MyD88 KO Mice Research Articles

MyD88-dependent expansion of an immature GR-1+CD11b+ population from donor bone marrow reduces early mortality after allogeneic hematopoietic stem cell transplantation (P2166)

Abstract Myeloid-derived suppressor cells coexpressing both CD11b and Gr-1 antigens are a heterogeneous population of immature myeloid cells. Activation of inflammatory signaling through MyD88, presumably through ligation of multiple TLRs, plays a key role in the expansion of MDSCs. We investigated how the MyD88-dependent expansion of MDSCs from donor BM contributes to protection of GVHD. Lethally irradiated B6D2F1 mice were transplanted with C57BL/6 WT or MyD88 KO mice TCD-BM together with WT spleen T cells. Morbidity and mortality of GVHD were significantly worse in recipients of KO TCD-BM. The expression of MDSCs in blood, MLN and liver was reduced in the recipients of KO TCD-BM, whereas the percentage of donor T cells infiltrating gut and liver was markedly higher. In parallel, the percentages of donor T cells to undergo apoptosis in response to alloantigens in vivo were significantly decreased in recipients of KO TCD-BM. Moreover, in vivo administration of isolated MDSCs to recipients of KO TCD-BM attenuated the severity of GVHD. Administration of LPS to donor mice expanded MDSCs in BM with enhanced expression of MyD88 mRNAs. Recipients of TCD-BM from WT mice injected LPS also showed attenuated GVHD severity. In summary, MyD88-dependent expansion of MDSCs population from donor non T-cell component appears to be critical for attenuating GVHD severity. Incomplete expansion of MDSCs in target organs correlates with decreased apoptosis and increased infiltration of donor T cells.

Induction of the specific allergic immune response is independent of proteases from the fungus Alternaria alternata

We have analyzed the importance of proteases for the induction of allergic responses against the mold Alternaria alternata. Responses induced in vivo with untreated or heat treated (protease inactivated) extracts were compared in BALB/c, C57BL/6, TLR4 KO, and MyD88 KO mice. In BALB/c mice, both extracts induced similar lung inflammation, upregulation of inflammatory mediators, Th2 cytokines, and Alternaria-specific antibodies. However heat inactivation abrogated polyclonal IgE production. Similar results were obtained in C57BL/6 albeit lung expression of some Th2 mediators was decreased in mice stimulated with the heat-treated extract. Treatment of the extract with protease inhibitors did not affect the induction of the allergic response either, except again for the polyclonal IgE response. Th2 responses and lung inflammation were readily induced in TLR4 knockout mice. In contrast, lung inflammation, Th2 responses, cytokine productions, and antibody synthesis were strongly suppressed in MyD88-deficient mice. Early lung IL-33 and IL-1-α expression were also suppressed. In conclusion, albeit some heat labile proteases are required for the stimulation of the polyclonal IgE secretion, fungal proteases, and TLR4 signaling are not required while MyD88 is essential for triggering the systemic immune response and for the development of lung allergic inflammation in response to Alternaria extracts.

Impact of infectious agents on trafficking of effector T cells is mediated by a polymorphic site of TLR2 and CD44 isoforms expression.

Impact of infectious agents on trafficking of effector T cells is mediated by a polymorphic site of TLR2 and CD44 isoforms expression.

Distinct IFN-Gamma Dependent Effect of the TLR5 Agonist, Flagellin On NK and CD4+ T Cell Immunity Against CMV

AbstractAbstract 255 Background:Opportunistic CMV infection remains a major clinical complication in allogeneic BMT (allo-BMT) recipients. We previously published that peri-transplant treatment of highly purified flagellin, a TLR5 agonist extracted from S. typhimurium reduced GvHD in lethally irradiated allo-BMT recipients by reducing early activation and proliferation of donor T cells. Flagellin-treated recipients had enhanced lymphoid immune reconstitution and were protected from lethal MCMV infection. CMV initiates innate immunity through the activation of Toll-like receptors (TLR) 9, 3 and 2 but direct enhancement of anti-CMV immunity through TLR5 has not been studied. Methods:To further explore the effects of a TLR5 agonist on anti-CMV immunity, we infected flagellin-treated C57BL/6 (B6), TLR5KO and Myd88KO mice (B6 background) with sub lethal dose (1×105pfu/mouse i.p) of MCMV. Anti-CMV immunity of NK and T-cells were determined by measuring surface activation markers on the NK and T-cells. Flagellin-treated and MCMV infected IL-12KO, IFN-γRKO and IDO KO mice were used to study the role of IL-12, IFN-γ and IDO on anti-CMV immunity. The relationship between the MCMV lethality and flagellin signaling in hematopoietic or epithelial tissues expressing TLR5 was further investigated by generating radiation chimeras, using WT mice engrafted with TLR5 KO bone marrow (BM) and TLR5 KO mice engrafted with WT B6 BM. Chimeric recipients with WT or TLR5KO hematopoietic cells (or the reverse) were then treated with flagellin or PBS and infected with a sub lethal dose of MCMV infection. Results:Without MCMV infection, flagellin treatment significantly increased numbers of NK cells (p= 0.001) and KLRG1+ NK cells (p=0.009) in WT B6 mice compared with the PBS-treated control mice. Following MCMV infection (day 3), there were significantly increased numbers of splenic NK cells (p= 0.01) and KLRG1+ NK cells (p=0.0008) in flagellin-treated mice compared with the control mice. Flagellin-treatment also enhanced viral clearance, tending to decrease liver viral loads in WT B6 mice. In contrast, MCMV-infected and flagellin-treated TLR5 KO or Myd88KO mice had significantly increased liver viral loads (p=00001) compared with the flagellin- and PBS-treated B6 mice on day 3 post MCMV infection. While flagellin-treatment increased the numbers and activation status of NK cells, it decreased the numbers of activated (CD69+, ICOS+ and KLRG1+) CD4+ T cells on day 3 post MCMV infection. On day 10 post MCMV infection, enhanced numbers of splenic CD8+ T cells and CMV-specific tetramer+ CD8+ T cells (p<0.05) were detected in flagellin-treated mice compared with controls. The numbers of splenic CD4+ T cells increased >2 fold (p= 0.008) associated with significantly increased numbers of splenic CD4+CD25+ Foxp3+ regulatory T cells (p<0.05) on day 10 post MCMV infection in flagellin-treated mice compared with the control mice. Flagellin-induced anti-CMV immunity required IFN-γ signaling as flagellin-treated IFN-γRKO mice had markedly increased susceptibility to MCMV infection whereas flagellin-treated IL-12 KO and IDO KO mice had significantly reduced liver viral loads compared to the PBS-treated IL-12 KO and IDO KO mice on day 10 post MCMV infection. To explore interaction of flagellin with TLR5+ host hematopoietic cells and TLR5+host gut epithelium on CMV immunity, we next infected radiation chimeras expressing either TLR5 on hematopoietic cells or on gut epithelium with sub lethal i.p dose of MCMV. Flagellin-treated TLR5 KO BM → B6 radiation chimeras (TLR5+ host epithelium) had increased mortality compared with other groups following MCMV infection indicating that TLR5+ hematopoietic cells are required for protective immunity against CMV infection.In summary, flagellin caused selective early increased activation of NK cells and reduced activation of CD4+ T cells in presence of MCMV infection. The protective effect of flagellin on anti-CMV immune responses are IFN-γ dependent but independent of IL-12 and IDO. The TLR5+ hematopoietic cells are required for the protective immunity against CMV infection. These data suggest the clinical importance of flagellin in modulating both innate and adaptive immunity against CMV infection and also as an alternative GvHD prophylaxis in allo-BMT recipients. Disclosures:No relevant conflicts of interest to declare.

Selected Commensals Mediate GvHD Via MyD88-Dependent Signaling in Non-Hematopoietic Cells.

Abstract 3005Graft-versus-host disease (GVHD) primarily affects the epithelial compartments of skin, liver, and gut and is thought to display tissue tropism for two main reasons: the conditioning regimen can selectively damage these epithelial cell compartments due to rapid cell turnover; and these epithelial organs have high exposure to host commensals and their products that promote tissue-specific inflammation. To understand how commensals mediate GVHD, we first tested the hypothesis that the disruption of the MyD88 signaling pathway, which is critical for host-microbe interaction, would alter GVHD outcomes. We created bone marrow chimeras that received a second allogeneic bone marrow transplant (BMT) and showed that MyD88 deficiency in recipient hematopoietic cells reduced BMT mortality, while deficiency in the non-hematopoietic cells (NHC) increased BMT mortality (p<0.05). These findings indicate the protective role of MyD88 expression in the latter compartment. Furthermore, deficiency of TLR6, but not TLR1,2,4, and 5, in NHC recapitulated similar BMT outcomes, suggesting regulation of MyD88 by TLR6 in our BMT model. Based on a hypothesis that MyD88 signaling on NHC promotes epithelial healing and barrier function, we first determined whether the level of bacterial translocation in MyD88KO BMT recipients are different. 16s DNA, a measurement of bacteria content, was significantly increased in livers of MyD88 knock-out (KO) transplant recipients compared to WT counterparts. We next decontaminated MyD88KO transplant recipients with a cocktail of broad spectrum antibiotics (Abx) prior to BMT and maintained these mice in sterile isolators. MyD88KO mice treated with Abx and untreated WT BMT recipients had similar survival; in contrast, untreated MyD88KO recipients died rapidly within the first 2 weeks following BMT (p<0.05). 16S DNA level was reduced in MyD88KO mice treated with Abx prior to BMT compared to their untreated MyD88KO counterpart. Furthermore, MyD88KO radiation controls treated with Abx had improved survival, similar to WT radiation controls, compared to untreated MyD88KO controls. These series of findings suggest a critical role of MyD88 in promoting barrier protection and healing via the NHC following BMT. Interestingly, similar Abx decontamination in WT BMT recipients did not change survival, suggesting that broad microbial decontamination in normal recipients without significant risk for bacterial translocation is not beneficial. Based on these findings, we next hypothesized that selective versus broad decontamination of commensal populations would improve GVHD outcomes following allogeneic BMT in WT mice. In MHC-matched and mismatched BMT murine models, we used different antibiotic cocktails to selectively remove subsets of commensals from recipients pre- and peri-BMT. Transplant recipients which received vancomycin (V) alone had significantly improved GVHD measures and survival compared to those that were administered more broad spectrum antibiotic cocktails (p <0.05). Flow cytometric analysis of the colon lamina propria and intraepithelial layer showed a significant increase of Foxp3-expressing lymphocytes in mice given V vs other groups, suggesting that the reduced GVHD in the former may be due to a higher regulatory cell population that suppresses local inflammation in the gut. In sum, our studies suggest a critical interaction between commensals and host cells via MyD88 signaling in mediating GVHD and BMT outcomes, and support consideration of selective versus broad microbial decontamination with BMT. Disclosures:No relevant conflicts of interest to declare.

T-Cell Dependency of T-Independent (TI) Antigens: Antibody Response to Blood Group A-Antigen Requires CD4 T-Cells and CD40/CD40L Interaction

Introduction: Polysaccharides are considered TI antigens capable of eliciting antibody secretion without T-cell help. TNP-Ficoll is used as a model TI (type 2) antigen, but detailed mechanisms of B-cell responses remain unclear. We studied a T-cell role in the antibody response to polysaccharides, using a clinically relevant ABO antigen in its natural form. Methods: Mice (4-8 week-old) received three standardized immunizations (days 0- 7- 14) with human blood group-A erythrocyte membranes, TNP-Ficoll or sperm whale myoglobin (SPWM; protein); some groups received first injection in complete Freund's adjuvant then two boosts without adjuvant. For T-cell depletion studies, anti-CD4 (clone GK1.5) or isotype control were injected on day -1-0- +1. To further characterize direct and indirect interaction of CD40/CD40L, CD40L KO mice were injected with anti-CD40 (clone IC10) or isotype control in order to activate or block CD40 on B cells; CD40 KO mice were injected with mouse rCD40-Fc fusion protein to activate T-cells which in turn may activate B-cells through soluble factors. TLR contribution was studied in MyD88 KO. Anti-A antibodies were measured in plasma at day -1-14-21 by hemagglutination and ABO-ELISA, and TNP and SPWM antibodies by ELISA. Results: Natural anti-A antibodies were undetectable on day -1 in all groups, whereas anti-TNP antibody was present in young mice. A-erythrocytes elicited a substantial antibody response, which was abrogated by CD4 T-cell depletion (fig 1), including IgM (fig 2), whereas of SPWM antibodies, only IgG was absent with normal or low IgM and IgA. Antibodies to TNP-Ficoll were comparable in CD4-depleted vs. control mice. Similarly there were no differences in response to secondary A-antigen immunization when CD4 cells were depleted in pre-immunized mice. Anti-A antibodies were significantly reduced in CD40L KO, and further diminished with anti-CD40 antibody; no reduction was seen in MyD88 KO mice. Conclusions: This study clearly demonstrates that antibody response to A-antigen requires CD4 T-cells. Although polysaccharides may not stimulate MHC class II-dependent T-cell help, the presence of activated CD4 T-cells and their interaction with B-cells through CD40-CD40L (fig 2) is critical for B-cells to become antibody secreting cells. These findings have major implications for ABO-incompatible and xenotransplants.[Figure 1][Figure 2]

Expression of myeloid differentiation factor 88 in neurons is not requisite for the induction of sickness behavior by interleukin-1β

BackgroundAnimals respond to inflammation by suppressing normal high-energy activities, including feeding and locomotion, in favor of diverting resources to the immune response. The cytokine interleukin-1 beta (IL-1β) inhibits normal feeding and locomotor activity (LMA) via its actions in the central nervous system (CNS). Behavioral changes in response to IL-1β are mediated by myeloid differentiation factor 88 (MyD88) in non-hematopoietic cells. It is unknown whether IL-1β acts directly on neurons or requires transduction by non-neuronal cells.MethodsThe Nestin-cre mouse was crossed with MyD88lox mice to delete MyD88 from neurons and glia in the CNS (MyD88ΔCNS). These mice were compared to total body MyD88KO and wild type (WT) mice. Mice had cannulae stereotactically placed in the lateral ventricle and telemetry transponders implanted into the peritoneum. Mice were treated with either intracerebroventricular (i.c.v.) IL-1β (10 ng) or vehicle. Food intake, body weight and LMA were continuously monitored for 24 h after treatment. I.c.v. tumor necrosis factor (TNF), a MyD88-independent cytokine, was used to control for normal immune development. Peripheral inflammation was modeled using intraperitoneal lipopolysaccharide (LPS). Groups were compared using two-way ANOVA with Bonferroni post-test. Efficacy of recombination was evaluated using tdTomato reporter mice crossed with the Nestin-cre mouse. MyD88 deletion was confirmed by Western blot.ResultsI.c.v. IL-1β treatment caused a significant reduction in feeding, body weight and LMA in WT mice. MyD88KO mice were protected from these changes in response to i.c.v. IL-1β despite having intact behavioral responses to TNF. Cre-mediated recombination was observed in neurons and astrocytes, but not microglia or endothelial cells. In contrast to MyD88KO mice, the behavioral responses of MyD88ΔCNS mice to i.c.v. IL-1β or intraperitoneal (i.p.) LPS were indistinguishable from those of WT mice.ConclusionSickness behavior is mediated by MyD88 and is dependent on the activity of cytokines within the brain. Our results demonstrate that MyD88 is not required in neurons or astrocytes to induce this behavioral response to IL-1β or LPS. This suggests that a non-Nestin expressing cell population responds to IL-1β in the CNS and transduces the signal to neurons controlling feeding and activity.

P099 Myd88 gene knockout inhibits the development of lupus-like disease in NZB/W F1 mice

The New Zealand Black (NZB) × New Zealand White (NZW) F1 hybrid female mice (NZB/WF1) develop a disease closely resembling human systemic lupus erythematosus (SLE), characterized by the production of antinuclear antibodies, severe glomerulonephritis and early mortality. Recently, innate immune responses have been reported to play an important role in the development of autoimmune disease. In this study we investigated the role of Myd88 protein (an important regulator of innate immune signaling pathways) in the pathogenesis of lupus-like disease in Myd88 knockout NZB/W F1 mice. The Myd88-deficient mice were backcrossed separately in the NZB and NZW backgroud. Heterozygous NZB as well as NZW of F10 backcross generation were intercrossed to achieve Myd88 -/- NZB/W F1 mice (KO) and Myd88 +/+ NZB/W F1 mice (WT). WT and Myd88 KO mice were compared for serum concentrations of anti-dsDNAantibody, biochemical markers of active disease (BUN, Cr, Alb, TP, T-CHO, protein urea), histological analysis of kidney pathology and mortality. Compared with WT mice, Myd88 KO mice featured significantly reduced serum concentrations of anti-dsDNAantibody, reduced levels of biochemical markers of active disease, reduced kidney pathology and reduced mortality. Our result indicates that innate immunity participates in the pathogenesis of lupus prone mice and Myd88 is its important regulator.

Deficiency in MyD88 Signaling Results in Decreased Antibody Responses to an Adeno-Associated Virus Vector in Murine Pompe Disease

We have previously shown that antibody and T cell responses limit the efficacy of an adeno-associated virus (AAV) pseudotype 8 (2/8) vector containing the universally active cytomegalovirus enhancer/chicken β-actin regulatory cassette (AAV2/8-CBhGAA) in treating murine Pompe disease. However, the innate immune responses to AAV2/8-CBhGAA are largely unknown. In this study, we investigated acute immune responses to AAV2/8-CBhGAA and the role of MyD88/TRIF signaling pathway in shaping adaptive immune responses to this vector. We showed here that a small and transient increase in CXCL-1 and IL-1β expression in livers of acid-α-glucosidase knockout (GAAKO) mice 6 h following injection with AAV2/8-CBhGAA. There was a robust antibody response to GAA in wild-type mice injected with this vector. In contrast, the anti-GAA IgG1 response was diminished in MyD88KO mice, and showed a trend toward a decrease in TRIFKO mice. In addition, the vector genome and GAA activity were significantly higher in MyD88KO livers compared with wild-type livers, suggesting reduced cytotoxic T cell responses. Importantly, elevated CD4+ T cells were detected by immunohistochemistry in MyD88KO livers. When adoptively transferred to wild-type mice, these CD4+ T cells have an ability to suppress antibody responses against AAV2/8-CBhGAA and to prevent further immunization against rhGAA. Our study suggests that the MyD88 deficiency leads to the suppression of deleterious immune responses to AAV2/8-CBhGAA, which has implications for gene therapy in Pompe disease.

Abstract 234: MyD88 and TRIF Mediate Divergent Responses Following Skeletal Muscle Ischemia

Introduction: We have shown that TLR2 mediates muscle regeneration and angiogenesis after muscle ischemia, independent of TLR4. Downstream signaling of TLR2 and TLR4 include MyD88 (TLR2 and 4) and TRIF (TLR4 and 3) pathways. We hypothesize that MyD88 is required for normal angiogenesis and muscle regeneration following ischemia while TRIF is not. Methods: MyD88 -/- (KO), TRIF KO and control C57B6 mice (WT) (N=4-7) underwent femoral artery ligation on the right hindlimb, and sham operation on the left. LDPI was performed at day 14 and anterior tibialis muscle was collected to quantify muscle necrosis and regeneration. Vascular structures were identified with CD31 staining. Results: MyD88 KO and WT mice demonstrated partial perfusion recovery by 14 days while TRIF KO had minimal recovery. Despite this, there were significantly fewer vascular structures in MyD88 KO mice while TRIF KO had the greatest (17.9±1.5 MyD88 KO; 55.3±6.8 WT; 69.1±5.2 TRIF KO; p&lt;0.01). TRIF KO exhibited marked muscle necrosis (43.1±19.9%) while WT and MyD88 KO had none (p&lt;0.01) (Figure). Instead, WT had large areas of regenerating myocytes (95.8 ± 0.96%; p &lt;0.01). Strikingly, MyD88 KO had almost no histologic evidence of ischemic injury with essentially normal muscle (85.3±12.6%, p&lt;0.01). Conclusions: Contrary to our hypothesis, TRIF appears to be required for normal muscle regeneration and functional angiogenesis after ischemia, while MyD88 is an important mediator of hypoxic injury. These results, along with our prior findings that muscle regeneration is independent of TLR4, suggest that TLR3 may play a key role in ischemic muscle recovery independent of angiogenesis.

The Role of MyD88 in the Adjuvant Activity of Neisseria meningitidis PorB (53.5)

Abstract Adjuvants enhance the response to poorly immunogenic antigens in vaccines. Our laboratory has shown by ELISA that Neisseria meningitidis outer membrane protein PorB acts as an adjuvant by eliciting a greater serum concentration of antigen-specific IgG when compared to immunization with antigen alone, with Ovalbumin as the antigen. PorB is a TLR2 ligand and signals through the adaptor protein MyD88. We compared the adjuvant effect of PorB to other adjuvants (Alum, MPL &amp; CpG) in WT &amp; MyD88 KO mice. PorB adjuvant activity was ablated in MyD88 KO mice and consistent with previous investigations: MPL &amp; CpG adjuvant activity was also MyD88-dependent while Alum acted independently of MyD88. In addition, we previously observed PorB had residual adjuvant activity in TLR2 KO mice. One hypothesis was that PorB could stimulate the Nalp3 inflammasome and induce IL-1β production without a second signal e.g. ATP. Therefore, bone marrow-derived macrophages were stimulated with PorB ± ATP. The IL-1β production was analyzed by ELISA and compared to IL-1β production from stimulation with other TLR ligands. We found that PorB required ATP in order to sufficiently produce IL-1β, disproving our hypothesis. Overall, our results show an importance for MyD88 in both the adjuvant pathway of PorB &amp; other TLR ligand-based adjuvants. One future direction for this work is MyD88 deletion in an antigen-presenting cell-specific manner to examine the role of each cell type in the adjuvant activity of PorB.

Role of innate signaling pathways in the regulation of antiviral IgG2c responses (108.9)

Abstract Long-term antibody responses provide protection from re-infection and recrudescence of persistent viruses. IgG2a/c is a predominant immunoglobulin isotype in most virus infections. Using a polyomavirus (PyV) mouse model, our lab has shown that MyD88 KO mice generate low levels of virus-specific IgG after the acute phase of infection, have reduced numbers of long-lived plasma cells and make very low levels of IgG2c, but normal levels of IgG1. Investigating the pathways upstream and downstream of MyD88, here we show that in response to PyV infection (i) Unc93B1 mutant mice, which are triple deficient in TLR3, 7 and 9 have diminished IgG2c, but normal IgG1 responses; (ii) TLR9 KO and TLR7 KO mice do not have greatly altered IgG isotype responses as observed in MyD88 KO mice or the Unc93B1 mutant mice, suggesting that these receptors play a redundant role; and (iii) mice lacking IRF5, a transcriptional regulator downstream of MyD88, have greatly diminished IgG2c and normal IgG1 responses, similar to MyD88 KO mice. The isotype switching defect observed in IRF5 KO mice is B cell intrinsic, as transfer of IRF5 KO B cells and WT T cells into RAG KO mice results in diminished antiviral IgG2c secretion compared to mice reconstituted with B6 B and T cells. Therefore, our studies suggest that TLR7/9-MyD88-IRF5 pathways in B cells play an essential role in the generation of IgG2a/c responses to virus infection.

Ageing and expression of TLR pathway associated genes in macrophages to Porphyromonas gingivalis challenge. (55.26)

Abstract Background: Periodontal disease is a chronic inflammatory disease that leads to progressive loss of soft and hard tissues supporting the teeth. Porphyromonas gingivalis (Pg) is a bacterium closely associated with periodontal disease. Toll-like receptors (TLR) and their intracellular signaling pathways play roles in host response to Pg. The focus of the current study was to define the expression profile of TLR pathway associated genes in macrophages (MØ) cultured with Pg, and to define changes in expression of these genes as a consequence of ageing. Methods: Bone marrow MØ from 2 months and1 year old wild type (WT), TLR2 knockout (KO), TLR4KO, MyD88KO and LPS2 mice were cultured with Pg. RNA was harvested from unchallenged and Pg challenged cells and expression of TLR pathway associated genes was measured by qPCR microarrays. Results: Pg challenge of WT MØ induced expression of a large set of TLR pathway associated genes. MØ gene expression data revealed 19, 48, 45 and 56 genes differentially regulated in TLR2KO, TLR4KO, MyD88KO and LPS2 mice respectively as compared with WT in response to Pg. We identified 19 genes that are commonly regulated in all four mutant genotypes. Age had little effect on the differential regulation of these genes by mutant MØ as compared with WT. Conclusions: Regulation of TLR pathway associated genes by MØ was highly responsive to Pg challenge. Pg challenge and mouse genotype had the strongest effects on gene expression in this model.

NALP3 inflammasome activation is MYD88-, but not TLR4-dependent and involves both parenchymal and bone marrow derived cells in non-alcoholic steatohepatitis (NASH)

Background and Aims: Endogenous danger signals from damaged hepatocytes in NASH might up-regulate and activate the inflammasome, that is a multiprotein complex that leads to IL-1ß secretion. We reported NALP3 inflammasome up-regulation activation in NASH. Here, we aimed to investigate the role of toll-like receptor 4 (TLR4) and the common TLR adaptor, MyD88 in the activation of NALP3 inflammasome. Furthermore, we aimed to investigate which cell types are involved in the inflammasome activation in the liver in NASH. Methods: Wild type (WT), TLR4- and MyD88-deficient (KO) mice, and WT mice transplanted with MyD88 KO bone marrow (WT/MyD88KO-BM) or MyD88KO mice with WT bone marrow (MyD88KO/WT-BM) received methionine-choline-deficient (MCD) or -supplemented (MCS) diet. Results: MCD diet-induced NASH resulted in up-regulation of TLR4, MyD88 and NALP3 inflammasome (NALP3, ASC, pro-caspase-1) mRNA, and mature IL-1β (17kDa) protein and caspase-1 activation in the liver compared to MCS diet feeding in WT mice. Total body deficiency in TLR4 although attenuated the MCD diet-induced liver injury and steatosis, but did not prevent the either the NALP3 up-regulation or the IL-1β production. MyD88 is a common adaptor molecule of most of the TLRs, including TLR4, but also TLR9. Total body deficiency in MyD88 prevented up-regulation of NALP3 mRNA and mature IL-1β production and significantly attenuated liver damage and steatosis in NASH. Selective MyD88-deficiency in bone marrow-derived or parenchymal cells prevented NALP3 mRNA up-regulation and attenuated IL-1β production in both chimeras (WT/MyD88KO-BM and MyD88KO/WT-BM)). Interestingly, WT/MyD88KO-BM mice were protected from MCD diet induced steatosis and liver damage, but liver injury was comparable between MCD diet-fed WT and MyD88KO/WT-BM mice suggesting that BM-derived cells contribute to NASH in a MyD88-dependent manner. Conclusions: We demonstrate for the first time that NALP3 inflammasome activation requires MyD88-dependent signaling in the MCD diet-induced NASH, but the deficiency of TLR4 is not enough to prevent the infammasome priming and activation. Finally, we show that both bone marrow-derived and parenchymal cells contribute to IL-1β production and the absence of MyD88 in bone marrow-derived cells attenuates steatohepatitis.

2261 SIGNIFICANCE AND THERAPEUTIC POTENTIAL OF TARGETING DELTA-LIKE 4 IN RENAL ISCHEMIA REPERFUSION INJURY

You have accessJournal of UrologyTransplantation & Vascular Surgery: Renal Transplantation, Vascular Surgery II1 Apr 20122261 SIGNIFICANCE AND THERAPEUTIC POTENTIAL OF TARGETING DELTA-LIKE 4 IN RENAL ISCHEMIA REPERFUSION INJURY Kiyohiko Hotta, Masayuki Sho, Ichiro Yamato, Hiroshi Harada, Hideo Yagita, Yoshiyuki Nakajima, and Katsuya Nonomura Kiyohiko HottaKiyohiko Hotta Sapporo, Japan More articles by this author , Masayuki ShoMasayuki Sho Kashihara, Japan More articles by this author , Ichiro YamatoIchiro Yamato Kashihara, Japan More articles by this author , Hiroshi HaradaHiroshi Harada Sapporo, Japan More articles by this author , Hideo YagitaHideo Yagita Tokyo, Japan More articles by this author , Yoshiyuki NakajimaYoshiyuki Nakajima Kashihara, Japan More articles by this author , and Katsuya NonomuraKatsuya Nonomura Sapporo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.2439AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Delta-Like 4 (DL4), which is one of Notch ligands, affects many differentiation processes and cell fate determination during embryonic and postnatal development. Furthermore, it regulates tumor angiogenesis and promotes tumor growth. Recently it has been recognized as a critical regulator in lots of immune responses. However, its role in ischemia reperfusion injury (IRI) remains unknown. In this study, we investigated the role of DLL4 in IRI. METHODS IRI was induced in male C57BL/6 mice and MyD88 KO mice. The right kidney was removed, and then left renal pedicle was clamped for 30 minutes. In a lethal model, the left kidney vessels of mice were clamped for 40 minutes. To clarify the precise functions, we employed anti-DLL4 blocking mAb (HMD4-1). RESULTS The HMD4-1 treatment significantly inhibited renal IRI as determined by serum creatinine level. Histological analysis revealed that severe tubular necrosis and massive cellular infiltrations were identified in the control kidney, while there were less necrosis and cellular infiltrations in the treated kidney. Furthermore, the treatment significantly inhibited the accumulation of both neutrophils and macrophages as determined by MPO and F4/80 staining, respectively. To address the underlying mechanisms, we evaluated local immune status. Real-time PCR analysis indicated that the treatment downregulated the expressions of several potent proinflammatory cytokines, chemokines, and adhesion molecules. Moreover, apoptosis, as measured by TUNEL staining, was also significantly inhibited. Importantly, while there was a decrease in renal tubules and collagen deposition in interstitial tissue in control kidneys at 30 days after reperfusion, tubular architecture was preserved and collagen deposition was less in the treated kidneys. Thus, DLL4 blockade attenuated the development of chronic tubular fibrosis. Furthermore, DLL4 blockade significantly prolonged the survival of lethally injured mice. Taken together, DLL4 blockade inhibited local immune activation as well as cellular infiltrations, thereby resulting in the inhibition of apoptosis and preservation of renal functions. Finally, to reveal signaling pathway of DLL4 in IRI, we used MyD88 KO mice. The protection induced by DLL4 blockade was no longer observed in MyD88 KO mice, indicating that DLL4 signaling was dependent on MyD88-mediated pathway in IRI. CONCLUSIONS Our data highlight for the first time the significance of DLL4 in the pathogenesis of IRI and also clinical potential of its targeting as a novel therapeutic strategy. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e912-e913 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kiyohiko Hotta Sapporo, Japan More articles by this author Masayuki Sho Kashihara, Japan More articles by this author Ichiro Yamato Kashihara, Japan More articles by this author Hiroshi Harada Sapporo, Japan More articles by this author Hideo Yagita Tokyo, Japan More articles by this author Yoshiyuki Nakajima Kashihara, Japan More articles by this author Katsuya Nonomura Sapporo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 3676: MyD88-Dependent Pathway Has a Dual Effect on Brain Injury after Cerebral Ischemia/Reperfusion in Mouse

Myeloid differentiation primary-response protein-88 (MyD88) is one of the intracellular adaptors in Toll-like receptor (TLR) signaling pathways, which regulates the activation of nuclear factor kappa B (NF-kappa-B) and transcription of other proinflammatory cytokines. We assessed the hypothesis that MyD88-dependent signaling mediates the inflammatory responses in ischemic brain and is implicated in the process of brain injury. A mouse model of cerebral ischemia/reperfusion (I/R) was induced by transient middle cerebral artery occlusion (tMCAO). MyD88 knockout (KO) mice and wild type control mice (WT, C57BL/6J) were assigned to I/R or sham-operated groups. The mice were killed at 4 hrs or 24 hrs after reperfusion following 60 minutes of MCAO. Using Nissl staining, brain infarct areas were evaluated and expressed as a percentage of the whole brain. Brain edema was evaluated and presented as the percentage of water content in the total weight of the brain tissue. The expression of mRNA was detected by real-time PCR, and the levels of inflammatory cytokines were measured by ELISA and Western Blots. The infarct size was analyzed by t-test. Brain edema and inflammatory response were analyzed by one way ANOVA with post hoc comparisons. Our results showed that the brain infarct size was not significantly different in MyD88KO mice compared with WT mice 24 hrs after cerebral I/R. However, brain edema (water content) significantly increased in MyD88KO mice compared with WT 24 hrs after ischemia (p&lt;0.05). The data also showed that the levels of mRNA of CD14, Pellino-1, cyclooxygenase-2 (COX-2), and Interferon beta-1 (INFb1) increased significantly 4 hrs after cerebral I/R in WT mice compared to sham-operated controls (p&lt;0.05). MyD88 deficiency inhibited the increased expression of Pellino-1 (p&lt;0.05) and COX-2 (p&lt;0.05), but not CD14. Twenty-four hrs after cerebral I/R, the protein levels of Interleukin - 1-beta, Interleukin - 6 (IL-6), p-IkappaB alpha, and the activity of NFkappaB, significantly increased in the ischemic brain in WT mice, and were inhibited in MyD88KO mice. Interestingly, VEGF significantly increased in MyD88KO mice compared with WT mice (p&lt;0.05). In addition, ZO-1, a tight-junction protein on the blood-brain barrier (BBB) decreased in MyD88KO mice compared with WT mice subjected to cerebral I/R. Our data demonstrates that MyD88 contributes to the activation of inflammatory responses after cerebral I/R. However, MyD88 reduces brain edema by mediating the expression of VEGF, and tight-junction proteins, which are important for maintaining BBB integrity. We concluded that the MyD88-dependent pathway has a dual effect on brain injury induced by cerebral I/R. Interpreting the results for the role of MyD88 dependent pathway based on brain injury from whole animal experiments is cautioned.

Pivotal Role of Toll-Like Receptors 2 and 4, Its Adaptor Molecule MyD88, and Inflammasome Complex in Experimental Tubule-Interstitial Nephritis

Tubule-interstitial nephritis (TIN) results in decreased renal function and interstitial inflammation, which ultimately leads to fibrosis. Excessive adenine intake can cause TIN because xanthine dehydrogenase (XDH) can convert this purine into an insoluble compound, which precipitates in the tubuli. Innate immune sensors, such as Toll-like receptors (TLR) and inflammasome complex, play a crucial role in the initiation of inflammation. The aim of this study was to evaluate the roles of TLR-2 and -4, Myd88 and inflammasome complex in an experimental model of TIN. Here, we show that wild-type (WT) mice fed adenine-enriched food exhibited significant renal dysfunction and enhanced cellular infiltration accompanied by collagen deposition. They also presented higher gene and protein expression of pro-inflammatory cytokines. In contrast, TLR-2, -4, MyD88, ASC and Caspase-1 KO mice showed renoprotection associated with expression of inflammatory molecules at levels comparable to controls. Furthermore, treatment of WT animals with allopurinol, an XDH inhibitor, led to reduced levels of uric acid, oxidative stress, collagen deposition and a downregulation of the NF-kB signaling pathway. We concluded that MyD88 signaling and inflammasome participate in the development of TIN. Furthermore, inhibition of XDH seems to be a promising way to therapeutically target the developing inflammatory process.

Anti-CD180 (RP105) Activates B Cells To Rapidly Produce Polyclonal Ig via a T Cell and MyD88-Independent Pathway

CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is unclear. We report that injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses, with the exception of IgA and IgG2b, with up to 50-fold increases in serum IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR knockout [KO]), CD40 KO, IL-4 KO, and MyD88 KO mice. Anti-CD180 rapidly increased both transitional and mature B cells, with especially robust increases in transitional B cell number, marginal zone B cell proliferation, and CD86, but not CD80, expression. In contrast, anti-CD40 induced primarily follicular B cell and myeloid expansion, with increases in expression of CD80 and CD95 but not CD86. The expansion of splenic B cells was due, in part, to proliferation and occurred in wild-type and TCR KO mice, whereas T cell expansion occurred in wild-type, but not in B cell-deficient, mice, indicating a direct role for B cells in CD180 stimulation in vivo. Combination of anti-CD180 with various MyD88-dependent TLR ligands biased B cell fate because coinjection diminished Ig production, but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells, but it increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus, CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.

Abstract 412: Role of Interferon Regulatory Factor (IRF)-3 in lung tumor microenvironment

Abstract Cytokines present in the tumor microenvironment influence whether a cancer progresses or is rejected. For example, IFNs promote anti-tumor immunity and thus contribute to tumor rejection whereas IL-10 and TGFb can inhibit anti-tumor immunity and thereby contribute to tumor survival. We find that IFNbeta levels are significantly up-regulated in the microenvironment surrounding lung tumors. In the B16 F10 mouse melanoma lung metastasis model, CD11c+ alveolar cells respond to tumor challenge by rapidly up-regulating their expression of IFNbeta. This induction of IFNbeta was totally absent in IRF3 KO mice but intact in MyD88 and TRIF KO mice. Challenge studies indicate that IRF3 KO mice develop more lung metastasis and have shorter survival than controls. These findings suggest that CD11c+ cells play an important role in tumor recognition, and that they mount an IRF3/IFNbeta pathway specific response that slows tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 412. doi:10.1158/1538-7445.AM2011-412

CD180 (RP105) intrinsically activates B cells to produce polyclonal Ig and integrates diverse TLR signals to modify cell fate (113.15)

Abstract CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is disputed. In this work, injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses except IgA and IgG2b, with up to 50-fold serum increases of IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR KO) and MyD88 KO mice. Expansion of splenic B cells occurred in WT and TCR KO mice whereas T cell expansion occurred in WT but not in B cell-deficient (μMT) mice, indicating a direct role for B cells but not T cells in CD180 stimulation in vivo. Combinations of anti-CD180 with various TLR ligands biased B cell fate as co-injection diminished Ig production but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells but increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.