To Toll-like Receptor Research Articles

The essential roles of memory B cells in the pathogenesis of systemic lupus erythematosus.

Emerging evidence indicates that memory B cells are dysfunctional in systemic lupus erythematosus (SLE). They are hyporesponsive to signalling through the B cell receptor (BCR) but retain responsiveness to Toll-like receptor (TLR) and type I interferon signalling, as well as to T cell-mediated activation via CD40-CD154. Chronic exposure to immune complexes of ribonucleoprotein (RNP)-specific autoantibodies and TLR-engaging or BCR-engaging cargo is likely to contribute to this partially anergic phenotype. TLR7 or TLR8 signalling and the resulting production of type I interferon, as well as the sustained activation by bystander T cells, fuel a positive feedforward loop in memory B cells that can evade negative selection and permit preferential expansion of anti-RNP autoantibodies. Clinical trials of autologous stem cell transplantation or of B cell-targeted monoclonal antibodies and chimeric antigen receptor (CAR) T cells have correlated replenishment of the memory B cell population with relapse of SLE. Moreover, the BCR hyporesponsiveness of memory B cells might explain the failure of non-depleting B cell-targeting approaches in SLE, including BTK inhibitors and anti-CD22 monoclonal antibodies. Thus, targeting of dysfunctional memory B cells might prove effective in SLE, while also avoiding the adverse events of broad-spectrum targeting of B celland plasma cell subsets that are not directly involved in disease pathogenesis.

Bach2 repression of CD36 regulates lipid-metabolism-linked effector functions in follicular B cells

The transcription repressor Bach2 plays a crucial role in shaping humoral immunity, but its cell-autonomous function remains elusive. Here, we reveal the mechanism by which Bach2 regulates effector cell maturation in peripheral B cells. In response to Toll-like receptor (TLR) agonists, Bach2 deficiency promotes the differentiation of follicular, but not marginal zone, B cells into effector cells, producing interleukin (IL)-6 and antibodies. This phenomenon is associated with changes in lipid metabolism, such as increases in CD36 expression, lipid influx, and fatty acid oxidation. Consistent with this, Bach2-deficient B cells exhibit elevated levels of mitochondrial oxidative stress, lipid peroxidation, and p38 activation. Mechanistically, Bach2 acts as a repressor of Cd36, and inhibition of CD36 or fatty acid oxidation reduces the differentiation of naive B cells into IL-6- and antibody-secreting cells. These results indicate Bach2 as a key metabolic checkpoint regulator crucial for maintaining a functionally quiescent state of follicular B cells.

Pancreatic Adenocarcinoma Up-Regulated Factor (PAUF) Transforms Human Monocytes into Alternative M2 Macrophages with Immunosuppressive Action

Tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) promote immune evasion, cancer cell proliferation, and metastasis. Ongoing research is focused on finding ways to prevent tumor growth by inhibiting TAM polarization, which has shown a correlation with unfavorable prognosis in clinical studies. Pancreatic adenocarcinoma up-regulated factor (PAUF) is a protein secreted from pancreatic cancer (PC) and acts as a TME modulator that affects the TME by acting on not only cancer cells but also stromal cells and immune cells. Tumor cells can evade the immune system by PAUF binding to Toll-like receptor (TLR) in monocytes, as this research shows. In this study, the examination centered around the recruitment of human monocytes by PAUF and the subsequent differentiation into macrophages. In an in vitro chemotaxis assay, PAUF induced chemotactic migration of TLR2-mediated monocytes. In addition, PAUF induced differentiation of monocytes into M2 macrophages, which was verified based on expressing surface markers and cytokines and morphological analysis. The inhibition of T cell proliferation and function was observed in differentiated M2 macrophages. To conclude, these findings indicate that PAUF functions as a promoter of cancer progression by regulating the recruitment and differentiation of macrophages within TMEs, ultimately causing immunosuppression.

Elevated levels of damage-associated molecular patterns HMGB1 and S100A8/A9 coupled with toll-like receptor-triggered monocyte activation are associated with inflammation in patients with myelofibrosis.

Inflammation plays a pivotal role in the pathogenesis of primary and post-essential thrombocythemia or post-polycythemia vera myelofibrosis (MF) in close cooperation with the underlying molecular drivers. This inflammatory state is induced by a dynamic spectrum of inflammatory cytokines, although recent evidence points to the participation of additional soluble inflammatory mediators. Damage-associated molecular patterns (DAMPs) represent endogenous signals released upon cell death or damage which trigger a potent innate immune response. We assessed the contribution of two prototypical DAMPs, HMGB1 and S100A8/A9, to MF inflammation. Circulating HMGB1 and S100A8/A9 were elevated in MF patients in parallel to the degree of systemic inflammation and levels increased progressively during advanced disease stages. Patients with elevated DAMPs had higher frequency of adverse clinical features, such as anemia, and inferior survival, suggesting their contribution to disease progression. Monocytes, which are key players in MF inflammation, were identified as a source of S100A8/A9 but not HMGB1 release, while both DAMPs correlated with cell death parameters, such as serum LDH and cell-free DNA, indicating that passive release is an additional mechanism leading to increased DAMPs. HMGB1 and S100A8/A9 promote inflammation through binding to Toll-like receptor (TLR) 4, whereas the former also binds TLR2. Monocytes from MF patients were shown to be hyperactivated at baseline, as reflected by higher CD11b and tissue factor exposure and increased expression levels of proinflammatory cytokines IL-1β and IL-6. Patient monocytes showed preserved TLR4 and TLR2 expression and were able to mount normal or even exacerbated functional responses and cytokine upregulation following stimulation of TLR4 and TLR2. Elevated levels of endogenous TLR ligands HMGB1 and S100A8/A9 coupled to the finding of preserved or hyperreactive TLR-triggered responses indicate that DAMPs may promote monocyte activation and cytokine production in MF, fueling inflammation. Plasma IL-1β and IL-6 were elevated in MF and correlated with DAMPs levels, raising the possibility that DAMPs could contribute to cytokine generation in vivo. In conclusion, this study highlights that, in cooperation with classic proinflammatory cytokines, DAMPs represent additional inflammatory mediators that may participate in the generation of MF inflammatory state, potentially providing novel biomarkers of disease progression and new therapeutic targets.

Cholesterol Efflux Decreases TLR4-Target Gene Expression in Cultured Macrophages Exposed to T. brucei Ghosts.

Trypanosoma brucei causes African trypanosomiasis in humans. Infection with T. brucei elicits a potent pro-inflammatory immune response within infected human hosts, and this response is thought to at least be partially due to Toll-like receptor (TLR) activation. In response to stimulation by lipopolysaccharide and other pathogen antigens, TLR4 translocates to lipid rafts, which induces the expression of pro-inflammatory genes. However, cholesterol efflux is acknowledged as anti-inflammatory due to promoting lipid raft disruption. In this study, we wanted to assess the impact of T. brucei "ghosts", which are non-viable T. brucei essentially devoid of intracellular contents, in stimulating macrophage TLR4 translocation to lipid rafts, and whether promoting cholesterol efflux in macrophages incubated with T. brucei ghosts attenuates TLR4-target gene expression. When cultured macrophages were exposed to T. brucei ghosts, we observed an increase in lipid raft TLR4 protein content, which suggests certain surface molecules of T. brucei serve as ligands for TLR4. However, pretreating macrophages with cholesterol acceptors before T. brucei ghost exposure decreased lipid raft TLR4 protein content and the expression of pro-inflammatory TLR4-target genes. Taken together, these results imply that macrophage cholesterol efflux weakens pro-inflammatory responses which occur from T. brucei infection via increasing macrophage lipid raft disruption.

Polygala tenuifolia willd. Extract alleviates LPS-induced acute lung injury in rats via TLR4/NF-κB pathway and NLRP3 inflammasome suppression

BackgroundAcute lung injury (ALI) has received considerable attention in the field of critical care as it can lead to high mortality rates. Polygala tenuifolia, a traditional Chinese medicine with strong expectorant properties, can be used to treat pneumonia. Owing to the complexity of its composition, the main active ingredient is not yet known. Thus, there is a need to identify its constituent compounds and mechanism of action in the treatment of ALI using advanced technological means. PurposeWe investigated the anti-inflammatory mechanism and constituent compounds with regard to the effect of P. tenuifolia Willd. extract (EPT) in lipopolysaccharide (LPS)-induced ALI in vivo and in vitro. MethodsThe UHPLC-Q-Exactive Orbitrap MS technology was used to investigate the chemical profile of EPT. Network pharmacology was used to predict the targets and pathways of action of EPT in ALI, and molecular docking was used to validate the binding of polygalacic acid to Toll-like receptor (TLR) 4. The main compounds were determined using LC-MS. A rat model of LPS-induced ALI was established, and THP-1 cells were stimulated with LPS and adenosine triphosphate (ATP) to construct an in vitro model. Pathological changes were observed using hematoxylin and eosin staining, Wright-Giemsa staining, and immunohistochemistry. The expression of inflammatory factors (NE, MPO, Ly-6 G, TNF-α, IL-1β, IL-6, and iNOS) was determined using enzyme-linked immunosorbent assay, real-time fluorescence quantitative polymerase chain reaction, and western blotting. The LPS + ATP-induced inflammation model in THP-1 cells was used to verify the in vivo experimental results. ResultsNinety-nine compounds were identified or tentatively deduced from EPT. Using network pharmacology, we found that TLR4/NF-κB may be a relevant pathway for the prevention and treatment of ALI by EPT. Polygalacic acid in EPT may be a potential active ingredient. EPT could alleviate LPS-induced histopathological lung damage and reduce the wet/dry lung weight ratio in the rat model of ALI. Moreover, EPT decreased the white blood cell and neutrophil counts in the bronchoalveolar lavage fluid and decreased the expression of genes and proteins of relevant inflammatory factors (NE, MPO, Ly-6 G, TNF-α, IL-1β, IL-6, and iNOS) in lung tissues. It also increased the expression of endothelial-type nitric oxide synthase expression. Western blotting confirmed that EPT may affect TLR4/NF-κB and NLRP3 signaling pathways in vivo. Similar results were obtained in THP-1 cells. ConclusionEPT reduced the release of inflammatory factors by affecting TLR4/NF-κB and NLRP3 signaling pathways, thereby attenuating the inflammatory response of ALI. Polygalacic acid is the likely compounds responsible for these effects.

Oxymatrine, a novel TLR2 agonist, promotes megakaryopoiesis and thrombopoiesis through the STING/NF-ĸB pathway

Radiation-induced thrombocytopenia (RIT) faces a perplexing challenge in the clinical treatment of cancer patients, and current therapeutic approaches are inadequate in the clinical settings. In this researsh, oxymatrine, a new molecule capable of healing RIT was screened out, and the underlying regulatory mechanism associated with magakaryocyte (MK) differentiation and thrombopoiesis was demonstrated. The capacity of oxymatrine to induce MK differentiation was verified in K-562 and Meg-01 cells in vitro. The ability to induce thrombopoiesis was subsequently demonstrated in Tg (cd41:eGFP) zebrafish and RIT model mice. In addition, we carried out network pharmacological prediction, drug affinity responsive target stability assay (DARTS) and cellular thermal shift assay (CETSA) analyses to explore the potential targets of oxymatrine. Moreover, the pathway underlying the effects of oxymatrine was determined by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, Western blot, and immunofluorescence. Oxymatrine markedly promoted MK differentiation and maturation in vitro. Moreover, oxymatrine induced thrombopoiesis in Tg (cd41:eGFP) zebrafish and accelerated thrombopoiesis and platelet function recovery in RIT model mice. Mechanistically, oxymatrine directly binds to toll-like receptor 2 (TLR2) and further regulates the downstream pathway stimulator of interferon genes (STING)/nuclear factor-kappaB (NF-κB), which can be blocked by C29 and C-176, which are specific inhibitors of TLR2 and STING, respectively. Taken together, we demonstrated that oxymatrine , a novel TLR2 agonist, plays a critical role in accelerating MK differentiation and thrombopoiesis via the STING/NF-κB axis, suggesting that oxymatrine is a promising candidate for RIT therapy.

Development of multi-epitope mRNA vaccine against Clostridioides difficile using reverse vaccinology and immunoinformatics approaches

Clostridioides difficile (C. difficile), as the major pathogen of diarrhea in healthcare settings, has become increasingly prevalent within community populations, resulting in significant morbidity and mortality. However, the therapeutic options for Clostridioides difficile infection (CDI) remain limited, and as of now, no authorized vaccine is available to combat this disease. Therefore, the development of a novel vaccine against C. difficile is of paramount importance. In our study, the complete proteome sequences of 118 strains of C. difficile were downloaded and analyzed. We found four antigenic proteins that were highly conserved and can be used for epitope identification. We designed two vaccines, WLcd1 and WLcd2, that contain the ideal T-cell and B-cell epitopes, adjuvants, and the pan HLA DR-binding epitope (PADRE) sequences. The biophysical and chemical assessments of these vaccine candidates indicated that they were suitable for immunogenic applications. Molecular docking analyses revealed that WLcd1 bonded with higher affinity to Toll-like receptors (TLRs) than WLcd2. Furthermore, molecular dynamics (MD) simulations, performed using Gmx_MMPBSA v1.56, confirmed the binding stability of WLcd1 with TLR2 and TLR4. The preliminary findings suggested that this multi-epitope vaccine could be a promising candidate for protection against CDI; however, experimental studies are necessary to confirm these predictions.

Susceptibility to innate immune activation in genetically-mediated myocarditis.

Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment for myocarditis is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis with biomarkers and pathological features indistinguishable from other forms of myocarditis. DSP-associated myocarditis can progress to dilated cardiomyopathy with heightened arrhythmia risk. To model the cardiomyocyte aspects of DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients and gene-edited healthy control hiPSC lines. Homozygous and heterozygous DSP disrupted EHTs were generated to contain 90% hiPSC-CMs and 10% healthy control human cardiac fibroblasts. We measured innate immune activation and function at baseline and in response to Toll-like receptor (TLR) stimulation in EHTs. At baseline, DSP-/- EHTs displayed a transcriptomic signature of immune activation which was mirrored by EHT cytokine release. Importantly, DSP-/- EHTs were hypersensitive to TLR stimulation demonstrating greater contractile function impairment compared to isogenic controls. Compared to homozygous DSP-/- EHTs, heterozygous DSP patient-derived EHTs had less functionally impairment but also displayed heightened sensitivity to TLR stimulation. When subjected to strain, heterozygous DSP EHTs developed greater functional deficit indicating reduced contractile reserve compared to healthy control. Colchicine or NFΚB inhibitors improved baseline force production and strain-induced force deficits in DSP EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Genetic reduction of DSP renders cardiomyocytes susceptible to innate immune activation and strain-dependent contractile deficits. EHTs replicate electrical and contractile phenotypes seen in human myocarditis implicating cytokine release as a key part of the myogenic susceptibility to inflammation. This heightened innate immune activation and sensitivity is a target for clinical intervention.

MyD88 Inhibition Attenuates Cerebral Ischemia-reperfusion Injury by Regulating the Inflammatory Response and Reducing Blood–brain Barrier Damage

Myeloid differentiation primary response gene 88 (MyD88), a downstream molecule directly linked to Toll-like receptor (TLRs) and IL1 receptor, has been implicated in ischemia-reperfusion injury across various organs. However, its role in cerebral ischemia-reperfusion injury (CIRI) remains unclear. Five transient middle cerebral artery occlusion (tMCAO) microarray datasets were obtained from the Gene Expression Omnibus (GEO) database. We screened these datasets for differentially expressed genes (DEGs) using the GSE35338 and GSE58720 datasets and performed weighted gene co-expression network analysis (WGCNA) using the GSE30655, GSE28731, and GSE32529 datasets to identify the core module related to tMCAO. A protein–protein interaction (PPI) network was constructed using the intersecting DEGs and genes in the core module. Finally, we identified Myd88 was the core gene. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) validated that TNFα, IL17, and MyD88 signaling pathways were significantly enriched in tMCAO. Subsequently, we investigated the mechanistic role of MyD88 in the tMCAO model using male C57BL/6 mice. MyD88 expression increased significantly 24 h after reperfusion. After intraperitoneal administration of TJ-M2010-5, a MyD88-specific inhibitor, during reperfusion, the infarction volumes in the mice were ameliorated. TJ-M2010-5 inhibits the activation of microglia and astrocytes. Moreover, it attenuates the upregulation of inflammatory cytokines TNFα, IL17, and MMP9 while preserving the expression level of ZO1 after tMCAO, thereby safeguarding against blood-brain barrier (BBB) disruption. Finally, our findings suggest that MyD88 regulates the IRAK4/IRF5 signaling pathway associated with microglial activation. MyD88 participates in CIRI by regulating the inflammatory response and BBB damage following tMCAO.

Design of multi-epitope chimeric vaccine against Monkeypox virus and SARS-CoV-2: A vaccinomics perspective.

The current era we experience is full with pandemic infectious agents that no longer threatens the major local source but the whole globe. Almost the most emerging infectious agents are severe acute respiratory syndrome coronavirus-2 (SARS CoV-2), followed by monkeypox virus (MPXV). Since no approved antiviral drugs nor licensed active vaccines are yet available, we aimed to utilize immunoinformatics approach to design chimeric vaccine against the two mentioned viruses. This is the first study to deal with design divalent vaccine against SARS-CoV-2 and MPXV. ORF8, E and M proteins from Omicron SARS-CoV-2 and gp182 from MPXV were used as the protein precursor from which multi-epitopes (inducing B-cell, helper T cells, cytotoxic T cells and interferon-ɣ) chimeric vaccine was contrived. The structure of the vaccine construct was predicted, validated, and docked to toll-like receptor-2 (TLR-2). Moreover, its sequence was also used to examine the immune simulation profile and was then inserted into the pET-28a plasmid for in silico cloning. The vaccine construct was probable antigen (0.543) and safe (non-allergen) with strong binding energy to TLR-2 (-1169.8 kcal/mol) and found to have significant immune simulation profile. In conclusion, the designed chimeric vaccine was potent and safe against SARS-CoV-2 and MPXV, which deserves further consideration.

Gut Microbiota Composition Correlates with Disease Severity in Myelodysplastic Syndrome.

The myelodysplastic syndrome (MDS) is a heterogeneous group of clonal disorders of hematopoietic progenitor cells related to ineffective hematopoiesis and an increased risk of transformation to acute myelogenous leukemia. MDS is divided into categories, namely lineage dysplasia (MDS-SLD), MDS with ring sideroblasts (MDS-RS), MDS with multilineage dysplasia (MDS-MLD), MDS with excess blasts (MDS-EB). The International Prognostic Classification System (IPSS) ranks the patients as very low, low, intermediate, high, and very high based on disease evolution and survival rates. Evidence points to toll-like receptor (TLR) abnormal signaling as an underlying mechanism of this disease, providing a link between MDS and immune dysfunction. Microbial signals, such as lipopolysaccharides from gram-negative bacteria, can activate or suppress TLRs. Therefore, we hypothesized that MDS patients present gut microbiota alterations associated with disease subtypes and prognosis. To test this hypothesis, we sequenced the 16S rRNA gene from fecal samples of 30 MDS patients and 16 healthy elderly controls. We observed a negative correlation between Prevotella spp. and Akkermansia spp. in MDS patients compared with the control group. High-risk patients presented a significant increase in the genus Prevotella spp. compared to the other risk categories. There was a significant reduction in the abundance of the genus Akkermansia spp. in high-risk patients compared with low- and intermediate-risk. There was a significant decrease in the genus Ruminococcus spp. in MDS-EB patients compared with controls. Our findings show a new association between gut dysbiosis and higher-risk MDS, with a predominance of gram-negative bacteria.

Abstract 6739: Efficacy and toxicity of microbial mimetic mesoporous silica nanoparticles in a mouse model of ovarian cancer

Abstract Ovarian cancer is predominately left undetected and untreated until the later stages of the disease where it becomes incredibly difficult to treat. Preclinical studies demonstrate that immunotherapy can be highly effective. Our lab has been working on an immunotherapy approach to treat women with ovarian cancer that utilizes mesoporous silica nanoparticles(MSN). MSN that present pathogen-associated molecular patterns (PAMPS) are able to significantly reduce tumor burden in mouse models of serous epithelial ovarian cancer when administered by intraperitoneal injection. These microbial mimetic particles are decorated with polyethyleneimine (PEI), CpG oligonucleotide, and monophosphoryl lipid A (MPLA). When in the presence of antigen presenting cells, they bind to Toll-like receptors (TLR) present on the cell surface and within the endosome. To better understand mechanisms underlying their efficacy, we injected fluorescent MSN-PEI-CpG-MPLA (100 or 500 nm) or unmodified MSN into mice with advanced ovarian cancer. The particles, regardless of the presence of TLR-agonists, were rapidly internalized by myeloid cells, with trafficking almost exclusively to immune clusters located in tumor-burdened adipose tissues when administered by intraperitoneal injection. Conversely, intravenous injection of MSN-PEI-CpG-MPLA led to predominate accumulation in liver, with no particles found in tertiary lymphoid clusters 24 hours post injection. Furthermore, unlike intraperitoneal injection, intravenous injection of MSN-PEI-CpG-MPLA was without therapeutic effect. Currently we are studying MSN safety and the impact of administration route and dose on efficacy and cytotoxicity. In summary, MSN, with and without TLR agonists, are trafficked by myeloid cells to tumor-burdened fat-associated lymphoid clusters when administered by intraperitoneal injection. Intraperitoneal injection of TLR-agonist modified MSN effectively reduces tumor burden in mice with ovarian cancer. Citation Format: Lorel Y. Medina, Rita E. Serda, Benjamin Marwedel, Melanie S. Jun, Josh Adogola. Efficacy and toxicity of microbial mimetic mesoporous silica nanoparticles in a mouse model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6739.

Elevated expression of interleukin-6 (IL-6) and serum amyloid A (SAA) in the skin and the serum of recessive dystrophic epidermolysis bullosa: Skin as a possible source of IL-6 through Toll-like receptor ligands and SAA.

The effect of persistent skin inflammation on extracutaneous organs and blood is not well studied. Patients with recessive dystrophic epidermolysis bullosa (RDEB), a severe form of the inherited blistering skin disorder, have widespread and persistent skin ulcers, and they develop various complications including anaemia, hyperglobulinaemia, hypoalbuminaemia and secondary amyloidosis. These complications are associated with the bioactivities of IL-6, and the development of secondary amyloidosis requires the persistent elevation of serum amyloid A (SAA) level. We found that patients with RDEB had significantly higher serum levels of IL-6 and SAA compared to healthy volunteers and patients with psoriasis or atopic dermatitis. Both IL-6 and SAA were highly expressed in epidermal keratinocytes and dermal fibroblasts of the skin ulcer lesions. Keratinocytes and fibroblasts surrounding the ulcer lesions are continuously exposed to Toll-like receptor (TLR) ligands, pathogen-associated and damage-associated molecular pattern molecules. Invitro, TLR ligands induced IL-6 expression via NF-κB in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs). SAA further induced the expression of IL-6 via TLR1/2 and NF-κB in NHEKs and NHDFs. The limitation of this study is that NHEKs and NHDFs were not derived from RDEB patients. These observations suggest that TLR-mediated persistent skin inflammation might increase the risk of IL-6-related systemic complications, including RDEB.

Pulsed electric field (PEF) activates immune activity in RAW 264.7 macrophages by altering pine nut peptide-TLR4 binding sites

The purpose of this study was to investigate the mechanism of PEF treatment on the regulation of pine nut peptide (VNAVLH) induced immune activity of RAW 264.7 based on C-terminal modification by molecular dynamics (MD). The results showed that PEF treatment can reduce the overall stability of pine nut peptides, enhance the flexibility of hydrophobic and positively charged residues in pine nut peptides, and make their residue structures more flexible. Active sites verification experiments showed that PEF treatment could improve the immune activity of histidine only when it was at C-terminal. Western blot and immunofluorescence analysis showed that PEF-treated VNAVLH could activate NF-κB pathway by promoting the nuclear translocation of NF-κB/p65 and the expression of IKKβ, and activate MAPK pathway by promoting the phosphorylation of JNK, ERK, and p38 in RAW 264.7. Furthermore, toll-like receptor 4 (TLR4), as the main receptor for VNAVLH to exert immune activity, exhibited a more prominent peptide-binding capacity and had more hydrogen-bonding sites to PEF-treated peptide compared to toll-like receptor 2 (TLR2). Therefore, enhanced immunoreactivity of VNAVLH may be due to the formation of a more stable complex with TLR4 after PEF treatment, which activates the MAPK/NF-κB signaling pathway causing an intracellular cascade response.

β-glucans from Agaricus bisporus mushroom products drive Trained Immunity.

Macrofungi, such as edible mushrooms, have been used as a valuable medical resource for millennia as a result of their antibacterial and immuno-modulatory components. Mushrooms contain dietary fibers known as β-glucans, a class of polysaccharides previously linked to the induction of Trained Immunity. However, little is known about the ability of mushroom-derived β-glucans to induce Trained Immunity. Using various powdered forms of the white button mushroom (Agaricus bisporus), we found that mouse macrophages pre-treated with whole mushroom powder (WMP) displayed enhanced responses to restimulation with TLR ligands, being particularly sensitive to Toll-like receptor (TLR)-2 stimulation using synthetic lipopeptides. This trained response was modest compared to training observed with yeast-derived β-glucans and correlated with the amount of available β-glucans in the WMP. Enriching for β-glucans content using either a simulated in-vitro digestion or chemical fractionation retained and boosted the trained response with WMP, respectively. Importantly, both WMP and digested-WMP preparations retained β-glucans as identified by nuclear magnetic resonance analysis and both displayed the capacity to train human monocytes and enhanced responses to restimulation. To determine if dietary incorporation of mushroom products can lead to Trained Immunity in myeloid cells in vivo, mice were given a regimen of WMP by oral gavage prior to sacrifice. Flow cytometric analysis of bone-marrow progenitors indicated alterations in hematopoietic stem and progenitor cells population dynamics, with shift toward myeloid-committed multi-potent progenitor cells. Mature bone marrow-derived macrophages derived from these mice displayed enhanced responses to restimulation, again particularly sensitive to TLR2. Taken together, these data demonstrate that β-glucans from common macrofungi can train innate immune cells and could point to novel ways of delivering bio-available β-glucans for education of the innate immune system.

Mesenchymal stem cell exosomes differentially regulate gene expression of mast cells

Emerging evidence indicates that the immunomodulatory effect of mesenchymal stem cells (MSCs) is primarily attributed to the paracrine pathway. As a key paracrine effector, MSC-derived exosomes are small vesicles that play an important role in cell-to-cell communication by carrying bioactive substances. We previously found that exosomes derived from tonsil-derived mesenchymal stem cells (T-MSCs) were able to effectively attenuate inflammatory responses in mast cells. Here we investigated how T-MSC exosomes impact mast cells in steady state, and how exposure of T-MSCs to Toll-like receptors (TLRs) ligands changes this impact. Transcriptomic analysis of HMC-1 cells, a human mast cell line, using DNA microarrays showed that T-MSC exosomes broadly regulate genes involved in the normal physiology of mast cells. TLR3 or TLR4 primed T-MSC exosomes impacted fewer genes involved in specific functions in mast cells. This distinguishable regulation also was apparent in the analysis of related gene interactions. Our results suggest that MSC exosomes maintain immune homeostasis in normal physiology and impact the inflammatory state by modulating mast cell transcription.

Engineered CpG-Loaded Nanorobots Drive Autophagy-Mediated Immunity for TLR9-Positive Cancer Therapy.

Smart nanorobots have emerged as novel drug delivery platforms in nanomedicine, potentially improving anti-cancer efficacy and reducing side effects. In this study, an intelligent tumor microenvironment-responsive nanorobot is developed that effectively delivers CpG payloads to Toll-like receptor 9 (TLR9)-positive tumors to induce autophagy-mediated cell death for immunotherapy. The nanorobots are fabricated by co-self-assembly of two amphiphilic triblock polymer peptides: one containing the matrix metallopeptidase 2 (MMP2)-cleaved GPLGVRGS motif to control the mechanical opening of the nanorobots and provide targeting capability for TLR-9-positive tumors and the other consisting of an arginine-rich GRRRDRGRS sequence that can condense nuclear acid payloads through electrostatic interactions. Using multiple tumor-bearing mouse models, it is investigated whether the intravenous injection of CpG-loaded nanorobots could effectively deliver CpG payloads to TLR-9-positive tumors and elicit anti-tumor immunity through TLR9 signaling and autophagy. Therefore, besides being a commonly used adjuvant for tumor vaccination, CpG-loaded nanorobots can effectively reprogram the tumor immunosuppressive microenvironment and suppress tumor growth and recurrence. This nanorobot-based CpG immunotherapy can be considered a feasible approach to induce anti-tumor immunity, showing great therapeutic potential for the future treatment of TLR9-positive cancers.

Glycogen for lysosome-targeted CpG ODNs delivery and enhanced cancer immunotherapy

CpG oligodeoxynucleotides (ODNs) strongly activate the immune system after binding to toll-like receptor 9 (TLR9) in lysosome, which demonstrated significant potential in cancer immunotherapy. However, their therapeutic efficacy is limited by drawbacks such as rapid degradation and poor cellular uptake. Although encouraging progress have been made on developing various delivery systems for CpG ODNs, safety risks of the synthetic nanocarriers as well as the deficient CpG ODNs release within lysosome remain big obstacles. Herein, we developed a novel nanovector for lysosome-targeted CpG ODNs delivery and enhanced cancer immunotherapy. Natural glycogen was simply aminated (NH2-Gly) through grafting with diethylenetriamine (DETA), which was spherical in shape with diameter of approximately 40 nm. NH2-Gly possessed good biocompatibility. Cationic NH2-Gly complexed CpG ODNs well and protected them from nuclease digestion. NH2-Gly significantly enhanced the cellular uptake of CpG ODNs. Efficient CpG ODNs release was observed in the presence of α-glucosidase that mimicking the environment of lysosome. Consequently, NH2-Gly/CpG complexes triggered potent antitumor immunity and effectively inhibit the tumor growth without causing any toxic effect or tissue damages. This work highlights the promise of glycogen for lysosome-targeted on-command delivery of CpG ODNs, which brings new hope for precision cancer immunotherapy.

Common Pollen Modulate Immune Responses against Viral-Like Challenges in Airway Coculture Model.

Recent research indicates that exposure to pollen increases the risk and severity of respiratory infections, while studies also suggest that it may possess a protective function. Our aim was to investigate how exposure to common pollen modifies airway cells' responses to viral- or bacterial-like challenges and vice versa. Cocultured A549 and THP-1 cells were exposed to three doses of four different pollens (Alnus glutinosa, Betula pendula, Phleum pratense, or Ambrosia artemisiifolia) and subsequently to Toll-like receptor (TLR) ligands mimicking bacterial and viral challenges (TLR3, TLR4, TLR7/8). The stimulation experiment was replicated in reverse order. Toxicological and immunological end points were analyzed. When cells were primed with pollen, especially with grass (P. pratense) or weed (A. artemisiifolia), the ability of cells to secrete cytokines in response to bacterial- and viral-like exposure was decreased. In contrast, cells primed with viral ligand TLR7/8 showed greater cytokine responses against pollen than cells exposed to ligands or pollen alone. Our results suggest that pollen exposure potentially weakens immune reactions to bacterial- or viral-like challenges by modulating cytokine production. They also indicate that TLR7/8-mediated viral challenges could elicit exaggerated immune responses against pollen. Both mechanisms could contribute to the acceleration and complication of infections during the pollen season.