IL-2 mRNA Research Articles

Subclinical Doses of Combined Fumonisins and Deoxynivalenol Predispose Clostridium perfringens-Inoculated Broilers to Necrotic Enteritis.

Fumonisins (FB) and deoxynivalenol (DON) are mycotoxins which may predispose broiler chickens to necrotic enteritis (NE). The objective of this study was to identify the effects of subclinical doses of combined FB and DON on NE. A total of 480 day-old male broiler chicks were divided into four treatment groups; 1) control group (basal diet + Clostridium perfringens); 2) necrotic enteritis group (basal diet + Eimeria maxima + C. perfringens); 3) FB + DON group (basal diet + 3 mg/kg FB + 4 mg/kg DON + C. perfringens); and 4) FB + DON + NE group (basal diet + 3 mg/kg FB + 4 mg/kg DON + E. maxima + C. perfringens). Birds in NE and FB + DON + NE groups received 2.5 × 103 E. maxima on day 14. All birds were inoculated with C. perfringens on days 19, 20, and 21. On day 35, birds in the NE, FB + DON, and FB + DON + NE groups had 242, 84, and 339 g lower BWG and a 19-, 2-, and 22-point increase in FCR respectively, than in the control group. Subclinical doses of FB + DON increased (p < 0.05) the NE lesion scores compared to the control group on day 21. On day 21, birds in the NE, FB + DON, and FB + DON + NE groups had increased (p < 0.05) serum FITC-D, lower (p < 0.05) jejunal tight junction protein mRNA, and increased (p < 0.05) cecal tonsil IL-1 mRNA compared to control group. On day 21, birds in the NE group had decreased (p < 0.05) villi height to crypt depth ratio compared to the control group and the presence of FB + DON in NE-induced birds further decreased the villi height to crypt depth ratio. Birds in the NE, FB + DON, and FB + DON + NE groups had increased (p < 0.05) C. perfringens, lower (p < 0.05) Lactobacillus loads in the cecal content, and a lower (p < 0.05) CD8+: CD4+ cell ratio in the cecal tonsils compared to the control group. It can be concluded that subclinical doses of combined FB and DON predispose C. perfringens-inoculated birds to NE, and the presence of FB + DON in NE-induced birds exacerbated the severity of NE.

A Nutritional Blend Suppresses the Inflammatory Response from Bronchial Epithelial Cells Induced by SARS-CoV-2

Even after virus elimination, numerous sequelae of coronavirus disease 2019 (COVID-19) persist. Based on accumulating evidence, large amounts of proinflammatory cytokines are released to drive COVID-19 progression, severity, and mortality, and their levels remain elevated after the acute phase of COVID-19, playing a central role in the disease’ sequelae. In this manner, bronchial epithelial cells are the first cells hyperactivated by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), leading to massive cytokine release, triggering the hyperactivation of leukocytes and other cells, and mediating COVID-19 sequelae. Therefore, proinflammatory cytokine production is initiated by the host. This in vitro study tested the hypothesis that ImmuneRecov™, a nutritional blend, inhibits the SARS-CoV-2-induced hyperactivation of human bronchial epithelial cells (BEAS-2B). BEAS-2B (5x104/mL/well) cells were cocultivated with 1 ml of blood from a SARS-CoV-2-infected patient for 4 h, and the nutritional blend (1 µg/mL) was added in the first minute of coculture. After 4 h, the cells were recovered and used for analyses of cytotoxicity with the (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay and the expression of the IL-1β, IL-6, and IL-10 mRNAs. The supernatant was collected to measure cytokine levels. SARS-CoV-2 incubation resulted in increased levels of IL-1β and IL-6 in BEAS-2B cells (p < 0.001). Treatment with the nutritional blend resulted in reduced levels of the proinflammatory cytokines IL-1β and IL-6 (p < 0.001) and increased levels of the anti-inflammatory cytokine IL-10 (p < 0.001). Additionally, the nutritional blend reduced the expression of the IL-1β and IL-6 mRNAs in SARS-CoV-2-stimulated cells and increased the expression of the IL-10 and IFN-γ mRNAs. In conclusion, the nutritional blend exerts important anti-inflammatory effects on cells in the context of SARS-CoV-2 infection.

β-Sitosterol inhibits ovalbumin-induced asthma-related inflammation by regulating dendritic cells

Aim To investigate the effects of β-sitosterol (B-SIT) and the underlying mechanisms of action in an ovalbumin-induced rat model of asthma. Methods The pathological and morphological changes in lung and tracheal tissues were observed by H&E, PAS, and Masson’s staining. The levels of IgE, TNF-α, and IFN-γ in the bronchoalveolar lavage fluid (BALF) and those of IL-6, TGF-β1, and IL-10 in serum were measured by ELISA. The relative expression levels of IL-5, IL-13, IL-21, CD11c, CD80, and CD86 mRNA in lung tissue were examined by RT-qPCR. Flow cytometry was performed to assess the levels of immune cells, including macrophages and neutrophils in spleen tissue and Th cells, Tc cells, NK cells, and DCs in peripheral blood. The protein expression levels of CD68, MPO, CD11c, CD80, and CD86 were detected by western blotting or immunohistochemistry. Results B-SIT improved the injury in OVA-induced pathology, decreased the levels of inflammatory factors of IgE, TNF-α, IL-6, TGF-β1, IL-5, IL-13, and IL-21 and increased the levels of IFN-γ and IL-10. In addition, B-SIT decreased the number of macrophages and neutrophils and the relative expression levels of CD68 and MPO in the spleen. Moreover, B-SIT increased the number of Th cells, Tc cells, NK cells, and DCs in peripheral blood and upregulated the levels of CD11c, CD80, and CD86 in the spleen and lung. Conclusion B-SIT improved symptoms in a rat model of asthma likely via the inhibition of inflammation by regulating dendritic cells.

Implantation of dedifferentiated fat cells ameliorated antineutrophil cytoplasmic antibody glomerulonephritis by immunosuppression and increases in tumor necrosis factor-stimulated gene-6

IntroductionThe implantation of dedifferentiated fat (DFAT) cells has been shown to exert immunosuppressive effects. To develop DFAT cell therapy for antineutrophil cytoplasmic antibody (ANCA) glomerulonephritis, the effects of the implantation of DFAT cells on ANCA glomerulonephritis were investigated in mice.MethodsPKH26-labeled DFAT cells (105) were infused through the posterior orbital venous plexus to investigate delivery of DFAT cells in ICR mice. DFAT cells (105) were also implanted in SCG mice as a model for ANCA glomerulonephritis. Expression of tumor necrosis factor-stimulated gene-6 (TSG-6) mRNA and protein in kidney was evaluated, and the expression of microRNAs associated with TSG-6 in plasma, lung and kidney was analyzed. Expressions of CD44, prostaglandin (PG) E2, interleukin (IL)-10, IL-1β, tumor necrosis factor (TNF)-α mRNAs, C–C motif chemokine ligand 17 (CCL-17) and monocyte chemoattractant protein (MCP)-1 proteins were measured in kidney from SCG mice implanted with DFAT cells.ResultsAfter their intravenous infusion, almost all DFAT cells were trapped in the lung and not delivered into the kidney. Implantation of DFAT cells in SCG mice suppressed glomerular crescent formation, decreased urinary protein excretions and increased expression of TSG-6 mRNA, protein and immunostaining in kidney from these mice. Increased expression of microRNA 23b-3p in plasma, kidney and lung; decreased expression of CD44 mRNA; and increased expression of PGE2 and IL-10 mRNAs were also observed in kidney from these mice. Implantation of DFAT cells also decreased the expression of TNF-α and MCP-1 proteins and increased that of CCL-17 protein in kidney from the SCG mice. Survival rates were higher in SCG mice implanted with DFAT cells than in SCG mice without implantation.ConclusionMechanisms underlying the effects of improvement of ANCA glomerulonephritis are associated with immunosuppressive effects by TSG-6 and the transition of M1–M2 macrophages, suggesting that implantation of DFAT cells may become a cell therapy for ANCA glomerulonephritis.

Acute sleep deprivation increases inflammation and aggravates heart failure after myocardial infarction.

Sleep disorders have been observed among patients with heart failure. The aim of this study was to investigate whether acute sleep deprivation (SD) aggravates left heart function. Male C57B/L6 mice were assigned to four experimental groups. Ligation of the left anterior descending branch (LAD) caused myocardial infarction (MI) in mice in the LAD group and the LAD+SD group, while mice in the sham and sham+SD groups underwent the same surgery without ligation. Echocardiography was performed before and 8 weeks after ligation of the LAD to evaluate the left ventricular internal diameter at diastole (LVIDd), left ventricular internal diameter at systole (LVIDs), ejection fraction (EF), and fractional shortening (FS). Seven days of sleep deprivation induced using the modified single platform method resulted in a lower EF and FS and a higher LVIDd and LVIDs, as well as increased expression of the IL-1β, IL-18, and IL-10 mRNAs in the left ventricular tissue of MI mice. ELISA also indicated higher levels of IL-1β and IL-10 in the LAD+SD group. It was concluded that acute sleep deprivation induced cardiovascular alterations in cardiac structure and function in HF mice, accompanied by increased levels of inflammatory cytokines.

Kaempferol-3-O-Rhamnoside Inhibits the Proliferation of Jurkat Cells Through Jun Amino-Terminal Kinase Signaling

Background: Indonesian herbal medicine has become target of new drugs against diseases, including cancer. The high incidence and mortality rate of cancer, anticancer resistance, and side effects of chemotherapy contribute to the urgency of researching novel anticancer drugs. A natural product from Schima wallichii Korth., an Indonesian herbal medicine empirically used for many diseases, has shown anticancer activity in MCF-7 and LNCaP cells. Objective: In this study, we investigated the antiproliferative mechanism of the active compound of S. wallichii, kaempferol-3-O-rhamnoside, against Jurkat cells. Methods: Treated cells were analyzed using a proliferation assay and real time-reverse transcriptase polymerase chain reaction for IL-2 mRNA measurement. The mechanism of antiproliferative activity was assesed by western blotting analysis for Mitogen Activated Protein Kinases (MAPKs). Results: Kaempferol-3-O-rhamnoside has an antiproliferative activity at IC50 of 76.3 μM and slightly inhibited IL-2 mRNA expression. The mechanism to inhibit Jurkat cells proliferation was through the stimulation of phosphorylated Jun amino-terminal kinase. Conclusions: The present study observed the molecular mechanism of antiproliferative activity of kaempferol-3-O-rhamnoside.

Distinct Phenotypes of Inflammation Associated Macrophages and Microglia in the Prefrontal Cortex Schizophrenia Compared to Controls.

Approximately 40% of people with schizophrenia are classified as having “high inflammation.” This subgroup has worse neuropathology than patients with “low inflammation.” Thus, one would expect the resident microglia and possibly monocyte-derived macrophages infiltrating from the periphery to be “activated” in those with schizophrenia with elevated neuroinflammation. To test whether microglia and/or macrophages are associated with increased inflammatory signaling in schizophrenia, we measured microglia- and macrophage-associated transcripts in the postmortem dorsolateral prefrontal cortex of 69 controls and 72 people with schizophrenia. Both groups were stratified by neuroinflammatory status based on cortical mRNA levels of cytokines and SERPINA3. We found microglial mRNAs levels were either unchanged (IBA1 and Hexb, p > 0.20) or decreased (CD11c, <62% p < 0.001) in high inflammation schizophrenia compared to controls. Conversely, macrophage CD163 mRNA levels were increased in patients, substantially so in the high inflammation schizophrenia subgroup compared to low inflammation subgroup (>250%, p < 0.0001). In contrast, high inflammation controls did not have elevated CD163 mRNA compared to low inflammation controls (p > 0.05). The pro-inflammatory macrophage marker (CD64 mRNA) was elevated (>160%, all p < 0.05) and more related to CD163 mRNA in the high inflammation schizophrenia subgroup compared to high inflammation controls, while anti-inflammatory macrophage and cytokine markers (CD206 and IL-10 mRNAs) were either unchanged or decreased in schizophrenia. Finally, macrophage recruitment chemokine CCL2 mRNA was increased in schizophrenia (>200%, p < 0.0001) and CCL2 mRNA levels positively correlated with CD163 mRNA (r = 0.46, p < 0.0001). Collectively, our findings support the co-existence of quiescent microglia and increased pro-inflammatory macrophages in the cortex of people with schizophrenia.

Effect of electroacupuncture on pyroptosis of synovial tissues in rats with knee osteoarthritis

To observe the effect of electroacupuncture (EA) on pyroptosis-related proteins in synovium of knee joint in rats with knee osteoarthritis(KOA), in order to explore its mechanism underlying improvement of KOA. Forty male SD rats were randomly divided into control， model, EA and medication groups, with 10 rats in each group. The KOA model was established by injecting 0.2 mL 4% papain solution into the right intra-articular cavity, followed by repeating the injection again on day 4 and 7 after the first injection. After successful modeling, rats of the EA group received EA stimulation of "Neixiyan" (EX-LE4) and "Dubi"(ST35) on the right limb for 15 min, once every day, 6 days per week, for a total of 4 weeks, and those of the medication group received gavage of celecoxib 24 mg/kg, once every day, 6 days per week, for a total of 4 weeks. The severity of dysfunction of the right knee was assessed by using Lequesne's score. Serum interleukin(IL)-1β and IL-18 contents were detected by ELISA. Histopathological changes of the synovium tissue of the right knee joint were observed to give score (synovial pathological score) after H.E. staining. The expression position and intensity of Nod-like receptor pyrin domain 3 (NLRP3) in syno-vial tissue were observed by immunohistochemistry. The expression levels of NLRP3, apoptosis-associated speck-like protein containing card (ASC), Caspase-1, Gasdermin D(GSDMD), IL-1β and IL-18 mRNAs and proteins (including GSDMD-N) in the synovial tissue of the right knee joint were detected by real-time fluorescence quantitative PCR and Western blot, separately. Compared with the control group, the model group had a significant increase in the Lequesne's score, synovial pathological score, serum IL-1β and IL-18 contents, and the expression levels of NLRP3, ASC, Caspase-1, GSDMD, IL-1β, IL-18 mRNAs and proteins and GSDMD-N protein (P<0.01). Whereas relevant to the model group, both the EA and medication groups had marked lower levels of Lequesne's score and synovial pathological score, serum IL-1β and IL-18 contents, and expression levels of NLRP3, ASC, Caspase-1, IL-1β, IL-18 mRNAs and proteins, GSDMD mRNA and GSDMD-N protein (P<0.01, P<0.05). Comparison between two intervention groups showed that the contents of serum IL-1β and IL-18, and the expression levels of IL-1β mRNA and protein were significantly higher in the EA group than in the medication group (P<0.05, P<0.01). No significant differences were found between the EA and medication groups in the Lequesne's score, synovial pathological score, NLRP3, ASC, Caspase-1 and IL-18 mRNAs and proteins, as well as GSDMD mRNA (P>0.05). EA can alleviate the inflammatory response of synovial tissues of knee joints in KOA rats, which may be related to its function in down-regulating the expression levels of synovial NLRP3, ASC, Caspase-1, IL-1β and IL-18 mRNAs and proteins, and GSDMD mRNA and GSDMD-N proteins, reducing the occurrence of pyroptosis.

Serum IL-5, POSTN and IL-33 levels in chronic rhinosinusitis with nasal polyposis correlate with clinical severity

BackgroundChronic rhinosinusitis (CRS) is a group of heterogeneous diseases characterized by epithelial inflammation and tissue eosinophilic infiltration. IL-5, POSTN, and IL-33 are important factors that act as chemoattractants for eosinophils, and a tissue-remodeling protein positively correlated with eosinophils in blood and mediators of eosinophilic infiltration. The aim of the study was to determine the expression of IL-5, POSTN and IL-33, at the gene and protein levels, in eosinophilic CRS with nasal polyps (CRSwNP) and without nasal polyps (CRSsNP), and to correlate this expression with clinical severity.Materials and methodsThe study included 40 CRSwNP patients and 53 CRSsNP patients and 40 control subjects. The expression of IL-5, POSTN and IL-33 mRNA was determined in sinonasal mucosal samples and in nasal polyp tissue by real-time PCR. Protein levels in the serum of CRSwNP patients were measured by ELISA. Computed tomography was evaluated according to Lund–Mackay scores, and visual analog scale scores were assessed.ResultsNP tissue demonstrated significantly higher IL-5 and POSTN mRNA expression than the sinonasal tissue in the CRSsNP and CRSwNP groups. CRS groups demonstrated elevated IL-33 mRNA expression in comparison to controls irrespective of the presence of NP. No correlation was found between IL-5, POSTN and IL-33 mRNA expression and disease severity. CRSwNP group demonstrated significantly higher serum IL-5, POSTN and IL-33 protein levels than controls, and this corresponds to disease severity.ConclusionSerum IL-5, POSTN and IL-33 levels may be important markers for classification of eosinophilic CRSwNP patients, along with disease severity.

Possible association of rotavirus IgG with cytokine expression levels and dyslipidemia in rotavirus-infected type 1 diabetic children

BackgroundRotavirus (RV) has been postulated as a viral trigger for the onset of autoimmune disorders, such as type 1 diabetes (T1D). This study aimed to examine the conceivable association of RV IgG with cytokine levels and dyslipidemia in the pathogenesis of pediatric T1D.MethodsThis study included 30 healthy controls and 80 children with T1D who were divided into two groups based on the time since their T1D diagnosis: newly diagnosed (ND ≤ 1 year; n = 30) and previously diagnosed (PD > 1 year; n = 50). ND and PD patients were also separated into negative and positive according to IgG detection (RV IgG−, ND−, and PD−; RV IgG+, ND+, and PD+).ResultsPositive polymerase chain reaction for RVs was evidenced in 7.5% of children with T1D. Anti-RV IgG was 30% and 36% in ND and PD, respectively, compared to healthy controls (2 of 30, 6.6%; P < 0.05). Fasting blood sugar and hemoglobin A1c significantly increased in PD+ compared to PD−. Interferon-γ and interleukin (IL)-15 levels significantly increased. IL-12 and IL-22 mRNA expression was upregulated in ND+ patients compared to that in ND− patients. IL-37 mRNA expression was significantly downregulated in ND− and ND+ patients compared to that in healthy controls. Total cholesterol and high- and low-density lipoprotein-cholesterol levels were significantly lower in PD+ than in PD−; whereas triglyceride levels were higher than those in healthy controls.ConclusionsThis study suggested that anti-RV IgG may have a role in the pathogenesis, development, and progression of T1D, and RV infections are implicated in dyslipidemia and inflammation status.

Abstract 2036: Multi-omic dissection of immunotherapy response groups in non-small cell lung cancer (NSCLC)

Abstract Immunotherapies have led to long term benefits in a subset of non-small cell lung cancer (NSCLC) patients. Developing predictive biomarkers of response to therapy is currently an unmet clinical need. The composition of the cellular content of the tumour microenvironment (TME) is an important characteristic driving treatment resistance. In this study, we utilised spatial transcriptomics methods, including Nanostring digital spatial profiling (DSP) and multiplex IHC, to define the tumour/stroma (compartment) specific proteome and transcriptome from a cohort of 2nd line immunotherapy treated NSCLC patients. We identified by mIHC that CD68+ macrophages with PD1+, FoxP3+ cells is significantly enriched in immunotherapy resistant tumours (p=0.012). Moreover, IL2 mRNA (p=0.001) in the stromal compartment was significantly increased in patients that were sensitive to ICI therapy, whilst CTLA-4 and IDO-1 were suppressed in responsive patients. Tumour compartment localised CD44 was depleted in the responder group and corresponded inversely with higher stromal expression of one of its ligands, SPP1 (p=0.008). Orthogonal validation of CD44 by multiplex immunofluorescence confirmed both its association with response and localisation to tumour cells rather than immune cell infiltrate. Cox survival analysis showed that tumour compartment CD44 expression was associated with a poorer prognosis (HR=1.61, p=0.01) whilst stromal localisation of CTLA-4 (HR=1.78, p=0.003) and ARG1 (HR=2.37, p=0.01) were associated with poorer outcome. Through a multi-omics approach, we demonstrate the utility of spatial mapping of NSCLC tumours and provide evidence for the role of a number of compartment-specific biomarkers which may aid in identifying those likely to respond to immunotherapy. Citation Format: Arutha Kulasinghe, James Monkman, Honesty Kim, Aaron Mayer, Ahmed Mehdi, Nicholas Matigian, Marie Cumberbatch, Milan Bhagat, Rahul Ladwa, Scott Mueller, Mark Adams, Ken O'Byrne. Multi-omic dissection of immunotherapy response groups in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2036.

Abstract CT183: First-in-human study of MEDI1191 (mRNA encoding IL-12) plus durvalumab in patients (pts) with advanced solid tumors

Abstract Background: IL-12 is a key mediator of antitumor immune response. In preclinical models, IT IL-12 mRNA led to IFNγ release and CD8+ T cell-dependent tumor regression and potentiated PD-L1 blockade. MEDI1191, a lipid nanoparticle-formulated mRNA encoding IL-12 delivered by IT injection, drives IL-12 production and enhances antitumor immune response with improved tolerability. We hypothesized that combining MEDI1191 with PD-L1 blockade would augment antitumor immunity in vivo. Here we report updated results from the dose-escalation phase of the first-in-human study of IT MEDI1191 and IV durvalumab (D; anti-PD-L1) for advanced/metastatic solid tumors (NCT03946800). Methods: In this multicenter, open-label study, MEDI1191 was dosed sequentially (seq, Part 1A) or concurrently (conc, Part 1B) with D. In Part 1A, MEDI1191 was given IT on Days 1 and 22 followed by D 1500 mg on day 43 and then Q4W IV. In Part 1B, MEDI1191 was given IT on Days 1, 29, 57 and then Q8W, along with D on Day 1 and then Q4W. Treatment continued until progression or unacceptable toxicity for up to 2 years. Eligible adult pts had any solid tumor with cutaneous or subcutaneous lesions suitable for IT injection and progression on standard therapy for recurrent/metastatic disease. Primary objectives were safety and tolerability and determination of maximum tolerated dose (MTD); secondary objectives included preliminary antitumor activity by RECIST v1.1. Results: Starting in May 2019, 31 pts received seq MEDI1191 with D (Part 1A cohorts 0.1-12 μg; n=20) or conc MEDI1191 with D (Part 1B cohorts 1.0-3.0 μg; n=11); 23 pts had received prior anti-PD-1/PD-L1 therapy. Most common tumor types were melanoma, n=8; head and neck cancer, n=4; and breast cancer, n=4. At the data cutoff of Dec 7, 2021, there were no dose-limiting toxicities and no MTD was identified. One pt (3.2%) had a Gr ≥3 MEDI1191-related AE (Gr 3 pyrexia, resolved within 24 hr) and 1 (3.2%) had a MEDI1191-related serious AE (SAE; Gr 2 confusion). Two pts had Gr 3 D-related AEs (6.5%, pyrexia also related to MEDI1191 and pruritus; each n=1); none had a D-related SAE. There were no Gr 4 related AEs. 3 pts had partial responses (PR): 1 with head and neck cancer (unconfirmed, off study) and 2 with anti-PD-1 resistant melanoma (1 confirmed, &amp;gt;12 months on treatment; 1 unconfirmed, off study); 10 pts had stable disease (including the 2 unconfirmed PRs). Among pts with available biomarker data (up to 3 μg in Part 1A and 1 μg in Part 1B), MEDI1191 increased serum IL-12 in 15/17 pts, increased CD8+ T cell tumor infiltration by &amp;gt;2-fold in 8/14 pts and increased tumor PD-L1 expression in 6/14 pts. Conclusions: IT MEDI1191 plus systemic anti-PD-L1 was safe and feasible. Preliminary antitumor efficacy and pharmacodynamics, including tumor CD8+ T cell recruitment, were consistent with expected mechanism of action. Pts with injectable deep visceral and superficial lesions are being recruited. Citation Format: Benedito A. Carneiro, Dmitriy Zamarin, Thomas Marron, Inderjit Mehmi, Sandip P. Patel, Vivek Subbiah, Anthony El-Khoueiry, David Grand, Kirema Garcia-Reyes, Sanjay Goel, Phillip Martin, Jixin Wang, Yuling Wu, Steven Eck, Benjamin Ridgway, Nairouz Elgeioushi, Jim Eyles, Nicholas Durham, Analia Azaro, Omid Hamid. First-in-human study of MEDI1191 (mRNA encoding IL-12) plus durvalumab in patients (pts) with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT183.

Influence of fermented feed additive on gut morphology, immune status, and microbiota in broilers

BackgroundThis study examined the effects of a solid-state fermented feed additive (FFA) on the small intestine histology/morphology, immunity and microbiota of broilers. Two hundred eighty-eight day-old Arbor Acre chicks, were randomly assigned to one of four groups (each group has 6 replicates, with each replicate containing 12 chickens). The negative control (NC; basal diet), the positive control (PC; basal diet +antibiotic 15 ppm), the fermented feed additive low dose (FFL; basal diet + 0.3 kg/t FFA), and the fermented feed additive high dose (FFH; 3 kg/t FFA) with Lactobacillus casei (L.casei).ResultsThe study found that the FFH and FFL groups gained more weight (1-21d) and the FFL and PC diets had better feed conversion ratio (P < 0.05) than the NC from 0-42d. The FFH group had higher villus height (P < 0.05) in the duodenum than the PC and villus height to crypt depth ratio VH/CD compared to PC and FFL groups. The FFL chickens had greater (P < 0.05) jejunal and ileal villus height than PC and NC groups respectively. The FFL group had a higher ileal VH/CD ratio (P < 0.05). Jejunum VH/CD was higher in FFL and FFH (P < 0.05) than PC (P < 0.05). FFH had a smaller thymus than NC (P < 0.05). FFA diets also increased IL-10 expression (P < 0.05). While IL-1 and TLR4 mRNA expression decreased (P < 0.05) compared to NC. The microbiota analysis showed that the microorganisms that have pathogenic properties such as phylum Delsulfobacterota and class Desulfovibriona and Negativicutes was also significantly reduced in the group treated with FFH and PC while microorganisms having beneficial properties like Lactobacillaceae family, Lactobacillus aviarus genus and Lactobacillus spp were also tended to increase in the FFH and FFL fermented feed groups compared to the PC and NC groups.ConclusionThese findings suggested that the FFA diet may modulate cecal microbiota by reducing pathogenic microorganisms such as phylum Delsulfobacterota and class Desulfovibriona and Negativicutes improve beneficial microorganisms like Lactobacillaceae family, Lactobacillus aviarus genus and Lactobacillus spp. While FFA diet also affect immunity, and gene expression related to immunity.

Enhanced production of IL-2 from anti-CD3 antibody-stimulated mouse spleen cells by artepillin C, a major component of Brazilian green propolis

ObjectivesIn this report, we attempt to clarify the immune modulatory effects of Brazilian green propolis (BGP) and its major component, artepillin C, on the cytokine production of anti-CD3 antibody-stimulated mouse spleen cells. We also estimate the physiological mechanism affecting artepillin C's upon the cells. MethodsMale C3H/HeN mouse spleen cells stimulated by antiCD3 monoclonal antibody were co-cultured with BGP, artepillin C, and HC030031, a transient receptor potential ankyrin 1 (TRPA1) Ca2+ channel antagonist. The synthesis of interferon (IFN)-γ, interleukin (IL)-6, IL-17, IL-4, IL-10, and IL-2 was assayed by enzyme-linked immunoassay. The expression of IL-2 mRNA and the protein product were examined by reverse transcription-quantitative polymerase chain reaction and Western blot analyses, respectively. ResultsThe production of IL-2 was markedly enhanced, while that of IL-4 and IL-10 was not significantly affected; by contrast, the production of IFN-γ, IL-6, and IL-17 was significantly reduced in the antibody-stimulated spleen cells treated with BGP at a non-cytostatic concentration. These effects were reproduced in the cells treated with artepillin C. The expression of IL-2 mRNA was unaffected; however, that of the protein was significantly enhanced in the artepillin C-treated cells compared to untreated control cells. The enhancement of protein expression and the production of IL-2 by artepillin C was significantly alleviated by adding HC030031. ConclusionsArtepillin C is an important regulator of cytokine synthesis from activated spleen cells. The agent specifically augmented the expression of IL-2 via the Ca2+-permeable cation channel, TRPA1, at least in part, at the translational or secretion levels.

OX40 Expression in Eosinophils Aggravates OVA-Induced Eosinophilic Gastroenteritis.

Background & AimsEosinophils are the main inflammatory effector cells that damage gastrointestinal tissue in eosinophilic gastrointestinal diseases (EGIDs). Activation of the OX40 pathway aggravates allergic diseases, such as asthma, but it is not clear whether OX40 is expressed in eosinophils to regulate inflammation in EGIDs. In this study, we assessed the expression and effect of OX40 on eosinophils in WT and Ox40-/- eosinophilic gastroenteritis (EGE) mice.MethodsEosinophil infiltration, ovalbumin (OVA)-specific Ig production, OX40 expression and inflammatory factor levels in the intestine and bone marrow (BM) were investigated to evaluate inflammation.ResultsWe confirmed that OVA-challenged mice produced high levels of Ox40, Mbp, Ccl11, Il5, Il4, Il13, and Il6 mRNA and a low level of Ifng mRNA in the intestine. Increased eosinophils were observed in intestinal and lymph tissues, accompanied by significantly upregulated OX40 and Type 2 cytokine production in eosinophils of EGE mice. Ox40 deficiency ameliorated OVA-induced inflammation, eosinophil infiltration, and cytokine production in the intestine. Consistently, Ox40-/ - eosinophils exhibited decreased proliferation and proinflammatory function. The stimulation of the agonistic anti-OX40 antibody, OX86, promoted the effect of OX40 on eosinophils. The present study also showed that Ox40 deficiency dampened the Traf2/6-related NF-κB signaling pathway in eosinophils.ConclusionsOX40 may play a critical role in the progress of OVA-induced EGE by promoting the maturation and function of eosinophils via the Traf2/6-related NF-κB signaling pathway.

Multi-omic and spatial dissection of immunotherapy response groups in non–small cell lung cancer (NSCLC).

8544 Background: Immune checkpoint inhibitors (ICI) have shown durable benefit in a subset of non-small cell lung cancer (NSCLC) patients. The composition of the tumour microenvironment (TME) is becomingly increasingly recognised as an important factor to predict response to therapy. Methods: Here, we applied digital spatial profiling of the tumour and stromal compartments from a 2nd line NSCLC ICI-treated cohort (n = 41 patient) and standard of care (SOC), platinum treated NSCLC cohort (n = 47), to identify tissue-based signatures of response to therapy. Results: We demonstrate by mIHC that the interaction of CD68+ macrophages with PD1+, FoxP3+ cells is significantly enriched in ICI refractory tumours (p = 0.012). Patients sensitive to ICI therapy expressed higher levels of IL2 receptor alpha (CD25, p = 0.028) within the tumour compartments, which corresponded with the increased expression of IL2 mRNA (p = 0.001) within their stroma. Immuno-inhibitory markers CTLA-4 (p = 0.021) and IDO-1 (p = 0.023) were supressed in ICI-responsive patients. Tumour CD44 (p = 0.02) was depleted in the response group and corresponded inversely with significantly higher stromal expression of one of its ligands, SPP1 (osteopontin, p = 0.008). Analysis of dysregulated transcripts indicated the potential inhibition of stromal interferon-gamma (IFNγ) activity, estrogen-receptor and Wnt-1 signalling activity within the tumour cells of ICI responsive patients. Cox survival analysis indicated tumour CD44 expression was associated with poorer prognosis (HR = 1.61, p = 0.01), consistent with its depletion in ICI sensitive patients. Similarly, stromal CTLA-4 (HR = 1.78, p = 0.003) and MDSC/M2 macrophage marker ARG1 (HR = 2.37, p = 0.01) were associated with poorer outcome while BAD (HR = 0.5, p = 0.01) appeared protective. The SOC cohort paralleled similar roles for immune checkpoints and pro-apoptotic markers, with LAG3 (HR = 3.81, p = 0.04) indicating poorer outcome, and BIM (HR = 0.16, p = 0.014) with improved outcome. Interestingly, stromal mRNA for E-selectin (HR = 652, p = 0.001), CCL17 (HR = 70, p = 0.006) and MTOR (HR = 1065, p = 0.008) were highly associated with poorer outcome in ICI treated patients, indicating pro-tumourigenic features in the tumour microenvironment that may facilitate ICI resistance. Conclusions: Through multi-modal approaches, we have dissected the characteristics of NSCLC treatment groups and provide evidence for the role of several markers including IL2, CD25, CD44 and SPP1 in the efficacy of current generations of ICI therapy.

The effects of dietary supplementation of α-lipoic acid on the growth performance, antioxidant capacity, immune response, and disease resistance of northern snakehead, Channa argus

The study was the first time to explore the positive effects of α-LA on growth performance, antioxidant capability, immunity, and disease resistance of northern snakehead (Channa argus). Five hundred and forty northern snakehead fish (initial body weight: 8.74 ± 0.12 g (mean ± SE)) were randomly allocated into six groups with three replicates each. Six diets supplemented with α-LA at doses of 0 (CON), 300 (LA300), 600 (LA600), 900 (LA900), 1200 (LA1200), and 1500 (LA1500) mg/kg were fed to northern snakehead for 8 weeks. The results demonstrated that, when compared with the control group, optimal dietary α-LA increased the weight gain (WG), protein efficiency ratio (PER), and specific growth rate (SGR) and reduced the feed conversion ratio (FCR) of the fish (P < 0.05). Also, optimal dietary α-LA enhanced the immune-related parameters and antioxidant enzyme parameters levels in the head kidney, spleen, and liver of northern snakehead (P < 0.05). Dietary α-LA upregulated the mRNA expression levels of anti-inflammatory cytokines (il10 and tgfβ) and antioxidant related genes (gst, gsh-px, gr and Cu/Zn sod), down-regulated the pro-inflammatory cytokines (il1β, il8, il12 and tnfα) mRNA levels in the liver, spleen and head kidney of the northern snakehead (P < 0.05). The above results demonstrated that optimal dietary α-LA showed enhancement effects on the growth, antioxidant and anti-inflammatory capability, and immune response of northern snakehead. The survival rates in all α-LA treatments were significantly raised after the challenge with Aeromonas veronii (P < 0.05). Based on the quadratic regression analysis of WG, GSH-Px, LYS, and il1β, the optimal dietary α-LA levels were estimated to be 737.0, 775.0, 890.0, and 916.7 mg/kg, respectively. Considering the overall responses in growth performance, antioxidant status, immune response, and inflammatory factors, the recommended dose of α‐LA in the diet of fish is 737.0–916.7 mg/kg.

Ripe Tomato Saponin Esculeoside A and Sapogenol Esculeogenin A Suppress CD4+ T Lymphocyte Activation by Modulation of Th2/Th1/Treg Differentiation.

We report that esculeoside A (EsA), a glycoside and a major component in ripe tomato fruit, ameliorated experimental dermatitis in mice. However, the underlying immunologic molecular mechanisms are unknown. The present study examined its underlying immune nutrition mechanism using concanavalin A (ConA)-blast mouse splenocyte primary culture. We found that EsA and its sapogenol esculeogenin A (Esg-A) concentration-dependently suppressed T-lymphoproliferation using CFSE-labeled flow-cytometry and water-soluble tetrazolium (WST) assay. Using ELISA and q-PCR methods, EsA/Esg-A showed profound decreases in T helper 2 (Th2)-relevant interleukin-4 (IL-4) secretion and mRNA expression, and GATA3 expression. Moreover, EsA/Esg-A suppressed CD4+ T-lymphocyte activation by decreasing IL-2 secretion and mRNA expression and CD25+ cell proportion. Further, EsA/Esg-A alleviated Treg suppressive activity by reducing IL-10 secretion, Foxp3 mRNA expression, and cell numbers. We suggest the immune nutrition function by tomato component, and highlight that EsA/Esg-A are capable of reducing CD4+ T-lymphocyte activation via a reduction in Th2-lymphocyte activity by modulation of Th2/Th1/Treg subunit differentiation.

Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy.

Cytokines are important immunotherapeutics with approved drugs for the treatment of human cancers. However, systemic administration of cytokines often fails to achieve adequate concentrations to immune cells in tumors due to dose-limiting toxicity. Thus, developing localized therapy that directly delivers immune-stimulatory cytokines to tumors may improve the therapeutic efficacy. In this study, we generated novel lipid nanoparticles (LNPs) encapsulated with mRNAs encoding cytokines including IL-12, IL-27 and GM-CSF, and tested their anti-tumor activity. We first synthesized ionizable lipid materials containing di-amino groups with various head groups (DALs). The novel DAL4-LNP effectively delivered different mRNAs in vitro to tumor cells and in vivo to tumors. Intratumoral injection of DAL4-LNP loaded with IL-12 mRNA was most potent in inhibiting B16F10 melanoma tumor growth compared to IL-27 or GM-CSF mRNAs in monotherapy. Furthermore, intratumoral injection of dual DAL4-LNP-IL-12 mRNA and IL-27 mRNA showed a synergistic effect in suppressing tumor growth without causing systematic toxicity. Most importantly, intratumoral delivery of IL-12 and IL-27 mRNAs induced robust infiltration of immune effector cells, including IFN-γ and TNF-α producing NK and CD8+ T cells into tumors. Thus, intratumoral administration of DAL-LNP loaded with IL-12 and IL-27 mRNA provides a new treatment strategy for cancer.

STING is an intrinsic checkpoint inhibitor that restrains the TH17 cell pathogenic program.

SUMMARYExternal and intrinsic factors regulate the transcriptional profile of T helper 17 (TH17) cells, thereby affecting their pathogenic potential and revealing their context-dependent plasticity. The stimulator of interferon genes (STING), a component of the intracellular DNA-sensing pathway, triggers immune responses but remains largely unexplored in T cells. Here, we describe an intrinsic role of STING in limiting the TH17 cell pathogenic program. We demonstrate that non-pathogenic TH17 cells express higher levels of STING than those activated under pathogenic conditions. Activation of STING induces interleukin-10 (IL-10) production in TH17 cells, decreasing IL-17A and IL-23R expression in a type I interferon (IFN)-independent manner. Mechanistically, STING-induced IL-10 production partially requires aryl hydrocarbon receptor (AhR) signaling, while the decrease of IL-17A expression occurs due to a reduction of Rorγt transcriptional activity. Our findings reveal a regulatory function of STING in the TH17 cell activation program, proposing it as a valuable target to limit TH17-cell-mediated inflammation.