 of septic ob/ob mice relative to untreated septic ob/ob 24 h after CLP (p = 0.028; Fig. 5b) but did not change serum IL-10 (Fig. 5c) suggesting that leptin regulates selected inflammatory processes. Icv leptin-treated ob/ob mice also had significantly reduced BUN (p = 0.044) compared to controls (Fig. 5d), indicating that CNS leptin can ameliorate sepsis-induced kidney damage. Systemic MCP-1 and KC levels were also significantly reduced in CNS leptin-treated mice (p = 0.017 and 0.023; Fig. 5e–f). To rule out the possibility that a fraction of the small amount of icv-infused leptin escaped into the periphery and reached immune-relevant targets via the circulation, we infused the same amount of leptin systemically using peripherally implanted mini-pumps, and there was no improvement in survival or immune parameters after CLP (Fig. 6). These findings therefore demonstrate for the first time that leptin administration directly into the CNS influences survival and the immune response to sepsis in a beneficial manner. In order to ensure that the beneficial effects of leptin on immune response and survival in sepsis are not just a consequence of altered systemic corticosterone levels, we performed an additional experiment where we chronically infused ICV leptin and measured food intake, body weight and corticosterone levels in mice. Seven days of infusion intracerebroventricular leptin infusion (1.0 μg/d) decreased daily food intake and body weight but did not alter circulating corticosterone levels significantly in C57/B6J and ob/ob mice (Fig. 7a–d). Taken together, these data show that the increase in survival of leptin-treated mice was not mediated via generation of corticosterone.
Pharmacological administration of leptin directly into the CNS of ob/ob mice rescues their survival in sepsis. In order to corroborate that finding, we used a genetic approach to test if endogenous CNS leptin action controls survival in sepsis. Specifically, we tested if survival of globally leptin receptor deficient (db/db) mice in sepsis would still differ from survival of wt littermates if leptin signaling was genetically reinstated exclusively in the brain. To rescue CNS leptin signaling only, we using neuron-specific enolase (NSE)- cre and Lepr(flox/flox) mice (Kowalski et al., 2001; Chua et al., 2004; de Luca et al., 2005). We compared these mice (which only have functioning leptin receptor signaling in the CNS - but not in any peripheral organs) with db/db mice (which have no leptin receptor signaling at all) and wt control mice. Survival of db/db mice with rescued leptin signaling in CNS neurons, which were however still lacking any leptin receptors in peripheral, specifically immune-competent, cells, did not differ from survival of wt littermates (p=0.18). However, these mice showed markedly increased survival as compared to db/db mice (p=0.0001) (Fig. 6). These observations were strikingly consistent with our pharmacological studies and confirmed that endogenous leptin signaling in the CNS is critical for normal survival rates in sepsis.
Restoring normal immune function to animals lacking leptin from birth is highly important and reveals a previously unknown function of this systemic cytokine. However, it cannot be inferred from those experiments whether a low level of leptin in the CNS has a permissive role or whether incrementing leptin action in normal animals would bestow a similar benefit. To assess this, we implanted icv minipumps containing leptin (1 μg/24hours) or vehicle (saline as control) into WT mice 3 days before performing CLP. Leptin increased survival rates relative to vehicle-infused controls (Fig. 8a). These unexpected findings were paralleled by changes in systemic immune parameters. Leptin administration into the CNS significantly decreased (p = 0.016) serum IL-6 in septic WT mice (Fig. 8b) and there was no change of IL-10 (Fig. 8c). Consistent with the lack of change of BUN in septic WT mice given systemic leptin (Fig. 3C), icv leptin also did not affect BUN (Fig. 8d). Interestingly, systemic MCP-1 and KC levels were significantly reduced in icv leptin-treated WT mice compared to the untreated septic WT mice (p = 0.05 and 0.03; Fig. 8e–f).
These data reveal that a specific CNS pathway controls systemic immune responses in a functionally relevant manner, conferring protection against sepsis. Specifically, leptin signaling in the CNS is required for efficient coordination of this CNS-controlled immune response in sepsis and is crucial to limit organ damage and prevent mortality; and incrementing leptin in the brain of normal animals enhances their ability to cope with sepsis.
The observations presented here reveal the existence of a specific CNS signaling system that controls systemic immune defense in a functionally relevant manner. This conclusion is based on a combination of pharmacological and genetic models and indicates that CNS leptin action is part of an important regulatory system controlling systemic immune defense. We observed that leptin deficiency is associated with an impaired immune response and lowered survival in a murine model of sepsis. Interestingly, leptin deficiency seems to specifically weaken neutrophil function by inhibition of p38 MAP kinase activation. Systemic leptin replacement corrects the immune response, but most importantly, pharmacological increase or genetic rescue of leptin signaling exclusively and specifically within the CNS is sufficient to improve mortality and cytokine profiles in sepsis. The beneficial effects of leptin that is directly administered into the CNS do not result from leakage of leptin to the periphery in that ob/ob mice, which were infused with the same low dose of leptin peripherally were not improved (Fig. 6). In contrast, higher doses of peripherally administered leptin in ob/ob mice were sufficient to modulate various important immune functions (Figs. 3 & 4) are likely to achieve those effects by crossing the blood brain barrier and acting at CNS leptin receptors. It seems intriguing that leptin receptor deficient db/db mice seem to show reduced survival while presumably leptin resistant DIO mice do not. One potential explanation for the difference in survival may be that db/db mice are completely leptin receptor deficient, while in DIO mice, the level of leptin receptor desensitization in response to chronic high fat diet exposure may be partial or tissue specific.
A considerable body of previously published observations suggest that leptin may regulate immune function (Lam and Lu, 2007) presumably via modulation of the autonomic nervous system. Mancuso and colleagues reported that leptin deficiency leads to weakened immune defense in mouse models of pneumonia and that leptin replacement provides benefits for the host defense in these models (Mancuso et al., 2002; Mancuso et al., 2006; Hsu et al., 2007). Other studies observed a weakened defense of leptin-deficient mice against mycobacterium tuberculosis and lipopolysaccharides (Faggioni et al., 1999; Wieland et al., 2005) as well as hepatic effects of listeria infection (Ikejima et al., 2005). Some of the first studies revealing a potential role of leptin in the modulation of immune function were reported by Lord and colleagues who demonstrated that declining leptin levels may be responsible for the diminished immune response in starvation (Lord et al., 1998). These observations were consistent with evidence generated decades before leptin was discovered that leptin (ob/ob mice) or leptin receptor deficiency (db/db mice) is associated with reduced T-cell function (Mandel and Mahmoud, 1978). However, all of these previous studies focused exclusively on putative peripheral effects of leptin, for example via leptin receptors on lymphocytes or macrophages (Gainsford et al., 1996; Caldefie-Chezet et al., 2001; Siegmund et al., 2004).
The resolution to a bacterial infection includes an immune response that contains and eliminates the pathogen. In contrast, a dysfunctional response allows for the systemic spread of bacteria and increased tissue damage. Our data suggest that leptin is an important contributor to that functional immune response to a septic challenge. In support of this, we observed that phagocytic activity was suppressed in neutrophils taken from septic leptin-deficient mice (Fig. 2b). We speculate that the increased bacterial load at the site of infection in the leptin-deficient mice was due largely to this decreased phagocytosis. Further, increased bacterial load at the site of infection leads to increased inflammation, resulting in increased tissue permeability (Schlag et al., 1991; Tschop et al., 2008). This would allow the pathogen to spread to the blood and organs. Among many actions in response to bacteria, tissues secrete chemokines. We further speculate that the increased serum KC and MIP-2 isolated from the leptin-deficient mice was due to the larger bacterial spread that was observed. Finally, we observed that neutrophils isolated from leptin-deficient mice had decreased neutrophil p38 activation. The p38 MAP kinase is involved in an intracellular kinase cascade that regulates stress-activated signal transduction. In response to certain stresses, like sepsis or proinflammatory cytokines, p38 MAP kinase becomes activated. Zu et al. (Zu et al., 1998) have found that p38 MAP kinase is required for neutrophil function in humans. Leptin is capable to inducing neutrophil locomotion by activation of p38 MAP kinase (Montecucco et al., 2006). Loss of p38 MAP kinase activation may represent an important mechanism responsible for proper neutrophil function such that tissue damage is increased.
Using a murine sepsis model along with selective pharmacological and genetic modulation of CNS leptin signaling we have demonstrated that the essential impact of leptin on immune defense mechanisms is mediated in the brain. We find that CNS leptin action improves survival, normalizes the response of chemokines IL-6, MCP-1 and KC to CLP-induced immune challenge and decreases BUN levels reflecting less organ damage. In principle, non-immune-specific actions of leptin could have contributed to our observations. However, with our study design, we did neither find thermoregulatory differences between treatment groups nor differences in body weight, while there were contemporaneous effects on immune function. One theoretical possibility for a non-specific action of leptin to contribute to its impact on immune function would be via its known role in the modulation of the hypothalamus-pituitary-adrenal (HPA) axis: ob/ob mice have increased circulating levels of corticosterone, which would suppress immune function. Systemic or peripheral leptin administration would act to normalize the HPA axis activity and thereby potentially promote immune function. However, icv leptin administration actually decreases secretion of cytokines and chemokines such as IL-6, MCP-1 and KC, and improves survival, even in WT mice, which have normal corticosterone levels and adrenal function. We therefore propose that leptin acts in the brain to directly regulate peripheral immune function thereby contribute better outcomes in infectious diseases or sepsis as compared to states of relative or total leptin deficiency. A CNS leptin-induced enhancement of systemic immune defense may be mediated by autonomic nervous system (ANS) signaling as suggested by studies focusing on cholinergic modulation of inflammatory pathways in sepsis (Tracey, 2007) and the known impact of CNS leptin action on peripheral metabolism via the efferent ANS. Based on this model, we consider it likely that large amounts of peripherally administered leptin cross the blood brain barrier (Banks, 2006) via active transport to achieve its effects on the immune system by acting in the CNS and affecting peripheral immune defense components via modulation of the ANS.
It has become clear from recent studies that stroke-induced or traumatic CNS injury leads to secondary immunodeficiency and significantly increases susceptibility to infection based on brain-specific mechanisms (Prass et al., 2003; Dziedzic et al., 2004; Meisel et al., 2005; Dirnagl et al., 2007). Understanding CNS injury-induced immunodepression (CIDS) may set the stage for the development of novel effective therapeutic strategies for eliminating a major determinant of mortality after CNS damage (Meisel et al., 2005). We speculate that lack of leptin action in the CNS may represent one important component of the molecular mechanisms leading to CIDS and that leptin analogues may offer potential pharmacological opportunities for the treatment of sepsis. Future studies will have to identify and dissect the likely complex neural circuitry and efferent connections specifically mediating central effects of leptin on systemic immune defense, thereby providing the basis for a new understanding of CIDS.
The concept of a specific and functionally relevant CNS control of peripheral immune defense, as implied by the present results, is consistent with a series of observations reported by Tracey and colleagues. Those authors have examined the role of the ANS in the regulation of immunity and have proposed the existence of a brain-based control of the inflammatory response to sepsis (Tracey, 2007). In their model is that the ANS detects the presence of inflammatory stimuli and in return modulates cytokine production as documented by pharmacological and electrophysiological studies. The vagus nerve communicates afferent signals to the brain as well as responsive efferent activity culminating in the release of acetylcholine (ACh) affecting macrophages via macrophage alpha7 subunits of nicotinic ACh receptors, and this in turn leads to decreased cytokine release (Wang et al., 2003). Autonomic dysfunction has been associated with human inflammatory diseases such as diabetes and sepsis suggesting that these diseases may actually be caused or exacerbated by an autonomic dysfunction. Interestingly, afferent as well as efferent vagal signaling has been shown to mediate components of leptin action (Nagashima et al., 2000; Buyse et al., 2001; Peters et al., 2006b; Peters et al., 2006a; Williams et al., 2007). While we are confident based on the available data that the autonomic nervous system plays a considerable role in the neuroendocrine control of systemic immune function and survival in sepsis, it is well known, that leptin can modulate the activity of the hypothalamus-pituitary-adrenal (HPA) axis. We therefore performed additional experiments where we were able to show that with the doses infused icv in our series of studies, not significant change in circulating corticosterone levels was caused. Therefore, it seems unlikely that our findings are a consequence of leptin induced HPA axis modulation.
Consistent with these pre-clinical data, the observation that several patients with congenital leptin and leptin receptor deficiency have died of sepsis suggests the clinical relevance of these findings. Further supporting the clinical relevance of our findings, it was recently reported that relatively low leptin in patients with sepsis is correlated with high mortality (Bracho-Riquelme et al., 2008). These clinical observations, together with our studies indicating that leptin treatment improves immune function and survival even in WT mice which are not genetically leptin-deficient, strongly supports a potential therapeutic value for leptin and its analogues in the treatment of sepsis and related disorders.
The role of leptin in the control of the immune response via the CNS has not been described previously. Here, we show that leptin is a critical factor in host resistance and that the lack of leptin contributes substantially to mortality and weakens systemic and local immune responding as an essential component of the physiological defense against bacteremia after CLP, a model of severe sepsis. Sepsis-induced organ damage is increased while neutrophil function is diminished by attenuation of p38 MAP kinase signaling. We further describe an important role of leptin in the CNS for regulating survival and systemic immune response in sepsis: Selective leptin administration into the CNS controls systemic immune response in a functionally relevant manner and to the extent of significant protection from sepsis. We conclude that leptin-dependent neurocircuitry in the CNS is required for efficient coordination of the immune response in sepsis in order to limit organ damage and prevent mortality.
This work was supported by funding from the National Institutes of Health R01 GM72760, NIDDK59630, NIDDK69987, and NIDDK56863 (SCW & MHT). We would like to thank Kim Brown, Paul T. Pfluger, and Randy Seeley for research assistance, helpful discussions and manuscript editing.
Commercial Interest Statement: Matthias Tschoep is a Scientific Advisory Board Member for Marcadia Biotech and Acylin Inc, and a Consultant for Ambrx Inc.