Associations between toll-like receptors and interleukin-4 in the lungs of patients with tuberculosis

Toll-like receptors (TLRs) are implicated in the intracellular killing of Mycobacterium tuberculosis, and their expression is modulated by interleukin-4 (IL-4) in vitro. Our aim was to examine the expression of TLRs at the site of pathology in tuberculous lung granulomas and to explore the effect of the immune response on TLR expression. Immunohistochemistry was performed on lung granulomas from nine patients with tuberculosis undergoing lobectomy for haemoptysis. All nine patients expressed all of the TLRs studied (TLRs 1-5 and 9), whereas only five out of the nine patients had any granulomas positive for IL-4. Statistical analysis of TLR and cytokine staining patterns in 183 individual granulomas from the nine patients revealed significant associations between pairs of receptors and IL-4. A positive association between TLR2 and TLR4 (P < 0.0001) and a negative association between TLR2 and IL-4 (P < 0.0001) was observed. The associations between TLRs 1, 5, and 9 were significantly different in IL-4-negative compared with IL-4-positive patients. In conclusion, TLRs are expressed by various cell types in the human tuberculous lung, and their expression patterns are reflected by differences in the immune response.     

Toll-like receptors 3 and 4 are expressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) are multipotent, nonhemopoietic progenitors that also possess regulatory activity on immune effector cells through different mechanisms. We demonstrate that human BM-derived MSCs expressed high levels of Toll-like receptors (TLRs) 3 and 4, which are both functional, as shown by the ability of their ligands to induce nuclear factor kappaB (NF-kappaB) activity, as well as the production of interleukin (IL)-6, IL-8, and CXCL10. Of note, ligation of TLR3 and TLR4 on MSCs also inhibited the ability of these cells to suppress the proliferation of T cells, without influencing their immunophenotype or differentiation potential. The TLR triggering effects appeared to be related to the impairment of MSC signaling to Notch receptors in T cells. Indeed, MSCs expressed the Notch ligand Jagged-1, and TLR3 or TLR4 ligation resulted in its strong downregulation. Moreover, anti-Jagged-1 neutralizing antibody and N[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of Notch signaling, hampered the suppressive activity of MSCs on T-cell proliferation. These data suggest that TLR3 and TLR4 expression on MSCs may provide an effective mechanism to block the immunosuppressive activity of MSCs and therefore to restore an efficient T-cell response in the course of dangerous infections, such as those sustained by double-stranded RNA viruses or Gram-negative bacteria, respectively.     

Toll-like receptors on hematopoietic progenitor cells stimulate innate immune system replenishment

Toll-like receptors (TLRs) are best known for their ability to recognize microbial or viral components and initiate innate immune responses. We showed here that TLRs and their coreceptors were expressed by multipotential hematopoietic stem cells, whose cell cycle entry was triggered by TLR ligation. TLR expression also extended to some of the early hematopoietic progenitors, although not the progenitor cells dedicated to megakaryocyte and erythroid differentiation. TLR signaling via the Myd88 adaptor protein drove differentiation of myeloid progenitors, bypassing some normal growth and differentiation requirements, and also drove lymphoid progenitors to become dendritic cells. CD14 contributed to the efficiency of lipopolysaccharide (LPS) recognition by stem and progenitor cells, and LPS interacted directly with the TLR4/MD-2 complex on these cells in bone marrow. Thus, the preferential pathogen-mediated stimulation of myeloid differentiation pathways may provide a means for rapid replenishment of the innate immune system during infection.     

TLR4 single-nucleotide polymorphisms alter mucosal cytokine and chemokine patterns in Mexican patients with Helicobacter pylori-associated gastroduodenal diseases

Helicobacter pylori is associated with peptic ulcer and gastric adenocarcinoma. Toll-like receptors (TLRs) participate in H. pylori recognition, and single-nucleotide polymorphisms (SNPs) in TLRs are associated with impaired immune response. We aimed to evaluate the association of TLR2/R753Q and TLR4/D299G/T399I SNPs with gastroduodenal diseases; and study the effect of SNPs on cytokine and chemokine expression in the gastric mucosa. Study included 450 Mexican patients with gastroduodenal diseases. SNPs in TLRs 2 and 4 genes were analyzed by allele-specific PCR. Cytokines and chemokines were assessed by qRT-PCR and immunoassay. TLR4/D299G/T399I polymorphisms were more frequent in duodenal ulcer and showed a trend in gastric cancer, when compared with non-atrophic gastritis. Patients with TLR4 polymorphisms expressed significantly lower levels of IL-1beta, IL-6, IL-8 and GRO-alpha; and higher levels of TNF-alpha, IL-10, MCP-1 and MIP-1alpha . SNPs in TLR4 gene had an association with severe H. pylori-associated disease and with modified pattern of inflammatory cytokines and chemokines in the gastric mucosa. These results suggest that TLR4 SNPs contributes importantly to the clinical outcome of H. pylori infection.     

Rapamycin suppresses TLR4-triggered IL-6 and PGE(2) production of colon cancer cells by inhibiting TLR4 expression and NF-kappaB activation

Toll-like receptor 4 (TLR4) signaling in tumor cells can mediate tumor cell immune escape and tumor progression, being regarded as one of the mechanisms for chronic inflammation in tumorigenesis and progression. So, intervention of TLR4-mediated immune escape and metastasis has been proposed as one of the approaches to cancer prevention and treatment. Rapamycin, an immunosuppressant agent widely used for treatment of autoimmune diseases and transplantation rejection, is recently used for cancer therapy. However, the underlying mechanisms remain to be fully understood. In the present study, we demonstrate that rapamycin can significantly inhibit TLR4-triggered IL-6 and PGE₂ production and invasion of colon cancer cells. Suppression of TLR4-induced IL-6 and PGE₂ production is responsible for the rapamycin-mediated decrease of TLR4-evoked invasion of colon cancer cells. Furthermore, disruption of NF-κB pathway contributes to the inhibition of TLR4-induced IL-6, PGE₂ production and invasion by rapamycin in colon cancer cells. Rapamycin can also downregulate TLR4 expression. Therefore, we demonstrate that rapamycin may abrogate TLR4-triggered tumor cell immune escape and invasion by downregulating TLR4 expression and inhibiting TLR4-activated NF-κB pathway, thus providing new mechanistic explanation for the antitumor effect of rapamycin.     

Toll-like receptors TLR2 and TLR4 initiate the innate immune response of the renal tubular epithelium to bacterial products

Renal tubular epithelial cells (TECs) respond diffusely to local infection, with the release of multiple cytokines, chemokines and other factors that are thought to orchestrate the cellular constituents of the innate immune response. We have investigated whether the Toll-like receptors TLR4 and TLR2, which are present on tubular epithelium and potentially detect a range of bacterial components, co-ordinate this inflammatory response acting through nuclear factor-kappa B (NF-kappaB). Primary cultures of TECs were grown from C57BL/6, C3H/HeN, C3H/HeJ, TLR2 and TLR4 knock-out mice. Cell monolayers were stimulated with lipopolysaccharide (LPS) and synthetic TLR2 and 4 agonists. The innate immune response was quantified by measurement of the cytokines tumour necrosis factor (TNF)-alpha and KC (IL-8 homologue) in cell supernatants by enzyme-linked immunosorbent assay. Cultured TECs grown from healthy mice produced the cytokines TNF-alpha and KC in response to stimulation by LPS and synthetic TLR2 and TLR4 agonists. Cells lacking the respective TLRs had a reduced response to stimulation. The TLR2- and TLR4-mediated response to stimulation was dependent on NF-kappaB signalling, as shown by curcumin pretreatment of TECs. Finally, apical stimulation of these TLRs elicited basal surface secretion of TNF-alpha and KC (as well as the reverse), consistent with the biological response in vivo. Our data highlight the potential importance of TLR-dependent mechanisms co-ordinating the innate immune response to upper urinary tract infection.     

Toll-like receptor 4 stimulation initiates an inflammatory response that decreases cardiomyocyte contractility

Toll-like receptors (TLRs) have been identified as primary innate immune receptors for the recognition of pathogen-associated molecular patterns by immune cells, initiating a primary response toward invading pathogens and recruitment of the adaptive immune response. TLRs, especially Toll-like receptor 4 (TLR4), can also be stimulated by host-derived molecules and are expressed in the cardiovascular system, thus acting as a possible key link between cardiovascular diseases and the immune system. TLR4 is involved in the acute myocardial dysfunction caused by septic shock and myocardial ischemia. We used wild-type (WT) mice, TLR4-deficient (TLR4-knockout [ko]) mice, and chimeras that underwent myeloablative bone marrow transplantation to dissociate between TLR4 expression in the heart (TLR4-ko/WT) and the immunohematopoietic system (WT/TLR4-ko). Following lipopolysaccharide (LPS) challenge (septic shock model) or coronary artery ligation, myocardial ischemia (MI) model, we found WT/TLR4-ko mice challenged with LPS or MI displayed reduced cardiac function, increased myocardial levels of interleukin-1β and tumor necrosis factor-α, and upregulation of mRNA encoding TLR4 prior to myocardial leukocyte infiltration. The cardiac function of TLR4-ko or WT/TLR4-ko mice was less affected by LPS and demonstrated reduced suppression by MI compared with WT. These results suggest that TLR4 expressed in the cardiomyocytes plays a key role in this acute phenomenon.     

Intact gram-negative Helicobacter pylori, Helicobacter felis, and Helicobacter hepaticus bacteria activate innate immunity via toll-like receptor 2 but not toll-like receptor 4

Molecular and genetic studies have demonstrated that members of the Toll-like receptor (TLR) family are critical innate immune receptors. TLRs are recognition receptors for a diverse group of microbial ligands including bacteria, fungi, and viruses. This study demonstrates that distinct TLRs are responsible for the recognition of Helicobacter lipopolysaccharide (LPS) versus intact Helicobacter bacteria. We show that the cytokine-inducing activity of Helicobacter LPS was mediated by TLR4; i.e., TLR4-deficient macrophages were unresponsive to Helicobacter pylori LPS. Surprisingly, the cytokine response to whole Helicobacter bacteria (H. pylori, H. hepaticus, and H. felis) was mediated not by TLR4 but rather by TLR2. Studies of HEK293 transfectants revealed that expression of human TLR2 was sufficient to confer responsiveness to intact Helicobacter bacteria, but TLR4 transfection was not sufficient. Our studies further suggest that cag pathogenicity island genes may modulate the TLR2 agonist activity of H. pylori as cagA+ bacteria were more active on a per-cell basis compared to cagA mutant bacteria for interleukin-8 (IL-8) cytokine secretion. Consistent with the transfection studies, analysis of knockout mice demonstrated that TLR2 was required for the cytokine response to intact Helicobacter bacteria. Macrophages from both wild-type and TLR4-deficient mice produced a robust cytokine secretion response (IL-6 and MCP-1) when stimulated with intact Helicobacter bacteria. In contrast, macrophages from TLR2-deficient mice were profoundly unresponsive to intact Helicobacter stimulation, failing to secrete cytokines even at high (100:1) bacterium-to-macrophage ratios. Our studies suggest that TLR2 may be the dominant innate immune receptor for recognition of gastrointestinal Helicobacter species.     

Toll-like receptor 2, 3, and 4 expression and function in human airway smooth muscle

BACKGROUND: Host defense against microbial pathogens is elicited through the innate immune system by means of Toll-like receptors (TLRs). Airway smooth muscle cells (ASMCs) display proinflammatory and immunomodulatory functions. ASMCs might participate in airway inflammatory responses associated with innate immune activation. OBJECTIVES: We determined the effects of cytokines, TLR ligands, and corticosteroids on TLR expression and function in human ASMCs. METHODS: Real-time PCR and flow cytometry were used to assess TLR mRNA and protein expression, respectively. ASMCs were stimulated with TLR ligands, and chemokine release was measured by means of ELISA. RESULTS: ASMCs expressed TLR1 to TLR10 mRNA, and TLR2 and TLR3 protein expression was demonstrated. TNF-alpha and double-stranded RNA (dsRNA; TLR3 ligand) were potent inducers of TLR2 and TLR3 mRNA expression, and both stimuli had additive or synergistic effects with IFN-gamma on TLR2 and TLR4, but not TLR3, mRNA expression. Peptidoglycan (TLR2 ligand) and LPS (TLR4 ligand) weakly enhanced TLR2 mRNA expression. Peptidoglycan, dsRNA, and LPS induced IL-8 and eotaxin release, with dsRNA being most potent. dsRNA also modulated cytokine-induced chemokine release in a differential manner. Dexamethasone inhibited cytokine- and ligand-induced TLR2, TLR3, and TLR4 expression and chemokine release. However, dexamethasone potentiated TLR2 expression induced by combined IFN-gamma and TNF-alpha stimulation. CONCLUSION: Expression of TLR2, TLR3, and TLR4 is regulated by cytokines and TLR ligands, and their activation mediates chemokine release in ASMCs. CLINICAL IMPLICATIONS: Proinflammatory responses mediated by activation of pathogen-recognition receptors in ASMCs might contribute to infectious exacerbations of airway inflammatory conditions, such as asthma and chronic obstructive pulmonary disease.     

Systemic Up-Regulation of TLR4 Causes Lipopolysaccharide-Induced Augmentation of Nasal Cytokine Release in Allergic Rhinitis

Background: Allergic rhinitis is a systemic disorder, and it is clinically well recognized that it can be aggravated by infection. Activation of the innate immune system constitutes a critical element in the process. Toll-like receptors (TLRs) comprise a part of the innate immune system, and lipopolysaccharide (LPS)-induced activation of TLR4 represents bacterial-induced interactions in various model systems. The present study examines how TLR2 and TLR4 expression is affected by symptomatic allergic rhinitis, and if LPS added upon allergen affects nasal cytokine release. Methods: In patients with pollen-induced allergic rhinitis and healthy non-allergic volunteers, nasal lavage (NAL), peripheral blood and bone marrow were sampled before and during the pollen season. TLR2 and TLR4 expression was determined flow cytometrically. Changes in the TLR receptor expression pattern were evaluated by a nasal challenge with allergen followed by LPS, or vice versa. Symptoms along with cells and cytokines in NAL were analyzed. Results: TLR4 expression increased in leukocytes in NAL, peripheral blood and bone marrow during symptomatic allergic rhinitis. A similar increase was seen for TLR2 in neutrophils in blood. Nasal challenge with allergen followed by LPS augmented the release of IL-4, IL-5, IL-10, IL-13, IFN-γ and TNF-α. Conclusion: A systemic up-regulation of TLR4 in symptomatic allergic rhinitis may explain why LPS preceded by allergen increases nasal cytokine release.     

Lipopolysaccharides from Periodontopathic Bacteria Porphyromonas gingivalis and Capnocytophaga ochracea Are Antagonists for Human Toll-Like Receptor 4

Toll-like receptors (TLRs) 2 and 4 have recently been identified as possible signal transducers for various bacterial ligands. To investigate the roles of TLRs in the recognition of periodontopathic bacteria by the innate immune system, a Chinese hamster ovary (CHO) nuclear factor-kappaB (NF-kappaB)-dependent reporter cell line, 7.7, which is defective in both TLR2- and TLR4-dependent signaling pathways was transfected with human CD14 and TLRs. When the transfectants were exposed to freeze-dried periodontopathic bacteria, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Capnocytophaga ochracea, and Fusobacterium nucleatum, and a non-oral bacterium, Escherichia coli, all species of the bacteria induced NF-kappaB-dependent CD25 expression in 7.7/huTLR2 cells. Although freeze-dried A. actinomycetemcomitans, F. nucleatum, and E. coli also induced CD25 expression in 7.7/huTLR4 cells, freeze-dried P. gingivalis did not. Similarly, lipopolysaccharides (LPS) extracted from A. actinomycetemcomitans, F. nucleatum, and E. coli induced CD25 expression in 7.7/huTLR4 cells, but LPS from P. gingivalis and C. ochracea did not. Furthermore, LPS from P. gingivalis and C. ochracea attenuated CD25 expression in 7.7/huTLR4 cells induced by repurified LPS from E. coli. LPS from P. gingivalis and C. ochracea also inhibited the secretion of interleukin-6 (IL-6) from U373 cells, the secretion of IL-1beta from human peripheral blood mononuclear cells, and ICAM-1 expression in human gingival fibroblasts induced by repurified LPS from E. coli. These findings indicated that LPS from P. gingivalis and C. ochracea worked as antagonists for human TLR4. The antagonistic activity of LPS from these periodontopathic bacteria may be associated with the etiology of periodontal diseases.     

树突状细胞分化成熟障碍在肿瘤免疫逃逸中的研究

大量研究证实,肿瘤病人体内存在广泛的免疫逃逸现象。目前所知肿瘤逃逸的机制很多,其中重要的一个就是树突状细胞的分化成熟异常导致的功能障碍。近来国内外研究发现许多因素能够影响肿瘤内树突状细胞的正常分化成熟和功能,这些因素包括一些免疫抑制性细胞因子（TGF-β,IL-10,IL-6,VEGF）,神经节苷脂,吲哚咹-2-3加双氧酶等等。     

Involvement of Toll-like receptors in the immune response of nasal polyp epithelial cells

Recognition systems employed by airway epithelial cells to respond to microbial exposure include the action of Toll-like receptors (TLRs). We investigated the presence and function of TLR2, 3, and 4 in primary cultures of human nasal polyp epithelial cells. dsRNA stimulation significantly enhanced the expression and secretion of RANTES, IP-10, IL-8, and GM-CSF. LPS also exhibited stimulatory action, but it was much weaker than dsRNA. Peptidoglycan had no significant stimulatory action on the genes. Flow cytometry showed that the nasal polyp epithelial cell mainly expressed TLR3 in an intracellular compartment, but expression of TLR2 and TLR4 was very low on both the cell surface and in the cell. The immune response of primary nasal polyp epithelial cells induced by TLR3 could not be blocked by anti-TLR3 antibody. Among the TLR ligands evaluated, dsRNA, the ligand for TLR3, mediated the strongest pro-inflammatory effects in primary nasal polyp epithelial cells.     

Toll-like receptors confer responsiveness to lipopolysaccharide from Porphyromonas gingivalis in human gingival fibroblasts

Gingival fibroblasts produce proinflammatory cytokines in response to lipopolysaccharide (LPS) from periodontopathic bacteria. Recently it has become evident that the human homologue of Drosophila Toll can transduce intracellular signaling by LPS stimulation. Toll-like receptors (TLRs) have been identified in myeloid cells; however, their role in nonmyeloid cells such as gingival fibroblasts has not been fully elucidated. Here, we report that human gingival fibroblasts constitutively express TLR2 and TLR4 and that their levels of expression are increased by stimulation with LPS from Porphyromonas gingivalis. Upregulated expression of interleukin-6 gene and protein in fibroblasts stimulated with LPS is inhibited by anti-TLR4 antibody. These findings suggest that TLRs may confer responsiveness to LPS in gingival fibroblasts.     

Toll-like receptors: balancing host resistance with immune tolerance

Recent advances in the field of Toll-like receptors (TLRs) have established their importance for acute innate immune responses and their fundamental requirement for initiation of CD4(+) T-cell responses. Current directions in the field have been towards understanding the role of cytokines secreted by professional antigen-presenting cells in response to TLR ligands and also pathological manifestations in terms of increased production of rheumatoid factors due to TLR ligation on B cells. Thus, it is becoming clear that TLRs are important for induction of adaptive immune responses, but misguided responses can lead to autoimmune pathology.