IL-12p70 Production Research Articles

A novel immunomodulating peptide with potential to complement oligodeoxynucleotide-mediated adjuvanticity in vaccination strategies.

The identification of adjuvants to improve vaccination efficacy is a major unmet need. One approach is to augment the functionality of dendritic cells (DCs) by using Toll-like receptor-9 (TLR9) agonists such as cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) as adjuvants. Another approach is adjuvant selection based on production of bioactive interleukin-12 (IL-12). We report a D-peptide isomer, designated D-15800, that induces monocyte differentiation to the DC phenotype in vitro and more effectively stimulates IL-12p70 production upon T cell receptor (TCR) activation than the L-isomer. In the absence of TCR activation and either IL-12p70 or interleukin-2 production, only D-15800 activates CD4+ T and natural killer cells. In the presence of CpG ODN, D-15800 synergistically enhances production of interferon-alpha (IFN-α). Taken together with its biostability in human serum and depot retention upon injection, co-delivery of D-15800 with TLR9 agonists could serve to improve vaccine efficacy.

Immunomodulatory effects of Fructus corni acidic polysaccharide and its selenium nanoparticles composites in hepatocellular carcinoma

Natural polysaccharides have promising application prospects in cancer immunotherapy due to their excellent biocompatibility and immunomodulatory effects. In this study, an acidic polysaccharide (FCP-3) from Fructus corni with good immunomodulatory effects was extracted. Stable polysaccharide‑selenium nanoparticles composites (FCP-SeNPs) were synthesized to enhance the immunoregulatory effects of FCP-3. The molecular weight and monosaccharide composition of FCP-3, as well as the morphological characteristics and chemical composition of FCP-SeNPs were analyzed. The immunological effects and anti-tumor activity of FCP-3 and FCP-SeNPs were explored. These results suggested that FCP-3 and FCP-SeNPs increased the production of NO, TNF-α and IL-12p70 in macrophages, and the ratio of CD4+/CD8+ T cells in peripheral blood, as well as indirectly promoted hepatoma cells apoptosis in vitro. After treatment with FCP-3 and FCP-SeNPs, tumor size was significantly controlled. The proliferation antigen staining (KI67) and apoptosis assay (TUNEL) also demonstrated an obvious inhibitory effect on tumor proliferation. Moreover, FCP-SeNPs showed stronger efficacy compared to FCP-3 alone. These findings demonstrated the promising potential of FCP-SeNPs as an immunoregulatory agent for hepatic carcinoma.

Secobutanolides Isolated from Lindera obtusiloba Stem and Their Anti-Inflammatory Activity.

In this study, a new secobutanolide, named secosubamolide B (3), along with three previously known secobutanolides (1, 2, and 4), were successfully isolated from a methanol extract of the stem of Lindera obtusiloba. The chemical structures of these compounds were elucidated through the analysis of spectroscopic data, and then compared with the existing literature to confirm their identities. Furthermore, the anti-inflammatory effect of these isolated compounds on bone marrow-derived dendritic cells stimulated by lipopolysaccharide (LPS) was evaluated. Compounds 1-3 showed the significant suppression of LPS-triggered IL-6 and IL-12 p40 production, with IC50 values between 1.8 and 24.1 µM. These findings may provide a scientific foundation for developing anti-inflammatory agents from L. obtusiloba.

Necrotic Change of Tunica Media Plays a Key Role in the Development of Coronary Artery Lesions in Kawasaki Disease.

Alarmins resulting from cell death or oxidative stress are involved in the development of Kawasaki disease (KD) vasculitis. In a previous study, we demonstrated the potential role of interleukin (IL)-33 as an alarmin in the development of KD vasculitis. Although edematous dissociation (necrotic change) of the tunica media is thought to be a major source of IL-33 in KD vasculitis, it has not yet been elucidated. We investigated the impact of IL-33 released from necrotic human coronary artery smooth muscle cells (HCASMCs) on human coronary artery endothelial cells (HCAECs) using a coculture assay. Subsequently, we evaluated the anti-inflammatory effects of anti-IL-33 and anti-suppression of tumorigenicity 2 (ST2) antibodies compared with conventional therapies of KD, such as high-dose IgG or anti-tumor necrosis factor (TNF)-α antibody. Primary necrosis of HCASMCs induced significant release of IL-33. In cocultures of necrotic HCASMCs with HCAECs, the necrotic HCASMCs significantly induced the production of various proinflammatory cytokines in the HCAECs. Anti-IL-33 and anti-ST2 antibodies exhibited unique inhibitory effects on the production of platelet-derived growth factor-BB or IL-12(p70) in HCAECs. There is potential involvement of edematous dissociation of the tunica media in the development of KD vasculitis. Furthermore, the distinctive anti-inflammatory effects of the anti-IL-33/ST2 axis drugs suggest novel therapeutic options for patients with refractory KD.

The effects of in vitro vitamin D treatment on glycolytic reprogramming of bone marrow-derived dendritic cells from Ldlr knock-out mouse

Dendritic cells (DCs) undergo glycolytic reprogramming, a metabolic conversion process essential for their activation. Vitamin D has been reported to affect the function of DCs, but studies in metabolic diseases are insufficient. This study investigates the effects of in vitro 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment on glycolytic reprogramming of bone marrow-derived dendritic cells (BMDCs) from control, obese, and atherosclerosis mice. Six-week-old male C57BL/6J mice were fed a control diet (CON) or a Western diet (WD), and B6.129S7-Ldlrtm1Her/J mice were fed a Western diet (LDLR−/−) for 16 weeks. BMDCs were cultured in a medium containing 1,25(OH)2D3 (10 nM) for 7 days and stimulated with lipopolysaccharide (LPS, 50 ng/mL) for 24 h. In mature BMDCs, 1,25(OH)2D3 treatment decreased basal and compensatory glycolytic proton efflux rates (glycoPER), the expression of surface markers related to immune function of DCs (MHC class II, CD80, and CD86), and IL-12p70 production. In addition, mTORC1 activation and nitric oxide (NO) production were suppressed by 1,25(OH)2D3 treatment in mature BMDCs. The effect of 1,25(OH)2D3 treatment on IL-12p70 production and mTORC1 activity in the LDLR−/− group was greater than in the CON group. These findings suggest that vitamin D can affect the metabolic environment of BMDCs by regulating glycolytic reprogramming as well as by inducing tolerogenic phenotypes of DCs.

Pediococcus pentosaceus MIANGUAN2 Alleviates Influenza Virus Infection by Modulating Gut Microbiota and Enhancing Short-Chain Fatty Acid Production.

Influenza, a severe respiratory disease caused by the influenza virus, has long been a prominent threat to human health. An increasing number of studies have demonstrated that oral administration with probiotics may increase the immune response to lung infection via the gut-lung axis leading to the alleviation of the pulmonary disease. In this study, we evaluated the effects of oral administration of Pediococcus pentosaceus MIANGUAN2 (MIANGUAN2) on influenza infection in a mouse model. Our results showed that oral administration of MIANGUAN2 significantly improved weight loss, lung index, and lung pathology, and decreased lung viral load of influenza-infected mice. Additionally, MIANGUAN2-treated mice showed significantly lower levels of TNF-α, IL-1β, IFN-γ, and IL-12p70 and higher production of IL-4 in the lung. In accordance with this, the transcriptome analysis of the lung indicated that MIANGUAN2-treated mice had reduced expression of inflammation markers, such as TNF, apoptosis, and the NF-Kappa B pathway. Furthermore, the administration of MIANGUAN2 restored the SCFAs profiles through regulating the gut microbiota. SCFA-producing bacteria, such as p_Firmicutes, f_Lachnospiraceae, and f_Ruminococcaceae, were enriched in the MIANGUAN2-treated group compared with PBS-treated group. Consistently, the concentrations of SCFAs in the MIANGUAN2 group were significantly higher than those in the PBS-treated group. In addition, the concentrations of SCFAs were positively correlated with SCFA-producing bacteria, such as Ruminococcus, while being negatively correlated with the virial titers and proinflammatory cytokines. In conclusion, this animal study suggests that Pediococcus pentosaceus MIANGUAN2 may alleviate the influenza infection by altering the gut microbiota composition and increasing the levels of gut microbiota-derived SCFAs.

#1699 The role of interleukin 12 cytokine family in autosomal dominant polycystic kidney disease physiopathology

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is an inherited, complex disease, characterized by cysts formation and phenotypic changes. ADPKD physiopathology is under research, the role of proinflammatory chemokines responsible for perpetuating the cycle of chronic inflammation leading to kidney damage being intensively studied. We aimed to explore the individualized contribution of IL-12 interleukin family members in ADPKD physiopathology. Method We developed a prospective, case control study, in Carol Davila Clinical Hospital of Nephrology, including a group of 66 ADPKD subjects (56.1 years old, men:women 35:32), and a group of 40 healthy subjects, similar as sex, mean age. ADPKD diagnosis was based on familial history, clinical exam and CT or MRI scan. We excluded from the study subjects that presented cysts in other organs except the kidney, with eGFR<60 mL/min/1.73 mp, with history of hematuria, cysts infection, urinary tract infection, renal lithiasis, with unstable blood pressure in the last 6 months. Laboratory tests: serum levels of monomers IL-12 p40 and IL-20 p35 and heterodimers IL-12 p70, IL-23, IL 35, assessed by ELISA method. Statistical analysis. All the results were analyzed using IBM SPSS Statistics 2015 and were presented as average value ± standard deviation. p < 0.05 was considered with statistical significance. Results In ADPKD patients, we detected altered production of IL-12 cytokine, with low production of IL-12 p35 and IL-35, respectively high production of IL-12 p70, IL-23 and IL-12 p40 versus control. IL-12 p35 and IL-35 had statistically significant lower levels in ADPKD patients with eGFR<90 ml/min/1.73 mp, when compared with ADPKD patients with eGFR>90 ml/min/1.73 mp (Table 1). IL-12 p70, IL-23 and IL-12 p40 had statistically significant higher levels in ADPKD patients with eGFR<90 ml/min/1.73 mp, when compared with ADPKD patients with eGFR>90 ml/min/1.73 mp (Table 1). IL-12p70, IL-23 and IL-12 p40 had a negative significant correlation with eGFR, while IL-12p35 and IL-35 had a positive significant correlation with eGFR. Though, IL-12p35 and IL-35 serum levels correlated negatively with IL-12p70, IL-23 and IL-12 p40 serum levels in ADPKD patients. Conclusion The results of the present study suggest that ADPKD is a cellular source of IL-12 family, cytokines produced by antigenic stimulation. IL-12 family programming is abnormal in ADPKD patients. We detected synergic and divergent action of IL-12 cytokine family members, relevant for ADPKD physiopathology.

High production of IL-12 by human dendritic cells stimulated with combinations of pattern-recognition receptor agonists

The cytokine IL-12p70 is crucial for T helper 1 (Th1) polarization and the generation of type 1 immunity required to fight cancer and pathogens. Therefore, strategies to optimize the production of IL-12p70 by human dendritic cells (DCs) may significantly improve the efficacy of vaccines and immunotherapies. However, the rules governing the production of IL-12p70 remain obscure. Here, we stimulated pattern recognition receptors (PRRs) representing five families of PRRs, to evaluate their ability to elicit high production of IL-12p70 by monocyte-derived DCs. We used ten well-characterized agonists and stimulated DCs in vitro with either single agonists or 27 different combinations. We found that poly(I:C), which engages the RNA-sensing PRRs TLR3 and MDA5, and LPS which stimulates TLR4, were the only agonists that could elicit notable IL-12p70 production when used as single ligands. We identified six different combinations of PRR agonists, all containing either the TLR3/MDA5 agonist poly(I:C) or the TLR7/8 agonist R848, that could synergize to elicit high production of IL-12p70 by human DCs. Five of the six combinations also triggered high production of the antiviral and antitumor cytokine IFNβ. Overall, the tested PRR ligands could be divided into three groups depending on whether they triggered production of both IL-12p70 and IFNβ, only one of the two, or neither. Thus, combinations of PRR agonists were found to increase the production of IL-12p70 by human DCs in a synergistic manner, and we identified six PRR agonist combinations that may represent strong adjuvant candidates, in particular for therapeutic cancer vaccines.

Abstract 6744: Activating anti-MDS immunity with polarized dendritic cell vaccines

Abstract Less than 50% of patients with higher-risk myelodysplastic syndromes (MDS) show clinical response to standard-of-care hypomethylating (HMA) therapy. Significant enthusiasm existed for immunotherapy/HMA combinations for these patients, but studies have failed to deliver on the initial promise. We have demonstrated that patients with MDS have a deficiency in antigen-presenting cells, called CD141Hi dendritic cells (DC). DC-deficient MDS patients have reduced response to HMAs, worse overall survival, and show less response to cancer vaccination. We hypothesized that restoration of this population using a novel DC vaccination approach - alpha-type-1 polarized dendritic cells (αDC1) - might compensate for this deficiency and restore immunity. To generate αDC1s, we collected monocytes from MDS patients and healthy donors (HD) and cultured them for 6 days in vitro in the presence of recombinant human GM-CSF and IL-4 followed by an additional 18-24 hours in the presence of αDC1 maturation factors (poly-I:C/IFNα/IFNγ/TNFα/IL-1β). Overall, we attempted to generate αDC1s from 15 MDS samples; 13/15 exhibited intermediate to very high-risk disease and 10/15 are currently receiving HMA therapy. We successfully cultured cells from 8/15 samples (53%). Of the 8 MDS samples, 5 were collected from patients receiving HMA therapy (62.5%). We were unable to successfully culture αDC1s from the other 5 patients receiving HMA therapy, and thus the effect of HMA therapy on aDC1 generation remains unclear. To test the function of MDS αDC1s, we focused on two signals necessary to activate immune responses: 1) the expression of co-stimulatory molecules and 2) the production of IL-12p70. Flow cytometric analysis on αDC1s and immature DCs from MDS samples revealed intra-patient variation in the fold-change surface expression of CD40/80/83/86 and HLA-DR proteins. Additionally, there was a trend towards decreased up-regulation of CD80 and CD86 in MDS specimens compared to HD. To assess production of IL-12p70, we stimulated immature DCs and αDC1s with CD40L and performed ELISA analysis on the media supernatant. We found that 3 out of 4 samples could produce IL-12p70 (range 27 - 5,480 pg/ml). To date, our results suggest that generation of MDS patient-derived αDC1 cells is feasible and additional work is being performed to fully characterize their ability to activate anti-MDS immune responses. Since αDC1s have an established safety profile in patients with solid tumors, we anticipate that we will be able to rapidly translate our results to the clinic. Citation Format: Daniel Alvarado, Jennifer Peresie, Adam Brinkman, Cheryl Allen, Weijian Jiang, Pawel Kalinski, Tiffany Tanaka, Elizabeth Griffiths, Michael Nemeth. Activating anti-MDS immunity with polarized dendritic cell vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6744.

Fungal polysaccharides from Inonotus obliquus are agonists for Toll-like receptors and induce macrophage anti-cancer activity

Fungal polysaccharides can exert immunomodulating activity by triggering pattern recognition receptors (PRRs) on innate immune cells such as macrophages. Here, we evaluate six polysaccharides isolated from the medicinal fungus Inonotus obliquus for their ability to activate mouse and human macrophages. We identify two water-soluble polysaccharides, AcF1 and AcF3, being able to trigger several critical antitumor functions of macrophages. AcF1 and AcF3 activate macrophages to secrete nitric oxide and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Combined with interferon-γ, the fungal polysaccharides trigger high production of IL-12p70, a central cytokine for antitumor immunity, and induce macrophage-mediated inhibition of cancer cell growth in vitro and in vivo. AcF1 and AcF3 are strong agonists of the PRRs Toll-like receptor 2 (TLR2) and TLR4, and weak agonists of Dectin-1. In comparison, two prototypical particulate β-glucans, one isolated from I. obliquus and one from Saccharomyces cerevisiae (zymosan), are agonists for Dectin-1 but not TLR2 or TLR4, and are unable to trigger anti-cancer functions of macrophages. We conclude that the water-soluble polysaccharides AcF1 and AcF3 from I. obliquus have a strong potential for cancer immunotherapy by triggering multiple PRRs and by inducing potent anti-cancer activity of macrophages.

P1229 The Role and Niche-Specific Adaptation of Malassezia in patients with Ulcerative colitis

Abstract Background Accumulating evidence has underscored the role of gut fungal microbiota (mycobiota) in the development of inflammatory bowel disease. We aimed to isolate a Malassezia strain directly from the human intestine mucosal surface from the patients with ulcerative colitis (UC) and investigated its genome and virulence in comparison with the same fungal species isolated from the human skin. Methods Mucosal lavage samples were collected separately from colonic areas with and without inflammation in patients with UC. Samples from healthy individuals (HT) were obtained in the same manner as from patients with UC at sigmoid or descending colon. Skin samples were taken from HT in our previous work. DNA was extracted from these lavage samples, and fungal isolation was conducted using PCR amplification with ITS4 and ITS5 primers. Comprehensive analysis and comparison of the genomes, transcriptomes, and virulence between M. globosa gut isolates and those of M. globosa strains isolated from the skin were performed. To determine the contribution of M. globosa gut isolates to exacerbating inflammation, 1107 fungal cells were orally gavaged to DSS-induced colitis mouse model for three days. Results Total 56 and 11 intestinal water-lavage samples from 29 UC patients and 11 HT were obtained respectively. The α- and b-diversities of mycobiota showed no significant differences between the groups, patients with UC vs. HT or the sites with inflammation vs. non-inflammation of the patient with UC. Malassezia was the fifth most frequently found fungal genus throughout the samples, and live fungal strains belong to 28 and 7 different species were isolated from the patients with UC and HT, respectively. The patients with UC tend to have higher frequency of M. globosa and M. restricta than HT in their gut mucosal surface with inflammation. Whole genome sequencing showed no specific genomic characteristics between gut-isolated M. globosa and skin-isolated M. globosa. However, gut-isolated M. globosa were suffered more from the higher oxygen levels than the skin isolates in different oxygen concentrations. In a mouse model, gut-isolated M. globosa exhibited a more pronounced exacerbation of DSS-induced colitis and elevated production of inflammatory cytokines, including TNF-a, IL-6, IL-12p40, IL-1b, and IL-18, while the skin isolates showed no difference compared to the negative control (Figure). Conclusion Our data shed new light on the pivotal role of M. globosa in the pathogenesis of UC, highlighting the potential influence of niche-specific adaptations on the virulence of this fungus. These findings provide critical insights into the complex interplay between the member of the gut mycobiota and host health.

Human In vitro Modeling Identifies Adjuvant Combinations that Unlock Antigen Cross-presentation and Promote T-helper 1 Development in Newborns, Adults and Elders

Adjuvants are vaccine components that can boost the type, magnitude, breadth, and durability of an immune response. We have previously demonstrated that certain adjuvant combinations can act synergistically to enhance and shape immunogenicity including promotion of Th1 and cytotoxic T-cell development. These combinations also promoted protective immunity in vulnerable populations such as newborns. In this study, we employed combined antigen-specific human in vitro models to identify adjuvant combinations that could synergistically promote the expansion of vaccine-specific CD4+ cells, induce cross-presentation on MHC class I, resulting in antigen-specific activation of CD8+ cells, and direct the balance of immune response to favor the production of Th1-promoting cytokines. A screen of 78 adjuvant combinations identified several T cell-potentiating adjuvant combinations. Remarkably, a combination of TLR9 and STING agonists (CpG + 2,3-cGAMP) promoted influenza-specific CD4+ and CD8+ T cell activation and selectively favored production of Th1-polarizing cytokines TNF and IL-12p70 over co-regulated cytokines IL-6 and IL-12p40, respectively. Phenotypic reprogramming towards cDC1-type dendritic cells by CpG + 2,3-cGAMP was also observed. Finally, we characterized the molecular mechanism of this adjuvant combination including the ability of 2,3-cGAMP to enhance DC expression of TLR9 and the dependency of antigen-presenting cell activation on the Sec22b protein important to endoplasmic reticulum-Golgi vesicle trafficking. The identification of the adjuvant combination CpG + 2,3-cGAMP may therefore prove key to the future development of vaccines against respiratory viral infections tailored for the functionally distinct immune systems of vulnerable populations such as older adults and newborns.

D-alanine Inhibits Murine Intestinal Inflammation by Suppressing IL-12 and IL-23 Production in Macrophages.

Free D-amino acids, which have different functions from L-amino acids, have recently been discovered in various tissues. However, studies on the potential interactions between intestinal inflammation and D-amino acids are limited. We examined the inhibitory effects of D-alanine on the pathogenesis of intestinal inflammation. We investigated serum D-amino acid levels in 40 patients with ulcerative colitis and 34 healthy volunteers. For 7 days [d], acute colitis was induced using dextran sulphate sodium in C57BL/6J mice. Plasma D-amino acid levels were quantified in mice with dextran sulphate sodium-induced colitis, and these animals were administered D-alanine via intraperitoneal injection. IFN-γ, IL-12p35, IL-17A, and IL-23p19 mRNA expression in the colonic mucosa was measured using real-time polymerase chain reaction [PCR]. In vitro proliferation assays were performed to assess naïve CD4+ T cell activation under Th-skewing conditions. Bone marrow cells were stimulated with mouse macrophage-colony stimulating factor to generate mouse bone marrow-derived macrophages. Serum D-alanine levels were significantly lower in patients with ulcerative colitis than in healthy volunteers. Dextran sulphate sodium-treated mice had significantly lower plasma D-alanine levels than control mice. D-alanine-treated mice had significantly lower disease activity index than control mice. IFN-γ, IL-12p35, IL-17A, and IL-23p19 mRNA expression levels were significantly lower in D-alanine-administered mice than in control mice. D-alanine suppressed naïve T cell differentiation into Th1 cells in vitro, and inhibited the production of IL-12p35 and IL-23p19 in bone marrow-derived macrophages. Our results suggest that D-alanine prevents dextran sulphate sodium-induced colitis in mice and suppresses IL-12p35 and IL-23p19 production in macrophages.

Novel Antibacterial Agents SAAP-148 and Halicin Combat Gram-Negative Bacteria Colonizing Catheters.

The antibiotic management of catheter-related infections (CRIs) often fails owing to the emergence of antimicrobial-resistant strains and/or biofilm/persister apparitions. Thus, we investigated the efficacy of two novel antimicrobial agents, i.e., the synthetic peptide SAAP-148 and the novel antibiotic halicin, against Gram-negative bacteria (GNB) colonizing catheters. The antibacterial, anti-biofilm, and anti-persister activities of both agents were evaluated against Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae strains. The enrolled strains were isolated from catheters and selected based on their resistance to at least three antibiotic classes and biofilm formation potential. Furthermore, the hemolysis and endotoxin neutralization abilities of these agents were explored. The bactericidal activity of both agents was reduced in urine and plasma as compared to buffered saline. In a dose-dependent manner, SAAP-148 and halicin reduced bacterial counts in 24 h preformed biofilms on silicone elastomer discs and eliminated persisters originating from antibiotic-exposed mature 7-day biofilms, with halicin being less effective than SAAP-148. Importantly, SAAP-148 and halicin acted synergistically on E. coli and K. pneumoniae biofilms but not on A. baumannii biofilms. The peptide, but not halicin, decreased the production of IL-12p40 upon exposure to UV-killed bacteria. This preliminary study showed that SAAP-148 and halicin alone/in combination are promising candidates to fight GNB colonizing catheters.

The nuclear receptor Nurr1 is preferentially expressed in human pro-inflammatory macrophages and limits their inflammatory profile.

Nurr1 is a member of the orphan nuclear receptor family NR4A (nuclear receptor subfamily 4 group A) that modulates inflammation in several cell lineages, both positively and negatively. Macrophages are key regulators of inflammatory responses, yet information about the role of Nurr1 in human macrophages is scarce. Here we examined Nurr1 expression and activity in steady state and activated human macrophages. Pro- and anti-inflammatory macrophages were generated in vitro by culture of blood monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Nurr1 expression was predominant in macrophages with the pro-inflammatory phenotype. Nurr1 activation with the agonists 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) or isoxazolo-pyridinone 7e (IP7e) did not globally modify the polarization status of pro-inflammatory macrophages, but they decreased their production of TNF, IL-1β, IL-6, IL-8, IL-12 p40, CCL2, IFN-β, and reactive oxygen species, with variable potencies. Conversely, Nurr1 deficient macrophages increased the expression of transcripts encoding inflammatory mediators, particularly that of IL6, IFNB1, and CCL2. Mechanistically, endogenous Nurr1 interacted with NF-κB p65 in basal conditions and upon lipopolysaccharide (LPS)-mediated activation. C-DIM12 stabilized those complexes in cells exposed to LPS and concurrently decreased NF-κB transcriptional activity and p65 nuclear translocation. Expression of high levels of Nurr1 was associated with a subset of dermal macrophages that display enhanced levels of TNF and lower expression of the anti-inflammatory marker CD163L1 in skin lesions from patients with bullous pemphigoid (BP), a chronic inflammatory autoimmune blistering disorder. These results suggest that Nurr1 expression is linked with the pro-inflammatory phenotype of human macrophages, both in vivo and in vitro, where it may constitute a brake to attenuate the synthesis of inflammatory mediators.

Sitagliptin Induces Tolerogenic Human Dendritic Cells

Sitagliptin, an anti-diabetic drug, is a dipeptidyl peptidase (DPP)-4/CD26 inhibitor with additional anti-inflammatory and immunomodulatory properties. In this study, we investigated for the first time the effect of sitagliptin on the differentiation and functions of human dendritic cells generated from monocytes (MoDCs) for 4 days using the standard GM-CSF/IL-4 procedure. LPS/IFN-γ treatment for an additional 24 h was used for maturation induction of MoDCs. Sitagliptin was added at the highest non-cytotoxic concentration (500 µg/mL) either at the beginning (sita 0d protocol) or after MoDC differentiation (sita 4d protocol). Sitagliptin impaired differentiation and maturation of MoDCs as judged with the lower expression of CD40, CD83, CD86, NLRP3, and HLA-DR, retention of CD14 expression, and inhibited production of IL-β, IL-12p70, IL-23, and IL-27. In contrast, the expression of CD26, tolerogenic DC markers (ILT4 and IDO1), and production of immunoregulatory cytokines (IL-10 and TGF-β) were increased. Generally, the sita 0d protocol was more efficient. Sitagliptin-treated MoDCs were poorer allostimulators of T-cells in MoDC/T-cell co-culture and inhibited Th1 and Th17 but augmented Th2 and Treg responses. Tolerogenic properties of sitagliptin-treated MoDCs were additionally confirmed by an increased frequency of CD4+CD25+CD127- FoxP3+ Tregs and Tr1 cells (CD4+IL-10+FoxP3-) in MoDC/T-cell co-culture. The differentiation of IL-10+ and TGF-β+ Tregs depended on the sitagliptin protocol used. A Western blot analysis showed that sitagliptin inhibited p65 expression of NF-kB and p38MAPK during the maturation of MoDCs. In conclusion, sitagliptin induces differentiation of tolerogenic DCs, and the effect is important when considering sitagliptin for treating autoimmune diseases and allotransplant rejection.

Evaluation of biocompatibility and angiogenic potential of extracellular matrix hydrogel biofunctionalized with the LL-37 peptide.

Biomaterials must allow revascularization for a successful tissue regeneration process. Biomaterials formulated from the extracellular matrix (ECM) have gained popularity in tissue engineering because of their superior biocompatibility, and due to their rheological properties, ECM-hydrogels can be easily applied in damaged areas, allowing cell colonization and integration into the host tissue. Porcine urinary bladder ECM (pUBM) retains functional signaling and structural proteins, being an excellent option in regenerative medicine. Even some small molecules, such as the antimicrobial cathelicidin-derived LL-37 peptide have proven angiogenic properties. The objective of this study was to evaluate the biocompatibility and angiogenic potential of an ECM-hydrogel derived from the porcine urinary bladder (pUBMh) biofunctionalized with the LL-37 peptide (pUBMh/LL37). Macrophages, fibroblasts, and adipose tissue-derived mesenchymal stem cells (AD-MSC) were exposed pUBMh/LL37, and the effect on cell proliferation was evaluated by MTT assay, cytotoxicity by quantification of lactate dehydrogenase release and the Live/Dead Cell Imaging assays. Moreover, macrophage production of IL-6, IL-10, IL-12p70, MCP-1, INF-γ, and TNF-α cytokines was quantified using a bead-based cytometric array. pUBMh/LL37 was implanted directly by dorsal subcutaneous injection in Wistar rats for 24 h to evaluate biocompatibility, and pUBMh/LL37-loaded angioreactors were implanted for 21 days for evaluation of angiogenesis. We found that pUBMh/LL37 did not affect cell proliferation and is cytocompatible to all tested cell lines but induces the production of TNF-α and MCP-1 in macrophages. In vivo, this ECM-hydrogel induces fibroblast-like cell recruitment within the material, without tissue damage or inflammation at 48 h. Interestingly, tissue remodeling with vasculature inside angioreactors was seen at 21 days. Our results showed that pUBMh/LL37 is cytologically compatible, and induces angiogenesis in vivo, showing potential for tissue regeneration therapies.

The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm.

The novel coronavirus pandemic, first reported in December 2019, was caused by the severe acute respiratory syndrome coronavirus2 (SARS-CoV-2). SARS-CoV-2 infection leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In response to viral infections, monocytes are recruited into the lung and subsequently differentiate into dendritic cells (DCs). DCs are critical players in the development of acute lung inflammation that causes ARDS. Here, we focus on the interaction of a specific SARS-CoV-2 open reading frame protein, ORF8, with DCs. We show that ORF8 binds to DCs, causes pre-maturation of differentiating DCs, and induces the secretion of multiple proinflammatory cytokines by these cells. In addition, we identified DC-SIGN as a possible interaction partner of ORF8 on DCs. Blockade of ORF8 leads to reduced production of IL-1β, IL-6, IL-12p70, TNF-α, MCP-1 (also named CCL2), and IL-10 by DCs. Therefore, a neutralizing antibody blocking the ORF8-mediated cytokine and chemokine response could be an improved therapeutic strategy against SARS-CoV-2.

CXCR1 drives the pathogenesis of EAE and ARDS via boosting dendritic cells-dependent inflammation

Chemokines secreted by dendritic cells (DCs) play a key role in the regulation of inflammation and autoimmunity through chemokine receptors. However, the role of chemokine receptor CXCR1 in inflammation-inducing experimental autoimmune encephalomyelitis (EAE) and acute respiratory distress syndrome (ARDS) remains largely enigmatic. Here we reported that compared with healthy controls, the level of CXCR1 was aberrantly increased in multiple sclerosis (MS) patients. Knockout of CXCR1 not only ameliorated disease severity in EAE mice but also suppressed the secretion of inflammatory factors (IL-6/IL-12p70) production. We observed the same results in EAE mice with DCs-specific deletion of CXCR1 and antibody neutralization of the ligand CXCL5. Mechanically, we demonstrated a positive feedback loop composed of CXCL5/CXCR1/HIF-1α direct regulating of IL-6/IL-12p70 production in DCs. Meanwhile, we found CXCR1 deficiency in DCs limited IL-6/IL-12p70 production and lung injury in LPS-induced ARDS, a disease model caused by inflammation. Overall, our study reveals CXCR1 governs DCs-mediated inflammation and autoimmune disorders and its potential as a therapeutic target for related diseases.

The Unfolded Protein Response Sensor IRE1 Regulates Activation of In Vitro Differentiated Type 1 Conventional DCs with Viral Stimuli.

Type 1 conventional dendritic cells (cDC1s) are leukocytes competent to coordinate antiviral immunity, and thus, the intracellular mechanisms controlling cDC1 function are a matter of intense research. The unfolded protein response (UPR) sensor IRE1 and its associated transcription factor XBP1s control relevant functional aspects in cDC1s including antigen cross-presentation and survival. However, most studies connecting IRE1 and cDC1 function are undertaken in vivo. Thus, the aim of this work is to elucidate whether IRE1 RNase activity can also be modeled in cDC1s differentiated in vitro and reveal the functional consequences of such activation in cells stimulated with viral components. Our data show that cultures of optimally differentiated cDC1s recapitulate several features of IRE1 activation noticed in in vivo counterparts and identify the viral analog Poly(I:C) as a potent UPR inducer in the lineage. In vitro differentiated cDC1s display constitutive IRE1 RNase activity and hyperactivate IRE1 RNase upon genetic deletion of XBP1s, which regulates production of the proinflammatory cytokines IL-12p40, TNF-α and IL-6, Ifna and Ifnb upon Poly(I:C) stimulation. Our results show that a strict regulation of the IRE1/XBP1s axis regulates cDC1 activation to viral agonists, expanding the scope of this UPR branch in potential DC-based therapies.