MyD88 is essential for clearance of Leishmania major: possible role for lipophosphoglycan and Toll-like receptor 2 signaling

Leishmania major is an obligate intracellular eukaryotic pathogen of mononuclear phagocytes. Invasive promastigotes gain entry into target cells by receptor-mediated phagocytosis, transform into non-motile amastigotes and establish in the phagolysosome. Glycosylphosphatidylinositol-anchored lipophosphoglycan (LPG) is a virulence factor and a major parasite molecule involved in this process. We observed that mice lacking the Toll-like receptor (TLR) pathway adaptor protein MyD88 were more susceptible to infection with L. major than wild-type C57BL/6 mice, demonstrating a central role for this innate immune recognition pathway in control of infection, and suggesting that L. major possesses a ligand for TLR. We sought to identify parasite molecules capable of activating the protective Toll pathway, and found that purified Leishmania LPG, but not other surface glycolipids, activate innate immune signaling pathways via TLR2. Activation of cytokine synthesis by LPG required the presence of the lipid anchor and a functional MyD88 adaptor protein. LPG also induced the expression of negative regulatory pathways mediated by members of thesuppressors of cytokine signaling family SOCS-1 and SOCS-3. Thus, the Toll pathway is required for resistance to L. major and LPG is a defined TLR agonist from this important human pathogen.     

Functional aspects of Toll‐like receptor/MyD88 signalling during protozoan infection: focus on Toxoplasma gondii

Toll‐like receptor (TLR)/MyD88 signalling has emerged as a major pathway of pathogen recognition in the innate immune system. Here, we review recent data that begin to show how this pathway controls the immune response to protozoan infection, with particular emphasis on the opportunistic pathogen Toxoplasma gondii. The various ways that the parasite activates and suppresses TLR/MyD88 signalling defines several key principals that illuminate the complexities of the host–pathogen interaction. We also speculate how TLR/MyD88 signalling might be exploited to provide protection against Toxoplasma, as well as other protozoa and infection in general.     

The early monocytic response to cytomegalovirus infection is MyD88 dependent but occurs independently of common inflammatory cytokine signals

Cytomegalovirus latently infects myeloid cells; however, the acute effects of the virus on this cell subset are poorly characterised. We demonstrate that systemic cytomegalovirus infection induced rapid activation of monocytes in the bone marrow, characterised by upregulation of CD69, CD11c, Ly6C and M‐CSF receptor. Activated bone marrow monocytes were more sensitive to M‐CSF and less sensitive to granulocyte‐monocyte colony stimulating factor in vitro, resulting in the generation of more macrophages and fewer dendritic cells, respectively. Monocyte activation was also observed in the periphery and resulted in significant accumulation of monocytes in the spleen. MyD88 expression was required within the haematopoietic compartment to initiate monocyte activation and recruitment. However, monocytes lacking MyD88 were activated and recruited in the presence of MyD88‐sufficient cells in mixed bone marrow chimeras, indicating that once initiated, the process was MyD88 independent. Interestingly, we found that monocyte activation occurred in the absence of the common inflammatory cytokines, namely type I interferons (IFNs), IL‐6, TNF‐α and IL‐1 as well as the NLRP3 inflammasome adaptor protein, ASC. We also excluded a role for the chemokine‐like protein MCK‐2 (m131/129) expressed by murine CMV. Taken together, these results challenge the notion that a single inflammatory cytokine mediates activation and recruitment of monocytes in response to infection.     

MyD88 is critical for the development of innate and adaptive immunity during acute lymphocytic choriomeningitis virus infection

We investigated the roles of Toll-like receptor 2 (TLR2) and myeloid differentiation factor 88 (MyD88) in the course of a lymphocytic choriomeningitis virus (LCMV) infection and revealed the following: (i) studies of transfected cells and murine peritoneal macrophages demonstrated that TLR2 and MyD88 are essential for the initial pro-inflammatory cytokine response (human IL-8, mouse IL-6) to LCMV; (ii) TLR2 knockout (KO) mice and MyD88 KO mice challenged with LCMV produced less IL-6 and monocyte chemotactic protein-1 in the serum than wild-type mice; (iii) in contrast to inflammatory cytokines, the production of type 1 IFN (IFN-alpha) in response to LCMV was MyD88 independent; (iv) MyD88 plays an essential role in antiviral CD8(+) T cell responses, CD8(+) T cells in MyD88 KO mice were defective in their expression of intracellular antiviral cytokines; and (v) the failure of MyD88 KO mice to activate CD8(+) T cells was accompanied by persistent viral infection in MyD88 KO mice. We demonstrate that TLR-mediated responses are important in the innate immune response to LCMV and that MyD88 is essential for the control of the LCMV infection and the maturation/activation of virus-specific CD8(+) T cells.     

Simultaneous deletion of MyD88 and Trif delays major histocompatibility and minor antigen mismatch allograft rejection

This study investigated whether ablation of all Toll-like receptor (TLR) signaling influenced skin allograft rejection across a complete donor/recipient mismatch of major histocompatibility and minor antigens. We predicted that defective TLR signaling would interfere with the activation of donor dendritic cells (DC) in vivo, by preventing DC activation in response to local environmental ("danger") signals. The ablation of TLR signals would therefore be associated with decreased activation of host T cells and decreased graft rejection. Using mice with deletions of the proximal TLR adapter proteins MyD88 or Trif, and those with simultaneous deletions of both MyD88 and Trif, we demonstrated that absence of both MyD88 and Trif adapter proteins prolonged skin graft survival, notably across a complete MHC and minor antigen barrier. Absence of MyD88 or Trif alone only had a modest effect on graft survival across even a minor MHC antigen difference. Prolonged survival of skin grafts from mice deficient in both MyD88 and Trif was associated with diminished migration of donor cells to draining lymph nodes and, subsequently, with delayed infiltration of host T cells into the grafted tissue.     

The effects of MyD88 deficiency on disease phenotype in dysferlin‐deficient A/J mice: role of endogenous TLR ligands

An absence of dysferlin leads to activation of innate immune receptors such as Toll‐like receptors (TLRs) and skeletal muscle inflammation. Myeloid differentiation primary response gene 88 (MyD88) is a key mediator of TLR‐dependent innate immune signalling. We hypothesized that endogenous TLR ligands released from the leaking dysferlin‐deficient muscle fibres engage TLRs on muscle and immune cells and contribute to disease progression. To test this hypothesis, we generated and characterized dysferlin and MyD88 double‐deficient mice. Double‐deficient mice exhibited improved body weight, grip strength, and maximum muscle contractile force at 6–8 months of age when compared to MyD88‐sufficient, dysferlin‐deficient A/J mice. Double‐deficient mice also showed a decrease in total fibre number, which contributed to the observed increase in the number of central nuclei/fibres. These results indicate that there was less regeneration in the double‐deficient mice. We next tested the hypothesis that endogenous ligands, such as single‐stranded ribonucleic acids (ssRNAs), released from damaged muscle cells bind to TLR‐7/8 and perpetuate the disease progression. We found that injection of ssRNA into the skeletal muscle of pre‐symptomatic mice (2 months old) resulted in a significant increase in degenerative fibres, inflammation, and regenerating fibres in A/J mice. In contrast, characteristic histological features were significantly decreased in double‐deficient mice. These data point to a clear role for the TLR pathway in the pathogenesis of dysferlin deficiency and suggest that TLR‐7/8 antagonists may have therapeutic value in this disease. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

MyD88 and TLR2, but not TLR4, are required for host defense against Cryptococcus neoformans

We investigated here the potential role of Toll-like receptors (TLR) and the adaptor protein MyD88 in innate immunity responses to Cryptococcus neoformans, a pathogenic encapsulated yeast. Peritoneal macrophages from MyD88(-/-) or TLR2(-/-) mice released significantly less TNF-alpha, compared with wild-type controls, after in vitro stimulation with whole yeasts. In contrast, no differences in TNF-alpha release were noted between macrophages from C3H/HeJ mice, which have a loss of function mutation in TLR4, relative to C3H/HeN controls. When MyD88- or TLR2-deficient mice were infected with low doses of the H99 serotype A strain, all of the control animals, but none of MyD88(-/-) and only 38% of the TLR2(-/-) animals survived, in association with higher fungal burden in the mutant mice. Both MyD88(-/-) and TLR2(-/-) animals showed decreased TNF-alpha, IL-12p40 and/or IFN-gamma expression in various organs during infection. No difference in susceptibility to experimental cryptococcosis was found between C3H/HeJ mice and C3H/HeN controls. In conclusion, our data indicate that TLR2 and MyD88, but not TLR4, critically contribute to anti-cryptococcal defenses through the induction of increased TNF-alpha, IL-12 and IFN-gamma expression.     

Experimental and natural infections in MyD88‐ and IRAK‐4‐deficient mice and humans

Most Toll‐like‐receptors (TLRs) and interleukin‐1 receptors (IL‐1Rs) signal via myeloid differentiation primary response 88 (MyD88) and interleukin‐1 receptor‐associated kinase 4 (IRAK‐4). The combined roles of these two receptor families in the course of experimental infections have been assessed in MyD88‐ and IRAK‐4‐deficient mice for almost fifteen years. These animals have been shown to be susceptible to 46 pathogens: 27 bacteria, eight viruses, seven parasites, and four fungi. Humans with inborn MyD88 or IRAK‐4 deficiency were first identified in 2003. They suffer from naturally occurring life‐threatening infections caused by a small number of bacterial species, although the incidence and severity of these infections decrease with age. Mouse TLR‐ and IL‐1R‐dependent immunity mediated by MyD88 and IRAK‐4 seems to be vital to combat a wide array of experimentally administered pathogens at most ages. By contrast, human TLR‐ and IL‐1R‐dependent immunity mediated by MyD88 and IRAK‐4 seems to be effective in the natural setting against only a few bacteria and is most important in infancy and early childhood. The roles of TLRs and IL‐1Rs in protective immunity deduced from studies in mutant mice subjected to experimental infections should therefore be reconsidered in the light of findings for natural infections in humans carrying mutations as discussed in this review.     

Stimulation of toll-like receptor 2 in mononuclear cells from HIV-infected patients induces chemokine responses: possible pathogenic consequences

Toll-like receptor 2 (TLR2) stimulation in monocytes may contribute to enhanced inflammation and viral replication in HIV infection. In the present study we examined if TLR2 stimulation could modulate chemokine responses in peripheral blood mononuclear cells (PBMC) from HIV-infected patients and healthy controls. Our main findings were, with similar qualitative patterns in both healthy controls and HIV-infected patients: (1) TLR2 stimulation induced up-regulation of several chemokines at the mRNA level as well as increased protein levels of macrophage inflammatory protein (MIP)-1alpha, interleukin (IL)-8 and regulated on activation, normal T cell expressed and secreted (RANTES); (2) TLR2 stimulation induced enhanced protein expression of CCR5 (a receptor for MIP-1alpha and RANTES) on monocytes; (3) In vitro stimulation with RANTES induced release of MIP-1alpha, MCP-1, IL-8 and interferon-gamma from PBMC. While increased levels of beta-chemokines possibly have antiviral effects, TLR2 stimulation may also promote a chemokine-driven inflammatory loop, potentially contributing to the immunopathogenesis of HIV infection.     

Translational mini-review series on Toll-like receptors: recent advances in understanding the role of Toll-like receptors in anti-viral immunity

(TLRs) respond to pathogens to initiate the innate immune response and direct adaptive immunity, and evidence to date suggests that they have a role in the detection of viruses. Many viral macromolecules have been shown to activate anti-viral signalling pathways via TLRs, leading to the induction of cytokines and interferons, while viruses also have means of not only evading detection by TLRs, but also of subverting these receptors for their own purposes. This review discusses the role of TLRs in the context of other known viral detection systems, and examines some of the often surprising results from studies using mice deficient in TLRs and their adaptors, in an attempt to unravel the particular contribution of TLRs to anti-viral immunity.     

Mechanisms of chemical-induced innate immunity in allergic contact dermatitis

Allergic contact dermatitis (ACD) is one of the most prevalent occupational skin diseases and causes severe and long-lasting health problems in the case of chronification. It is initiated by an innate inflammatory immune response to skin contact with low molecular weight chemicals that results in the priming of chemical-specific, skin-homing CD8(+) Tc1/Tc17 and CD4(+) Th1/Th17 cells. Following this sensitization step, T lymphocytes infiltrate the inflamed skin upon challenge with the same chemical. The T cells then exert cytotoxic function and secrete inflammatory mediators to produce an eczematous skin reaction. The recent characterization of the mechanisms underlying the innate inflammatory response has revealed that contact allergens activate innate effector mechanisms and signalling pathways that are also involved in anti-infectious immunity. This emerging analogy implies infection as a potential trigger or amplifier of the sensitization to contact allergens. Moreover, new mechanistic insights into the induction of ACD identify potential targets for preventive and therapeutic intervention. We summarize here the latest findings in this area of research.     

Regulation of cytokine responses by seasonality of vitamin D status in healthy individuals

Phage display technology has been utilized to select target molecules against circulating antibodies. The aims of this study were to isolate a peptide that binds with serum from Crohn's disease (CD) patients and to examine its diagnostic and pathogenic significance. A phage display library was constructed using cDNA from Caco-2 cells. Affinity selection using this cDNA library and serum samples from patients with CD was then performed. Phage clones that specifically reacted with the CD sera were then selected using a phage enzyme-linked immunosorbent assay (ELISA). After the DNA sequences of the selected phages were determined and converted to amino acid sequences, the synthesized peptides were examined using an ELISA. The effect of the synthesized peptides on cytokine release from cultured blood mononuclear cells was investigated. An ELISA analysis for TCP-353 demonstrated that while 61·7% of the samples from CD patients were seroreactive, seroreactivity was less common among patients with ulcerative colitis (7·3%), acute colitis (0%) or colon cancer (11·4%) and among normal subjects (2·8%). The induction of interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α release, but not IL-10 release, in response to TCP-353 peptide was enhanced in CD mononuclear cells only. We isolated a novel peptide that specifically binds to CD sera and stimulates the proinflammatory responses of CD mononuclear cells. TCP-353 may have diagnostic, pathogenic and therapeutic significance with regard to the treatment of CD.     

Enhanced natural killer activity and production of pro-inflammatory cytokines in mice selected for high acute inflammatory response (AIRmax)

Strains of mice with maximal and minimal acute inflammatory responsiveness (AIRmax and AIRmin, respectively) were developed through selective breeding based on their high- or low-acute inflammatory responsiveness. Previous reports have shown that AIRmax mice are more resistant to the development of a variety of tumours than AIRmin mice, including spontaneous metastasis of murine melanoma. Natural killer activity is involved in immunosurveillance against tumour development, so we analysed the number and activity of natural killer cells (CD49b(+)), T-lymphocyte subsets and in vitro cytokine production by spleen cells of normal AIRmax and AIRmin mice. Analysis of lymphocyte subsets by flow cytometry showed that AIRmax mice had a higher relative number of CD49b(+) cells than AIRmin mice, as well as cytolytic activity against Yac.1 target cells. The number of CD3(+) CD8(+) cells was also higher in AIRmax mice. These findings were associated with the ability of spleen cells from AIRmax mice in vitro to produce higher levels of the pro-inflammatory cytokines tumour necrosis factor-alpha, interleukin-12p40 and interferon-gamma but not the anti-inflammatory interleukin-10. Taken together, our data suggest that the selective breeding to achieve the AIRmax and AIRmin strains was able to polarize the genes associated with cytotoxic activity, which can be responsible for the antitumour resistance observed in AIRmax mice.     

Ageing and Toll-like receptor expression by innate immune cells in chronic human schistosomiasis

There has been no systematic study of the immune response of individuals aged over 60 years living in Schistosomiasis mansoni-endemic areas, although senescence is reportedly associated with susceptibility to infection and progressive decline in immune function. We have shown previously, in two endemic areas in Minas Gerais, Brazil, that the frequency of individuals aged over 60 years with chronic schistosomiasis is no longer negligible. Moreover, several elderly individuals who have always lived in these endemic areas stay protected from infection. An important question for studies of ageing and disease control in developing countries is which differences in the immunological profile of these negatively tested (non-infected) individuals can account for their resistance to either infection or reinfection. We show, in the present study, that non-infected (negative) elderly individuals develop innate immune mechanisms of protection that replace the age-associated decline in T cell function. Non-infected elderly individuals from endemic areas of schistosome infection present an increase in the frequency of the natural killer (NK) CD56(low) subset of NK cells expressing Toll-like receptors (TLR)-1, -2, -3 and -4 as determined by flow cytometry analysis. In addition, the proportion of dendritic cells expressing TLR-1 is elevated as well as the frequency of monocytes expressing TLR-1 and -4. These results suggest that TLR expression by cells of the innate immune system may be related to the negative status of infection in some elderly individuals who are constantly exposed to S. mansoni. Developing mechanisms of protection from infection may represent a biomarker for healthy ageing in this population.     

CD300a and CD300f differentially regulate the MyD88 and TRIF-mediated TLR signalling pathways through activation of SHP-1 and/or SHP-2 in human monocytic cell lines

CD300a, a membrane protein expressed on myeloid lineages and specific subsets of CD4(+) T cells, has been reported to have inhibitory activities in cellular activation. However, the role of CD300a in Toll-like receptor (TLR) -mediated macrophage activation has not been investigated. The human monocytic cell lines THP-1 and U937 were stimulated with various TLR ligands after triggering of CD300a with specific monoclonal antibody. Interestingly, CD300a blocked TLR4-mediated and TLR9-mediated expression of pro-inflammatory mediators without affecting TLR3-mediated events. In contrast, CD300f, another member of the CD300 family, blocked the activation of cells induced by all TLR ligands. A transient transfection assay using luciferase reporter gene under the regulation of nuclear factor-κB binding sites indicated that co-transfection of CD300f blocked reporter expression induced by over-expression of both myeloid differentiation factor 88 (MyD88) and toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF), whereas CD300a blocked only MyD88-induced events. Synthetic peptides representing immunoreceptor tyrosine-based inhibitory motifs of CD300a or CD300f mimicked the differential inhibition patterns of their original molecules. The use of various signalling inhibitors and Western blotting analysis revealed that TLR9/MyD88-mediated signalling was regulated mainly by SH2-containing tyrosine phosphatase 1 (SHP-1), which could be activated by CD300a or CD300f. In contrast, regulation of the TLR3/TRIF-mediated pathway required the combined action of SHP-1 and SHP-2, which could be accomplished by CD300f but not CD300a. These data indicate that CD300a and CD300f regulate the MyD88 and TRIF-mediated TLR signalling pathways through differential activation of SHP-1 and SHP-2.     

Interleukin‐10 and interferon‐γ modulate surface expression of fractalkine‐receptor (CX3CR1) via PI3K in monocytes

The membrane‐anchored form of the chemokine fractalkine (CX₃CL1) has been identified as a novel adhesion molecule that interacts with its specific receptor (CX₃CR1) expressed in monocytes, T cells and natural killer cells to induce adhesion. In addition, CX₃CL1 can be cleaved from the cell membrane to induce chemotaxis of CX₃CR1‐expressing leucocytes. Recently, marked variations in CX₃CR1 monocyte expression have been observed during several pathological conditions. Regulation of CX₃CR1 in monocytes during basal or inflammatory/anti‐inflammatory conditions is poorly understood. The aim of this study was therefore to examine CX₃CR1 expression during monocyte maturation and the effect of soluble mediators on this process. We found that basal expression of CX₃CR1 in fresh monocytes was reduced during culture, and that lipopolysacchairde accelerated this effect. In contrast, interleukin‐10 and interferon‐γ treatment abrogated CX₃CR1 down‐modulation, through a phosphatidylinositol 3 kinase‐dependent pathway. Most importantly, CX₃CR1 membrane expression correlated with monocyte CX₃CL1‐dependent function. Taken together, our data demonstrate that CX₃CR1 expression in monocytes can be modulated, and suggest that alterations in their environment are able to influence CX₃CL1‐dependent functions, such as chemotaxis and adhesion, leading to changes in the kinetics, composition and/or functional status of the leucocyte infiltrate.