Biological importance of the two Toll-like receptors, TLR2 and TLR4, in macrophage response to infection with Candida albicans

The aim of this study was to assess the role of TLR2, TLR4 and MyD88 accessory molecule in the effector and secretory response of macrophages to viable microbial agents. Using TLR-deleted macrophage cell lines generated from the bone marrow of genetically engineered mice (TLR4 gene-deficient, MyD88- and TLR2-knockout mice) and wild-type control mice, we found that TLR2-deleted macrophages exhibit increased ability to contain Candida albicans infection compared to TLR2+/+ counterpart. In contrast, both MyD88-/- and TLR4-/- macrophages retain levels of functional activity comparable to that of the respective wild-type MyD88+/+ and TLR4+/+ controls. The difference in anticandidal effector functions observed between TLR2-/- and TLR2+/+ macrophages is abrogated upon opsonization of the fungal target and interestingly is not observed when using other microbial targets, such as Streptococcus pneumoniae and Helicobacter pylori. When tested for secretory response to C. albicans, TLR2-deleted macrophages show a pattern of cytokine production similar to that of TLR2+/+ controls. Finally, flow cytometry analysis reveals that TLR2-deleted macrophages express only TLR4, while, as expected, TLR2+/+ macrophages are both TLR2 and TLR4 positive; in no cases, modulation of such markers occurs in macrophages exposed to C. albicans infection. In conclusion, these data indicate that TLR2 and TLR4 have different biological relevance, in which TLR2 but not TLR4, is involved in the accomplishment of macrophage-mediated anticandidal activity, while the secretory response to C. albicans appears to be TLR4 but not TLR2-dependent.     

Candida albicans induces selective development of macrophages and monocyte derived dendritic cells by a TLR2 dependent signalling

As TLRs are expressed by haematopoietic stem and progenitor cells (HSPCs), these receptors may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that in in vitro defined conditions inactivated yeasts and hyphae of Candida albicans induce HSPCs proliferation and differentiation towards the myeloid lineage by a TLR2/MyD88 dependent pathway. In this work, we showed that C. albicans invasive infection with a low virulence strain results in a rapid expansion of HSPCs (identified as LKS cells: Lin(-) c-Kit(+) Sca-1(+) IL-7Rα(-)), that reach the maximum at day 3 post-infection. This in vivo expansion of LKS cells in TLR2(-/-) mice was delayed until day 7 post- infection. Candidiasis was, as expected, accompanied by an increase in granulopoiesis and decreased lymphopoiesis in the bone marrow. These changes were more pronounced in TLR2(-/-) mice correlating with their higher fungal burden. Accordingly, emigration of Ly6C(high) monocytes and neutrophils to spleen was increased in TLR2(-/-) mice, although the increase in macrophages and inflammatory macrophages was completely dependent on TLR2. Similarly, we detected for the first time, in the spleen of C. albicans infected control mice, a newly generated population of dendritic cells that have the phenotype of monocyte derived dendritic cells (moDCs) that were not generated in TLR2(-/-) infected mice. In addition, C. albicans signalling through TLR2/MyD88 and Dectin-1 promotes in vitro the differentiation of Lin(-) cells towards moDCs that secrete TNF-α and are able to kill the microorganism. Therefore, our results indicate that during infection C. albicans can directly stimulate progenitor cells through TLR2 and Dectin-1 to generate newly formed inflammatory macrophages and moDCs that may fulfill an essential role in defense mechanisms against the pathogen.     

Masking of typical TLR4 and TLR5 ligands modulates inflammation and resolution by Helicobacter pylori

Helicobacter pylori is a paradigm of chronic bacterial infection and is associated with peptic ulceration and malignancies. H. pylori uses specific masking mechanisms to avoid canonical ligands from activating Toll-like receptors (TLRs), such as lipopolysaccharide (LPS) modification and specific flagellin sequences that are not detected by TLR4 and TLR5, respectively. Thus, it was believed for a long time that H. pylori evades TLR recognition as a crucial strategy for immune escape and bacterial persistence. However, recent data indicate that multiple TLRs are activated by H. pylori and play a role in the pathology. Remarkably, H. pylori LPS, modified through changes in acylation and phosphorylation, is mainly sensed by other TLRs (TLR2 and TLR10) and induces both pro- and anti-inflammatory responses. In addition, two structural components of the cag pathogenicity island-encoded type IV secretion system (T4SS), CagL and CagY, were shown to contain TLR5-activating domains. These domains stimulate TLR5 and enhance immunity, while LPS-driven TLR10 signaling predominantly activates anti-inflammatory reactions. Here, we discuss the specific roles of these TLRs and masking mechanisms during infection. Masking of typical TLR ligands combined with evolutionary shifting to other TLRs is unique for H. pylori and has not yet been described for any other species in the bacterial kingdom. Finally, we highlight the unmasked T4SS-driven activation of TLR9 by H. pylori, which mainly triggers anti-inflammatory responses.     

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.     

NK cell tolerance to TLR agonists mediated by regulatory T cells after polymicrobial sepsis

As sensors of infection, innate immune cells are able to recognize pathogen-associated molecular patterns by receptors such as TLRs. NK cells present in many tissues contribute to inflammatory processes, particularly through the production of IFN-γ. They may display a protective role during infection but also a detrimental role during sterile or infectious systemic inflammatory response syndrome. Nevertheless, the exact status of NK cells during bacterial sepsis and their capacity directly to respond to TLR agonists remain unclear. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors. The aim of this study was to gain insight into TLR2/TLR4/TLR9 expression on/in murine NK cells at the protein level and determine if their agonists were able to induce cytokine production. We show, by flow cytometry, a strong intracellular expression of TLR2 and a low of TLR4 in freshly isolated murine spleen NK cells, similar to that of TLR9. In vitro, purified NK cells respond to TLR2, TLR4, and TLR9 agonists, in synergy with activating cytokines (IL-2, IL-15, and/or IL-18), and produce proinflammatory cytokines (IFN-γ and GM-CSF). Finally, we explored the possible tolerance of NK cells to TLR agonists after a polymicrobial sepsis (experimental peritonitis). For the first time, to our knowledge, NK cells are shown to become tolerant in terms of proinflammatory cytokines production after sepsis. We show that this tolerance is associated with a reduction of the CD27(+)CD11b(-) subset in the spleen related to the presence of regulatory T cells and mainly mediated by TGF-β.     

TLR9 contributes to antiviral immunity during gammaherpesvirus infection

The human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and EBV cause important infections. As pathogenetic studies of the human infections are restricted, murine gammaherpesvirus 68 serves as a model to study gammaherpesvirus pathogenesis. TLRs are a conserved family of receptors detecting microbial molecular patterns. Among the TLRs, TLR9 recognizes unmethylated CpG DNA motifs present in bacterial and viral DNA. The aim of this study was to assess the role of TLR9 in gammaherpesvirus pathogenesis. Upon stimulation with murine gammaherpesvirus 68, Flt3L-cultured bone marrow cells (dendritic cells) from TLR9-/- mice secreted reduced levels of IL-12, IFN-alpha, and IL-6, when compared with dendritic cells from wild-type mice. Intranasal infection of TLR9-/- and wild-type mice did not reveal any differences during lytic and latent infection. In contrast, when infected i.p., TLR9-/- mice showed markedly higher viral loads both during lytic and latent infection. Thus, we show for the first time that TLR9 is involved in gammaherpesvirus pathogenesis and contributes to organ-specific immunity.     

Divergent Toll-like receptors in catfish (Ictalurus punctatus): TLR5S, TLR20, TLR21

Toll-like receptors (TLR) mediate pathogen recognition in vertebrate species through detection of conserved microbial ligands. Families of TLR molecules have been described from the genomes of the teleost fish model species zebrafish and Takifugu, but much research remains to characterize the full length sequences and pathogen specificities of individual TLR members in fish. While the majority of these pathogen receptors are conserved among vertebrate species with clear orthologues present in fish for most mammalian TLRs, several interesting differences are present in the TLR repertoire of teleost fish when compared to that of mammals. A soluble form of TLR5 has been reported from salmonid fish and Takifugu rubripes which is not present in mammals, and a large group of TLRs (arbitrarily numbered 19-23) was identified from teleost genomes with no easily discernible orthologues in mammals. To better understand these teleost adaptations to the TLR family, we have isolated, sequenced, and characterized the full-length cDNA and gene sequences of TLR5S, TLR20, and TLR21 from catfish as well as studied their expression pattern in tissues. We also mapped these genes to bacterial artificial chromosome (BAC) clones for genome analysis. While TLR5S appeared to be common in teleost fish, and TLR21 is common to birds, amphibians and fish, TLR20 has only been identified in zebrafish and catfish. Phylogenetic analysis of catfish TLR20 indicated that it is closely related to murine TLR11 and TLR12, two divergent TLRs about which little is known. All three genes appear to exist in catfish as single copy genes.     

Expression of TLR2 and TLR4 in murine small intestine during postnatal development

The important role played by the gut microbiota in host immunity is mediated, in part, through toll-like receptors (TLRs). We evaluated the postnatal changes in expression of TLR2 and TLR4 in the murine small intestine and assessed how expression is influenced by gut microbiota. The expression of TLR2 and TLR4 in the murine small intestine was highly dynamic during development. The changes were especially profound during the suckling period, with the maximal mRNA levels detected in the mid-suckling period. Immunohistochemical and flow-cytometric analyses indicated that the changes in TLR2 and TLR4 expression involve primarily epithelial cells. The germ-free mice showed minor changes in TLR2/TLR4 mRNA and TLR2 protein during the suckling period. This study demonstrated that the postnatal expression of TLR2 and TLR4 in small intestinal epithelial cells is dynamic and depends on the presence of commensal intestinal microbiota.     

Signalling through TLR2/MyD88 induces differentiation of murine bone marrow stem and progenitor cells to functional phagocytes in response to Candida albicans

We have previously demonstrated that inactivated yeasts and hyphae of Candida albicans induce in vitro the proliferation of murine haematopoietic stem and progenitor cells (HSPCs, sorted as LKS cells: Lin^- c-Kit⁺ Sca-1⁺) as well as their differentiation to lineage-positive cells, through a MyD88-dependent pathway. In this work, we have found that this process is mainly mediated by TLR2, and that expanding cells express myeloid and not lymphoid markers. Incubation of long-term repopulating HSCs (Lin^- CD105⁺ and Sca-1⁺) with C. albicans yeasts resulted in their proliferation and up regulation of the common myeloid progenitors (CMPs) markers, CD34 and FcγRII/III, by a TLR2/MyD88-dependent signalling pathway. In addition, this TLR2/MyD88 signalling promotes the differentiation of CMPs and granulocyte and macrophage progenitors (GMPs) into cells with the morphology of macrophages and neutrophils, characterized by an increase in the expression of CD11b, F4/80 and Ly6G, independently of the presence of growth and differentiation factors. These differentiated cells were able to phagocytose C. albicans yeasts and to produce proinflammatory cytokines. In conclusion, C. albicans may be sensed by TLRs on haematopoietic stem and progenitor cells to promote the host capability for rapidly replenishing myeloid cells that constitute the first line of defence against C. albicans .     

Both viable and killed Candida albicans cells induce in vitro production of TNF-alpha and IFN-gamma in murine cells through a TLR2-dependent signalling

The in vitro production of TNF-alpha and IFN-gamma in response to Candida albicans was investigated in wild type, TLR2-/- and TLR4-/- murine cells. TLR2-/- resident peritoneal macrophages showed a strong impairment of TNF-alpha production in response to viable and non-viable (heat-killed, antimycotic-treated and formaldehyde-fixed) yeasts and hyphae (germ tube-bearing cells) of the high virulence C. albicans ATCC 26555 strain, as compared with macrophages from wild-type and TLR4-/- mice. The in vitro production of IFN-gamma was investigated in murine splenocytes obtained three days after intravenous injection with the low virulence, non-germinative C. albicans PCA2 strain, and again, TLR2-/- splenocytes showed a strong impairment of the in vitro production of IFN-gamma in response to non-viable (heat-killed, antimycotic-treated and formaldehyde-fixed) C. albicans ATCC 26555 yeasts, as compared with splenocytes of TLR4-/- and wild type mice. These results indicate that the TLR2-mediated recognition of C. albicans leading to a proinflammatory Th1 host response appears to be well conserved in killed C. albicans cells, regardless of the inactivating treatment employed.     

TLR1/TLR2 heterodimers play an important role in the recognition of Borrelia spirochetes

After infection with Borrelia species, the risk for developing Lyme disease varies significantly between individuals. Recognition of Borrelia by the immune system is mediated by pattern recognition receptors (PRRs), such as TLRs. While TLR2 is the main recognition receptor for Borrelia spp., little is known about the role of TLR1 and TLR6, which both can form functionally active heterodimers with TLR2. Here we investigated the recognition of Borrelia by both murine and human TLR1 and TLR6. Peritoneal macrophages from TLR1- and TLR6- gene deficient mice were isolated and exposed to Borrelia. Human PBMCs were stimulated with Borrelia with or without specific TLR1 and TLR6 blocking using specific antibodies. Finally, the functional consequences of TLR polymorphisms on Borrelia-induced cytokine production were assessed. Splenocytes isolated from both TLR1-/- and TLR6-/- mice displayed a distorted Th1/Th2 cytokine balance after stimulation with B.burgdorferi, while no differences in pro-inflammatory cytokine production were observed. In contrast, blockade of TLR1 with specific neutralizing antibodies led to decreased cytokine production by human PBMCs after exposure to B.burgdorferi. Blockade of human TLR6 did not lead to suppression of cytokine production. When PBMCs from healthy individuals bearing polymorphisms in TLR1 were exposed to B.burgdorferi, a remarkably decreased in vitro cytokine production was observed in comparison to wild-type controls. TLR6 polymorphisms lead to a minor modified cytokine production. This study indicates a dominant role for TLR1/TLR2 heterodimers in the induction of the early inflammatory response by Borrelia spirochetes in humans.     

Role of TLR1 and TLR6 in the host defense against disseminated candidiasis

Toll-like receptor-1 (TLR1) and TLR6 are receptors of the TLR family that form heterodimers with TLR2. The role of TLR1 and TLR6 for the recognition of the fungal pathogen Candida albicans was investigated. TLR1 is not involved in the recognition of C. albicans, and TLR1 knock-out (TLR1-/-) mice showed a normal susceptibility to disseminated candidiasis. In contrast, recognition of C. albicans by TLR6 modulated the balance between Th1 and Th2 cytokines, and TLR6 knock-out mice displayed a defective production of IL-10 and an increased IFN-gamma release. Production of the monocyte-derived cytokines tumor necrosis factor, IL-1, and IL-6 was normal in TLR6-/- mice, and this was accompanied by a normal susceptibility to disseminated candidiasis. In conclusion, TLR6 is involved in the recognition of C. albicans and modulates the Th1/Th2 cytokine balance, but this results in a mild phenotype with a normal susceptibility of TLR6-/- mice to Candida infection.     

Toll-like receptor (TLR)2 and TLR3 sensing is required for dendritic cell activation, but dispensable to control Schistosoma mansoni infection and pathology

Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs) and in the induction of immune responses. However, relatively little is known about their functions in innate/acquired responses to complex eukaryotic microorganisms, including helminth parasites. That Schistosoma mansoni eggs activate myeloid DCs through TLR2 and TLR3 has been shown by us and others, but the consequences of this combined activation are still unknown. We show that the engagement of both TLR2 and TLR3 by schistosome eggs is important for the production of inflammatory cytokines and interferon-stimulated genes, such as some chemokines, by DCs. Strikingly, DCs sensitized with ovalbumin in the presence of parasite eggs dramatically reduce the release of Th2-type cytokines by ovalbumin-specific T lymphocytes, an effect that fully depends on TLR3. Finally, although TLR2 and TLR3 have no role in host resistance and in egg-induced granuloma formation in S. mansoni-infected mice, they individually and additionally increase the Th1/Th2 balance of the immune response. Thus, TLR2 and TLR3 sensing is required to shape the immune response during murine schistosomiasis, but is dispensable to control infection and pathology.     

Toll-like receptors and innate antifungal responses

The mammalian Toll-like receptors (TLRs) are homologues of Drosophila Toll and constitute a novel protein family involved in the mediation of innate immunity and the activation of adaptive immunity. Analysis of infection with human pathogenic fungi Candida albicans and Aspergillus fumigatus implicated TLR2 and TLR4 in elicitation of immune responses. Cryptococcus neoformans is recognized by a process that uses TLR4. C. albicans induces immunostimulation through causative agents, such as mannan or its structural derivatives (e.g. phospholipomannan), which are recognized by the immune system as pathogen-associated molecular patterns and are located in the cell wall of fungi. Secreted aspartic proteinases represent a key virulence factor that contributes to the ability of C. albicans to cause mucosal and disseminated infections, and might be a further potential stimulator of TLRs. Simultaneous activation of other pattern recognition receptors collaborating with TLRs illustrates the cooperation of various chains within ligand-specific receptor complexes for the recognition of fungal pathogens and their cell wall components.     

Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection

The roles of Toll-like receptor (TLR) 2 and TLR4 in the host inflammatory response to infection caused by Chlamydia trachomatis have not been elucidated. We examined production of TNF-alpha and IL-6 in wild-type TLR2 knockout (KO), and TLR4 KO murine peritoneal macrophages infected with the mouse pneumonitis strain of C. trachomatis. Furthermore, we compared the outcomes of genital tract infection in control, TLR2 KO, and TLR4 KO mice. Macrophages lacking TLR2 produced significantly less TNF-alpha and IL6 in response to active infection. In contrast, macrophages from TLR4 KO mice consistently produced higher TNF-alpha and IL-6 responses than those from normal mice on in vitro infection. Infected TLR2-deficient fibroblasts had less mRNA for IL-1, IL-6, and macrophage-inflammatory protein-2, but TLR4-deficient cells had increased mRNA levels for these cytokines compared with controls, suggesting that ligation of TLR4 by whole chlamydiae may down-modulate signaling by other TLRs. In TLR2 KO mice, although the course of genital tract infection was not different from that of controls, significantly lower levels of TNF-alpha and macrophage-inflammatory protein-2 were detected in genital tract secretions during the first week of infection, and there was a significant reduction in oviduct and mesosalpinx pathology at late time points. TLR4 KO mice responded to in vivo infection similarly to wild-type controls and developed similar pathology. TLR2 is an important mediator in the innate immune response to C. trachomatis infection and appears to play a role in both early production of inflammatory mediators and development of chronic inflammatory pathology.     

Differential Toll-Like Receptor (TLR) mRNA Expression Patterns during Chicken Embryological Development

Toll-like receptors (TLRs), important components of innate immune response, play a pivotal role in early recognition of pathogen as well as in the initiation of robust and specific adaptive immune response. In the present study, the expression profile of chicken TLRs (TLR2A, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21) in various chicken embryonic tissues during embryo development was examined by real-time PCR assay. All the TLR mRNAs were expressed in whole embryonic tissue as early as 3rd embryonic day (ED). Four of the seven TLRs (TLR2, TLR3, TLR4, and TLR7) mRNA expressions were significantly (P < 0.01) higher at 12ED relative to expression at 3 ED, whereas TLR15 mRNA expression was significantly (P < 0.01) higher on 7ED and TLR5 and 21 were highly expressed on 18 ED. Among all the TLRs investigated TLR4 mRNA was the highest expressed and TLR15 mRNA expression was the lowest in all tissues during chicken embryo development. Tissue wise analysis of mRNA expression of TLRs showed that liver expressed significantly (P < 0.01) higher levels of most of the genes (TLR2, TLR4, and TLR21). However no significant difference was found in TLR15 mRNA expression among the tissues during development. Our results suggest the innate preparedness of chicken embryos and also a possible role for TLRs in the regulation of chicken embryo development that needs to be further evaluated.     

Aspergillus fumigatus induces innate immune responses in alveolar macrophages through the MAPK pathway independently of TLR2 and TLR4

Aspergillus fumigatus causes invasive aspergillosis in immunosuppressed patients. In the immunocompetent host, inhaled conidia are cleared by alveolar macrophages. The signaling pathways of the alveolar macrophage involved in the clearance of A. fumigatus are poorly understood. Therefore, we investigated the role of TLRs in the immune response against A. fumigatus and their contribution to the signaling events triggered in murine alveolar macrophages upon infection with A. fumigatus conidia. Specifically, we examined the MAPKs and NF-kappaB activation and cytokine signaling. Our investigations revealed that immunocompetent TLR2, TLR4, and MyD88 knockout mice were not more susceptible to invasive aspergillosis as compared with wild-type mice and that the in vitro phosphorylation of the MAPKs ERK and p38 was not affected in TLR2, TLR4, or MyD88 knockout mice following stimulation with conidia. In vivo experiments suggest that ERK was an essential MAPK in the defense against A. fumigatus, whereas the activation of NF-kappaB appeared to play only a secondary role. In conclusion, our findings demonstrate that TLR2/4 recognition and MyD88 signaling are dispensable for the clearance of A. fumigatus under immunocompetent situations. Furthermore, our data stress the important role of ERK activation in innate immunity to A. fumigatus.