T cell-independent eradication of experimental glioma by intravenous TLR7/8-agonist-loaded nanoparticles.

Glioblastoma, the most common and aggressive primary brain tumor type, is considered an immunologically "cold" tumor with sparse infiltration by adaptive immune cells. Immunosuppressive tumor-associated myeloid cells are drivers of tumor progression. Therefore, targeting and reprogramming intratumoral myeloid cells is an appealing therapeutic strategy. Here, we investigate a ß-cyclodextrin nanoparticle (CDNP) formulation encapsulating the Toll-like receptor 7 and 8 (TLR7/8) agonist R848 (CDNP-R848) to reprogram myeloid cells in the glioma microenvironment. We show that intravenous monotherapy with CDNP-R848 induces regression of established syngeneic experimental glioma, resulting in increased survival rates compared with unloaded CDNP controls. Mechanistically, CDNP-R848 treatment reshapes the immunosuppressive tumor microenvironment and orchestrates tumor clearing by pro-inflammatory tumor-associated myeloid cells, independently of T cells and NK cells. Using serial magnetic resonance imaging, we identify a radiomic signature in response to CDNP-R848 treatment and ultrasmall superparamagnetic iron oxide (USPIO) imaging reveals that immunosuppressive macrophage recruitment is reduced by CDNP-R848. In conclusion, CDNP-R848 induces tumor regression in experimental glioma by targeting blood-borne macrophages without requiring adaptive immunity.

Effects of Lysophosphatidylcholine on Intestinal Health of Turbot Fed High-Lipid Diets.

An 8-week feeding trial was conducted, where turbot were fed four experimental diets, containing different LPC levels (0%, 0.1%, 0.25%, and 0.5%, named LPC0, LPC0.1, LPC0.25, and LPC0.5, respectively). The intestinal morphology results showed that there were no widened lamina propria and mixed inflammatory cells in the LPC-supplemented groups. Dietary LPC remarkably decreased the expression of TLRs (TLR3, TLR8, TLR9, and TLR22), MyD88, and signaling molecules (NF-κB, JNK, and AP-1). Similarly, diets with LPC supplementation markedly depressed the gene expression of NF-κB and JNK signaling pathway downstream genes (TNF-α, IL-1ß, Bax, Caspase9, and Caspase-3). Furthermore, dietary LPC modified the intestinal microbial profiles, increasing the relative abundance of short-chain fatty acids-producers, lactic acid bacteria, and digestive enzyme-producing bacteria. Predictive functions of intestinal microbiota showed that turbot fed LPC diets had a relatively higher abundance of functions, such as lipid metabolism and immune system, but a lower abundance of functions, such as metabolic diseases and immune system diseases. The activities of intestinal acid phosphatase and alkaline phosphatase were also increased by dietary LPC. In conclusion, LPC supplementation could regulate the intestinal mucosal barrier via the TLR signaling pathway and alter the intestinal microbiota profile of turbot fed high-lipid diets.

Bovine colostrum promoted ileal health in newborn lambs at 24 h after birth: insight from intestinal morphology and innate immunity.

The contribution of colostrum to passive immunity transfer and intestinal protection in newborn ruminants is well known; however, it is currently unclear how colostrum intake affects intestinal innate immunity. We investigated the effects of bovine colostrum intake on ileal morphology, expression of genes involved in intestinal innate immunity, and serum concentrations of inflammatory cytokines in newborn lambs. Twenty-seven newborn male Hu lambs were used, of which 18 were bottle-fed either bovine colostrum (C24h; n = 9) or bovine mature milk (M24h; n = 9) within the first 2 h after birth at an intake of approximately 8% of BW; the remaining nine lambs did not receive any feeding (N24h). Blood and ileal tissue samples were collected after the lambs were slaughtered at 24 h after birth. Ileal villus height and villus height-to-crypt depth ratio were significantly higher in C24h than those in N24h and M24h lambs (P < 0.01). Messenger RNA (mRNA) abundance of toll-like receptor (TLR)-2, TLR3, TLR4, TLR6, TLR7, TLR8 and tumour necrosis factor alpha in the ileum was lower in C24h than that in N24h lambs (P < 0.05). Moreover, C24h lambs had a lower TLR3 mRNA abundance (P < 0.01) and a trend of lower TLR6 (P = 0.06) and interleukin 1 beta (P = 0.08) expression compared with those in M24h lambs. We also observed strong positive correlations of tumour necrosis factor alpha expression with that of TLR2 (r = 0.71; P < 0.001), TLR4 (r = 0.88; P < 0.001) and TLR8 (r = 0.83; P < 0.001). Interestingly, the expression of barrier-related molecules, including mucin-13, lysozyme, claudin (CLDN)-1, CLDN2, CLDN4, CLDN7, CLDN12, occludin, zonula occluden-1 and junctional adhesion molecule-1, was consistently lower in C24h lambs than that in N24h and M24h lambs (P < 0.05). These results indicated that the beneficial roles of colostrum intake on intestinal protection in newborn lambs were associated with low TLR expression, which was reflected by improved intestinal development and reduced inflammatory response. Further studies using fluorescence in situ hybridisation and immunohistochemical methods are needed to further explore the mechanisms underlying the lower expression of intestinal barrier-related molecules due to colostrum feeding.

In Situ Tumor Vaccination with Calcium-Linked Degradable Coacervate Nanocomplex Co-Delivering Photosensitizer and TLR7/8 Agonist to Trigger Effective Anti-Tumor Immune Responses.

In situ anti-tumor vaccination is an attractive type of cancer immunotherapy which relies on the effectiveness of dendritic cells (DCs) to engulf tumor antigens, become activated, and present antigens to T cells in lymphoid tissue. Here, a multifunctional nanocomplex based on calcium crosslinked polyaspartic acid conjugated to either a toll-like receptor (TLR)7/8 agonist or a photosensitizer is reported. Intratumoral administration of the nanocomplex followed by laser irradiation induces cell killing and hence generation of a pool of tumor-associated antigens, with concomitant promotion of DCs maturation and expansion of T cells in tumor-draining lymph nodes. Suppression of tumor growth is observed both at the primary site and at the distal site, thereby hinting at successful induction of an adaptive anti-tumor response. This strategy holds promise for therapeutic application in a pre-operative and post-operative setting to leverage to mutanome of the patient's own tumor to mount immunological memory to clear residual tumor cells and metastasis.

Sustained IL-4 priming of macrophages enhances the inflammatory response to TLR7/8 ligand R848.

Macrophages (Mϕ) are highly plastic, and can acquire a variety of functional phenotypes depending on the presence of different stimuli in their local environment. Mφ stimulated by interleukin (IL)-4 induce an alternative activation state and function as anti-inflammatory cells and promote tissue repair. However, there is overwhelming evidence that IL-4 can play a role in promoting inflammation. In asthma and allergic inflammation, IL-4 mediates proinflammatory responses that lead to tissue damage. Thus the effect of IL-4 on the outcome of the immune responses is greatly influenced by other cofactors and cytokines present in the microenvironment. R848 (resiquimod), a TLR7/8 agonist is a novel vaccine adjuvant, triggering a strong Th1-skewed response but its efficacy as a vaccine adjuvant shows variable results. It is not currently known whether the presence of IL-4 can dampen or enhance immunity in response to TLR7 agonists. In the present study, we sought to investigate the impact of IL-4-induced Mφ polarization on the outcome of R848 stimulation. The activation marker expression and production of cytokines were measured in murine spleen-derived Mφ. Protein expression levels of innate recognition molecules and transcription factors involved, including retinoic-acid inducible gene I, mitochondrial antiviral signaling protein, stimulator of interferon genes (STING), and IFN regulatory factors were evaluated in activated Mφ. These play a crucial role in the control of viral replication and optimal CD8+ T cell priming. We report that sustained priming with IL-4 alone promotes an antiviral response in Mφ, and enhances proinflammatory responses to R848 treatment. This highlights the need for better understanding of IL-4 proinflammatory functions and its potential use as a broad-acting antiviral in combination with R848 may be used in combination with other therapies to target the innate arm of immunity against emerging infections.

Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that commonly affects multiple joints of the body. Currently, there is no permanent cure to the disease, but it can be managed with several potent drugs that cause serious side effects on prolonged use. Traditional remedies are considered promising for the treatment of several diseases, particularly chronic conditions, because they have lower side effects compared to synthetic drugs. In folklore, the rhizome of Alpinia calcarata Roscoe (Zingiberaceae) is used as a major ingredient of herbal formulations to treat RA. Phytoconstituents reported in A. calcarata rhizomes are diterpenoids, sesquiterpenoid, flavonoids, phytosterol, and volatile oils. The present study is intended to understand the molecular-level interaction of phytoconstituents present in A. calcarata rhizomes with RA molecular targets using computational approaches. A total of 30 phytoconstituents reported from the plant were used to carry out docking with 36 known targets of RA. Based on the docking results, 4 flavonoids were found to be strongly interacting with the RA targets. Further, molecular dynamics simulation confirmed stable interaction of quercetin with 6 targets (JAK3, SYK, MMP2, TLR8, IRAK1, and JAK1), galangin with 2 targets (IRAK1 and JAK1), and kaempferol (IRAK1) with one target of RA. Moreover, the presence of these three flavonoids was confirmed in the A. calcarata rhizome extract using LC-MS analysis. The computational study suggests that flavonoids present in A. calcarata rhizome may be responsible for RA modulatory activity. Particularly, quercetin and galangin could be potential development candidates for the treatment of RA. Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

A TLR7/8 Agonist-Including DOEPC-Based Cationic Liposome Formulation Mediates Its Adjuvanticity Through the Sustained Recruitment of Highly Activated Monocytes in a Type I IFN-Independent but NF-κB-Dependent Manner.

Novel adjuvants, such as Toll-like receptors (TLRs) agonists, are needed for the development of new formulations able to circumvent limitations of current vaccines. Among TLRs, TLR7/8 agonists represent promising candidates, as they are well described to enhance antigen-specific antibody responses and skew immunity toward T helper (TH) 1 responses. We find here that the incorporation of the synthetic TLR7/8 ligand 3M-052 in a cationic DOEPC-based liposome formulation shifts immunity toward TH1 responses and elicits strong and long-lasting germinal center and follicular T helper cell responses in adult mice. This reflects the prolonged recruitment of innate cells toward the site of immunization and homing of activated antigen-loaded monocytes and monocyte-derived dendritic cells toward draining lymph nodes. We further show that this adjuvanticity is independent of type I IFN but NF-κB-dependent. Overall, our data identify TLR7/8 agonists incorporated in liposomes as promising and effective adjuvants to enhance TH1 and germinal center responses.

Discovery of potent, orally bioavailable in vivo efficacious antagonists of the TLR7/8 pathway.

Antagonism of the Toll-like receptors (TLRs) 7 and TLR8 has been hypothesized to be beneficial to patients suffering from autoimmune conditions. A phenotypic screen for small molecule antagonists of TLR7/8 was carried out in a murine P4H1 cell line. Compound 1 was identified as a hit that showed antagonistic activity on TLR7 and TLR8 but not TLR9, as shown on human peripheral blood mononuclear cells (hPBMCs). It was functionally cross reactive with mouse TLR7 but lacked oral exposure and had only modest potency. Chemical optimization resulted in 2, which showed in vivo efficacy following intraperitoneal administration. Further optimization resulted in 8 which had excellent in vitro activity, exposure and in vivo activity. Additional work to improve physical properties resulted in 15, an advanced lead that had favorable in vitro and exposure properties. It was further demonstrated that activity of the series tracked with binding to the extracellular domain of TLR7 implicating that the target of this series are endosomal TLRs rather than downstream signaling pathways.

Optimization of 8-oxoadenines with toll-like-receptor 7 and 8 activity.

Toll-like receptors 7 and 8 (TLR7/8) agonists are potent immunostimulants that are attracting considerable interest as vaccine adjuvants. We recently reported the synthesis of a new series of 2-O-butyl-8-oxoadenines substituted at the 9-position with various linkers and N-heterocycles, and showed that TLR7/8 selectivity, potency and cytokine induction could be modulated by varying the alkyl linker length and the N-heterocyclic ring. In the present study, we further optimized the oxoadenine scaffold by investigating the effect of different substituents at the 2-position of the oxoadenine on TLR7/8 potency/selectivity, cytokine induction and DC maturation in human PBMCs. The results show that introducing a 1-(S)-methylbutoxy group at the 2-position of the oxoadenine significantly increased potency for TLR7/8 activity, cytokine induction and DC maturation.

Datura Metel L. Ameliorates Imiquimod-Induced Psoriasis-Like Dermatitis and Inhibits Inflammatory Cytokines Production through TLR7/8-MyD88-NF-κB-NLRP3 Inflammasome Pathway.

BACKGROUND: Psoriasis is a chronic, immune-mediated inflammatory skin disease, and the inflammatory response plays an important role in its development and progression. Datura metel L. is a traditional Chinese medicine that exhibited a significant therapeutic effect on psoriasis in our previous study due to its remarkable anti-inflammatory effect. Meanwhile, the mechanism underlying its effects on psoriasis is still unclear. METHODS: An imiquimod-induced psoriasis-like dermatitis mouse model was constructed to evaluate the protective effect of the effective part of Datura metel L. (EPD), which was verified by evaluations of the Psoriasis Area and Severity Index (PASI) score. Hematoxylin and eosin (H&E) staining, immunohistochemical examination, enzyme-linked immunosorbent assay (ELISA), and Western blot were used to measure the inflammatory cytokines and the protein expression associated with the Toll-like receptor 7- myeloid differentiation primary response gene 88-nuclear Factor-κB-nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3 (TLR7/8-MyD88-NF-κB-NLRP3) inflammasome pathway. RESULTS: EPD significantly decreased the PASI, reduced epidermal thickness, and decreased the proliferation and differentiation of epidermal cells in psoriasis-like dermatitis C57BL/6 mice induced by imiquimod (IMQ). Furthermore, EPD reduced the infiltration of CD3+ cells to psoriatic lesions, as well as ameliorated the elevations of intercellular adhesion molecule 1 (ICAM-1) and inhibited the production of imiquimod-induced inflammatory cytokines, including IL-1ß, IL-2, IL-6, IL-10, IL-12, IL-17, IL-22, IL-23, tumor necrosis factor-α (TNF-α), monocyte chemotactic protein 1 (MCP-1), and interferon-γ (IFN-γ). Besides, EPD decreased the imiquimod-induced expression levels of TLR7, TLR8, TRAF6, MyD88, p-IKKα, p-IKBα, p-NF-κB, NLRP3, apoptosis-associated speck-like protein contained a caspase recruitment domain (ASC), cysteinyl aspartate specific proteinase 1 (caspase-1), and IL-1ß. CONCLUSION: This study demonstrated that EPD exhibited a protective effect on an imiquimod-induced psoriasis mice model by inhibiting the inflammatory response, which might be ascribed to the inhibition of the TLR7/8-MyD88-NF-κb-NLRP3 inflammasome pathway.

Formulation and preclinical evaluation of a toll-like receptor 7/8 agonist as an anti-tumoral immunomodulator.

The toll-like receptor 7 and 8 (TLR7/8) agonist Resiquimod (R848) has been recognized as a promising immunostimulator for the treatment of cutaneous cancers in multiple clinical trials. However, systemic administration of R848 often results in strong immune-related toxicities while having limited therapeutic effects to the tumor. In the present study, a prodrug-based nanocarrier delivery system was developed that exhibited high therapeutic efficiency. R848 was conjugated to α-tocopherol to constitute an R848-Toco prodrug, followed by formulating with a tocopherol-modified hyaluronic acid (HA-Toco) as a polymeric nano-suspension. In vitro evaluation showed that the delivery system prolonged the release kinetics while maintaining TLR agonist activities. When administered subcutaneously, the nano-suspension formed a depot at the injection site, inducing localized immune responses without systemic expansion. This formulation also suppressed tumor growth and recruited immune cells to the tumor in a murine model of head and neck cancer. In a preclinical canine study of spontaneous mast cell tumors, the treatment led to a 67% response rate (three partial remissions and one complete remission).

Decryption of Active Constituents and Action Mechanism of the Traditional Uighur Prescription (BXXTR) Alleviating IMQ-Induced Psoriasis-Like Skin Inflammation in BALB/c Mice.

Bai Xuan Xia Ta Re Pian (BXXTR) is a traditional Uighur medicine ancient prescription in China widely used in the treatment of psoriasis, presenting a high curative rate and few side effects. Given that the active constituents and action mechanism still remain unclear, the aim of this study is to explore the potential active constituents and mechanism of antipsoriasis of BXXTR. Psoriasis-like lesions model in BALB/c mice was induced by Imiquimod (IMQ), including five treatment groups: control group, IMQ-treated group, IMQ-ACITRETIN group (Positive control group), IMQ-BXXTR low dose group, IMQ-BXXTR medium dose group and IMQ-BXXTR high dose group. The Psoriasis Area and Severity Index (PASI) score, skin and ear thickness, and histologic section were collected. The differentially expressed genes were determined by using RNAseq technology and the relevant pathways were analyzed by KEGG database. The ELISA kit and western blot assays were used to detect the related protein expression levels. In addition, the chemical constituents of BXXTR were determined by UPLC-TOF-MS analysis and the potential active constituents were predicted by SEA DOCK and Gene Ontology (GO). The data demonstrated that BXXTR significantly alleviated IMQ-induced psoriasis. RNA-seq analysis showed that BXXTR induced the expression levels of 31 genes; the KEGG analysis suggested that BXXTR could significantly change IL-17-related inflammatory pathways. The ELISA kit confirmed that the expression level of IL-17A protein was significantly reduced. 75 compounds of BXXTR were determined by UPLC-TOF-MS analysis, 11 of 75 compounds were identified as potential active compounds by similarity ensemble approach docking (SEA DOCK) and Gene Ontology (GO). BXXTR reduced the severity of skin lesions by inhibiting IL-17-related inflammatory pathways. The results indicated that BXXTR could suppress psoriasis inflammation by multiple-constituents-regulated multiple targets synergistically. Collectively, this study could provide important guidance for the elucidation of the active constituents and action mechanism of BXXTR for the treatment of psoriasis.

Gender Difference and Dietary Supplemental Vitamin B6: Impact on Colon Luminal Environment.

Colon diseases can be affected by several factors such as gender difference and dietary supplemental vitamin B6 (B6). The nutritional status of B6 is affected by gender difference, leading us to hypothesize that gender difference affects colon luminal environment, which is dependent on B6 status. To investigate this hypothesis, we fed male and female rats a diet containing 1 mg, 7 mg, or 35 mg pyridoxine HCl/kg diet for 6 wk. We found significantly higher fecal mucin levels in female rats compared to those in male rats. Supplemental B6 significantly increased fecal mucins and was particularly profound in the female rats. The abundances of cecal and fecal Akkermansia muciniphila (mucin degrader) were unaffected. The fecal mucin levels were significantly correlated with colonic free threonine and serine and with gene expression of colon MUC16, implying that the combined effect of gender and dietary B6 on fecal mucins was mediated by the alteration in the levels of such amino acids and MUC16 expression. This study further showed the significant effects of gender difference on colonic free amino acids such as threonine, ornithine, asparagine/aspartate ratio, and glutamine/glutamate ratio, cecal and fecal Lactobacillus spp. levels, and colonic gene expressions of MUC16 and TLR8, the factors relating to colon health and diseases. Therefore, our findings suggest that gender difference and dietary B6 may have an impact on colon diseases by modulating these parameters.

Natural Modulators of Endosomal Toll-Like Receptor-Mediated Psoriatic Skin Inflammation.

Psoriasis is a chronic inflammatory autoimmune disease that can be initiated by excessive activation of endosomal toll-like receptors (TLRs), particularly TLR7, TLR8, and TLR9. Therefore, inhibitors of endosomal TLR activation are being investigated for their ability to treat this disease. The currently approved biological drugs adalimumab, etanercept, infliximab, ustekinumab, ixekizumab, and secukizumab are antibodies against effector cytokines that participate in the initiation and development of psoriasis. Several immune modulatory oligonucleotides and small molecular weight compounds, including IMO-3100, IMO-8400, and CPG-52364, that block the interaction between endosomal TLRs and their ligands are under clinical investigation for their effectiveness in the treatment of psoriasis. In addition, several chemical compounds, including AS-2444697, PF-05387252, PF-05388169, PF-06650833, ML120B, and PHA-408, can inhibit TLR signaling. Although these compounds have demonstrated anti-inflammatory activity in animal models, their therapeutic potential for the treatment of psoriasis has not yet been tested. Recent studies demonstrated that natural compounds derived from plants, fungi, and bacteria, including mustard seed, Antrodia cinnamomea extract, curcumin, resveratrol, thiostrepton, azithromycin, and andrographolide, inhibited psoriasis-like inflammation induced by the TLR7 agonist imiquimod in animal models. These natural modulators employ different mechanisms to inhibit endosomal TLR activation and are administered via different routes. Therefore, they represent candidate psoriasis drugs and might lead to the development of new treatment options.

Paeonol ameliorates imiquimod-induced psoriasis-like skin lesions in BALB/c mice by inhibiting the maturation and activation of dendritic cells.

Paeonol, an active component derived from the traditional Chinese medicine Cortex Moutan, possesses anti-inflammatory, analgesic, antioxidant and anti-allergic properties. Psoriasis is a chronic, recurrent, inflammatory dermatosis accompanied by excessive activation of Toll­like receptors (TLRs) in dendritic cells (DCs), which are primarily responsible for initiating an immune response. We investigated the effect of paeonol on inflammation in an imiquimod (IMQ)-induced psoriasis-like mouse model and murine bone marrow-derived dendritic cells (BMDCs) stimulated by R848. Mice were intragastrically administered 100 mg/kg (high), 50 mg/kg (medium) and 25 mg/kg (low) paeonol, respectively. We evaluated inflammation of psori-asis­like lesions based on histological changes, protein levels of myeloid differentiation factor 88 (MyD88) and TLR8 in skin lesions by western blotting, and levels of CD11c+ DCs in skin by immunoassay and in spleens by flow cytometry. Inflammatory cytokines [interleukin (IL)-23, IL-12 and IL-1ß] in skin lesions and BMDCs were also assessed by RT-PCR and ELISA. Application of paeonol decreased IMQ-induced keratinocyte proliferation, and infiltration of CD3+ cells, while the treatment ameliorated CD11c+ cells in the spleen and skin, and reduced MyD88 and TLR8 proteins in skin lesions. Paeonol inhibited IMQ-induced mRNA expression of IL-23, but not IL-12 and IL-1ß in BMDCs, along with significantly lower levels of DCs expressing MHCⅡ, CD80 and CD86 in vitro. These results indicate that paeonol suppresses the maturation and activation of DCs by decreasing MyD88 and TLR8 proteins in the TLR7/8 signaling pathway which finally alleviates psoriasis­like skin lesions. The TLR7/8 signaling pathway in DCs provides an important insight into the mechanism of psoriasis, and paeonol may be a potent therapeutic drug for psoriasis.

Nanoformulation of synergistic TLR ligands to enhance vaccination against Entamoeba histolytica.

Diarrheal infectious diseases represent a major cause of global morbidity and mortality. There is an urgent need for vaccines against diarrheal pathogens, especially parasites. Modern subunit vaccines rely on combining a highly purified antigen with an adjuvant to increase their efficacy. In the present study, we evaluated the ability of a nanoliposome adjuvant system to trigger a strong mucosal immune response to the Entamoeba histolytica Gal/GalNAc lectin LecA antigen. CBA/J mice were immunized with alum, emulsion or liposome based formulations containing synthetic TLR agonists. A liposome formulation containing TLR4 and TLR7/8 agonists was selected based on its ability to generate intestinal IgA, plasma IgG2a/IgG1, IFN-γ and IL-17A. Immunization with a mucosal prime followed by a parenteral boost generated a high mucosal IgA response that inhibited adherence of parasites to mammalian cells. Inclusion of the immune potentiator all-trans retinoic acid in the regimen further improved the mucosal IgA response. Immunization protected from infection with up to 55% efficacy. Our results show that a nanoliposome delivery system containing TLR agonists is a promising prospect for the development of vaccines against enteric pathogens, especially when a multifaceted immune response is desired.

Evaluation of Lipopeptides as Toll-like Receptor 2 Ligands.

BACKGROUND: Peptide-based vaccines are considered to be the next generation of modern immunizations, as they are safe, easy to produce and well-defined. However, due to their weak immunogenic effect, it is important to first develop an appropriate adjuvant for peptide-based vaccines. OBJECTIVE: The aim of this work was to synthesize a series of four adjuvanting moieties as alkyne derivatives, incorporating dipalmitoyl serine (DPS), 1,3-diglyceride (DG), two hexadecane lipoamino acids (diLAA), and 2,3-dipalmitoyl-S-glycerylcysteine (Pam2Cys). Next aim was to synthesize and attach the azide derivative of biotinylated J14 peptide (model B-cell epitope) to the alkynes through copper- catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. Final aim was to test the ability of the final biotin labeled conjugates to directly interact with in vitro expressed TLR2 and 8 using AlphaScreen proximity assay. METHOD: All of the peptides were synthesized by manual stepwise solid phase peptide synthesis (SPPS) on rink amide MBHA resin using HATU/DIPEA Fmoc-chemistry. The target compounds were synthesized in a solution phase using CuAAC reaction. RESULTS: Pam2Cys analogue bound to TLR2 as expected. Analogues of DPS and C16-LAA showed also affinity to TLR2, while it did not bind to the control protein (TLR8), demonstrating ability of the DPS and C16-LAA to be recognized by TLR2. CONCLUSION: Four alkyne derivatives of lipids were successfully synthesized and coupled to a biotinylated J14 peptide to give a series of self-adjuvanting ligands. These ligands showed different affinity to TLR2 upon testing by AlphaScreen assay. The DPS derivative showed the most promising affinity in comparison to the standard TLR2 agonist, Pam2Cys.

Adsorption of a synthetic TLR7/8 ligand to aluminum oxyhydroxide for enhanced vaccine adjuvant activity: A formulation approach.

For nearly a century, aluminum salts have been the most widely used vaccine adjuvant formulation, and have thus established a history of safety and efficacy. Nevertheless, for extremely challenging disease targets such as tuberculosis or HIV, the adjuvant activity of aluminum salts may not be potent enough to achieve protective efficacy. Adsorption of TLR ligands to aluminum salts facilitates enhanced adjuvant activity, such as in the human papilloma virus vaccine Cervarix®. However, some TLR ligands such as TLR7/8 agonist imidazoquinolines do not efficiently adsorb to aluminum salts. The present report describes a formulation approach to solving this challenge by developing a lipid-based nanosuspension of a synthetic TLR7/8 ligand (3M-052) that facilitates adsorption to aluminum oxyhydroxide via the structural properties of the helper lipid employed. In immunized mice, the aluminum oxyhydroxide-adsorbed formulation of 3M-052 enhanced antibody and TH1-type cellular immune responses to vaccine antigens for tuberculosis and HIV.

Suppressive effect of ß,ß-dimethylacryloyl alkannin on activated dendritic cells in psoriasis by the TLR7/8 pathway.

ß,ß-dimethylacryloyl alkannin (DMA) is a key component of Lithospermum and possesses good efficacy for treating psoriasis. DMA inhibits activated dendritic cells (DCs), but the mechanism is unknown. Therefore, this study aimed to explore the modulation of the TLR7/8 pathway by DMA in psoriasis-activated DCs. Models of psoriasis-like skin lesions were established using BALB/c mice; 8 mice were treated with DMA (2.5mg/kg). Bone marrow cells were isolated and induced into DCs using R848, a TLR7/8 agonist. Splenic CD11c+ cells were detected by flow cytometry. Skin CD11c+ cells were detected by immunofluorescence. TLR7, TLR8, MYD88, and IRAKM proteins were detected by Western blot. The effects of DMA on surface molecules of DCs were observed by flow cytometry. mRNA expression of inflammatory factors was detected by qRT-PCR. Secreted cytokines were detected by cytometric bead array. Compared with the model group, psoriasis-like skin lesions were alleviated by DMA, the splenic CD11c+ cells were significantly decreased (P<0.01), and CD11c+ cell numbers in skin lesions were decreased (P<0.01). Expression levels of TLR7, MYD88, and IRAKM were significantly decreased (P<0.05). R848-stimulated DCs showed increased expression of I-A/I-E, CD80, and CD86 (P<0.01), increased IL-23 and IL-1ß mRNA and secretion (P<0.05), and increased TLR7, TLR8, MYD88, and IRAKM expression (P<0.01); DMA inhibited all of these effects of the TLR7/8 pathway activation by R848 (P<0.05). In conclusion, DMA could inhibit psoriasis-activated DCs via the TLR7/8 pathway.

Vitamin D [1,25(OH)2D3] Differentially Regulates Human Innate Cytokine Responses to Bacterial versus Viral Pattern Recognition Receptor Stimuli.

Vitamin D plays multiple roles in regulation of protective and maladaptive immunity. Although epidemiologic studies link poor in vivo 25(OH)D status to increased viral respiratory infections, we poorly understand how vitamin D affects viral pattern recognition receptor (PRR)-driven cytokine production. In this study, we hypothesized that the biologically active metabolite of vitamin D, 1,25(OH)2D3, inhibits human proinflammatory and anti-inflammatory innate cytokine responses stimulated by representative bacterial or viral PRR ligands. Fresh PBMCs or CD14(+) monocytes were stimulated with TLR4, TLR7/8-selective ligands, or respiratory syncytial virus (RSV) ± 1,25(OH)2D3. Proinflammatory and anti-inflammatory responses resulting from TLR4 stimulation were inhibited ∼50% in the presence of 1,25(OH)2D3. Conversely, its usage at physiologic through pharmacologic concentrations inhibited neither proinflammatory nor anti-inflammatory responses evoked by viral PRR ligands or infectious RSV. This differential responsiveness was attributed to the finding that TLR7/8, but not TLR4, stimulation markedly inhibited vitamin D receptor mRNA and protein expression, selectively reducing the sensitivity of viral PRR responses to modulation. 1,25(OH)2D3 also enhanced expression of IkBa, a potent negative regulator of NF-κB and cytokine production, in TLR4-stimulated monocytes while not doing so upon TLR7/8 stimulation. Thus, 1,25(OH)2D3 inhibits both proinflammatory and a broad panel of anti-inflammatory responses elicited by TLR4 stimulation, arguing that the common view of it as an anti-inflammatory immune response modifier is an oversimplification. In viral responses, it consistently fails to modify TLR7/8- or RSV-stimulated innate cytokine production, even at supraphysiologic concentrations. Collectively, the data call into question the rationale for increasingly widespread self-medication with vitamin D supplements.