Functional MyD88 Research Articles

Growth, metastasis, and expression of CCL2 and CCL5 by murine mammary carcinomas are dependent upon Myd88

Previously we reported that lipopolysaccharide (LPS) treatment of murine mammary carcinomas resulted in decreased growth of the tumors. Here we show the decreased growth following LPS treatment was mediated through effects downstream of TLR4 and Myd88. Perhaps more notably, simply reducing TLR4 or Myd88 levels was sufficient to slow tumor growth rates. Moreover, reduced levels of Myd88 correlated with a significant reduction in lung metastasis as well as decreased CCL2 and CCL5 expression. To determine whether inhibiting Myd88 function could also alter tumor growth and chemokine expression we used a Myd88 homodimerization inhibitory peptide. Indeed, inhibiting Myd88 function in four different murine mammary carcinomas as well as the human breast cancer cell line MDA-MB-231 led to decreased growth as well as CCL2 and CCL5 expression. These data imply that Myd88 is important for growth and metastasis of breast cancer, and expression of at least two proinflammatory chemokines.

Growth and constitutive expression of CCL2 and CCL5 by murine mammary carcinomas are dependent upon Myd88 function (165.6)

Abstract Previously we reported that lipopolysaccharide (LPS) treatment of murine mammary carcinomas resulted in decreased growth of the tumors in vivo, but not in vitro. Here we explored whether the effects of LPS were dependent upon TLR4 and Myd88 using RNA interference. Two clones with reduced TLR4 mRNA and protein expression and two clones with reduced Myd88 mRNA and protein expression were generated, as well as a clone expressing shRNA specific for lacZ as a control. Significantly, tumors with reduced TLR4 or Myd88 expression no longer exhibited decreased growth rates in vivo following LPS treatment indicating that the decreased growth rate of the 4T1 tumor following LPS treatment was mediated through effects downstream of TLR4 and Myd88. Perhaps more significantly, tumor cells with reduced levels of TLR4 or Myd88 exhibited slower growth rates in vitro and in vivo relative to the parental 4T1 and lacZ controls, and reducing Myd88 expression was sufficient to decrease CCL2 and CCL5 expression. To determine whether inhibiting Myd88 function was enough to alter tumor growth and CCL2 and CCL5 expression we used a Myd88 homodimerization inhibitory peptide. The data revealed that inhibiting Myd88 function was sufficient enough to impair growth of the tumor cells, and decrease constitutive expression of CCL2 and CCL5. These data imply that Myd88 is necessary for growth of murine mammary carcinomas, and expression of at least two proinflammatory chemokines.

TLR4 Recognizes Pseudallescheria boydii Conidia and Purified Rhamnomannans

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.

MTA1 Coregulator Regulates LPS Response via MyD88-dependent Signaling*

Although metastasis tumor antigen 1 (MTA1) contributes to the responsiveness of macrophages to LPS, the underlying mechanism remains unknown. Here, we investigated the role of MTA1 in the regulation of expression and function of MyD88, a proximal component of NF-κB signaling. We discovered that MTA1 targets MyD88 and that MyD88 is a NF-κB-responsive gene in LPS-stimulated macrophages. We found that MTA1 is required for MyD88-dependent stimulation of NF-κB signaling and expression of proinflammatory cytokines such as IL-1β, MIP2, and TNF-α as MTA1 depletion leads to a substantial reduction in the expression of NF-κB target genes. In addition, LPS-mediated stimulation of MyD88 transcription was accompanied by an enhanced recruitment of MTA1, RNA polymerase II, and p65RelA complex to the NF-κB consensus sites in the MyD88 promoter. Interestingly, the recruitment of both MTA1 and MyD88 expression is effectively blocked by NF-κB inhibitor parthenolide. Selective knockdown of MyD88 by a dominant negative mutant of MyD88 or selective siRNA also impairs the ability of LPS to stimulate the NF-κB target genes. These findings reveal an inherent coregulatory role of MTA1 upon the expression of MyD88 and suggest that MTA1 regulation of MyD88 may constitute at least one of the mechanisms by which MTA1 stimulates LPS-induced NF-κB signaling in stimulated macrophages.

Dual function of MyD88 in RAS signaling and inflammation, leading to mouse and human cell transformation

Accumulating evidence points to inflammation as a promoter of carcinogenesis. MyD88 is an adaptor molecule in TLR and IL-1R signaling that was recently implicated in tumorigenesis through proinflammatory mechanisms. Here we have shown that MyD88 is also required in a cell-autonomous fashion for RAS-mediated carcinogenesis in mice in vivo and for MAPK activation and transformation in vitro. Mechanistically, MyD88 bound to the key MAPK, Erk, and prevented its inactivation by its phosphatase, MKP3, thereby amplifying the activation of the canonical RAS pathway. The relevance of this mechanism to human neoplasia was suggested by the finding that MyD88 was overexpressed and interacted with activated Erk in primary human cancer tissues. Collectively, these results show that in addition to its role in inflammation, MyD88 plays what we believe to be a crucial direct role in RAS signaling, cell-cycle control, and cell transformation.

A Unique Feature of Toll/IL-1 Receptor Domain-Containing Adaptor Protein Is Partially Responsible for Lipopolysaccharide Insensitivity in Zebrafish with a Highly Conserved Function of Myd88

MyD88 and Toll/IL-1R domain-containing adaptor protein (TIRAP) are required for the TLR4 response to LPS stimulation in mammals, but the functions of the two adaptors and their involvement in zebrafish insensitivity to LPS remains unknown. We present a functional analysis of zebrafish Myd88 and Tirap and suggest that Myd88 is more important than Tirap for the activation of Tlr-mediated NF-kappaB, which may be a novel mechanism of Myd88-dependent TLR signaling in teleosts. Zebrafish Tirap lacks the phosphatidylinositol 4,5-bisphosphate binding motif required for human TIRAP location and has leucine at position 233 rather than the conserved proline of human TIRAP, as well as 105 additional aa at the N terminus. Overexpression of zebrafish Tirap in HEK293T cells did not activate NF-kappaB and IFN-beta, but slightly activated NF-kappaB in carp leukocyte cells. Zebrafish Myd88 alone strongly induced the activation of NF-kappaB and IFN-beta both in HEK293T and carp leukocyte cells. The function of Myd88 was dependent on its cellular location and the proline in the Toll/IL-1R domain. Although zebrafish Tirap was distributed throughout the cell rather than localized to the cytoplasmic membrane, its impaired ability to activate downstream Tlr molecules was unlikely to be related to its location because chimera TIRAP with a human TIRAP N terminus and membrane-binding domain also did not activate NF-kappaB. However, the mutation of leucine to proline increased the ability of Tirap to activate NF-kappaB. We suggest that the zebrafish Tirap needs a longer N terminus to perform its function and could be partially responsible for the resistance to LPS in zebrafish.

Expression of human β-defensin-2 gene induced by CpG-DNA in human B cells

Defensins have a broad range of antimicrobial activity against bacteria, fungi, and viruses. The expression of human β-defensin-2 (hBD-2) is prevalently observed in epithelial cells and is induced by bacterial infection. Here, we have shown that the expression of the hBD-2 gene and release of hBD-2 protein into the medium is up-regulated in response to CpG-DNA in human B cell line RPMI 8226. The induction of hBD-2 was dependent on CG sequence and phosphorothioate backbone-modification. This was also confirmed in primary human lymphocytes. To shed light on the molecular mechanism involved in hBD-2 induction by CpG-DNA, we examined the contribution of the NF-κB signaling pathway in RPMI 8226 cells. Suppression of MyD88 function and inhibition of NF-κB nuclear localization blocked hBD-2 induction. The NF-κB pathway inhibitors also abolished hBD-2 induction. These results may contribute to a better understanding on the therapeutic effects of CpG-DNA against infectious diseases.

Identification and characterization of TLR8 and MyD88 homologs in Atlantic salmon (Salmo salar)

Toll-like receptor 8 (TLR 8) belongs to a subgroup of the TLR family that recognizes nucleic acids and that is involved in the protection against viruses. In mammals, TLR7 and 8 have been characterized as receptors for viral and synthetic single-stranded RNA. Here we describe the cloning of a TLR8 homolog in Atlantic salmon (Salmo salar) and its proximal adaptor protein MyD88. The mRNA expression of SsTLR8 was tissue-restricted and its highest level was detected in the spleen while SsMyD88 was expressed in all of the tested organs. SsTLR8 and SsMyD88 mRNAs were up-regulated in TO cells treated with recombinant IFN α1 and IFN γ. In vivo, the expression of SsTLR8 was not significantly affected following challenge with salmon alphavirus subtype 3 (SAV3). By contrast, infection with SAV3 up-regulated SsMyD88 transcripts on day 7 post-challenge and the expression remained elevated at day 28. The SsMyD88 expression in vivo paralleled type I IFN expression. In vitro stimulation of salmon head kidney leukocytes with CpG ODNs and IFN γ also up-regulated SsMyD88 mRNA. Furthermore, ectopic expression of SsMyD88 in HEK cells was able to activate a NF-κB reporter construct indicating that the cloned salmon molecule was a functional MyD88 homologue.

Synergistic BM-DC activation and immune induction by the oral vaccine vector Streptococcus gordonii and exogenous tumor necrosis factor

Streptococcus gordonii, a potential mucosal vaccine delivery vector, is proficient at colonizing murine oral mucosa; however, it often fails to elicit significant antibody titers against its vaccine antigen payloads. The poor response may be due to an inability of S. gordonii to elicit cytokines needed to suppress mucosal tolerance; exogenously supplied cytokines, such as TNF, could overcome this effect. To test this, murine bone marrow-derived dendritic cells (BM-DCs) were stimulated with UV-killed S. gordonii PM14, that surface expresses a fragment of the immunodominant S1 subunit of pertussis toxin. Peptidoglycan (PGN), lipoteichoic acid (LTA), lipoprotein (LP), and DNA were also isolated from the bacteria, and used to stimulate BM-DCs. Stimulation with TNF, S. gordonii, PGN, LTA, or LP all resulted in increased surface expression of MHCII, CD80, and CD86, compared to unstimulated BM-DCs. Stimulation with S. gordonii elicited IL-6, IL-10, and IL-12p70 production from the BM-DCs, while stimulation with the bacterial components induced some or all of the three cytokines. When BM-DCs were simultaneously stimulated with S. gordonii and TNF, a marginal increase in surface marker upregulation was observed, and the two stimuli synergized to elicit substantially greater quantities of IL-6, IL-10, and IL-12p70. Synergy between TNF and the purified bacterial components was also observed. The effect of TNF was abolished when BM-DCs were obtained from mice deficient for either TNFR1 or TNFR2, and cytokine induction by S. gordonii was entirely dependent on functional MyD88. Synergistic IL-10 induction by S. gordonii and TNF was not observed in TLR-2 −/− BM-DCs, and TNF was found to cause TLR-2 upregulation, providing at least a partial mechanism for the observed synergy. When S. gordonii and TNF were used to immunize mice, a more robust anti- S. gordonii IgG response was elicited as compared to immunization with S. gordonii alone. However, the addition of TNF did not result in stronger responses against the antigenic insert (S1 fragment) in immunized mice. These findings collectively demonstrate that TNF is able to prime BM-DCs to better respond to S. gordonii, through a mechanism at least partially involving TLR-2 upregulation.

Characterization analysis and polymorphism detection of the porcine Myd88 gene

The myeloid differentiation primary response protein 88 (Myd88) is an essential adaptor protein, which mediates in all Toll-like receptor (TLR) members signal transduction, except for TLR3. In this study, the 4464 bp genomic sequence of porcine Myd88 was first isolated, whereupon tissue distribution, chromosome mapping and single nucleotide polymorphism (SNP) were analyzed. Our results revealed that porcine Myd88 gene, which was located at chromosome 13 linked with marker S0288 (distance = 40 cR; LOD = 8.66), was widely expressed in all the examined tissues. There were 16 potential SNPs in the isolated genome fragment. SNP 797T/C in the first intron was studied, with no significant association being found between the genotype and immune traits in pigs (p > 0.05). The porcine Myd88 protein contained both the death domain (DD) and the Toll/IL-1 receptor domain (TIR). Leu residues, essential for its structure, were the most abundant encountered in the DD. The TIR contained two conserved motifs which may play important roles in the Myd88 function.

TLR Adaptor MyD88 Is Essential for Pathogen Control during OralToxoplasma gondiiInfection but Not Adaptive Immunity Induced by a Vaccine Strain of the Parasite

TLR adaptor MyD88 activation is important in host resistance to Toxoplasma gondii during i.p. infection, but the function of this signaling pathway during oral infection, in which mucosal immunity assumes a predominant role, has not been examined. In this study, we show that MyD88(-/-) mice fail to control the parasite and succumb within 2 wk of oral infection. Early during infection, T cell IFN-gamma production, recruitment of neutrophils and induction of p47 GTPase IGTP (Irgm3) in the intestinal mucosa were dependent upon functional MyD88. Unexpectedly, these responses were MyD88-independent later during acute infection. In particular, CD4(+) T cell IFN-gamma reached normal levels independently of MyD88, despite continued absence of IL-12 in these animals. The i.p. vaccination of MyD88(-/-) mice with an avirulent T. gondii uracil auxotroph elicited robust IFN-gamma responses and protective immunity to challenge with a high virulence T. gondii strain. Our results demonstrate that MyD88 is required to control Toxoplasma infection, but that the parasite can trigger adaptive immunity without the need for this TLR adaptor molecule.

LPS-induced TNF-alpha factor (LITAF)-deficient mice express reduced LPS-induced cytokine: Evidence for LITAF-dependent LPS signaling pathways.

Previously we identified a transcription factor, LPS-Induced TNF-alpha Factor (LITAF), mediating inflammatory cytokine expression in LPS-induced processes. To characterize the role of LITAF in vivo, we generated a macrophage-specific LITAF-deficient mouse (macLITAF(-/-)). Our data demonstrate that in macrophages (i) several cytokines (such as TNF-alpha, IL-6, sTNF-RII, and CXCL16) are induced at lower levels in macLITAF(-/-) compared with LITAF(+/+) control macrophages; (ii) macLITAF(-/-) mice are more resistant to LPS-induced lethality. To further identify LITAF signaling pathways, we tested mouse TLR-2(-/-), -4(-/-), and -9(-/-) and WT peritoneal macrophages exposed to LPS. Using these cells, we now show that LITAF expression can be induced after challenge either with LPS from Porphyromonas gingivalis via agonism at TLR-2, or with LPS from Escherichia coli via agonism at TLR-4, both requiring functional MyD88. We also show that, in response to LPS, the MyD88-dependent LITAF pathway differs from the NF-kappaB pathway. Furthermore, using a kinase array, p38alpha was found to mediate LITAF phosphorylation and the inhibition of p38alpha with a p38-specific inhibitor (SB203580) blocked LITAF nuclear translocation and reduced LPS-induced TNF-alpha protein levels. Finally, macLITAF(-/-) macrophages rescued by LITAF cDNA transfection restored levels of TNF-alpha similar to those observed in WT cells. We conclude that LITAF is an important mediator of the LPS-induced inflammatory response that can be distinguished from NF-kappaB pathway and that p38alpha is the specific kinase involved in the pathway linking LPS/MyD88/LITAF to TNF.

Identification of a Novel Toll-Like Receptor-Independent Immunoadjuvant Pathway That Depends upon Programmed Cell Death.

Molecules of microbial origin, and synthetic derivatives of these molecules, have long been used for their immuno-adjuvant effect, and as the key sensors of microbial infection, Toll-like receptors (TLRs) are thought to be essential for adjuvanticity. To the contrary, we now demonstrate the existence of a robust, TLR-independent pathway for adjuvant effect: one that is actually far stronger than the TLR-dependent pathway. Activation of Toll-like receptors (TLRs) and the subsequent production of cytokines such as type I interferon leads to the maturation of dendritic cells (DCs) with upregulation of MHC molecules and costimulatory molecules such as CD40, CD80 and CD86, allowing for optimal interaction between DCs and T-cells. We have determined that TLR signal transduction is minimally dependent upon two adapter proteins, MyD88 and TRIF. In compound homozygous mutant (DKO) mice that lack functional MyD88 and TRIF, there is complete abrogation of all TLR signaling. Such animals therefore comprise a unique model with which to study TLR-independent immune responses. We have now used DKO mice to determine whether an adaptive immune response can be obtained in the absence of TLR signaling. As expected, adjuvanticity obtained via “classical” microbial adjuvants such as complete Freund's adjuvant or LPS was completely absent in DKO mice. However, subcutaneous administration of syngeneic murine cells expressing ovalbumin and rendered apoptotic by exposure to ultraviolet light resulted in a strong T-cell response in vivo, with impressive production of interferon-g by CD8+ cells and efficient killing of EL-4 cells that expressed CD8-specific OVA peptides, both in wildtype and DKO mice. Adjuvanticity was observed only in the context of apoptosis, in that living cells, not exposed to ultraviolet light before injection, induced little or no response. Moreover, the mixture of the protein antigen with apoptotic cells was insufficient to induce an adaptive immune response; rather, only cells that expressed the protein prior to induction of apoptosis were stimulatory. These results indicate the existence of a specific, cell death-dependent mechanism for adjuvanticity that is TLR-independent and induced by endogenous molecules. We propose that this new adjuvant pathway is of fundamental importance to immune responses at large. We believe that it is required for initiation of the adaptive immune response witnessed in the context of allograft rejection, graft-versus-host disease, and autoimmune diseases as well.

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.

A universal role for MyD88 in TLR/IL-1R-mediated signaling.

The MyD88 adapter protein links members of the toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) superfamily to the downstream activation of nuclear factor-kappaB and mitogen-activated protein kinases. Although originally identified as a myeloid-differentiation marker, MyD88 is now known to play an essential role in the innate immune response of insects and mammals. The generation of MyD88-deficient mice, as well as the identification of MyD88-related proteins and regulators of MyD88 signaling, has revealed new and important insights into the function of MyD88.