Mouse Interleukin Research Articles

Epigenetic mechanisms of Nsd1-mediated histone methylation modifications in chondrocyte ferroptosis in knee osteoarthritis.

Knee osteoarthritis (KOA) is a degenerative joint disease characterized by pain, stiffness, and impaired mobility, with current therapies offering limited efficacy. This study investigates the epigenetic role of nuclear receptor-binding SET domain protein 1 (NSD1) in KOA pathogenesis. A KOA mouse model was established, and adenoviral vectors were employed to upregulate Nsd1 and inhibit SRY-box transcription factor 9 (Sox9), followed by histopathological assessments. We examined changes in cell morphology, proliferation, viability, and ferroptosis-related markers. The expression of Nsd1, Sox9, and acyl-CoA synthetase long-chain family member 4 (Acsl4) was analyzed, along with the enrichment of Nsd1 and dimethylated lysine 36 of histone 3 (H3K36me2) on the Sox9 promoter and Sox9 on the Acsl4 promoter. Additionally, the binding relationship between Sox9 and the Acsl4 promoter sequence was analyzed. Our results revealed that Nsd1 expression was reduced in KOA mouse tissues and interleukin (IL)-1β-stimulated chondrocytes. Nsd1 upregulation alleviated KOA, promoted chondrocyte proliferation and viability, and inhibited ferroptosis. Mechanistically, Nsd1 enhanced H3K36me2 to upregulate Sox9 expression, which in turn suppressed Acsl4 expression and ferroptosis. Sox9 inhibition partially reversed the protective effect of Nsd1 overexpression. In summary, Nsd1 upregulation mitigates chondrocyte ferroptosis and ameliorates KOA by modulating H3K36me2 to upregulate Sox9 and downregulate Acsl4 expression.

Oncolytic Tanapoxvirus Variants Expressing mIL-2 and mCCL-2 Regress Human Pancreatic Cancer Xenografts in Nude Mice.

Pancreatic ductal adenocarcinoma (PDAC) is the fifth leading cause of cancer-related death and presents the lowest 5-year survival rate of any form of cancer in the US. Only 20% of PDAC patients are suitable for surgical resection and adjuvant chemotherapy, which remains the only curative treatment. Chemotherapeutic and gene therapy treatments are associated with adverse effects and lack specificity/efficacy. In this study, we assess the oncolytic potential of immuno-oncolytic tanapoxvirus (TPV) recombinants expressing mouse monocyte chemoattractant protein (mMCP-1 or mCCL2) and mouse interleukin (mIL)-2 in human pancreatic BxPc-3 cells using immunocompromised and CD-3+ T-cell-reconstituted mice. Intratumoral treatment with TPV/∆66R/mCCL2 and TPV/∆66R/mIL-2 resulted in a regression in BxPc-3 xenograft volume compared to control in immunocompromised mice; mCCL-2 expressing TPV OV resulted in a significant difference from control at p < 0.05. Histological analysis of immunocompromised mice treated with TPV/∆66R/mCCL2 or TPV/∆66R/mIL-2 demonstrated multiple biomarkers indicative of increased severity of chronic, active inflammation compared to controls. In conclusion, TPV recombinants expressing mCCL2 and mIL-2 demonstrated a therapeutic effect via regression in BxPc-3 tumor xenografts. Considering the enhanced oncolytic potency of TPV recombinants demonstrated against PDAC in this study, further investigation as an alternative or combination treatment option for human PDAC may be warranted.

Medicinal evaluation and molecular docking study of osajin as an anti-inflammatory, antioxidant, and antiapoptotic agent against sepsis-associated acute kidney injury in rats

Despite efforts to find effective drugs for sepsis-associated acute kidney injury (SA-AKI), mortality rates in patients with SA-AKI have not decreased. Our study evaluated the protective effects of isoflavone osajin (OSJ) on SA-AKI in rats by targeting inflammation, oxidative stress, and apoptosis, which represent the cornerstones in the pathophysiological mechanism of SA-AKI. Polymicrobial sepsis was induced in rats via the cecal ligation and puncture (CLP) technique. Markers of oxidative stress were evaluated in kidney tissues using biochemical methods. The expression of interleukin-33 (IL-33), 8-hydroxydeoxyguanosine (8-OHdG), caspase-3, and kidney injury molecule-1 (KIM-1) was evaluated as indicators of inflammation, DNA damage, apoptosis, and SA-AKI respectively in the kidney tissues using immunohistochemical and immunofluorescent detection methods. The CLP technique significantly (p < 0.001) increased lipid peroxidation (LPO) levels and significantly (p < 0.001) decreased the activities of superoxide dismutase and catalase in kidney tissues. In the renal tissues, strong expression of IL-33, 8-OHdG, caspase-3, and KIM-1 was observed with severe degeneration and necrosis in the tubular epithelium and intense interstitial nephritis. In contrast, the administration of OSJ significantly (p < 0.001) reduced the level of LPO, markedly improved biomarkers of antioxidant status, decreased the levels of serum creatinine and urea, lowered the expression of IL-33, 8-OHdG, caspase-3, and KIM-1 and alleviated changes in renal histopathology. A promising binding score was found via a molecular docking investigation of the OSJ-binding mode with mouse IL-33 (PDB Code: 5VI4). Therefore, OSJ protects against SA-AKI by suppressing the IL-33/LPO/8-OHdG/caspase-3 pathway and improving the antioxidant system.

An antibody to IL-1 receptor 7 protects mice from LPS-induced tissue and systemic inflammation.

Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the IL-1 Family, is a key mediator ofautoinflammatory diseases associated with the development of macrophage activation syndrome (MAS).High levels of IL-18 correlate with MAS and COVID-19 severity and mortality, particularly in COVID-19patients with MAS. As an inflammation inducer, IL-18 binds its receptor IL-1 Receptor 5 (IL-1R5), leadingto the recruitment of the co-receptor, IL-1 Receptor 7 (IL-1R7). This heterotrimeric complex subsequentlyinitiates downstream signaling, resulting in local and systemic inflammation. We reported earlier the development of a novel humanized monoclonal anti-human IL-1R7 antibody whichspecifically blocks the activity of human IL-18 and its inflammatory signaling in human cell and wholeblood cultures. In the current study, we further explored the strategy of blocking IL-1R7 inhyperinflammation in vivo using animal models. We first identified an anti-mouse IL-1R7 antibody that significantly suppressed mouse IL-18 andlipopolysaccharide (LPS)-induced IFNg production in mouse splenocyte and peritoneal cell cultures. Whenapplied in vivo, the antibody reduced Propionibacterium acnes and LPS-induced liver injury and protectedmice from tissue and systemic hyperinflammation. Importantly, anti-IL-1R7 significantly inhibited plasma,liver cell and spleen cell IFNg production. Also, anti-IL-1R7 downregulated plasma TNFa, IL-6, IL-1b,MIP-2 production and the production of the liver enzyme ALT. In parallel, anti-IL-1R7 suppressed LPSinducedinflammatory cell infiltration in lungs and inhibited the subsequent IFNg production andinflammation in mice when assessed using an acute lung injury model. Altogether, our data suggest that blocking IL-1R7 represents a potential therapeutic strategy to specificallymodulate IL-18-mediated hyperinflammation, warranting further investigation of its clinical application intreating IL-18-mediated diseases, including MAS and COVID-19.

Oncolytic Tanapoxvirus Recombinants Expressing Flagellin C or Mouse Interleukin-2 Are Capable of Regressing Human Triple-Negative Breast Cancer Xenografts in Immuno-Competent BALB/c Nude Mice.

Triple-negative breast cancer (TNBC) in humans is the most aggressive and deadly form of BC. Although TNBCs are about 15 percent of the total number of BC cases, they are associated with the highest mortalities. Current treatment options are limited, and most modalities are toxic and have not increased the 5-year survival rates of TNBC. Many oncolytic viruses are emerging as potential therapies for TNBC. In this study, two Tanapoxvirus (TPV) recombinants, one expressing FliC and the other expressing mouse interleukin-2 (mIL-2), were assessed for their efficacy in an immuno-competent xenograft mouse model. MDA-MB-231 tumors were planted in BALB/c nude mice, treated, made immuno-competent via adoptive transfer of splenocytes from healthy BALB/c donors, and then monitored for 40 days. TPV/Δ2L/66R/FliC and TPV/Δ66R/mIL-2 demonstrated significant tumor reduction (p = 0.01602 and p = 0.03890, respectively) compared to the reconstituted control (RC), whereas wtTPV did not. Pathological analyses of treated tumors revealed cells consistent with lymphocyte and plasma cell morphology in reconstituted mice treated with TPV recombinants. Anti-viral plaque reduction assays conducted using harvested serum from treated animals indicated the presence of anti-TPV antibodies in mice reconstituted and treated with TPV that were missing from immune-deficient nude mice, including those exposed to TPV and of statistically equivalent serum concentrations to normal BALB/c mice immunized against TPV. The results suggest immuno-deficient BALB/c nude mice can become immuno-competent via adoptive transfer of splenocytes from genetically identical donors and allow for testing of tumor xenografts in a competent model system. The TPV recombinants tested should be further studied for the potential treatment of human TNBC.

Development of Recombinant Oncolytic rVSV-mIL12-mGMCSF for Cancer Immunotherapy.

Anti-cancer therapy based on oncolytic viruses (OVs) is a targeted approach that takes advantage of OVs' ability to selectively infect and replicate in tumor cells, activate the host immune response, and destroy malignant cells over healthy ones. Vesicular stomatitis virus (VSV) is known for its wide range of advantages: a lack of pre-existing immunity, a genome that is easily amenable to manipulation, and rapid growth to high titers in a broad range of cell lines, to name a few. VSV-induced tumor immunity can be enhanced by the delivery of immunostimulatory cytokines. The targeted cytokine delivery to tumors avoids the significant toxicity associated with systemic delivery while also boosting the immune response. To demonstrate this enhanced effect on both tumor growth and survival, a novel recombinant VSV (rVSV)-mIL12-mGMCSF, co-expressing mouse IL-12 (interleukin-12) and GM-CSF (granulocyte-macrophage colony-stimulating factor), was tested alongside rVSV-dM51-GFP (rVSV-GFP) that was injected intratumorally in a syngeneic in vivo C57BL/6 mouse model infused subcutaneously with B16-F10 melanoma cells. The pilot study tested the effect of two viral injections 4 days apart and demonstrated that treatment with the two rVSVs resulted in partial inhibition of tumor growth (TGII of around 40%) and an increased survival rate in animals from the treatment groups. The effect of the two VSVs on immune cell populations will be investigated in future in vivo studies with an optimized experimental design with multiple higher viral doses, as a lack of this information presents a limitation of this study.

Over-expression of IL-33 enhances rabies virus early antigen presentations and cellular immune responses in mice

Human inactivated rabies virus (RABV) vaccines have been widely used worldwide over 30 years. The mechanisms of humoral immunity elicited by previously reported rabies candidate vaccines have been fully investigated, but little is known about the cellular immunity profiles. Herein, the recombinant RABV rLBNSE-IL-33 overexpressing the mouse interleukin-33 (IL-33) proliferated well in Neuro-2a cells and had no effects with the parent virus on growth kinetic in vitro and viral pathogenicity in mice. The rLBNSE-IL-33 experienced more antigen presentations by MHC-II on DCs and activated more CD4+ T cells which helped recruit more CD19+CD40+ B cells in blood and promote rapid and robust IgG1 antibodies responses at initial infection stage compared with the parent rLBNSE strain. Simultaneously, the rLBNSE-IL-33 were also presented by MHC-I to CD8+ T cells which contributed to produce high levels of IgG2a. The rLBNSE-IL-33 elicited significantly high levels of RABV-specific IFN-γ secreting memory CD4+ T cells, more RABV-specific IL-4 and IFN-γ secreting memory CD8+ T cells in spleens at early infection stage in mice. Altogether, overexpression of IL-33 in rLBNSE-IL-33 enhanced early antigen presentation, markedly promote CD4+, memory CD4+ and CD8+ T cells-mediated responses and provided a 100 % protection from lethal RABV challenge in mice. These findings provided an alternative novel therapy and vaccine strategy in future.

Aminooxy acetic acid suppresses Th17-mediated psoriasis-like skin inflammation by inhibiting serine metabolism.

Background: Psoriasis is a common chronic inflammatory skin disease characterized by an external red rash that is caused by abnormal proliferation and differentiation of keratinocytes and immune T cells. This study aimed to elucidate the role of aminooxy acetic acid (AOA) in alleviating psoriasis from the perspective of immunology and metabolomics. Therefore, contributing to the development of new drugs as candidates for psoriasis treatment. Methods: To investigate the symptom-alleviating effects and the related mechanisms of AOA on the treatment of psoriasis, we used a 12-O-tetradecanoylphorbol-13-acetate-induced psoriasis-like skin mouse model and interleukin (IL)-17-stimulated human keratinocytes. Results: The results showed that AOA ameliorated psoriasis-related symptoms and decreased inflammation-associated antimicrobial peptides and T-helper 17 (Th17)-associated cytokines in a mouse model of psoriasis. Furthermore, AOA inhibited the activation of mechanistic target of rapamycin (mTOR) by suppressing serine metabolism-related genes. Importantly, mTOR inhibition ameliorated psoriatic disease by affecting the differentiation of various T cells and normalizing the Th17/regulatory T (Treg) cell balance. In addition, IL-17-stimulated human keratinocytes showed the same results as in the in vivo experiments. Conclusion: Taken together, these results suggest that targeting the serine metabolism pathway in the treatment of psoriasis is a novel strategy, and that AOA could be utilized as a novel biologic to treat psoriasis.

Multiple Administration Routes, Including Intramuscular Injection, of Oncolytic Tanapoxvirus Variants Significantly Regress Human Melanoma Xenografts in BALB/c Nude Mice Reconstituted with Splenocytes from Normal BALB/c Donors.

Human melanoma is the most aggressive form of skin cancer and is responsible for the most deaths of all skin cancers. Localized tumors, and those which have limited spread, have 5-year survival rates of over 90%, with those numbers steadily rising over the past decade. However, metastatic melanomas have a sharp decrease in 5-year survival rates and are still an area of need for new, successful therapies. Immuno-oncolytic viruses (OVs) have emerged as a promising class of immunovirotherapy that can potentially address this disease. The Food and Drug Administration in the United States has approved one oncolytic herpes simplex virus expressing granulocyte-macrophage colony-stimulating factor (Talimogene Laherparepvec) for the treatment of metastatic melanoma, and others could soon follow for this and other cancers. In previous studies, Tanapoxvirus (TPV) recombinants expressing mouse interleukin-2 (mIL-2) and another expressing bacterial flagellin from Salmonella typhimurium (FliC) have demonstrated anti-tumor efficacy in nude mouse models. TPV replicates only in humans and monkeys, including tumor cells, which makes the use of syngeneic tumor models impossible for the study of this OV in a standard immunocompetent system. In this study, TPV/Δ66R/mIL-2 and TPV/Δ2L/Δ66R/FliC were tested for their ability to treat human melanoma xenografts (SK-MEL3) in a BALB/c nude mouse model reconstituted with splenocytes from genetically compatible, normal BALB/c donor mice. Two SK-MEL3 tumors were transplanted into each flank of BALB/c nude mice, and the larger tumor was treated intratumorally (IT) with virus or mock injection. In one set of animals, mice received adoptive transfers of splenocytes from BALB/c mice on day 4 to reconstitute their immune systems and allow for adaptive immune responses to occur in a xenograft model. Direct IT injection of TPV/Δ66R/mIL-2 led to significantly greater rates of tumor regression compared to reconstituted control (RC) mice in the primary and distant tumor sites, whereas TPV/Δ2L/Δ66R/FliC treatment led to significantly greater rates of tumor regression in distant tumor sites only. A second experiment used TPV/Δ66R/mIL-2 to test whether TPV could be administered intravenously (IV), intramuscularly (IM), or both routes simultaneously to exert similar anti-tumor effects in an indirectly treated tumor. A single SK-MEL3 tumor was transplanted into one flank of BALB/c nude mice and was treated either into the tail vein, the nearest rear leg to the tumor, or both. All mice then received adoptive transfers of splenocytes in the same way as previously described on day 4 and received an additional TPV treatment on day 14. The results demonstrated that TPV/Δ66R/mIL-2 treatment IV or IM had significantly greater rates of tumor regression than RC-treated mice but failed to exert this effect when both routes were used simultaneously. Data obtained through these experiments support the continued development of Tanapoxvirus for the treatment of human melanoma and using immune reconstitution to create intact adaptive immunity in a xenograft context, which can allow other tropism-limited OVs to be studied against human cancers.

Distinct hypoxic molecular mechanisms differentially regulate human vs.mouse Interleukin 1β gene expression in monocytes/macrophages

Abstract Interleukin 1β (IL-1β) is an important cytokine, whose regulation is distinct from that of Tumor Necrosis Factor (TNF). We previously reported that LPS/TLR4 initiates hypoxia in mice, with HIF-1α binding the murine Il1b gene, suggesting that TLR4 directly activates Trained Innate Immune Memory (TIIM). To address whether human monocyte IL1B gene regulation parallels that of mouse, mRNA and chromatin immunoprecipitation (ChIP) kinetics, with small molecule inhibitors under either normoxia or hypoxia, were evaluated. IL1B transcription kinetics in Lipopolysaccharide (LPS)-treated human THP-1 monocytes revealed an Immediate-Early (IE) phase of IL1B gene induction, requiring Spi1/PU.1, NF-κB and C/EBPβ transcription factors (TF), followed by a late-phase of lower-level continuous non-tolerized TIIM expression that depends upon hypoxia-inducible TFs. We observed that Spi1/PU.1 is required for IL1B gene transcription, performing a critical role in inducing the promoter for IE expression via a TAF1-independent mechanism. Late HIF-2α-dependent IL1B expression is due to LPS pseudo-hypoxic stabilization of the labile HIF factor. In contrast to IE activation, late-phase transcription revealed minimal dependence on NF-κB due to human-specific competition for an overlapping NF-κB/HIF enhancer element. Our studies reveal that IL1B in naïve THP-1 human monocytes binds HIF-2α, not HIF-1α, while PMA treatment morphologically polarizes THP-1 macrophages to bind both HIF isoforms at IL1B. Additionally, bioinformatic screening revealed other immunologically important genes regulated by similar mechanisms, providing insight for novel therapeutic targeting of transcription factors other than NF-κB. Interleukin Foundation, Denver Colorado

Extraction of Extracelluar Vesicles Derived from Mycobacterium tuberculosis and Their Effect on the Production of Reactive Oxygen Species and Expression of Inflammatory Factors in Mouse Bone Marrow-Derived Dendritic Cells

To isolate extracellular vesicles (EVs) from Mycobacterium tuberculosis ( Mtb), to examine their morphology, particle size, and distribution, to study the effect of EVs derived from Mtb ( Mtb-EVs) on intracellular reactive oxygen species (ROS) production and cytokine secretion in dendritic cells (DCs), and to make preliminary exploration of Mtb-EVs' effect on the immune regulation of DCs. Mtb-EVs were obtained by ultrafiltration concentration and the protein concentration was determined by BCA assay. The morphology of Mtb-EVs was observed through negative staining electron microscopy (EM). The particle size distribution and concentration of Mtb-EVs were determined by nanoparticle tracking analysis (NTA). Mouse bone marrow was isolated through sterile procedures and mice myeloid DCs were induced and amplified by the combined use of recombinant mouse granulocyte-macrophage colony-stimulating factor (rm GM-CSF) and recombinant mouse interleukin-4 (rm IL-4). Then, morphological and immunophenotypic characterization was performed. After that, the DCs were treated with Mtb-EVs at different concentrations and CCK-8 assay was done to measure their effect on the survival rate of DCs and to identify the appropriate stimulation concentration for subsequent experimental procedures. The intracellular ROS levels of DCs were evaluated with DCFH-DA fluorescence probe and the cytokine secretion of DCs was determined by ELISA. EM observation showed that Mtb-EVs isolated by ultrafiltration concentration were spherical vesicles of varied sizes, all being approximately 100 nm in diameter and displaying typical morphology. NTA results from NanoSight nanoparticle tracker showed that the peak particle size was 98.5 nm, that the average particle size was 110.2 nm, and that the particle size was mainly distributed between 68.4-155.7 nm. Mtb-EVs that were smaller than 250 nm accounted for 98.39% of the total. Mouse myeloid DCs directionally induced and amplified in vitro displayed typical DC phenotype and morphology, and the purity exceeded 85%. EM verified the abundance of microvilli and radial protuberance on the surface of DCs, which had uniform cytoplasm and clear nuclear membrane. Loaded with Mtb-EVs at different concentrations, including 10 2, 10 3, and 10 4 particles/cell, the DCs had significantly upregulated levels of intracellular ROS ( P<0.05). In addition, Mtb-EVs induced the release of IL-1β and IL-6 in a dose-dependent manner ( P<0.05). We established in the study a technical process for the extraction of Mtb-EVs by ultrafiltration concentration and obtained Mtb-EVs with sound morphology, high purity, and concentrated particle size distribution. Furthermore, Mtb-EVs can upregulate the intracellular ROS level in DCs and induce the release of IL-1β and IL-6 in a dose-dependent manner.

Immunopathological Assessment of Hydatid Cyst and Cysticercus Tenuicollis Sonicated Protoscoilces Antigens in Mice

The present study was designed to investigate the cross-protection (protective immunity) between Hydatid cyst and Bladder worm and evaluate the immunologic response of both humerol and cellular immunity in mice. To achieve these goals, 120 mice were used and equally divided into four groups immunized subcutaneously with 2 doses of antigen at the first and 14 days of the experiment. Mice in the first group (n=30) were immunized with 0.3 ml of hydatid cyst sonicated protoscolex antigen. Those in the second group (n=30) were immunized s/c with 0.3 ml of Cysticercus tenuicollis sonicated protscolex antigen. The third group (n=30) was immunized with 0.3 ml of both antigens (0.15 + 0.15), and the fourth group was a control group in which the mice were intraperitoneally injected with 0.2 ml of phosphate buffer solution. At the end of the experiment (30 days), blood samples were taken from the hearts of mice in all groups after being anesthetized by intramuscular injection of Ketamine 60 mg/kg, and Xylazine 12 mg/kg for the assessment of mouse Interleukin-12, IgG, and tumor necrosis alpha levels. The skin test results 24 hours (day 28) post-immunization showed an increase in the skin thickness against both antigens in the treatments, compared to the control. However, there was a decrease at 48 hours (day 29) post-examination in all groups. The results of TNFα titer showed higher titer in the third group, compared to the first, second, and fourth groups. Interleukin 12 concentration showed a higher titer in the third group than in the first, second, and fourth groups. The IgG concentration showed higher titer in the third group compared to the first, second, and fourth groups. In conclusion, immunopathological studies have shown that Ags used in the study, induce humoral and cellular immunity, compared to each Ag alone, and the mixed antigens were much more immunogenic. This cross-reactivity and synergistic interactions between the two parasites may be the cause of their antigenic activities.

IL-33 Deficiency Attenuates Lung Inflammation by Inducing Th17 Response and Impacting the Th17/Treg Balance in LPS-Induced ARDS Mice via Dendritic Cells.

The characteristic pathophysiological feature of acute respiratory distress syndrome (ARDS) is a dysregulated inflammatory response. T helper 17 (Th17) cells in the lung are inflammatory cells that contribute to pulmonary inflammatory cascades. In addition, Th17/regulatory T cells (Treg cells) also play an important role in the inflammatory process. Dendritic cells (DCs) can regulate the differentiation of CD4+ T cells, including Th17 and Treg cells. Recent evidence revealed that interleukin-33 (IL-33) signaling could activate and mature DCs. Therefore, the aim of this study was to investigate the effects of IL-33 on inflammation and immunoregulation by inducing the Th17 response and influencing the Th17/Treg balance in LPS-induced ARDS. IL-33 gene knockout mice and the administration of recombinant mouse IL-33 (rmIL-33) were used to investigate the role of IL-33 and the underlying mechanisms in an LPS-induced ARDS model. Hematoxylin and eosin (H&E) staining, wet/dry (W/D) weight ratios, cell counts, and the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-17 (IL-17), and interleukin-10 (IL-10) in bronchoalveolar lavage fluid (BALF) were investigated. The levels of IL-33, orphan nuclear receptor gamma t (RORγt), and forkhead transcription factor protein 3 (FOXP3) protein in lung tissue were evaluated by Western blotting. The mRNA expression levels of IL-33 and RORγt were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Th17 and Treg cell frequencies were determined by flow cytometry. The levels of IL-6 in the supernatant in a dendritic cell culture system were examined by ELISA. Increased expression of IL-33 was observed in mice with LPS-induced ARDS. IL-33 deficiency significantly inhibited inflammation and attenuated LPS-induced ARDS, whereas pretreatment with rmIL-33 aggravated pulmonary inflammatory response. Furthermore, depletion of IL-33 inhibited Th17 cells, significantly decreased RORγt mRNA and protein expression and IL-17 levels in BALF, and led to less differentiation of T cells into Th17 cells than Treg cells. Moreover, IL-33-/- DCs secreted less IL-6 and IL-23 than normal control DCs. IL-33 deficiency alleviated lung injury in the LPS-induced ARDS model, which was closely related to suppressing Th17 responses and regulating the Th17/Treg balance. The expansion of Th17 cells and imbalance in Th17/Treg cells may be associated with IL-6 and IL-23 secreted from IL-33-activated DCs.

A retrospective study on effectiveness of combined recombinant human interferon-α-1b, interleukin-2, and thalidomide for the treatment of acute myeloid leukemia in various disease states.

Interferon-α-1b, interleukin-2 combined with thalidomide (ITI) improved the outcome and prognosis of some acute myeloid leukemia (AML) patients, but the cases was insufficient. This study observed the efficacy and safety of this regimen in the treatment of numbers of AML patients in various disease states. Starting in January 2014, patients with AML (n=188) were treated with ITI regimen, including 60 refractory/relapses patients in group A, 40 patients in group B remained minimal residual disease-positive (MRD) or changed from negative to positive again after consolidation therapy, and 88 patients in group C with initial complete remission of AML received the ITI treatment after routine consolidation therapy. Bone marrow, fusion gene and MRD were detected to judge the curative effect and the adverse reactions were observed. The remission rate, MRD status and long-term survival of three groups were analyzed. An AML mouse model was constructed to observe the anti-leukemia effect of the three drugs in vivo. Sixty patients with primary AML who were unable to receive chemotherapy, or with relapsed/refractory AML, showed a total response rate of 28.3% (17/60) after receiving the ITI regimen. Forty patients with morphologically complete remission and MRD-positive achieved a response rate of 77.5% (31/40); the MRD converted to negative in 19 patients and was mitigated in 12 patients. Among 88 patients with initial complete remission, 11 failed to maintain the negative MRD, and the relapse rate was 12.5%, which was significantly lower than that of the non-maintenance treatment group (54.3%). In the mouse model, interferon, interleukin-2, and thalidomide exerted an anti-leukemia effect, prolonged the survival time of the mice, and the anti-leukemia effect was further enhanced after administration of the combination ITI regimen. For suitable patients, hematopoietic stem cell transplantation is still strong recommended. The ITI regimen may be an effective option for patients with AML who cannot tolerate conventional chemotherapy, including those with relapsed/refractory disease, those with a complete remission status but are MRD-positive, or those who require maintenance treatment after consolidation therapy. However, a rigorous clinical randomized controlled trial and more in-depth mechanism exploration are still needed to verify this conclusion.

Digital Protein Detection in Bulk Solutions.

Quick and accurate molecular diagnostics in protein detection can greatly benefit medicine in disease diagnosis and lead to positive patient outcomes. However, specialized equipment used in clinical laboratories often comes with trade-offs between operation and function serving a single role for very specific needs. For example, to achieve high analytical sensitivity and specificity, instruments such as high-performance liquid chromatography and/or liquid chromatography–mass spectrometry use a complex instrument design and require thorough training of the users. On the other hand, simple tests such as protein detection in urinary tract infection using dip-stick assays provide very quick results but suffer from poor analytical sensitivity. Here, we present an application study for the 3D particle counter technology, which is based on optical confocal detection in order to scan large sample volumes (0.5–3 mL) in glass cuvettes, that aims to close the gap between analytical sensitivity and turnover assay time and simplify protein detection by adopting bead-based immunoassays. Combining the 3D particle counter technology with bead-based immunoassays, a subpicomolar limit of detection—ranging from 119 to 346 fM—was achieved within 3.5-hour assay time for recombinant mouse interleukin 6 detection. As an alternative instrument to a flow cytometer, the 3D particle counter takes advantages of bead-based immunoassays and provides unique accessibility and flexibility for users.

STING-induced regulatory B cells compromise NK function in cancer immunity.

An immunosuppressive tumour microenvironment is a major obstacle in the control of pancreatic and other solid cancers1-3. Agonists of the stimulator of interferon genes (STING) protein trigger inflammatory innate immune responses to potentially overcome tumour immunosuppression4. Although these agonists hold promise as potential cancer therapies5, tumour resistance to STING monotherapy has emerged in clinical trials and the mechanism(s) is unclear5-7. Here we show that the administration of five distinct STING agonists, including cGAMP, results in an expansion of human and mouse interleukin (IL)-35+ regulatory B cells in pancreatic cancer. Mechanistically, cGAMP drives expression of IL-35 by B cells in an IRF3-dependent but type I interferon-independent manner. In several preclinical cancer models, the loss of STING signalling in B cells increases tumour control. Furthermore, anti-IL-35 blockade or genetic ablation of IL-35 in B cells also reduces tumour growth. Unexpectedly, the STING-IL-35 axis in B cells reduces proliferation of natural killer (NK) cells and attenuates the NK-driven anti-tumour response. These findings reveal an intrinsic barrier to systemic STING agonist monotherapy and provide a combinatorial strategy to overcome immunosuppression in tumours.

Inhibition of osteoclastogenesis by interleukin-33 administration in the periodontal ligament under mechanical loading.

The molecular mechanisms mediating external root resorption are poorly understood. Interleukin-33 (IL-33) expression increased remarkably in the periodontal ligament (PDL) under orthodontic loading. The IL-33-driven responses are delicately cell type- and tissue context-dependent. It is unknown how IL-33 act on osteoclastogenesis in the context of root surface. This study aimed to investigate the effect of IL-33 on osteoclastogenesis in the PDL under mechanical loading. C57BL/6J mice were treated with injections of phosphate buffer saline (PBS) or recombinant mouse IL-33 (rmIL-33, 6μl, 30 μg/ml), and subjected to models of orthodontic tooth movement. Tartrated resistant acid phosphates (TRAP)-positive cells and IL-33 expressions were examined in the PDL. IL-33 release from human PDL cells (hPDLCs) was detected by ELISA. Cementoblast-like (OCCM-30) cells were cultured in the presence of rmIL-33 to examine the release of osteoclast-regulatory proteins. The effects of rmIL-33 on osteoclastogenesis were examined in vitro in cultures of bone marrow macrophages (BMMs) and in BMMs-OCCM-30 cocultures. Expressions of osteoclast-specific or -related genes and proteins were investigated in BMMs-OCCM-30 cocultures treated with or without rmIL-33, in the presence or absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) neutralizing antibody. Interleukin-33 expressions were upregulated in the PDL under orthodontic loading. Static compressive force enhanced expression and release of IL-33 from hPDLCs. Administration of rmIL-33 resulted in reduced number of TRAP-positive cells in the PDL, and inhibited osteoclast differentiation from BMMs in vitro. OCCM-30 cells had varied osteoprotegerin (OPG) / receptor activator for nuclear factor-κB ligand (RANKL) secretion and increased release of GM-CSF under rmIL-33 stimulation. Treatment with rmIL-33 in BMMs-OCCM-30 cocultures resulted in inhibited differentiation and decreased activity of osteoclasts, and these effects were partially reversed by GM-CSF neutralizing antibody. Interleukin-33 inhibits osteoclastogenesis in the PDL under orthodontic loading. The anti-osteoclastogenic effects were mediated partly by directly affecting osteoclast precursors and partly by cementoblast-mediated release of GM-CSF.

Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor.

AimsOsteoarthritis (OA) is a common degenerative joint disease characterized by chronic inflammatory articular cartilage degradation. Long noncoding RNAs (lncRNAs) have been previously indicated to play an important role in inflammation-related diseases. Herein, the current study set out to explore the involvement of lncRNA H19 in OA.MethodsFirstly, OA mouse models and interleukin (IL)-1β-induced mouse chondrocytes were established. Expression patterns of IL-38 were determined in the synovial fluid and cartilage tissues from OA patients. Furthermore, the targeting relationship between lncRNA H19, tumour protein p53 (TP53), and IL-38 was determined by means of dual-luciferase reporter gene, chromatin immunoprecipitation, and RNA immunoprecipitation assays. Subsequent to gain- and loss-of-function assays, the levels of cartilage damage and proinflammatory factors were further detected using safranin O-fast green staining and enzyme-linked immunosorbent assay (ELISA) in vivo, respectively, while chondrocyte apoptosis was measured using Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) in vitro.ResultsIL-38 was highly expressed in lentivirus vector-mediated OA mice. Meanwhile, injection of exogenous IL-38 to OA mice alleviated the cartilage damage, and reduced the levels of proinflammatory factors and chondrocyte apoptosis. TP53 was responsible for lncRNA H19-mediated upregulation of IL-38. Furthermore, it was found that the anti-inflammatory effects of IL-38 were achieved by its binding with the IL-36 receptor (IL-36R). Overexpression of H19 reduced the expression of inflammatory factors and chondrocyte apoptosis, which was abrogated by knockdown of IL-38 or TP53.ConclusionCollectively, our findings evidenced that upregulation of lncRNA H19 attenuates inflammation and ameliorates cartilage damage and chondrocyte apoptosis in OA by upregulating TP53, IL-38, and by activating IL-36R.Cite this article: Bone Joint Res 2022;11(8):594–607.

Application of Pharmacokinetic/Pharmacodynamic Modeling to Bridge Mouse Antitumor Efficacy and Monkey Toxicology Data for Determining the Therapeutic Index of an Interleukin-10 Fc Fusion Protein

Pharmacokinetic/pharmacodynamic (PK/PD) modeling was performed to quantitatively integrate preclinical pharmacology and toxicology data for determining the therapeutic index (TI) of an interleukin-10 (IL-10) fragment crystallizable (Fc) fusion protein. Mouse Fc fused with mouse IL-10 (mFc-mIL-10) was studied in mice for antitumor efficacy, and the elevation of interleukin-18 (IL-18) was examined as a PD biomarker. The in vivo mFc-mIL-10 EC50 for the IL-18 induction was estimated to be 2.4 nM, similar to the in vitro receptor binding affinity (Kd) of 3.2 nM. The IL-18 induction was further evaluated in cynomolgus monkeys, where the in vivo induction EC50 by a human IL-10 human Fc-fusion protein (hFc-hIL-10) was 0.08 nM vs. 0.3 nM measured as the in vitro Kd. The extent of the IL-18 induction correlated with mouse antitumor efficacy and was used to connect mouse efficacy to that in monkeys. The PD-based efficacious dose projected in monkeys was comparable to the results obtained using a PK-based method in which mouse efficacious exposure was targeted and corrected for affinity differences between the species. Furthermore, PK/PD relationships were developed for anemia and thrombocytopenia in monkeys treated with hFc-hIL-10, with thrombocytopenia predicted to be dose-limiting toxicity. Using quantitative pharmacology and toxicology information obtained through modeling work in the same species, the TI of hFc-hIL-10 in monkeys was determined to be 2.4 (vs. PD-based efficacy) and 1.2–3 (vs. PK-based efficacy), indicating a narrow safety margin. The model-based approaches were proven valuable to the developability assessment of the IL-10 Fc-fusion protein.

Abstract 1122: Selinexor synergizes with IL-12 to inhibit tumor growth in syngeneic mouse models of melanoma

Abstract Background: Melanoma is the most deadly skin cancer and represents 4.6% of all cancers diagnosed within the US (American Cancer Society). Risk factors for melanoma are almost entirely related to pale skin color and sunburns. Selinexor is a clinically approved oral inhibitor of nuclear exportation protein Exportin 1 (XPO1/CRM1). In preclinical studies, selinexor significantly inhibited growth of A375 and CHL-1 human melanoma cell lines in vivo at well tolerated doses (Yang et al. 2014). Interleukin 12 (IL-12) is a cytokine produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells in response to antigenic stimulation. IL-12 drives the differentiation of T-cells into T helper (Th1) cells, in addition to stimulating T-cell and NK cell activity, specifically through the activation of interferon-γ (IFN-γ). IL-12 exerts antitumor activity through IFN-γ-dependent and independent mechanisms, which include modulation of the immune system and anti-angiogenesis (Zhang et al. 2016). The objective of this study was to compare the impact and scheduling of recombinant mouse IL-12 alone and in combination with selinexor on the growth of murine B16F10 melanoma cells grown as subcutaneous tumors in C57B/6 mice. Methods: Sixty, 6-7 weeks old female C57B/6 mice were inoculated subcutaneously in the left flank with 1 x 105 B16F10 cells. Treatment was initiated when the tumors reached a mean volume of 100 mm3 (standard deviation ± 6.3 mm3). Mice were allocated to 6 groups of 10 such that mean tumor volume in each group was within the range of 11.8 to 13.4 mm3, then treated with: oral selinexor (10mg/kg, twice weekly on day 0 and 2) combined with a pre-dose of subcutaneous IL-12 (1µg/kg, once weekly on day -2); selinexor with post-dose IL-12 (1µg/kg, once weekly on day 3); selinexor alone; IL-12 alone (day -2); IL-12 alone (day 3); or vehicle. Animal weight was recorded daily, and tumors were measured 3 days a week to record percent of tumor growth inhibition (%TGI). Results: Both selinexor and IL-12 demonstrated single agent activity against B16F10 tumor in vivo. All treatment groups showed significant tumor growth inhibition as compared to vehicle. The group receiving selinexor with pre-dose IL-12 showed the largest tumor inhibition (%TGI=92%). Significant weight loss compared to vehicle occurred only in the single agent selinexor group (p=0.039). Conclusions: Synergistic anti-cancer activity of selinexor and IL-12 warrants further clinical investigation. Further mechanistic studies to understand the effects of IL-12 priming on increasing the effectiveness of selinexor will be undertaken. Citation Format: Shira Orr, Leah Henegar, Christopher J. Walker, Feng Wang, Trinayan Kashyap, Marie Maloof, Kathleen Martyn, Michael G. Kauffman, Sharon Shacham, Yosef Landesman. Selinexor synergizes with IL-12 to inhibit tumor growth in syngeneic mouse models of melanoma [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 1122.