Dietary Chitin Particles Called Mimetic Fungi Ameliorate Colitis in Toll-Like Receptor 2/CD14- and Sex-Dependent Manners.

Chitin is a natural N-acetylglucosamine polymer and a major structural component of fungal cell walls. Dietary chitin is mucoadhesive; anti-inflammatory effects of chitin microparticles (CMPs; 1- to 10-µm diameters) have been demonstrated in models of inflammatory bowel disease (IBD). The goals of this study were to assess (i) whether CMPs among various chitin preparations are the most effective against colitis in male and female mice and (ii) whether host chitin-binding Toll-like receptor 2 (TLR2) and CD14 are required for the anti-inflammatory effect of chitin. We found that colitis in male mice was ameliorated by CMPs and large chitin beads (LCBs; 40 to 70 µm) but not by chitosan (deacetylated chitin) microparticles, oligosaccharide chitin, or glucosamine. In fact, LCBs were more effective than CMPs. In female colitis, on the other hand, CMPs and LCBs were equally and highly effective. Neither sex of TLR2-deficient mice showed anti-inflammatory effects when treated with LCBs. No anti-inflammatory effect of LCBs was seen in either CD14-deficient males or females. Furthermore, an in vitro study indicated that when LCBs and CMPs were digested with stomach acidic mammalian chitinase (AMC), their size-dependent macrophage activations were modified, at least in part, suggesting reduced particle sizes of dietary chitin in the stomach. Interestingly, stomach AMC activity was greater in males than females. Our results indicated that dietary LCBs were the most effective preparation for treating colitis in both sexes; these anti-inflammatory effects of LCBs were dependent on host TLR2 and CD14.

Phagocytosis-mediated M1 activation by chitin but not by chitosan.

Chitin particles have been used to understand host response to chitin-containing pathogens and allergens and are known to induce a wide range of polarized macrophage activations, depending, at least in part, on particle size. Nonphagocytosable particles larger than a macrophage induce tissue repair M2 activation. In contrast, phagocytosable chitin microparticles (CMPs, 1-10 µm diameters) induce M1 macrophages that kill intracellular microbes and damage tissues. However, chitosan (deacetylated) microparticles (de-CMPs, 1-10 µm) induce poor M1 activation. Toll-like receptor 2 (TLR2) and associated coreceptors in macrophages appear to be required for the M1 activation. To understand the exact mechanism of phagocytosis-mediated M1 activation by chitin, we isolated macrophage proteins that bind to CMPs during early phagocytosis and determined that TLR1, TLR2, CD14, late endosomal/lysosomal adaptor MAPK and mechanistic target of rapamycin activator 1 (LAMTOR1), Lck/Yes novel tyrosine kinase (Lyn), and ß-actin formed phagosomal CMP-TLR2 clusters. These proteins were also detected in TLR2 phagosomal clusters in macrophages phagocytosing de-CMPs, but at relatively lower levels than in the CMP-TLR2 clusters. Importantly, CMP-TLR2 clusters further recruited myeloid differentiation primary response gene 88 (MyD88) and Toll-IL-1 receptor-containing adaptor protein (TIRAP) and phosphorylated Lyn, whereas neither the adaptors nor phosphorylated Lyn was detected in the de-CMP clusters. The results indicate that the acetyl group played an obligatory, phagocytosis-dependent role in the initiation of an integrated signal for TLR2-mediated M1 activation.

Glucocorticoid inhibition of leptin- and lipopolysaccharide-induced interleukin-6 production in obesity.

Obesity is considered a chronic inflammatory condition that enhances the risk of numerous inflammatory diseases, including diabetes and cardiovascular disease. Glucocorticoids (GCs) and synthetic therapeutic GCs are anti-inflammatory agents, but the exact functions of GCs in obesity-related inflammation are unknown. Therefore, the objective of this study was to examine the inhibitory effect of an exogenous GC (dexamethasone, DEX) on leptin- and lipopolysaccharide (LPS)-induced IL-6 production by peripheral blood mononuclear cells (PBMCs) ex vivo in obese subjects compared to normal-weight subjects. Blood samples were drawn from 14 obese (BMI>30 kg/m(2)) and 14 normal-weight (BMI<25 kg/m(2)) subjects. Plasma cortisol, TNF-α and IL-6 levels, and insulin resistance (HOMA-IR) were quantified. Subjects' PBMCs (1×10(6) cells/mL) were isolated and cultured with leptin (18.75 and 250 ng/mL) or LPS (10ng/mL) in the presence of DEX (0, 10(-8), 10(-7), and 10(-6) M), a synthetic GC, for 24 h; IL-6 levels and GC sensitivity (IC50) were assessed in the cultured supernatants. No differences in the plasma cortisol levels were found between the two groups. We found that obese subjects showed greater leptin- and LPS-induced IL-6 production compared to normal-weight subjects. The suppressive effect of DEX on leptin- and LPS-induced IL-6 production (IC50) was not different between the two groups. However, the IC50 of DEX for LPS-induced was correlated with BMI, waist circumference, and hip circumference. These findings suggest that reduced GC sensitivity may be an important mechanism in the up-regulation of selected obese inflammation.

Cholera toxin induces a shift from inactive to active cyclooxygenase 2 in alveolar macrophages activated by Mycobacterium bovis BCG.

Intranasal vaccination stimulates formation of cyclooxygenases (COX) and release of prostaglandin E(2) (PGE(2)) by lung cells, including alveolar macrophages. PGE(2) plays complex pro- or anti-inflammatory roles in facilitating mucosal immune responses, but the relative contributions of COX-1 and COX-2 remain unclear. Previously, we found that Mycobacterium bovis BCG, a human tuberculosis vaccine, stimulated increased release of PGE(2) by macrophages activated in vitro; in contrast, intranasal BCG activated no PGE(2) release in the lungs, because COX-1 and COX-2 in alveolar macrophages were subcellularly dissociated from the nuclear envelope (NE) and catalytically inactive. This study tested the hypothesis that intranasal administration of BCG with cholera toxin (CT), a mucosal vaccine component, would shift the inactive, NE-dissociated COX-1/COX-2 to active, NE-associated forms. The results showed increased PGE(2) release in the lungs and NE-associated COX-2 in the majority of COX-2(+) macrophages. These COX-2(+) macrophages were the primary source of PGE(2) release in the lungs, since there was only slight enhancement of NE-associated COX-1 and there was no change in COX-1/COX-2 levels in alveolar epithelial cells following treatment with CT and/or BCG. To further understand the effect of CT, we investigated the timing of BCG versus CT administration for in vivo and in vitro macrophage activations. When CT followed BCG treatment, macrophages in vitro had elevated COX-2-mediated PGE(2) release, but macrophages in vivo exhibited less activation of NE-associated COX-2. Our results indicate that inclusion of CT in the intranasal BCG vaccination enhances COX-2-mediated PGE(2) release by alveolar macrophages and further suggest that the effect of CT in vivo is mediated by other lung cells.

Induction of proinflammatory mediators by CHI3L1 is reduced by chitin treatment: decreased tumor metastasis in a breast cancer model.

Disseminated metastasis accounts for over 90% of breast cancer deaths. Recently, elevated serum levels of a glycoprotein known as chitinase-3 like-protein-1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with metastatic breast cancer. In this study, we show that there are increased levels of CHI3L1 in plasma of tumor-bearing mice and that both tumor cells and immune cells express and secrete CHI3L1. However, the biological and physiological functions of CHI3L1 are still unclear. We demonstrate that while CHI3L1 has an inhibitory role in the expression of interferon-gamma (IFN-γ), CHI3L1 up-regulates pro-inflammatory mediators, C-chemokine ligand 2 (CCL2), chemokine CX motif ligand 2 (CXCL2) and matrix metalloproteinase-9 (MMP-9) all of which contribute to tumor growth and metastasis. We found that in vitro inhibition of CHI3L1 by siRNA suppressed the production of CCL2, CXCL2 and MMP-9 by macrophages. In vivo treatment of mammary tumor-bearing mice with chitin (ß-(1-4)-poly-N-acetyl D-glucosamine), a TH(1) adjuvant and a ligand for CHI3L1, promoted immune effector functions with increased production of IFN-γ and decreased CCL2, CXCL2 and MMP-9 expression. In vivo administration of chitin to mammary tumor-bearing mice significantly decreased lung metastasis. These studies show that CHI3L1 plays a role in tumor progression and that chitin can inhibit the pleiotropic effects of CHI3L1 giving support to the idea that CHI3L1 is a useful therapeutic target for treatment of breast cancer.

Persistent inactivation of macrophage cyclooxygenase-2 in mycobacterial pulmonary inflammation.

The induction of cyclooxygenase-2 (COX-2) in tissue macrophages (MØ) increases prostaglandin E(2) (PGE(2)) release, potentially down-regulating granulomatous inflammation. In response to Mycobacteria, local MØ express COX-2, which is either nuclear envelope (NE)-associated or NE-dissociated. Persistent mycobacterial pulmonary inflammation is characterized by alveolar MØ expressing NE-dissociated (inactive) COX-2 without release of PGE(2). In this study, we examined COX-2 in alveolar MØ after intranasal exposure to heat-killed Mycobacterium bovis BCG (HK-BCG). After administration, whole lungs of C57Bl/6 mice were lavaged with saline; COX-2 expression and PGE(2) release by alveolar MØ and tumor necrosis factor (TNF)-alpha and nitric oxide levels in the lung lavage were monitored. Normal alveolar MØ had undetectable levels of COX-2 on Western blots. However, 1 day after intranasal administration, almost all alveolar MØ had phagocytosed HK-BCG and expressed NE-dissociated COX-2 without any increase in the release of PGE(2). At 28 days after intranasal administration, 68% of alveolar MØ still contained both BCG and the NE-dissociated form of COX-2. NE-associated (active) COX-2 was not observed in alveolar MØ. In contrast, 7 days after intraperitoneal injection of HK-BCG, peritoneal MØ containing HK-BCG were no longer detected. At 28 days after intranasal administration, TNF-alpha and nitrite levels in the lung lavage fluid were significantly higher than those in controls. Our results indicate that mycobacterial pulmonary inflammation is associated with suppressed PGE(2) production by alveolar MØ, with expression of COX-2 dissociated from the NE.

Capture of heat-killed Mycobacterium bovis bacillus Calmette-Guérin by intelectin-1 deposited on cell surfaces.

Intelectin is an extracellular animal lectin found in chordata. Although human and mouse intelectin-1 recognize galactofuranosyl residues included in cell walls of various microorganisms, the physiological function of mammalian intelectin had been unclear. In this study, we found that human intelectin-1 was a serum protein and bound to Mycobacterium bovis bacillus Calmette-Guérin (BCG). Human intelectin-1-binding to BCG was inhibited by Ca(2+)-depletion, galactofuranosyl disaccharide, ribose, or xylose, and was dependent on the trimeric structure of human intelectin-1. Although monomeric, mouse intelectin-1 bound to BCG, with its C-terminal region contributing to efficient binding. Human intelectin-1-transfected cells not only secreted intelectin-1 into culture supernatant but also expressed intelectin-1 on the cell surface. The cell surface intelectin-1 was not a glycosylphosphatidylinositol-anchored membrane protein. Intelectin-1-transfected cells captured BCG more than untransfected cells, and the BCG adherence was inhibited by an inhibitory saccharide of intelectin-1. Intelectin-1-preincubated cells took up BCG more than untreated cells, but the adhesion of intelectin-1-bound BCG was the same as that of untreated BCG. Mouse macrophages phagocytosed BCG more efficiently in medium containing mouse intelectin-1 than in control medium. These results indicate that intelectin is a host defense lectin that assists phagocytic clearance of microorganisms.

Chitin protects the gut epithelial barrier in a protochordate model of DSS-induced colitis.

The gastrointestinal tract of Ciona intestinalis, a solitary tunicate that siphon-filters water, shares similarities with its mammalian counterpart. The Ciona gut exhibits other features that are unique to protochordates, including certain immune molecules, and other characteristics, e.g. chitin-rich mucus, which appears to be more widespread than considered previously. Exposure of Ciona to dextran sulphate sodium (DSS) induces a colitis-like phenotype similar to that seen in other systems, and is characterized by alteration of epithelial morphology and infiltration of blood cells into lamina propria-like regions. DSS treatment also influences the production and localization of a secreted immune molecule shown previously to co-localize to chitin-rich mucus in the gut. Resistance to DSS is enhanced by exposure to exogenous chitin microparticles, suggesting that endogenous chitin is critical to barrier integrity. Protochordates, such as Ciona, retain basic characteristics found in other more advanced chordates and can inform us of uniquely conserved signals shaping host-microbiota interactions in the absence of adaptive immunity. These simpler model systems may also reveal factors and processes that modulate recovery from colitis, the role gut microbiota play in the onset of the disease, and the rules that help govern the reestablishment and maintenance of gut homeostasis.

Heat-killed BCG induces biphasic cyclooxygenase 2+ splenic macrophage formation--role of IL-10 and bone marrow precursors.

Previous studies have shown that prostaglandin E(2) (PGE(2)) release by splenic F4/80(+) cyclooxygenase (COX)-2(+) macrophages (MØ) isolated from mice, treated with mycobacterial components, plays a major role in the regulation of immune responses. However, splenic MØ, isolated from untreated mice and treated in vitro with lipopolysaccharide and interferon-gamma, express COX-1 and COX-2 within 1 day but release only minimal amounts of PGE(2) following elicitation with calcium ionophore A23187. For further characterization of in vivo requirements for development of PGE(2)-releasing MØ (PGE(2)-MØ), C57Bl/6 [wild-type (WT)], and interleukin (IL)-10-deficient (IL-10(-/-)) mice were treated intraperitoneally with heat-killed Mycobacterium bovis bacillus Calmette-Guerin (HK-BCG). One day following injection, COX-2 was induced in splenic MØ of both mouse strains. However, PGE(2) biosynthesis by these MØ was not increased. Thus, expression of COX-2 is not sufficient to induce PGE(2) production in vivo or in vitro. In sharp contrast, 14 days after HK-BCG treatment, PGE(2) release by COX-2(+) splenic MØ increased as much as sevenfold, and a greater increase was seen in IL-10(-/-) cells than in WT cells. To further determine whether the 14-day splenic PGE(2)-MØ could be derived from bone marrow precursors, we established a chimera in which bone marrow cells were transfused from green fluorescent protein (GFP)-transgenic donors to WT mice. Donors and recipients were treated with HK-BCG simultaneously, and marrow transfusion was performed on Days 1 and 2. On Day 14 after BCG treatment, a significant number of spleen cells coexpressed COX-2 and GFP, indicating that bone marrow-derived COX-2(+) MØ may be responsible for the increased PGE(2) production.

Exercise reduced pentraxin 3 levels produced by endotoxin-stimulated human peripheral blood mononuclear cells in obese individuals.

The purpose of this study was to determine whether obesity would reduce the capacity of peripheral blood mononuclear cells (PBMCs) to produce the anti-inflammatory protein pentraxin 3 (PTX3) in response to ex vivo stimulation with lipopolysaccharide (LPS), and if acute aerobic exercise would enhance this PTX3 production capacity. In addition, the inter-relationships of LPS-induced PTX3 with the inflammatory cytokines (interleukin 6 [IL-6], IL-10, and tumor necrosis factor alpha) were examined. Twenty-one healthy subjects (10 obese and 11 normal-weight) performed an acute bout of aerobic exercise at 75% VO2max. The capacity of PBMCs to produce PTX3 ex vivo following LPS stimulation was the same in obese and normal-weight subjects at rest, and decreased equally in both subject groups following acute aerobic exercise. This is in contrast to plasma PTX3, which is lower in obese subjects at rest and increased equally in both obese and normal-weight subjects following exercise. In addition, ex vivo PTX3 production was positively associated with IL-6 and IL-10 in response to acute aerobic exercise ( r = 0.686, P = 0.020; r = 0.744, P = 0.009, respectively) in normal-weight, but not in obese individuals ( r = 0.429, P = 0.249; r = 0.453, P = 0.189, respectively). These findings indicate that concentrations of PTX3 observed in plasma are relatively independent of those produced by PBMCs ex vivo and the mechanisms associated with PTX3-mediated anti-inflammatory signaling may differ during obesity. Impact statement Our laboratory has previously demonstrated that obese individuals present with lower plasma concentrations of the anti-inflammatory protein pentraxin 3 (PTX3), whereas acute aerobic exercise increases plasma PTX3 levels similarly compared to normal-weight individuals. As a follow-up, the present study demonstrates that PBMCs isolated from obese and normal-weight individuals produce comparable amounts of PTX3 ex vivo in response to lipopolysaccharide (LPS). Furthermore, given that acute aerobic exercise reduced the ex vivo production of PTX3 in both groups, our results clearly indicate that plasma PTX3 levels are relatively independent of those produced by PBMCs ex vivo. In addition, our findings suggest that the mechanisms associated with PTX3-mediated production of the anti-inflammatory cytokine interleukin 10 may be impaired in obese individuals, and thus provides a key finding necessary for the elucidation of PTX3's role in the mediation of anti-inflammatory profiles and the subsequent amelioration of inflammatory disease during obesity.

Splenic PGE2-releasing macrophages regulate Th1 and Th2 immune responses in mice treated with heat-killed BCG.

Hosts infected with low doses of mycobacteria develop T helper cell type 1 (Th1) immunity, but at relatively higher doses, a switch to Th2 immunity occurs. Prostaglandin E2 (PGE2) is a proposed mediator of the Th1-to-Th2 shift of immune responses, and mycobacterial products induce PGE2-releasing macrophages (PGE2-MØ) in the mouse spleen in a dose-dependent manner. Splenic PGE2-M Ø from Balb/c mice, given 0.01 or 1 mg heat-killed (HK) Mycobacterium bovis bacillus Calmette-Guerin (BCG) intraperitoneally (i.p.), were characterized by the ex vivo release of PGE2 (>10 ng/10(6) cells), cytokine production, and expression of PGG/H synthase (PGHS)-1, PGHS-2, cytosolic PGE synthase (PGES), and microsomal PGES-1. At Day 14 after the treatment, mice treated with 1 mg, but not 0.01 mg, BCG had increased levels of PGHS-2+ PGE2-MØ, total serum immunoglobulin E (IgE), and serum IgG1 antibodies (Th2 responses) against heat shock protein 65 and purified protein derivative. Cultures of spleen cells isolated from these mice expressed interleukin (IL)-4 and IL-10 in recall responses. Treatment of mice receiving 1 mg BCG with NS-398 (a PGHS-2 inhibitor, 10 mg/kg i.p., daily) resulted in enhanced interferon-gamma (IFN-gamma) production with reduced IL-4 and IL-10 production in recall responses. This treatment also resulted in decreased total serum IgE levels. Treatment of C57Bl/6 mice with HK-BCG (0.5 mg dose) also induced a mixture of Th1 and Th2 responses, although IFN-gamma production was markedly increased, and IL-4 was decreased compared with Balb/c mice. Thus, our results indicate that by 14 days following treatment of mice with high doses of HK-BCG, splenic PGE2-MØ formation is associated with a PGHS-2-dependent shift from Th1-to-Th2 immune responses.

Differential effects of IL-10 on prostaglandin H synthase-2 expression and prostaglandin E2 biosynthesis between spleen and bone marrow macrophages.

Different populations of mononuclear phagocytes (MO) show considerable diversity of cellular function including prostaglandin E2 (PGE2) biosynthesis. Certain bacterial components enhance PGE2 biosynthesis differentially in selected populations of MO. Interleukin (IL)-10 is proposed to inhibit modulation of PGE2 biosynthesis by down-regulating prostaglandin G/H synthase-2 (PGHS-2) expression. To assess whether IL-10 regulates PGE2 biosynthesis and PGHS-2 expression, splenic and bone marrow MO were isolated from IL-10-deficient (IL-10(-/-)), C57Bl/6 [wild-type (WT) control], and Balb/c (comparison control) mice and were treated with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma) as a model of bacterial inflammation. LPS-induced PGHS-2 expression was similar for splenic MO isolated from the three strains of mice. However, PGE2 released by LPS-treated splenic MO was significantly higher in IL-10(-/-) and Balb/c than in WT cells. In the presence of LPS and IFN-gamma, PGHS-2 expression and PGE2 release by IL-10(-/-) and Balb/c splenic MO were enhanced compared with stimulation with LPS alone or IFN-gamma alone. However, there was no significant increase in PGE2 release from WT splenic MO treated with LPS plus IFN-gamma despite increased PGHS-2 expression. In sharp contrast, PGHS-2 expression and PGE2 release by bone marrow MO were greatly enhanced in IL-10(-/-) cells compared with control cells. Our results indicate that IL-10 regulation of MO PGE2 biosynthesis and PGHS-2 expression is compartment-dependent and that PGE2 production is not linked directly to PGHS-2 levels. Furthermore, our findings emphasize strain-specific differences between C57Bl/6 and Balb/c mice, and Balb/c appears more similar to the IL-10(-/-) than to the C57Bl/6 with respect to prostanoid production.

The impact of acute aerobic exercise on chitinase 3-like protein 1 and intelectin-1 expression in obesity.

Chitinase 3-like 1 (CHI3L1) and intelectin 1 (ITLN-1) recognize microbial N-acetylglucosamine polymer and galactofuranosyl carbohydrates, respectively. Both lectins are highly abundant in plasma and seem to play pro- and anti-inflammatory roles, respectively, in obesity and inflammatory-related illnesses. The aim of this study was to examine whether plasma levels of these lectins in obese subjects are useful for monitoring inflammatory conditions immediately influenced by acute aerobic exercise. Plasma interleukin-6, a pro-inflammatory cytokine, was also examined. Twenty-two (11 obese and 11 normal-weight) healthy subjects, ages 18-30 years, were recruited to perform a 30 min bout of acute aerobic exercise at 75% VO2max. We confirmed higher baseline levels of plasma CHI3L1, but lower ITLN-1, in obese subjects than in normal-weight subjects. The baseline levels of CHI3L1 were negatively correlated with cardiorespiratory fitness (relative VO2max). However, when controlled for BMI, the relationship between baseline level of CHI3L1 and relative VO2max was no longer observed. While acute aerobic exercise elicited an elevation in these parameters, we found a lower ITLN-1 response in obese subjects compared to normal-weight subjects. Our study clearly indicates that acute aerobic exercise elicits a pro-inflammatory response (e.g. CHI3L1) with a lower anti-inflammatory effect (e.g. ITLN-1) in obese individuals. Furthermore, these lectins could be predictors of outcome of exercise interventions in obesity-associated inflammation.

Lipopolysaccharide-binding protein and leptin are associated with stress-induced interleukin-6 cytokine expression ex vivo in obesity.

Obesity is associated with enhanced inflammation and mental stress, but limited information has addressed the potential additive effect of psychological stress on obesity-associated inflammation. This study examined whether obese subjects would elicit a greater host immune response (IL-6 mRNA and cytokine) to lipopolysaccharide (LPS) in response to mental stress. Blood samples for LPS-stimulated IL-6 mRNA and cytokine were collected prior to and following mental stress. Results showed that obese subjects elicited a greater LPS-induced IL-6 along with its mRNA expression following mental stress compared to normal-weight subjects. Stress-induced IL-6 cytokine response to LPS was correlated with the baseline levels of plasma LPS binding protein (LBP) and leptin. These findings are consistent with the idea that endogenous inflammatory agents (e.g., LBP and leptin), often elevated with obesity, enhance inflammatory responses to psychological stress.

Acute aerobic exercise mediates G protein-coupled receptor kinase 2 expression in human PBMCs.

AIMS: G protein-coupled receptor kinase 2 (GRK2), a cytosolic enzyme desensitizing G protein-couple receptors (e.g., ß-adrenergic receptors [ß-ARs]), is involved in regulation of hypertension, congestive heart failure, and inflammatory response. Since cellular GRK2 levels change quickly in response to exogenous/endogenous stimuli, this study examined whether GRK2 levels in human peripheral blood mononuclear cells (PBMCs) would increase during acute aerobic exercise and be associated with plasma IL-6 and cardiorespiratory fitness levels. MAIN METHODS: Eighteen subjects (8 men and 10 women), ages 18 to 30 years, were recruited to perform a 30-minute bout of acute aerobic exercise at 75% VO2max. KEY FINDINGS: Our results demonstrated that women exhibited significantly greater exercise-induced GRK2 expression in PBMCs compared to men. IL-6 modulation is independent of GRK2 expression. Furthermore, the percent change in GRK2 expression was negatively correlated with cardiorespiratory fitness levels (relative VO2max), but not plasma IL-6. SIGNIFICANCE: Acute aerobic exercise induces a greater GRK2 expression in women than men, while increased cardiorespiratory fitness is associated with exercise-induced GRK2 expression in PBMCs. Gender could be a contributor to regulate this GRK2 responsiveness to acute aerobic exercise.

Dust mite-induced asthma in cynomolgus monkeys.

Animal models exhibiting high homology with humans at the genetic and pathophysiological levels will facilitate identification and validation of gene targets underlying asthma. In the present study, a nonhuman primate model of allergic asthma was developed by sensitizing cynomolgus monkeys to dust mite antigen. Sensitization elevated allergen-specific serum IgE and IgG levels, and peripheral blood mononuclear cells isolated from sensitized animals released IL-4, IL-5, and IL-10, but not IFN-gamma. Aerosolized allergen decreased dynamic compliance and induced airway inflammation and hyperresponsiveness to aerosolized histamine. Albuterol and dexamethasone inhibited the airway constriction and allergen-induced inflammation, respectively. Airway wall remodeling that included goblet cell hyperplasia, basement membrane thickening, and smooth muscle hypertrophy was particularly evident in neonatally sensitized animals. In contrast to animals sensitized as adults, neonatally sensitized animals exhibited increased sensitivity to adenosine and larger allergen-induced changes in airway resistance and dynamic compliance. These results demonstrate that sensitization of cynomolgus monkeys with dust mite induces asthmalike symptoms, some of which may be dependent on age at the time of sensitization.

Brain-derived neurotrophic factor expression ex vivo in obesity.

Obesity is associated with an increased risk in neurodegenerative diseases. To counteract the neuronal damage, the human body increases brain-derived neurotrophic factor (BDNF) expression, leading to neuronal survival and plasticity. Recently, peripheral blood mononuclear cells (PBMCs) have been found to release BDNF as a potential neuroprotective role of inflammation. Therefore, the purpose of this study was to examine whether lipopolysaccharide (LPS)-induced PBMC activation would lead to differences in BDNF and inflammatory responses between obese and non-obese subjects. Thirty-one subjects (14 obese and 17 non-obese), ages 18 to 30years, were recruited. PBMCs were cultured for 24h with 10ng/mL LPS. BDNF, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured in both plasma and cell culture supernatants. Our results did not illustrate any differences in plasma BDNF levels between obese and non-obese groups. However, obese subjects elicited a greater plasma IL-6 production, which was positively associated with plasma BDNF. Furthermore, LPS-induced PBMCs expressed significantly higher BDNF and IL-6 levels in obese subjects compared to the non-obese subjects. Finally, these BDNF levels were positively correlated with IL-6 response ex vivo. These findings suggest that under a high inflammatory state, PBMCs produce greater BDNF and IL-6 expression which may play a collaborative role to protect against neuronal damage associated with obesity.

Chitin enhances obese inflammation ex vivo.

Infection has been implicated as a co-risk factor for obesity, but the mechanism remains uncertain. Elevated levels of plasma chitinase 3-like 1 (CHI3L1) are found in obese individuals. Since CHI3L1 is produced by activated immune cells including macrophages and recognizes microbial N-acetylglucosamine polymer (chitin), we asked whether the plasma CHI3L1 protein change in obese individuals might alter their innate immune response to chitin. Thirty-six subjects (15 obese and 21 non-obese), ages 18-30 years, were recruited. Peripheral blood mononuclear cells (PBMCs) were cultured with chitin microparticles (CMP; 1-10 µm) for 24h; tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and CHI3L1 in the culture supernatants were measured. We chose CMP, since neither large chitin beads (40-100 µm), chitosan microparticles (1-10 µm), nor soluble chitin induced the cytokine/CHI3L1 production by PBMCs isolated from non-obese PBMCs ex vivo. We found that the quantity of IL-6, but not TNF-α or CHI3L1, induced by CMP was significantly correlated with plasma IL-6, BMI, waist/hip circumferences, fasting plasma insulin, and insulin resistance. These findings suggest that chitin, a substrate of CHI3L1, further promotes obese inflammation in a size- and chemical composition- dependent manner.

Role of PPARγ in COX-2 activation in mycobacterial pulmonary inflammation.

Preliminary studies show that intranasal (i.n.) administration of BCG in mice induces M1 activation of alveolar macrophages (M∅) that increase TNF-α production and cyclooxygenase-2 (COX-2) expression but reduce constitutive peroxisome proliferator-activated receptor gamma (PPARγ) expression. However, COX-2 is catalytically inactive for prostaglandin E(2) release, unlike COX-2 that is active in M1 activation in vitro by BCG. In this study, we determined the role of PPARγ for BCG-induced M1 activation in vivo and in vitro. We found that treatment of mice with GW9662, a PPARγ antagonist, prior to i.n. BCG, partially restored PPARγ expression, and decreased TNF-α production and COX-2 expression. But COX-2 was still inactive. The decreased effects on TNF-α and COX-2 were also observed when alveolar M∅ were treated in vitro with GW9662/BCG, but COX-2 was still active. Our results indicate that PPARγ upregulates M1 activation of alveolar M∅, but inactive COX-2 formation is independent of PPARγ in mycobacterial pulmonary inflammation.

Chitin microparticles for the control of intestinal inflammation.

BACKGROUND: Chitin is a polymer of N-acetylglucosamine with the ability to regulate innate and adaptive immune responses. However, the detailed mechanisms of chitin-mediated regulation of intestinal inflammation are only partially known. METHODS: In this study chitin microparticles (CMPs) or phosphate-buffered saline (PBS) were orally administered to acute and chronic colitis models every 3 days for 6 consecutive weeks beginning at weaning age. The effects of this treatment were evaluated by histology, cytokine production, coculture study, and enteric bacterial analysis in dextran sodium sulfate (DSS)-induced colitis or T-cell receptor alpha (TCRα) knockout chronic colitis models. RESULTS: Histologically, chitin-treated mice showed significantly suppressed colitis as compared with PBS-treated mice in both animal models. The production of interferon-gamma (IFN-γ) was upregulated in the mucosa of chitin-treated mice compared with control mice. The major source of IFN-γ-producing cells was CD4+ T cells. In mouse dendritic cells (DCs) we found that CMPs were efficiently internalized and processed within 48 hours. Mesenteric lymph nodes (MLNs) CD4+ T cells isolated from chitin-treated mice produced a 7-fold higher amount of IFN-γ in the culture supernatant after being cocultured with DCs and chitin as compared with the control. Proliferation of carboxyfluorescein succinimidyl ester (CFSE)(low) CD4+ T cells in MLNs and enteric bacterial translocation rates were significantly reduced in chitin-treated mice when compared with the control. In addition, CMPs improved the imbalance of enteric bacterial compositions and significantly increased interleukin (IL)-10-producing cells in noninflamed colon, indicating the immunoregulatory effects of CMPs in intestinal mucosa. CONCLUSIONS: CMPs significantly suppress the development of inflammation by modulating cytokine balance and microbial environment in colon.