Sterile-α- and Armadillo Motif-Containing Protein Inhibits the TRIF-Dependent Downregulation of Signal Regulatory Protein α To Interfere with Intracellular Bacterial Elimination in Burkholderia pseudomallei-Infected Mouse Macrophages

Burkholderia pseudomallei, the causative agent of melioidosis, evades macrophage killing by suppressing the TRIF-dependent pathway, leading to inhibition of inducible nitric oxide synthase (iNOS) expression. We previously demonstrated that virulent wild-type B. pseudomallei inhibits the TRIF-dependent pathway by upregulating sterile-α- and armadillo motif-containing protein (SARM) and by inhibiting downregulation of signal regulatory protein α (SIRPα); both molecules are negative regulators of Toll-like receptor signaling. In contrast, the less virulent lipopolysaccharide (LPS) mutant of B. pseudomallei is unable to exhibit these features and is susceptible to macrophage killing. However, the functional relationship of these two negative regulators in the evasion of macrophage defense has not been elucidated. We demonstrated here that SIRPα downregulation was observed after inhibition of SARM expression by small interfering RNA in wild-type-infected macrophages, indicating that SIRPα downregulation is regulated by SARM. Furthermore, this downregulation requires activation of the TRIF signaling pathway, as we observed abrogation of SIRPα downregulation as well as restricted bacterial growth in LPS mutant-infected TRIF-depleted macrophages. Although inhibition of SARM expression is correlated to SIRPα downregulation and iNOS upregulation in gamma interferon-activated wild-type-infected macrophages, these phenomena appear to bypass the TRIF-dependent pathway. Similar to live bacteria, the wild-type LPS is able to upregulate SARM and to prevent SIRPα downregulation, implying that the LPS of B. pseudomallei may play a crucial role in regulating the expression of these two negative regulators. Altogether, our findings show a previously unrecognized role of B. pseudomallei-induced SARM in inhibiting SIRPα downregulation-mediated iNOS upregulation, facilitating the ability of the bacterium to multiply in macrophages.     

Mycoplasma genitalium-Derived Lipid-Associated Membrane Proteins Activate NF-κB through Toll-Like Receptors 1, 2, and 6 and CD14 in a MyD88-Dependent Pathway

Mycoplasma genitalium is a leading pathogen of nongonoccocal chlamydia-negative urethritis, which has been implicated directly in numerous other genitourinary and extragenitourinary tract pathologies. The pathogenesis of infection is attributed in part to excessive immune responses. M. genitalium-derived lipid-associated membrane proteins (LAMPs) are a mixture of bacterial lipoproteins, exposed at the surface of mycoplasma, that are potent inducers of the host innate immune system. However, the interaction of M. genitalium-derived LAMPs as pathogenic agents with Toll-like receptors (TLRs) and the signaling pathways responsible for active inflammation and NF-κB activation have not been fully elucidated. In this study, LAMPs induced the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in a dose-dependent manner. Blocking assays showed that TLR2- and CD14-neutralizing antibodies reduced the expression of TNF-α and IL-6 in THP-1 cells. Furthermore, LAMP-induced NF-κB activation was increased in 293T cells transfected with TLR2 plasmid. The activity of NF-κB was synergically augmented by cotransfected TLR1, TLR6, and CD14. Additionally, LAMPs were shown to inhibit NF-κB expression by cotransfection with dominant-negative MyD88 and TLR2 plasmids. These results suggest that M. genitalium-derived LAMPs activate NF-κB via TLR1, TLR2, TLR6, and CD14 in a MyD88-dependent pathway.Mycoplasmas are the smallest of the known self-replicating parasitic microorganisms, and they lack a cell wall in cell-free medium. They are pervasive in nature, humans, and animals, but most mycoplasmas are nonpathogenic to the host (36). Lipid-associated membrane proteins (LAMPs) are a mixture of bacterial lipoproteins that are expressed on the surface, and they are the main structures of interaction with the host cells (37). They have been demonstrated to be biologically active and are the most potent initiator of inflammatory reactions in mycoplasma infection (9, 11, 34). Since Mycoplasma genitalium was first isolated from humans in 1981 (51), it has been considered an important pathogen that could emerge in sexually transmitted diseases, including acute endometritis, salpingitis, mucopurulent cervicitis, tubal factor infertility, and pelvic inflammatory disease, as well as a range of other pathologies, such as arthritis, AIDS progression, chronic fatigue, and autoimmune disorders (4, 10, 19). Recent evidence has demonstrated that M. pneumoniae-derived LAMPs can induce NF-κB activation in human acute monocytic leukemia cell lines (THP-1 cells) (42, 43).Toll-like receptors (TLRs) play an essential role in initiating the inflammatory reactions, such as NF-κB activation (2, 22, 26). To date, 13 types of human TLRs have been identified and shown to be critical for signaling transduction in response to a number of pathogen-associated molecular patterns (PAMPs) (8, 44). PAMPs are recognized by different TLRs to form heteromeric complexes, among which the family of TLRs prominently features, for the cell to distinguish successfully among different pathogens. Among TLRs, TLR2, in tandem with TLR1 or TLR6, has been identified as a receptor that is important to the innate immune response against several gram-positive bacteria and for cellular signaling by components of gram-positive bacteria, such as peptidoglycan, lipoteichoic acid, and lipoproteins. The receptors transmit signaling pathways via interleukin-1R (IL-1R)-associated signal molecules, including myeloid differentiation factor 88 (MyD88), IL-1R-activated kinase, tumor necrosis factor (TNF) receptor-associated factor 6, and mitogen-activated protein kinases, leading to the activation of NF-κB and activating protein 1 (AP-1), which in turn triggers the expression of many proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-8 (1, 20, 29, 47). MyD88 serves as the essential adaptor for other IL-1/TLR family members, including IL-1R and IL-18R. The activation of the intracellular pathway through MyD88 results in the activation of NF-κB. CD14 is a glycosylphosphatidylinositol surface-anchored molecule expressed in many kinds of cells as another accessory receptor of TLR2. It lacks an intracellular segment and functions as a coreceptor for numerous bacterial products, including peptidoglycan, lipopolysaccharide (LPS), and bacterial lipoproteins, thereby facilitating signaling through other receptors (23). Several studies have suggested that TLR2 functions as a signaling receptor for LPS in the presence of CD14 (50). Recently published data support the possibility that the multifunctional B class scavenger receptor CD36 also is involved in TLR2 transmembrane signal transducing (16). However, data available also have underscored the current lack of a complete understanding of the molecular mechanisms that link M. genitalium infection to the activation of the innate immune system, which is essential to the induction of the inflammation.The LAMPs from Mycoplasma fermentans and Mycoplasma penetrans activate human immunodeficiency virus long terminal repeats through TLRs (41). To further understand how LAMPs activate the immune system of the cell, including THP-1 cells and human embryonic kidney cells (HEK293T cells), the present study was designed to investigate the interaction of M. genitalium-derived LAMPs with TLRs and CD14 and to clarify the role of MyD88 in activating the NF-κB signaling pathway.     

Propofol Attenuates Airway Inflammation in a Mast Cell-Dependent Mouse Model of Allergic Asthma by Inhibiting the Toll-like Receptor 4/Reactive Oxygen Species/Nuclear Factor κB Signaling Pathway

Propofol, an intravenous anesthetic agent widely used in clinical practice, is the preferred anesthetic for asthmatic patients. This study was designed to determine the protective effect and underlying mechanisms of propofol on airway inflammation in a mast cell-dependent mouse model of allergic asthma. Mice were sensitized by ovalbumin (OVA) without alum and challenged with OVA three times. Propofol was given intraperitoneally 0.5 h prior to OVA challenge. The inflammatory cell count and production of cytokines in the bronchoalveolar lavage fluid (BALF) were detected. The changes of lung histology and key molecules of the toll-like receptor 4 (TLR4)/reactive oxygen species (ROS)/NF-κB signaling pathway were also measured. The results showed that propofol significantly decreased the number of eosinophils and the levels of IL-4, IL-5, IL-6, IL-13, and TNF-α in BALF. Furthermore, propofol significantly attenuated airway inflammation, as characterized by fewer infiltrating inflammatory cells and decreased mucus production and goblet cell hyperplasia. Meanwhile, the expression of TLR4, and its downstream signaling adaptor molecules—–myeloid differentiation factor 88 (MyD88) and NF-κB, were inhibited by propofol. The hydrogen peroxide and methane dicarboxylic aldehyde levels were decreased by propofol, and the superoxide dismutase activity was increased in propofol treatment group. These findings indicate that propofol may attenuate airway inflammation by inhibiting the TLR4/MyD88/ROS/NF-κB signaling pathway in a mast cell-dependent mouse model of allergic asthma.     

Lipopolysaccharide Could Be Internalized into Human Peripheral Blood Mononuclear Cells and Elicits TNF-α Release,but not via the Pathway of Toll-Like Receptor 4 on the Cell Surface

Lipopolysaccharide (LPS), the principal component of the outer membrane of Gram-negative bacteria, stimulates various cell types to release numerous proinflammatory mediators such as TNF-alpha, IL-6 and IL-12, which may damage cells and lead to organ injury, even sepsis and septic shock. Toll-like receptor 4 (TLR4) has been identified as the receptor involved in the recognition of LPS, but the role of LPS uptake in activating signal transduction remains controversial. In the present study, TNF-alpha was used as a marker of macrophages/ monocytes activated by LPS, and CQ was used as an inhibitor of endosome mature in order to definitude what stage of the signal transduction elicited by LPS was interrupted. We found that there indeed existed internalization of LPS and internalization partially participated in LPS signaling since CQ inhibited cytokine release, and decreased accumulation of FITC-LPS in hPBMCs. In contrast, anti-hTLR4 antibody could decrease cytokine release, but had no inhibition on accumulation of FITC-LPS. This result revealed that inhibition of cytokine release was related to reduction of FITC-LPS accumulation in the cells. But TLR4 on the cell surface couldn't participate in internalization of LPS. Thus, LPS signaling and internalization couldn't be viewed as mutually independent processes.     

Lipopolysaccharide Could Be Internalized into Human Peripheral Blood Mononuclear Cells and Elicit TNF-α Release,but not via the Pathway of Toll-Like Receptor 4 on the Cell Surface

Lipopolysaccharide(LPS),the principal component of the outer membrane of Gram-negative bacteria,stimulatesvarious cell types to release numerous proinflammatory mediators such as TNF-α,IL-6 and IL-12,which maydamage cells and lead to organ injury,even sepsis and septic shock.Toll-like receptor 4(TLR4) has beenidentified as the receptor involved in the recognition of LPS,but the role of LPS uptake in activating signaltransduction remains controversial.In the present study,TNF-α was used as a marker of macrophages/monocytes activated by LPS,and CQ was used as an inhibitor of endosome mature in order to definitude whatstage of the signal transduction elicited by LPS was interrupted.We found that there indeed existedinternalization of LPS and internalization partially participated in LPS signaling since CQ inhibited cytokinerelease,and decreased accumulation of FITC-LPS in hPBMCs.In contrast,anti-hTLR4 antibody coulddecrease cytokine release,but had no inhibition on accumulation of FITC-LPS.This result revealed thatinhibition of cytokine release was related to reduction of FITC-LPS accumulation in the cells.But TLR4 on thecell surface couldn't participate in internalization of LPS.Thus,LPS signaling and internalization couldn't beviewed as mutually independent processes.Cellular & Molecular Immunology.2004;1(5):373-377.     

Effects of the Neuropeptides Substance P, Calcitonin Gene-Related Peptide and α-Melanocyte-Stimulating Hormone on the IL-8/IL-8 Receptor System in a Cultured Human Keratinocyte Cell Line and Dermal Fibroblasts

The neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP) and -melanocyte-stimulating hormone (-MSH) are known to be able to regulate the production of cytokines in the skin. Since IL-8 plays an important role in cutaneous inflammation, the effects of SP, CGRP and -MSH on the IL-8/IL-8 receptor (IL-8RA) systems of these cell types were studied. Cultures of human dermal fibroblasts and an immortalized keratinocyte cell line HaCaT were treated with 10^–8 M SP, CGRP or -MSH. The results demonstrated that these neuropeptides have different effects on the IL-8 and IL-8RA expressions of the cells. SP and CGRP upregulated the IL-8RA mRNA expression in HaCaT cells, but had no influence on their IL-8 production, whereas, -MSH had no effect on either the IL-8 or the IL-8RA mRNA expression in HaCaT cells. In contrast, -MSH resulted in a time-dependent induction of the IL-8 mRNA expression in dermal fibroblasts. This induction was already detectable after 6 h, and after 12 h there was a 5-fold change in comparison with the controls. The IL-8 content of the supernatant was also increased, with a maximum at 48 h after -MSH treatment. The data established in the present study support the notion that neuropeptides can directly modulate the IL-8/IL-8RA system of keratinocytes and fibroblasts.