Effect of sp3/sp2 carbon ratio and hydrodynamic size on the biodistribution kinetics of nanodiamonds in mice via intravenous injection.

BACKGROUND: Nanodiamonds (NDs) have gained a rapidly growing interest in biomedical applications; however, little is known regarding their biokinetics owing to difficulties in measurements and limited synthesis/purification technologies. In this study, we investigated the distribution kinetics of detonation-synthesized NDs in mice via intravenous injection to evaluate the parameters that determine the behavior of the particles. We prepared two distinctive NDs that controlled the sp3/sp2 carbon ratio and particle size by coating them with serum proteins. The four control samples were intravenously injected into mice, and tissue distribution and clearance were evaluated at 30 min and 1, 7, and 28 days post-injection. RESULTS: The sp3/sp2 carbon ratio showed no correlation with the organ distribution of the NDs. However, hydrodynamic size showed an excellent correlation with organ distribution levels: a negative correlation in the liver and positive correlations in the spleen and lungs. Furthermore, the deposition levels of NDs in the lung suggest that particles smaller than 300 nm could avoid lung deposition. Finally, a similar organ distribution pattern was observed in mice injected with carbon black nanoparticles controlled hydrodynamic size. CONCLUSIONS: In conclusion, the tissue distribution of NDs is modulated not by the sp3/sp2 carbon ratio but by the hydrodynamic size, which can provide helpful information for targeting the tissue of NDs. Furthermore, the organ distribution pattern of the NDs may not be specific to NDs but also can apply to other nanoparticles, such as carbon black.

Water extract of Artemisia scoparia Waldst. & Kitam suppresses LPS-induced cytokine production and NLRP3 inflammasome activation in macrophages and alleviates carrageenan-induced acute inflammation in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia scoparia Waldst. & Kitam (A. scoparia) is a perennial herbal plant that is widely used as a folk remedy in Asian countries. Several studies have demonstrated that A. scoparia has various physiological effects, including anti-inflammation, anti-hypertension, anti-obesity, anti-hepatotoxicity, and anti-oxidant effects. AIM OF THE STUDY: The objective of the present study was to examine the anti-inflammatory effects of water extract of A. scoparia (WAS). MATERIALS AND METHODS: Murine bone marrow-derived macrophages (BMDMs), human monocyte THP-1 and murine fibroblast 3T3-L1 cells were used for the in vitro experiments. Cell viability and cytokine production were determined by the MTT assay and ELISA, respectively. RT-PCR was performed to determine iNOS gene expression and the Griess reaction was used to measure nitrite levels. iNOS protein expression, activation of NF-κB and MAPKs, and cleavage of caspase-1 and IL-1ß were determined by Western blot analysis. A carrageenan-induced mouse model of acute inflammation was used in the in vivo experiments. RESULTS: Pretreatment with WAS concentration-dependently suppressed gene expression and IL-6, TNF-α, CXCL1 and iNOS protein levels in BMDMs stimulated with LPS. In addition, pretreatment with WAS inhibited LPS-induced production of IL-6 and TNF-α in THP-1 cells and CXCL1 in 3T3-L1. Furthermore, LPS induced phosphorylation of p65 in BMDMs, and this induction was dramatically suppressed by WAS pretreatment. We further investigated whether WAS regulates activation of the NLRP3 inflammasome, which is known to be essential for IL-1ß processing. WAS inhibited the production of IL-1ß, but not IL-6, in response to adenosine triphosphate (ATP) and monosodium uric acid (MSU) crystals in LPS-primed BMDMs. Cleavage of caspase-1 and IL-1ß was also reduced by WAS. We finally evaluated the in vivo anti-inflammatory effects of WAS in a mouse model of carrageenan-induced acute inflammation. Subcutaneous administration of WAS reduced production of the inflammatory cytokines IL-6, TNF-α, CXCL1, and IL-1ß. Recruitment of immune cells, mostly neutrophils, was also reduced by administration of WAS. Infiltration of inflammatory cells and edema in the submucosa of air pouch tissues were markedly improved in the WAS-treated groups. CONCLUSIONS: Our results indicate that WAS possesses potent anti-inflammatory properties. These findings suggest that A. scoparia is a candidate functional food targeting several inflammatory diseases.

The sp3/sp2 carbon ratio as a modulator of in vivo and in vitro toxicity of the chemically purified detonation-synthesized nanodiamond via the reactive oxygen species generation.

Nanodiamonds have been suggested as biocompatible materials and are suitable for various biomedical applications, but little is known about how to synthesize safer nanodiamonds. Herein, seven different detonation-synthesized nanodiamonds (DNDs) with sequential sp3/sp2 carbon ratios were assembled by controlling the chemical purification parameters and the role of sp3/sp2 carbon ratio on the toxicity of DNDs was investigated. Carbon black and nickel oxide nanoparticles were used as reference particles. The intrinsic reactive oxygen species (ROS) generation potential of DNDs was estimated by a 2'7'-dichlorofluorescein diacetate (DCFH-DA) assay, and these values showed a good negative correlation with the sp3/sp2 carbon ratios, which implies that ROS generation increased as the sp3/sp2 carbon ratio decreased. As a model to investigate inflammogenic potential of DND samples, a rat intratracheal instillation model was used as the lung is very sensitive to nanoparticle exposures. The sp3/sp2 carbon ratios or the estimated values of ROS generation potential showed excellent linear correlations with the number of neutrophils and pro-inflammatory cytokines in bronchoalveolar lavage fluid at 24 h after instillation. Treatment of DND samples to THP-1 derived macrophages also showed that the sp3/sp2 carbon ratios or the estimated values of ROS generation potential were closely related with the toxicity endpoints such as cell viability and pro-inflammatory cytokines. Taken together, these data demonstrate that the sp3/sp2 carbon ratio is the key determinant for the toxicity of DNDs, which can be a useful tool for the safer-by-design approach of DNDs and the safety assessment of carbon nanoparticles.

Ureaplasma Urealyticum Infection Contributes to the Development of Pelvic Endometriosis Through Toll-Like Receptor 2.

Endometriosis is a chronic gynecological disorder, characterized by the presence of ectopic endometrial tissue outside the uterine cavity. Among several hypotheses, Sampson's theory of retrograde menstruation is still applicable. Recent studies have reported the importance of inflammation among endometrial tissue, the peritoneum, and immune cells. However, less is known regarding the role of bacterial infection in the pathophysiology of endometriosis. We hypothesized that Ureaplasma urealyticum infection might contribute to the development of endometriosis by inducing the production of inflammatory mediators by peritoneal mesothelial cells (PMCs), possibly through TLR2. Hence, our objective was to reveal whether PMC infection by U. urealyticum is associated with endometriosis. Moreover, we aimed to demonstrate the molecular mechanism involved in this relationship. We developed a new infection-induced mouse model of endometriosis with wild type and Tlr2-deficient mice. Based on the in vivo mouse model, U. urealyticum-infected mice showed significantly increased numbers and sizes of ectopic endometriotic lesions. U. urealyticum upregulated not only the production of IL-6, CXCL1, and CCL2, but also the expression of ICAM-1, VCAM-1, and MMP2 in murine PMCs. Similarly, endometrial stromal cells dose-dependently produced IL-6, CXCL1, and CCL2 in response to U. urealyticum infection. The series of inflammatory responses in PMCs was mediated mainly through TLR2. The phosphorylation of ERK and JNK was observed when U. urealyticum was added to PMCs and knock out of Tlr2 inhibited these MAPKs phosphorylation. Based on our co-culture study, U. urealyticum-infected PMCs exhibited significantly increased attachment to ESCs compared with uninfected PMCs. Collectively, U. urealyticum infection promotes the development of endometriosis by increasing inflammatory mediators, adhesion molecules, and MMP-2 expression in PMCs through TLR2 signaling. Through our results, we present a theory that infection-induced pelvic inflammation contributes to the initiation and progression of endometriosis. Appropriate treatment of reproductive tract infection may decrease the prevalence of endometriosis.

Nucleotide-Binding Oligomerization Domain 2 Contributes to Limiting Growth of Mycobacterium abscessus in the Lung of Mice by Regulating Cytokines and Nitric Oxide Production.

Mycobacterium abscessus is a prominent cause of pulmonary infection in immunosuppressed patients and those with cystic fibrosis. Nucleotide-binding oligomerization domain (NOD) 2 is a cytosolic receptor which senses a bacterial peptidoglycan component, muramyl dipeptide (MDP). Although nucleotide-binding oligomerization domain 2 (NOD2) contributes to protect host against various microbial infections, it is still unclear whether NOD2 is essential to regulate host immune responses against M. abscessus infection. In this study, we sought to clarify the role of NOD2 and the underlying mechanism in host defense against M. abscessus infection. Mice were infected intranasally with M. abscessus and sacrificed at indicated time points. Bacterial survival, cytokines production, and pathology in the lungs were determined. Bone marrow-derived macrophages were used to clarify cellular mechanism of NOD2-mediated immune response. Bacterial clearance was impaired, and pathology was more severe in the lungs of NOD2-deficient mice compared with the wild-type mice. In macrophages, NOD2-mediated activation of p38 and JNK were required for production of proinflammatory cytokines and nitric oxide (NO) and expression of iNOS in response to M. abscessus. NO was critical for limiting intracellular growth of the pathogen. Intranasal administration of MDP reduced in vivo bacterial replication and thus improved lung pathology in M. abscessus-infected mice. This study offers important new insights into the potential roles of the NOD2 in initiating and potentiating innate immune response against M. abscessus pulmonary infection.

NLRP3 inflammasome mediates interleukin-1ß production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice.

Acinetobacter baumannii is a multi-drug resistant, Gram-negative bacteria and infection with this organism is one of the major causes of mortality in intensive care units. Inflammasomes are multiprotein oligomers that include caspase-1, and their activation is required for maturation of interleukin-1ß (IL-1ß). Inflammasome signalling is involved in host defences against various microbial infections, but the precise mechanism by which A. baumannii activates inflammasomes and the roles of relevant signals in host defence against pulmonary A. baumannii infection are unknown. Our results showed that NLRP3, ASC and caspase-1, but not NLRC4, are required for A. baumannii-induced production of IL-1ß in macrophages. An inhibitor assay revealed that various pathways, including P2X7R, K+ efflux, reactive oxygen species production and release of cathepsins, are involved in IL-1ß production in macrophages in response to A. baumannii. Interleukin-1ß production in bronchoalveolar lavage (BAL) fluid was impaired in NLRP3-deficient and caspase-1/11-deficient mice infected with A. baumannii, compared with that in wild-type (WT) mice. However, the bacterial loads in BAL fluid and lungs were comparable between WT and NLRP3-deficient or caspase-1/11-deficient mice. The severity of lung pathology was reduced in NLRP3- deficient, caspase-1/11- deficient and IL-1-receptor-deficient mice, although the recruitment of immune cells and production of inflammatory cytokines and chemokines were not altered in these mice. These findings indicate that A. baumannii leads to the activation of NLRP3 inflammasome, which mediates IL-1ß production and lung pathology.

Anti-Helicobacter pylori activity of crude N-acetylneuraminic acid isolated from glycomacropeptide of whey.

Helicobacter pylori colonizes the gastric mucosa of about half of the world's population, causing chronic gastritis and gastric cancer. An increasing emergence of antibiotic-resistant H. pylori arouses demand on alternative non-antibiotic-based therapies. In this study, we freshly prepared crude N-acetylneuraminic acid obtained from glycomacropeptide (G-NANA) of whey through a neuraminidase-mediated reaction and evaluated its antibacterial ability against H. pylori and H. felis. Overnight cultures of the H. pylori were diluted with fresh media and different concentrations (1-150 mg/mL) of crude G-NANA were added directly to the culture tube. Bacterial growth was evaluated by measuring the optical density of the culture medium and the number of viable bacteria was determined by a direct count of the colony forming units (CFU) on agar plates. For the in vivo study, mice were orally infected with 100 µL (5×10(8) cfu/mL) of H. felis four times at a day's interval, accompanied by a daily administration of crude G-NANA or vehicle. A day after the last infection, the mice were daily administered the crude G-NANA (0, 75, and 300 mg/mL) for 10 days and euthanized. Their stomachs were collected and bacterial colonization was determined by quantitative real-time PCR. Crude G-NANA inhibited H. pylori's growth and reduced the number of viable bacteria in a dose-dependent manner. Furthermore, crude G-NANA inhibited bacterial colonization in the mice. These results showed that crude G-NANA has antibacterial activity against Helicobacter and demonstrated its therapeutic potential for the prevention of chronic gastritis and gastric carcinogenesis induced by Helicobacter infection in humans.

Withaferin A inhibits inflammatory responses induced by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

Periodontitis is a progressive chronic inflammatory disease and a major cause of tooth loss in humans. As a withanolides, withaferin A (WA) is known to exhibit strong anti­inflammatory activity. The present study examined whether WA inhibited inflammatory responses in macrophages in response to two representative periodontal pathogens, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Murine bone marrow­derived macrophages (BMDMs) were used in this study and cytokine production in culture supernatants was measured by enzyme­linked immunosorbent assays. Western blot analysis was performed to determine the activation of nuclear factor­κB and mitogen­activated protein kinases (MAPKs) and the expression of inducible nitric oxide synthase (iNOS), toll­like receptor (TLR) 2 and TLR4. The production of nitric oxide (NO) was determined by the Griess reaction. WA treatment was shown to decrease interleukin (IL)­6 and tumor necrosis factor (TNF)­α production in BMDMs in response to F. nucleatum and A. actinomycetemcomitans in a dose­dependent manner. The phosphorylation of IκB­α and MAPKs (p38, extracellular signal­regulated kinases and c­Jun N­terminal kinases) induced by F. nucleatum and A. actinomycetemcomitans was also inhibited by WA. F. nucleatum and A. actinomycetemcomitans induced iNOS expression and NO production in BMDMs, which was inhibited by WA in a dose­dependent manner. WA also reduced endogenous and induced expression of TLR2 and TLR4 in these cells. These results suggest that WA may be a potential therapeutic agent or preventive additive for periodontitis control.

Nod2-mediated recognition of the microbiota is critical for mucosal adjuvant activity of cholera toxin.

Cholera toxin (CT) is a potent adjuvant for inducing mucosal immune responses. However, the mechanism by which CT induces adjuvant activity remains unclear. Here we show that the microbiota is critical for inducing antigen-specific IgG production after intranasal immunization. After mucosal vaccination with CT, both antibiotic-treated and germ-free (GF) mice had reduced amounts of antigen-specific IgG, smaller recall-stimulated cytokine responses, impaired follicular helper T (TFH) cell responses and reduced numbers of plasma cells. Recognition of symbiotic bacteria via the nucleotide-binding oligomerization domain containing 2 (Nod2) sensor in cells that express the integrin CD11c (encoded by Itgax) was required for the adjuvanticity of CT. Reconstitution of GF mice with a Nod2 agonist or monocolonization with Staphylococcus sciuri, which has high Nod2-stimulatory activity, was sufficient to promote robust CT adjuvant activity, whereas bacteria with low Nod2-stimulatory activity did not. Mechanistically, CT enhanced Nod2-mediated cytokine production in dendritic cells via intracellular cyclic AMP. These results show a role for the microbiota and the intracellular receptor Nod2 in promoting the mucosal adjuvant activity of CT.

Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1ß and Apoptotic Cell Death.

Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1ß (IL-1ß), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1ß secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1ß. Processing and release of both caspase-1 and IL-1ß were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1ß as well as to promote apoptotic cell death.

Nucleotide-binding oligomerization domain 2 (NOD2) activation induces apoptosis of human oral squamous cell carcinoma cells.

OBJECTIVES: Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. MATERIALS AND METHODS: NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. RESULTS: The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. CONCLUSIONS: Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC.

The role of nucleotide-binding oligomerization domain 1 during cytokine production by macrophages in response to Mycobacterium tuberculosis infection.

Tuberculosis due to Mycobacterium tuberculosis infection is a leading cause of death worldwide. Recognition of this pathogen is crucial for the activation of innate and adaptive immune responses. Nucleotide-binding oligomerization domain (Nod)1 and Nod2 are cytoplasmic receptors that can detect unique muropeptides of bacterial peptidoglycan. Nod2 is critical for the initiation of the host immune response against M. tuberculosis infection, however the role of Nod1 remains largely unknown. We investigated the role of Nod1 with respect to cytokine production by bone marrow-derived macrophages (BMDMs) in response to M. tuberculosis infection. Production of proinflammatory cytokines, such as IL-6, TNF-α, and IL-1ß were induced in BMDMs; cytokine levels were not affected by a deficiency in Nod1. Activation of NF-κB and MAPKs was also comparable between wild-type and Nod1-deficient BMDMs. Levels of IL-6 and IL-1ß were reduced in Nod1/Nod2 double-deficient BMDMs to a greater extent than in Nod2-deficient cells. Furthermore, when signaling of Toll-like receptors (TLRs) was inhibited by lipopolysaccharide pre-treatment, cytokine production was diminished in Nod1-deficient BMDMs. Our results indicate that Nod1 cooperates with Nod2 or TLRs to produce cytokines in macrophages in response to M. tuberculosis infection.

Inhibitory effect of withaferin A on Helicobacter pylori­induced IL­8 production and NF­κB activation in gastric epithelial cells.

Withaferin A (WA), a withanolide purified from Withania somnifera, has been known to exert anti-inflammatory effects. The present study sought to determine the effects of WA on Helicobacter (H.) pylori-mediated inflammation in the AGS gastric epithelial cell line. Cellular production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) was measured by ELISA. Western blot analysis was performed to determine the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) as well as hypoxia-inducible factor 1α stabilization. Bacterial growth was also examined by measuring the optical density. Pre-treatment or co-treatment with WA efficiently reduced IL-8 production by AGS cells in response to H. pylori infection. H. pylori-induced activation of NF-κB, but not MAPKs, was also inhibited by pre-treatment of WA in the cells. However, WA did not affect VEGF production and HIF-1α stabilization induced by H. pylori in AGS cells. In addition, WA did not influence the growth of H. pylori, suggesting that the anti-inflammatory effect of WA was not due to any bactericidal effect. These findings indicate that WA is a potential preventive or therapeutic agent for H. pylori-mediated gastric inflammation.

Decreased expression of Toll-like receptor 4 and 5 during progression of prostate transformation in transgenic adenocarcinoma of mouse prostate mice.

Chronic inflammation has been considered an important risk factor for development of prostate cancer. Toll-like receptors (TLRs) recognize microbial moieties or endogenous molecules and play an important role in the triggering and promotion of inflammation. In this study, we examined whether expression of TLR4 and TLR5 was associated with progression of prostate transformation in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The expression of TLR4 and TLR5 was evaluated by immunohistochemisty in formalin-fixed paraffin-embedded prostate tissue from wild-type (WT) and TRAMP mice. Normal prostate tissue from WT mice showed strong expression of TLR4 and TLR5. However, TLR4 expression in the prostate tissue from TRAMP mice gradually decreased as pathologic grade became more aggressive. TLR5 expression in the prostate tissue from TRAMP mice also decreased in low-grade prostate intraepithelial neoplasia (PIN), high-grade PIN and poorly differentiated adenocarcinoma. Overall, our results suggest that decreased expression of TLR4 and TLR5 may contribute to prostate tumorigenesis.

Immune cells in the female reproductive tract.

The female reproductive tract has two main functions: protection against microbial challenge and maintenance of pregnancy to term. The upper reproductive tract comprises the fallopian tubes and the uterus, including the endocervix, and the lower tract consists of the ectocervix and the vagina. Immune cells residing in the reproductive tract play contradictory roles: they maintain immunity against vaginal pathogens in the lower tract and establish immune tolerance for sperm and an embryo/fetus in the upper tract. The immune system is significantly influenced by sex steroid hormones, although leukocytes in the reproductive tract lack receptors for estrogen and progesterone. The leukocytes in the reproductive tract are distributed in either an aggregated or a dispersed form in the epithelial layer, lamina propria, and stroma. Even though immune cells are differentially distributed in each organ of the reproductive tract, the predominant immune cells are T cells, macrophages/dendritic cells, natural killer (NK) cells, neutrophils, and mast cells. B cells are rare in the female reproductive tract. NK cells in the endometrium significantly expand in the late secretory phase and further increase their number during early pregnancy. It is evident that NK cells and regulatory T (Treg) cells are extremely important in decidual angiogenesis, trophoblast migration, and immune tolerance during pregnancy. Dysregulation of endometrial/decidual immune cells is strongly related to infertility, miscarriage, and other obstetric complications. Understanding the immune system of the female reproductive tract will significantly contribute to women's health and to success in pregnancy.

Synergistic effect of muramyl dipeptide with heat shock protein 70 from Mycobacterium tuberculosis on immune activation.

Heat shock protein 70 from Mycobacterium tuberculosis (Mtb Hsp70) has been known to modulate immune response including dendritic cell activation. Muramyl dipeptide (MDP) is an immunoreactive derivative of peptidoglycan from all Gram-negative and Gram-positive bacteria and recognized to be responsible for function of Freund's complete adjuvant. In this study, we evaluated effect of MDP on in vitro activation of bone marrow derived dendritic cells (BMDCs) and in vivo production of cytokines and chemokines induced by Mtb Hsp70. MDP treatment with Mtb Hsp70 dramatically increased production of IL-6, IL-12p40 and TNF-α in BMDCs compared with Mtb Hsp70 alone whereas these effects were abolished in Nod2-deficient BMDCs. Phosphorylation of IκB-α and ERK and impairment of phagocytosis, which is an indicator of DC maturation were enhanced by MDP co-treatment with Mtb hsp70 in BMDCs. In addition, ability of Mtb Hsp70-stimulated BMDCs to induce IFN-γ productions of T cells was increased by MDP co-treatment. Finally, intraperitoneal injection of MDP with Mtb Hsp70 dramatically increased production of IL-6, CXCL-1 and CCL2 in serum compared with Mtb hsp70 injection. Our study showed the synergistic effects of MDP with Mtb Hsp70 on DCs and in vivo immune activation. The use of MDP with Mtb Hsp70 to induce immune activation may provide an effective strategy for vaccination to treat cancer and protect against pathogens.

Critical role of TRIF and MyD88 in Mycobacterium tuberculosis Hsp70-mediated activation of dendritic cells.

As a potent immune regulator, heat shock protein 70 derived from Mycobacterium tuberculosis (Mtb Hsp70) has adjuvant effect and activates immune cells such as macrophages and dendritic cells (DCs). Although Toll-like receptors (TLRs) are known to involve in DCs activation by Mtb Hsp70, there is still a controversy and the underlying mechanism is not well understood. In this study, we examined whether TRIF and MyD88, the core adaptor molecules for TLRs signaling, regulate Mtb Hsp70-induced DCs activation. Although Mtb Hsp70 produced substantial level of cytokines (IL-6, IL-12p40, and TNF-α) in TRIF-deficient DCs in a dose-dependent manner, each level was significantly lower than that in WT cells. The cytokines production was almost abolished in MyD88-deficient DCs. Consistent with cytokine results, Mtb Hsp70-induced activation of NF-κB and MAPKs was also impaired in both TRIF- and MyD88-deficient DCs, as compared with WT cells. Inhibitor assay revealed that NF-κB, ERK, and JNK, but not p38, regulate Mtb Hsp70-induced production of cytokines. In addition, the up-regulation of co-stimulatory molecules and MHC class II was mostly TRIF-dependent in DCs in response Mtb Hsp70, whereas MyD88 was only partially involved. Finally, mixed leukocytes reaction (MLR) assay revealed that both TRIF and MyD88 are critical for DCs ability promoted by Mtb Hsp70 to differentiate naïve T cells into effector T cells of producing IFN-γ. Our findings suggest that both TRIF and MyD88 are essential for the activation and maturation of DCs in response to Mtb Hsp70.

Helicobacter pylori induces vascular endothelial growth factor production in gastric epithelial cells through hypoxia-inducible factor-1α-dependent pathway.

BACKGROUND: Although Helicobacter pylori have been known to induce vascular endothelial growth factor (VEGF) production in gastric epithelial cells, the precise mechanism for cellular signaling is incompletely understood. In this study, we investigated the role of bacterial virulence factor and host cellular signaling in VEGF production of H. pylori-infected gastric epithelial cells. MATERIALS AND METHODS: We evaluated production of VEGF, activation of nuclear factor nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPKs) and hypoxia-inducible factor-1α (HIF-1α) stabilization in gastric epithelial cells infected with H. pylori WT or isogenic mutants deficient in type IV secretion system (T4SS). RESULTS: H. pylori induced VEGF production in gastric epithelial cells via both T4SS-dependent and T4SS-independent pathways, although T4SS-independent pathway seems to be the dominant signaling. The inhibitor assay implicated that activation of NF-κB and MAPKs is dispensable for H. pylori-induced VEGF production in gastric epithelial cells. H. pylori led to HIF-1α stabilization in gastric epithelial cells independently of T4SS, NF-κB, and MAPKs, which was essential for VEGF production in these cells. N-acetyl-cysteine (NAC), a reactive oxygen species (ROS) inhibitor, treatment impaired H. pylori-induced HIF-1α stabilization and VEGF production in gastric epithelial cells. CONCLUSION: We defined the important role of ROS-HIF-1α axis in VEGF production of H. pylori-infected gastric epithelial cells, and bacterial T4SS has a minor role in H. pylori-induced VEGF production of gastric epithelial cells.

Nod2 and Rip2 contribute to innate immune responses in mouse neutrophils.

Nod-like receptors are a family of innate immune receptors that link cytosolic sensing of microbial and danger stimuli to the activation of immune responses. Two Nod-like receptor family members, Nod1 and Nod2, recognize bacterial peptidoglycan and activate immune responses via nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). The function of Nod1 and Nod2 has been largely studied in macrophages, but the role of these receptors in other innate immune cells remains unclear. In this study, we examined the function of Nod1 and Nod2 in innate immune responses of neutrophils. Mice were injected intraperitoneally with thioglycollate, and then peritoneal neutrophils were isolated 4 hr after injection. Tri-DAP and muramyl-dipeptide (MDP) were used as Nod1 and Nod2 agonists, respectively. The level of cytokines [interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α)] and chemokines (CXCL1 and CCL2) was increased by MDP, but not Tri-DAP in wild-type (WT) neutrophils. Increased production of cytokines and chemokines with MDP was abolished in Nod2- and Rip2-deficient neutrophils. MDP also induced the activation of NF-κB and MAPK in WT neutrophils, but not in Nod2- and Rip2-deficient cells. Flow cytometry analysis showed that L-selectin shedding was induced by MDP in WT neutrophils, but not in Nod2- and Rip2-deficient cells. MDP and Toll-like receptor (TLR) agonists (Pam3 CSK4 and lipopolysaccharide) exerted synergistic effects on the production of IL-6 and CXCL1 in neutrophils. Moreover, Nod2 and TLR4 cooperated to produce IL-6, TNF-α, CXCL1 and CCL2 in neutrophils in response to Gram-negative bacteria. Our findings suggest that the Nod2-Rip2 axis may contribute to the innate immune response of neutrophils against bacterial infection.

Diverse Toll-like receptors mediate cytokine production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.