Lower Levels Of Proinflammatory Cytokines Research Articles

P089 Identification of the first protein kinase inhibitors that switch macrophages from pro-inflammatory to anti-inflammatory cytokine production

Introduction Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation and resolution of inflammation. Classically-activated macrophages produce pro-inflammatory mediators that help to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages produce high levels of anti-inflammatory molecules, such as interleukin (IL)-10, and low levels of pro-inflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Methods Bone-marrow derived macrophages were treated with protein kinase inhibitors prior to stimulation with Toll-like receptor (TLR) agonists. mRNA synthesis was measured by qPCR and cytokine secretion was measured using the BIOPLEX system. To identify the relevant substrate, the effects of the protein kinase inhibitors on the phosphoproteome were measured by LC-MS/MS. Subsequently, the repertoire of phosphorylation sites were mapped and their effects on protein function were analyzed using luciferase assays. Finally, the targets of the protein kinase inhibitors were validated in mouse knock-outs. Results We have discovered a novel mechanism that restricts the formation of regulatory macrophages. This has led us to identify the first small-molecule inhibitors of protein kinases capable of inducing all the hallmarks of regulatory macrophages, including the production of high levels of IL-10 and IL-1ra. The molecular mechanism underlying the effects of these compounds will be presented. Conclusion The remarkable effects of these protein kinase inhibitors on macrophage function suggest a novel approach for the development of anti-inflammatory drugs.

Biofilm and planktonicEnterococcus faecaliselicit different responses from host phagocytesin vitro

Enterococcus faecalis is a commensal organism of the gastrointestinal tract but can also cause serious opportunistic infections. In addition to high levels of antibiotic resistance, the ability to form biofilms on abiotic surfaces and on in-dwelling devices within the host complicates treatment strategies and successful outcomes of antibiotic therapy. Despite rapid advances made in recent years in understanding the genomics and virulence of this organism, much remains to be learned regarding the host response to enterococcal infections. In this study, we investigated the interaction of RAW264.7 macrophages and JAWS II dendritic cells with biofilm and planktonic E.faecalis, in vitro. Specifically, we compared phagocytosis, intracellular survival, secretion of proinflammatory cytokines, and the activation and maturation of phagocytes. Our results revealed that both macrophages and dendritic cells phagocytize biofilm mode cells at levels equal to or better than their planktonic counterparts. Internalized biofilm bacteria showed relatively greater survival at 24h in macrophages than in dendritic cells and led to slightly higher expression of phagocyte activation markers. Macrophages infected with biofilm cells also secreted lower levels of proinflammatory cytokines studied. Overall, these results suggest that biofilm E.faecalis may be better adapted to overcome host defenses in vivo.

VIP Deficient Mice Exhibit Resistance to Lipopolysaccharide Induced Endotoxemia with an Intrinsic Defect in Proinflammatory Cellular Responses

Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide with immunomodulatory properties. The administration of this peptide has been shown to have beneficial effects in murine models of inflammatory diseases including septic shock, rheumatoid arthritis, multiple sclerosis (MS) and Crohn's disease. However, the role of the endogenous peptide in inflammatory disease remains obscure because VIP-deficient mice were recently found to exhibit profound resistance in a model of MS. In the present study, we analyzed the response of female VIP deficient (KO) mice to intraperitoneal lipopolysaccharide (LPS) administration. We observed significant resistance to LPS in VIP KO mice, as evidenced by lower mortality and reduced tissue damage. The increased survival was associated with decreased levels of proinflammatory cytokines (TNFα, IL-6 and IL-12) in sera and peritoneal suspensions of these mice. Moreover, the expression of TNFα and IL-6 mRNA was reduced in peritoneal cells, spleens and lungs from LPS-treated VIP KO vs. WT mice, suggesting that the resistance might be mediated by an intrinsic defect in the responsiveness of immune cells to endotoxin. In agreement with this hypothesis, peritoneal cells isolated from VIP KO naive mice produced lower levels of proinflammatory cytokines in response to LPS in vitro. Finally, decreased NF-κB pathway activity in peritoneal cells was observed both in vivo and in vitro, as determined by assay of phosphorylated I-κB. The results demonstrate that female VIP KO mice exhibit resistance to LPS-induced shock, explainable in part by the presence of an intrinsic defect in the responsiveness of inflammatory cells to endotoxin.

TLR9 and MyD88 Are Crucial for the Development of Protective Immunity to Malaria

Effective resolution of malaria infection by avoiding pathogenesis requires regulated pro- to anti-inflammatory responses and the development of protective immunity. TLRs are known to be critical for initiating innate immune responses, but their roles in the regulation of immune responses and development of protective immunity to malaria remain poorly understood. In this study, using wild-type, TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) mice infected with Plasmodium yoelii, we show that TLR9 and MyD88 regulate pro/anti-inflammatory cytokines, Th1/Th2 development, and cellular and humoral responses. Dendritic cells from TLR9(-/-) and MyD88(-/-) mice produced significantly lower levels of proinflammatory cytokines and higher levels of anti-inflammatory cytokines than dendritic cells from wild-type mice. NK and CD8(+) T cells from TLR9(-/-) and MyD88(-/-) mice showed markedly impaired cytotoxic activity. Furthermore, mice deficient in TLR9 and MyD88 showed higher Th2-type and lower Th1-type IgGs. Consequently, TLR9(-/-) and MyD88(-/-) mice exhibited compromised ability to control parasitemia and were susceptible to death. Our data also show that TLR9 and MyD88 distinctively regulate immune responses to malaria infection. TLR9(-/-) but not MyD88(-/-) mice produced significant levels of both pro- and anti-inflammatory cytokines, including IL-1β and IL-18, by other TLRs/inflammasome- and/or IL-1R/IL-18R-mediated signaling. Thus, whereas MyD88(-/-) mice completely lacked cell-mediated immunity, TLR9(-/-) mice showed low levels of cell-mediated immunity and were slightly more resistant to malaria infection than MyD88(-/-) mice. Overall, our findings demonstrate that TLR9 and MyD88 play central roles in the immune regulation and development of protective immunity to malaria, and have implications in understanding immune responses to other pathogens.

Critical role of TLR4 in human metapneumovirus mediated innate immune responses and disease pathogenesis (169.21)

Abstract Human metapneumovirus (hMPV), described for the first time in 2001, has become one of the main viral pathogens responsible for acute respiratory tract infections in children, elderly and immunocompromised patients. The mammalian Toll-like receptors (TLR) were identified as critical regulators of innate immunity to a variety of microbes, including bacteria and fungi. Respiratory syncytial virus (RSV) fusion protein has been shown to activate TLR4, but their role in other viral infections, such as hMPV, has been largely unexplored. In this study, we examine the in vivo innate immune response in TLR4-deficient (C57BL/10ScNJ) mice and wild-type (C57BL/10SnSnJ) mice challenged with hMPV virus to address the role of TLR4 in the innate immune response to this respiratory virus infection. Our results demonstrate that mice lacking TLR4 loose less body weight compared to wild type mice. When inflammatory mediators was measured in bronchoalveolar lavage at different days post-infection, significantly lower levels of proinflammatory cytokines (IL-6, TNF-α) immunomodulatory cytokines (IL-12 p40, IL-17) and chemokines (MCP-1, MIP-1α) were detected. Further, consistent with bodyweight loss, TLR4 deficient mice show better pulmonary function demonstrated by reduced airway obstruction and airway hyperresponsiveness. The results of this study indicate that TLR4 is important for activation of the innate immune response to hMPV infection however it can also contribute to disease pathogenesis.

Pharmacokinetics, tissue distribution and immunomodulatory effect of intralipid formulation of nystatin in mice

We developed a novel lipid formulation of nystatin suitable for parenteral administration, nystatin-intralipid (NYT-IL), with antifungal activity and reduced toxicity in mice. We investigated the pharmacokinetics, tissue distribution and immunomodulatory effect of NYT-IL in mice. Nystatin levels in serum and organs were determined using HPLC after NYT-IL or nystatin administration in mice. The levels of the pro-inflammatory cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) and the anti-inflammatory cytokine interleukin 10 (IL-10) produced by splenocytes from mice injected with NYT-IL or nystatin were evaluated by an ELISA assay. Injection of NYT-IL resulted in similar levels and similar kinetics of nystatin in serum, higher concentrations in the liver and lower concentrations in the kidneys, in comparison with nystatin injection. Injection of mice with NYT-IL yielded higher levels of IL-10 than that of nystatin, whereas the levels of TNF-α and IFN-γ induced by NYT-IL were lower than those elicited by nystatin. Since polyene treatment is associated with nephrotoxicity, lower levels of nystatin in the kidneys following NYT-IL injection suggest the possibility of reduced toxicity. As the acute infusion-related adverse effects associated with polyene treatment are considered to be induced by pro-inflammatory cytokines, a higher level of anti-inflammatory and lower levels of pro-inflammatory cytokines elicited by NYT-IL administration suggest the possibility of amelioration of such effects. In summary, the altered pharmacokinetics, tissue distribution and immune response due to the use of this intralipid formulation of nystatin merit further research towards the development of a therapeutic agent against invasive mycoses.

A Francisella tularensis Live Vaccine Strain That Improves Stimulation of Antigen-Presenting Cells Does Not Enhance Vaccine Efficacy

Vaccination is a proven strategy to mitigate morbidity and mortality of infectious diseases. The methodology of identifying and testing new vaccine candidates could be improved with rational design and in vitro testing prior to animal experimentation. The tularemia vaccine, Francisella tularensis live vaccine strain (LVS), does not elicit complete protection against lethal challenge with a virulent type A Francisella strain. One factor that may contribute to this poor performance is limited stimulation of antigen-presenting cells. In this study, we examined whether the interaction of genetically modified LVS strains with human antigen-presenting cells correlated with effectiveness as tularemia vaccine candidates. Human dendritic cells infected with wild-type LVS secrete low levels of proinflammatory cytokines, fail to upregulate costimulatory molecules, and activate human T cells poorly in vitro. One LVS mutant, strain 13B47, stimulated higher levels of proinflammatory cytokines from dendritic cells and macrophages and increased costimulatory molecule expression on dendritic cells compared to wild type. Additionally, 13B47-infected dendritic cells activated T cells more efficiently than LVS-infected cells. A deletion allele of the same gene in LVS displayed similar in vitro characteristics, but vaccination with this strain did not improve survival after challenge with a virulent Francisella strain. In vivo, this mutant was attenuated for growth and did not stimulate T cell responses in the lung comparable to wild type. Therefore, stimulation of antigen-presenting cells in vitro was improved by genetic modification of LVS, but did not correlate with efficacy against challenge in vivo within this model system.

Differences in Acinetobacter baumannii Strains and Host Innate Immune Response Determine Morbidity and Mortality in Experimental Pneumonia

Despite many reports documenting its epidemicity, little is known on the interaction of Acinetobacter baumannii with its host. To deepen our insight into this relationship, we studied persistence of and host response to different A. baumannii strains including representatives of the European (EU) clones I–III in a mouse pneumonia model. Neutropenic mice were inoculated intratracheally with five A. baumannii strains and an A. junii strain and at several days morbidity, mortality, bacterial counts, airway inflammation, and chemo- and cytokine production in lungs and blood were determined. A. baumannii RUH875 and RUH134 (EU clone I and II, respectively) and sporadic strain LUH8326 resulted in high morbidity/mortality, whereas A. baumannii LUH5875 (EU clone III, which is less widespread than clone I and II) caused less symptoms. A. baumannii type strain RUH3023T and A. junii LUH5851 did not cause disease. All strains, except A. baumannii RUH3023T and A. junii LUH5851, survived and multiplied in the lungs for several days. Morbidity and mortality were associated with the severity of lung pathology and a specific immune response characterized by low levels of anti-inflammatory (IL-10) and specific pro-inflammatory (IL-12p40 and IL-23) cytokines at the first day of infection. Altogether, a striking difference in behaviour among the A. baumannii strains was observed with the clone I and II strains being most virulent, whereas the A. baumannii type strain, which is frequently used in virulence studies appeared harmless.

The Effects of Fish Oil Supplementation on Markers of Inflammation in Chronic Kidney Disease Patients

One prevalent characteristic of all stages of chronic kidney disease (CKD) is excessive production of proinflammatory cytokines. Fish oil (FO) supplementation has been reported to lower levels of proinflammatory cytokines. The benefits of FO for an extensive range of populations and a variety of health concerns are apparent, yet the anti-inflammatory benefits for nondialysis CKD patients are not as well documented. Therefore, the purpose of this study was to investigate the effects of the daily consumption of FO (1,400 mg eicosapentaenoic acid + 1,000 mg docosahexaenoic acid) on interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) for 8weeks in nondialysis CKD patients. In this double-blind, randomized, placebo-controlled intervention, the effect of 8weeks of FO administration on IL-1β, IL-6, and TNF-α levels in nondialysis CKD patients were evaluated. Thirty-one nondialysis CKD patients (17 = FO; 14 = placebo) randomly received either FO dietary supplementation 2.4 g/day (1,400 mg eicosapentaenoic acid + 1,000 mg docosahexaenoic acid) or placebo (safflower oil) for 8 weeks. IL-1β, IL-6, and TNF-α were all measured as markers of inflammation. One-way analysis of variance revealed no significant differences in IL-6 (P = .06), IL-1β (P = .18), and TNF-α (P = .20) between groups in pretest values. Additionally, no pretest differences existed between groups for age (P = .549), weight (P = .324), waist circumference (P = .086), gender (P = .591), and ethnicity (P = .875). Covariance was calculated using compliance, age, gender, ethnicity, body weight, and waist circumference as covariates. No significant differences were discovered between groups after FO supplementation for IL-6 (P = .453) and TNF-α (P = .242). A significant difference was discovered for IL-1β (P = .050) with lower levels in the FO group. The results of this study are in agreement with some previous studies that suggest that FO supplementation has no effect on plasma proinflammatory cytokines TNF-α or IL-6, but does have an effect on IL-1β in nondialysis CKD patients.

Deletion of C/EBP homologous protein (Chop) in C57Bl/6 mice dissociates obesity from insulin resistance

Endoplasmic reticulum (ER) stress has been implicated in the development of type 2 diabetes, via effects on obesity, insulin resistance and pancreatic beta cell health. C/EBP homologous protein (CHOP) is induced by ER stress and has a central role in apoptotic execution pathways triggered by ER stress. The aim of this study was to characterise the role of CHOP in obesity and insulin resistance. Metabolic studies were performed in Chop ( -/- ) and wild-type C57Bl/6 mice, and included euglycaemic-hyperinsulinaemic clamps and indirect calorimetry. The inflammatory state of liver and adipose tissue was determined by quantitative RT-PCR, immunohistology and macrophage cultures. Viability and absence of ER stress in islets of Langerhans was determined by electron microscopy, islet culture and quantitative RT-PCR. Systemic deletion of Chop induced abdominal obesity and hepatic steatosis. Despite marked obesity, Chop ( -/- ) mice had preserved normal glucose tolerance and insulin sensitivity. This discrepancy was accompanied by lower levels of pro-inflammatory cytokines and less infiltration of immune cells into fat and liver. These observations suggest that insulin resistance is not induced by fat accumulation per se, but rather by the inflammation induced by ectopic fat. CHOP may play a key role in the crosstalk between excessive fat deposition and induction of inflammation-mediated insulin resistance.

Serology and cytokine profiles in patients infected with the newly discovered Bundibugyo ebolavirus

A new species of Ebolavirus, Bundibugyo ebolavirus, was discovered in an outbreak in western Uganda in November 2007. To study the correlation between fatal infection and immune response in Bundibugyo ebolavirus infection, viral antigen, antibodies, and 17 soluble factors important for innate immunity were examined in 44 patient samples. Using Luminex assays, we found that fatal infection was associated with high levels of viral antigen, low levels of pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-6, TNF-α, and high levels of immunosuppressor cytokines like IL-10. Also, acute infected patients died in spite of generating high levels of antibodies against the virus. Thus, our results imply that disease severity in these patients is not due to the multi-organ failure and septic shock caused by a flood of inflammatory cytokines, as seen in infections with other Ebolavirus species.

Interleukin (IL)-4, IL-13, and IL-17A differentially affect the profibrotic and proinflammatory functions of fibrocytes from asthmatic patients.

Fibrocytes contribute to the fibrotic changes most frequently observed in forms of asthma where inflammation is driven by T helper type 2 (Th2) cells. The mechanisms that regulate the profibrotic function of asthmatic fibrocytes are largely unknown. We isolated circulating fibrocytes from patients with allergen-exacerbated asthma, who showed the presence of fibrocytes, together with elevated concentrations of interleukin (IL)-4 and IL-13 and slightly increased concentrations of the Th17 cell-derived IL-17A, in induced sputum. Fibrocytes stimulated with IL-4 and IL-13 produced high levels of collagenous and non-collagenous matrix components and low levels of proinflammatory cytokines. Conversely, fibrocytes stimulated with IL-17A proliferated and released proinflammatory factors that may promote neutrophil recruitment and airway hyperresponsiveness. IL-17A also indirectly increased α-smooth muscle actin but not collagen expression in fibrocytes. Thus, fibrocytes may proliferate and express a predominant profibrotic or proinflammatory phenotype in asthmatic airways depending on the local concentrations of Th2- and Th17-derived cytokines.

CCL17 Promotes Intestinal Inflammation in Mice and Counteracts Regulatory T Cell–Mediated Protection From Colitis

Priming of T cells by dendritic cells (DCs) in the intestinal mucosa and associated lymphoid tissues helps maintain mucosal tolerance but also contributes to the development of chronic intestinal inflammation. Chemokines regulate the intestinal immune response and can contribute to pathogenesis of inflammatory bowel diseases. We investigated the role of the chemokine CCL17, which is expressed by conventional DCs in the intestine and is up-regulated during colitis. Colitis was induced by administration of dextran sodium sulfate (DSS) to mice or transfer of T cells to lymphopenic mice. Colitis activity was monitored by body weight assessment, histologic scoring, and cytokine profile analysis. The direct effects of CCL17 on DCs and the indirect effects on differentiation of T helper (Th) cells were determined in vitro and ex vivo. Mice that lacked CCL17 (Ccl17(E/E) mice) were protected from induction of severe colitis by DSS or T-cell transfer. Colonic mucosa and mesenteric lymph nodes from Ccl17-deficient mice produced lower levels of proinflammatory cytokines. The population of Foxp3(+) regulatory T cells (Tregs) was expanded in Ccl17(E/E) mice and required for long-term protection from colitis. CCR4 expression by transferred T cells was not required for induction of colitis, but CCR4 expression by the recipients was required. CCL17 promoted Toll-like receptor-induced secretion of interleukin-12 and interleukin-23 by DCs in an autocrine manner, promoted differentiation of Th1 and Th17 cells, and reduced induction of Foxp3(+) Treg cells. The chemokine CCL17 is required for induction of intestinal inflammation in mice. CCL17 has an autocrine effect on DCs that promotes production of inflammatory cytokines and activation of Th1 and Th17 cells and reduces expansion of Treg cells.

Differences in Efficacy and Cytokine Profiles following Echinocandin or Liposomal Amphotericin B Monotherapy or Combination Therapy for Murine Pulmonary or Systemic Aspergillus flavus Infections

Given the recent increase in aspergillosis caused by species other than Aspergillus fumigatus, micafungin, caspofungin, and liposomal amphotericin B (L-AmBi) were investigated as monotherapy or combination therapy for murine systemic or pulmonary Aspergillus flavus infection. Treatment for 3 or 6 days was begun at 24 h (intravenous [i.v.], 2.8 × 10(4) conidia) or 2 h (intranasal, 4.1 × 10(6) to 6.75 × 10(6) conidia) postchallenge as follows: 5 or 10 mg/kg L-AmBi, 10 mg/kg caspofungin, 15 mg/kg micafungin, L-AmBi plus echinocandin, L-AmBi on days 1 to 3 and echinocandin on days 4 to 6, or echinocandin on days 1 to 3 and L-AmBi on days 4 to 6. Mice were monitored for survival, fungal burden, serum or tissue cytokines, and lung histopathology. In the systemic infection, micafungin or caspofungin was more effective than L-AmBi in prolonging survival (P < 0.05), and L-AmBi was associated with significantly elevated serum levels of interleukin-6 (IL-6), macrophage inflammatory protein 1α (MIP-1α), and IL-12 (P < 0.05). In contrast, L-AmBi was significantly more effective than the echinocandins in reducing fungal growth in most tissues (P < 0.05). Concomitant therapies produced significantly enhanced survival, reduction in fungal burden, and low levels of proinflammatory cytokines, while antagonism was seen with some sequential regimens. In comparison, in the pulmonary infection, L-AmBi was significantly better (P < 0.05) than caspofungin or the combination of L-AmBi and caspofungin in prolonging survival and reducing lung fungal burden. Caspofungin stimulated high lung levels of IL-1α, tumor necrosis factor alpha (TNF-α), and IL-6, with extensive tissue damage. In summary, systemic A flavus infection was treated effectively with L-AmBi plus micafungin or caspofungin provided that the drugs were administered concomitantly and not sequentially, while pulmonary A. flavus infection responded well to L-AmBi but not to caspofungin.

Implications of long-term medication of oral steroids and antimalarial drugs in primary Sjögren's syndrome

BackgroundImmunomodulating drugs are commonly used in treating patients with autoimmune diseases but with very different outcomes. We aimed to investigate differences in cytokine and autoantibody levels with regard to patient characteristics in patients with primary Sjögren's syndrome (pSS) receiving oral steroids or antimalarial drugs (AM) after a longer period of time. MethodsSerum samples from 141 patients fulfilling the revised EU–US criteria and 99 healthy controls were analysed for 25 cytokines and 8 autoantibodies. ResultsAM-patients had lowered levels of IL-5, IL-10, IL-13 and IFN-γ, though non-significantly. Use of prednisolone was associated with reduced levels of IL-15, IL-2, IL-4, IL-12p40, TNF-α, MIP-1α and MIP-1β (p<0.05), and a trend towards decreased levels of IL-1RA and IL-1β was observed. No associations were seen between AM and antibody levels. Significantly higher protein levels of anti-Ro-52 and anti-Ro-60 were observed in the patients taking prednisolone (p<0.05). The proportion of patients positive for anti-Ro-52 and anti-La-48 did not differ significantly in the groups taking and not taking prednisolone, but a difference was seen for anti-Ro-60 (p<0.05). ConclusionsPrednisolone is a potent anti-inflammatory and immunosuppressive drug commonly used in autoimmune diseases. Our study shows that oral steroids are associated with reduced levels of several pro-inflammatory cytokines, but increased levels of pSS specific autoantibodies. The association between steroid use and increased antibody levels is not readily explained by known steroid effects, and should therefore be confirmed in further studies. Lower levels of pro-inflammatory cytokines indicate a beneficial effect of oral steroids in this patient group.

Differential immune response of adipocytes to virulent and attenuated Mycobacterium tuberculosis

Mycobacterium tuberculosis ( M. tb) takes advantage of various cell types, allowing it to remain in the host for long periods. Because adipocytes have been proposed as niches for dormant M. tb in the latent state, understanding the interaction of virulent M. tb with adipocytes is important. We compared changes in cytokine secretion from 3T3-L1 murine adipocytes infected with virulent M. tb H37Rv (V- M. tb) and attenuated M. tb H37Ra (A- M. tb) strains. Both strains maintained non-replicating states within adipocytes until 10 days post-infection. Adipocytes infected with V- M. tb secreted lower levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12p40, IL-6, and IL-17, and lower levels of nitric oxide than those infected with A- M. tb. In contrast, the anti-inflammatory cytokines, IL-10 and IL-4, were markedly induced in V -M. tb-infected adipocytes versus those infected with A -M. tb at an early time point. Heat-killed or formalin-fixed bacteria induced lower levels of cytokines and no difference was observed between strains. Moreover, V- M. tb induced a high level of necrosis versus A -M. tb in conjunction with increased levels of LHD. These results suggest that V- M. tb regulates cytokine expression in its favor, increasing cytokines necessary for immune evasion and decreasing those required for protective immunity.

Cinnamon extract suppresses experimental colitis through modulation of antigen-presenting cells

To investigate the anti-inflammatory effects of cinnamon extract and elucidate its mechanisms for targeting the function of antigen presenting cells. Cinnamon extract was used to treat murine macrophage cell line (Raw 264.7), mouse primary antigen-presenting cells (APCs, MHCII(+)) and CD11c(+) dendritic cells to analyze the effects of cinnamon extract on APC function. The mechanisms of action of cinnamon extract on APCs were investigated by analyzing cytokine production, and expression of MHC antigens and co-stimulatory molecules by quantitative real-time PCR and flow cytometry. In addition, the effect of cinnamon extract on antigen presentation capacity and APC-dependent T-cell differentiation were analyzed by [H(3)]-thymidine incorporation and cytokine analysis, respectively. To confirm the anti-inflammatory effects of cinnamon extract in vivo, cinnamon or PBS was orally administered to mice for 20 d followed by induction of experimental colitis with 2,4,6 trinitrobenzenesulfonic acid. The protective effects of cinnamon extract against experimental colitis were measured by checking clinical symptoms, histological analysis and cytokine expression profiles in inflamed tissue. Treatment with cinnamon extract inhibited maturation of MHCII(+) APCs or CD11c(+) dendritic cells (DCs) by suppressing expression of co-stimulatory molecules (B7.1, B7.2, ICOS-L), MHCII and cyclooxygenase (COX)-2. Cinnamon extract induced regulatory DCs (rDCs) that produce low levels of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, IL-12, interferon (IFN)-γ and tumor necrosis factor (TNF)-α] while expressing high levels of immunoregulatory cytokines (IL-10 and transforming growth factor-β). In addition, rDCs generated by cinnamon extract inhibited APC-dependent T-cell proliferation, and converted CD4(+) T cells into IL-10(high) CD4(+) T cells. Furthermore, oral administration of cinnamon extract inhibited development and progression of intestinal colitis by inhibiting expression of COX-2 and pro-inflammatory cytokines (IL-1β, IFN-γ and TNF-α), while enhancing IL-10 levels. Our study suggests the potential of cinnamon extract as an anti-inflammatory agent by targeting the generation of regulatory APCs and IL-10(+) regulatory T cells.

HIV‐Exposed Seronegative Commercial Sex Workers Show a Quiescent Phenotype in the CD4+T Cell Compartment and Reduced Expression of HIV‐Dependent Host Factors

Studies of human immunodeficiency virus (HIV)-exposed seronegative individuals are crucial to inform vaccine design. In the present study we demonstrated that HIV-exposed seronegative commercial sex workers produce lower levels of proinflammatory cytokines at baseline than HIV-negative control subjects. We also showed that CD4+ T cells of HIV-exposed seronegative commercial sex workers have a characteristically lower level of gene expression that can be seen in differentially expressed genes and systems crucial for HIV replication, such as the T cell receptor pathway and previously identified HIV dependency factors. This apparent lowered activation results in a phenomenon we term "immune quiescence," which may contribute to host resistance to HIV.

Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome

Aims: The proinflammatory cytokines IL-1β and IL-18 are central players in the pathogenesis of inflammatory bowel disease (IBD). In response to a variety of microbial components and crystalline substances, both cytokines are processed via the caspase-1 activating multi-protein complex, the NLRP3 inflammasome. Here, we examined the role of the NLRP3 inflammasome in experimental colitis induced by dextran sulfate sodium (DSS). Methods: IL-1β production in response to dextran sulfate sodium (DSS) was studied in macrophages of wild-type (WT), caspase-1–/–, NLRP3–/–, ASC–/–, cathepsin B–/– or cathepsin L–/– mice. Colitis was induced in C57BL/6 and NLRP3–/– mice by oral DSS administration. A clinical disease activity score was evaluated daily. Histological colitis severity and expression of cytokines were determined in colonic tissue. Results: Macrophages incubated with DSS in vitro secreted high levels of IL-1β in a caspase-1-dependent manner. IL-1β secretion was abrogated in macrophages lacking NLRP3, ASC or caspase-1, indicating that DSS activates caspase-1 via the NLRP3 inflammasome. Moreover, IL-1β secretion was dependent on phagocytosis, lysosomal maturation, cathepsin B and L, and reactive oxygen species (ROS). After oral administration of DSS, NLRP3–/– mice developed a less severe colitis than WT mice and produced lower levels of proinflammatory cytokines in colonic tissue. Pharmacological inhibition of caspase-1 with pralnacasan achieved a comparable level of mucosal protection as NLRP3 deficiency. Conclusions: We identified the NLRP3 inflammasome as a critical check-point of intestinal inflammation in the DSS colitis model. The NLRP3 inflammasome may serve as a novel target for the development of therapeutics for patients with IBD.

Microglia in the degenerating brain are capable of phagocytosis of beads and of apoptotic cells, but do not efficiently remove PrPSc, even upon LPS stimulation.

Despite the phagocytic machinery available to microglia the aberrant amyloid proteins produced during Alzheimer's and prion disease, amyloid-β and PrPSc, are inefficiently cleared. We have shown that microglia in the ME7 model of prion disease show morphological evidence of activation, synthesize low levels of pro-inflammatory cytokines and are primed to produce exaggerated responses to subsequent inflammatory challenges. Whether these microglia engage in significant phagocytic activity in the disease per se, or upon subsequent inflammatory challenge is not clear. In the present study we show transcriptional activation of a large number of scavenger receptors (SRs), matrix metalloproteinases (MMPs), oxidative enzymes, and cathepsins in ME7 animals. Hippocampally-injected inert latex beads (6 μm) are efficiently phagocytosed by microglia of ME7 prion-diseased animals, but not by microglia in normal animals. Stimulation of ME7 animals with systemic bacterial endotoxin (lipopolysaccharide, LPS) induced further increases in SR-A2, MMP3, and urokinase plasminogen activator receptor (uPAR) but decreased, or did not alter, transcription of most phagocytosis-related genes examined and did not enhance clearance of deposited PrPSc. Furthermore, intracerebral injection with LPS (0.5 μg) induced marked microglial production of IL-1β, robust cellular infiltration and marked apoptosis but also did not induce further clearance of PrPSc. These data indicate that microglia in the prion-diseased brain are capable of phagocytosis per se, but show limited efficacy in removing PrPSc even upon marked escalation of CNS inflammation. Furthermore, microglia/macrophages remain IL-1β-negative during phagocytosis of apoptotic cells. The data demonstrate that phagocytic activity and pro-inflammatory microglial phenotype do not necessarily correlate. © 2010 Wiley-Liss, Inc.