Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells

Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.     

Toll-like Receptor 11 (TLR11) Prevents Salmonella Penetration into the Murine Peyer Patches

Toll-like receptors (TLRs) are key molecular sensors used by the mammalian innate immune system to detect microorganisms. Although TLR functions in colonic immune homeostasis and tolerance to commensal bacteria have been intensively researched, the precise roles of different TLRs in response to pathogen infection in the gut remain elusive. Peyer patches are the major entrance of Salmonella infection and antigen transportation in intestine. Here, we report that, in contrast to TLR5 as a “carrier of Salmonella,” TLR11 works as a “blocker of Salmonella” to prevent highly invasive Salmonella from penetrating into the murine Peyer patches and spreading systemically. TLR11 plays an important role in mediating TNF-α induction and systemic inflammation in response to Salmonella infection. Remarkably, in mice lacking TLR11, apparent hemorrhages at Peyer patches are induced by highly invasive Salmonella, a phenotype resembling human Salmonella infection. Therefore, our results indicate a potentially important role for TLR11 in preventing murine intestinal infection and modulating antigen transportation in the gut and imply an important role for various TLRs in cooperation with tight control of pathogens penetrating into Peyer patches. The TLR11 knock-out mouse can serve as a good animal model to study Salmonella infection.     

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.     

Spectroscopic and kinetic studies of Nor1, a cytochrome P450 nitric oxide reductase from the fungal pathogen Histoplasma capsulatum

The fungal respiratory pathogen Histoplasma capsulatum evades the innate immune response and colonizes macrophages during infection. Although macrophage production of the antimicrobial effector nitric oxide (NO) restricts H. capsulatum growth, the pathogen is able to establish a persistent infection. H. capsulatum contains a P450 nitric oxide reductase homologue (NOR1) that may be important for detoxifying NO during infection. To characterize the activity of this putative P450 enzyme, a 404 amino acid fragment of Nor1p was expressed in Escherichia coli and purified to homogeneity. Spectral characterization of Nor1p indicated that it was similar to other fungal P450 nitric oxide reductases. Nor1p catalyzed the reduction of NO to N₂O using NADH as the direct reductant. The K_M for NO was determined to be 20 μM and the k_cat to be 5000 min⁻¹. Together, these results provide evidence for a protective role of a P450 nitric oxide reductase against macrophage-derived NO.     

果蝇先天性免疫研究进展

果蝇是生命科学与人类疾病研究的重要模式生物,虽然不具有人类高度专一的获得性免疫,但也有对病原微生物感染作出快速有效反应的先天性免疫应答系统,主要包括体液免疫,细胞免疫和黑化反应。文章结合国外最新研究,详细介绍果蝇体液免疫中控制抗菌肽合成的Toll信号通路和Imd信号通路中涉及的蛋白及其相互作用,并对果蝇细胞免疫中的吞噬、包埋功能和黑化反应作简要阐述。研究表明,果蝇的Toll和Imd信号通路分别与人类的TLR4和TNRF-1信号通路存在着惊人的相似之处,说明果蝇与人类在免疫调控通路方面可能存在着共同的进化起源。     

FcγR-driven Release of IL-6 by Macrophages Requires NOX2-dependent Production of Reactive Oxygen Species

Activation of the FcγR via antigen containing immune complexes can lead to the generation of reactive oxygen species, which are potent signal transducing molecules. However, whether ROS contribute to FcγR signaling has not been studied extensively. We set out to elucidate the role of NADPH oxidase-generated ROS in macrophage activation following FcγR engagement using antigen-containing immune complexes. We hypothesized that NOX2 generated ROS is necessary for propagation of downstream FcγR signaling and initiation of the innate immune response. Following exposure of murine bone marrow-derived macrophages (BMDMs) to inactivated Francisella tularensis (iFt)-containing immune complexes, we observed a significant increase in the innate inflammatory cytokine IL-6 at 24 h compared with macrophages treated with Ft LVS-containing immune complexes. Ligation of the FcγR by opsonized Ft also results in significant ROS production. Macrophages lacking the gp91^phox subunit of NOX2 fail to produce ROS upon FcγR ligation, resulting in decreased Akt phosphorylation and a reduction in the levels of IL-6 compared with wild type macrophages. Similar results were seen following infection of BMDMs with catalase deficient Ft that fail to scavenge hydrogen peroxide. In conclusion, our findings demonstrate that ROS participate in elicitation of an effective innate immune in response to antigen-containing immune complexes through FcγR.     

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.     

Influence of microRNA deregulation on chaperone-mediated autophagy and α-synuclein pathology in Parkinson's disease

The presence of α-synuclein aggregates in the characteristic Lewy body pathology seen in idiopathic Parkinson''s disease (PD), together with α-synuclein gene mutations in familial PD, places α-synuclein at the center of PD pathogenesis. Decreased levels of the chaperone-mediated autophagy (CMA) proteins LAMP-2A and hsc70 in PD brain samples suggests compromised α-synuclein degradation by CMA may underpin the Lewy body pathology. Decreased CMA protein levels were not secondary to the various pathological changes associated with PD, including mitochondrial respiratory chain dysfunction, increased oxidative stress and proteasomal inhibition. However, decreased hsc70 and LAMP-2A protein levels in PD brains were associated with decreases in their respective mRNA levels. MicroRNA (miRNA) deregulation has been reported in PD brains and we have identified eight miRNAs predicted to regulate LAMP-2A or hsc70 expression that were reported to be increased in PD. Using a luciferase reporter assay in SH-SY5Y cells, four and three of these miRNAs significantly decreased luciferase activity expressed upstream of the lamp-2a and hsc70 3′UTR sequences respectively. We confirmed that transfection of these miRNAs also decreased endogenous LAMP-2A and hsc70 protein levels respectively and resulted in significant α-synuclein accumulation. The analysis of PD brains confirmed that six and two of these miRNAs were significantly increased in substantia nigra compacta and amygdala respectively. These data support the hypothesis that decreased CMA caused by miRNA-induced downregulation of CMA proteins plays an important role in the α-synuclein pathology associated with PD, and opens up a new avenue to investigate PD pathogenesis.     

“Reframing” dopamine signaling at the intersection of glial networks in the aged Parkinsonian brain as innate Nrf2/Wnt driver: Therapeutical implications

Dopamine (DA) signaling via G protein‐coupled receptors is a multifunctional neurotransmitter and neuroendocrine–immune modulator. The DA nigrostriatal pathway, which controls the motor coordination, progressively degenerates in Parkinson''s disease (PD), a most common neurodegenerative disorder (ND) characterized by a selective, age‐dependent loss of substantia nigra pars compacta (SNpc) neurons, where DA itself is a primary source of oxidative stress and mitochondrial impairment, intersecting astrocyte and microglial inflammatory networks. Importantly, glia acts as a preferential neuroendocrine–immune DA target, in turn, counter‐modulating inflammatory processes. With a major focus on DA intersection within the astrocyte–microglial inflammatory network in PD vulnerability, we herein first summarize the characteristics of DA signaling systems, the propensity of DA neurons to oxidative stress, and glial inflammatory triggers dictating the vulnerability to PD. Reciprocally, DA modulation of astrocytes and microglial reactivity, coupled to the synergic impact of gene–environment interactions, then constitute a further level of control regulating midbrain DA neuron (mDAn) survival/death. Not surprisingly, within this circuitry, DA converges to modulate nuclear factor erythroid 2‐like 2 (Nrf2), the master regulator of cellular defense against oxidative stress and inflammation, and Wingless (Wnt)/β‐catenin signaling, a key pathway for mDAn neurogenesis, neuroprotection, and immunomodulation, adding to the already complex “signaling puzzle,” a novel actor in mDAn–glial regulatory machinery. Here, we propose an autoregulatory feedback system allowing DA to act as an endogenous Nrf2/Wnt innate modulator and trace the importance of DA receptor agonists applied to the clinic as immune modifiers.     

A third-generation mouse model of Alzheimer's disease shows early and increased cored plaque pathology composed of wild-type human amyloid β peptide

We previously developed single App knock-in mouse models of Alzheimer''s disease (AD) harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (App^NL-G-F and App^NL-F mice, respectively). These models showed Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner. The former model exhibits extensive pathology as early as 6 months, but is unsuitable for investigating Aβ metabolism and clearance because the Arctic mutation renders Aβ resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-Aβ antibodies. The latter model may take as long as 18 months for the pathology to become prominent, which leaves an unfulfilled need for an Alzheimer''s disease animal model that is both swift to show pathology and useful for antibody therapy. We thus utilized mutant Psen1 knock-in mice into which a pathogenic mutation (P117L) had been introduced to generate a new model that exhibits early deposition of wild-type human Aβ by crossbreeding the App^NL-F line with the Psen1^P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new third-generation mouse model showed more cored plaque pathology and neuroinflammation than App^NL-G-F mice and will help accelerate the development of disease-modifying therapies to treat preclinical AD.     

Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

The extent of the innate immune response is regulated by many positively and negatively acting signaling proteins. This allows for proper activation of innate immunity to fight infection while ensuring that the response is limited to prevent unwanted complications. Thus mutations in innate immune regulators can lead to immune dysfunction or to inflammatory diseases such as arthritis or atherosclerosis. To identify novel innate immune regulators that could affect infectious or inflammatory disease, we have taken a comparative genomics RNAi screening approach in which we inhibit orthologous genes in the nematode Caenorhabditis elegans and murine macrophages, expecting that genes with evolutionarily conserved function also will regulate innate immunity in humans. Here we report the results of an RNAi screen of approximately half of the C. elegans genome, which led to the identification of many candidate genes that regulate innate immunity in C. elegans and mouse macrophages. One of these novel conserved regulators of innate immunity is the mRNA splicing regulator Eftud2, which we show controls the alternate splicing of the MyD88 innate immunity signaling adaptor to modulate the extent of the innate immune response.     

Porphyromonas gingivalis Peptidoglycans Induce Excessive Activation of the Innate Immune System in Silkworm Larvae

Porphyromonas gingivalis, a pathogen that causes inflammation in human periodontal tissue, killed silkworm (Bombyx mori, Lepidoptera) larvae when injected into the blood (hemolymph). Silkworm lethality was not rescued by antibiotic treatment, and heat-killed bacteria were also lethal. Heat-killed bacteria of mutant P. gingivalis strains lacking virulence factors also killed silkworms. Silkworms died after injection of peptidoglycans purified from P. gingivalis (pPG), and pPG toxicity was blocked by treatment with mutanolysin, a peptidoglycan-degrading enzyme. pPG induced silkworm hemolymph melanization at the same dose as that required to kill the animal. pPG injection increased caspase activity in silkworm tissues. pPG-induced silkworm death was delayed by injecting melanization-inhibiting reagents (a serine protease inhibitor and 1-phenyl-2-thiourea), antioxidants (N-acetyl-l-cysteine, glutathione, and catalase), and a caspase inhibitor (Ac-DEVD-CHO). Thus, pPG induces excessive activation of the innate immune response, which leads to the generation of reactive oxygen species and apoptotic cell death in the host tissue.     

MyD88 expression by CNS-resident cells is pivotal for eliciting protective immunity in brain abscesses

MyD88 KO (knockout) mice are exquisitely sensitive to CNS (central nervous system) infection with Staphylococcus aureus, a common aetiological agent of brain abscess, exhibiting global defects in innate immunity and exacerbated tissue damage. However, since brain abscesses are typified by the involvement of both activated CNS-resident and infiltrating immune cells, in our previous studies it has been impossible to determine the relative contribution of MyD88-dependent signalling in the CNS compared with the peripheral immune cell compartments. In the present study we addressed this by examining the course of S. aureus infection in MyD88 bone marrow chimaera mice. Interestingly, chimaeras where MyD88 was present in the CNS, but not bone marrow-derived cells, mounted pro-inflammatory mediator expression profiles and neutrophil recruitment equivalent to or exceeding that detected in WT (wild-type) mice. These results implicate CNS MyD88 as essential in eliciting the initial wave of inflammation during the acute response to parenchymal infection. Microarray analysis of infected MyD88 KO compared with WT mice revealed a preponderance of differentially regulated genes involved in apoptotic pathways, suggesting that the extensive tissue damage characteristic of brain abscesses from MyD88 KO mice could result from dysregulated apoptosis. Collectively, the findings of the present study highlight a novel mechanism for CNS-resident cells in initiating a protective innate immune response in the infected brain and, in the absence of MyD88 in this compartment, immunity is compromised.     

Endoplasmic Reticulum Stress Pathway Required for Immune Homeostasis Is Neurally Controlled by Arrestin-1

In response to pathogen infection, the host innate immune system activates microbial killing pathways and cellular stress pathways that need to be balanced because insufficient or excessive immune responses have deleterious consequences. Recent studies demonstrate that two G protein-coupled receptors (GPCRs) in the nervous system of Caenorhabditis elegans control immune homeostasis. To investigate further how GPCR signaling controls immune homeostasis at the organismal level, we studied arrestin-1 (ARR-1), which is the only GPCR adaptor protein in C. elegans. The results indicate that ARR-1 is required for GPCR signaling in ASH, ASI, AQR, PQR, and URX neurons, which control the unfolded protein response and a p38 mitogen-activated protein kinase signaling pathway required for innate immunity. ARR-1 activity also controlled immunity through ADF chemosensory and AFD thermosensory neurons that regulate longevity. Furthermore, we found that although ARR-1 played a key role in the control of immunity by AFD thermosensory neurons, it did not control longevity through these cells. However, ARR-1 partially controlled longevity through ADF neurons.     

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.     

Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis

Trichomonas vaginalis commonly causes vaginitis and perhaps cervicitis in women and urethritis in men and women. Macrophages are important immune cells in response to T. vaginalis infection. In this study, we investigated whether human macrophages could be involved in inflammation induced by T. vaginalis. Human monocyte-derived macrophages (HMDM) were co-cultured with T. vaginalis. Live, opsonized-live trichomonads, and T. vaginalis lysates increased proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6 by HMDM. The involvement of nuclear factor (NF)-κB signaling pathway in cytokine production induced by T. vaginalis was confirmed by phosphorylation and nuclear translocation of p65 NF-κB. In addition, stimulation with live T. vaginalis induced marked augmentation of nitric oxide (NO) production and expression of inducible NO synthase (iNOS) levels in HMDM. However, trichomonad-induced NF-κB activation and TNF-α production in macrophages were significantly inhibited by inhibition of iNOS levels with L-NMMA (NO synthase inhibitor). Moreover, pretreatment with NF-κB inhibitors (PDTC or Bay11-7082) caused human macrophages to produce less TNF-α. These results suggest that T. vaginalis stimulates human macrophages to produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-α, and NO. In particular, we showed that T. vaginalis induced TNF-α production in macrophages through NO-dependent activation of NF-κB, which might be closely involved in inflammation caused by T. vaginalis.     

Mice lacking inducible nitric oxide synthase develop exacerbated hepatic inflammatory responses induced by Plasmodium berghei NK65 infection

Infection of mice with Plasmodium berghei NK65 represents a well-recognized malaria model in which infection is accompanied by an intense hepatic inflammatory response. Enzyme-inducible nitric oxide synthase is an important regulator of inflammation and leukocyte recruitment in microvessels, but these functions have yet to be evaluated in experimental malaria. In this study, we assessed the involvement of inducible nitric oxide synthase in inflammatory responses to murine experimental malaria induced by P. berghei NK65. We observed that wild type (WT) and nitric oxide synthase (iNOS)-deficient mice (iNOS^−/−) mice showed similar levels of parasitemia following P. berghei NK65 infection, although infected iNOS^−/− mice presented early mortality. Inducible nitric oxide synthase deficiency led to increased leukocyte rolling and adhesion to the liver in iNOS^−/− mice relative to the WT animals, as observed via intravital microscopy. Infected iNOS^−/− mice also exhibited increased hepatic leukocyte migration and subsequent liver damage, which was associated with high serum levels of the cytokines TNF-α, IL-6 and IL-10. Our data suggest potential role for the iNOS enzyme as a regulator of hepatic inflammatory response induced by P. berghei NK65-infection, and its absence leads to exacerbated inflammation and sequential associated-hepatic damage in the animals.     

Anti-leishmanial Effects of Trinitroglycerin in BALB/C Mice Infected with Leishmania major via Nitric Oxide Pathway

This study investigated whether trinitroglycerine (TNG) as nitric oxide (NO) releasing agent had anti-leishmanial effects and mediated pathology in BALB/c mice infected with Leishmania major. Cutaneous leishmaniasis (CL), a zoonotic infection caused by leishmania protozoa is still one of the health problems in the world and in Iran. NO is involved in host immune responses against intracellular L. major, and leishmania killing by macrophages is mediated by this substance. Moreover, application of CL treatment with NO-donors has been recently indicated. In our study, TNG was used for its ability to increase NO and to modify CL infection in mice, in order to evaluate NO effects on lesion size and formation, parasite proliferation inside macrophages, amastigote visceralization in target organs, and NO induction in plasma and organ suspensions. Data obtained in this study indicated that TNG increased plasma and liver-NO, reduced lesion sizes, removed amastigotes from lesions, livers, spleens, and lymph nodes, declined proliferation of amastigotes, hepatomegaly, and increased survival rate. However, TNG reduced spleen-NO and had no significant effects on spelenomegaly. The results show that TNG therapy reduced leishmaniasis and pathology in association with raised NO levels. TNG had some antiparasitic activity by reduction of positive smears from lesions, livers, spleens, and lymph nodes, which could emphasize the role of TNG to inhibit visceralization of L. major in target organs.