Macrophages polarization is mediated by the combination of PRR ligands and distinct inflammatory cytokines

Macrophages recognize microbes through Pattern Recognition Receptors (PRRs), and then release pro-inflammatory and anti-inflammatory cytokines. Recent studies have highlighted that collaboration between different PRRs. However, these studies have neglected the crosstalk between various PRRs on macrophages. In the present study, we investigated the interplay of nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (NOD1, NOD2) and TLRs (TLR1, 2, 3, 4, 5, 6, 7, 8) in terms of macrophage activation, the expression and production of cytokines. The macrophages were stimulated with a single PRR ligand or a combination of TLR and NOD ligands. After 8 h of incubation, the mRNA expression of interleukin-1β (IL-1β), IL-4, IL-6, IL-10, IL-12p35, IL-12p40, IL-13, and interferon-γ (IFN-γ) was evaluated. The production of these cytokines was also measured. NOD2 synergized with TLR3 agonists on enhancement of IL-10 release. However, the combination of NOD1 with TLR3 ligands showed little effect on IL-10 production. Moreover, NOD2 inhibited the percentages of CD11b + F4/80 + cells activated by TLR3 agonist.     

Heat shock up-regulates expression of Toll-like receptor-2 and Toll-like receptor-4 in human monocytes via p38 kinase signal pathway

Summary Heat stress can alert innate immunity by inducing stress proteins such as heat-shock proteins (HSPs). However, it remains unclear whether heat stress affects the activation of antigen-presenting cell (APC) in response to pathogen-associated molecule patterns (PAMPs) by directly regulating pathogen recognition receptors (PRRs). As an important kind of PRRs, Toll-like receptors (TLRs) play critical roles in the activation of immune system. In this study, we demonstrated that heat shock up-regulated the expression of HSP70 as well as TLR2 and TLR4 in monocytes. The induction of TLRs was prior to that of HSP70, which suggesting the up-regulation of TLR2 and TLR4 might be independent of the induction of HSP70. Heat shock activated p38 kinase, extracellular signal-related kinase (ERK) and nuclear factor-kappa B (NF-kappaB) signal pathways in monocytes. Pretreatment with specific inhibitor of p38 kinase, but not those of ERK and NF-kappaB, inhibited heat shock-induced up-regulation of TLR2 and TLR4. This indicates that p38 pathway takes part in heat shock-induced up-regulation of TLR2 and TLR4. Heat shock also increased lipoteichoic acid- or lipopolysaccharide-induced interleukin-6 production by monocytes. These results suggest that the p38 kinase-mediated up-regulation of TLR2 and TLR4 might be involved in the enhanced response to PAMP in human monocytes induced by heat shock.     

Innate immunity networks during infection with Borrelia burgdorferi

Abstract

The recognition of Borrelia species represents a complex process in which multiple components of the immune system are involved. In this review, we summarize the interplay between the host innate system and Borrelia spp., from the recognition by pattern recognition receptors (PRRs) to the induction of a complex network of proinflammatory mediators. Several PRR families are crucial for recognition of Borrelia spp., including Toll-like receptors (TLRs) and Nucleotide Oligomerization Domain (NOD)-like receptors (NLRs). TLR-2 is crucial for the recognition of outer surface protein (Osp)A from Borrelia spp. and together with TLR8 mediates phagocytosis of the microorganism and production of type I interferons. Intracellular receptors such as TLR7, TLR8 and TLR9 on the one hand and the NLR receptor NOD2 on the other hand, represent the second major recognition system of Borrelia. PRR-dependent signals induce the release of pro-inflammatory cytokines such as interleukin-1 and T-helper-derived cytokines, which are thought to mediate the inflammation during Lyme disease. Understanding the regulation of host defense mechanisms against Borrelia has the potential to lead to the discovery of novel immunotherapeutic targets to improve the therapy against Lyme disease.     

FAK-mediated activation of ERK for eosinophil migration: a novel mechanism for infection-induced allergic inflammation

Bacterial and viral infections often induce the exacerbation of allergic diseases. In this study, we investigated the activation of human eosinophils by different microbial products via Toll-like receptors (TLRs). The underlying intracellular mechanism involving activation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK), an integrin-associated focal adhesion molecule, was also examined. Seven TLR ligands were studied for their abilities in promoting survival, modulating the expression of adhesion molecules and facilitating chemotactic migration of eosinophils. While peptidoglycan (PGN) (TLR2 ligand) showed the most prominent effects, flagellin (TLR5 ligand) and imiquimod R837 (TLR7 ligand) were also effective in activating eosinophils. However, little or no effect was observed for double-stranded polyinosinic-polycytidylic acid (TLR3 ligand), ultra-purified LPS (TLR4 ligand), single-stranded RNA (ssRNA) (TLR8 ligand) and CpG-DNA (TLR9 ligand). Further investigation confirmed that PGN, flagellin and R837 commonly transmitted signals through ERK activation that required prior phosphorylation of tyrosine 925, but not tyrosine 577, on FAK. Moreover, the inhibition of ERK activation by selective inhibitor PD98059 and FAK expression by FAK-specific RNA interference could significantly abolish the stimulatory effects induced by PGN, flagellin and R837. Taken together, our findings indicate the involvement of FAK-dependent activation of ERK1 in TLR-mediated eosinophil stimulation. A potential role of eosinophils was also suggested in exacerbating allergic inflammation in response to microbial infections.     

The crosstalk between TLR2 and NOD2 in Aspergillus fumigatus keratitis

Innate immunity is considered to be critical in the pathogenesis of fungal keratitis. Pattern recognition receptors (PRRs) recognize conserved microbial structures called pathogen-associated molecular patterns (PAMPS), thereby initiating the innate immunity. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (leucine-rich repeat-containing receptors, NLRs) are two major PRR families. The crosstalk between TLR2 and NOD2 is not completely understood, and their interrelationship in Aspergillus fumigates keratitis is still unclear. To our surprise, we found herein that NOD2 and TLR2 were increased by A. fumigatus conidia in immortalized human corneal epithelial cells (HCECs). In addition, NOD2 expression was up-regulated by its agonist muramyl dipeptide (MDP), along with receptor interacting protein 2 (RIP2), nuclear factor κB (NFκB)-p65, inhibitor of NFκB (IκB)-α, and multiple inflammatory cytokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor α (TNF-α). Interestingly, zymosan, a TLR2 agonist, promoted the expression of NOD2 and RIP2 in a TLR2-dependent manner. Furthermore, we demonstrated that the increased expression of NOD2 and RIP2 caused by A. fumigatus conidia occurred in part through a TLR2-dependent pathway. However, zymosan pretreatment decreased NOD2 and RIP2 expression along with the MDP induced secretion of inflammatory cytokines in HCECs. In agreement, NOD2 knockdown by small interfering RNA (siRNA) reduced the release of IL-6, IL-8 and TNF-α induced by A. fumigatus conidia. These findings suggest the existence of complex interactions between TLR2 and NOD2 in HCECs inflammatory response against A. fumigatus infection.     

Toll‐like receptor polymorphism in host immune response to infectious diseases: A review

Immunopolymorphism is considered as an important aspect behind the resistance or susceptibility of the host to an infectious disease. Over the years, researchers have explored many genetic factors for their role in immune surveillance against infectious diseases. Polymorphic characters in the gene encoding Toll‐like receptors (TLRs) play profound roles in inducing differential immune responses by the host against parasitic infections. Protein(s) encoded by TLR gene(s) are immensely important due to their ability of recognizing different types of pathogen associated molecular patterns (PAMPs). This study reviews the polymorphic residues present in the nucleotide or in the amino acid sequence of TLRs and their influence on alteration of inflammatory signalling pathways promoting either susceptibility or resistance to major infectious diseases, including tuberculosis, leishmaniasis, malaria and filariasis. Population‐based studies exploring TLR polymorphisms in humans are primarily emphasized to discuss the association of the polymorphic residues with the occurrence and epidemiology of the mentioned infectious diseases. Principal polymorphic residues in TLRs influencing immunity to infection are mostly single nucleotide polymorphisms (SNPs). I602S (TLR1), R677W (TLR2), P554S (TLR3), D299G (TLR4), F616L (TLR5), S249P (TLR6), Q11L (TLR7), M1V (TLR8), G1174A (TLR9) and G1031T (TLR10) are presented as the major influential SNPs in shaping immunity to pathogenic infections. The contribution of these SNPs in the structure‐function relationship of TLRs is yet not clear. Therefore, molecular studies on such polymorphisms can improve our understanding on the genetic basis of the immune response and pave the way for therapeutic intervention in a more feasible way.     

Expression and function of the Fas receptor on human blood and tissue eosinophils

The interaction between activated T cells and eosinophils has been proposed to play an important role in the pathogenesis of allergic diseases. T cell-derived cytokines such as interleukin-5 and granulocyte/macrophage colony-stimulating factor inhibit eosinophil apoptosis and may therefore contribute to the development of tissue and blood eosinophilia in these disorders. Withdrawal of these cytokines leads to eosinophil apoptosis in vitro. In contrast, the mechanisms which actively induce apoptosis in eosinophils are at present not completely understood. In this study, we demonstrate that freshly isolated human eosinophils express mRNA and protein for the Fas receptor. Using anti-Fas monoclonal antibody (mAb), we show that Fas activation accelerates apoptotic eosinophil death in vitro. Moreover, treatment of nasal polyps ex vivo with anti-Fas mAb decreased eosinophilic tissue inflammation. However, we observed that blood as well as tissue eosinophils derived from some eosinophilic donors do not express functional Fas receptors, although Fas protein is normally expressed in these cells. This implies that the susceptibility of the Fas receptor is a matter of regulation in eosinophils as previously observed in other systems. These data suggest that Fas ligand/Fas interactions are involved in the regulation of eosinophil apoptosis and that defects in this system could contribute to the accumulation of these cells in allergic and asthmatic diseases.     

CD45 negatively regulates tumour necrosis factor and interleukin-6 production in dendritic cells

CD45 is known to regulate signalling through many different surface receptors in diverse haemopoietic cell types. Here we report for the first time that CD45-/- bone marrow dendritic cells (BMDC) are more activated than CD45+/+ cells and that tumour necrosis factor (TNF) and interleukin-6 (IL-6) production by BMDC and splenic dendritic cells (sDC), is increased following stimulation via Toll-like receptor (TLR)3 and TLR9. Nuclear factor-kappaB activation, an important downstream consequence of TLR3 and TLR9 signalling, is also increased in CD45-/- BMDC. BMDC of CD45-/- mice also produce more TNF and IL-6 following stimulation with the cytokines TNF and interferon-alpha. These results show that TLR signalling is increased in CD45-/- dendritic cells and imply that CD45 is a negative regulator of TLR and cytokine receptor signalling in dendritic cells.     

Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce surface expression of interleukin-2 receptor p55-chain and CD4 by human eosinophils.

In this report it is shown by immunofluorescence analysis, biochemical analysis and mRNA hybridization that human eosinophils express surface CD4 and interleukin-2 receptor (IL-2R) (CD25) when exposed to eosinophil activators granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3. Although the functional role of eosinophil CD4/CD25 expression has to be elucidated, it will be of interest in further studies to investigate whether in vivo induction of these molecules occurs in association with certain disease processes such as the hypereosinophilic syndrome or in immunological responses during allergic and helminthic parasitic diseases.     

T-helper 2 cytokines attenuate senescent eosinophil activation by the CXCR4 ligand stromal-derived factor-1α (CXCL12)

BACKGROUND: Different chemokine receptors have been suggested to play a pivotal role in allergic diseases and therefore to be relevant for the activation of effector cells and propagation of the inflammatory response. The CXC chemokine receptor CXCR4 has recently been found on the surface of eosinophils implicating a role in allergic diseases. OBJECTIVE: The aim of this study was to investigate the functional expression of CXCR4 on senescent eosinophils. Moreover, we questioned whether the cytokine profile--T-helper (Th)1 and Th2 cytokines--affect the activation of eosinophils via the CXCR4 that could be important for the different phases of the allergic reaction. METHODS: CXCR4 expression on human eosinophils was analysed by flow cytometry and RT-PCR. Functional analyses of intracellular calcium fluxes, actin polymerization, release of reactive oxygen species and, chemotaxis were carried out using spectrofluorometry, flow cytometry, chemiluminescence and modified Boyden chamber technique. RESULTS: Whole blood and freshly isolated eosinophils weakly express CXCR4 surface protein. Incubation in culture medium without addition of cytokines for 24 h always lead to strong CXCR4 surface expression that paralleled with stromal-derived factor-1alpha (CXCL12)-induced eosinophil activation. Stimulation of eosinophils with CXCL12 leads to an internalization of CXCR4, which could be prevented by phenylarsine oxide. Co-incubation of eosinophils with Th2 cytokines such as IL-3, IL-4, IL-5, IL-13, and granulocyte macrophage-colony stimulating factor prevented the expression of CXCR4 and affected eosinophil activation after stimulation with the CXCR4 ligand CXCL12. From these cytokines, IL-3 was the only cytokine completely inhibited intracellular calcium fluxes and chemotaxis of eosinophils in response to CXCL12. CONCLUSION: Senescent eosinophils express functional CXCR4 receptors, which are prevented by Th2 cytokines that are found in the early phase of allergic reaction. Therefore, CXCR4 activation of eosinophils seems to be important in the chronic phase of allergic reaction, which is dominated by a Th1 cytokine profile.     

Aspergillus antigen-induced eosinophil differentiation in a murine model.

Eosinophilia is a prominent feature of the cellular response in allergic and parasitic diseases. Allergic bronchopulmonary aspergillosis due to colonization of the lungs of some asthmatics with Aspergillus fumigatus is characterized by high levels of serum immunoglobulin E and peripheral blood (PB) and lung eosinophilia. This study investigates the role of eosinophils in the pathogenesis of allergic bronchopulmonary aspergillosis by using a mouse model. BALB/c mice were immunized intranasally and intraperitoneally with A. fumigatus antigens (Ag), and the eosinophils in PB and bone marrow (BM) were enumerated. Eosinophilopoiesis in BM cultures was studied in the presence of murine recombinant interleukin-5 (mrIL-5) and supernatants from pokeweed mitogen-stimulated spleen cells as the source of eosinophil differentiation factors. Eosinophils were quantitated by direct counting and by estimating eosinophil peroxidase activity. The results indicate that the percentage of eosinophils in the PB (5.77 +/- 1.17) and the BM (11.19 +/- 4.31) of mice exposed to A. fumigatus Ag was higher than in controls (PB, 2.42 +/- 0.76; BM, 5.12 +/- 2.79; P less than 0.01 for both). Similarly, a significant increase in eosinophils was observed in the BM population from mice exposed to A. fumigatus Ag compared with that in controls when cultured with murine recombinant interleukin-5 (23.13 +/- 7.14 versus 13.77 +/- 5.79, P less than 0.01), indicating that the mice exposed to A. fumigatus Ag had significantly greater numbers of eosinophil precursors in their BM. This study demonstrates that A. fumigatus Ag may be involved in the in vivo commitment of stem cells in the eosinophil differentiation pathway.     

Toll‐like receptors: The role in bladder cancer development,progression and immunotherapy

Bladder cancer is one of the leading causes of death worldwide. The main immune mechanisms which lead to bladder cancer development or treatment outcomes have yet to be elucidated. Toll‐like receptors (TLRs) play key roles against cancer. TLRs are expressed both on immune cells and on tumour cells and drive immune responses in progression as well as treatment of cancer. Identification of signalling pathways via TLRs could revolutionize further improvement of therapeutic strategies against cancers in the future. According to the recent studies, TLRs agonists are effective immunostimulants and have important role in induction of immune responses with immunotherapeutic potential against several diseases including cancer. They play an important role in the bladder urothelium as a part of immune defence against uropathogens. On the other hand, decreased TLRs expression was found in bladder tumours, particularly in non‐muscle‐invasive ones. Bacillus Calmette‐Guerin (BCG) (agonist of TLR2 and TLR4) is approved by US FDA for immunotherapy of bladder cancer. Despite high efficiency, immunotherapy with BCG may cause toxicity and adverse effects. Nowadays, in vitro and in vivo studies have been conducted to find alternative options for non‐responder patients. Studies on TLR agonists for bladder cancer treatment have shown promising results. In this review, we discuss recent data about mechanisms played by TLRs in bladder cancer developments as well as therapeutic application of TLR agonists in cancer treatment.     

IgA is a more potent inducer of NADPH oxidase activation and degranulation in blood eosinophils than IgE

Human eosinophils can mediate both beneficial and detrimental responses in parasitic and allergic diseases. Binding of aggregated immunoglobulin to Fc receptors on eosinophils mediates important defence processes, including generation of activated oxygen species resulting from NADPH oxidase activation, and eosinophil peroxidase release following degranulation. The abilities of a matched set of IgA, IgG and IgE antibodies to elicit such responses in blood-derived eosinophils were compared using a chemiluminescence assay. IgA and IgG, but not IgE, were found to trigger NADPH oxidase activation and degranulation in eosinophils. This non-responsiveness to IgE did not result from receptor blockade by endogenous IgE since no blood-derived IgE was detectable on freshly isolated eosinophils. Moreover, while cross-linking of FcalphaRI by specific mAbs triggered NADPH oxidase activation and degranulation in blood-derived eosinophils, equivalent cross-linking of FcvarepsilonRI or FcvarepsilonRII did not elicit such responses. Therefore IgA is more potent at eliciting activated oxygen species release and degranulation in eosinophils than IgE, suggesting that the importance of IgA in eosinophil activation in immune defence and allergy may have been underestimated.     

Toll样受体2和Toll样受体4在免疫应答衣原体感染中的作用

衣原体是重要的人类病原体,其能够导致多种疾病的发生.由衣原体引起的许多人类疾病被认为是免疫病理学介导的.已经证明Toll样受体(TLRs)是多种病原体感染的主要模式识别受体( PRRs),在起始固有免疫应答,建立适应性免疫应答中发挥着重要作用.在TLR家族中,TLR2和TLR4与衣原体感染的相关性研究备受关注,在识别衣原体感染、调节宿主的早期免疫应答、炎症反应和病理形成中执行着关键性的作用.研究TLR2和TLR4在免疫应答衣原体感染中的作用可以更好地理解TLRs介导的分子免疫机制,可能有助于研发免疫治疗的分子靶标,最终有效预防、控制衣原体感染引起的疾病.     

The dendritic cell mannose receptor mediates allergen internalization and maturation involving notch 1 signalling

Dendritic cells (DCs) have been shown recently to play a key role in inducing and mediating T helper type 2 (Th2) responses associated with atopic disease. These responses are mediated in part by ligation to different Toll‐like receptors (TLRs) and C‐type lectins, e.g. the mannose receptor (MR), depending upon the DC subset involved and the respective microenvironments. Because ovalbumin (OVA) (which is structurally related to various allergens) can engage the MR, we can use OVA stimulation as a model for understanding the roles of both TLRs and the MR in allergic inflammatory responses. We examined TLR‐ and MR‐mediated responses from mouse bone marrow‐derived DCs in the context of antigen recognition and presentation in addition to examining the relationship between notch 1, TLRs and MR signalling pathways. This work demonstrated that OVA‐mediated signalling up‐regulated both TLR‐2 and MR and that MR RNA interference (RNAi) but not TLR2 RNAi inhibited DC internalization of fluorescein isothiocyanate–OVA. Furthermore, MR RNAi inhibited OVA‐ and house dust mite allergen extract‐induced DC maturation and MR RNAi and TLR2 RNAi influenced DC interleukin‐12 production independently. Finally, we demonstrated that blocking notch 1 signalling inhibited both notch 1 and TLR‐2 expression but not MR expression levels. However, MR RNAi inhibited the expression of MR, TLR‐2 and notch 1. These results indicate that MR is the primary receptor mediating the internalization of environmental allergen glycoproteins. In addition, TLR‐2 and notch 1 play important roles in DC maturation and antigen presentation and signals originating from the MR and TLR‐2 receptors converge with the notch 1 signalling pathway.