Electron tomography of negatively stained complex viruses: application in their diagnosis

Pattern recognition receptors are a key component of the first line host defense against infection, recognizing specific microbial products. We hypothesize that monocyte hyporesponsiveness in human sepsis is associated with a downregulation of the pattern recognition receptors Toll-like receptor (TLR)-2 and TLR4. Protein expression of CD14, TLR2 and TLR4 on blood monocytes was examined using flow cytometry from 29 patients with sepsis and 14 healthy controls. In addition LPS stimulated TNF-α and IL-10 production was studied in a 24 hour whole blood assay. We found an increased expression of CD14, TLR2 and TLR4 in patients with sepsis compared to controls (p < 0.01). In patients with sepsis, death was associated with significant lower CD14 and TLR2 expression at admission (CD14: 25.7 +- 19.1 vs 39.1 +- 17.3 mean fluorescence intensity [MFI], p = 0.02; TLR2: 21.8 +- 9.4 vs. 30.9 +- 9.6, p = 0.01). At 72 hours the TLR2 expression on monocytes was associated with the IL-10 inducibility after LPS stimulation (r = 0.52, p = 0.02) and the CD14 expression with the IL-6, IL-10 and TNF inducibility. We conclude that septic patients are characterized by an increased expression of CD14, TLR2 and TLR4 on monocytes compared to controls. Death is associated with downregulation of TLR2 and CD14 expression on monocytes correlating with reduced cytokine inducibility. We suggest that CD14 and TLR2 are a key factor in monocyte hyporesponsibility during severe sepsis.     

Quercetin's influence on exercise-induced changes in plasma cytokines and muscle and leukocyte cytokine mRNA.

Trained male cyclists (n = 40) ingested quercetin (Q; n = 20) (1,000 mg/day) or placebo (P; n = 20) supplements under randomized, double-blinded methods for 3 wk before and during a 3-day period in which subjects cycled for 3 h/day at approximately 57% maximal work rate. Blood samples were collected before and after each exercise session and assayed for plasma IL-6, IL-10, IL-1ra, IL-8, TNF-alpha, and monocyte chemoattractant protein 1, and leukocyte IL-10, IL-8, and IL-1ra mRNA. Muscle biopsies were obtained before and after the first and third exercise sessions and assayed for NF-kappaB and cyclooxygenase-2 (COX-2), IL-6, IL-8, IL-1beta, and TNF-alpha mRNA. Postexercise increases in plasma cytokines did not differ between groups, but the pattern of change over the 3-day exercise period tended to be lower in Q vs. P for IL-8 and TNF-alpha (P = 0.094 for both). mRNA increased significantly postexercise for each cytokine measured in blood leukocyte and muscle samples. Leukocyte IL-8 and IL-10 mRNA were significantly reduced in Q vs. P (interaction effects, P = 0.019 and 0.012, respectively) with no other leukocyte or muscle mRNA group differences. Muscle NF-kappaB did not increase postexercise and did not differ between Q and P. Muscle COX-2 mRNA increased significantly postexercise but did not differ between Q and P. In summary, 1 g/day quercetin supplementation by trained cyclists over a 24-day period diminished postexercise expression of leukocyte IL-8 and IL-10 mRNA, indicating that elevated plasma quercetin levels exerted some effects within the blood compartment. Quercetin did not, however, influence any of the muscle measures, including NF-kappaB content, cytokine mRNA, or COX-2 mRNA expression across a 3-day intensified exercise period.     

Intracellular monocyte and serum cytokine expression is modulated by exhausting exercise and cold exposure

This study tested the hypothesis that exercise elicits monocytic cytokine expression and that prolonged cold exposure modulates such responses. Nine men (age, 24.6 +/- 3.8 y; VO(2 peak), 56.8 +/- 5.6 ml. kg(-1). min(-1)) completed 7 days of exhausting exercise (aerobic, anaerobic, resistive) and underwent three cold, wet exposures (CW). CW trials comprised 相似文献   

Activated T lymphocytes regulate hyaluronan binding to monocyte CD44 via production of IL-2 and IFN-gamma

Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-alpha. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-gamma, and TNF-alpha were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-alpha, anti-IL-2, and anti-IFN-gamma Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-alpha induced high levels of monocyte HA binding. Likewise, IFN-gamma augmented TNF-alpha-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4(+) T cells in these assays. Fixed, PMA/ionomycin-activated CD4(+) T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-gamma and anti-TNF-alpha Abs blocked fixed PMA/ionomycin-activated CD4(+) T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-gamma, and a role for monocyte-derived TNF-alpha in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.     

IL-13 acutely augments HIV-specific and recall responses from HIV-1-infected subjects in vitro by modulating monocytes

We show in this study that acute exposure of PBMCs derived from HIV-infected subjects to IL-13 results in increased recall T cell lymphoproliferative responses against HIV-1 p24 (n = 30, p < 0.0001) and other recall Ags (influenza, n = 43, p < 0.0001; purified protein derivative tuberculin, n = 6, p = 0.0299). This effect is due to a mechanism that acutely targets APC function in the adherent monocyte subset, as shown by the expansion of CD4(+) T cell responses following coculture of IL-13-treated enriched CD14(+) monocytes with donor-matched enriched CD4(+) T cells and Ag. Exposure to IL-13 over 18-72 h resulted in a significant enhancement of monocyte endocytosis (n = 11, p = 0.0005), CD86 expression (n = 12, p = 0.001), and a significant decrease in spontaneous apoptosis (n = 8, p = 0.008). Moreover, IL-13 exposure induced a significant decrease of significantly elevated constitutive levels of PBMC-secreted TNF-alpha (n = 14, p < 0.001) and IL-10 (n = 29, p < 0.001) within 18 h of exposure ex vivo, also reflected by decreased gene expression in the adherent cell population. Our data show that IL-13 is able to acutely enhance the function of the CD14(+) cell subset toward supporting Ag-specific cell-mediated responses in chronic HIV-1 infection.     

alpha1-Antitrypsin regulates CD14 expression and soluble CD14 levels in human monocytes in vitro

The recognition of bacterial lipopolysaccharide (LPS) is principally mediated by either membrane-bound or soluble form of the glycoprotein CD14 and CD14-associated signal transducer, toll-like receptor 4 (TLR4). Recent findings indicate that the serine protease inhibitor, alpha1-antitrypsin (AAT), may not only afford protection against proteolytic injury, but may also neutralize microbial activities and affect regulation of innate immunity. We postulated that AAT affects monocyte responses to LPS by regulating CD14 expression and soluble CD14 release. Here we show that a short-term (up to 2h) monocyte exposure to AAT alone or in combination with LPS leads to a remarkable induction of CD14 levels. In parallel, a short-term (2h) cell exposure to AAT/LPS significantly enhances LPS-induced NF kappaB (p50 and p65) activation in conjunction with increased TNFalpha, IL-1 beta and IL-8 release. In contrast, longer term incubation (18 h) of monocytes with combined AAT/LPS results in a significant reduction in expression of both CD14 and TLR4, inhibition of LPS-induced TNFalpha, IL-1 beta and IL-8 mRNA and protein expression. These findings provide evidence that AAT is an important regulator of CD14 expression and release in monocytes and suggest that AAT may be involved in LPS neutralization and prevention of over-activation of monocytes in vivo.     

Effects of exercise on weight loss and monocytes in obese mice

Obesity causes innate immune dysfunction, contributing to increased disease risk. Weight loss from a combination of caloric restriction and exercise is the most effective treatment of obesity. We compared forced and voluntary exercise as weight-loss treatments in diet-induced obese (DIO) mice and assessed the effects of weight loss on monocyte concentration and cell-surface expression of Toll-like receptor (TLR) 2, TLR4, CD80, and CD86. DIO CD1 male mice were allocated randomly to 1 of 3 groups (n = 6 per group): voluntary wheel running (VEX); forced treadmill running (FEX); and sedentary (S). A fourth (control) group (CN, n = 6) of nonDIO mice was included also. During the 8-wk weight-loss treatment, all 4 groups consumed a low-fat (10% fat) diet. Nonlethal saphenous vein blood samples collected at baseline, week 4, and week 8 were analyzed by flow cytometry to assess monocyte concentration and functional receptor expression. The VEX and FEX groups lost significantly more body weight (36% and 27%, respectively) over the 8 wk of treatment than did other groups. VEX mice ran 4.4 times more than did FEX animals. VEX mice had higher monocyte concentrations (48% and 58%, respectively) than did the CN and FEX groups. Compared with baseline, week 8 cell-surface expression of TLR2 (22%), TLR4 (33%), and CD86 (18%) was increased in VEX mice. At week 4, CD80 expression was 42% greater for VEX than S mice. The present study confirms that short-term exercise and low-fat diet consumption cause significant weight loss and altered immune profiles.     

Hormonal modulation of interleukin-6, tumor necrosis factor and associated receptor secretion in postmenopausal women

Hormone replacement therapy (HRT) reduces the risk for osteoporosis but transiently increases cardiovascular risk for some postmenopausal women. This study investigated the hypothesis that these risks are associated with HRT-induced changes in mononuclear cell secretion of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and associated soluble receptors. Compared to the untreated condition (n=8), estrogen therapy (n=7) and estrogen+progestin therapy (n=7) both caused 2-fold elevations in TNF-alpha secretion. IL-6 secretion was increased (48%, P=0.04) only by estrogen+progestin therapy. Although soluble receptor secretion was not different among groups, soluble TNF receptor type I and IL-6 receptor secretion were inversely related to plasma follicle stimulating hormone (P<0.05). Both therapies reduced plasma osteocalcin (a marker for osteoporosis) by approximately 50% (P<0.002). Plasma C-reactive protein (CRP, a marker for cardiovascular risk) was 3-fold higher in women receiving only estrogen, compared to untreated women (P=0.01), and twice as high as those receiving estrogen+ progestin (P=0.045). Simple linear relationships were not observed between cytokine secretion and these markers, but a significant HRT/TNF-alpha interaction with osteocalcin (P=0.022) and an HRT/IL-6 interaction with CRP (P =0.016) indicated more complex relationships between hormone replacement, cytokine activity, and health risks associated with menopause.     

Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses

Leukocyte responsiveness to LPS is dependent upon CD14 and receptors of the Toll-like receptor (TLR) family. Neutrophils respond to LPS, but conflicting data exist regarding LPS responses of eosinophils and basophils, and expression of TLRs at the protein level in these granulocyte lineages has not been fully described. We examined the expression of TLR2, TLR4, and CD14 and found that monocytes expressed relatively high levels of cell surface TLR2, TLR4, and CD14, while neutrophils also expressed all three molecules, but at low levels. In contrast, basophils expressed TLR2 and TLR4 but not CD14, while eosinophils expressed none of these proteins. Tested in a range of functional assays including L-selectin shedding, CD11b up-regulation, IL-8 mRNA generation, and cell survival, neutrophils responded to LPS, but eosinophils and basophils did not. In contrast to previous data, we found, using monocyte depletion by negative magnetic selection, that neutrophil responses to LPS were heavily dependent upon the presence of a very low level of monocytes, and neutrophil survival induced by LPS at 22 h was monocyte dependent. We conclude that LPS has little role in the regulation of peripheral blood eosinophil and basophil function, and that, even in neutrophils, monocytes orchestrate many previously observed leukocyte LPS response patterns.     

Construction of a sensitive pyrogen-testing cell model by site-specific knock-in of multiple genes

A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.     

Neisseria meningitidis can induce pro-inflammatory cytokine production via pathways independent from CD14 and toll-like receptor 4

Fulminant meningococcal sepsis (FMS) is considered the prototypical Gram-negative sepsis. Lipopolysaccharide (LPS) is thought to be the main toxic element that induces pro-inflammatory cytokine production after interaction with CD14 and toll-like receptor 4 (TLR4). However, there is increasing evidence that LPS is not the sole toxic element of meningococci. The aim of the present study was to determine the role of CD14 and TLR4 in pro-inflammatory cytokine induction by meningococci. To this end, cytokine induction by isolated meningoccal LPS, wild-type N. meningitidis H44/76 (LPS+-meningococci) matched for concentrations of LPS and LPS-deficient N. meningitidis H44/76lpxA (LPS - -meningococci) was studied in human PBMCs and murine peritoneal macrophages (PMs). Pre-incubation of PBMCs with WT14, a monoclonal antibody against CD14, abolished TNF-alpha and IL-1beta induction by E. coli LPS, while cytokine induction by meningococcal LPS was only partially inhibited. When LPS+- and LPS - -meningococci at higher concentrations were used as stimuli, anti-CD14 had a minimal effect. In C3H/HeJ murine PMs, devoid of a functional TLR4, minimal IL-1alpha, IL-6 and TNF-alpha production was seen after stimulation with 10 ng/mL E. coli or meningococcal LPS. However, at higher concentrations (1000 ng LPS/mL) the production of TNF-alpha, but not IL-1alpha or IL-6, occurred also independently of TLR4. The expression of a functional TLR4 in murine PMs had no effect on the cytokine induction by LPS+- or LPS - -meningococci. It is concluded that pro-inflammatory cytokine induction by N. meningitidis can occur independently of CD14 and TLR4.     

TNF-alpha sensitizes HT-29 colonic epithelial cells to intestinal lactobacilli

The ability of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) to influence epithelial interleukin (IL)-8 responses to the intestinal bacterium Lactobacillus plantarum 299v was analyzed in the human HT-29 colonic epithelial cell line. In the absence of TNF-alpha, IL-8 mRNA expression was not detectable by Northern blot analysis in HT-29 cells alone or in HT-29 cells co-cultured with L. plantarum 299v. However, TNF-alpha induced IL-8 mRNA expression, and co-culture of TNF-alpha-treated HT-29 cells with L. plantarum 299v significantly increased IL-8 mRNA expression above levels induced by TNF-alpha alone in an adhesion-dependent manner. The increase in IL-8 mRNA expression was not observed in TNF-alpha-treated HT-29/L. plantarum 299v co-cultures using heat-killed lactobacilli or when L. plantarum adhesion was prevented using mannoside or a trans-well membrane. Paradoxically, IL-8 secretion was decreased in TNF-alpha-treated HT-29 cells with L. plantarum 299v relative to cells treated with TNF-alpha alone. TNF-alpha-mediated responsiveness to L. plantarum 299v was further investigated by analyzing expression of a coreceptor for bacterial cell wall products CD14. HT-29 cells expressed CD14 mRNA and cell-surface CD14; however, TNF-alpha did not alter CD14 mRNA or cell-surface expression, and blockade of CD14 with monoclonal antibody MY4 did not alter the IL-8 response to L. plantarum 299v in TNF-alpha-treated HT-29 cells. These results indicate that although TNF-alpha sensitizes HT-29 epithelial cells to intestinal lactobacilli, the bacteria exert a protective effect by downregulating IL-8 secretion.     

Exercise accelerates cutaneous wound healing and decreases wound inflammation in aged mice

The purpose of this study was to determine the effect of exercise on wound healing and inflammation in young (3 mo) and old (18 mo) female BALB/cByJ mice. Mice were assigned to either exercise or sedentary control (control) groups. The exercise group mice were run on a motorized treadmill at a moderate intensity 30 min/day for 8 days. All mice were given four full-thickness dermal wounds, and the rate of wound closure was assessed daily for 10 days. Four months later, the aged mice were rerandomized to treatment, wounded again in different locations, and wounds were harvested at 1, 3, or 5 days postwounding. Wound tissue was analyzed for IL-1beta, IL-6, keratinocyte chemoattractant (KC), monocyte chemoattractant protein-1 (MCP-1), and TNF-alpha protein. Myeloperoxidase (MPO) activity and F4/80 mRNA were assessed as an indirect measure of neutrophil and macrophage content, respectively. There was a trend (P = 0.10) for exercise to reduce wound size in young mice, and exercise significantly (P < 0.05) decreased wound size in old mice. TNF-alpha, KC, and MCP-1 were significantly (P < 0.05) lower in wounds from exercised old mice compared with control. No group differences were found for wound IL-1beta or IL-6, MPO activity, or F4/80 mRNA. Our data suggest that exercise accelerates the wound healing process in old mice. This improved healing response in the old mice may be the result of an exercise-induced anti-inflammatory response in the wound.     

Cytokine gene expression in human skeletal muscle during concentric contraction: evidence that IL-8, like IL-6, is influenced by glycogen availability

To determine the expression and induction of cytokines in human skeletal muscle during concentric contractions, eight males performed 60 min of bicycle exercise, with either a normal (Con) or reduced (Lo Gly) preexercise intramuscular glycogen content. Muscle biopsy samples were obtained before and after exercise and analyzed for glycogen and the mRNA expression of 13 cytokines. Resting muscle glycogen was higher (P < 0.05) in Con compared with Lo Gly and was reduced (P < 0.05) to 102 +/- 32 vs. 17 +/- 5 mmol U glycosyl/kg dry mass for Con and Lo Gly, respectively. We detected mRNA levels in human skeletal muscle for five cytokines, namely interleukin (IL)-1 beta, IL-6, IL-8, IL-15, and tumor necrosis factor-alpha. However, muscle contraction increased (P < 0.05) the mRNA expression of IL-6 and IL-8 alone. In addition, the fold change for both IL-8 and IL-6 was markedly higher (P < 0.05) in Lo Gly compared with Con. Given these results, we analyzed venous blood samples, obtained before and during exercise, for IL-6 and IL-8. Plasma IL-6 was not different at rest, and although the circulating concentration of this cytokine increased (P < 0.05) it increased to a greater extent (P < 0.05) throughout exercise in Lo Gly. In contrast, plasma IL-8 was not affected by exercise or treatment. These data demonstrate that cytokines are not ubiquitously expressed in skeletal muscle and that only IL-6 and IL-8 mRNA are increased during contraction of this mode and duration. Furthermore, the mRNA abundance of IL-6 and IL-8 appears to be influenced by glycogen availability in the contracting muscle.     

Differential pattern recognition receptor expression but stereotyped responsiveness in rat spleen dendritic cell subsets

Dendritic cells (DC) are a heterogeneous population of APC endowed with specific functions. The nature of the DC subset involved in the course of an immune response to a specific pathogen might be important for inducing the appropriate effectors. In addition, each DC subset might also exhibit intrinsic functional plasticity. In the rat, spleen DC can be separated into three morphological and phenotypical distinct subsets, namely CD4+, CD4-, and plasmacytoid DC (pDC), whose frequencies are strain dependent. We correlated the expression of TLR and nucleotide-binding oligomerization domain 2 (NOD2) in these DC subsets to their in vitro responsiveness to specific ligands. CD4- DC expressed high levels of TLR1, 2, 3, and 10 mRNA, low TLR4, 5, 6, 7, and 9, and very low, if any, TLR8. pDC had a restricted repertoire characterized by high TLR7 and 9. CD4+ DC expressed all TLR and 10-fold higher levels of NOD2 mRNA than CD4- and pDC. Upon stimulation by TLR and NOD2 ligands, each DC subset responded in quite a stereotyped fashion. TLR2/6, 3, 4, 5, 9, and NOD2 triggering induced CD4- DC to mature and produce high IL-12p40, low IL-10, and TNF-alpha. TLR7/8 and 9 triggering induced pDC to mature and produce copious amounts of IL-6, IL-12p40, and TNF-alpha and low IFN-alpha. CD4+ DC were very poor producers of inflammatory cytokines. This study suggests that the nature of spleen DC responses to pathogens is dependent on subset specific-stimulation rather than intrinsic plasticity.     

Toll-like receptor 2 participates in mediation of immune response in experimental pneumococcal meningitis

Heterologous expression of Toll-like receptor (TLR)2 and CD14 in Chinese hamster ovary fibroblasts was reported to confer responsiveness to pneumococcal peptidoglycan. The present study characterized the role of TLR2 in the host immune response and clinical course of pneumococcal meningitis. Pneumococcal infection of mice caused a significant increase in brain TLR2 mRNA expression at both 4 and 24 h postchallenge. Mice with a targeted disruption of the TLR2 gene (TLR2-/-) showed a moderate increase in disease severity, as evidenced by an aggravation of meningitis-induced intracranial complications, a more pronounced reduction in body weight and temperature, and a deterioration of motor impairment. These symptoms were associated with significantly higher cerebellar and blood bacterial titers. Brain expression of the complement inhibitor complement receptor-related protein y was significantly higher in infected TLR2-/- than in wild-type mice, while the expression of the meningitis-relevant inflammatory mediators IL-1beta, TNF-alpha, IL-6, macrophage-inflammatory protein (MIP)-2, inducible NO synthase, and C3 was similar in both genotypes. We first ectopically expressed single candidate receptors in HEK293 cells and then applied peritoneal macrophages from mice lacking TLR2 and/or functional TLR4 for further analysis. Overexpression of TLR2 and TLR4/MD-2 conferred activation of NF-kappaB in response to pneumococcal exposure. However, pneumococci-induced TNF-alpha release from peritoneal macrophages of wild-type and TLR2/functional TLR4/double-deficient mice did not differ. Thus, while TLR2 plays a significant role in vivo, yet undefined pattern recognition receptors contribute to the recognition of and initiation of the host immune defense toward Streptococcus pneumoniae infection.     

C-C chemokine ligand 2/monocyte chemoattractant protein-1 directly inhibits NKT cell IL-4 production and is hepatoprotective in T cell-mediated hepatitis in the mouse

T cell-mediated liver diseases are associated with elevated serum levels of C-C chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1). However, the extent to which the actions of CCL2/MCP-1 contribute to the pathogenesis of T cell-mediated hepatitis remains incompletely understood. Con A-induced hepatitis is a liver-specific inflammation mediated by activated T cells and is driven by an up-regulation of the hepatic expression of TNF-alpha, IFN-gamma, and IL-4. The present study examined the role of CCL2/MCP-1 in the pathogenesis of T cell-mediated hepatitis induced by Con A administration in the mouse. We demonstrate a novel hepatoprotective role for CCL2/MCP-1 during Con A-induced hepatitis, because CCL2/MCP-1 neutralization strikingly enhanced hepatic injury, both biochemically and histologically, after Con A administration. Furthermore, CCL2/MCP-1 neutralization was associated with a significant reduction in the hepatic levels of TNF-alpha and IFN-gamma, but with a significant increase in hepatic IL-4 levels. Moreover, IL-4 production and CCR2 expression by Con A-stimulated CD3(+)NK1.1(+) T cells was significantly reduced by rMCP-1 treatment in vitro. In summary, we propose that CCL2/MCP-1 fulfills a novel anti-inflammatory role in T cell-mediated hepatitis by inhibiting CD3(+)NK1.1(+) T cell-derived IL-4 production through direct stimulation of its specific receptor CCR2. These findings may have direct clinical relevance to T cell-mediated hepatitis.     

IL-8 and IDO expression by human gingival fibroblasts via TLRs

Human gingival fibroblasts (HGFs), a predominant cell type in tooth-supporting structure, are presently recognized for their active role in the innate immune response. They produce a variety of inflammatory cytokines in response to microbial components such as LPS from the key periodontal pathogen, Porphyromonas gingivalis. In this study, we demonstrated that HGFs expressed mRNA of TLRs 1, 2, 3, 4, 5, 6, and 9, but not TLRs 7, 8, and 10. Stimulation of HGFs with highly purified TLR2 ligand (P. gingivalis LPS), TLR3 ligand (poly(I:C)), TLR4 ligand (Escherichia coli LPS), and TLR5 ligand (Salmonella typhimurium flagellin) led to expression of IL-8 and IDO. A potent TLR 9 ligand, CpG oligodeoxynucleotide 2006 had no effect, although HGFs showed a detectable TLR9 mRNA expression. No significant enhancement on IL-8 or IDO expression was observed when HGFs were stimulated with various combinations of TLR ligands. Surprisingly, the TLR9 ligand CpG oligodeoxynucleotide 2006 was able to specifically inhibit poly(I:C)-induced IL-8 and IDO expression. TNF-alpha enhanced TLR ligand-induced IL-8 production in HGFs, whereas IFN-gamma enhanced TLR ligand-induced IDO expression. HGF production of IDO in response to P. gingivalis LPS, IFN-gamma, or the two in combination inhibited T cell proliferation in MLRs. The observed T cell inhibition could be reversed by addition of either 1-methyl-dl-tryptophan or l-tryptophan. Our results suggest an important role of HGFs not only in orchestrating the innate immune response, but also in dampening potentially harmful hyperactive inflammation in periodontal tissue.     

Limited contribution of Toll-like receptor 2 and 4 to the host response to a fungal infectious pathogen, Cryptococcus neoformans

The present study was designed to elucidate the role of Toll-like receptor (TLR) 2 and TLR4 in the host response to Cryptococcus neoformans. Both TLR2 knockout (KO) and TLR4KO mice produced interleukin-1beta (IL-1beta), IL-6, IL-12p40 and tumor necrosis factor-alpha (TNF-alpha) in sera and cleared this fungal pathogen from infected lungs at a comparable level to control littermate (LM) mice. Synthesis of these cytokines was not significantly different in the lungs of these KO mice and LM mice, although IL-1beta, IL-6 and IL-12p40 tended to be lower in TLR2KO, but not TLR4KO, mice than in controls. In addition, there was no significant reduction detected in the synthesis of IL-12 and TNF-alpha by bone marrow-derived dendritic cells from TLR2KO and TLR4KO mice upon stimulation with live yeast cells. Finally, HEK293 cells expressing either TLR2/dectin-1 or TLR4/MD2/CD14 did not respond to C. neoformans in the activation of nuclear factor kappa B (NFkappaB) detected by a luciferase assay. Our results suggest that TLR2 and TLR4 do not or only marginally contribute to the host and cellular response to this pathogen.