TLR Agonists Research Articles

GelMA microneedle-loaded bio-derived nanovaccine shows therapeutic potential for gliomas

ABSTRACT Glioma is the most common primary malignant tumor of the central nervous system in adults. Although immunotherapy, especially tumor vaccines, has made some progress in the treatment of gliomas compared with surgery and radiotherapy. However, the lack of specific or relevant tumor antigens severely limits the further development of tumor vaccines. Here, we report a bio-derived vaccine (TMV@CpG) derived from glioma cell membrane vesicles and carrying TLR9 agonist CpG as adjuvant, which was loaded onto the GelMA microneedle to obtain the microneedle vaccine (MN-TMV@CpG). Microneedle vaccine fully utilize the innate immune cells rich in the skin, inducing stronger cellular immune responses. In subcutaneous tumor models, MN-TMV@CpG reversed the immune-suppressing microenvironment of tumor, and effectively inhibited tumor progression. In an intracranial tumor model, MN-TMV@CpG significantly prolonged the survival duration and induced stronger immune memory responses in tumor bearing mice when combined with anti-PD1 mAb. These results suggest that bio-derived nanovaccines can be used as a potential antitumor immunotherapy strategy.

Anti-Inflammatory Effects of SGLT1 Synthetic Ligand in In Vitro and In Vivo Models of Lung Diseases

Background. Several research findings suggest that sodium–glucose co-transporter 1 (SGLT1) is implicated in the progression and control of infections and inflammation processes at the pulmonary level. Moreover, our previous works indicate an engagement of SGLT1 in inhibiting the inflammatory response induced in intestinal epithelial cells by TLR agonists. In this study, we report the anti-inflammatory effects observed in the lung upon engagement of the transporter, and upon the use of glucose and BLF501, a synthetic SGLT1 ligand, for the treatment of animal models of lung inflammation, including a model of allergic asthma. Methods. In vitro experiments were carried out on human pneumocytes stimulated with LPS from Pseudomonas aeruginosa and co-treated with glucose or BLF501, and the production of IL-8 was determined. The anti-inflammatory effect associated with SGLT1 engagement was then assessed in in vivo models of LPS-induced lung injury, as well as in a murine model of ovalbumin (OVA)-induced asthma, treating mice with aerosolized LPS and the synthetic ligand. After the treatments, lung samples were collected and analyzed for morphological alterations by histological examination and immunohistochemical analysis; serum and BALF samples were collected for the determination of several pro- and anti-inflammatory markers. Results. In vitro experiments on human pneumocytes treated with LPS showed significant inhibition of IL-8 production. The results of two in vivo experimental models, mice exposed to aerosolized LPS and OVA-induced asthma, revealed that the engagement of glucose transport protein 1 (SGLT1) induced a significant anti-inflammatory effect in the lungs. In the first model, the acute respiratory distress induced in mice was abrogated by co-treatment with the ligand, with almost complete recovery of the lung morphology and physiology. Similar results were observed in the OVA-induced model of allergic asthma, both with aerosolized and oral BLF501, suggesting an engagement of SGLT1 expressed both in intestinal and alveolar cells. Conclusions. Our results confirmed the engagement of SGLT1 in lung inflammation processes and suggested that BLF501, a non-metabolizable synthetic ligand of the co-transporter, might represent a drug candidate for therapeutic intervention against lung inflammation states.

Alveolar epithelial cells shape lipopolysaccharide-induced inflammatory responses and reprogramming of alveolar macrophages.

Alveolar macrophages (AMs) are sentinels in the airways, where they sense and respond to invading microbes and other stimuli. Unlike macrophages in other locations, AMs can remain responsive to Gram-negative lipopolysaccharides (LPS) after they have responded to LPS in vivo (they do not develop "endotoxin tolerance"), suggesting that the alveolar microenvironment may influence their responses. Although alveolar epithelial cells (AECs) normally limit AMs' innate responses, preventing inflammation induced by harmless antigens in the lung, how AECs influence the innate responses of AMs to infectious agents has been uncertain. Here we report that (1) after exposure to aspirated (intranasal instillation) LPS, AMs increase their responses to TLR agonists and elevate their phagocytic and bactericidal activities in mice; (2) Aspirated LPS pre-exposure increases host resistance to pulmonary infection caused by Gram-negative bacteria and the protection effect lasts for at least 35 days; (3) LPS stimulation of AECs both increases AMs' innate immune responses and prevents AMs from developing tolerance in vitro; (4) Upon LPS stimulation, AMs secreted TNF-α induces AECs to release GM-CSF, which potentiates AMs' response. These experiments have revealed a previously unappreciated role that AECs may play in boosting the innate responses of AMs and promoting resistance to pulmonary infections.

Virulence‐Free Reconstituted Synthetic Nanopathogen Empowered by Timely‐Activating TLR Agonist Promotes Heterologous Cancer Immunotherapy with Depletion of Tumor‐Specific Treg Cells

AbstractThe heterologous immunity of live pathogens leads to the emergence of this approach as a pivotal component in cancer immunotherapy. However, virulence, inflammatory‐related toxicity, and the induction of Treg cells after treatment hinder their clinical translation. Here, to exploit the heterologous immunity of live pathogens without risks, a virulence‐free reconstituted synthetic nanopathogen (RSnP) is developed by the cell wall skeleton of disrupted Mycobacterium bovis and the incorporation of timely‐activating Toll‐like receptor 7/8 agonists of which multifaceted activities can be promoted by endolysosomal enzymes. Immunization with RSnP, even without tumor‐specific antigens, exhibits potent antitumor efficacy against melanoma, breast cancer, and bladder cancer by promoting antitumor effector cells (CD8+ T cells, NK cells, M1 macrophages, and Th17 cells) and proinflammatory cytokines (IL‐12p70, TNF‐α, and IL‐6) while simultaneously mitigating immunosuppressive myeloid cells (MDSCs and M2 macrophages) in the tumor microenvironment, surpassing the therapeutic efficacies of approved live‐BCG drug and mRNA vaccine. The increase in CCR8+Foxp3+ Treg cells induced to counteract RSnP treatment can be attenuated by anti‐CCR8 antibody, a depletion antibody for tumor‐specific Treg cells, to synergize therapeutic efficacy with relieved autoimmunity. RSnP can be a therapeutic nanomedicine platform across heterologous cancers and emerging infectious virus variants with minimized toxicity.

NK cell membrane/MnO2 hybrid nanoparticle-adjuvanted intranasal vaccines synergistically boost protective immunity against H1N1 influenza infection

Intranasal vaccines hold a huge promise for preventing influenza infection, however, their development is compromised due to mucosal barriers and limited systemic and mucosal immunity. Herein, a novel biomimetic nano-adjuvant based on NK cell membrane (MNK)/MnO2 hybrid nanoparticles (NLipoMn) was developed for intranasal administration of H1N1 inactivated influenza vaccine (IIV). NK cell membrane, served as TLR4 agonist was fused with cationic liposomes encapsulating STING agonist MnO2 NPs (LipoMn) to yield NLipoMn, which inherited versatile characteristics to prolong the nasal retention of IIV. Encouragingly, CCL20 secretion was enhanced by NLipoMn-activated the TLR4/NF-κB pathway in mucosal endothelial cells, thereby promoting submucosal dendritic cells (DCs) recruitment for IIV uptake. Moreover, NLipoMn-IIV synergistically harnessed TLR4 and STING signaling pathway to augment STING activation though NLipoMn-activated the TLR4/NF-κB pathway, which demonstrated a superiority to elicit strong DCs maturation, CD8+ T cells activation, and high levels of antigen-specific IgG, antigen-specific IgA, cytokines secretion, resulting in a robust systemic and mucosal immune responses. Notably, intranasal immunization with NLipoMn-IIV elicited an effective immune protection against homologous and heterologous H1N1 influenza virus infection. This study provides a promising adjuvant candidate to develop needle-free intranasal vaccines that are active against influenza and other respiratory viral infection.

Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages

Abstract Objectives M2-polarized macrophages and interleukin (IL)-6 significantly alter the tumor microenvironment and promote the malignant behaviors of tumor cells. This study aimed to establish M2-type macrophages from THP-1 cells, which are human leukemia monocytes, and investigate the significance of toll-like receptor 2 (TLR2) signaling in IL-6 production. Methods THP-1 cells were treated with phorbol 12-myristate 13-acetate, IL-4, and IL-13 to stimulate their differentiation into M2 macrophages. Cell differentiation was confirmed by cytokine production, marker expression, and morphological alterations. Treatment with TLR agonists induced TLR stimulation in M2 macrophages. Subsequently, secretion and expression levels of IL-6 in M2 macrophages were measured using enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, and western blotting. Results Myeloid differentiation factor 88, tumor necrosis factor-associated factor 6, and IL-1 receptor-associated kinase-1/4 signaling pathways contributed to IL-6 production upon TLR2 activation in M2 macrophages. While both TLR2 and TLR4 activated NF-κB in M2 macrophages, IL-6 production was mainly dependent on TLR2, not TLR4, suggesting the involvement of major mechanisms other than NF-κB in IL-6 production. Notably, TLR2-stimulated extracellular signal-regulated kinase (ERK) was necessary for abundant IL-6 production, indicating that TLR2-mediated ERK signaling plays an essential role in M2 macrophages. Conclusions These results highlight the significance of TLR2 signaling in IL-6 production by M2 macrophages and provide insights into the underlying regulatory mechanisms.

Toll-Like Receptor 2 Attenuates the Formation and Progression of Angiotensin II-Induced Abdominal Aortic Aneurysm in ApoE−/− Mice

Introduction: We demonstrated Toll-like receptor (TLR) 4 in the pathogenesis of angiotensin II (AngII)-mediated abdominal aortic aneurysm (AAA) formation. Here, we study TLR2 in the AAA formation. Methods: Male ApoE−/− and ApoE−/−TLR2−/− mice were treated with AngII. Mice were injected with the TLR2 agonist Pam3CSK4. The incidence and severity of AAA were determined. MCP-1, MCP-5, RANTES, CXCL10, CCR5, and CXCR3 were analyzed. M1 and M2 macrophages in the aorta were detected by flow cytometry. Results: These studies demonstrated an increase in AAA formation in TLR2−/− mice and a decrease by Pam3CSK4. Pam3CSK4 decreased the ratio of M1/M2 and the levels of RANTES, CXCL10, CCR5, and CXCR3. Furthermore, Pam3CSK4 treatment 1 week following AngII retarded the progression of AAA. Conclusion: These data demonstrated a protective effect of TLR2 signaling on AAA in association with a decrease in the ratio of M1 to M2 macrophages and the expression of chemokines and their receptors. Furthermore, the treatment of Pam3CSK4 after AngII demonstrated a marked retardation of lesion progression. Given the fact that most AAA patients are detected late in the disease process, these findings suggest that TLR2 stimulation may play a therapeutic role in retarding disease progression.

A TLR7 Agonist Conjugated to a Nanofibrous Peptide Hydrogel as a Potent Vaccine Adjuvant.

Toll-like receptors (TLRs) recognize pathogen- and damage-associated molecular patterns and, in turn, trigger the release of cytokines and other immunostimulatory molecules. As a result, TLR agonists are increasingly being investigated as vaccine adjuvants. Many of these agonists are small molecules that quickly diffuse away from the vaccination site, limiting their co-localization with antigens and, thus, their effect. Here, the small-molecule TLR7 agonist 1V209 is conjugated to a positively-charged multidomain peptide (MDP) hydrogel, K2, which was previously shown to act as an adjuvant promoting humoral immunity. Mixing the 1V209-conjugated K2 50:50 with the unfunctionalized K2 produces hydrogels that retain the shear-thinning and self-healing physical properties of the original MDP while improving the solubility of 1V209 more than 200-fold compared to the unconjugated molecule. When co-delivered with ovalbumin as a model antigen, 1V209-functionalized K2 produces a robust Th2 immune response and an antigen-specific Th1 immune response superior to alum, a widely used vaccine adjuvant. Together, these results suggest that K2 MDP hydrogels functionalized with 1V209 are a promising adjuvant for vaccines against infectious diseases, especially those benefiting from a combined Th1 and Th2 immune response.

Long-term Immunity of a Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Irradiated by Gamma Rays in Mice.

COVID-19 vaccines effectively prevent symptomatic infection and severe disease, including hospitalization and death. However, unequal vaccine distribution during the pandemic, especially in low- and middle-income countries, has led to the emergence of vaccine-resistant strains. This underscores the need for alternative, safe, and thermostable vaccine platforms, such as dissolved microneedle array patches (MAP) delivering a subunit vaccine, which eliminate the need for cold chain and trained healthcare personnel. This study demonstrates that the SARS-CoV-2 S1 monomer with RS09, a TLR-4 agonist peptide, serves as an optimal protein subunit immunogen. This combination stimulates a stronger S1-specific immune response, resulting in binding to the membrane-bound spike on the cell surface and ACE2-binding inhibition, compared to the monomer S1 alone or trimer S1, regardless of RS09. MAP delivery of the rS1RS09 subunit vaccine elicited higher and longer-lasting immunity compared to conventional intramuscular injection. S1-specific IgG levels remained significantly elevated for up to 70 weeks post-administration. Additionally, different doses of 5, 15, and 45 μ g of MAP vaccines induced robust and sustained Th2-prevalent immune responses, suggesting a dose-sparing effect and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants at 15 and 45 μ g dose. Moreover, gamma irradiation as a terminal sterilization method did not significantly affect immunogenicity, with irradiated vaccines maintaining comparable efficacy to non-irradiated ones. The stability of MAP vaccines was evaluated after long-term storage at room temperature and refrigeration for 19 months, showing minimal protein degradation. Further, after an additional one-month of storage at elevated temperature (42°C), rS1RS09 in both non-irradiated and irradiated MAP degraded less than 3%, while the liquid subunit vaccine degraded over 23%. Overall, these results indicate that gamma irradiation sterilized MAP-rS1RS09 vaccines maintain stability during extended storage without refrigeration, supporting their potential for mass production and widespread use in global vaccination efforts.

WIF-1 contributes to lupus-induced neuropsychological deficits via the CRYAB/STAT4-SHH axis

BackgroundNeuropsychiatric systemic lupus erythematosus (NPSLE) often manifests as cognitive deterioration, with activated microglia and blood-brain barrier (BBB) disruption implicated in these neurological complications. Wnt-inhibitory factor-1 (WIF-1), a secreted protein, has been detected in the cerebrospinal fluid (CSF) of NPSLE patients. However, the contribution of WIF-1 in contributing to lupus cognitive impairment remains poorly understood.MethodsUsing MRL/MpJ-Faslpr (MRL/lpr) lupus-prone mice and TLR7 agonist imiquimod (IMQ)-induced lupus mice, recombinant WIF-1 protein (rWIF-1) and adeno-associated virus (AAV) encoding sh-WIF-1 were administered via intracerebroventricular injection. Behavioral tests, histopathological examinations, flow cytometry, and molecular biology techniques were employed to investigate the underlying mechanisms.ResultsMicroinjection of rWIF-1 exacerbated cognitive deficits and mood abnormalities, increased BBB leakage and neuronal degeneration, and caused aberrant activation of microglia and synaptic pruning in the hippocampus. Conversely, lupus mice injected with AAV-shWIF-1 exhibited significant remission. In vitro, rWIF-1 induced overactivation of microglia with an increased CD86+ pro-inflammatory subpopulation, upregulated phagocytic activity, and excessive synaptic engulfment, contributing to increased BBB permeability. Furthermore, WIF-1 exerted its biological effects through the CRYAB/STAT4 pathway, transcriptionally decreasing SHH production. We also identified that symmetric dimethylarginine (SDMA) could alleviate rWIF-1-induced microglial activation and BBB damage, thereby restoring SHH levels.ConclusionsIn conclusion, WIF-1 exacerbates lupus-induced cognitive dysfunction in mice by triggering aberrant microglial activation and BBB disruption through the CRYAB/STAT4-SHH axis, highlighting the potential therapeutic effects of SDMA for the treatment of NPSLE.

MINCLE and TLR9 agonists synergize to induce Th1/Th17 vaccine memory and mucosal recall in mice and non-human primates

Development of new vaccines tailored for difficult-to-target diseases is hampered by a lack of diverse adjuvants for human use, and none of the currently available adjuvants induce Th17 cells. Here, we develop a liposomal adjuvant, CAF®10b, that incorporates Mincle and Toll-like receptor 9 agonists. In parallel mouse and non-human primate studies comparing to CAF® adjuvants already in clinical trials, we report species-specific effects of adjuvant composition on the quality and magnitude of the responses. When combined with antigen, CAF®10b induces Th1 and Th17 responses and protection against a pulmonary infection with Mycobacterium tuberculosis in mice. In non-human primates, CAF®10b induces higher Th1 responses and robust Th17 responses detectable after six months, and systemic and pulmonary Th1 and Th17 recall responses, in a sterile model of local recall. Overall, CAF®10b drives robust memory antibody, Th1 and Th17 vaccine-responses via a non-mucosal immunization route across both rodent and primate species.

Differences in Susceptibility to SARS-CoV-2 Infection Among Transgenic hACE2-Hamster Founder Lines.

Animal models that are susceptible to SARS-CoV-2 infection and develop clinical signs like human COVID-19 are desired to understand viral pathogenesis and develop effective medical countermeasures. The golden Syrian hamster is important for the study of SARS-CoV-2 since hamsters are naturally susceptible to SARS-CoV-2. However, infected hamsters show only limited clinical disease and resolve infection quickly. In this study, we describe development of human angiotensin-converting enzyme 2 (hACE2) transgenic hamsters as a model for COVID-19. During development of the model for SARS-CoV-2, we observed that different hACE2 transgenic hamster founder lines varied in their susceptibility to SARS-CoV-2 lethal infection. The highly susceptible hACE2 founder lines F0F35 and F0M41 rapidly progress to severe infection and death within 6 days post-infection (p.i.). Clinical signs included lethargy, weight loss, dyspnea, and mortality. Lethality was observed in a viral dose-dependent manner with a lethal dose as low as 1 × 100.15 CCID50. In addition, virus shedding from highly susceptible lines was detected in oropharyngeal swabs on days 2-5 p.i., and virus titers were observed at 105.5-6.5 CCID50 in lung and brain tissue by day 4 p.i.. Histopathology revealed that infected hACE2-hamsters developed rhinitis, tracheitis, bronchointerstitial pneumonia, and encephalitis. Mortality in highly susceptible hACE2-hamsters can be attributed to neurologic disease with contributions from the accompanying respiratory disease. In contrast, virus challenge of animals from less susceptible founder lines, F0M44 and F0M51, resulted in only 0-20% mortality. To demonstrate utility of this SARS-CoV-2 infection model, we determined the protective effect of the TLR3 agonist polyinosinic-polycytidylic acid (Poly (I:C)). Prophylactic treatment with Poly (I:C) significantly improved survival in highly susceptible hACE2-hamsters. In summary, our studies demonstrate that hACE2 transgenic hamsters differ in their susceptibility to SARS-CoV-2 infection, based on the transgenic hamster founder line, and that prophylactic treatment with Poly (I:C) was protective in this COVID-19 model of highly susceptible hACE2-hamsters.

RIPK2 is crucial for the microglial inflammatory response to bacterial muramyl dipeptide but not to lipopolysaccharide.

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is a kinase that plays an essential role in the modulation of innate and adaptive immune responses. As a downstream signaling molecule for nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs), it is implicated in the signaling triggered by recognition of microbe-associated molecular patterns by NOD1/2 and TLRs. Upon activation of these innate immune receptors, RIPK2 mediates the release of pro-inflammatory factors by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). However, whether RIPK2 is essential for downstream inflammatory signaling following the activation of NOD1/2, TLRs, or both remains controversial. In this study, we examined the role of RIPK2 in NOD2- and TLR4-dependent signaling cascades following stimulation of microglial cells with bacterial muramyl dipeptide (MDP), a NOD2 agonist, or lipopolysaccharide (LPS), a TLR4 agonist. We utilized a highly specific proteolysis targeting chimera (PROTAC) molecule, GSK3728857A, and found dramatic degradation of RIPK2 in a concentration- and time-dependent manner. Importantly, the PROTAC completely abolished MDP-induced increases in iNOS and COX-2 protein levels and pro-inflammatory gene transcription of Nos2 , Ptgs2 , Il-1β , Tnfα , Il6 , Ccl2 , and Mmp9 . However, increases in iNOS and COX-2 proteins and pro-inflammatory gene transcription induced by the TLR4 agonist, LPS, were only slightly attenuated with the GSK3728857A pretreatment. Further findings revealed that the RIPK2 PROTAC completely blocked the phosphorylation and activation of p65 NF-κB and p38 MAPK induced by MDP, but it had no effects on the phosphorylation of these two mediators triggered by LPS. Collectively, our findings strongly suggest that RIPK2 plays an essential role in the inflammatory responses of microglia to bacterial MDP but not to LPS.

Neoadjuvant vidutolimod and nivolumab in high-risk resectable melanoma: A prospective phase II trial

Intratumoral TLR9 agonists and anti-PD-1 produce clinical responses and broad immune activation. We conducted a single-arm study of neoadjuvant TLR9 agonist vidutolimod combined with anti-PD-1 nivolumab in high-risk resectable melanoma. In 31 evaluable patients, 55% major pathologic response(MPR) was observed, meeting primary endpoint. MPR was associated with necrosis, and melanophagocytosis with increased CD8+ tumor-infiltrating lymphocytes and plasmacytoid dendritic cells (pDCs) in the tumor microenvironment, and increased frequencies of Ki67+CD8+ Tcells peripherally. MPRs had an enriched pre-treatment gene signature of myeloid cells, and response to therapy was associated with gene signatures of immune cells, pDCs, phagocytosis, and macrophage activation. MPRs gut microbiota were enriched for Gram-negative bacteria belonging to the Bacteroidaceae and Enterobacteriaceae families and the small subgroup of Gram-negative Firmicutes. Our findings support that combined vidutolimod and nivolumab stimulates a broad anti-tumor immune response and is associated with distinct baseline myeloid gene signature and gut microbiota. ClinicalTrials.gov identifier: NCT03618641.

Intratumoral vidutolimod in combination with PD-1 blockade in locoregionally advanced melanoma

Combinatorial immunotherapy may improve the efficacy of neoadjuvant checkpoint inhibitors in locoregionally advanced melanoma. In this issue of Cancer Cell, Davar and colleagues report a promising phase 2 neoadjuvant trial of the TLR9 agonist vidutolimod in combination with nivolumab. Analyses suggest a unique myeloid expression signature is associated with response.

A Polysaccharide-Based Oral-Vaccine Delivery System and Adjuvant for the Influenza Virus Vaccine.

Influenza virus enters the host body through the mucosal surface of the respiratory tract. An efficient immune response at the mucosal site can interfere with virus entry and prevent infection. However, formulating oral vaccines and eliciting an effective mucosal immune response including at respiratory mucosa presents numerous challenges including the potential degradation of antigens by acidic gastric fluids and the risk of antigen dilution and dispersion over a large surface area of the gut, resulting in minimal antigen uptake by the immune cells. Additionally, oral mucosal vaccines have to overcome immune tolerance in the gut. To address the above challenges, in the current study, we evaluated inulin acetate (InAc) nanoparticles (NPs) as a vaccine adjuvant and antigen delivery system for oral influenza vaccines. InAc was developed as the first polysaccharide polymer-based TLR4 agonist; when tailored as a nanoparticulate vaccine delivery system, it enhanced antigen delivery to dendritic cells and induced a strong cellular and humoral immune response. This study compared the efficacy of InAc-NPs as a delivery system for oral vaccines with Poly (lactic-co-glycolic acid) (PLGA) NPs, utilizing influenza A nucleoprotein (Inf-A) as an antigen. InAc-NPs effectively protected the encapsulated antigen in both simulated gastric (pH 1.1) and intestinal fluids (pH 6.8). Moreover, InAc-NPs facilitated enhanced antigen delivery to macrophages, compared to PLGA-NPs. Oral vaccination studies in Balb/c mice revealed that InAc-Inf-A NPs significantly boosted the levels of Influenza virus-specific IgG and IgA in serum, as well as total and virus-specific IgA in the intestines and lungs. Furthermore, mice vaccinated with InAc-Inf-A-NPs exhibited notably higher hemagglutination inhibition (HI) titers at mucosal sites compared to those receiving the antigen alone. Overall, our study underscores the efficacy of InAc-NPs in safeguarding vaccine antigens post-oral administration, enhancing antigen delivery to antigen-presenting cells, and eliciting higher virus-neutralizing antibodies at mucosal sites following vaccination.

Lipopolysaccharide Induces Trained Innate Immune Tolerance in the Heart Through Interferon Signaling in a Model of Stress-Induced Cardiomyopathy.

Although the ability of the heart to adapt to environmental stress has been studied extensively, the molecular and cellular mechanisms responsible for cardioprotection are not yet fully understood. We administered Toll-like receptor (TLR) agonists or a diluent to wild-type mice and assessed their potential to induce cardiac protection against injury from a high intraperitoneal dose of isoproterenol (ISO) administered 7 days later. Cardioprotective effects were analyzed through serum cardiac troponin I levels, immune cell profiling via flow cytometry, echocardiography, and multiomic single-nuclei RNA and ATAC sequencing. Pretreatment with the TLR4 agonist lipopolysaccharide (LPS), but not TLR1/2 or TLR3 agonists, conferred cardioprotection against ISO, as demonstrated by reduced cardiac troponin I leakage, decreased inflammation, preservation of cardiac structure and function, and improved survival. Remarkably, LPS-induced tolerance was reversed by β-glucan treatment. Multiomic analysis showed that LPS-tolerized hearts had greater chromatin accessibility and upregulated gene expression compared to hearts treated with LPS and β-glucan (reverse-tolerized). The LPS tolerance was associated with upregulation of interferon response pathways across various cell types, including cardiac myocytes and stromal cells. Blocking both type 1 and type 2 interferon signaling eliminated LPS-induced tolerance against ISO, while pretreatment with recombinant type 1 and 2 interferons conferred cardiac protection. Multiomic sequencing further revealed enhanced cytoprotective signaling in interferon-treated hearts. Analysis of cell-cell communication networks indicated increased autocrine signaling by cardiac myocytes, as well as greater paracrine signaling between stromal cells and myeloid cells, in LPS-tolerized versus reverse-tolerized hearts. LPS pretreatment confers cardiac protection against ISO-induced injury through TLR4 mediated type 1 and 2 interferon signaling, consistent with trained innate immune tolerance. The observation that LPS-induced protection in cardiac myocytes involves both cell-autonomous and non-cell-autonomous mechanisms underscores the complexity of innate immune tolerance in the heart, warranting further investigation into this cardioprotective phenotype. What is new?: The Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) confers cardiac protection against isoproterenol-mediated injury in a manner consistent with trained innate immune tolerance, which is reversed by β-glucan treatment.Activation of type 1 and 2 interferon signaling, which is downstream of Toll-like receptor 4, is necessary and sufficient for LPS-induced cardiac protection.LPS-tolerized hearts show heightened autocrine signaling by cardiac myocytes and, to a greater degree, increased cell-cell communication between cardiac myocytes and stromal and myeloid cells compared to reverse-tolerized hearts.What are the clinical implications?: TLR4 and interferon signaling play key roles in the establishment of cardiac protection and LPS-induced trained innate immune tolerance.The protective effects of LPS are mediated by cell-autonomous and non-cell-autonomous mechanisms, suggesting that a deeper understanding of the molecular and cellular signatures of innate immune tolerance is required for the development of targeted approaches to modulate trained innate immunity, and consequently cytoprotection, in the heart.

Remimazolam ameliorates postoperative cognitive dysfunction after deep hypothermic circulatory arrest through HMGB1-TLR4-NF-κB pathway

BackgroundPostoperative cognitive dysfunction (POCD) is a complication of deep hypothermic circulatory arrest (DHCA). Various amounts of neurologic dysfunctions have been shown after DHCA, which has often been attributed to systemic inflammatory response syndrome and cerebral ischemia/reperfusion injury. Remimazolam is one of the commonly used anesthetic drugs with protective actions against inflammatory diseases, such as sepsis and cerebral ischemia/reperfusion injury. Here, we determined the protective effect and potential mechanism of action of remimazolam against neuronal damage after DHCA. MethodsA rat model of DHCA was established, and a gradient dosage of remimazolam was administered during cardiopulmonary bypass (CPB). The cognitive function of rats was evaluated by Morris water maze. Hematoxylin and eosin and TUNEL staining were performed to assess hippocampus tissue injury and neuronal apoptosis. Inflammatory cytokines concentration were analyzed by enzyme-linked immunosorbent assay. The protein expression was analyzed using automated electrophoresis western analysis and immunohistochemical analysis. ResultsThe appropriate dosage of remimazolam reduced histologic injury, neuronal apoptosis, microglia activation, and secondary inflammatory cascades, as well as the downregulation of the expression of the HMGB1-TLR4-NF-κB pathway after DHCA, improved the memory and learning abilities in DHCA rats. Further, administration of a TLR4 antagonist TAK-242 had a similar effect to remimazolam, while the TLR4 agonist LPS attenuated the effect of remimazolam. ConclusionsRemimazolam could ameliorate POCD after DHCA through the HMGB1-TLR4-NF-κB signaling pathway.

Regional lymph node changes on breast MRI in patients with early-stage breast cancer receiving neoadjuvant chemo-immunotherapy

Abstract Purpose Establishing breast MRI imaging patterns associated with neoadjuvant immunotherapy is needed to monitor response. We analyzed serial breast MRIs in patients receiving neoadjuvant chemo-immunotherapy on the I-SPY2 clinical trial. Methods Patients with stage 2–3 HER2-negative breast cancer were randomized to weekly paclitaxel (control), weekly paclitaxel and pembrolizumab, or weekly paclitaxel, pembrolizumab and intra-tumoral injection of SD-101, a TLR9 agonist. All patients received AC. Regional lymph nodes were retrospectively evaluated on breast MRI at baseline, 3, 12 and 20 weeks by a single blinded radiologist. MRIs were assessed for development of new regional lymphadenopathy, or increase in the longest diameter or cortical thickness of the largest abnormal regional lymph node. Results Between 12/2015 and 4/2021, a total of 43 patients enrolled in the control (n = 16) and paclitaxel + pembrolizumab ± SD-101 (n = 27) arms. 12 of 27 patients (44.4%) receiving chemo-immunotherapy experienced increased lymphadenopathy within the first 12 weeks compared to 1 of 16 patients (6.3%) in the control group (p = 0.014). Most patients with increased lymphadenopathy were in the SD101/pembro arm (n = 10, p = 0.002). Increased lymphadenopathy was observed despite concomitant decrease in breast tumor size at all time points. 11 of 12 patients with increased lymphadenopathy had pathologically negative nodes at surgery. There was no association between lymphadenopathy and lower residual cancer burden or immune-related toxicity. Conclusions The combination of neoadjuvant paclitaxel and pembrolizumab ± SD101 intratumoral injection was associated with early increases in regional lymphadenopathy on MRI despite decreased breast tumor size. Increased lymphadenopathy was not associated with node positive disease at surgery.

Salmonella Detection in Food Using a HEK-hTLR5 Reporter Cell-Based Sensor.

The development of a rapid, sensitive, specific method for detecting foodborne pathogens is paramount for supplying safe food to enhance public health safety. Despite the significant improvement in pathogen detection methods, key issues are still associated with rapid methods, such as distinguishing living cells from dead, the pathogenic potential or health risk of the analyte at the time of consumption, the detection limit, and the sample-to-result. Mammalian cell-based assays analyze pathogens' interaction with host cells and are responsive only to live pathogens or active toxins. In this study, a human embryonic kidney (HEK293) cell line expressing Toll-Like Receptor 5 (TLR-5) and chromogenic reporter system (HEK dual hTLR5) was used for the detection of viable Salmonella in a 96-well tissue culture plate. This cell line responds to low concentrations of TLR5 agonist flagellin. Stimulation of TLR5 ligand activates nuclear factor-kB (NF-κB)-linked alkaline phosphatase (AP-1) signaling cascade inducing the production of secreted embryonic alkaline phosphatase (SEAP). With the addition of a ρ-nitrophenyl phosphate as a substrate, a colored end product representing a positive signal is quantified. The assay's specificity was validated with the top 20 Salmonella enterica serovars and 19 non-Salmonella spp. The performance of the assay was also validated with spiked food samples. The total detection time (sample-to-result), including shortened pre-enrichment (4 h) and selective enrichment (4 h) steps with artificially inoculated outbreak-implicated food samples (chicken, peanut kernel, peanut butter, black pepper, mayonnaise, and peach), was 15 h when inoculated at 1-100 CFU/25 g sample. These results show the potential of HEK-DualTM hTLR5 cell-based functional biosensors for the rapid screening of Salmonella.