MyD88-independent Signaling Research Articles

Rutin, a natural flavonoid glycoside, ameliorates zearalenone induced liver inflammation via inhibiting lipopolysaccharide gut leakage and NF-κB signaling pathway in mice

Zearalenone (ZEN) poses a potential threat on human and animal health partly through the nuclear factor (NF)-κB signaling pathway. In silico study suggested that rutin effective against TLR4 and NF-κB. A wetting test was designed to evaluate the effect and underlying mechanism of rutin in alleviating ZEN-induced inflammation in animals. Twenty-four female mice were randomly divided into 4 groups: control (basal diet), ZEN group (basal diet + ZEN), rutin group (basic diet + rutin), Z + R group (basal diet + rutin + ZEN). Results showed that rutin effectively alleviated ZEN-induced inflammation and damage of liver and jejunum in mice. Rutin addition reduced the content of lipopolysaccharide (LPS) in serum and liver mainly by improving the intestinal barrier function resulted from the production increase of short-chain fatty acids (SCFA). In sum, this study showed that rutin alleviated ZEN-induced liver inflammation and injury by modulating the gut microbiota, increasing the production of SCFA and improving intestinal barrier function, leading to the decrease of LPS in liver and the inhibition of MyD88 independent NF-κB signaling pathway in mice. Specifically, these findings may provide useful insights into the screening of functional natural compounds and its action mechanism to alleviate ZEN induced liver inflammation.

The podocytes’ inflammatory responses in experimental GN are independent of canonical MYD88-dependent toll-like receptor signaling

Podocytes form the kidney filtration barrier and continuously adjust to external stimuli to preserve their integrity even in the presence of inflammation. It was suggested that canonical toll-like receptor signaling, mediated by the adaptor protein MYD88, plays a crucial role in initiating inflammatory responses in glomerulonephritis (GN). We explored the influence of podocyte-intrinsic MYD88 by challenging wild-type (WT) and podocyte-specific Myd88 knockout (MyD88pko) mice, with a model of experimental GN (nephrotoxic nephritis, NTN). Next-generation sequencing revealed a robust upregulation of inflammatory pathways and changes in cytoskeletal and cell adhesion proteins in sorted podocytes from WT mice during disease. Unchallenged MyD88pko mice were healthy and showed no proteinuria, normal kidney function and lacked morphological changes. During NTN, MyD88pko exhibited a transient increase in proteinuria in comparison to littermates, while histological damage, podocyte ultrastructure in STED imaging and frequencies of infiltrating immune cells by flow cytometry were unchanged. MYD88-deficiency led to subtle changes in the podocyte transcriptome, without a significant impact on the overall podocyte response to inflammation, presumably through MYD88-independent signaling pathways. In conclusion, our study reveals a comprehensive analysis of podocyte adaptation to an inflammatory environment on the transcriptome level, while MYD88-deficiency had only limited impact on the course of GN suggesting additional signaling through MYD88-independent signaling.

Abstract 554: Monocyte Toll-like Receptor 4 (tlr4) Signaling Is Essential For Post Thrombotic Upa Expression

Background: Leukocyte expression of Toll-like receptor 4 (TLR4) increases with transformation to chronic deep venous thrombosis (DVT) & in non-resolving thrombi, but TLR4 function during thrombus clearance is unclear. We hypothesized TLR4 signaling to regulate expression of fibrinolytic factors in monocytes & macrophages (MO/MFs) for VT resolution. Methods: Small interfering RNA (siRNA) knockdown (KD) of TLR4 & disruption of Myd88-dependent/independent pathways occurred in immortalized MFs (RAW264.7), with quantitative real-time PCR (qRT-PCR) for a panel of fibrinolytic factors & immunoblotting for urokinase (uPA). Tlr4-/- mice & wild-type controls underwent IVC stasis/ligation, yielding VT at 2h, 2d, & 8d. Murine/chronic human thrombi underwent immunofluorescent staining for indicated targets & murine thrombi weight-length ratios were found. Splenic & Bone marrow-derived MFs from Tlr4 Cre mice were immunoblotted for uPA. Results: TLR4 silencing in RAW264.7 cells yielded suppression of uPA mRNA (Fig 1B) & protein levels (not shown). TRIF KD yielded decreased uPA mRNA (Fig 1C) & protein levels (not shown), implicating Myd88-independent signaling in MO/MF uPA production. Human (not shown) & murine chronic thrombi staining saw TLR4 co-localization with MF marker CCR2 (Fig 1D). Global knockout (KO) of murine TLR4 did not affect early thrombogenesis (Fig 1E left), with delayed VT resolution seen from 2d (Fig 1E right). Decreased uPA protein levels were observed in Tlr4 KO mice (Fig 1F). Conclusions: TLR4 signaling stimulates in vivo VT resolution. Expression of fibrinolytic factor uPA is enforced in MO/MFs by TLR4-Myd88-independent signaling, a potential target for VT resolution.

TIRAP, TRAM, and Toll-Like Receptors: The Untold Story.

Toll-like receptors (TLRs) are the most studied receptors among the pattern recognition receptors (PRRs). They act as microbial sensors, playing major roles in the regulation of the innate immune system. TLRs mediate their cellular functions through the activation of MyD88-dependent or MyD88-independent signaling pathways. Myd88, or myeloid differentiation primary response 88, is a cytosolic adaptor protein essential for the induction of proinflammatory cytokines by all TLRs except TLR3. While the crucial role of Myd88 is well described, the contribution of other adaptors in mediating TLR signaling and function has been underestimated. In this review, we highlight important results demonstrating that TIRAP and TRAM adaptors are also required for full signaling activity and responses induced by most TLRs.

TLR9 triggers MyD88-independent anti-inflammatory signaling in lupus

Activation of Toll-like receptor 7 (TLR7) can induce lupus in mice, whereas activation of TLR9 can prevent it, even though both receptors interact with myeloid differentiation primary response gene 88 (MyD88) for downstream signaling. How TLR9 triggers anti-inflammatory responses in autoimmunity is unclear. Leibler et al. recently reported that TLR9 initiates anti-inflammatory signaling and inhibits lupus pathogenesis in a MyD88-independent but ligand-dependent manner.

Schisandrin B Attenuates Diabetic Cardiomyopathy by Targeting MyD88 and Inhibiting MyD88-Dependent Inflammation.

Diabetes manifests as chronic inflammation and leads to the development diabetic cardiomyopathy (DCM). Targeting key proteins in inflammatory signaling may provide new therapy for DCM. In this study, the authors explore the pharmacological effects and mechanisms of Schisandrin B (SchB), a natural compound with anti-inflammatory activity against DCM. It is shown that SchB prevents high-level glucose (HG)-induced hypertrophic and fibrotic responses in cultured cardiomyocytes. RNA sequencing and inflammatory qPCR microarray show that SchB mainly affects myeloid differentiation primary response 88 (MyD88)-dependent inflammatory gene expression in HG-challenged cardiomyocytes. Further studies indicate that SchB directly binds to and inhibits MyD88 activation, but does not alter MyD88-independent Toll-like receptor signaling invivo and invitro. Inhibiting or silencing MyD88 is associated with reduced levels of HG-induced inflammatory cytokines and myocardial injuries invitro. Treatment of type1 and type2 diabetic mice with SchB protects heart function, reduces myocardial injuries, and decreases secretion of inflammatory cytokines. Cardiomyocyte-specific MyD88 knockout also protects mice against cardiac inflammation and injury in type1 diabetic mice. In conclusion, these studies show that cardiomyocyte MyD88 plays an apathogenetic role in DCM and SchB specifically targets MyD88 to reduce inflammatory DCM.

Adaptive Evolution in TRIF Leads to Discordance between Human and Mouse Innate Immune Signaling.

The TIR domain-containing adapter inducing IFN-β (TRIF) protein is an innate immune system protein that mediates the MyD88-independent toll-like receptor response pathway in mice and humans. Previously, we identified positive selection at seven distinct residues in mouse TRIF (mTRIF), as compared with human and other mammalian orthologs, thus predicting protein functional shift in mTRIF. We reconstructed TRIF for the most recent common ancestor of mouse and human, and mutated this at the seven sites to their extant mouse/human states. We overexpressed these TRIF mutants in immortalized human and mouse cell lines and monitored TRIF-dependent cytokine production and gene expression induction. We show that optimal TRIF function in human and mouse is dependent on the identity of the positively selected sites. These data provide us with molecular data relating observed differences in response between mouse and human MyD88-independent signaling in the innate immune system with protein functional change.

P38 MAPK activation by Toxoplasma gondii secretory effector protein GRA24: Role in cytokine regulation and induction of protective immunity

Abstract The apicomplexan Toxoplasma gondii induces strong protective immunity in part dependent upon recognition by Toll-like receptors (TLR) 11 and 12 operating in conjunction with MyD88 in the murine host. However, these TLR are not present in humans, prompting us to investigate MyD88-independent signaling pathways of resistance. We show here that parasite dense granule protein GRA24 induces p38 MAPK activation and subsequent IL-12 production in bone marrow derived mouse macrophages (BMDM). Furthermore, Toxoplasma triggers two waves of p38 MAPK phosphorylation in MyD88+/+ and MyD88−/− BMDM. Using the uracil auxotrophic Type I T. gondii strain cps1-1, we demonstrate that the late response does not require active parasite proliferation, but strictly depends upon GRA24. By i. p. inoculation with cps1-1 and cps1-1:Δgra24, we identified unique subsets of chemokines and cytokines that were up and down regulated by GRA24. We demonstrate that cps1-1 triggers a strong host-protective GRA24-dependent Th1 response in the absence of MyD88. To gain deeper insight into genes regulated by GRA24, we employed qPCR to evaluate expression of transcripts for 84 cytokine and chemokine genes in BMDM infected with GRA24-positive compared to GRA24-negative parasites. Our results identify a specific signature of genes regulated by this dense granule protein. In conjunction, using qPCR we evaluated the impact of GRA24 on a panel of macrophage long noncoding RNA (lncRNA) regulated by Toxoplasma infection. Taken together, our data identify GRA24 as a major mediator of p38 MAPK activation, induction of IL-12 and other immune mediators, as well as protective immunity that operates independently of the TLR/MyD88 cascade.

Sexually Dimorphic Role of Toll-like Receptor 4 (TLR4) in High Molecular Weight Hyaluronan (HMWH)-induced Anti-hyperalgesia

High molecular weight hyaluronan (HMWH), a prominent component of the extracellular matrix binds to and signals via multiple receptors, including cluster of differentiation 44 (CD44) and toll-like receptor 4 (TLR4). We tested the hypothesis that, in the setting of inflammation, HMWH acts at TLR4 to attenuate hyperalgesia. We found that the attenuation of prostaglandin E2 (PGE2)-induced hyperalgesia by HMWH was attenuated by a TLR4 antagonist (NBP2-26245), but only in male and ovariectomized female rats. In this study we sought to evaluated the role of the TLR4 signaling pathway in anti-hyperalgesia induced by HMWH in male rats. Decreasing expression of TLR4 in nociceptors, by intrathecal administration of an oligodeoxynucleotide (ODN) antisense to TLR4 mRNA, also attenuated HMWH-induced anti-hyperalgesia, in male and ovariectomized female rats. Estrogen replacement in ovariectomized females reconstituted the gonad-intact phenotype. The administration of an inhibitor of myeloid differentiation factor 88 (MyD88), a TLR4 second messenger, attenuated HMWH-induced anti-hyperalgesia, while an inhibitor of the MyD88-independent TLR4 signaling pathway did not. Since it has previously been shown that HMWH-induced anti-hyperalgesia is also mediated, in part by CD44 we evaluated the effect of the combination of ODN antisense to TLR4 and CD44 mRNA. This treatment completely reversed HMWH-induced anti-hyperalgesia in male rats. Our results demonstrate a sex hormone-dependent, sexually dimorphic involvement of TLR4 in HMWH-induced anti-hyperalgesia, that is MyD88 dependent. PerspectiveThe role of TLR4 in anti-hyperalgesia induced by HMWH is a sexually dimorphic, TLR4 dependent inhibition of inflammatory hyperalgesia that provides a novel molecular target for the treatment of inflammatory pain.

Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages.

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-β and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-β pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.

Heme Induced Platelet Mitochondrial Oxidant Production Regulates Thrombospondin-1 Release from Platelets

Background: Sickle Cell Disease (SCD) patients develop chronic vasculopathy leading to conditions such as pulmonary hypertension (PH), a major cause of morbidity and mortality. Vasculopathy is tightly associated with hemolysis in SCD. We and others have shown that in SCD, hemolysis activates platelets, and activated platelets are implicated in vasculopathy through their release of vasoactive molecules, such as thrombosponding-1 (TSP1), from storage granules. In SCD, free heme is accumulated in the circulation as a consequence of oxidative degradation of hemoglobin secondary to intravascular hemolysis. Under homeostatic conditions, albumin, haptoglobin, hemopexin, and high- and low-density lipoproteins clear plasma heme from the circulation. However excessive hemolysis can overwhelm these clearance mechanisms, resulting in elevated levels of free heme, which can lead to platelet activation, aggregation and thrombus formation. The role of activated platelets in these thrombotic events is well studied, but the mechanisms by which heme induces platelet activation and platelet-dependent signaling to the vessel wall remain unclear. We previously showed that hemolysis induces platelet mitochondrial reactive oxygen species (mtROS) production, but the mechanism by which heme induces mtROS production and downstream consequences are less clear. We hypothesize that heme induces platelet mtROS through Toll-like Receptor 4 (TLR4) signaling, which regulates platelet activation and granule secretion. Methods: Washed platelets were prepared from whole blood collected from healthy human volunteers in 0.32% sodium citrate anticoagulant (n=6). Platelets were treated with heme (2.5µM) in presence or absence of TLR4 neutralizing antibody, IRAK1/4 inhibitor, TBK1/IKKε inhibitor, MitoTEMPO or DS16570511 (Mitochondrial calcium uniporter inhibitor). Platelet activation was measured using flow cytometry by staining for CD62P (P-selectin) on the platelet surface and the active confirmation of GPIIb/IIIa. Platelet mtROS was measured by fluorescence spectroscopy using MitoSOX Red. The granule secretion function of platelets was measured by assessing TSP-1 and Platelet Factor 4 (PF4) levels in platelet releasates by ELISA. Results: We found that heme activates platelets and induces mtROS production, which can be mitigated by TLR4 neutralizing antibody, a molecule that makes platelet surface TLR4 unavailable to heme. In heme-treated platelets, pharmacological inhibition of the interleukin-1 receptor kinase-4 (IRAK4), a key kinase recruited by the TLR4 adaptor protein MyD88, had no significant effect on platelet activation. In contrast, inhibition of tank binding kinase-1 (TBK1), a TLR4 effector molecule independent of MyD88 signaling, significantly attenuated heme-mediated platelet activation and mtROS production. Scavenging mtROS with MitoTEMPO did not alter platelet activation, instead it decreased the TSP1 release from platelets, indicating heme induced platelet mtROS tightly regulates the granule secretion function of platelets. In addition, blocking mitochondrial calcium uptake by Mitochondrial calcium uniporter (MCU) inhibitor significantly decreased mtROS production and TSP-1 release from heme treated platelets. Conclusion: Our data demonstrate that heme activates human platelets and induces mtROS production via a TLR4 -MyD88-independent signaling pathway. In addition, heme also induces mitochondrial calcium uptake that enhances mtROS production in platelets. Heme induced platelet mtROS tightly regulates platelet granule secretion function. Ongoing studies are further elucidating this pathway and determining the role of mtROS in heme-dependent platelet function. These studies will provide a mechanistic link between hemolysis and thrombosis, as well as platelet-vascular wall signaling. These studies suggest that platelet mtROS may potentially be a novel therapeutic target to prevent thrombotic events and vascular dysfunction in hemolytic disorders including SCD. Disclosures No relevant conflicts of interest to declare.

Heme Induces Platelet mtROS Production via MyD88-independent TLR4 Signaling that Regulates Thrombospondin-1 Release from Platelets

Heme Induces Platelet mtROS Production via MyD88-independent TLR4 Signaling that Regulates Thrombospondin-1 Release from Platelets

Lipopolysaccharide-induced DC-SIGN/TLR4 crosstalk activates NLRP3 inflammasomes via MyD88-independent signaling in gastric epithelial cells

Abnormal pattern recognition receptor (PRR) signaling plays an important role in gastric mucosal damage caused by stomach microbiota; however, the underlying molecular mechanisms remain obscure. Here, we show that DC-SIGN, a surface phenotype marker of dendritic cells, is overexpressed in gastric epithelial cells facing LPS stimulation. NLRP3 expression in gastric epithelial cells are significantly increased and related to the degree of LPS stimulation. Furthermore, DC-SIGN could interact with TLR4, promote NLRP3 and related genes expression via MyD88-independent signaling pathway and regulate the secretion of IL-1β and IL-18 in gastric epithelial cells. The results of flow cytometry analysis show that DC-SIGN primarily mediates Th1 differentiation when co-cultured with gastric epithelial cells. These results reveal that LPS-induced DC-SIGN expression modulates NLRP3 inflammasomes formation via MyD88-independent TLR4 signaling in gastric epithelial cell, and induces a Th1-predominant host immune response,these findings may indicate a new function of DC-SIGN in non-immune cells, and elucidate the diversity role of gastric epithelial cells in mechanism of immune damage caused by microbial flora.

MyD88-dependent and -independent signalling via TLR3 and TLR4 are differentially modulated by Δ9-tetrahydrocannabinol and cannabidiol in human macrophages

Toll-like receptors (TLRs) are sensors of pathogen-associated molecules that trigger inflammatory signalling in innate immune cells including macrophages. All TLRs, with the exception of TLR3, promote intracellular signalling via recruitment of the myeloid differentiation factor 88 (MyD88) adaptor, while TLR3 signals via Toll-Interleukin-1 Receptor (TIR)-domain-containing adaptor-inducing interferon (IFN)-β (TRIF) adaptor to induce MyD88-independent signalling. Furthermore, TLR4 can activate both MyD88-dependent and –independent signalling (via TRIF). The study aim was to decipher the impact of the highly purified plant-derived (phyto) cannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), when delivered in isolation and in combination (1:1), on MyD88-dependent and -independent signalling in macrophages. We employed the use of the viral dsRNA mimetic poly(I:C) and endotoxin lipopolysaccharide (LPS), to induce viral TLR3 and bacterial TLR4 signalling in human Tamm-Horsfall protein-1 (THP-1)-derived macrophages, respectively. TLR3/TLR4 stimulation promoted the activation of interferon (IFN) regulatory factor 3 (IRF3) and TLR4 promoted the activation of nuclear factor (NF)-κB signalling, with downstream production of the type I IFN-β, the chemokines CXCL10 and CXCL8, and cytokine TNF-α. THC and CBD (both at 10 μM) attenuated TLR3/4-induced IRF3 activation and induction of CXCL10/IFN-β, while both phytocannabinoids failed to impact TLR4-induced IκB-α degradation and TNF-α/CXCL8 expression. The role of CB1, CB2 and PPARγ receptors in mediating the effect of THC and CBD on MyD88-independent signalling was investigated. TLRs are attractive therapeutic targets given their role in inflammation and initiation of adaptive immunity, and data herein indicate that both CBD and THC preferentially modulate TLR3 and TLR4 signalling via MyD88-independent mechanisms in macrophages. This offers mechanistic insight into the role of phytocannabinoids in modulating cellular inflammation.

Ferric Heme Induces Platelet Activation and mtROS Production through a MyD88-independent TLR4 Signaling Pathway

Ferric Heme Induces Platelet Activation and mtROS Production through a MyD88-independent TLR4 Signaling Pathway

Lipopolysaccharide enhances DNA-induced IFN-β expression and autophagy by upregulating cGAS expression in A549 cells.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is a newly identified cytosolic DNA sensor, but its function in lung epithelial cells is relatively unknown. In the present study, the effects of lipopolysaccharide (LPS) on the expression and function of cGAS in the A549 lung epithelial cell line was investigated. The cells were treated with LPS at different concentrations (e.g., 100, 200, 400 and 800 ng/ml), and the cGAS expression levels were examined via western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The cells were pretreated with LPS, followed by E. coli DNA transfection using Lipofectamine® 3000. After 24 h, interferon (IFN)-β production was measured using ELISA and the expression of the autophagic markers, microtubule-associated proteins 1A/1B light chain 3 and sequestosome-1, were determined using western blot analysis. The cells were also pretreated with either a toll-like receptor (TLR) 4 inhibitor, a serine/threonine-protein kinase TBK1 (TBK1) inhibitor or an nuclear factor (NF)-κB inhibitor, followed by LPS treatment, and the cGAS expression levels were examined via western blot analysis and RT-qPCR. The result showed that LPS treatment upregulated cGAS expression in a dose-dependent manner. E. coli DNA treatment could induce IFN-β production and autophagy via cGAS, which was enhanced by LPS pretreatment. The effect of LPS on cGAS expression was suppressed by treatment with a TLR4 inhibitor, a TBK1 inhibitor and an NF-κB inhibitor. In conclusion, LPS enhances DNA-induced IFN-β production and autophagy by upregulating cGAS expression through the myeloid differentiation primary response protein MyD88-independent TLR4 signaling pathway in A549 cells.

Abstract 2563: Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies

Abstract Recent clinical trials using chimeric antigen receptors (CAR) T cells have demonstrated tremendous success in eradicating hematologic malignancies. Notwithstanding the excitement generated by CAR T cell therapy, its clinical efficacy has not been effectively translated to the context of solid tumors; the physical barriers of solid malignancies and the immunosuppressive conditions at the tumor site hinder the efficacy of CAR T cells. Macrophages have the ability to infiltrate almost every tissue and frequently are recruited into tumors. Therefore, macrophages are an attractive vehicle for CAR therapy and could help solve current challenges that CAR T cells face in the treatment of solid tumors. MOTO-CAR cells are monocyte-derived human macrophages that are genetically modified by a lentiviral or adenoviral approach to express a synthetic tumor-targeting receptor and to secrete cytokines, ligands or chemokine receptors. MOTO-CAR receptors are composed of a single-chain variable fragment (ScFv) that binds to a specific tumor target, a hinge to link it to a transmembrane domain, and an engineered Toll/Interleukin-1 receptor (TIR) signaling domain. When the ScFv binds to the tumor cell via its tumor target, an activation signal is transmitted. Myd88 dependent and independent signaling cascades are elicited, activating the macrophage and polarizing it towards a proinflammatory phenotype to eliminate cancer cells in a selective way. We previously reported the expression of Thymidine Kinase 1 (TK1) on the cell membrane of the non-small cell lung carcinoma NCI-H460 and A549 cell lines. The in vitro function of TK1 MOTO-CAR cells was evaluated against these cancer cell lines, using GFP-based phagocytosis and killing assays. Additionally, cell migration and interaction was recorded using time-lapse video with a confocal microscope. Upon co-culturing, with its target TK1 specific MOTO-CARs showed a nearly 4-fold increase in killing activity when compared with the controls (p<0.01). MOTO-CAR cells were produced through a lentiviral approach with around 30% of the cells expressing MOTO-CARs and with an adenoviral approach using the Ad5f35 vector with an efficiency of 70-80% of cells being transduced. Furthermore, after transduction MOTO-CAR cells showed a consistent M1 phenotype expressing high levels of CD14, CD80, CD206 and low levels of CD163. Time-lapse videos showed migration and clustering of MOTO-CAR cells around H460 GFP + cells. Moreover, cell death was observed upon contact of MOTO-CAR cells with target cells as well as phagocytic activity. In vivo testing using an orthotopic NOD scid gamma mice model is in progress. Our preclinical data show evidence that human macrophages are a suitable vehicle for CAR therapy and have the potential to successfully extrapolate the clinical efficacy of CAR therapy to the context of solid tumors. Citation Format: Edwin J. Velazquez, John E. Lattin, Taylor D. Brindley, Zachary Z. Reinstein, Roger Chu, Lu Liu, Evita G. Weagel, Michelle H. Townsend, Kiara V. Whitley, Eliza L. Lawrence, Brandon T. Garcia, Scott Weber, Richard A. Robison, Kim L. O'Neill. Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2563.

The inhibition of RANKL expression in fibroblasts attenuate CoCr particles induced aseptic prosthesis loosening via the MyD88-independent TLR signaling pathway

Periprosthetic osteolysis and aseptic loosening are mainly caused by wear particles (Ps) that are generated from friction interfaces. However, the mechanisms underlying the development of aseptic loosening remain unclear. Therefore, we aimed toclarify how the myeloid differentiation factor 88 (MyD88)-independent Toll-like receptor (TLR) signaling pathway mediates cobalt and chromium (CoCr)-Ps-induced osteolysis. We quantified the expression levels of TLRs, MyD88, RANKL, and inflammatory factors in patients experiencing aseptic loosening after primary total hip arthroplasty (THA) with metal-on-metal (MoM) bearings and hip osteoarthritis (hOA). We observed the in vitro and in vivo levels of RANKL, TLRs, and MyD88 in fibroblasts challenged with CoCr Ps by applying shMyD88 interference lentivirus vectors to block the MyD88-independent TLR pathway. The levels of TLRs, MyD88, RANKL, and inflammatory factors in the revision THA (rTHA) with MoM group were higher than those in the hOA group. Our data collectively revealed that inhibiting MyD88 expression could reduce osteoclastogenesis in vitro and CoCr-Ps-induced osteolysis in vivo. Our findings suggested that osteoclastogenesis is promoted by the CoCr-Ps-induced expression of RANKL in fibroblasts and that MyD88 is a potential target in the treatment of wear Ps-induced osteolysis.

A malaria protein factor induces IL-4 expression by dendritic cells via TLR/MyD88-independent signaling, promoting Th2 development

Abstract Dendritic cells (DCs) and cytokines produced by DCs play crucial roles in inducing and regulating pro/anti-inflammatory and Th1/Th2 responses. While DCs are known to produce Th1-promoting IL-12 in response to malaria and other pathogenic infections, it is widely thought that DCs do not produce Th2-promoting IL-4. Here, we show that a protein factor of malaria parasites induces IL-4 responses by CD11chiMHCIIhiCD3ɛ−CD49b−CD19− FcɛRI− DCs via MyD88-independent signaling. Malaria parasite-activated wild type DCs induced IL-4 responses by T cells. Interestingly, lethal parasites, Plasmodium falciparum and P. berghei ANKA, induced IL-4 response primarily by CD8α− DCs, whereas nonlethal P. yoelii induced IL-4 by both CD8α+ and CD8α− DCs. In both P. Berghei ANKA- and P. yoelii-infected mice, IL-4-expressing CD8α− DCs did not express IL-12 and a distinct population expressed IL-12. Further, in P berghei ANKA-infection, CD8α+ DCs expressed IL-12 but not IL-4, whereas in P. yoelii infection CD8α+ DCs expressed IL-4 but not IL-12. This differential IL-4 and IL-12 responses by DC subpopulations may contribute to differential Th1 and Th2 development and different clinical outcomes in lethal and nonlethal malaria. Our results for the first time demonstrated that a malaria protein factor induces IL-4 production by DCs, revealing the molecular mechanism that initiate the promotes Th2 development.

MyD88 in antigen-presenting cells is not required for CD4+ T-cell responses during peptide nanofiber vaccination.

Self-assembled peptide nanofibers raise significant antibody and T cell responses without adjuvants, but the mechanism by which they achieve this has not been fully elucidated. Myeloid differentiation primary response gene 88 (MyD88) previously has been shown to be critical for the antibody response to antigens presented by peptide nanofibers. The present study sought to determine the cell subset in which MyD88 is essential for T cell responses. Mice deficient in MyD88 or CD11c+ cells had severely attenuated T cell responses. However, mice lacking MyD88 in only CD11c+ cells remained capable of internalizing, processing, and presenting nanofiber-derived epitopes to stimulate T cell responses. The necessity of inflammasome pathway was ruled out. Using adoptive transfer models where MyD88 was eliminated in CD4+ T cells or in the host, we observed that deficiency only in T cells or only in the host had no impact on the T cell response to nanofiber vaccines. Therefore, knocking out MyD88 in either antigen presenting cells (APCs) or CD4 T cells could not compromise the CD4 T cell responses, suggesting that self-assembled peptide nanofibers trigger redundant MyD88-dependent and MyD88-independent signaling pathways in APCs and T cells. Similar redundancy has been observed for other adjuvants, and this is discussed.