Engagement Of Toll-like Receptors Research Articles

Carbohydrate-Lectin Interactions Reprogram Dendritic Cells to Promote Type 1 Anti-Tumor Immunity.

Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) is thought to shape immunity by directing T cell function. Controlling downstream responses, however, remains a major challenge. A critical goal in advancing vaccine development involves the identification of receptors that drive type 1 cellular immunity. The immune system monitors cells for aberrant glycosylation (a sign of a foreign entity), but potent activation occurs when a second signal, such as single-stranded RNA or lipopolysaccharide, is present to activate TLR signaling. To exploit dual signaling, we engineered a glycan-costumed virus-like particle (VLP) vaccine that displays a DC-SIGN-selective aryl mannose ligand and encapsulates TLR7 agonists. These VLPs deliver programmable peptide antigens to induce robust DC activation and type 1 cellular immunity. In contrast, VLPs lacking this critical DC-SIGN ligand promoted DC-mediated humoral immunity, offering limited tumor control. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, significantly inhibiting tumor growth in a murine melanoma model. The tailored VLPs also afforded protection against the reintroduction of tumor cells. Thus, DC lectin-driven immune reprogramming, combined with the modular programmability of VLP platforms, provides a promising framework for directing cellular immunity to advance cancer immunotherapies and vaccines.

The Effect of Exosomes Isolated from Poly (I:C) Treated Human Wharton's Jelly Mesenchymal Stem Cells on CD4+CD25+Foxp3+ Regulatory T Cells.

Mesenchymal stem cells (MSCs) are a potential cell therapy candidate for autoimmune and inflammatory diseases due to their multilineage capacity and immune modulating function. MSCs exert immunomodulatory effects on target cells through the secretion of exosomes. Inflammatory conditions such as Toll-like receptors (TLRs) engagement can change the biological functions and immunomodulatory activities of MSCs and the contents of exosomes derived from MSCs are changed. Regulatory T-cells (Treg) are crucial for maintaining immune cell homeostasis and self-tolerance. Our study aimed to investigate the impact of isolated exosomes from hWJ-MSCs that were treated with Poly (I:C) on regulatory CD4 CD25 Foxp3 T-cells. MSCs were harvested from human umbilical cord Wharton's Jelly by explant method. Stem cells were treated by Polyinosinic-polycytidylic acid sodium salt (Poly (I:C)) for 48 hours. Exosomes were extracted from supernatant of cells and Scanning electron microscopy (SEM) and Dynamic light scattering (DLS) were performed for them. Peripheral blood mononuclear cells (PBMCs) isolated from the healthy donors were stimulated with PHA (Phytohemagglutinin) and co-cultured with Poly (I:C) treated hWJ-MSCs derived exosome and untreated hWJ-MSCs derived exosome or without hWJ-MSCs-derived exosome for 6 days. Then, frequency of CD4+CD25+ Foxp3+ regulatory T cells was measured by flow cytometry. Our results showed that exosomes isolated from Poly (I:C) treated hWJ-MSCs significantly increased frequency of CD4+CD25+ Foxp3+ regulatory T cells compared to the untreated hWJ-MSCs derived exosome group and control group. Stimulation by TLR3 improved the anti-inflammatory features of exosomes that were derived from hWJ-MSCs by increasing the frequency of Treg cells.

RNA aggregates harness the danger response for potent cancer immunotherapy

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

Adiponectin Attenuates Splenectomy-Induced Cognitive Deficits by Neuroinflammation and Oxidative Stress via TLR4/MyD88/NF-κb Signaling Pathway in Aged Rats.

Perioperative neurocognitive disorder (PND) is a common adverse event after surgical trauma in elderly patients. The pathogenesis of PND is still unclear. Adiponectin (APN) is a plasma protein secreted by adipose tissue. We have reported that a decreased APN expression is associated with PND patients. APN may be a promising therapeutic agent for PND. However, the neuroprotective mechanism of APN in PND is still unclear. In this study, 18 month old male Sprague-Dawley rats were assigned to six groups: the sham, sham + APN (intragastric (i.g.) administration of 10 μg/kg/day for 20 days before splenectomy), PND (splenectomy), PND + APN, PND + TAK-242 (intraperitoneal (i.p.) administration of 3 mg/kg TAK-242), and PND + APN + lipopolysaccharide (LPS) (i.p. administration of 2 mg/kg LPS). We first found that APN gastric infusion significantly improved learning and cognitive function in the Morris water maze (MWM) test after surgical trauma. Further experiments indicated that APN could inhibit the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κb) p65 pathway to decrease the degree of oxidative damage (malondialdehyde (MDA) and superoxide dismutase (SOD)), microglia-mediated neuroinflammation (ionized calcium binding adapter molecule 1 (IBA1), caspase-1, tumor necrosis factor (TNF)-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6)), and apoptosis (p53, Bcl2, Bax, and caspase 3) in hippocampus. By using LPS-specific agonist and TAK-242-specific inhibitor, the involvement of TLR4 engagement was confirmed. APN intragastric administration exerts a neuroprotective effect against cognitive deficits induced by peripheral trauma, and the possible mechanisms include the inhibition of neuroinflammation, oxidative stress, and apoptosis, mediated by the suppression of the TLR4/MyD88/NF-κb signaling pathway. We propose that oral APN may be a promising candidate for PND treatment.

Toll-like receptor expression on young and old hematopoietic cell types

Abstract Hematopoiesis is an essential process that produces white and red blood cells within the body. As the body ages, we have observed defects in the hematopoietic process including an increase in the number of stem cells, skewing of white blood cells down the myeloid lineage, and a decrease in the production of new B and T cells. Since hematopoietic aging skews the production of professional immune cells, the elderly population has a disadvantage in recognizing and clearing infections compared to young individuals. Immune cells use a common bacterial and viral recognition system, which includes toll-like receptors (TLRs), to identify common pathogen-associated molecular patterns (PAMPs) that bacteria and viruses produce. Upon exposure, immune cells will activate to complete two functions: to direct myeloid differentiation for acute responses and to recognize and eliminate the pathogens via the TNFα or type 1 interferon (IFN-1) signaling pathway. Deficiencies in aging hematopoiesis, IFN-1 expression, and the TLR responses associated with IFN-1 in immune cells have been described in both mice and humans. The expression of these TLRs and any age-associated changes in that expression, as well as downstream signaling, are not as well established. Our goal is to identify which hematopoietic cells express the four TLRs associated with IFN-1 (TLR-3, −4, −7, −9) and if aging leads to changes in receptor expression and/or downstream signaling in human immune cells. Using flow cytometry, we evaluated receptor expression and the downstream effects of TLR engagement. The data reveals age-associated changes in IFN-1-related innate immune signaling pathways. Supported by grants from NIH (R00 AG055717)

Potential role of EBV and Toll-like receptor 9 ligand in patients with systemic lupus erythematosus.

SLE is a multisystem autoimmune disease characterized by multiple immunological abnormalities including production of autoantibodies. While the etiology of SLE is largely unknown, it is generally accepted that both genetic and environmental factors contribute to disease risk and immune dysregulation. Production of IFN-α is important for protecting the host against infections; however, over stimulation of innate immune pathways can induce autoimmune disease. Environmental factors, particularly Epstein-Barr virus (EBV), have been proposed to play an important role in SLE disease. Improper engagement of Toll-like receptor (TLR) pathways by endogenous or exogenous ligands may lead to the initiation of autoimmune responses and tissue injury. EBV is shown to be a potent stimulant of IFN-α by TLR signaling cascades. Given the highlighted role of IFN-α in SLE pathogenesis and potential role of EBV infection in this disease, the present study is aimed at exploring the in vitro effects of EBV infection and CPG (either alone or in combination) on IFN-α. We also examined the expression level of CD20 and BDCA-4 and CD123 in PBMCs in 32 SLE patients and 32 healthy controls. Our results showed PBMCs treated with CPG-induced higher levels of IFN-α and TLR-9 gene expression fold change compared to cells treated with either EBV or EBV-CPG. Moreover, PBMCs treated with CPG produced significantly higher IFN-α concentration in supernatant compared to cells treated with EBV but not EBV-CPG. Our results further highlight the potential role of EBV infection and TLRs in SLE patients although more studies are warranted to ascertain the global imprint that EBV infection can have on immune signature in patients with SLE.

Macrophage transcription factor TonEBP promotes systemic lupus erythematosus and kidney injury via damage-induced signaling pathways

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by autoreactive B cells and dysregulation of many other types of immune cells including myeloid cells. Lupus nephritis (LN) is a common target organ manifestations of SLE. Tonicity-responsive enhancer-binding protein (TonEBP, also known as nuclear factor of activated T-cells 5 (NFAT5)), was initially identified as a central regulator of cellular responses to hypertonic stress and is a pleiotropic stress protein involved in a variety of immunometabolic diseases. To explore the role of TonEBP, we examined kidney biopsy samples from patients with LN. Kidney TonEBP expression was found to be elevated in these patients compared to control patients - in both kidney cells and infiltrating immune cells. Kidney TonEBP mRNA was elevated in LN and correlated with mRNAs encoding inflammatory cytokines and the degree of proteinuria. In a pristane-induced SLE model in mice, myeloid TonEBP deficiency blocked the development of SLE and LN. In macrophages, engagement of various toll-like receptors (TLRs) that respond to damage-associated molecular patterns induced TonEBP expression via stimulation of its promoter. Intracellular signaling downstream of the TLRs was dependent on TonEBP. Therefore, TonEBP can act as a transcriptional cofactor for NF-κB, and activated mTOR-IRF3/7 via protein-protein interactions. Additionally, TonEBP-deficient macrophages displayed elevated efferocytosis and animals with myeloid deficiency of TonEBP showed reduced Th1 and Th17 differentiation, consistent with macrophages defective in TLR signaling. Thus, our data show that myeloid TonEBP may be an attractive therapeutic target for SLE and LN.

ECSIT is essential for RANKL-induced stimulation of mitochondria in osteoclasts and a target for the anti-osteoclastogenic effects of estrogens.

Estrogens inhibit bone resorption and preserve bone mass, at least in part, via direct effects on osteoclasts. The binding of RANKL, the critical cytokine for osteoclast differentiation, to its receptor in osteoclast precursor cells of the monocyte lineage recruits the adaptor protein TRAF6 and activates multiple signaling pathways. Early effects of RANKL include stimulation of mitochondria. 17β-estradiol (E2) prevents the effects of RANKL on mitochondria and promotes mitochondria mediated apoptotic cell death. However, the molecular mechanisms responsible for the actions of RANKL and estrogens on mitochondria remain unknown. Evolutionarily Conserved Signaling Intermediate in Toll Pathway (ECSIT) is a complex I-associated protein that regulates immune responses in macrophages following the engagement of Toll-like receptors, which also recruit TRAF6. Here, we examined whether ECSIT could be implicated in the rapid effects of RANKL and E2 on osteoclast progenitors. Bone marrow-derived macrophages (BMMs) from C57BL/6 mice were cultured with RANKL (30 ng/ml) with or without E2 (10-8 M). ECSIT-TRAF6 interaction was evaluated by co-immunoprecipitation and ECSIT levels in mitochondria and cytosolic fractions by Western blot. ShRNA lentivirus particles were used to knockdown ECSIT. Osteoclasts were enumerated after tartrate-resistant acid phosphatase staining. Oxygen consumption and extracellular acidification rates were measured with Seahorse XFe96 Analyzer. ATP, lactate, and NAD/NADH were measured with commercial assay kits. NADH oxidation to NAD was used to evaluate Complex I activity. Total and mitochondrial ROS, and mitochondrial membrane potential were measured with H2DCFDA, MitoSOX, and TMRM probes, respectively. Degradation of DEVD-AFC was used to measure Caspase-3 activity. We found that RANKL promoted ECSIT-TRAF6 interaction and increased the levels of ECSIT in mitochondria. E2 abrogated these effects of RANKL. Silencing of ECSIT decreased osteoclast differentiation and abrogated the inhibitory effects of E2 on osteoclastogenesis. Loss of ECSIT decreased complex I activity, oxygen consumption, NAD+/NADH redox ratio, and ATP production and increased mitochondrial ROS. In the absence of ECSIT, the stimulatory actions of RANKL on complex I activity and all other markers of oxidative phosphorylation, as well as their inhibition by E2, were prevented. Instead, RANKL stimulated apoptosis of osteoclast progenitors. These findings suggest that dysregulated mitochondria cause a switch in RANKL signaling from pro-survival to pro-apoptotic. In addition, our results indicate that ECSIT represents a central node for the early effects of RANKL on mitochondria and that inhibition of ECSIT-mediated mitochondria stimulation might contribute to the bone protective actions of estrogens.

Oxidized Mitochondrial DNA Engages TLR9 to Activate the NLRP3 Inflammasome in Myelodysplastic Syndromes.

Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, including NLRP3 inflammasome driven pyroptotic cell death. We recently reported that the danger-associated molecular pattern (DAMP) oxidized mitochondrial DNA (ox-mtDNA) is diagnostically increased in MDS plasma although the functional consequences remain poorly defined. We hypothesized that ox-mtDNA is released into the cytosol, upon NLRP3 inflammasome pyroptotic lysis, where it propagates and further enhances the inflammatory cell death feed-forward loop onto healthy tissues. This activation can be mediated via ox-mtDNA engagement of Toll-like receptor 9 (TLR9), an endosomal DNA sensing pattern recognition receptor known to prime and activate the inflammasome propagating the IFN-induced inflammatory response in neighboring healthy hematopoietic stem and progenitor cells (HSPCs), which presents a potentially targetable axis for the reduction in inflammasome activation in MDS. We found that extracellular ox-mtDNA activates the TLR9-MyD88-inflammasome pathway, demonstrated by increased lysosome formation, IRF7 translocation, and interferon-stimulated gene (ISG) production. Extracellular ox-mtDNA also induces TLR9 redistribution in MDS HSPCs to the cell surface. The effects on NLRP3 inflammasome activation were validated by blocking TLR9 activation via chemical inhibition and CRISPR knockout, demonstrating that TLR9 was necessary for ox-mtDNA-mediated inflammasome activation. Conversely, lentiviral overexpression of TLR9 sensitized cells to ox-mtDNA. Lastly, inhibiting TLR9 restored hematopoietic colony formation in MDS BM. We conclude that MDS HSPCs are primed for inflammasome activation via ox-mtDNA released by pyroptotic cells. Blocking the TLR9/ox-mtDNA axis may prove to be a novel therapeutic strategy for MDS.

Determining Endosomal Toll-Like Receptors Gene Expression in NK Cells After Stimulation with Specific Agonists.

Poor knowledge is currently available about the biology of Toll-like receptors (TLRs) in natural killer (NK) cells. This is particularly due to the old belief that NK cells are unable to specifically eliminate microbes without presensitization. On the contrary, it has been clearly demonstrated that not only they can be activated through the engagement of Toll-like receptors (TLRs) by microbial molecules, but also that this interaction induces NK cells to release cytokines that, in turn, activate other cells of both innate and adaptive immunity. For this reason, immunotherapy based on local infusion of TLRs ligands is currently considered as a novel potential strategy to treat solid tumors. Here, we provide a protocol to efficiently stimulate NK cells via endosomal TLRs agonists and to determine endosomal TLRs gene expression level. This protocol can be used for in vitro investigation into endosomal TLRs function in NK cells under different conditions.

Evaluation of toll-like receptors 2 and 4 polymorphism and intestinal microbiota in children with food allergies.

Mutual regulation between immune system and gut microbiota is achieved through several mechanisms including the engagement of toll-like receptors (TLRs) which is expressed on numerous cell types. In this study we aimed to explore the association between food allergies and TLR gene polymorphisms in association with gut microbiota. Toll-like receptors polymorphism frequencies and some bacteria in the gut microbiota in 130 infants aged 1-24 months with egg and/or milk allergy in a prospective cohort were compared with 110 non-food allergic controls. Four candidate polymorphisms (TLR2 rs1898830/rs5743708 and TLR4 rs4986790/rs4986791) were genotyped by allelic discrimination polymerase chain rection (PCR) method. Gut microbiota analysis was achieved by using high-throughput sequencing. The TLR4 rs4986790 (Asp299Gly) single nucleotide polymorphism (SNP) major/minor allele frequency was 0.788/0.212 in food allergy patients and 0.719/0.280 in controls (p=0.017). There was a statistically significant difference between groups in terms of genotype frequencies (AA, AG, GG). Gut microbiota analysis revealed increased Firmicutes phylum in stool of the patients with food allergy. Except for TLR4 rs4986791 (Thr399lle) allele, the other TLR polymorphisms were not associated with food allergies in children. When the bacteria in the intestinal microbiota and TLR2 and TLR4 gene polymorphisms were compared; we determined a statistically significant increase in Bifidobacterium concentration in the intestinal microbiota in TLR4 rs4986791 CT heterozygous genotype (p=0.004). This study demonstrated a partial role of TLR4 gene polymorphism and gut microbiota in the development of food allergies. Future work in this area will be required to clarify the roles of different microbial strains that modulate gut microbiota composition and function in conjunction with TLR transcription pathways.

The Latest Approach of Immunotherapy with Endosomal TLR Agonists Improving NK Cell Function: An Overview.

Toll-like receptors (TLRs) are the most well-defined pattern recognition receptors (PRR) of several cell types recognizing pathogens and triggering innate immunity. TLRs are also expressed on tumor cells and tumor microenvironment (TME) cells, including natural killer (NK) cells. Cell surface TLRs primarily recognize extracellular ligands from bacteria and fungi, while endosomal TLRs recognize microbial DNA or RNA. TLR engagement activates intracellular pathways leading to the activation of transcription factors regulating gene expression of several inflammatory molecules. Endosomal TLR agonists may be considered as new immunotherapeutic adjuvants for dendritic cell (DC) vaccines able to improve anti-tumor immunity and cancer patient outcomes. The literature suggests that endosomal TLR agonists modify TME on murine models and human cancer (clinical trials), providing evidence that locally infused endosomal TLR agonists may delay tumor growth and induce tumor regression. Recently, our group demonstrated that CD56bright NK cell subset is selectively responsive to TLR8 engagement. Thus, TLR8 agonists (loaded or not to nanoparticles or other carriers) can be considered a novel strategy able to promote anti-tumor immunity. TLR8 agonists can be used to activate and expand in vitro circulating or intra-tumoral NK cells to be adoptively transferred into patients.

CAF08 adjuvant enables single dose protection against respiratory syncytial virus infection in murine newborns

Respiratory syncytial virus is a leading cause of morbidity and mortality in children, due in part to their distinct immune system, characterized by impaired induction of Th 1 immunity. Here we show application of cationic adjuvant formulation CAF08, a liposomal vaccine formulation tailored to induce Th 1 immunity in early life via synergistic engagement of Toll-like Receptor 7/8 and the C-type lectin receptor Mincle. We apply quantitative phosphoproteomics to human dendritic cells and reveal a role for Protein Kinase C-δ for enhanced Th1 cytokine production in neonatal dendritic cells and identify signaling events resulting in antigen cross-presentation. In a murine in vivo model a single immunization at birth with CAF08-adjuvanted RSV pre-fusion antigen protects newborn mice from RSV infection by induction of antigen-specific CD8+ T-cells and Th1 cells. Overall, we describe a pediatric adjuvant formulation and characterize its mechanism of action providing a promising avenue for development of early life vaccines against RSV and other respiratory viral pathogens.

Nucleic Acid Sensing and Systemic Lupus Erythematosus: The Danger of Self.

Abstract This Pillars of Immunology article is a commentary on “Chromatin–IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors,” a pivotal article written by E. A. Leadbetter, I. R. Rifkin, A. M. Hohlbaum, B. C. Beaudette, M. J. Shlomchik and A. Marshak-Rothstein, and published in Nature in 2002. https://www.nature.com/articles/416603a.

Immunization With Lipopolysaccharide-Activated Dendritic Cells Generates a Specific CD8+ T Cell Response That Confers Partial Protection Against Infection With Trypanosoma cruzi.

Lipopolysaccharide (LPS) induces the activation of dendritic cells (DCs) throughout the engagement of toll-like receptor 4. LPS-activated DCs show increased capacity to process and present pathogen-derived antigens to activate naïve T cells. DCs-based vaccines have been successfully used to treat some cancer types, and lately transferred to the field of infectious diseases, in particular against HIV. However, there is no vaccine or DC therapy for any parasitic disease that is currently available. The immune response against Trypanosoma cruzi substantially relies on T cells, and both CD4+ and CD8+ T lymphocytes are required to control parasite growth. Here, we develop a vaccination strategy based on DCs derived from bone marrow, activated with LPS and loaded with TsKb20, an immunodominant epitope of the trans-sialidase family of proteins. We extensively characterized the CD8+ T cell response generated after immunization and compared three different readouts: a tetramer staining, ELISpot and Activation-Induced Marker (AIM) assays. To our knowledge, this work shows for the first time a proper set of T cell markers to evaluate specific CD8+ T cell responses in mice. We also show that our immunization scheme confers protection against T. cruzi, augmenting survival and reducing parasite burden in female but not male mice. We conclude that the immunization with LPS-activated DCs has the potential to prime significant CD8+ T cell responses in C57BL/6 mice independently of the sex, but this response will only be effective in female, possibly due to mice sexual dimorphisms in the response generated against T. cruzi.

Abstract 3502: AXA-042 - a novel systemic TLR2/6 agonist for anti-tumor therapy

Abstract Background: Treatment approaches that engage both the innate and adaptive immune response have the potential to transform anti-cancer therapy, especially in settings of checkpoint inhibitor insensitivity or acquired resistance. Toll-like receptors (TLRs) are a class of targets that mediate the initial cellular response to external pathogens or endogenous alarmins, activating downstream pro-inflammatory cascades and leading to the activation and recruitment of key innate subsets. TLR2 is a cell surface receptor, expressed predominantly on macrophages, dendritic cells (DC), neutrophils and subsets of NK and T cells. TLR2 signaling plays important role in NK and T cell cytolytic cell functions, as well as DC and macrophage activation. AXA-042 is a novel synthetic TLR2/6 agonist designed for systemic delivery to re-engage the innate immune response to help overcome tumour immune escape. Methods: In vitro potency and selectivity of AXA-042 was assessed across a panel of human and mouse HEK-blue TLR reporter assays. AXA-042 activity was profiled relative to other TLR agonists in human PBMC cytokine release assays. AXA-042 in vivo efficacy was evaluated in the EMT6 and CT26 syngeneic tumour models. The Nanostring nCounter™ Mouse PanCancer Immune Profiling Panel was used to identify AXA-042 tumour-localized and peripheral response signatures. Results: AXA-042 demonstrated potent and selective activity in murine and human TLR2/6 reporter assays and a differential PBMC cytokine release profile relative to other TLR agonists in vitro. Systemic delivery (QW, IV or IP) of AXA-042 significantly inhibited EMT6 and CT26 tumour growth, which was accompanied by an increase in circulating plasma levels of IL6, IL12 and CXCL10. Combination with anti-PD-1 Ab further potentiated anti-tumour response, resulting in approximately 30-40% tumour regressions. AXA-042 anti-tumour effects were accompanied by increased activation of the tumour-infiltrating myeloid cells, including DCs and macrophages, and a reduction in the M2 TAM subset. Nanostring analysis confirmed TLR and IFNγ pathway engagement and revealed an induction of gene signatures associated with macrophage, DC and NK functions. Longitudinal studies to characterize whole blood biomarkers associated with response are currently in progress. Conclusion: AXA-042 was well tolerated in vivo and demonstrated innate response engagement and anti-tumour efficacy as monotherapy and in combination with anti-PD-1 immunotherapy. AXA-042 has completed GLP toxicology studies and initiation of Phase 1/1b trial is planned for early 2022. Citation Format: Anna V. Galkin, Francesca Mercuri, Nicholas West, Ping Zhang, Weiguang Zeng, Grant McLachlan, Michael Bettess, Ian Holmes, David Jackson, Chris Smith, Christophe Demaison, Tobias Bald, Phil Kearney. AXA-042 - a novel systemic TLR2/6 agonist for anti-tumor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3502.

Innate Immune Response to Dengue Virus: Toll-like Receptors and Antiviral Response.

Dengue is a mosquito-borne viral disease caused by the dengue virus (DENV1-4). The clinical manifestations range from asymptomatic to life-threatening dengue hemorrhagic fever (DHF) and/or Dengue Shock Syndrome (DSS). Viral and host factors are related to the clinical outcome of dengue, although the disease pathogenesis remains uncertain. The innate antiviral response to DENV is implemented by a variety of immune cells and inflammatory mediators. Blood monocytes, dendritic cells (DCs) and tissue macrophages are the main target cells of DENV infection. These cells recognize pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs). Pathogen recognition is a critical step in eliciting the innate immune response. Toll-like receptors (TLRs) are responsible for the innate recognition of pathogens and represent an essential component of the innate and adaptive immune response. Ten different TLRs are described in humans, which are expressed in many different immune cells. The engagement of TLRs with viral PAMPs triggers downstream signaling pathways leading to the production of inflammatory cytokines, interferons (IFNs) and other molecules essential for the prevention of viral replication. Here, we summarize the crucial TLRs’ roles in the antiviral innate immune response to DENV and their association with viral pathogenesis.

Abstract P3-03-07: Single-institution retrospective analysis of lymph node (LN) change on breast MRI in patients with high risk early-stage breast cancer receiving neoadjuvant chemotherapy with and without immunotherapy on the ISPY-2 TRIAL

Abstract Background: The I-SPY2 TRIAL has shown that the addition of pembrolizumab to standard neoadjuvant chemotherapy results in improved rates of estimated pathologic complete response (pCR) for hormone-receptor positive (HR+) as well as triple negative breast cancer (TNBC). I-SPY2 has tested a number of novel immunotherapy combinations including the combination of pembrolizumab and SD-101, a synthetic oligonucleotide with cytidine-phospho-guanosine (CpG) motifs that stimulates plasmacytoid dendritic cells (pDC) through engagement of toll-like receptor 9 (TLR9). Biomarkers of response to immunotherapy may be different than chemotherapy, and pathologic response may not fully reflect the benefit from checkpoint inhibitors. Early markers of response to immunotherapy are needed to minimize the risk of immune-related toxicity in patients who are unlikely to benefit. The objective of this study is to assess the change in regional lymph nodes (LNs) by breast MRI in I-SPY2 patients receiving neoadjuvant chemotherapy +/- immunotherapy, and to explore if changes in regional LNs correlate with pCR and residual cancer burden (RCB). Methods: This is a retrospective study of the prospective multicenter I-SPY2 adaptive neoadjuvant trial investigating all patients enrolled at UCSF in 3 study arms between Dec 2015 and April 2021: 1) Control (weekly paclitaxel 80 mg/m2 x 12 weeks followed by AC x 4); 2) weekly paclitaxel + pembrolizumab 200 mg IV every 3 weeks, followed by AC x 4; 3) weekly paclitaxel + every 3 week pembrolizumab + intra-tumoral SD-101 weekly x 4 then every 3 weeks x 2 followed by AC x 4. Serial pre-operative breast MRIs were performed in all patients at baseline, 3 weeks, 12 weeks, and 20 weeks. A single breast radiologist blinded to treatment arm reviewed all MRIs for all patients included in this study and assessed longest diameter and cortical thickness of the largest ipsilateral axillary LN, number and location of abnormal LNs, and development of new abnormal LNs over time. Rates of pCR and RCB were evaluated in patients who showed evidence of LN growth and/or development of new lymphadenopathy (LAD) compared to those who did not. A two-sample test for equality of proportions to measure the statistical significance at alpha=.5 between control and immunotherapy arms and LN change was used. Results: A total of 43 patients were included, of whom 16 were in the control group, 11 received pembrolizumab, and 16 received pembrolizumab + SD-101. Median age was 45 years, mean tumor size was 4.8 cm, and 60% of tumors were HR+HER2-negative with the remaining 40% being TNBC. Baseline patient and tumor characteristics were similar between the 3 study arms. LN enlargement and/or development of new LAD over time was significantly more common in patients receiving immunotherapy than patients in the control arm (48.1% versus 6.3%, p=0.006). This was seen in both HR+ and TN subtypes. While there was a numerically higher rate of pCR/RCB-1 pathology in patients with increased and/or new LAD compared to those without (64% vs 51%), this difference was not statistically significant. The association of LN change and immune-related toxicities will be reported. Conclusion: The addition of immunotherapy to standard neoadjuvant chemotherapy in the I-SPY2 trial was associated with an increase in size of ipsilateral axillary LN and/or development of new LAD on serial breast MRI imaging during the course of neoadjuvant treatment. These changes were not associated with worse pathologic response at surgery and should not be assumed to be due to disease progression. Whether these changes could reflect immunotherapy benefit needs to be investigated in a larger trial with longer follow-up. Citation Format: Anika Christofferson, Elissa Price, Rita A Mukhtar, Amrita Basu, Laura Esserman, A Jo Chien. Single-institution retrospective analysis of lymph node (LN) change on breast MRI in patients with high risk early-stage breast cancer receiving neoadjuvant chemotherapy with and without immunotherapy on the ISPY-2 TRIAL [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-03-07.

Bacillus anthracis induces NLRP3 inflammasome activation and caspase-8–mediated apoptosis of macrophages to promote lethal anthrax

Lethal toxin (LeTx)-mediated killing of myeloid cells is essential for Bacillus anthracis, the causative agent of anthrax, to establish systemic infection and induce lethal anthrax. The "LeTx-sensitive" NLRP1b inflammasome of BALB/c and 129S macrophages swiftly responds to LeTx intoxication with pyroptosis and secretion of interleukin (IL)-1β. However, human NLRP1 is nonresponsive to LeTx, prompting us to investigate B. anthracis host-pathogen interactions in C57BL/6J (B6) macrophages and mice that also lack a LeTx-sensitive Nlrp1b allele. Unexpectedly, we found that LeTx intoxication and live B. anthracis infection of B6 macrophages elicited robust secretion of IL-1β, which critically relied on the NLRP3 inflammasome. TNF signaling through both TNF receptor 1 (TNF-R1) and TNF-R2 were required for B. anthracis-induced NLRP3 inflammasome activation, which was further controlled by RIPK1 kinase activity and LeTx-mediated proteolytic inactivation of MAP kinase signaling. In addition to activating the NLRP3 inflammasome, LeTx-induced MAPKK inactivation and TNF production sensitized B. anthracis-infected macrophages to robust RIPK1- and caspase-8-dependent apoptosis. In agreement, purified LeTx triggered RIPK1 kinase activity- and caspase-8-dependent apoptosis only in macrophages primed with TNF or following engagement of TRIF-dependent Toll-like receptors. Consistently, genetic and pharmacological inhibition of RIPK1 inhibited NLRP3 inflammasome activation and apoptosis of LeTx-intoxicated and B. anthracis-infected macrophages. Caspase-8/RIPK3-deficient mice were significantly protected from B. anthracis-induced lethality, demonstrating the invivo pathophysiological relevance of this cytotoxic mechanism. Collectively, these results establish TNF- and RIPK1 kinase activity-dependent NLRP3 inflammasome activation and macrophage apoptosis as key host-pathogen mechanisms in lethal anthrax.

Adult-onset mast cell activation syndrome following scombroid poisoning: a case report and review of the literature

BackgroundMast cells are closely associated with epithelium, serving as sentinels responsible for the recognition of tissue injury and coordination of the initial inflammatory response. Upon detection of the injured cell content, mast cells then tailor the release of preformed and newly produced chemical mediators to the detected challenge, via an array of pathogen receptors. In addition to immunoglobulin E receptor-triggered mast cell activation, commonly referred to as allergic or atopic disorders, non-immunoglobulin E receptor mediated mast cell activation follows engagement of toll-like receptors, immunoglobulin G receptors, and complement receptors. Upon containment of the extrinsic challenge, acute inflammation is downregulated, and repair of the injured tissue ensues. The mast cell compartments must return to a baseline steady state to remain tolerant towards self-antigens and harmless entities, including environmental conditions, to prevent unnecessary immune activation and chronic hypersensitivity disorders. Over the past 50 years, an increasing number of patients are experiencing episodes of aberrant mast cell activation, not associated with allergen-specific mast cell disease or systemic mastocytosis. This led to proposed diagnostic criteria of mast cell activation syndrome. Mast cell activation syndrome is a heterogeneous disorder, defined by a combination of (1) recurrent symptoms typical of mast cell activation, (2) an increase of validated mast cell derived mediators, and (3) response to treatment with mast cell stabilizing or mast cell mediator-targeted therapies. Onset of mast cell activation syndrome ostensibly reflects the loss of tolerance in the mast cell compartment to nonthreatening entities and nonhazardous environmental conditions. The etiology of chronic mast cell dysregulation and associated intolerance to self-antigens or harmless entities is not well understood, but a growing number of studies point to exposure of the epithelial borders, which leads to inappropriate or excessive mast cell activation or impaired resolution of acute inflammation following neutralization of the identified pathogen.Case presentationHere we present a case of adult onset mast cell activation syndrome following scombroid poisoning. Scombroid toxicity is usually a self-limited illness, but there are individuals who have been shown to have severe symptoms or persistent illness following histamine fish poisoning. We describe a 74-year-old Caucasian woman, with a history of drug-induced urticaria, who developed a constellation of hypersensitivity illnesses consistent with the diagnosis of mast cell activation syndrome after ingestion of tainted fish.ConclusionMast cell activation disease causes problems of increased complexity in children and adults. The increased prevalence and severity of mast cell activation disease has been attributed to dramatic changes in our lifestyles and modern living environments. These changes likely impact the integrity of the epithelial barriers, leading to loss of tolerance in the mast cell compartment. Here, we present a case of a nonatopic, 74-year-old female who developed mast cell activation disease after exposure to a potent environmental toxin. Mast cell activation disease commonly involves several organ systems, with patients often referred to a succession of different specialists. This results in delayed diagnosis and suboptimal care. Instead, early recognition of mast cell activation disease would lead to better outcomes. We review the literature, describing the diagnostic criteria for mast cell activation disorders that can improve recognition of this multiorgan system syndrome. Further research is needed into the interaction of epithelial barrier disruption and the dysregulation of the immune system.