Synthetic TLR4 Research Articles

Akkermansia muciniphila phospholipid induces homeostatic immune responses

Multiple studies have established associations between human gut bacteria and host physiology, but determining the molecular mechanisms underlying these associations has been challenging1–3. Akkermansia muciniphila has been robustly associated with positive systemic effects on host metabolism, favourable outcomes to checkpoint blockade in cancer immunotherapy and homeostatic immunity4–7. Here we report the identification of a lipid from A. muciniphila’s cell membrane that recapitulates the immunomodulatory activity of A. muciniphila in cell-based assays8. The isolated immunogen, a diacyl phosphatidylethanolamine with two branched chains (a15:0-i15:0 PE), was characterized through both spectroscopic analysis and chemical synthesis. The immunogenic activity of a15:0-i15:0 PE has a highly restricted structure–activity relationship, and its immune signalling requires an unexpected toll-like receptor TLR2–TLR1 heterodimer9,10. Certain features of the phospholipid’s activity are worth noting: it is significantly less potent than known natural and synthetic TLR2 agonists; it preferentially induces some inflammatory cytokines but not others; and, at low doses (1% of EC50) it resets activation thresholds and responses for immune signalling. Identifying both the molecule and an equipotent synthetic analogue, its non-canonical TLR2–TLR1 signalling pathway, its immunomodulatory selectivity and its low-dose immunoregulatory effects provide a molecular mechanism for a model of A. muciniphila’s ability to set immunological tone and its varied roles in health and disease.

Abstract 3034: Purine nucleoside phosphorylase regulates metabolic and immune checkpoints

Abstract Purine nucleoside phosphorylase (PNP) functions as a central regulator of nucleotide metabolism by catalyzing the degradation of purine nucleosides guanosine and deoxyguanosine. PNP inactivation has been paradoxically associated with both immunodeficiency and autoimmunity in humans but the genetic and molecular determinants of these divergent outcomes are poorly defined and whether they reflect cell type specific mechanisms is currently unknown. Moreover, the mechanisms underlying the clinical efficacy of PNP inhibitors in a subset of patients with hematological malignancies are yet to be determined, thereby severely limiting the potential of this promising new class of metabolic targeted therapies. To address these gaps in knowledge we performed a systematic evaluation of the metabolic, immunological and cell-context phenotypic effects of targeting PNP using a clinical-stage inhibitor with picomolar affinity and excellent oral bioavailability. Here we show that across cancer types PNP inhibition is synthetically lethal with sterile alpha motif and HD domain-containing protein 1 (SAMHD1) deficiency by triggering massive dGTP pool expansion, and impairing DNA replication. These cytotoxic effects require the expression of deoxycytidine kinase (dCK) which mediates the intracellular trapping of deoxyguanosine and expansion of dGTP pools, and are opposed by environmental deoxycytidine produced by bone marrow stromal cells in a SAMHD1 and nucleoside transporter dependent manner. This resistance mechanism to PNP inhibition can be counteracted by the deoxycytidine catabolizing enzyme cytidine deaminase (CDA). We further show that in addition to, and independent of its dCK and CDA-dependent cytotoxic effects in SAMHD1 deficient cells, PNP inhibition elevates serum inflammatory cytokines and cytokine transcript levels in secondary lymphoid organs. Using scRNAseq we identify CD19+ B-cells and CSF1R+ macrophages, which express the pattern recognition receptor TLR7, as the drivers of these alterations. TLR7 activation in these cell types is promoted by guanosine accumulation which functions as a ligand for TLR7 alongside uridine-containing ssRNA. Importantly, TLR7 stimulation achieved by PNP inhibition initiates transcriptional alterations that are quantitatively and qualitatively unique from synthetic TLR7 agonists, such as the production of type I interferons. We additionally demonstrate that PNP inactivation stimulates germinal center responses in secondary lymphoid organs via the expansion of activated B-cells and T follicular helper cells. In summary, we demonstrate that by modulating multiple metabolic and immune checkpoints in a multigenic, cell type and environment dependent manner, pharmacological PNP inhibition elicits specific outcomes which can be leveraged for the development of novel therapeutic applications. Citation Format: Evan R. Abt, Khalid Rashid, Thuc M. Le, Hailey R. Lee, Amanda L. Creech, Ting-Ting Wu, Thomas Mehrling, Shanta Bantia, Caius G. Radu. Purine nucleoside phosphorylase regulates metabolic and immune checkpoints [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 3034.

Pre-treatment with a novel synthetic TLR4 agonist prior to challenge with mouse-adapted H1N1 and H2N3 influenza strains reduced morbidity and mortality in BALB/c mice

Abstract According to the World Health Organization, influenza causes 3–5 million cases of severe illness and 250,000 – 500,000 deaths annually despite the availability of a seasonal vaccine. As an alternative therapy for use in high-risk groups and environments, we evaluated the efficacy of prophylactic treatment with a novel synthetic TLR4 agonist in BALB/c mice. Animals were anesthetized and treated with a TLR4 agonist intranasally (IN) 0 to 28 days prior to a lethal challenge with either H1N1 or H3N2 mouse-adapted human influenza. Animals were monitored daily for weight loss, decreased body temperature, body score, and mortality. Initial dose-response experiments demonstrated that mice treated with 50 μg or 10 μg of our novel TLR4 agonist in an aqueous formulation were similarly protected from H3N2 influenza challenge with respect to weight loss and survival. However, initial weight loss data demonstrated that 50 μg was not as well tolerated as 10 μg. The 10 μg dose was therefore selected as the lead dose in subsequent experiments. In a series of formulation comparison experiments, the lead formulation resulted in marked improvement in both percent weight loss and survival compared to initial TLR4 agonist formulations. Animals treated with all formulations of our novel synthetic TLR4 agonist 0, 7, or 14 days prior to infection resulted in 80–90% survival. The lead formulation also resulted 80–90% survival when given prophylactically 21 or 28 days prior to infection compared to 0–20% survival in untreated mice. Overall, our results show that pre-treatment with our novel synthetic TLR4 agonist prior to exposure to mouse-adapted H1N1 and H2N3 influenza strains reduced morbidity and mortality in infected mice for up to 28 days after treatment. This work was supported by NIH grant 5R44AI136081-03

Assessing Immunogenicity of Vaccines and Adjuvant Combinations for Q Fever

Abstract There is an unmet need for developing a safer, effective, single-dose vaccine for Coxiella burnetii conferring prolonged resistance, due to its high infectivity and biothreat potential. C. burnetii causes Q fever, which acutely causes fever-like symptoms but may manifest chronically and escalate into pneumonia and endocarditis. Prior sensitization has been documented to exacerbate the current vaccine’s reactogenicity and there have been considerable efforts in developing alternatives. Our lab has been studying the effects of combinations of adjuvants and their synergistic effects in eliciting antibody and T cell responses. Here we describe an effective adjuvant combination using synthetic TLR4 agonist monophosphoryl lipid A (MPLA), TLR9 agonist CpG ODN 1018, and Addavax, a squalene oil-in-water emulsion. In vitro assays were performed to assess the immunogenicity of these adjuvants in the context of NFκB activation and inducible nitric oxide synthase (iNOS) production. Model antigens from C. burnetii were selected based on previous protein microarray data and formulated with varying adjuvant combinations. These formulations were administered to C57BL/6 mice and assessed for immunogenicity. Sera collected at regular intervals were screened against protein microarrays to monitor the dynamics and isotypes of the antibody response. Here we observed robust, durable and antigen-specific T cell and antibody responses. Our top vaccine formulations were administered to Hartley guinea pigs in a challenge study. Initial data demonstrate specific IgG production against immunizing antigens. Downstream analysis for this data set is currently underway. Supported by DTRA contract HDTRA1-16-C-0009 DTRA grants HDTRA1-18-1-0035 and HDTRA1-18-1-0036

Intranasal administration of a novel synthetic TLR4 agonist suppresses allergen-specific Th2 responses in mice

Abstract Allergen-agnostic immunotherapies to treat seasonal allergic rhinitis are not currently available. Antihistamines are the most common form of seasonal allergic rhinitis treatment – minimizing symptoms instead of addressing underlying immune dysfunction. Allergen specific immunotherapies (AIT) are also available for seasonal allergic rhinitis but require a known allergen and long-term treatment regiments that can be costly and take several years to complete. Here we report a novel synthetic TLR4 agonist that prevented Th2 allergic responses in an OVA-sensitized mouse model. This novel synthetic TLR4 agonist has improved stability compared to other TLR4 agonists used as vaccine adjuvants or immunotherapeutics. After sensitizing Balb/c mice to OVA using IP injections of OVA + alum, intranasal treatment with the novel TLR4 agonist at one, three, or seven days prior to intranasal OVA challenge reduced or ameliorated OVA-specific Th2 allergic responses. Specifically, intranasal TLR4 agonist treatment reduced OVA-induced airway hypersensitivity, eosinophil influx, and Th2 T cell cytokine secretion – hallmarks of Th2 allergic immune responses. Additionally, we have identified a particular formulation of our novel synthetic TLR4 agonist which has shown greater efficacy, stability, and reduced toxicity profile compared to initial formulations. These data suggest this novel synthetic TLR4 agonist can potentially treat the underlying immune dysfunction associated with allergic responses for a durable cure of seasonal allergic rhinitis. Supported by NIH contract HHSN272201800036C

Adjuvanted multivalent vaccine targeting clinically relevant Pf bacteriophage creates cross-reactive antibodies

Abstract Pseudomonas aeruginosa (Pa) is a bacterial pathogen associated with chronic diabetic wounds as well as potentially deadly lung infections in cystic fibrosis patients. Pa is listed by the WHO as “Priority 1” in the Priority Pathogens list and results in hundreds of deaths each year. Pa diversity presents a challenge in developing a vaccine or therapeutic capable of resolving infections caused by different Pa clinical isolates. Filamentous Pf phages (a virus) infect the majority of Pa strains. Additionally, the presence Pf phage is a key virulence factor contributing to Pa infections. Pf is a filamentous-lysogenic phage that is primarily comprised of repeats of coat protein (CoaB). A consensus peptide derived from the coat protein was identified and used as the antigen for our phage vaccine targeted vaccine. This conjugate vaccine targeting Pf phage induced protection against Pa wound challenges in mice. Additionally, our team has implemented the use of a novel TLR4 agonist to further enhance antibody response against Pf coat protein intact Pf phage virions. Based on their amino acid sequence homology, clinical isolates of Pa and associated Pf phage were divided into two major clades. We prepared CRM-197 conjugates using the consensus peptides from both clades. Vaccination with these multivalent conjugates in combination with a novel synthetic TLR4 agonist (adjuvant) boosts antibody response against the peptides and promotes antibody cross-recognition between peptides. Creating a safe and effective vaccine that can mount a robust immune response against both clades of Pf phage is an important advancement towards the development and clinical testing of phage based vaccine targeting deadly Pa infections. Supported by R01 AI138981-01

Cationic liposomes containing a TLR4 agonist promote the efficient development of cellular immunity against SARS-CoV-2 Spike protein in a subunit vaccine.

Abstract Subunit vaccines require an adjuvant, as protein antigens alone provoke a very poor immune response. Liposomes can be used to deliver an adjuvant, particularly when it is an amphipathic molecule such as a toll-like receptor (TLR) 4 agonist. TLR4 engagement causes the release of cytokines known to skew the immune response towards Th1-mediated cellular immunity, making it attractive for use in viral vaccines. Additionally, liposomes formulated with a TLR4 agonist can be neutral, positive, or negative in charge, further affecting the immune response. We tested the effect of liposome composition and charge on the immune response against the SARS-CoV-2 Spike protein following vaccination with recombinant Spike trimers and liposomes containing a synthetic TLR4 agonist in mice. Anti-Spike antibody titers were modestly boosted by neutral liposomes, but charged liposomes increased it further. Control groups given liposomes without the TLR4 agonist demonstrated that cationic liposomes alone boosted antibody titers, but gave a strikingly different T cell response. These liposomes evoked a strongly Th2-associated immune profile, with high IgG1 levels and T cells that released IL-5 when stimulated ex vivo. In contrast, the TLR4-stimulating cationic liposomes resulted in high IgG2c levels, along with strong production of IFNγ, some IL-17, and no IL-5 by restimulated T cells. Interestingly, despite similar antibody titers, serum from groups given the TLR4-stimulating liposomes more effectively prevented Spike binding to ACE-2 than that of the control groups in pseudoneutralization assays. These data show a beneficial effect of TLR4-stimulating cationic liposomes on the anti-viral cellular immune response in subunit vaccines. Supported by a supplement to NIH Adjuvant Discovery Contract 75N93019C00045

An adjuvanted zoster vaccine elicits potent cellular immune responses in mice without QS21

Herpes zoster (HZ) is caused by reactivation of latent varicella-zoster virus (VZV) when VZV-specific cellular immunity is insufficient to control reactivation. Currently, Shingrix, which contains the VZV gE protein and GSK’s AS01B adjuvant composed of liposomes formulated with cholesterol, monophosphoryl lipid A (MPL) and QS21, is used for prevention of HZ. However, reactogenicity to Shingrix is common leading to poor patient compliance in receiving one or both shots. Here, we evaluated the immunogenicity of a newly formulated gE protein-based HZ vaccine containing Second-generation Lipid Adjuvant (SLA), a synthetic TLR4 ligand, formulated in an oil-in-water emulsion (SLA-SE) without QS21 (gE/SLA-SE). In VZV-primed mouse models, gE/SLA-SE-induced gE-specific humoral and cellular immune responses at comparable levels to those elicited by Shingrix in young mice, as both gE/SLA-SE and Shingrix induce polyfunctional CD4+ T-cell responses. In aged mice, gE/SLA-SE elicited more robust gE-specific T-cell responses than Shingrix. Furthermore, gE/SLA-SE-induced T-cell responses were sustained until 5 months after immunization. Thus, QS21-free, gE/SLA-SE is a promising candidate for development of gE-based HZ vaccines with high immunogenicity—particularly when targeting an older population.

Intratumoral expression of IL-12 from lentiviral or RNA vectors acts synergistically with TLR4 agonist (GLA) to generate anti-tumor immunological memory.

Systemic interleukin-12 (IL12) anti-tumor therapy is highly potent but has had limited utility in the clinic due to severe toxicity. Here, we present two IL12-expressing vector platforms, both of which can overcome the deficiencies of previous systemic IL12 therapies: 1) an integrating lentiviral vector, and 2) a self-replicating messenger RNA formulated with polyethyleneimine. Intratumoral administration of either IL12 vector platform resulted in recruitment of immune cells, including effector T cells and dendritic cells, and the complete remission of established tumors in multiple murine models. Furthermore, concurrent intratumoral administration of the synthetic TLR4 agonist glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE) induced systemic memory T cell responses that mediated complete protection against tumor rechallenge in all survivor mice (8/8 rechallenged mice), whereas only 2/6 total rechallenged mice treated with intratrumoral IL12 monotherapy rejected the rechallenge. Taken together, expression of vectorized IL12 in combination with a TLR4 agonist represents a varied approach to broaden the applicability of intratumoral immune therapies of solid tumors.

Novel TLR4 adjuvant elicits protection against homologous and heterologous Influenza A infection

Influenza A virus (IAV) is a leading cause of respiratory disease worldwide often resulting in hospitalization or death. In this study, TLR4 immunostimulatory molecules, Bacterial Enzymatic Combinatorial Chemistry (BECC) 438 and BECC470 were found to be superior IAV vaccine adjuvants when compared to the classic adjuvant alhydrogel (alum) and Phosphorylated Hexa-Acyl Disaccharide (PHAD), a synthetic TLR4 agonist. BECC molecules allow for antigen sparing of a recombinant HA (rHA) protein, elicit a more balanced IgG1/IgG2a response, and were protective in a prime only dosing schedule. Importantly, BECC molecules afford protection from a heterologous IAV strain demonstrating that a cross-protective influenza vaccine is possible when the antigen is effectively adjuvanted.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.

Combinations of alum and synthetic toll-like receptor agonists as adjuvants for CoVID-19 vaccines

Abstract Aluminum-based adjuvants (“alum”) are the most widely-used adjuvants for human vaccines, with an extensive history of safety and efficacy. Alum is known to elicit high antibody titers by driving a Th2-based immune response, which may not be optimal for many viral infections, such as SARS-CoV-2. Therefore, combining the strong safety and efficacy attributes of alum with a Th1-polarizing adjuvant could improve immunity against viral antigens. Here we test the combination of alum with synthetic TLR4- and TLR7/8 -ligands in vaccines against SARS-CoV-2. The combination of alum and TLR agonists resulted in efficient adjuvantation of both humoral and cell-mediated immunity. Alum adsorption studies showed low association between the small, positively-charged receptor binding domain (RBD) antigen and aluminum hydroxide (Alhydrogel) and high adsorption to negatively charged aluminum phosphate (Adju-phos). However, Adju-phos and Alhydrogel both enhanced immunity to the SARS-CoV-2 RBD antigen, suggesting antigen adsorption may not be required for the immune enhancing effects of alum. These data demonstrated that adjuvants combining alum with a TLR agonist result in an improved anti-viral immune response. However, the ability of alum to adsorb to an antigen may not predict its ability to effectively adjuvant.

Inclusion of an Optimized Plasmodium falciparum Merozoite Surface Protein 2-Based Antigen in a Trivalent, Multistage Malaria Vaccine.

Plasmodium falciparum merozoite surface protein (PfMSP)2 is a target of parasite-neutralizing Abs. Inclusion of recombinant PfMSP2 (rPfMSP2) as a component of a multivalent malaria vaccine is of interest, but presents challenges. Previously, we used the highly immunogenic PfMSP8 as a carrier to enhance production and/or immunogenicity of malaria vaccine targets. In this study, we exploited the benefits of rPfMSP8 as a carrier to optimize a rPfMSP2-based subunit vaccine. rPfMSP2 and chimeric rPfMSP2/8 vaccines produced in Escherichia coli were evaluated in comparative immunogenicity studies in inbred (CB6F1/J) and outbred (CD1) mice, varying the dose and adjuvant. Immunization of mice with both rPfMSP2-based vaccines elicited high-titer anti-PfMSP2 Abs that recognized the major allelic variants of PfMSP2. Vaccine-induced T cells recognized epitopes present in both PfMSP2 and the PfMSP8 carrier. Competition assays revealed differences in Ab specificities induced by the two rPfMSP2-based vaccines, with evidence of epitope masking by rPfMSP2-associated fibrils. In contrast to aluminum hydroxide (Alum) as adjuvant, formulation of rPfMSP2 vaccines with glucopyranosyl lipid adjuvant-stable emulsion, a synthetic TLR4 agonist, elicited Th1-associated cytokines, shifting production of Abs to cytophilic IgG subclasses. The rPfMSP2/8 + glucopyranosyl lipid adjuvant-stable emulsion formulation induced significantly higher Ab titers with superior durability and capacity to opsonize P. falciparum merozoites for phagocytosis. Immunization with a trivalent vaccine including PfMSP2/8, PfMSP1/8, and the P. falciparum 25 kDa sexual stage antigen fused to PfMSP8 (Pfs25/8) induced high levels of Abs specific for epitopes in each targeted domain, with no evidence of antigenic competition. These results are highly encouraging for the addition of rPfMSP2/8 as a component of an efficacious, multivalent, multistage malaria vaccine.

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.

Prophylactic efficacy against Mycobacterium tuberculosis using ID93 and lipid-based adjuvant formulations in the mouse model.

An estimated 10 million people developed tuberculosis (TB) disease in 2019 which underscores the need for a vaccine that prevents disease and reduces transmission. The aim of our current studies is to characterize and test a prophylactic tuberculosis vaccine comprised of ID93, a polyprotein fusion antigen, and a liposomal formulation [including a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant, GLA) and QS-21] in a preclinical mouse model of TB disease. Comparisons of the ID93+GLA-LSQ vaccines are also made to the highly characterized ID93+GLA-SE oil-in-water emulsion adjuvant, which are also included these studies. The recent success of vaccine candidate M72 combined with adjuvant AS01E (GlaxoSmithKline Biologicals) in reducing progression to active disease is promising and has renewed excitement for experimental vaccines currently in the TB vaccine pipeline. The AS01E adjuvant contains monophosphoryl lipid A (MPL) and QS-21 (a saponin) in a liposomal formulation. While AS01E has demonstrated potent adjuvant activity as a component of both approved and experimental vaccines, developing alternatives to this adjuvant system will become important to fill the high demand envisioned for future vaccine needs. Furthermore, replacement sources of potent adjuvants will help to supply the demand of a TB vaccine [almost one-quarter of the world’s population are estimated to have latent Mycobacterium tuberculosis (Mtb) according to the WHO 2019 global TB report], addressing (a) cost of goods, (b) supply of goods, and (c) improved efficacy of subunit vaccines against Mtb. We show that both ID93+GLA-SE (containing an emulsion adjuvant) and ID93+GLA-LSQ (containing a liposomal adjuvant) induce ID93-specific TH1 cellular immunity including CD4+CD44+ T cells expressing IFNγ, TNF, and IL-2 (using flow cytometry and intracellular cytokine staining) and vaccine-specific IgG2 antibody responses (using an ELISA). In addition, both ID93+GLA-SE and ID93+GLA-LSQ effectively decrease the bacterial load within the lungs of mice infected with Mtb. Formulations based on this liposomal adjuvant formulation may provide an alternative to AS01 adjuvant systems.

A51 ENTERIC PARASITE INFECTION-INDUCED ALTERATION OF THE GUT MICROBIOTA REGULATES INTESTINAL GOBLET CELL BIOLOGY AND MUCIN PRODUCTION VIA TLR2 SIGNALING

Abstract Background Goblet cells (GCs) are the major source of mucin which are the main components of the mucus layer that represents the front line of innate defense in the gastrointestinal (GI) tract. Hyperplasia of GCs and increased mucin production are observed in many enteric nematode infections such as Trichuris muris infection. Increased mucin production contributes to parasite clearance by trapping in mucus and inhibiting motility. The GI tract contains trillions of commensal microbes, and these microbes control mucin production from GCs by activating different signaling cascades. During nematode parasite infection due to the coexistence of parasites and microbiota in close proximity of GCs in gut, it is likely that this nematode-microbiota interaction plays an important role in mucin production. Toll-like receptors (TLRs), components of the innate immune system, sense gut microbiota stimuli. The human GC-like cell line, LS174T, expresses TLR2 mRNA which was enhanced by stimulation with synthetic TLR2 ligands. We hypothesize T. muris-induced altered microbiota modulates GC response and mucin production via TLR2 signaling. Aims To elucidate the role of T. muris-induced altered gut microbiota in the regulation of intestinal GC response and mucin production via host TLR2 signaling. Methods C57BL/6 mice were infected by gavage with ~300 T. muris eggs and infectivity was confirmed by worm burden. Microbiota was analyzed by 16s rRNA sequencing. Colonic GCs response, mucins and TLR2 expression and cytokines production were assessed in germ-free (GF) mice receiving non-infected and T. muris-infected microbiota (collected on day 36 post-infection to exclude worms). Muc2 and Muc5ac expression were assessed in wild-type (WT) and TLR2 deficient (TLR2-/-) mice transplanted with T. muris-infected microbiota following antibiotic treatment. Results We observed a difference in microbial composition between non-infected and T. muris infected mice. Transfer of T. muris-infected microbiota into GF mice significantly increased GC numbers and TLR2 expression as well as up-regulated Muc2 and Muc5ac expression and IL-4, IL-13 production compared to GF mice with non-infected microbiota. Antibiotic-treated TLR2-/- mice after receiving microbiota from T. muris-infected mice showed significantly decreased expression of Muc2 and Muc5ac compared to antibiotic-treated WT mice receiving the same microbiota. Conclusions T. muris-induced altered microbiota influences intestinal GC response and mucin production via TLR2. In addition to enhancing our understanding on the interaction of parasite with resident microbiota in host defense, this study provides new information on TLR2 based innate signaling in the regulation of GC biology and mucin production. Funding Agencies NSERC

Glyco-Nanoadjuvants: Sugar Structures on Carriers of a Small Molecule TLR7 Ligand Affect Their Immunostimulatory Activities.

Toll-like receptors (TLRs) are pattern recognition receptors that activate innate immunity, and their ligands are promising adjuvants for vaccines and immunotherapies. Small molecule TLR7 ligands are ideal vaccine adjuvants as they induce not only proinflammatory cytokines but also type I interferons. However, their application has only been approved for local administration due to severe systemic immune-related adverse events. In a previous study, we prepared the gold nanoparticles coimmobilized with synthetic small molecule TLR7 ligand, 1V209, and α-mannose (1V209-αMan-GNPs). 1V209-αMan-GNPs were selectively delivered via a cell surface sugar-binding protein, mannose receptor, which enabled selective delivery of TLR7 ligands to immune cells. Besides the mannose receptor, immune cells express various sugar-binding proteins such as macrophage galactose binding lectins and sialic acid-binding immunoglobulin-type lectins and recognize distinct sugar structures. Hence, in the present study, we investigated whether sugar structures on GNPs affect the efficiency and selectivity of intracellular delivery and subsequent immunostimulatory potencies. Five neutral sugars and two sialosides were selected and each sugar was coimmobilized with 1V209 onto GNPs (1V209-SGNPs) and their innate immunostimulatory potencies were compared to that of 1V209-αMan-GNPs. The in vitro study using mouse bone marrow derived dendritic cells (BMDCs) demonstrated that α-glucose, α-N-acetylglucosamine, or α-fucose immobilized 1V209-SGNPs increased interleukin-6 and type I interferon release similar to that of 1V209-αMan-GNPs, whereas galacto-type sugar immobilized 1V209-SGNPs predominantly enhanced type I interferon release. In contrast, sialoside immobilized 1V209-SGNPs did not enhance the potency of 1V209. In the in vivo immunization study using ovalbumin as a model antigen, neutral sugar immobilized 1V209-SGNPs induced comparable T helper-1 immune response to that of 1V209-αMan-GNPs and by 10-fold higher than that of sialoside immobilized 1V209-SGNPs. These results indicate that the sugar structures on 1V209-SGNPs affect their immunostimulatory activities, and functionalization of the carrier particles is important to shape immune responses.

Cyclic Di-Adenosine Monophosphate: A Promising Adjuvant Candidate for the Development of Neonatal Vaccines

Underdeveloped immunity during the neonatal age makes this period one of the most dangerous during the human lifespan, with infection-related mortality being one of the highest of all age groups. It is also discussed that vaccination during this time window may result in tolerance rather than in productive immunity, thus raising concerns about the overall vaccine-mediated protective efficacy. Cyclic di-nucleotides (CDN) are bacterial second messengers that are rapidly sensed by the immune system as a danger signal, allowing the utilization of these molecules as potent activators of the immune response. We have previously shown that cyclic di-adenosine monophosphate (CDA) is a potent and versatile adjuvant capable of promoting humoral and cellular immunity. We characterize here the cytokine profiles elicited by CDA in neonatal cord blood in comparison with other promising neonatal adjuvants, such as the imidazoquinoline resiquimod (R848), which is a synthetic dual TLR7 and TLR8 agonist. We observed superior activity of CDA in eliciting T helper 1 (Th1) and T follicular helper (TfH) cytokines in cells from human cord blood when compared to R848. Additional in vivo studies in mice showed that neonatal priming in a three-dose vaccination schedule is beneficial when CDA is used as a vaccine adjuvant. Humoral antibody titers were significantly higher in mice that received a neonatal prime as compared to those that did not. This effect was absent when using other adjuvants that were reported as suitable for neonatal vaccination. The biological significance of this immune response was assessed by a challenge with a genetically modified influenza H1N1 PR8 virus. The obtained results confirmed that CDA performed better than any other adjuvant tested. Altogether, our results suggest that CDA is a potent adjuvant in vitro on human cord blood, and in vivo in newborn mice, and thus a suitable candidate for the development of neonatal vaccines.

Oral administration of PEGylated TLR7 ligand ameliorates alcohol-associated liver disease via the induction of IL-22

Effective therapies for alcohol-associated liver disease (ALD) are limited; therefore, the discovery of new therapeutic agents is greatly warranted. Toll-like receptor 7 (TLR7) is a pattern recognition receptor for single-stranded RNA, and its activation prevents liver fibrosis. We examined liver and intestinal damage in Tlr7-/- mice to determine the role of TLR7 in ALD pathogenesis. In an alcoholic hepatitis (AH) mouse model, hepatic steatosis, injury, and inflammation were induced by chronic binge ethanol feeding in mice, and Tlr7 deficiency exacerbated these effects. Because these results demonstrated that endogenous TLR7 signaling activation is protective in the AH mouse model, we hypothesized that TLR7 activation may be an effective therapeutic strategy for ALD. Therefore, we investigated the therapeutic effect of TLR7 agonistic agent, 1Z1, in the AH mouse model. Oral administration of 1Z1 was well tolerated and prevented intestinal barrier disruption and bacterial translocation, which thus suppressed ethanol-induced hepatic injury, steatosis, and inflammation. Furthermore, 1Z1 treatment up-regulated the expression of antimicrobial peptides, Reg3b and Reg3g, in the intestinal epithelium, which modulated the microbiome by decreasing and increasing the amount of Bacteroides and Lactobacillus, respectively. Additionally, 1Z1 up-regulated intestinal interleukin (IL)-22 expression. IL-22 deficiency abolished the protective effects of 1Z1 in ethanol-induced liver and intestinal damage, suggesting intestinal IL-22 as a crucial mediator for 1Z1-mediated protection in the AH mouse model. Collectively, our results indicate that TLR7 signaling exerts protective effects in the AH mouse model and that a TLR7 ligand, 1Z1, holds therapeutic potential for the treatment of AH.

IFIT Proteins Are Involved in CXCL10 Expression in Human Glomerular Endothelial Cells Treated with a Toll-Like Receptor 3 Agonist

Introduction: Various viruses including a novel coronavirus (SARS-CoV-2) can infect the kidney. When viruses invade the glomeruli from the bloodstream, glomerular endothelial cells (GECs) initiate the innate immune reactions. We investigated the expression of interferon (IFN)-induced protein with tetratricopeptide repeats (IFIT) 1/2/3, antiviral molecules, in human GECs treated with a toll-like receptor (TLR) 3 agonist. Role of IFIT1/2/3 in the expression of C-X-C motif chemokine ligand 10 (CXCL10) was also examined. Methods: Human GECs were cultured and stimulated with polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 agonist. Real-time qPCR, Western blotting, and ELISA were used to examine the expression of IFIT1/2/3, IFN-β, and CXCL10. RNA interference against IFN-β or IFIT1/2/3 was also performed. Results: Expression of IFIT1/2/3 and CXCL10 was induced by poly IC in GECs. The inductions were inhibited by RNA interfering of IFN-β. Knockdown of IFIT1/2/3 decreased the CXCL10 expression. Knockdown of IFIT3 decreased the expression of IFIT1 and IFIT2 proteins. Conclusion: IFIT1/2/3 and CXCL10 were induced by poly IC via IFN-β in GECs. IFIT1/2/3 may increase the expression of CXCL10 which induces lymphocyte chemotaxis and may inhibit the replication of infected viruses. These molecules may play a role in GEC innate immune reactions in response to viruses.