Mycobacterium tuberculosis RpfE promotes simultaneous Th1‐ and Th17‐type T‐cell immunity via TLR4‐dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4⁺ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4⁺ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4^?/? mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.     

Leishmania expressed lipophosphoglycan interacts with Toll‐like receptor (TLR)‐2 to decrease TLR‐9 expression and reduce anti‐leishmanial responses

Two different Toll‐like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR‐2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR‐9 has been shown to promote a host‐protective response. However, whether there is a relationship between the interaction of LPG and TLR‐2, on one hand, with the effect of TLR‐9, on the other hand, remains unknown. In this study, we report that in‐vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR‐9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti‐LPG as well as anti‐TLR‐2 antibodies prevents this reduction of TLR‐9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR‐9 expression, which is shown to be mediated by transforming growth factor (TGF)‐β and interleukin (IL)‐10. Finally, in‐vitro treatment of macrophages with anti‐LPG and/or anti‐TLR‐2 antibodies before infection reduces the number of amastigotes in macrophages and co‐treatment of mice with anti‐TLR‐2 antibodies and cytosine–phosphate–guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)‐γ‐predominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR‐2 reduce anti‐leishmanial responses via cytokine‐mediated decrease of TLR‐9 expression.     

Toll‐like receptors and CD40 modulate each other's expression affecting Leishmania major infection

Toll‐like receptors (TLRs) recognize pathogen‐associated molecular patterns and results in innate immune system activation that results in elicitation of the adaptive immune response. One crucial modulator of the adaptive immune response is CD40. However, whether these molecules influence each other's expression and functions is not known. Therefore, we examined the effects of TLRs on CD40 expression on macrophages, the host cell for the protozoan parasite L eishmania major. While polyinosinic‐polycytidylic acid [poly (I:C)], a TLR‐3 ligand, lipopolysaccharide (LPS), a TLR‐4 ligand, imiquimod, a TLR‐7/8 ligand and cytosine–phosphate–guanosine (CpG), a TLR‐9 ligand, were shown to enhance CD40 expression, CD40 stimulation enhanced only TLR‐9 expression. Therefore, we tested the synergism between CD40 and CpG in anti‐leishmanial immune response. In L eishmania‐infected macrophages, CpG was found to reduce CD40‐induced extracellular stress‐regulated kinase (ERK)1/2 activation; with the exception of interleukin (IL)‐10, these ligands had differential effects on CD40‐induced IL‐1α, IL‐6 and IL‐12 production. CpG significantly enhanced the anti‐leishmanial function of CD40 with differential effects on IL‐4, IL‐10 and interferon (IFN)‐γ production in susceptible BALB/c mice. Thus, we report the first systematic study on CD40–TLR cross‐talk that regulated the experimental L . major infection.     

The CD20 homolog Ms4a8a integrates pro‐ and anti‐inflammatory signals in novel M2‐like macrophages and is expressed in parasite infection

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor‐associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a⁺ macrophages in M2‐associated infectious pathologies. In late‐stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll‐like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL‐4). LPS/dexamethasone/IL‐4‐induced Ms4a8a⁺ BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF‐κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL‐4‐stimulated Ms4a8a⁺ BMDMs and in peritoneal macrophages from late‐stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2‐like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.     

Characterization of innate immune signalling receptors in virus‐induced acute asthma

Background The role of toll‐like receptors (TLRs) and innate immune activation in clinical asthma exacerbations and their relationship to virus infection are unclear. Objective This study aimed to characterize TLR expression and innate immune activity during virus infection in acute asthma. Methods Subjects with acute asthma, stable asthma and healthy controls were recruited and underwent spirometry and sputum induction with isotonic saline. Selected sputum was dispersed with dithiothreitol and total and differential leucocyte counts were performed. Selected sputum was also used for quantitative real‐time PCR for TLR2, TLR3, TLR4, IL‐10 and IP‐10mRNA expression. Sputum supernatant was used for the measurement of innate immune markers, including IL‐8, matrix metalloproteinase‐9 and neutrophil elastase activity. Viruses were detected using real‐time and gel‐based PCR. Results Sputum TLR2 mRNA expression was up‐regulated in both acute and stable asthma compared with healthy controls and decreased 4–6 weeks after acute exacerbation. Sputum TLR2 mRNA expression was elevated in viral, compared with non‐viral, acute asthma. Sputum TLR3 mRNA expression was similar in controls, stable and acute asthma. However, in acute asthma, subjects with virus‐induced acute asthma had significantly higher sputum TLR3 mRNA expression. Induced sputum gene expression for IP‐10 and IL‐10 were increased in viral, compared with non‐viral, acute asthma. In virus‐induced acute asthma, levels of IP‐10 and IL‐10 mRNA expression were correlated with the mRNA expression of TLR2 and TLR3. Conclusions and Clinical Relevance Virus‐induced acute asthma leads to specific induction of TLR2, TLR3, IP‐10 and IL‐10, suggesting that signalling via TLRs may play an important role in mediating airway inflammation, via both innate and adaptive pathways, in virus‐induced exacerbations. These mediators may provide potential treatment targets for virus‐induced asthma. They may also be useful in diagnosing the nature of acute asthma exacerbations and monitoring treatment responses, which would be useful in the clinical management of asthma exacerbations. Cite this as: L. G. Wood, J. L. Simpson, P. A. B. Wark, H. Powell and P. G. Gibson, Clinical & Experimental Allergy, 2011 (41) 640–648.     

Activation of human basophils by combined toll‐like receptor‐ and FcεRI‐triggering can promote Th2 skewing of naive T helper cells

Basophils are mostly known for their involvement in allergic reactions. Recent studies in mice indicate a role for basophils in the induction of adaptive immunity, especially T helper 2 (Th2) responses. Therefore, it would be highly important to understand how basophils respond to pathogen‐associated molecules, such as ligands for toll‐like receptors (TLRs), and if the basophils could promote Th2 responses via these stimuli. To this end, the activation of basophils via TLRs in combination with activation via IgE was studied, as well as its effect on T helper cell skewing. Using quantitative PCR, we demonstrated the presence of mRNA for TLRs 1–8 in human basophils. Basophils responded to TLR triggering with differential cytokine production, but not with degranulation. Simultaneous triggering of TLRs and IgE led to synergy in production of IL‐4, IL‐8, IL‐13, and RANTES. Furthermore, the synergistic effects on basophils mediated by IgE and TLR‐4 triggering allowed robust Th2 skewing upon activation of naïve human CD4⁺ T cells. Our data show that human basophils respond to TLR ligands in synergy with IgE‐mediated activation and that the cytokines produced can promote Th2 differentiation. These results indicate a role for basophils in the regulation of T‐cell responses in humans.     

M2000 (β‐D‐Mannuronic Acid) as a Novel Antagonist for Blocking the TLR2 and TLR4 Downstream Signalling Pathway

To date, selective blockade of Toll‐like receptor (TLR) signalling has been developed as a new approach for treatment for many inflammatory diseases. As β‐D‐mannuronic acid (M2000) has been known as an anti‐inflammatory molecule in several experimental models, we investigated the antagonistic effects of M2000 on TLR2 and TLR4 downstream signalling transduction pathway in human embryonic kidney (HEK) 293 cell lines overexpressing TLR2/CD14 and the TLR4/MD2/CD14 complex, respectively. M2000 effectively inhibited mRNA expression of MyD88 and p65, major subunit of nuclear factor‐κB, in HEK293 cells stimulated by lipoteichoic acid (LTA, a TLR2 agonist) and lipopolysaccharide (LPS, a TLR4 agonist) with no evidence of cytotoxicity. In addition, M2000 also suppressed LTA and LPS‐induced production of TNF‐α and IL‐6 inflammatory cytokines in these cells. Furthermore, the results revealed that M2000 had no significant effect on Tollip mRNA expression as a negative regulator of TLR signalling in aforesaid cells. Overall, these data point to M2000 inhibitory effect on Toll‐like receptor (TLR) 2, 4 signalling in HEK293 cells. This information might provide new insights into the possible roles of this small drug in order to introduce it as a TLR signalling pathway inhibitor. However, more studies are needed to confirm β‐D‐mannuronic acid antagonistic effects including the effects of M2000 on peritoneal isolated macrophages and also on blood cells in patients with inflammatory diseases such as ankylosing spondylitis.     

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4⁺ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8^hiIFN‐γ^null in freshly isolated to CXCL8^loIFN‐γ^hi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4⁺ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.     

RIP2/RICK‐Dependent Cytokine Production Upon Yersinia enterocolitica Infection in Macrophages with TLR4 Deficiency

Receptor‐interacting protein 2 (RIP2) is a caspase recruitment domain (CARD)‐containing serine/threonine kinase that is activated by NOD1 or NOD2 recognition of their ligands and essential for the activation of NF‐κB and mitogen‐activated protein kinase (MAPK). RIP2 has been known to play an important role in innate immune responses against certain bacterial infection. However, the role and interplay of RIP2 with TLR signalling on cytokine production in macrophages against Yersinia enterocolitica infection remains poorly understood. In the present study, we examined whether RIP2 is essential for Yersinia‐induced production of cytokines in macrophages. Our results showed that naïve RIP2‐deficient macrophages produced similar level of IL‐6, TNF‐α and IL‐10 upon Y. enterocolitica infection compared with wild‐type macrophages. However, the production of IL‐6, TNF‐α and IL‐10 by Y. enterocolitica was impaired in RIP2‐deficient macrophages after lipopolysaccharide (LPS) pretreatment, a TLR4‐tolerant condition. In addition, RIP2 inhibitors, SB203580, PP2, and gefitinib, reduced IL‐6 production in TLR4‐deficient macrophages in response to Y. enterocolitica, whereas they did not affect the cytokines production in WT cells. These results demonstrate that RIP2 may play an important role in proinflammatory cytokine production in macrophages at the absence of TLR signalling.     

TLR2 activation in corneal stromal cells by Staphylococcus aureus‐induced keratitis

The Toll‐Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus‐induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air‐interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub‐groups exposed to UV‐killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL‐8 mRNA expressions were analyzed by quantitative real‐time RT‐PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL‐8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial‐corneal tissue interactions and their inflammatory consequences.     

Activation profile of Toll‐like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes for the production of inflammatory cytokines and autoreactive antibodies. Animal studies of SLE have indicated that Toll‐like receptors (TLR) are important in the pathogenesis of murine lupus. In the present clinical study, differential protein expressions of TLR‐1–9 of monocytes and different lymphocyte subsets from patients with SLE and normal control subjects were determined by flow cytometry. Results showed that the expression of intracellular TLRs (TLR‐3, ‐8, ‐9) and extracellular TLRs (TLR‐1, ‐2, ‐4, ‐5, ‐6) were elevated in monocytes, CD4⁺ T lymphocytes, CD8⁺ T lymphocytes and B lymphocytes of SLE patients compared to control subjects (all P < 0·001). Moreover, cell surface expression of TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes, and TLR‐6 on B lymphocytes, were correlated positively with SLE disease activity index (SLEDAI) (TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes: r = 0·536, P = 0·04; r = 0·713, P = 0·003; TLR‐6 in B lymphocytes: r = 0·572, P = 0·026). In concordance with the above results, there is an observable increased relative induction (%) of inflammatory cytokine interleukin (IL)‐1β, IL‐6, IL‐10 and IL‐12, chemokines CCL2, CXCL8, CCL5 and CXCL10 from peripheral blood mononuclear cells (PBMC) upon differential stimulation by PolyIC (TLR‐3 ligand), lipopolysaccharide (TLR‐4 ligand), peptidoglycan (TLR‐2 ligand), flagellin (TLR‐5 ligand), R837 (TLR‐7 ligand) and CpG DNA (TLR‐9 ligand) in SLE patients compared to controls. These results suggest that the innate immune response for extracellular pathogens and self‐originated DNA plays immunopathological roles via TLR activation in SLE.