Toll-like Receptor Ligands Research Articles

Intracellular Sphingosine‐1‐Phosphate is Involved in Lung Cancer Cells Proliferation

BackgroundSphingosine‐1‐phosphate (S1P) is a membrane‐derived bioactive phospholipid exerting a multitude of effects on respiratory cell physiology and pathology through five S1P receptors (S1PR1‐5). Higher levels of S1P have been registered in a broad range of respiratory diseases, including inflammatory disorders and cancer, although its exact role is still elusive.ObjectivesThe aim of our study was to understand the role of S1P in healthy versus lung adenocarcinoma cells after Toll‐Like Receptors (TLRs) activation, well‐known modulators of sphingolipid metabolism.MethodsWe stimulated healthy fibroblast and lung adenocarcinoma cells with TLRs ligands and S1P, to compare physiological vs pathological S1P role. Pro‐inflammatory cytokines release, proliferation rate and the expression/activity of the mediators involved in the S1P signaling were evaluated.ResultsWe found that the activation of TLR9, but not of TLR4, was involved in the generation of S1P by means of ceramidase and sphingosine kinase I/II (SPHK I/II) activation in tumor cells but not in non‐cancerous cells. Through an inside‐out signaling, newly formed S1P was extruded from the cells through its transporter SPNS2, supporting TLR9/NF‐kB‐mediated TNF‐α release mainly via membrane S1PR3. These results were not observed in healthy fibroblasts, implying that this pathway was mainly involved in pathological conditions. Moreover, we found that S1PR3 and SPHK II were overexpressed in the nucleus of lung adenocarcinoma cells and that were associated to S1P‐mediated proliferation of tumor cells.ConclusionThese results prove that the endogenous TLR9‐induced S1P can on one side favor pro‐inflammatory mechanisms in the tumor microenvironment via the activation of cell surface receptors, but on the other tumor progression via the nuclear S1PR3/SPHK II axis, highlighting a novel molecular mechanism that identifies S1P as one of the crucial mediators for lung carcinogenesis‐associated inflammatory processes.

Aggregated Alpha‐Synuclein Activates Pro‐Inflammatory NFKB Signaling Pathways Through TLR‐Dependent and Independent Mechanisms in Peripheral Monocytic Cells

Toll‐like receptors (TLRs) are pattern recognition molecules that induce inflammatory signaling cascades upon recognition of pathogen or damage associated molecules. In addition to peripheral immune cells including monocytes, TLRs are broadly expressed on cells of the central nervous system (CNS) including microglia, astrocytes, and neurons and their activation induces production of pro‐inflammatory mediators inducing neuroinflammation and neurodegeneration in the chronic state. Prolonged neuroinflammation in the substantia nigra (SN) leading to accumulation of alpha‐synuclein fibril (α‐syn) containing Lewy‐bodies (LBs) and ultimately death of dopaminergic neurons is a pathological hallmark of Parkinson’s disease (PD) and is thought to be a major mechanism driving disease progression. TLRs are an integral component of the feed‐forward neuroinflammatory loop where damage from neurodegeneration generates TLR ligands that further amplify the inflammatory circuit. Importantly, aggregate fibrillar α‐syn can interact with TLRs on microglia in the brain, of which TLR2 and 4 are best characterized; however, the extent to which fibrillar α‐syn interacts with peripheral immune cells including monocytes is poorly understood. Peripheral inflammation may play a causal role in inducing and perpetuating neuroinflammation and accumulation of fibrillar α‐syn has been reported at several peripheral sites including the gut and liver in PD. To model the peripheral immune response to fibrillar α‐syn we exposed THP‐1 monocytic cells in vitro to determine inflammatory signaling pathways altered by α‐syn. Compared to monomeric α‐syn, preformed‐fibrillar (PFF) α‐syn displays overt inflammatory gene upregulation and pathway activation as assessed by RNA‐Seq. Both MyD88 and TRIF mediated TLR signaling pathways were broadly overrepresented in the PFF condition as well as RNAK/RANKL signaling supported by large increases in NFKB signaling and pro‐inflammatory cytokine production including TNF and IL‐1B. These results indicate that α‐syn may act broadly across all TLRs and other pro‐inflammatory receptors such as RANK.

Characterization of Dosage Levels for In Ovo Administration of Innate Immune Stimulants for Prevention of Yolk Sac Infection in Chicks.

Innate immune stimulants, especially toll-like receptor (TLR) ligands and agonists, are the main players in the initiation of innate immunity and have been widely studied as alternatives to antibiotics to control infection. In the present study, we characterized the dosage levels of various innate immune stimulants, including unmethylated cytosine-phosphate-guanosine dinucleotide -containing oligodeoxynucleotides (CpG ODN), polyinosinic-polycytidylic acid (poly I:C), cyclic polyphosphazene 75B (CPZ75B), avian beta-defensin 2 (ABD2), and combinations of these reagents given in ovo. Data derived from a series of animal experiments demonstrated that the in ovo administration of 10–50 µg CpG ODN/embryo (on embryonic day 18) is an effective formulation for control of yolk sac infection (YSI) due to avian pathogenic Escherichia coli (E. coli) in young chicks. Amongst the different combinations of innate immune stimulants, the in ovo administration of CpG ODN 10 µg in combination with 15 µg of poly I:C was the most effective combination, offering 100% protection from YSI. It is expected that the introduction of these reagents to management practices at the hatchery level may serve as a potential replacement for antibiotics for the reduction of early chick mortality (ECM) due to YSI/colibacillosis.

Titanium Surface Characteristics Induce the Specific Reprogramming of Toll-like Receptor Signaling in Macrophages.

Most of the research on titanium-based dental implants (Ti-discs) is focused on how they are able to stimulate the formation of new tissue and/or cytotoxic studies, with very scarce data on their effects on functional responses by immunocompetent cells. In particular, the link between the rewiring of innate immune responses and surface biomaterials properties is poorly understood. To address this, we characterize the functional response of macrophage cultures to four different dental titanium surfaces (MA: mechanical abrasion; SB + AE: sandblasting plus etching; SB: sandblasting; AE: acid etching). We use different Toll-like receptor (TLR) ligands towards cell surface receptors (bacterial lipopolysaccharide LPS for TLR4; imiquimod for TLR7; synthetic bacterial triacylated lipoprotein for TLR2/TLR1) and endosomal membrane receptor (poly I:C for TLR3) to simulate bacterial (cell wall bacterial components) or viral infections (dsRNA and ssRNA). The extracellular and total LDH levels indicate that exposure to the different Ti-surfaces is not cytotoxic for macrophages under resting or TLR-stimulated conditions, although there is a tendency towards an impairment in macrophage proliferation, viability or adhesion under TLR4, TLR3 and TLR2/1 stimulations in SB discs cultures. The secreted IL-6 and IL-10 levels are not modified upon resting macrophage exposure to the Ti-surfaces studied as well as steady state levels of iNos or ArgI mRNA. However, macrophage exposure to MA Ti-surface do display an enhanced immune response to TLR4, TLR7 or TLR2/1 compared to other Ti-surfaces in terms of soluble immune mediators secreted and M1/M2 gene expression profiling. This change of characteristics in cellular phenotype might be related to changes in cellular morphology. Remarkably, the gene expression of Tlr3 is the only TLR that is differentially affected by distinct Ti-surface exposure. These results highlight the relevance of patterned substrates in dental implants to achieve a smart manipulation of the immune responses in the context of personalized medicine, cell-based therapies, preferential lineage commitment of precursor cells or control of tissue architecture in oral biology.

TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae.

Porphyromonas gingivalis, a keystone oral pathogen implicated in development and progression of periodontitis, may also contribute to the pathogenicity of diseases such as arthritis, atherosclerosis, and Alzheimer’s. P. gingivalis is a master manipulator of host immune responses due to production of a large variety of virulence factors. Among these, P. gingivalis peptidilarginine deiminase (PPAD), an enzyme unique to P. gingivalis, converts C-terminal Arg residues in bacterium- and host-derived proteins and peptides into citrulline. PPAD contributes to stimulation of proinflammatory responses in host cells and is essential for activation of the prostaglandin E2 (PGE2) synthesis pathway in gingival fibroblasts. Since P. gingivalis is recognized mainly by Toll-like receptor-2 (TLR2), we investigated the effects of PPAD activity on TLR2-dependent host cell responses to P. gingivalis, as well as to outer membrane vesicles (OMVs) and fimbriae produced by this organism. Using reporter cell lines, we found that PPAD activity was required for TLR2 activation by P. gingivalis cells and OMVs. We also found that fimbriae, an established TLR2 ligand, from wild-type ATCC 33277 (but not from its isogenic PPAD mutant) enhanced the proinflammatory responses of host cells. Furthermore, only fimbriae from wild-type ATCC 33277, but not from the PPAD-deficient strains, induced cytokine production and stimulated expression of genes within the PGE2 synthesis pathway in human gingival fibroblasts via activation of the NF-ĸB and MAP kinase-dependent signaling pathways. Analysis of ten clinical isolates revealed that type I FimA is preferable for TLR2 signaling enhancement. In conclusion, the data strongly suggest that both PPAD activity and fimbriae are important for TLR2-dependent cell responses to P. gingivalis infection.

Glyco-Nanoadjuvants: Impact of Linker Length for Conjugating a Synthetic Small-Molecule TLR7 Ligand to Glyco-Nanoparticles on Immunostimulatory Effects.

Immunotherapy has become a powerful clinical strategy for treating infectious diseases and cancer. Synthetic small-molecule toll-like receptor 7 (TLR7) ligands are attractive candidates as immunostimulatory agents for immunotherapy. TLR7 is mainly localized in intracellular endosomal compartments so that the formulation of their small-molecule ligands with macromolecules enhances endocytic uptake of TLR7 ligands and improves the pharmaceutical properties. Previously, we demonstrated that gold nanoparticles co-immobilized with a TLR7 ligand derivative, that is, a conjugate of synthetic small-molecule TLR7 ligand (1V209) and thioctic acid (TA) via 4,7,10-trioxa-1,13-tridecanediamine, and α-mannose (1V209-αMan-GNPs: glyco-nanoadjuvants) significantly enhances immunostimulatory effects. In the present study, we designed a second-generation glyco-nanoadjuvant that possesses a poly(ethylene glycol) (PEG) chain as a spacer between 1V209 and GNPs and investigated the impact of linker length in 1V209 derivatives on the immunostimulatory activities. We used different chain lengths of PEG (n = 3, 5, 11, or 23) as spacers between 1V209 and thioctic acid to prepare four 1V209-αMan-GNPs. In the in vitro study using primary mouse bone-marrow-derived dendritic cells, 1V209-αMan-GNPs that immobilized with longer 1V209 derivatives, especially the 1V209 derivative possessing PEG23 (1V209-PEG23-TA), showed the highest potency toward induction both for interleukin-6 and type I interferon production than those derivatives with shorter PEG chains. Furthermore, 1V209-αMan-GNPs that immobilized with 1V209-PEG23-TA showed significantly higher adjuvant effects for inducing both humoral and cell-mediated immune responses against ovalbumin in the in vivo immunization study. These results indicate that the linker length for immobilizing small-molecule TLR7 ligand on the GNPs significantly affects the adjuvant activity of 1V209-αMan-GNPs and that 1V209-αMan-GNPs immobilized with 1V209-PEG-23-TA could be superior adjuvants for immunotherapies.

Recent advances in cancer immunotherapy: Modulation of tumor microenvironment by Toll-like receptor ligands.

Immunotherapy is considered a promising approach for cancer treatment. An important strategy for cancer immunotherapy is the use of cancer vaccines, which have been widely used for cancer treatment. Despite the great potential of cancer vaccines for cancer treatment, their therapeutic effects in clinical settings have been limited. The main reason behind the lack of significant therapeutic outcomes for cancer vaccines is believed to be the immunosuppressive tumor microenvironment (TME). The TME counteracts the therapeutic effects of immunotherapy and provides a favorable environment for tumor growth and progression. Therefore, overcoming the immunosuppressive TME can potentially augment the therapeutic effects of cancer immunotherapy in general and therapeutic cancer vaccines in particular. Among the strategies developed for overcoming immunosuppression in TME, the use of toll-like receptor (TLR) agonists has been suggested as a promising approach to reverse immunosuppression. In this paper, we will review the application of the four most widely studied TLR agonists including agonists of TLR3, 4, 7, and 9 in cancer immunotherapy.

Toll-like receptor agonist combinations augment mouse T-cell anti-tumor immunity via IL-12- and interferon ß-mediated suppression of immune checkpoint receptor expression

ABSTRACT We previously found that activated CD8+ T-cells increase expression of PD-1, which can be attenuated in the presence of specific Toll-like receptor (TLR) agonists, mediated by IL-12 secreted by professional antigen-presenting cells. While these CD8+ T-cells had greater anti-tumor activity, T-cells stimulated by different TLR had different gene expression profiles. Consequently, we sought to determine whether combinations of TLR agonists might further affect the expression of T-cell checkpoint receptors and improve T-cell anti-tumor immunity. Activation of CD8+ T-cells in the presence of specific TLR ligands resulted in decreased expression of PD-1, LAG-3, and CD160, notably with combinations of TLR1/2, TLR3, and TLR9 agonists. Immunization of E.G7-OVA or TRAMP-C1 tumor-bearing mice with peptide or DNA vaccines, co-administered with combination of TLR3 and TLR9 agonists, showed greater suppression of tumor growth. The anti-tumor effect of TLR1/2 and/or TLR9, but not TLR3, was abrogated in IL-12KO mice. RNA sequencing of TLR-conditioned CD8+ T-cells revealed IL-12 pathway activation, and type 1 IFN pathway activation following TLR3 stimulation. Our results provide a mechanistic rationale for the choice of optimal combinations of TLR ligands to use as adjuvants to improve the efficacy of anti-tumor vaccines.

Chronic Alcohol Exposure Among People Living with HIV Is Associated with Innate Immune Activation and Alterations in Monocyte Phenotype and Plasma Cytokine Profile.

Despite advances in antiretroviral therapy, chronic immune activation continues to be observed among individuals with well-controlled HIV viral loads, and is associated with non-AIDS defining morbidities among people living with HIV. Alcohol use disorder impacts a significant proportion of individuals living with HIV, and alcohol exposure is known to damage the intestinal epithelium which may increase translocation of pathogens and their molecular products, driving systemic immune activation and dysregulation. The aim of this study was to determine if adults living with HIV with well-controlled viral loads, who also suffer from alcohol use disorder with and without hepatitis C virus co-infection (n=23), exhibit evidence of advanced systemic immune activation, intestinal damage, and microbial translocation, as compared to adults living with HIV who are not exposed to chronic alcohol or other substances of abuse (n=29). The impact of a 1-month intervention to treat alcohol-use disorder was also examined. Alcohol-use disorder was associated with evidence of advanced innate immune activation, alterations in monocyte phenotype including increased expression of Toll-like receptor 4, increased burden of stimulatory ligands for Toll-like receptor 4, and alterations in plasma cytokine signature, most notably elevations in soluble CD40 ligand and transforming growth factor beta. Alcohol-associated immune activation was more pronounced among individuals with hepatitis C virus co-infection. Although the 1-month intervention to treat alcohol use disorder did not result in significant reductions in the interrogated indicators of immune activation, our findings suggest that chronic alcohol exposure is a major modifiable risk factor for chronic immune activation and dysregulation among people-living with HIV.

Promoting mechanism of serum amyloid a family expression in mouse intestinal epithelial cells

Serum amyloid A (SAA) is an acute phase inflammatory protein that we previously described as a robust biomarker of colorectal inflammation in patients with ulcerative colitis (UC) in clinical remission. However, what induces SAA expression in UC remains unclear. This study demonstrates that SAA is significantly expressed in the intestinal tract of UC mouse models when compared with C-reactive protein, another inflammatory biomarker. Moreover, interleukin-6 and tumor necrosis factor-α were found to promote SAA1 expression, as were Toll-like receptor ligands flagellin and lipopolysaccharide. Furthermore, results suggested that the nuclear factor-kappa B (NF-κB) pathway may be involved in the promotion of SAA1 expression by flagellin, which was inhibited by treatment with 5-aminosalicylic acid (5-ASA). Therefore, the flagellin/NF-κB/SAA1 axis may represent one of the mechanisms by which 5-ASA suppresses intestinal inflammation.

Evidence for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19

A key component of severe COVID-19 is a “cytokine storm” i.e., the excessive expression of unneeded cytokines. Previous studies suggest that SARS-CoV-2 proteins can induce macrophages to secrete pro-inflammatory cytokines; a process that may involve Toll-like receptors (TLRs). Glycogen synthase kinase-3 (GSK-3) has been implicated in TLR signal transduction and a selective GSK-3 inhibitor, termed COB-187, dramatically attenuates cytokine expression induced by the TLR ligand lipopolysaccharide (LPS). In the present study, we provide evidence that the SARS-CoV-2 spike protein (S) and the S2 subunit (S2) induce production of CXCL10 (a chemokine elevated in severe COVID-19) by a human macrophage cell line. Further, we report that two clinically relevant GSK-3 inhibitors and COB-187 attenuate S and S2 protein-induced CXCL10 production. Combined, our observations provide impetus for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19.

A Novel Toll-Like Receptor 2 Agonist Protects Mice in a Prophylactic Treatment Model Against Challenge With Bacillus anthracis.

Current therapies for anthrax include the use of antibiotics (i.e., doxycycline, and ciprofloxacin), an anthrax vaccine (BioThrax) and Bacillus anthracis-specific, monoclonal antibody (mAb) (i.e., Raxibacumab and obiltoxaximab). In this study, we investigated the activity of immunomodulators, which potentiate inflammatory responses through innate immune receptors. The rationale for the use of innate immune receptor agonists as adjunctive immunomodulators for infectious diseases is based on the concept that augmentation of host defense should promote the antimicrobial mechanism of the host. Our aim was to explore the anti-B. anthracis effector function of Toll-like receptor (TLR) agonists using a mouse model. Amongst the six TLR ligands tested, Pam3CSK4 (TLR1/2 ligand) was the best at protecting mice from lethal challenge of B. anthracis. We then evaluated the activity of a novel TLR2 ligand, DA-98-WW07. DA-98-WW07 demonstrated enhanced protection in B. anthracis infected mice. The surviving mice that received DA-98-WW07 when re-challenged with B. anthracis 20 days post the first infection showed increased survival rate. Moreover, ciprofloxacin, when treated in adjunct with a suboptimal concentration of DA-98-WW07 demonstrated augmented activity in protecting mice from B. anthracis infection. Taken together, we report the prophylactic treatment potential of DA-98-WW07 for anthrax and the utility of immunomodulators in combination with an antibiotic to treat infections caused by the B. anthracis bacterium.

ABIN1 is a signal-induced autophagy receptor that attenuates NF-κB activation by recognizing linear ubiquitin chains.

Linear ubiquitin chains play pivotal roles in immune signaling by augmenting NF-κB activation and suppressing programmed cell death induced by various stimuli. A20-binding inhibitor of NF-κB 1 (ABIN1) binds to linear ubiquitin chains and attenuates NF-κB activation and cell death induction. Although interactions with linear ubiquitin chains are thought to play a role in ABIN1-mediated suppression of NF-κB and cell death, the underlying molecular mechanisms remain unclear. Here, we show that upon stimulation by Toll-like receptor (TLR) ligands, ABIN1 is phosphorylated on Ser 83 and functions as a selective autophagy receptor. ABIN1 recognizes components of the MyD88 signaling complex via interaction with linear ubiquitin chains conjugated to components of the complex in TLR signaling, which leads to autophagic degradation of signaling proteins and attenuated NF-κB signaling. Our current findings indicate that phosphorylation and linear ubiquitination also play a role in downregulation of signaling via selective induction of autophagy.

Homeostatic and Pathophysiological Regulation of Toll-like Receptor 4 Signaling by GM3 Ganglioside Molecular Species

Chronic inflammation plays an important role in the pathogenesis of obesity and metabolic disorders. In obesity, pattern-recognition receptors in innate immune system, such as Toll-like receptor 4 (TLR4), cause chronic inflammation through prolonged activation by various endogenous ligands, including fatty acids and its metabolites. Gangliosides and other glycosphingolipids are important metabolites of fatty acids and saccharides. GM3, the simplest ganglioside comprising α2,3-sialyllactose, is expressed in insulin-sensitive peripheral tissues such as liver and adipose tissue, and furthermore secreted abundantly into serum. It has been shown that GM3 regulates the signal transduction of insulin receptor in adipose tissue as a component of membrane microdomains, and elevation in GM3 level causes insulin resistance. However, the homeostatic and pathophysiological functions of extracellularly secreted GM3 are poorly understood. We recently reported that GM3 species with differing fatty acid structures act as pro- and anti-inflammatory endogenous TLR4 ligands. GM3 with very long-chain fatty acid (VLCFA) and α-hydroxyl VLCFA strongly enhanced TLR4 activation. Conversely, GM3 with long-chain fatty acid (LCFA) and ω-9 unsaturated VLCFA inhibited TLR4 activation, counteracting the VLCFA species. GM3 interacted with the extracellular complex of TLR4 and promoted dimerization/oligomerization. In obesity and metabolic disorders, VLCFA species were increased in serum and adipose tissue, whereas LCFA species was relatively decreased; their imbalances were correlated to disease progression. Our findings suggest that GM3 species are disease-related endogenous TLR4 ligands, and "glycosphingolipid sensing" by TLR4 controls the homeostatic and pathological roles of innate immune signaling.

Immunomodulation by probiotics and prebiotics in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most prevalent primary malignancy in patients suffering from chronic liver diseases and cirrhosis. Recent attention has been paid to the involvement of the gut-liver axis (GLA) in HCC pathogenesis. This axis results from a bidirectional, anatomical and functional relationship between the gastrointestinal system and the liver. Moreover, the complex network of interactions between the intestinal microbiome and the liver plays a crucial role in modulation of the HCC-tumor microenvironment, contributing to the pathogenesis of HCC by exposing the liver to pathogen-associated molecular patterns, such as bacterial lipopolysaccharides, DNA, peptidoglycans and flagellin. Indeed, the alteration of gut microflora may disturb the intestinal barrier, bringing several toll-like receptor ligands to the liver thus activating the inflammatory response. This review explores the new therapeutic opportunities that may arise from novel insights into the mechanisms by which microbiota immunomodulation, represented by probiotics, and prebiotics, affects HCC through the GLA.

Incorporation of Toll-Like Receptor Ligands and Inflammasome Stimuli in GM3 Liposomes to Induce Dendritic Cell Maturation and T Cell Responses.

Cancer vaccination aims to activate immunity towards cancer cells and can be achieved by delivery of cancer antigens together with immune stimulatory adjuvants to antigen presenting cells (APC). APC maturation and antigen processing is a subsequent prerequisite for T cell priming and anti-tumor immunity. In order to specifically target APC, nanoparticles, such as liposomes, can be used for the delivery of antigen and adjuvant. We have previously shown that liposomal inclusion of the ganglioside GM3, an endogenous ligand for CD169, led to robust uptake by CD169-expressing APC and resulted in strong immune responses when supplemented with a soluble adjuvant. To minimize the adverse effects related to a soluble adjuvant, immune stimulatory molecules can be incorporated in liposomes to achieve targeted delivery of both antigen and adjuvant. In this study, we incorporated TLR4 (MPLA) or TLR7/8 (3M-052) ligands in combination with inflammasome stimuli, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) or muramyl dipeptide (MDP), into GM3 liposomes. Incorporation of TLR and inflammasome ligands did not interfere with the uptake of GM3 liposomes by CD169-expressing cells. GM3 liposomes containing a TLR ligand efficiently matured human and mouse dendritic cells in vitro and in vivo, while inclusion of PGPC or MDP had minor effects on maturation. Immunization with MPLA-containing GM3 liposomes containing an immunogenic synthetic long peptide stimulated CD4+ and CD8+ T cell responses, but additional incorporation of either PGPC or MDP did not translate into stronger immune responses. In conclusion, our study indicates that TLRL-containing GM3 liposomes are effective vectors to induce DC maturation and T cell priming and thus provide guidance for further selection of liposomal components to optimally stimulate anti-cancer immune responses.

ENAMPT Is a Novel Damage-associated Molecular Pattern Protein That Contributes to the Severity of Radiation-induced Lung Fibrosis.

The paucity of therapeutic strategies to reduce the severity of radiation-induced lung fibrosis (RILF), a life-threatening complication of intended or accidental ionizing radiation exposure, is a serious unmet need. We evaluated the contribution of eNAMPT (extracellular nicotinamide phosphoribosyltransferase), a damage-associated molecular pattern (DAMP) protein and TLR4 (Toll-like receptor 4) ligand, to the severity of whole-thorax lung irradiation (WTLI)-induced RILF. Wild-type (WT) and Nampt+/- heterozygous C57BL6 mice and nonhuman primates (NHPs, Macaca mulatta) were exposed to a single WTLI dose (9.8 or 10.7 Gy for NHPs, 20 Gy for mice). WT mice received IgG1 (control) or an eNAMPT-neutralizing polyclonal or monoclonal antibody (mAb) intraperitoneally 4 hours after WTLI and weekly thereafter. At 8-12 weeks after WTLI, NAMPT expression was assessed by immunohistochemistry, biochemistry, and plasma biomarker studies. RILF severity was determined by BAL protein/cells, hematoxylin and eosin, and trichrome blue staining and soluble collagen assays. RNA sequencing and bioinformatic analyses identified differentially expressed lung tissue genes/pathways. NAMPT lung tissue expression was increased in both WTLI-exposed WT mice and NHPs. Nampt+/- mice and eNAMPT polyclonal antibody/mAb-treated mice exhibited significantly attenuated WTLI-mediated lung fibrosis with reduced: 1) NAMPT and trichrome blue staining; 2) dysregulated lung tissue expression of smooth muscle actin, p-SMAD2/p-SMAD1/5/9, TGF-β, TSP1 (thrombospondin-1), NOX4, IL-1β, and NRF2; 3) plasma eNAMPT and IL-1β concentrations; and 4) soluble collagen. Multiple WTLI-induced dysregulated differentially expressed lung tissue genes/pathways with known tissue fibrosis involvement were each rectified in mice receiving eNAMPT mAbs.The eNAMPT/TLR4 inflammatory network is essentially involved in radiation pathobiology, with eNAMPT neutralization an effective therapeutic strategy to reduce RILF severity.

Transcutaneous Administration of Imiquimod Promotes T and B Cell Differentiation into Effector Cells or Plasma Cells.

We are interested in promoting the development of transcutaneous immunization using microneedle technology and attempting to apply an adjuvant with transcutaneous immunization to improve the efficacy and reduce the amount of antigen and number of administrations needed. In this study, we collected basic information to help elucidate the mechanism responsible for the transcutaneous adjuvant activity of imiquimod (IMQ), which is a ligand of toll-like receptor (TLR) 7. In mouse groups administered ovalbumin (OVA), the OVA-specific IgG antibody titer of the IMQ-adjuvanted group was higher than that of the group administered OVA alone. No immune response bias due to transcutaneous IMQ administration was observed in terms of IgG1 (T helper cell [Th]2-type IgG subclass) and IgG2c (Th1-type IgG subclass) antibody titers. After the initial immunization, the IMQ-adjuvanted group showed increased migration of Langerhans cells to draining lymph nodes (dLNs) and active proliferation of OVA-specific CD4+ T cells. Transcutaneously administered IMQ did not affect the direction of CD4+ T cell differentiation, while promoted B cell activation and germinal center (GC) B cell differentiation. Immune staining revealed greater GC formation in the dLNs with the IMQ-adjuvanted group than in the OVA-alone group. In the secondary immune response, effector T cells increased in the dLNs and spleen, and effector memory T cells also increased in the spleen in the IMQ-adjuvanted group. In addition, our results suggested that the administration of IMQ enhanced B cell differentiation into plasma cells and GC B cells in the dLNs and spleen. In this study, we partially clarified the mechanism underlying the adjuvant activity of transcutaneously administered IMQ, which is required for the practical application of transcutaneous immunization with IMQ.

Inflammation Is Present, Persistent and More Sensitive to Proinflammatory Triggers in Celiac Disease Enterocytes.

Celiac disease (CD) is a chronic inflammatory disease caused by a genetic predisposition to an abnormal T cell-mediated immune response to the gluten in the diet. Different environmental proinflammatory factors can influence and amplify the T cell-mediated response to gluten. The aim of this manuscript was to study the role of enterocytes in CD intestinal inflammation and their response to different proinflammatory factors, such as gliadin and viruses. Intestinal biopsies from CD patients on a gluten-containing (GCD-CD) or a gluten-free diet (GFD-CD) as well as biopsies from potential CD patients (Pot-CD) before the onset of intestinal lesions and controls (CTR) were used to investigate IL-1β and IL-6 mRNA levels in situ. Organoids from CD patients were used to test the levels of NF-κB, ERK, IL-6, and IL-1β by Western blot (WB), ELISA, and quantitative PCR. The Toll-like receptor ligand loxoribine (Lox) and gliadin peptide P31-43 were used as proinflammatory stimuli. In CD biopsies inflammation markers IL-1β and IL-6 were increased in the enterocytes, and also in Pot-CD before the onset of the intestinal lesion and in GFD-CD. The inflammatory markers pNF-κB, pERK, IL-1β, and IL-6 were increased and persistent in CD organoids; these organoids were more sensitive to P31-43 and Lox stimuli compared with CTR organoids. Taken together, these observations point to constitutive inflammation in CD enterocytes, which are more sensitive to inflammatory stimuli such as food components and viruses.

In situ vaccination using unique TLR9 ligand K3-SPG induces long-lasting systemic immune response and synergizes with systemic and local immunotherapy

Although checkpoint inhibitors (CPIs) have changed the paradigm of cancer therapy, low response rates and serious systemic adverse events remain challenging. In situ vaccine (ISV), intratumoral injection of immunomodulators that stimulate innate immunity at the tumor site, allows for the development of vaccines in patients themselves. K3-SPG, a second-generation nanoparticulate Toll-like receptor 9 (TLR9) ligand consisting of K-type CpG oligodeoxynucleotide (ODN) wrapped with SPG (schizophyllan), integrates the best of conventional CpG ODNs, making it an ideal cancer immunotherapy adjuvant. Focusing on clinical feasibility for pancreaticobiliary and gastrointestinal cancers, we investigated the antitumor activity of K3-SPG-ISV in preclinical models of pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC). K3-SPG-ISV suppressed tumor growth more potently than K3-ISV or K3-SPG intravenous injections, prolonged survival, and enhanced the antitumor effect of CPIs. Notably, in PDAC model, K3-SPG-ISV alone induced systemic antitumor effect and immunological memory. ISV combination of K3-SPG and agonistic CD40 antibody further enhanced the antitumor effect. Our results imply that K3-SPG-based ISV can be applied as monotherapy or combined with CPIs to improve their response rate or, conversely, with CPI-free local immunotherapy to avoid CPI-related adverse events. In either strategy, the potency of K3-SPG-based ISV would provide the rationale for its clinical application to puncturable pancreaticobiliary and gastrointestinal malignancies.