IL-12p70 Secretion Research Articles

Sirtuin 1 regulates the phenotype and functions of dendritic cells through Ido1 pathway in obesity

Sirtuin 1 (SIRT1) is a class III histone deacetylase (HDAC3) that plays a crucial role in regulating the activation and differentiation of dendritic cells (DCs) as well as controlling the polarization and activation of T cells. Obesity, a chronic inflammatory condition, is characterized by the activation of immune cells in various tissues. We hypothesized that SIRT1 might influence the phenotype and functions of DCs through the Ido1 pathway, ultimately leading to the polarization towards pro-inflammatory T cells in obesity. In our study, we observed that SIRT1 activity was reduced in bone marrow-derived DCs (BMDCs) from obese animals. These BMDCs exhibited elevated oxidative phosphorylation (OXPHOS) and increased extracellular acidification rates (ECAR), along with enhanced expression of class II MHC, CD86, and CD40, and elevated secretion of IL-12p40, while the production of TGF-β was reduced. The kynurenine pathway activity was decreased in BMDCs from obese animals, particularly under SIRT1 inhibition. SIRT1 positively regulated the expression of Ido1 in DCs in a PPARγ-dependent manner. To support these findings, ATAC-seq analysis revealed that BMDCs from obese mice had differentially regulated open chromatin regions compared to those from lean mice, with reduced chromatin accessibility at the Sirt1 genomic locus in BMDCs from obese WT mice. Gene Ontology (GO) enrichment analysis indicated that BMDCs from obese animals had disrupted metabolic pathways, including those related to GTPase activity and insulin response. Differential expression analysis showed reduced levels of Pparg and Sirt1 in BMDCs from obese mice, which was challenged and confirmed using BMDCs from mice with conditional knockout of Sirt1 in dendritic cells (SIRT1∆). This study highlights that SIRT1 controls the metabolism and functions of DCs through modulation of the kynurenine pathway, with significant implications for obesity-related inflammation.

Polysaccharides from a Fermented Beverage Induce Nitric Oxide and Cytokines in Murine Macrophage Cell Line.

Super Ohtaka®, a fermented beverage of plant extracts, is prepared from approximately 50 kinds of fruits and vegetables. Natural fermentation is mainly performed by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp.). Four water-soluble polysaccharide fractions were obtained from Super Ohtaka® by dialysis, ion exchange chromatography, and gel filtration chromatography; these fractions were designated as OEP1-1, OEP1-2, OEP2, and OEP3. OEP1-1 is a polysaccharide composed solely of glucose. The other fractions contained polysaccharides composed of glucose, galactose, mannose, and a small amount of arabinose. OEP2 and OEP3 contained phosphorus, which was not detected in OEP1-1 and OEP1-2. Furthermore, the immunomodulatory activity of the polysaccharides was investigated in murine macrophage cell lines. OEP2 and OEP3 significantly induced nitric oxide (NO) secretion by macrophages in a dose-dependent manner (concentration range of 4 to 100 µg/mL). When the concentration of OEP3 was 100 µg/mL, NO production was almost identical to lipopolysaccharide (LPS; 10 ng/mL) used as a positive control. Notably, OEP3 induced NO secretion more strongly than OEP2. This trend was also observed for TNF-α, IL-1β, IL-6, and IL-12 p40 secretion. Overall, our in vitro studies on polysaccharides isolated from Super Ohtaka® suggest that the fermented beverage stimulates macrophages and activates the immune system.

Lutein Suppresses the Maturation and Function of Bone Marrow-Derived Dendritic Cells

Lutein, nonivamide, and baicalein, organic compounds found in a variety of plants, are known to exert anti-inflammatory effects in animal cells. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) and link the innate and adaptive immune systems. DCs can be directed into fully mature APCs by exposure to bacterial or viral components, resulting in inflammatory situations. The manipulation of DC maturation provides a strategy for the treatment of allergic and inflammatory diseases. In this study, we evaluated the effects of lutein, nonivamide, and baicalein on the maturation and activation of DCs. Compared to nonivamide and baicalein, lutein significantly and dose-dependently reduced the levels of maturation-associated cell surface markers, including CD40, co-stimulatory molecule CD86, and major histocompatibility complex class II (I-Ab) molecule in lipopolysaccharide (LPS)-stimulated DCs. Lutein also decreased IL-12p40 and IL-6 gene expression and secretion in LPS-stimulated DCs. Furthermore, lutein significantly enhanced the endocytic ability of LPS-stimulated DCs. These results demonstrated that lutein may exhibit immunosuppressive activity by inhibiting the phenotypic and functional maturation of DCs, and provide new evidence for the value of lutein in the search for novel therapeutic agents in the treatment of inflammatory diseases.

P103 Natural compounds in Ulcerative Colitis and Irritable Bowel Syndrome: Insights from immune-metabolic profiles

Abstract Background Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) represent chronic and occasionally disabling conditions, necessitating innovative treatment strategies. Covering 14% of Ireland's landscape, bogland ecosystems house unique plant biodiversity, yielding biologically active secondary metabolites. Informed by traditional medicinal knowledge and in vitro screening for immunomodulatory potential, three plant extracts (NTP0226A, NTP0127B, NTP0206EA) were selected for exploration of their potential to treat IBD and IBS within the "Unlocking Nature’s Pharmacy from Bogland Species" project. Methods Patients undergoing colonoscopy were prospectively recruited in three cohorts: healthy controls, IBS, and ulcerative colitis (UC). Recto-sigmoid biopsies were collected and cultured for 24 h with treatments (Infliximab, NTP0226A, NTP0127B, NTP0206EA). Colonic tissue explants underwent real-time energy metabolism profiling and quantification of 10 inflammatory mediators using Seahorse Xfe24 analyser and multiplex ELISA, respectively. P values <0.05 were considered significant in analyses. Results Twenty-seven patients were recruited (6 healthy controls, 6 IBS, and 15 UC). UC patient explants exhibited elevated glycolytic metabolism and increased IL-4, IL-6, and IL-12p70 secretion compared to non-UC patients. No differences were observed between healthy controls and IBS patients. Linear regression did not show correlation between age and mitochondrial bioenergetics. There was no change in metabolism profiles of any cohort following treatment with Infliximab or the 3 novel extracts. In the UC tissue, treatment with Infliximab reduced IL-12p70 secretion (p=0.0098), this reduction was not seen in the other 2 cohorts. NTP0206EA reduced IL-10 secretion in the UC cohort (p=0.0255). In the IBS cohort; NTP0127B reduced IL-10 secretion (p=0.0048) and NTP0226A reduced IL-4 (p=0.004) and IL-6 (p=0.0219) secretion. Principal component analysis demonstrated reduced separation between the immune-metabolic profiles of UC patients and healthy controls following treatment with NTP0226A (Figure 1). Conclusion Using an explant model, we have shown that UC patients have a distinct immune-metabolic profile modifiable by a standard IBD therapy and novel plant extracts. Blockade of IL-6 trans-signalling has recently shown promise as an effective treatment target in IBD. Our natural extract NTP0226A reduced IL-6 secretion in IBS patients and is a promising candidate for therapeutic exploration. These findings contribute to our understanding of the complex interplay in the colonic microenvironment and highlight potential avenues for novel therapeutic interventions.

MCT1 gene silencing enhances the immune effect of dendritic cells on cervical cancer cells.

Dendritic cells (DCs) are a key class of immune cells that migrate to the draining lymph nodes and present processed antigenic peptides to lymphocytes after being activated by external stimuli, thereby establishing adaptive immunity. Moreover, DCs play an important role in tumor immunity. The aim of the study was to investigate whether MCT1 gene silencing in DCs affects their ability to mount an immune response against cervical cancer cells. We silenced the expression of MCT1 in DCs from mouse bone marrow (BM) by infection with adenovirus. The surface antigen profile of DCs was analyzed by flow cytometry and cytokine secretion was evaluated using enzyme-linked immunosorbent assay (ELISA) following sodium lactate (sLA) exposure and lipopolysaccharide (LPS) stimulation. Then, various groups of DC-induced cytotoxic T lymphocytes (CTLs) were prepared and their cytotoxicity against U14 was tested. Without sLA exposure, silencing MCT1 did not affect the expression of CD1a, CD80, CD83, CD86, and major histocompatibility complex class II (MHCII) in DCs after LPS challenge. Similar results were found for interleukin (IL)-6, IL-12 p70 and tumor necrosis factor alpha (TNF-α). After sLA exposure, silencing MCT1 significantly decreased the expression of CD1a, CD80, CD83, CD86, and MHCII in DCs after the LPS challenge, as well as the secretion of IL-6, IL-12 p70 and TNF-α. In addition, sLA exposure significantly reduced the toxicity and inhibited the proliferation of DC-induced CTLs compared to U14 cells in vitro and in vivo. However, silencing MCT1 significantly attenuated the changes caused by sLA exposure. At the same time, in the absence of sLA, silencing MCT1 did not affect the toxicity nor inhibit the proliferation of DC-induced CTLs on U14 cells. Lactate exposure reduces the immune effect of DCs on cervical cancer cells, but MCT1 gene silencing attenuates these alterations.

Structural Optimization and Biological Evaluation of Isoxazolo[5,4-d]pyrimidines as Selective Toll-Like Receptor 7 Agonists.

Toll-like receptors (TLRs) are components of innate immunity that play a crucial role in several diseases, including chronic inflammatory and infectious diseases, autoimmune diseases, and cancer. In particular, TLR7 has been identified as a key player in the innate immune response against viral infections and small-molecule TLR7 agonists have shown potential for vaccine therapy, for treatment of asthma and allergies, and as anticancer drugs. Inspired by our previous discovery of selective TLR7 agonists, our goal was to develop and introduce a new chemotype of TLR7 agonists by replacing the quinazoline ring with a new heterocycle isoxazolo[5,4-d]pyrimidine. Here, we report design, optimized synthesis, and structure-activity relationship studies of a novel class of TLR7 agonists based on the 6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine-4-amine scaffold that demonstrate high selectivity and low micromolar potencies. The best-in-class agonist 21a, with an EC50 value of 7.8 μM, also proved to be noncytotoxic and induced secretion of cytokines, including IL-1β, IL-12p70, IL-8, and TNF-α, indicating its potential to modulate the immune response.

ADAM9 drives the immunosuppressive microenvironment by cholesterol biosynthesis-mediated activation of IL6-STAT3 signaling for lung tumor progression.

Chronic inflammation associated with lung cancers contributes to immunosuppressive tumor microenvironments, reducing CD8+ T-cell function and leading to poor patient outcomes. A disintegrin and metalloprotease domain 9 (ADAM9) promotes cancer progression. Here, we aim to elucidate the role of ADAM9 in the immunosuppressive tumor microenvironment. A bioinformatic analysis of TIMER2.0 was used to investigate the correlation of ADAM9 and to infiltrate immune cells in the human lung cancer database and mouse lung tumor samples. Flow cytometry, immunohistochemistry, and RNA sequencing (RNA-seq) were performed to investigate the ADAM9-mediated immunosuppressive microenvironment. The coculture system of lung cancer cells with immune cells, cytokine array assays, and proteomic approach was used to investigate the mechanism. By analyzing the human LUAD database and the mouse lung cancer models, we showed that ADAM9 was associated with the immunosuppressive microenvironment. Additionally, ADAM9 released IL6 protein from cancer cells to inhibit IL12p40 secretion from dendritic cells, therefore leading to dendritic cell dysfunction and further affecting T-cell functions. Proteomic analysis indicated that ADAM9 promoted cholesterol biosynthesis and increased IL6-STAT3 signaling. Mechanistically, ADAM9 reduced the protein stability of LDLR, resulting in reduced cholesterol uptake and induced cholesterol biosynthesis. Moreover, LDLR reduction enhanced IL6-STAT3 activation. We reveal that ADAM9 has a novel biological function that drives the immunosuppressive tumor microenvironment by linking lung cancer's metabolic and signaling axes. Thus, by targeting ADAM9 an innovative and promising therapeutic opportunity was indicated for regulating the immunosuppression of lung cancer.

Alteration of Immunoregulatory Patterns and Survival Advantage of Key Cell Types in Food Allergic Children.

All allergic responses to food indicate the failure of immunological tolerance, but it is unclear why cow's milk and egg (CME) allergies resolve more readily than reactivity to peanuts (PN). We sought to identify differences between PN and CME allergies through constitutive immune status and responses to cognate and non-cognate food antigens. Children with confirmed allergy to CME (n = 6) and PN (n = 18) and non-allergic (NA) (n = 8) controls were studied. Constitutive secretion of cytokines was tested in plasma and unstimulated mononuclear cell (PBMNC) cultures. Blood dendritic cell (DC) subsets were analyzed alongside changes in phenotypes and soluble molecules in allergen-stimulated MNC cultures with or without cytokine neutralization. We observed that in allergic children, constitutively high plasma levels IL-1β, IL-2, IL-4, IL-5 and IL-10 but less IL-12p70 than in non-allergic children was accompanied by the spontaneous secretion of sCD23, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12p70, IFN-γ and TNF-α in MNC cultures. Furthermore, blood DC subset counts differed in food allergy. Antigen-presenting cell phenotypic abnormalities were accompanied by higher B and T cell percentages with more Bcl-2 within CD69+ subsets. Cells were generally refractory to antigenic stimulation in vitro, but IL-4 neutralization led to CD152 downregulation by CD4+ T cells from PN allergic children responding to PN allergens. Canonical discriminant analyses segregated non-allergic and allergic children by their cytokine secretion patterns, revealing differences and areas of overlap between PN and CME allergies. Despite an absence of recent allergen exposure, indication of in vivo activation, in vitro responses independent of challenging antigen and the presence of unusual costimulatory molecules suggest dysregulated immunity in food allergy. Most importantly, higher Bcl-2 content within key effector cells implies survival advantage with the potential to mount abnormal responses that may give rise to the manifestations of allergy. Here, we put forward the hypothesis that the lack of apoptosis of key immune cell types might be central to the development of food allergic reactions.

Forodesine Enhances Immune Responses through Guanosine-Mediated TLR7 Activation while Preventing Graft-versus-Host Disease.

Recent evidence indicates that specific types of nuclear acids, including guanosine and its derivatives, act as natural ligands for TLR7. This led us to hypothesize that purine nucleoside phosphorylase inhibitors not only can induce apoptosis of T cells but also can lead to TLR7 activation by accumulation of guanine nucleosides, in particular under systemic inflammation, where damaged tissues release a large amount of nucleotides. We demonstrate in the present study that a purine nucleoside phosphorylase inhibitor, forodesine, can reduce the disease severity and prolong the survival in a xenogeneic mouse model of graft-versus-host disease (GVHD). Guanine nucleosides were undetectable in mice during GVHD but increased significantly following forodesine treatment. Our invitro experiments showed that forodesine enhanced guanosine-mediated cytokine production from APCs, including alveolar macrophages and plasmacytoid dendritic cells, through TLR7 signaling. Forodesine also enhanced Ag-presenting capacity, as demonstrated by increased CD8+ T cell proliferation and higher secretion of IFN-γ and IL-12p40 in an MLR with plasmacytoid dendritic cells. Furthermore, forodesine stimulated IFN-γ production from activated T cells in the presence of a low concentration of guanosine while inhibiting their proliferation and inducing apoptotic cell death. Although forodesine ameliorated GVHD severity, mice treated with forodesine showed significantly higher levels of multiple proinflammatory cytokines and chemokines in plasma, suggesting invivo upregulation of TLR7 signaling. Our study suggests that forodesine may activate a wide range of immune cells, including T cells, through TLR7 stimulation while inhibiting GVHD by inducing apoptosis of T cells, after allogeneic hematopoietic stem cell transplant.

Enterococcus faecium HDRsEf1 Promotes Systemic Th1 Responses and Enhances Resistance to SalmonellaTyphimurium Infection.

The gut microbiota is known to regulate the immune system and thereby influence susceptibility to infection. In this study, we observed that the administration of Enterococcus faecium HDRsEf1 (HDRsEf1) led to an improvement in the development of the immune system. This was evidenced by an increase in both the spleen index and the area of spleen white pulp. Specifically, the proportion of T helper (Th) 1 cells and the production of IFN-γ and IL-12 were significantly increased in the spleens of mice treated with HDRsEf1. In agreement with the in vivo results, we found that Th1-related cytokines, including IFN-γ and IL-12p70, were strongly induced in splenocytes treated with HDRsEf1. In addition, Th1 cell activation and high-level secretion of IL-12p70 were also confirmed by coculture of CD4+ T cells with bone marrow-derived dendritic cells treated with HDRsEf1. Moreover, the employment of HDRsEf1 was identified to augment resilience against systemic infection provoked by S. Typhimurium and stimulate the expression of the genes for TNFα and iNOS in the initial stage of infection, signifying that reinforced Th1 cells and IL-12 might activate macrophages for antibacterial safeguards. In summary, our study suggests that HDRsEf1 could act as an effective immunobiotic functional agent, promoting systemic Th1 immunological responses and priming defenses against infection.

Syringaldehyde ameliorates mouse arthritis by inhibiting dendritic cell maturation and proinflammatory cytokine secretion.

Syringaldehyde (SD), a kind of flavonoid polyphenolic small molecule compound, has the antioxidant and anti-inflammatory properties. But it is unknown whether SD has properties on the treatment of rheumatoid arthritis (RA) by modulating dendritic cells (DCs). We explored the effect of SD on the maturation of DCs in vitro and in vivo. The results showed that SD significantly down-regulated the expression of CD86, CD40 and MHC II, decreased the secretion of TNF-α, IL-6, IL-12p40 and IL-23, and increased IL-10 secretion and antigen phagocytosis in vitro induced by lipopolysaccharides in a dose-dependent manner through reducing the activation of MAPK/NF-κB signaling pathways. SD also significantly inhibited the expression of CD86, CD40 and MHC II on DCs in vivo. Moreover, SD suppressed the expression of CCR7 and the in vivo migration of DCs. In arthritis mouse models induced by λ-carrageenan and complete Freund's adjuvant, SD significantly alleviated paw and joint oedema, reduced the levels of pro-inflammatory cytokines TNF-α and IL-6 and increased the level of IL-10 in serum. Interestingly, SD significantly decreased the numbers of type I helper T cells (Th1), Th2, Th17 and Th17/Th1-like (CD4+IFN-γ+IL-17A+), but increased the numbers of regulatory T cells (Tregs) in spleens of mice. Importantly, the numbers of CD11c+IL-23+ and CD11c+IL-6+ cells were negatively correlated with the numbers of Th17 and Th17/Th1-like. These results suggested that SD ameliorated mouse arthritis through inhibiting the differentiation of Th1, Th17 and Th17/Th1-like and promoting the generation of Tregs via regulation of DC maturation.

Application of phosphorylcholine derivative as mucosal adjuvant enhancing mucosal immune responses in the upper respiratory tract

ObjectiveA phosphorylcholine (PC)-derivative with high binding ability (PCDB) was intranasally administered to mice with ovalbumin (OVA), and immune responses were investigated to determine whether PCDB has antigenicity and adjuvanticity. MethodsBALB/c mice were intranasally immunized with PCDB coupled with OVA, unbound PCDB plus OVA, cholera toxin (CT) plus OVA, OVA alone, and PCDB alone. Then, the production of OVA- and PC-specific antibodies in external secretions and serum, and the secretion of cytokines such as IL-4 and IFN-γ from splenic mononuclear cells by stimulation with PCDB and OVA were examined. Furthermore, the secretion of IL-12p40 from CD11c+ cells following stimulation with PCDB was observed to clarify the adjuvant effect of PCDB through TLR4. ResultsIntranasal immunization with PCDB plus OVA increased OVA- and PC-specific IgA in external secretions and OVA- and PC-specific antibodies in the serum. The analysis of IgG subclasses specific to OVA and PC showed a higher production of IgG1 than IgG2, and the secretion of both IL-4 and IFN-γ was enhanced. However, IL-12p40 secretion from CD11c+ cells was increased and OVA-specific IgE production was not promoted by PCDB stimulation. ConclusionIntranasal administration of the protein antigen with PCDB enhanced immune responses specific to the mixed antigen and PC. Although PCDB acted to bias the immune response toward the Th2-type, antigen-specific IgE production did not increase. These findings suggest that PCDB has the potential to be a mucosal vaccine with both adjuvanticity and antigenicity without causing side effects due to type I allergy.

Tolerogenic dendritic cells protect against acute kidney injury

Ischemia reperfusion injury is a common precipitant of acute kidney injury that occurs following disrupted perfusion to the kidney. This includes blood loss and hemodynamic shock, as well as during retrieval for deceased donor kidney transplantation. Acute kidney injury is associated with adverse long-term clinical outcomes and requires effective interventions that can modify the disease process. Immunomodulatory cell therapies such as tolerogenic dendritic cells remain a promising tool, and here we tested the hypothesis that adoptively transferred tolerogenic dendritic cells can limit kidney injury. The phenotypic and genomic signatures of bone marrow-derived syngeneic or allogeneic, Vitamin-D3/IL-10-conditioned tolerogenic dendritic cells were assessed. These cells were characterized by high PD-L1:CD86, elevated IL-10, restricted IL-12p70 secretion and a suppressed transcriptomic inflammatory profile. When infused systemically, these cells successfully abrogated kidney injury without modifying infiltrating inflammatory cell populations. They also provided protection against ischemia reperfusion injury in mice pre-treated with liposomal clodronate, suggesting the process was regulated by live, rather than reprocessed cells. Co-culture experiments and spatial transcriptomic analysis confirmed reduced kidney tubular epithelial cell injury. Thus, our data provide strong evidence that peri-operatively administered tolerogenic dendritic cells have the ability to protect against acute kidney injury and warrants further exploration as a therapeutic option. This technology may provide a clinical advantage for bench-to-bedside translation to affect patient outcomes.

ADAM9 drives immunosuppressive environment for lung tumor progression

Abstract Cancer cells develop the immune suppressive tumor environment, but the underlying molecular mechanism for it is still elusive. Here, we demonstrated that ADAM9 contributed to lung tumor progression and promoted IFN-related gene expression in lung cancer cells that participated in immune regulation in the tumor environment. ADAM9 knockout (KO) lung cancer cells reduced lung cancer metastasis with a decrease in neutrophil infiltration and an increase in CD8+ T cell infiltration. By performing the genome-wide approach to explore ADAM9-regulated genes in lung cancer cells, we revealed that ADAM9 regulated cytokine production and lymphocyte activation. It enhanced the master transcription factor of IFN signaling, STAT1 activation in lung cancer cells. Moreover, ADAM9 promoted IL6/IL6 receptor-STAT3 axis signaling that lead to suppression of IL12p40 secretion and MHC class II expression in dendritic cells and macrophages. Notably, we found that type I IFN related-proteins and steroid biosynthesis-related proteins were related to ADAM9-mediated STAT signaling activation by LC-MS/MS analysis. We demonstrated that ADAM9 created an immune suppressive environment by regulating IFN signaling for tumor progression. Supported by grants from NHRI (NHRI-EX112-11219BI)

Geobarrettin D, a Rare Herbipoline-Containing 6-Bromoindole Alkaloid from Geodia barretti

Geobarrettin D (1), a new bromoindole alkaloid, was isolated from the marine sponge Geodia barretti collected from Icelandic waters. Its structure was elucidated by 1D, and 2D NMR (including 1H-15N HSQC, 1H-15N HMBC spectra), as well as HRESIMS data. Geobarrettin D (1) is a new 6-bromoindole featuring an unusual purinium herbipoline moiety. Geobarrettin D (1) decreased secretion of the pro-inflammatory cytokine IL-12p40 by human monocyte derived dendritic cells, without affecting secretion of the anti-inflammatory cytokine IL-10. Thus, compound 1 shows anti-inflammatory activity.

Clearance of bacteria from lymph nodes in sheep immunized with Brucella suis S2 vaccine is associated with M1 macrophage activation

Ovine brucellosis is a global zoonotic disease of sheep caused by Brucella melitensis, which inflicts a significant burden on human and animal health. Brucella suis strain S2 (B. suis S2) is a smooth live attenuated vaccine for the prevention of ovine brucellosis in China. However, no previous studies have assessed the immunogenicity of B. suis S2 vaccine after oral immunization in sheep. Here, we attempted to evaluate the ovine immune response over the course of B. suis S2 immunization and to identify in vivo predictors for vaccine development. Body temperature, serum Brucella antibodies, serum cytokines (IL-12p70 and interferon [IFN]-γ), and bacterial load in the mandibular lymph nodes (LN), superficial cervical LN, superficial inguinal LN, and spleen were investigated to determine the safety and efficacy of the vaccine. The abnormal body temperature of sheep occurred within 8 days post-infection (dpi). Brucella suis S2 persisted for a short time (< 21 dpi) in the mandibular LN. The highest level of IL-12p70 was observed at 9 dpi, whereas serum IFN-γ levels peaked at 12 dpi. Transcriptome analysis and quantitative reverse transcription PCR were performed to determine gene expression profiles in the mandibular LN of sheep. Antigen processing and presentation pathway was the dominant pathway related to the dataset. Our studies suggest that the immune response in ovine LN resembled type 1 immunity with the secretion of IL-12p70 and IFN-γ after B.suis S2 immunization and the vaccine may eliminate Brucella via stimulation of M1 macrophages through the course of Th cells.

TLR-5 ligand conjugated with Per a 10 and T cell peptides potentiates Treg/Th1 response through PI3K/mTOR axis.

Novel strategies are needed to enhance efficacy of immunotherapy for allergic diseases. The present study evaluates role of conjugating flagellin with allergen, Per a 10 and its T cell epitopes in DC mediated polarization. Fusion proteins (FPer a 10, FPT1, FPT2 and FPT3) were sub-cloned, expressed and purified to homogeneity. FlaA fused proteins showed TLR-5 binding in TLR5+HEK293 cells as observed by enhanced NF-κB dual luciferase assay and IL-8 secretion in culture supernatant. Fusion proteins upregulate co-stimulatory molecules CD80, CD83 and CD86 on BMDCs and enhanced IL-6, IL-β, IL-12p70 and IL-10 secretion. Inhibition studies suggest fusion proteins mediate IL-10 secretion via activation of PI3K/mTOR/NF-κB pathway. FPT1 and FPT3 deviate Th2 cytokine milieu towards Th1/Treg by upregulating the expression of IFN-γ, IL-12p70, IL-10 and TGF-β in BMDCs co-culture with allergen specific T cells. Flow cytometric examination of BMDC:T cell co-culture revealed CD4+ T cell differentiation towards Th1/Treg by FPT1 and FPT3. Thus, our results indicate that flagellin fused with T cell peptides are efficient immunomodulators and may possess potential for allergy therapeutics.

Cytokine profile in &lt;i&gt;in vitro&lt;/i&gt; mouse macrophage culture infected with &lt;i&gt;Bacillus anthracis&lt;/i&gt; spores with varying plasmid composition

Bacillus anthracis, the causative agent of anthrax, is able to exist both in environmental conditions (soil) and in the macroorganism. The manifestation of pathogenic properties of B. anthracis strains is determined by relevant plasmid composition, because the main toxin and the capsule-related virulence factors are located in bacterial plasmid. Modeling anthrax infection in vitro in macrophage culture might reveal an influence of individual B. anthracis strain characteristics on infection and development of infectious process. The aim of this study was to analyze cytokine level during infection of in vitro macrophage cell cultures with spores of anthrax microbe strains bearing varying plasmid composition. The dependence of the macrophage cell cytokine profile on the plasmid composition of B. anthracis strains was revealed while modeling anthrax infection in vitro. The presence of the toxin-producing plasmid pXO1 in anthrax microbe strains has a powerful stimulating effect on the production of macrophages J774A cell line cytokines. B. anthracis strains lacking the pXO1 plasmid virtually stimulated no production of IL-1, caused very low secretion of IL-1, IL-6, MCP-1, MIP-1, MIP-1, IL-12 (p70) and active G-CSF products. The low cytokine response of macrophage cells infected with monoplasmid strains bearing only the capsule-forming plasmid was due not only to the absence of a binary toxin, but also to disturbed regulation of capsule production associated with the absence of the atxA gene. The capsule, along with lethal and edematous toxins, belongs to the main virulence factors of B. anthracis, but strains lacking the pXO1 virulence plasmid, had its production impaired, because the main regulator of capsule synthesis is the atxA gene localized on the pXO1 plasmid being positively regulated by the acpA and acpB genes, so that strains lacking the toxin-forming plasmid, even in the presence of the encapsulation plasmid, elicit a weak cytokine response in infected cells. Diplasmid strains of B. anthracis, due to produced main virulence factors a two-component toxin and a capsule, enforce macrophages (in the experiment) to actively produce IL-1, IL-6, MCP-1, G-CSF, MIP-1; MIP-1, IL-12 (p70). Strains with moderate virulence and capable of capsulation in air virtually did not differ from highly virulent strains in terms of their effect on in vitro macrophage culture.

Chronic Inflammation Drives p53 Mutant Clonal Hematopoiesis Via Activating the NLRP1 Inflammasome

Individuals with somatic TP53 mutations manifest clonal hematopoiesis and are at high risk of developing hematological malignancies. However, how environmental factors influence the expansion of p53 mutant hematopoietic stem and progenitor cells (HSPCs) is unclear. We found that pI:pC or LPS treatment promotes the clonal expansion of p53 mutant HSPCs in transplantation assays. To determine the impact of LPS on HSPC function in vivo, we treated p53+/+ and p53R248W/+ mice with LPS or PBS and then performed competitive transplantation assays. LPS-treated WT BM cells show decreased engraftment in both primary and secondary transplantation assays, whereas LPS-treated p53 mutant BM cells continued to show increased engraftment. To understand how LPS-induced chronic inflammation promotes p53 mutant HSPC expansion, we examined the impact of mutant p53 on cytokine secretion from BM derived macrophages. We found that p53 mutant macrophages show increased secretion of multiple pro-inflammatory cytokines, including IL-1β, IL-1α, IL-12 (p70), KC, VEGF, IP-10, CXCL-9, LIX, and MCP-1. To further investigate how chronic inflammation promotes p53 mutant HSPC expansion, we performed ATAC-seq assays in LSKs to identify differential regions of chromatin accessibility. Notably, we found that mutant p53 increases the chromatin accessibility to inflammasome genes, including Nlrp1a and Nlrp1b. Inflammasomes are multiprotein complexes that activate Caspase-1 and increase the release of pro-inflammatory cytokines such as IL-1β, leading to caspase-1-dependent death, known as pyroptosis. We found that loss of NLRP1 significantly decreased the secretion of multiple pro-inflammatory cytokines, including IL-1β, IL-1α, IL-6, MCP-1, KC, M-CSF, RANTES, LIX, GM-CSF G-CSF, TNF-α and IL-12 (p40), from macrophages expressing mutant p53. Loss of NLRP1 significantly decreases the repopulating potential of HSPCs expressing mutant p53 in vivo. To further understand how chronic inflammation may confer a competitive advantage to p53 mutant HSPCs, we performed in vitro cell competition assays. We found that WT CD45.1+ cells co-cultured with p53 mutant cells show decreased replating potential compared to WT CD45.1+ cells co-cultured with WT CD45.2+ cells. We measured the levels of cytokines in the condition media utilizing cytokine arrays and found increased levels of IL-1β, IL-6, CXCL-9, and IL-4 in mutant cell cultures compared to that of the WT cells. We found that WT CD45.1+ cells co-cultured with mutant cells show increased pyroptosis. Importantly, we found that the enhanced pyroptosis of WT CD45.1+ cells seen in the mutant group is IL-1β dependent. We found that IL-1β treatment decreases the replating potential of WT BM cells, but not that of p53 mutant cells. To determine the mechanism by which p53 mutant HSPCs are resistant to IL-1β treatment, we performed RNA sequencing in p53+/+ and p53R248W/+ LSKs treated with IL-1β (10 ng/ml) or PBS in vitro for 90 minutes. GSEA analysis revealed that hematopoietic stem and progenitor cell differentiation genes are significantly upregulated in p53 mutant LSKs compared to WT LSKs following IL-1β stimulation. Notably, we found that p53 mutant LSKs treated with IL-1β are enriched with MYC target genes. Furthermore, blocking of IL-1β using IL-1β neutralizing antibody decreases the fitness of p53 mutant HSPCs. Our studies will likely establish both the NLRP1 inflammasome and the pro-inflammatory cytokine IL-1β as novel potential therapeutic targets for preventing clonal hematopoiesis development and treating hematological malignancies with TP53 mutations.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune–metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune–metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1β was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1β, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1β). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1β (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.