Publications

Screening of causative genes for fibrosis development in mice.

<i>Salmonella</i>‐induced SipB‐independent cell death requires Toll‐like receptor‐4 signalling via the adapter proteins Tram and Trif

Summary Salmonella enterica serovar typhimurium ( S . typhimurium ) is an intracellular pathogen that causes macrophage cell death by at least two different mechanisms. Rapid cell death is dependent on the Salmonella pathogenicity island‐1 protein SipB whereas delayed cell death is independent of SipB and occurs 18–24 hr post infection. Lipopolysaccharide (LPS) is essential for the delayed cell death. LPS is the main structural component of the outer membrane of Gram‐negative bacteria and is recognized by Toll‐like receptor 4, signalling via the adapter proteins Mal, MyD88, Tram and Trif. Here we show that S . typhimurium induces SipB‐independent cell death through Toll‐like receptor 4 signalling via the adapter proteins Tram and Trif. In contrast to wild type bone marrow derived macrophages (BMDM), Tram –/– and Trif –/– BMDM proliferate in response to Salmonella infection.

Lipocalin 2 Bolsters Innate and Adaptive Immune Responses to Blood-Stage Malaria Infection by Reinforcing Host Iron Metabolism

Plasmodium parasites multiply within host erythrocytes, which contain high levels of iron, and parasite egress from these cells results in iron release and host anemia. Although Plasmodium requires host iron for replication, how host iron homeostasis and responses to these fluxes affect Plasmodium infection are incompletely understood. We determined that Lipocalin 2 (Lcn2), a host protein that sequesters iron, is abundantly secreted during human (P. vivax) and mouse (P. yoeliiNL) blood-stage malaria infections and is essential to control P. yoeliiNL parasitemia, anemia, and host survival. During infection, Lcn2 bolsters both host macrophage function and granulocyte recruitment and limits reticulocytosis, or the expansion of immature erythrocytes, which are the preferred target cell of P. yoeliiNL. Additionally, a chronic iron imbalance due to Lcn2 deficiency results in impaired adaptive immune responses against Plasmodium parasites. Thus, Lcn2 exerts antiparasitic effects by maintaining iron homeostasis and promoting innate and adaptive immune responses.

Functional V region formation during in vitro culture of a murine immature B precursor cell line

No abstract is provided for this article.

Requirement for TLR9 in the Immunomodulatory Activity of <i>Propionibacterium acnes</i>

Propionibacterium acnes (formerly Corynebacterium parvum) is part of the human flora and, as such, is associated with several human pathologies. It possesses strong immunomodulatory activities, which makes this bacterium interesting for prophylactic and therapeutic vaccination. The bacterial component(s) and the host receptor(s) involved in the induction of these activities are poorly understood. We show in this study that TLR9 is crucial in generating the characteristic effects of killed P. acnes priming in the spleen, such as extramedullary hemopoiesis and organ enlargement, and granuloma formation in the liver. Furthermore, the ability to overproduce TNF-α and IFN-γ in response to LPS, lipid A, synthetic lipopeptide Pam3CysK4, or whole killed bacteria was present in P. acnes-primed wild-type, but not TLR9−/−, mice. Finally, P. acnes priming failed to induce enhanced resistance to murine typhoid fever in TLR9−/− mice. Thus, TLR9 plays an essential role in the induction of immunomodulatory effects by P. acnes. Because IFN-γ is a key mediator of these effects, and enhanced IFN-γ mRNA expression was absent in spleen and liver of P. acnes-primed TLR9−/− mice, we conclude that TLR9 is required for the induction of IFN-γ by P. acnes.

<i>Plasmodium</i> <i>berghei</i>Infection in Mice Induces Liver Injury by an IL-12- and Toll-Like Receptor/Myeloid Differentiation Factor 88-Dependent Mechanism

Malaria, caused by infection with Plasmodium spp., is a life cycle-specific disease that includes liver injury at the erythrocyte stage of the parasite. In this study, we have investigated the mechanisms underlying Plasmodium berghei-induced liver injury, which is characterized by the presence of apoptotic and necrotic hepatocytes and dense infiltration of lymphocytes. Although both IL-12 and IL-18 serum levels were elevated after infection, IL-12-deficient, but not IL-18-deficient, mice were resistant to liver injury induced by P. berghei. Neither elevation of serum IL-12 levels nor liver injury was observed in mice deficient in myeloid differentiation factor 88 (MyD88), an adaptor molecule shared by Toll-like receptors (TLRs). These results demonstrated a requirement of the TLR-MyD88 pathway for induction of IL-12 production during P. berghei infection. Hepatic lymphocytes from P. berghei-infected wild-type mice lysed hepatocytes from both uninfected and infected mice. The hepatocytotoxic action of these cells was blocked by a perforin inhibitor but not by a neutralizing anti-Fas ligand Ab and was up-regulated by IL-12. Surprisingly, these cells killed hepatocytes in an MHC-unrestricted manner. However, CD1d-deficient mice that lack CD1d-restricted NK T cells, were susceptible to liver injury induced by P. berghei. Collectively, our results indicate that the liver injury induced by P. berghei infection of mice induces activation of the TLR-MyD88 signaling pathway which results in IL-12 production and activation of the perforin-dependent cytotoxic activities of MHC-unrestricted hepatic lymphocytes.

TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-β

No abstract is provided for this article.

Immunoadjuvant effects of polyadenylic:polyuridylic acids through TLR3 and TLR7

Double-stranded RNA (dsRNA) is produced upon viral infection and can activate innate immunity. Polyinosinic:polycytidylic acids [poly(I:C)] is a synthetic mimetic of dsRNA and functions through an endosomal receptor, Toll-like receptor (TLR) 3 or cytosolic receptors. Another type of dsRNA, polyadenylic:polyuridylic acids [poly(A:U)], can also act as an immune adjuvant, but it remains unclear how it exhibits its adjuvant effects. Here, we have characterized the adjuvant effects of poly(A:U). Poly(A:U) could induce both IFN-alpha and IL-12p40 from murine bone marrow dendritic cells (DCs). Poly(A:U)-induced IFN-alpha production depended on a DC subset, plasmacytoid dendritic cell (pDC), and required TLR7. IL-12p40 was also produced by poly(A:U)-stimulated pDC in a TLR7-dependent manner. In addition to pDC, conventional dendritic cell (cDC) also produced IL-12p40 in response to poly(A:U). This IL-12p40 induction resulted from two cDC subsets, CD24(high) cDC and CD11b(high) cDC in a TLR3- and TLR7-dependent manner, respectively. In vivo injection of poly(A:U) with antigen led to clonal expansion of and IFN-gamma production from antigen-specific CD8(+) T cells. Consistent with the in vitro findings, TLR3 and TLR7 were required for the clonal T-cell expansion. Notably, TLR3, rather than TLR7, was critical for generating IFN-gamma-producing CD8(+) T cells. CD8(+) T-cell responses induced by poly(A:U) were independent of type I IFN signaling. Our results demonstrate that poly(A:U) functions as an in vivo immunoadjuvant mainly through TLR3 and TLR7.

Differential Roles of MDA5 and RIG-I in the Recognition of RNA Viruses.

No abstract is provided for this article.

Toll-Like Receptors

Biological and clinical aspects of

33 Tony, H-P., Phillips, N.E. and Parker, E..C. (1985)J. Exp. Med. 162, 1695 1708 34 Kupfer, A. and Singer, S.J. (1989) J. Exp. Med. 170, 1697--1713 35 Watanabe, M., Wegma, D.R., Ochi, A. and Hozumi, N. (1986) Proc. Natl Acad. Sci. USA 83, 5247 5251 36 Bonnefoy, J.Y., Guillot, O., Spits, H. etal. (1988) J. Exp. Med. 167, 57 72 37 Lee, W.T., Rao, M and Conrad, D.H (1987) ./. Immunoi 139,1191 1198 38 MacLennan, I.C.M. and Gray, D. (1936)Immunol. Rev. 91, 61 85 39 Liu, Y-J., Joshua, D.E., Williams, G.T. et al. (1989)Nature 342,929 931 40 Reynes, M., Aubert, J-P., Cohen, J.H.M. etal. (1985) J. Imrnunol. 135, 2687 2694 41 Johnson, G.D., MacLennan, I.C.M., Ling, N.R. and Hardie, L. (1987)in Leukocyte I-yping III (McM0chael, A.J. et al., eds). p. 387, Oxford University Press 42 Sellheyer, K., <~ .... ;~ -, ~;J9 R. and Steln, 11. (1989) C/in £xp Immunol. 78, 431 436 43 Swendeman, S. and 1-horley Lawson, D A (1987) EMBO J 6,1637 1642 44 Gordon, J., Cairns, J.A, Millsum, M.J., Gillb, S. and Guy, G.R. (1988) Eur. 2. Irnmunol. 18, 156! 1565 45 Uchibayashi, N, Kikutani, H., Barsumian. E_L. et al (1989) J. Immunol. 142, 3901 3908 46 Fanger, M.W., Shen, L., Ora,iano, R.F. and Guyre, P.M (1989) Immunol. Today 1 O, 92 99 47 Kikutani, H., Invi, S., Sato, R. etal. (1986) Ce1147, 651 665 48 Phillips, N.E. and Parker, D C (1984)J. Immunol. 132, 627 632 49 Miettinen, H.M., Rose, JK ar,4 Mellman, I (1989) Cell 58, 317 327

Bruton's tyrosine kinase phosphorylates Toll-like receptor 3 to initiate antiviral response

Toll-like receptor 3 (TLR3) mediates antiviral response by recognizing double-stranded RNA. Its cytoplasmic domain is tyrosine phosphorylated upon ligand binding and initiates downstream signaling via the adapter TIR-containing adaptor inducing interferon–β (TRIF). However, the kinase responsible for TLR3 phosphorylation remains unknown. We show here that Bruton's tyrosine kinase (BTK)-deficient macrophages failed to secrete inflammatory cytokines and IFN-β upon TLR3 stimulation and were impaired in clearing intracellular dengue virus infection. Mutant mice were also less susceptible to d -galactosamine/p(I:C)-induced sepsis. In the absence of BTK, TLR3-induced phosphoinositide 3-kinase (PI3K), AKT and MAPK signaling and activation of NFκB, IRF3, and AP-1 transcription factors were all defective. We demonstrate that BTK directly phosphorylates TLR3 and in particular the critical Tyr759 residue. BTK point mutations that abrogate or led to constitutive kinase activity have opposite effects on TLR3 phosphorylation. Loss of BTK also compromises the formation of the downstream TRIF/receptor-interacting protein 1 (RIP1)/TBK1 complex. Thus, BTK plays a critical role in initiating TLR3 signaling.

Toll-Like Receptor 4 Mediates Tolerance in Macrophages Stimulated with<i>Toxoplasma gondii-</i>Derived Heat Shock Protein 70

Peritoneal macrophages (PMs) from toll-like receptor 4 (TLR4)-deficient and wild-type (WT) mice were responsive to recombinant Toxoplasma gondii -derived heat shock protein 70 (rTgHSP70) and natural TgHSP70 (nTgHSP70) in NO release, but those from TLR2-, myeloid differentiation factor 88 (MyD88)-, and interleukin-1R-associated kinase 4 (IRAK4)-deficient mice were not. Polymyxin B did not inhibit PM activation by TgHSP70 and nTgHSP70 from WT and TLR4-deficient mice, while it inhibited PM activation by lipopolysaccharide. Pretreatment of PMs from WT but not from TLR4-deficient mice with rTgHSP70 resulted in suppression of NO release on restimulation with rTgHSP70. Similarly, pretreatment of PMs from WT but not TLR4-deficient mice with nTgHSP70 resulted in suppression of NO release on restimulation with nTgHSP70. Polymyxin B did not inhibit rTgHSP70- and nTgHSP70-induced tolerance of PMs from TLR4-deficient mice. Furthermore, PMs from WT mice increased suppressor of cytokine-signaling-1 (SOCS-1) expression after restimulation with rTgHSP70, while those from TLR4-deficient mice did not. Phosphorylation of JNK and I-κBα occurred in rTgHSP70-induced tolerance of PMs from TLR4-deficient mice, but not in that from WT mice. These data indicated that TgHSP70 signaling mechanisms were mediated by TLR2, MyD88, and IRAK4, but not by TLR4. On the other hand, signaling of TgHSP70-induced tolerance was mediated by TLR4, and the expression of SOCS-1 suppressed the TLR2 signaling pathway.

Deficiencies of Myeloid Differentiation Factor 88, Toll-Like Receptor 2 (TLR2), or TLR4 Produce Specific Defects in Macrophage Cytokine Secretion Induced by<i>Helicobacter pylori</i>

Helicobacter pylori is a gram-negative microaerophilic bacterium that colonizes the gastric mucosa, leading to disease conditions ranging from gastritis to cancer. Toll-like receptors (TLRs) play a central role in innate immunity by their recognition of conserved molecular patterns on bacteria, fungi, and viruses. Upon recognition of microbial components, these TLRs associate with several adaptor molecules, including myeloid differentiation factor 88 (MyD88). To investigate the contribution of the innate immune system to H. pylori infection, bone marrow-derived macrophages from mice deficient in TLR2, TLR4, TLR9, and MyD88 were infected with H. pylori SS1 and SD4 for 24 or 48 h. We demonstrate that MyD88 was essential for H. pylori induction of all cytokines investigated except alpha interferon (IFN-alpha). The secretion of IFN-alpha was substantially increased from cells deficient in MyD88. H. pylori induced interleukin-12 (IL-12) and IL-10 through TLR4/MyD88 signaling. In addition, H. pylori induced less IL-6 and IL-1beta in TLR2-deleted macrophages, suggesting that the MyD88 pathway activated by TLR2 stimulation is responsible for H. pylori induction of the host proinflammatory response (IL-6 and IL-1beta). These observations are important in light of a recent report on IL-6 and IL-1beta playing a role in the development of H. pylori-related gastric cancer. In conclusion, our study demonstrates that H. pylori activates TLR2 and TLR4, leading to the secretion of distinct cytokines by macrophages.

Pillars Article: Cutting Edge: Toll-Like Receptor 4 (TLR4)-Deficient Mice Are Hyporesponsive to Lipopolysaccharide: Evidence for TLR4 as the Lps Gene Product. J. Immunol. 1999. 162: 3749-3752.

No abstract is provided for this article.

Contribution of TIR domain-containing adapter inducing IFN-β-mediated IL-18 release to LPS-induced liver injury in mice

Background/Aims After treatment with heat-killed Propionibacterium acnes mice show dense hepatic granuloma formation. Such mice develop liver injury in an interleukin (IL)-18-dependent manner after challenge with a sublethal dose LPS. As previously shown, LPS-stimulated Kupffer cells secrete IL-18 depending on caspase-1 and Toll-like receptor (TLR)-4 but independently of its signal adaptor myeloid differentiation factor 88 (MyD88), suggesting importance of another signal adaptor TIR domain-containing adapter inducing IFN-β (TRIF). Nalp3 inflammasome reportedly controls caspase-1 activation. Here we investigated the roles of MyD88 and TRIF in P. acnes-induced hepatic granuloma formation and LPS-induced caspase-1 activation for IL-18 release. Methods Mice were sequentially treated with P. acnes and LPS, and their serum IL-18 levels and liver injuries were determined by ELISA and ALT/AST measurement, respectively. Active caspase-1 in LPS-stimulated Kupffer cells was determined by Western blotting. Results Macrophage-ablated mice lacked P. acnes-induced hepatic granuloma formation and LPS-induced serum IL-18 elevation and liver injury. Myd88 −/− Kupffer cells, but not Trif −/− cells, exhibited normal caspase-1 activation upon TLR4 engagement in vitro. Myd88 −/− mice failed to develop hepatic granulomas after P. acnes treatment and liver injury induced by LPS challenge. In contrast, Trif −/− mice normally formed the hepatic granulomas, but could not release IL-18 or develop the liver injury. Nalp3 −/− mice showed the same phenotypes of Trif −/− mice. Conclusions Propionibacterium acnes treatment MyD88-dependently induced hepatic granuloma formation. Subsequent LPS TRIF-dependently activated caspase-1 via Nalp3 inflammasome and induced IL-18 release, eventually leading to the liver injury.

TLR5 functions as an endocytic receptor to enhance flagellin‐specific adaptive immunity

Innate immune activation via TLR induces dendritic cell maturation and secretion of inflammatory mediators, generating favorable conditions for naïve T-cell activation. Here, we demonstrate a previously unknown function for TLR5, namely that it enhances MHC class-II presentation of flagellin epitopes to CD4(+) T cells and is required for induction of robust flagellin-specific adaptive immune responses. Flagellin-specific CD4(+) T cells expanded poorly in TLR5-deficient mice immunized with flagellin, a deficiency that persisted even when additional TLR agonists were provided. Flagellin-specific IgG responses were similarly depressed in the absence of TLR5. In marked contrast, TLR5-deficient mice developed robust flagellin-specific T-cell responses when immunized with processed flagellin peptide. Surprisingly, the adaptor molecule Myd88 was not required for robust CD4(+) T-cell responses to flagellin, indicating that TLR5 enhances flagellin-specific CD4(+) T-cell responses in the absence of conventional TLR signaling. A requirement for TLR5 in generating flagellin-specific CD4(+) T-cell activation was also observed when using an in vitro dendritic cell culture system. Together, these data uncover an Myd88-independent function for dendritic cell TLR5 in enhancing the presentation of peptides to flagellin-specific CD4(+) T cells.

Toll-IL-1 Receptor Domain-Containing Adaptor Protein Is Critical for Early Lung Immune Responses against<i>Escherichia coli</i>Lipopolysaccharide and Viable<i>Escherichia coli</i>

Pulmonary bacterial diseases are a leading cause of mortality in the U.S. Innate immune response is vital for bacterial clearance from the lung, and TLRs play a critical role in this process. Toll-IL-1R domain-containing adaptor protein (TIRAP) is a key molecule in the TLR4 and 2 signaling. Despite its potential importance, the role of TIRAP-mediated signaling in lung responses has not been examined. Our goals were to determine the role of TIRAP-dependent signaling in the induction of lung innate immune responses against Escherichia coli LPS and viable E. coli, and in lung defense against E. coli in mice. LPS-induced neutrophil sequestration; NF-κB translocation; keratinocyte cell-derived chemokine, MIP-2, TNF-α, and IL-6 expression; histopathology; and VCAM-1 and ICAM-1 expression were abolished in the lungs of TIRAP−/− mice. A cell-permeable TIRAP blocking peptide attenuated LPS-induced lung responses. Furthermore, immune responses in the lungs of TIRAP−/− mice were attenuated against E. coli compared with TIRAP+/+ mice. TIRAP−/− mice also had early mortality, higher bacterial burden in the lungs, and more bacterial dissemination following E. coli inoculation. Moreover, we used human alveolar macrophages to examine the role of TIRAP signaling in the human system. The TIRAP blocking peptide abolished LPS-induced TNF-α, IL-6, and IL-8 expression in alveolar macrophages, whereas it attenuated E. coli-induced expression of these cytokines and chemokines. Taken together, this is the first study illustrating the crucial role of TIRAP in the generation of an effective early immune response against E. coli LPS and viable E. coli, and in lung defense against a bacterial pathogen.