Dopaminergic Control of Inflammation and Glycemia in Sepsis and Diabetes

Most preclinical treatments for sepsis failed in clinical trials in part because the experimental models of sepsis were performed on healthy animals that do not mimic septic patients. Here, we report that experimental diabetes worsens glycemia, inflammation, and mortality in experimental sepsis. Diabetes increases hyperglycemia, systemic inflammation, and mortality in sepsis. Diabetes exacerbates serum tumor necrosis factor (TNF) levels in sepsis by increasing splenic TNF production. Both serum from diabetic mice and glucose increase cytokine production in splenocytes. Anti-inflammatory treatments cannot control hyperglycemia and are less effective in diabetic patients. By contrast, dopaminergic agonist type-1, fenoldopam, attenuates hyperglycemia, and systemic inflammation in diabetic septic mice by inhibiting splenic p65NF-kB phosphorylation. Fenoldopam inhibits TNF production in splenocytes even at high glucose concentrations and inhibits the canonical NF-kB pathway by inhibiting p65RelA and p50NF-kB1 phosphorylation without affecting the non-canonical NF-kB proteins. Treatment with fenoldopam rescues diabetic mice from established polymicrobial peritonitis even when the treatment is started after the onset of sepsis. These results suggest that dopaminergic agonists can control hyperglycemia, systemic inflammation and provide therapeutic advantages for treating diabetic patients with sepsis in a clinically relevant time frame.

Most preclinical treatments for sepsis failed in clinical trials in part because the experimental models of sepsis were performed on healthy animals that do not mimic septic patients. Here, we report that experimental diabetes worsens glycemia, inflammation, and mortality in experimental sepsis. Diabetes increases hyperglycemia, systemic inflammation, and mortality in sepsis. Diabetes exacerbates serum tumor necrosis factor (TNF) levels in sepsis by increasing splenic TNF production. Both serum from diabetic mice and glucose increase cytokine production in splenocytes. Anti-inflammatory treatments cannot control hyperglycemia and are less effective in diabetic patients. By contrast, dopaminergic agonist type-1, fenoldopam, attenuates hyperglycemia, and systemic inflammation in diabetic septic mice by inhibiting splenic p65NF-kB phosphorylation. Fenoldopam inhibits TNF production in splenocytes even at high glucose concentrations and inhibits the canonical NF-kB pathway by inhibiting p65RelA and p50NF-kB1 phosphorylation without affecting the non-canonical NF-kB proteins. Treatment with fenoldopam rescues diabetic mice from established polymicrobial peritonitis even when the treatment is started after the onset of sepsis. These results suggest that dopaminergic agonists can control hyperglycemia, systemic inflammation and provide therapeutic advantages for treating diabetic patients with sepsis in a clinically relevant time frame.
Keywords: diabetic sepsis, inflammation mediators, dopaminergic agonist, murine sepsis, phosphorylation inTrODUcTiOn The regulation of the immune system and inflammation is critical for survival both from a physio logical and a clinical perspective. Probably one of the most characteristic examples is sepsis, a major clinical challenge in modern medicine killing around 250,000 patients every year and accounting for 9.3% of overall deaths in the United States (1)(2)(3)(4)(5)(6). Sepsis was originally defined as a systemic infection and its diagnosis required the confirmation of bacterial infection. Thus, initial strategies focused on designing effective antibiotics to control the infection. New generations of antibiotics are more effective controlling infections, but sepsis still causes around 1/3 deaths in hospitalized patients with high mortality rates in the ICU ranking from 30 to 60% depending on the clinical study and the organ failure (3)(4)(5)(6). In addition to the infection, septic is also characterized by detrimental systemic inflammatory responses that become more dangerous than the original infection and cause organ damage and lethal multiple organ failures (7)(8)(9)(10)(11). The inhibition of specific inflamma tory cytokines such as a tumor necrosis factor (TNF), migration inhibitory factor (MIF), or high mobility group box (HMGB)1 provided promising results in experimental sepsis (7,(10)(11)(12)(13), but, they failed in the clinical tri als for sepsis (14). One explanation is that sepsis is not produced by a single cytokine and thus, successful treatments for sepsis may require inhibiting multiple cytokines. Therefore, recent efforts focus on designing therapeutic strategies that control multiple inflammatory factors and rescue patients from established sepsis in a clinically relevant time frame.
Most preclinical strategies that provided promising results in experimental models of sepsis, failed in clinical trials (7,10). Indeed, more than 100 randomized clinical trials tested whether inhibition of inflammatory factors improves survival in sepsis. With one shortlived exception, none of these clinical trials have resulted in new treatments (15). Xigris [activated protein C (APC), drotrecoginalpha, DrotAA] was the only drug approved by the FDA for treating severe sepsis, as it improved survival by 6% in the 2001 PROWESS trial. However, Xigris increased the risk of severe hemorrhage in septic patients, and Eli Lily withdrew it from the market in 2011 due to the lack of beneficial effects in the PROWESSSHOCK trial (16). Currently, there is no treat ment for severe sepsis approved by the FDA and present therapies are mostly supportive. Current studies indicate that the pathology of sepsis is a complex process with both immune and metabolic alterations, and most septic patients have preexisting conditions with metabolic and immune alterations that contribute to mul tiple organ failure in sepsis (17)(18)(19)(20)(21)(22)(23)(24)(25). Thus, one potential reason for the failure of these clinical trials is that the preclinical studies focused on healthy animals that did not mimic the preexisting conditions of septic patients (26). The CDC reported that 7 in 10 septic patients had chronic diseases requiring frequent medical care or required hospital services 30 days before sepsis admission (27). Indeed, around 1/3 of septic patients are diabetic, and hyper glycemia increases 90day mortality in septic patients (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). There is a huge population of prediabetic patients and as many as 50% of diabetic patients can be asymptomatic and remain undiagnosed (30). Diabetic patients also represent an additional challenge because insulin treatment is not effective during sepsis (31). Sepsis is characterized by detrimental inflammatory and hyperglycemic responses to infection (32), and this combina tion is associated with higher mortality rates over 40% (32)(33)(34). Despite the use of new generations of antibiotics and regardless of their higher susceptibility to infection, diabetic patients have a higher mortality rate in sepsis. Thus, recent efforts focus on identifying the mechanisms connecting metabolic and immune alterations and their clinical implications in infectious and infla mmatory disorders. Here, we analyze how experimental diabetes affects sepsis and the efficacy of antiinflammatory treatments for sepsis.

MaTerials anD MeThODs chemicals and reagents
LPS (Escherichia coli 0111:B4), streptozotocin, glucose, dopa mine hydrochloride, and fenoldopam were purchased from SigmaAldrich ® (Saint Louis, MO, USA). The glucose measuring strips were purchased from PharmaTech Solutions, Inc. (Westlake Village, CA, USA). Pentobarbital sodium was purchased from Diamondback (Scottsdale, AZ, USA); ketamine from Henry Schein animal health (Dublin, OH, USA); xylazine from Akorn animal health (Lake Forest, IL, USA), and enrofloxacin from Bayer Healthcare (Shawnee Mission, KS, USA). Streptozotocin was injected (STZ; i.p., 50 mg/kg) at 10 and 5 days before the experiment as previously reported (35,36). Treatment with fen oldopam (Fen; 10 mg/kg/dose; i.p.) was administered at 6 and 1 h before LPS or CLP in most experiments. Treatment with fenoldopam was started 15 h after CLP and given every 12 h for 3 days in the survival experiments.

animal experiments
All experimental procedures adhered to The Guide for the Care and Use of Laboratory Animals by the National Academy of Sciences and published by the National Institutes of Health (Copyright© 1996 by the National Academy of Sciences), and were approved by the Institutional Animal Care & Use Committee of the Rutgers New Jersey Medical School. 6-8weekold (≈25 ± 5 g) BALB/c male mice obtained from Charles River Laboratories (Wilmington, MA, USA) were maintained in a controlled envi ronment, room temperature 70-75 F, air humidity 40-70%, 12h light/dark cycle, with free access to food and water (ad libitum) until experimentation. Animals were randomly distributed for the experimental treatments, and the investigators were blinded to the treatments.

experimental sepsis
Endotoxemia and cecal ligation and puncture (CLP) were performed as we previously described in Nat Med (37) with the modifications described in Nat Med (38). Endotoxemia: Endotoxin (E. coli LPS 0111:B4; Sigma Chemical, Saint Louis, MO, USA) was dissolved in sterile, pyrogenfree PBS (Gibco ® : Life Technologies, Grand Island, NY, USA), and sonicated for 20 min immediately before use. Animals received a LD50 dose of LPS (10 mg/kg, i.p.). CLP: animals were anesthetized with pentobarbital sodium (50 mg/kg, i.p.; Diamondback, Scottsdale, AZ, USA). Animals underwent to a standard CLP procedure with 25-50% average mortality as we described in Nat Med (37,38). Briefly, an abdominal incision, of approximately 1.0 cm, was performed to expose and ligate the cecum at 5.0 mm from the cecal tip away from the ileocecal valve. The ligated cecal stump was punctured only once with a 22gauge needle, and the stool was extruded (approx. 1.0 mm) to ascertain patency of puncture. The abdominal wound was closed in two layers, peritoneum and fascia separately, to prevent leakage of fluid. All animals received antibiotic (Enrofloxacin 2.5 mg/kg, s.c.; Baytril ® , Bayer Health Care™, Swanee Mission, KA, USA) dis solved in 0.9% normal saline immediately after surgery and every 12 h for 3 days, 0.5 mL/dose. splenectomy Animals were anesthetized with rodent cocktail 100mg/kg ketamine; 20mg/kg xylazine; intraperitoneal. Anesthesia was confirmed by the absence of withdrawal reflex to toe pinch. Splenectomy was performed 3 days before the experimental pro cedure as we described in J Exp Med (39). Right after surgery, all animals received antibiotic (Enrofloxacin 2.5 mg/kg, s.c) dissolved in 0.9% normal saline immediately after surgery and every 12 h for 3 days. Anesthetized animals were subjected to an abdominal incision on the epigastrium and mesogastrium. The spleen was exposed by gentle retraction of the stomach to the side. The three main branches of the spleen artery were stabilized with nylon thread, ligated and cut. The spleen was removed and the wound was closed with sutures; catgut for the abdominal wall, and nylon thread for the skin.

cell cultures
Primary culture of splenocytes and peritoneal macrophages were performed as we previously described (39). Murine RAW264.7 cells (ATCC, Manassas, VA, USA) were cultured as we previously described (37). Cells were transferred onto a 24well polysty rene culture plates at 3 × 10 5 cells/well and incubated overnight. Cells were washed with PBS and incubated overnight with 5% serumfree DMEM medium. Cells were incubated with DMEM, no glucose (ThermoFisher, SKU# 11966025) supplemented with the indicated concentrations of glucose. Alternatively, cells were incubated directly on serum without dilution from normal or diabetic mice for 3 h prior LPS challenge. Cells were lysed in lysis buffer with protease inhibitor (CelLyticMT and Protease Inhibitor Cocktail P8340; 1:100 v/v, both from SigmaAldrich, Saint Louis, MO, USA) and centrifuged at 12,000× g for 20 min at 4°C for NFkB analyses. The conditioned supernatant was used for TNF analyses.

Blood, Organ, and cell analyses
Serum samples were obtained by clotting the blood for 2 h at room temperature, and centrifuged at 2,000× g for 15 min at 4°C. Organs were collected and immediately homogenized in 4°C PBS. Samples were normalized to protein concentration and TNF was analyzed by ELISA (Affymettrix Inc, San Diego, CA, USA). Glucose was analyzed from the mouse tail tip blood using the Genstrip (PharmaTech Solutions Inc., Westlake Village, CA, USA) and the Onetouch UltraMini glucometer (LifeScan Inc., Milpitas, CA, USA). TNF in the culture cells was analyzed at 3 h postLPS. TNF levels in the serum and organs were analyzed at 90 min after the LPS treatment. Cell samples for NFkB analyses were normalized to protein concentration and their activa tion and binding to DNA was analyzed using the TransAM DNABinding ELISA (Active Motif; Cambridge, MA, USA). Phosphorylation of p65NFkB protein at serine 536 was ana lyzed by ELISA using the specific (Total/Phospho) Multispecies InstantOne™ ELISA Kit (Cat# 858608311; ThermoFisher, Waltham, MA, USA).

statistical analyses
All tests were performed using the GraphPad Prism Software ® (GraphPad Software, La Jolla, CA, USA). The sample size was determined using standard deviation values and power analyses of our previous studies on the vagal stimulation (39,40). All data in the figures are expressed as the mean ± SEM. The student's ttest (Mann-Whitney U test) was used to compare mean values between two experimental groups. Analyses of three or more groups were performed using the oneway ANOVA with multiple pairwise comparisons. The time courses and pairwise comparisons were analyzed with the twoway ANOVA for repeated measures. Pair comparisons in ANOVA nonparametric tests were post hoc adjusted with Tukey test (in equal sample sizes) or Bonferroni's for multiple hypothesis testing. Normality and homogeneity of variance were confirmed using the Kolmogorov-Smirnov ana lysis. Statistical analyses of survival were determined using the logrank (Mantel-Cox) test. n = sample size per group. p < 0.05 are considered statistically significant and represented as follows: # Student's ttest, + oneway ANOVA, *twoway ANOVA, and § survival logrank test.

Diabetes Worsening inflammation and survival in sepsis
Given the high incidence of diabetes in septic patients, we first analyzed the effects of experimental diabetes on glycemia, inflammation and survival in sepsis. Diabetes was induced with streptozotocin, the standard and most common method for experimental diabetes (35,36,41). Treatment with streptozotocin increases blood glucose levels in mice by itself before the septic challenge ( Figure 1A). By contrast, diabetes did not induce serum TNF levels before the septic challenge ( Figure 1B). Furthermore, diabetes increases both hyperglycemia and serum TNF levels during endotoxemia (Figures 1A,B). Time course analyses show that diabetic and control nondiabetic animals have similar kinetics with glycemia and serum TNF levels peaks around 1.5 h and return to baseline after 3-4 h postLPS. Next, we analyzed whether diabetes affects survival in different experimental models of sepsis including endotoxemia and polymicrobial peritonitis. Diabetes worsens survival in endotoxemic mice ( Figure 1C). Diabetes also worsens the survival of mice with polymicrobial peritonitis induced by CLP, the standard experimental model to induce polymicrobial peritonitis ( Figure 1D) (4,42,43). Unlike endotoxemia induced by LPS, CLP causes both polymicrobial infection (induced by the cecal puncture) and inflammation (induced by both the infection and the necrotic tissue of the cecal ligation) (4, 44). Diabetes decreased acute survival but the late deaths did not occur among control or STZ treated mice. These results indicate that diabetes worsens hyperglycemia, systemic inflammation and survival in experimental sepsis.

Diabetes enhancing splenic TnF Production
Next, we studied how experimental diabetes increases systemic inflammation in sepsis by analyzing TNF production in the organs. Bacterial endotoxin induces TNF production in all the organs but the highest TNF concentrations were found in the spleen (Figure 2A). Likewise, diabetic endotoxic mice have similar TNF levels in all the other organs but around twofold higher splenic TNF levels than nondiabetic endotoxic mice. These results sug gest that diabetes increases serum TNF levels by increasing TNF production in the spleen. Thus, we analyzed whether the spleen is essential for the higher serum TNF levels in diabetic mice by performing surgical splenectomy 3 days prior endotoxemia. Diabetic mice have around twofold higher serum TNF levels  than nondiabetic sham mice, but both diabetic and nondiabetic endotoxemic mice have similar serum TNF levels after splenec tomy ( Figure 2B). These results show that the higher serum TNF levels in diabetic mice are due to the higher TNF production in the spleen. Next, we analyzed whether splenectomy affects hyperglycemia. Splenectomy rendered diabetic and nondiabetic mice with similar hyperglycemia in endotoxemia ( Figure 2C). Given that the higher serum TNF levels of diabetic mice are mainly due to higher splenic TNF production, we analyzed whether splenocytes from diabetic mice produce more TNF than those from nondiabetic mice. Primary culture of spleno cytes from diabetic or nondiabetic mice produce similar TNF levels when challenged with LPS ( Figure 2D). Thus, we analyzed whether the serum from diabetic mice enhances TNF production in splenocytes from nondiabetic mice. We isolated primary cul ture of splenocytes from normal mice and incubated them with serum from diabetic or nondiabetic mice before the endotoxic challenge. Serum from diabetic mice increases TNF production in primary culture of splenocytes from normal nondiabetic mice ( Figure 2E). Thus, we reasoned that higher levels of glucose in the serum of diabetic mice enhance TNF production in spleno cytes. We incubated primary culture of splenocytes from normal mice with different concentrations of glucose before the endo toxic challenge. Higher glucose concentrations enhance TNF production in normal splenocytes ( Figure 2F). These results indi cate that higher glucose concentrations in the serum of diabetic mice enhance the cytokine production in splenocytes and thereby worsens the prognosis of sepsis.

Dopaminergic Type-1 agonist inhibiting TnF Production at high glucose concentrations
Given that hyperglycemia increases inflammation and worsens the prognosis of septic patients (17)(18)(19)(20)(21)(22)(23)(24), we reasoned that it may interfere with the efficacy of antiinflammatory strategies for sepsis. We previously reported that electrical stimulation of the vagus nerve attenuates serum TNF levels in endotoxemia by acti vating the adrenal medulla to produce dopamine (38). Thus, we analyzed the potential of dopamine to inhibit TNF production in splenocytes. Dopamine inhibits TNF production in splenocytes in a concentrationdependent manner with a half maximal effec tive concentration (EC50) of 0.12 ± 0.3 µM (Figure 3A). Then, we analyzed whether extracellular glucose levels interfere with the potential of dopamine to inhibit TNF production in splenocytes. Low concentration of dopamine (0.1 µM) inhibits TNF produc tion in splenocytes by around 55% and 70% at 100 and 300 mg/dL of glucose, respectively (Figure 3B). High concentration of   dopamine (1 µM) inhibits TNF production in splenocytes by around 70% regardless of the concentration of glucose. Given that dopamine has multiple side effects as a pleiotropic factor signaling through multiple receptors, we used specific agonists to identify the receptors modulating cytokine production in sple nocytes. According to IUPHAR nomenclature, dopamine signals through D1 and D2like dopamine receptors. D1like receptors include D1 and D5 dopaminergic receptors (42,43,45). D2like receptors include D2, D3, and D4 dopaminergic receptors (46). Fenoldopam is a wellcharacterized D1like agonist, whereas pergolide is the canonical D2like agonist (42,43,45). D2like agonist, pergolide fails to inhibit TNF production in splenocytes ( Figure 3C). By contrast, D1like agonist, fenoldopam inhibits LPSinduced TNF production in splenocytes even at high glucose concentrations ( Figure 3D). Low concentration of fenoldopam (0.1 µM) inhibits TNF production by 35 and 50% at 100 and 300 mg/dL of glucose, respectively. High concentration of fen oldopam (1 µM) inhibits TNF production in splenocytes by 60 and 75% at 100 and 300 mg/dL of glucose, respectively. These differences were not due to changes in the expression of the recep tors, and glucose did not affect the expression of dopaminergic receptor1 or 5 (D1R, D5R) ( Figure 3E). These results indicate that dopaminergic type1 agonists such as fenoldopam can inhibit TNF production in splenocytes both at normal and high concentrations of glucose.

Dopaminergic control of the nF-kB Pathway in sepsis
Next, we analyzed how fenoldopam inhibits TNF production in macrophages. Given the heterogeneity of cell types in primary culture of splenocytes, we focused on homogeneous cultures of RAW264.7 macrophage cells similar as we previously des cribed (37). Bacterial endotoxin induces TNF production in a concentrationdependent manner, and high concentrations of glucose enhance TNF production in RAW264.7 cells similar to that described in splenocytes ( Figure 4A). Fenoldopam also inhibits TNF production in RAW264.7 cells in a concentration dependent manner similar to that described in splenocytes ( Figure 4B). Given that NFkB proteins are key transcriptional factors regulating inflammatory cytokines in macrophages, we analyzed whether glucose affects the potential of fenoldopam to regulate their binding to DNA (47). Fenoldopam (1 µM) inhibits LPSinduced p65RelA activation and binding to DNA with higher efficacy at high glucose concentrations ( Figure 4C). Likewise, fenoldopam (1 µM) also inhibits p50NFkB1 activation and binding to DNA at high glucose concentrations ( Figure 4D). We also analyzed the specificity of this inhibition and their potential to regulate the noncanonical NFkB proteins. Neither extracel lular glucose levels nor fenoldopam affects the DNAbinding of the noncanonical NFkB proteins RelB, p52NFkB2 and cRel (Figures 4E-G). These results indicate that D1like dopamine receptor agonists can inhibit the canonical NFkB pathway by inhibiting both p65RelA and p50NFkB1 activation and DNA binding even at high glucose concentrations.

Dopaminergic control of sepsis With Diabetes
We next reasoned that dopaminergic agonists may have clinical implications for treating sepsis. Previous studies on sepsis are performed in experimental models of sepsis with "healthy" mice that do not mimic the preexisting conditions of septic patients (48). Given that around 1/3 of septic patients are diabetic and hyperglycemia increases 90day mortality in septic patients (17)(18)(19)(20)(21)(22)(23)(24), we analyzed whether fenoldopam attenuates systemic inflammation in experimental sepsis with diabetes. Treatment with fenoldopam attenuates serum TNF levels in endotoxemic mice with diabetes ( Figure 5A). Then, we analyzed different organs to find that the most significant effects of fenoldopam were in the spleen by inhibiting TNF production by around 50% (Figure 5A). Given that NFkB proteins are regulated by phosphorylation, we also analyzed p65NFkB phosphorylation in the organs of septic mice with diabetes ( Figure 5B). Again, the most significant effects were found in the spleen where endo toxin increases p65NFkB phosphorylation at serine 536 by over fourfold, and fenoldopam inhibits this phosphorylation by over threefold in the spleen without affecting the lung or liver. Fenoldopam also attenuates hyperglycemia in both diabetic and nondiabetic mice ( Figure 5C). Thus, we analyzed whether fenoldopam can improve survival in septic mice with diabetes. Treatment with fenoldopam at 6 and 2 h prior the LPS challenge improves survival in endotoxemic mice with diabetes ( Figure 5D). Next, we analyzed whether fenoldopam can improve survival in diabetic mice with polymicrobial peritonitis induced by CLP, the standard experimental model to induce polymicrobial infec tion peritonitis (4,44). Treatment with fenoldopam, started 15 h after the CLP, improves survival of diabetic mice with established polymicrobial peritonitis (Figure 5E). These results show that dopaminergic agonists can control systemic inflammation and improves the survival of diabetic mice in polymicrobial peritonitis.

DiscUssiOn
Sepsis is a major clinical and scientific challenge in modern medicine with over 100 unsuccessful clinical trials (15). Many preclinical strategies improved survival in experimental animal models of sepsis but failed in clinical trials (7,10). One explana tion is that most experimental models of sepsis are performed on healthy animals that do not mimic the preexisting conditions of septic patients. Indeed, over 72% of the septic patients had chronic diseases requiring frequent medical care or required hospital services within 30 days before sepsis admission (27). This combination is associated with the highest mortality rates over 40% (32). Diabetes is a leading comorbidity in sepsis, around 1/3 of septic patient are diabetic, and hyperglycemia increases 90day mortality in septic patients (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Thus, experimental models of sepsis using "healthy" animals with "normal" blood glucose levels and physiological functions may not mimic the actual responses observed in septic patients. In our study, experi mental diabetes was induced with streptozotocin, the standard method for experimental diabetes described in the literature (35,36,41). Streptozotocin induces both type1 and type2 insulinresistant diabetes, causes DNA alkylation and activates poly ADPribosylation, leading to cellular NAD + and ATP depletion and the formation of superoxide radicals (49). Strep tozotocin is a more specific, stable and reliable experimental model of diabetes that is neither diet dependent nor causes renal toxicity like alloxan (49). Our results show that diabetes incre ases serum glucose levels and induces hyperglycemia, but not detectable serum TNF levels by itself before the septic challenge. The effects of diabetes in sepsis are controversial. Some studies indicate that diabetic patients have functional immune deficiency and, they are less efficient in bacterial clearance (50)(51)(52). Likewise, experimental studies indicated that alloxandiabetic mice are highly susceptible to polymicrobial sepsis due to downregulation of CXCR2 in neutrophils, preventing their migration to the focus of infection (53). However, despite the use of new generations of antibiotics, diabetic patients have a higher mortality rate in sepsis suggesting pathogenic effects regardless of their susceptibility to infection. Regardless of the infection, our results indicate that diabetes exacerbates both hyperglycemic and TNF responses to bacterial endotoxin. These results concur with previous studies indicating that diabetes exacerbates systemic inflammation and induces a persistent systemic inflammation in experimental sepsis (54)(55)(56)(57). All these studies show that both type1 and type2 diabetic animals have exacerbated hyperglycemia, and produc tion of both pro (TNF, IL1, IL6, MCP1) and antiinflammatory (IL10) cytokines in experimental sepsis. Our results also indicate that these exacerbated glycemic and inflammatory responses of diabetic mice worsen their survival in sepsis. Similar studies reported that alloxaninduced diabetes also worsens mice survival in polymicrobial peritonitis by preventing neutrophil migration to the focus of the infection (53). Regardless of the susceptibility to infection, our results indicate that diabetes increases mortality   in endotoxemia but also in polymicrobial peritonitis induced by CLP even when the mice were treated with antibiotics to mimic clinical standards. Likewise, clinical studies show that diabetes and hyperglycemia increase morbidity and mortality in sepsis even using antibiotics (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). These results are not only relevant to sepsis, but an important clinical consideration as diabetic patients have exacerbated glycemia and inflammatory responses in critical conditions such as hemorrhage, ischemia and trauma.
Our results show that diabetes exacerbates the inflammatory responses to bacterial endotoxin by increasing TNF production in the spleen. Diabetic mice have around twofold higher splenic TNF levels than nondiabetic mice. However, both diabetic and nondiabetic mice have similar serum TNF levels after splenectomy. Of note, splenectomy increases hyperglycemia in nondiabetic but not in diabetic mice, rendering diabetic and nondiabetic mice with similar hyperglycemia in endotoxemia. Given that diabetes worsens systemic inflammation due to higher TNF production in the spleen, we analyzed the effects of diabetes at the cellular level in primary culture of splenocytes. Primary culture of splenocytes from either diabetic or nondiabetic mice produce similar TNF levels when challenged with bacterial endo toxin. These results suggest that acute diabetes does not produce a defect in the response of splenocytes to endotoxin. It remains to be determined if chronic diabetes, which is more clinically relevant, affects this response. However, serum from diabetic mice increases TNF production in primary culture of normal splenocytes. Our results indicate that glucose increases TNF production in splenocytes showing that hyperglycemia directly affects the inflammatory responses to bacterial endotoxin and contributes to systemic inflammation in sepsis.
Previous studies have focused on whether preexisting condi tions such as diabetes increase susceptibility to infections and sepsis. Here, we analyzed whether diabetes affects the effectivity of potential treatments for sepsis. We previously reported that electrical vagal stimulation attenuates serum TNF levels in endo toxemia by activating the adrenal medulla to produce dopamine (38). The present study shows that dopamine (0.1 µM) inhibits TNF production in splenocytes even when cultured at high glucose concentration. Thus, dopamine may provide therapeutic advantages for treating diabetic patients with sepsis or other cri tical conditions. Both dopamine and norepinephrine are com monly used in critically ill patients to restore tissue perfusion (58,59). Although dopamine is more effective in improving renal hemodynamics (60), it has significant side effects, increases the risk of tachyarrhythmia (58,59), and worsens survival in septic animals (61). We reasoned that these effects can be mediated by different dopaminergic receptors, and thus specific dopaminergic agonists may avoid the unspecific side effects. Given that dopamine signals through D1 and D2like dopamine receptors (62)(63)(64)(65). D1like receptors include D1 and D5 dopaminergic receptors. D2like receptors include D2, D3, and D4 dopaminergic recep tors (46). D2like agonist, pergolide did not affect TNF produ ction. By contrast, D1like agonist, fenoldopam inhibits TNF production in splenocytes similar to that reported by dopamine. Low concentration of fenoldopam (0.1 µM) inhibits TNF pro duction in splenocytes cultured at high glucose concentration. At the cellular level, fenoldopam inhibits the activation of the canonical NFkB pathway by preventing both p65RelA and p50NFkB1 activation and binding to DNA. These effects are specific as neither extracellular glucose levels nor fenoldopam affects the activation of the noncanonical NFkB proteins RelB, p52NFkB2 and cRel. Thus, fenoldopam can inhibit TNF pro duction in splenocytes at high glucose concentrations, and can provide therapeutic advantages for treating diabetic patients with sepsis.
In vivo, treatment with fenoldopam attenuates serum TNF levels in diabetic mice with sepsis by inhibiting splenic TNF production. Given that NFkB proteins are regulated by phos phorylation, we also analyzed p65NFkB phosphorylation in the organs of the septic mice. Again, the most significant effects were found in the spleen where endotoxin increases p65NFkB phosphorylation at serine 536 by over fourfold, and fenoldopam inhibits this phosphorylation by over threefold in the spleen without affecting the lung or liver. This specific inhibition of the canonical NFkB pathway in the spleen can have clinical implica tions because NFkB modulates cytokine production the spleen, and it protects parenchyma cells from cytotoxicity in other organs (66)(67)(68). The most characteristic example is that p65RelA (69)(70)(71) and IKKβ (72-74) knockout mice exhibit massive fetal hepato cyte apoptosis and embryonic death. These studies indicate that p65RelA can prevent hepatocyte apoptosis (73,74), and thus ubiquitous NFkB inhibition may not generate an overall benefi cial effect especially in the liver, unless the therapy targets specific organs or immune cells (75). Therefore, fenoldopam may provide therapeutic advantages for diabetic patients with sepsis due to its potential to specifically inhibit NFkB in the spleen. Fur thermore, fenoldopam also attenuates hyperglycemia. These results have significant clinical implications because although hyperglycemia is especially relevant in diabetic patients, sepsis and other critical conditions such as hemorrhage, ischemia and trauma induce insulin resistant hyperglycemia in both diabetic and nondiabetic patients (76,77). Sepsis is a complex process, and successful therapeutic treatments for sepsis may require controlling both immune and metabolic alterations. Given that hyperglycemia worsens systemic inflammation, organ function and mortality in sepsis (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), dopaminergic agonists such as fenoldopam may provide therapeutic advantages for both meta bolic and immune alterations in sepsis and other critical condi tions that induces insulin resistant hyperglycemia. Treatment with fenoldopam, started 15 h after the CLP, improves survival of diabetic mice with established polymicrobial peritonitis. By comparison with other strategies, administration of antiTNF antibodies increased the mortality when administered after cecal perforation (78). Antimacrophage MIF antibodies (79) or lysophosphatidylcholine (80) are ineffective if administered more than 8 or 10 h after the induction of peritonitis (79,81). Our stu dies indicate that diabetes and glycemia affect the pathogenesis of sepsis and the efficacy of antiinflammatory strategies. These results warrant further studies in other experimental models of diabetes and in other experimental groups including aging population Together, these results suggest that dopaminergic agonist type 1 can control systemic inflammation and provide therapeutic advantages for treating diabetic patients with sepsis in a clinically relevant time frame.

DaTa aVailaBiliTY sTaTeMenTs
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

eThics sTaTeMenT
All experimental procedures adhered to The Guide for the Care and Use of Laboratory Animals by the National Academy of Sciences and published by the National Institutes of Health