Metabolic rewiring of macrophages by CpG potentiates clearance of cancer cells and overcomes tumor-expressed CD47-mediated 'don't-eat-me' signal.

Macrophages enforce antitumor immunity by engulfing and killing tumor cells. Although these functions are determined by a balance of stimulatory and inhibitory signals, the role of macrophage metabolism is unknown. Here, we study the capacity of macrophages to circumvent inhibitory activity mediated by CD47 on cancer cells. We show that stimulation with a CpG oligodeoxynucleotide, a Toll-like receptor 9 agonist, evokes changes in the central carbon metabolism of macrophages that enable antitumor activity, including engulfment of CD47+ cancer cells. CpG activation engenders a metabolic state that requires fatty acid oxidation and shunting of tricarboxylic acid cycle intermediates for de novo lipid biosynthesis. This integration of metabolic inputs is underpinned by carnitine palmitoyltransferase 1A and adenosine tri-phosphate citrate lyase, which, together, impart macrophages with antitumor potential capable of overcoming inhibitory CD47 on cancer cells. Our findings identify central carbon metabolism to be a novel determinant and potential therapeutic target for stimulating antitumor activity by macrophages.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) restores immune regulatory functions of airway macrophages of patients with asthma.

Allergic asthma is characterized by the polarization of Th2 cells and impaired immune regulation. Macrophages occupy the largest proportion of airway immune cells. This study aims to discover the mechanism that hinders the immune regulatory functions of airway macrophages. In this study, macrophages were isolated from cells in bronchoalveolar lavage fluids (BALF) collected from asthma patients and normal control (NC) subjects. The results indicated that macrophages occupied the largest portion of the cellular components in BALF. The frequency of IL-10+ macrophage was significantly lower in asthma patients than in NC subjects. The expression of IL-10 in macrophages of BALF was associated with the levels of asthma-related parameters. The immune-suppressive functions of BALF M0 cells were defective in asthma patients. The inducibility of IL-10 expression was impaired in BALF macrophages of asthma patients, which could be restored by exposing to CpG. In conclusion, the induction of IL-10 in macrophages of BALF in asthma patients was impaired, and it could be restored by exposure to CpG.

Potential of HMGB-inhibitory oligodeoxynucleotide ISM ODN to neutrophil recruitment in mouse model of hepatitis.

High-mobility group box 1 (HMGB1) is a nucleotide-binding chromatin protein that has also been characterized as a prototypical damage-associate molecular pattern. It triggers inflammatory responses upon release from damaged or dying cells. In fact, HMGB1 has been linked to the induction of many inflammatory diseases through immune cell activation including neutrophil recruitment. In this study, we examined the impact of HMGB1-binding inhibitory oligodeoxynucleotide (ISM ODN) on the development of hepatitis using a murine model of the disease. Our results indicate that ISM ODN effectively suppresses pathological features of hepatitis, including neutrophil accumulation. This study therefore may offer clinical insight into the treatment of hepatitis and possibly other inflammatory diseases.

Advancing targeted combination chemotherapy in triple negative breast cancer: nucleolin aptamer-mediated controlled drug release.

BACKGROUND: Triple-negative breast cancer (TNBC) is a recurrent, heterogeneous, and invasive form of breast cancer. The treatment of TNBC patients with paclitaxel and fluorouracil in a sequential manner has shown promising outcomes. However, it is challenging to deliver these chemotherapeutic agents sequentially to TNBC tumors. We aim to explore a precision therapy strategy for TNBC through the sequential delivery of paclitaxel and fluorouracil. METHODS: We developed a dual chemo-loaded aptamer with redox-sensitive caged paclitaxel for rapid release and non-cleavable caged fluorouracil for slow release. The binding affinity to the target protein was validated using Enzyme-linked oligonucleotide assays and Surface plasmon resonance assays. The targeting and internalization abilities into tumors were confirmed using Flow cytometry assays and Confocal microscopy assays. The inhibitory effects on TNBC progression were evaluated by pharmacological studies in vitro and in vivo. RESULTS: Various redox-responsive aptamer-paclitaxel conjugates were synthesized. Among them, AS1411-paclitaxel conjugate with a thioether linker (ASP) exhibited high anti-proliferation ability against TNBC cells, and its targeting ability was further improved through fluorouracil modification. The fluorouracil modified AS1411-paclitaxel conjugate with a thioether linker (FASP) exhibited effective targeting of TNBC cells and significantly improved the inhibitory effects on TNBC progression in vitro and in vivo. CONCLUSIONS: This study successfully developed fluorouracil-modified AS1411-paclitaxel conjugates with a thioether linker for targeted combination chemotherapy in TNBC. These conjugates demonstrated efficient recognition of TNBC cells, enabling targeted delivery and controlled release of paclitaxel and fluorouracil. This approach resulted in synergistic antitumor effects and reduced toxicity in vivo. However, challenges related to stability, immunogenicity, and scalability need to be further investigated for future translational applications.

Additional use of α-IgM antibodies potentiates CpG ODN2006-induced B cell activation by targeting mainly naïve and marginal zone-like B cells.

CpG ODN2006 is widely used as a potent B cell stimulant in vitro and in vivo. However, it shows a deficit in targeting naïve B cells in vitro. In this study, we investigated whether α-IgM can support ODN2006-induced effects on B cells to obtain enhanced activation with focus on different B cell subsets. Our results delineated robust B cell activation, shown by increased activation marker expression and cytokine secretion by each agent alone, and further augmented when used in combination. Interestingly, α-IgM targeted mainly naïve and marginal zone-like B cells, thus complementing the pronounced effects of ODN2006 on memory B cells and achieving optimal activation for all B cell subsets. Taken together, combining ODN2006 and α-IgM is beneficial for in vitro activation including all B cell subsets. Furthermore, our results suggest that α-IgM could enhance efficacy of ODN2006 in vivo with further need of investigation.

Improvement of the Nuclease Resistance and Immunostimulatory Activity of CpG Oligodeoxynucleotides by Conjugation to Sugar-Immobilized Gold Nanoparticles.

Adjuvants are essential substances for vaccines and immunotherapies that enhance antigen-specific immune responses. Single-stranded oligodeoxynucleotides containing an unmethylated CpG motif (CpG ODNs) are agonistic ligands for toll-like receptor 9 that initiate an innate immune response. They represent promising adjuvants for antiviral and antitumor immunotherapies; however, CpG ODNs have some limitations, such as poor nuclease resistance and low cell membrane permeability. Therefore, an effective formulation is needed to improve the nuclease resistance and immunostimulatory effects of CpG ODNs. Previously, we demonstrated the selective delivery of a small molecule toll-like receptor 7 ligand to immune cells through sugar-binding receptors using sugar-immobilized gold nanoparticles (SGNPs), which significantly enhanced the potency of the ligand. In this study, we examined SGNPs as carriers for partially phosphorothioated A-type CpG ODN (D35) and an entirely phosphorothioated B-type CpG ODN (K3) and evaluated the functionality of the sugar moiety on SGNPs immobilized with CpG ODN. SGNPs immobilized with D35 (D35-SGNPs) exhibited improved nuclease resistance and the in vitro and in vivo potency was significantly higher compared with that of unconjugated D35. Furthermore, the sugar structure on the GNPs was a significant factor in enhancing the cell internalization ability, and enhanced intracellular delivery of D35 resulted in improving the potencies of the A-type CpG ODN, D35. SGNPs immobilized with K3 (K3-SGNPs) exhibited significantly higher induction activities for both humoral and cellular immunity compared with unconjugated K3 and D35-SGNPs. On the other hand, sugar structure on K3-SGNPs did not affect the immunostimulatory effects. These results indicate that the sugar moiety on K3-SGNPs primarily functions as a hydrophilic dispersant for GNPs and the formulation of K3 to SGNPs contributes to improving the immunostimulatory activity of K3. Because our CpG ODN-SGNPs have superior induction activities for antigen-specific T-cell mediated immune responses, they may be effective adjuvants for vaccines and immunotherapies.

Tumor-Targeted Codelivery of CpG and siRNA by a Dual-Ligand-Functionalized Curdlan Nanoparticle.

The simultaneous delivery of CpG oligonucleotide along with short interfering RNA (siRNA) has the potential to significantly boost the anticancer impact of siRNA medications. Our previous research demonstrated that Curdlan nanoparticles functionalized with adenosine are capable of selectively delivering therapeutic siRNA to cancerous cells through endocytosis mediated by adenosine receptors. Herein, we synthesized a dual-ligand-functionalized Curdlan polymer (denoted by CuMAN) to simultaneously target tumor cells and tumor-associated macrophages (TAMs). CuMAN nanoparticles containing CpG and siRNA demonstrated enhanced uptake by B16F10 tumor cells and bone marrow-derived macrophages, which are facilitated by AR on tumor cells and mannose receptor on macrophages. This led to increased release of pro-inflammatory cytokines in both in vitro and in vivo settings. The synergistic effect of CpG on TAMs and RNAi on tumor cells mediated by the CuMAN nanoparticle not only suppressed the tumor growth but also strongly inhibited the lung metastasis. Our findings indicate that the CuMAN nanoparticle has potential as an effective dual-targeting delivery system for nucleic acid therapeutics.

Depletion of regulatory T cells enhancing the anti-tumor effect of in situ vaccination in solid tumors.

The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the clinical treatment for tumor. However, the low response rate of ICIs remains the major obstacle for curing patients and effective approaches for patients with primary or secondary resistance to ICIs remain lacking. In this study, immune stimulating agent unmethylated CG-enriched (CpG) oligodeoxynucleotide (ODN) was locally injected into the tumor to trigger a robust immune response to eradicate cancer cells, while anti-CD25 antibody was applied to remove immunosuppressive regulatory T cells, which further enhanced the host immune activity to attack tumor systematically. The combination of CpG and anti-CD25 antibody obtained notable regression in mouse melanoma model. Furthermore, rechallenge of tumor cells in the xenograft model has resulted in smaller tumor volume, which demonstrated that the combinational treatment enhanced the activity of memory T cells. Remarkably, this combinational therapy presented significant efficacy on multiple types of tumors as well and was able to prevent relapse of tumor partially. Taken together, our combinational immunotherapy provides a new avenue to enhance the clinical outcomes of patients who are insensitive or resistant to ICIs treatments.

Characterization of the impact of dietary immunostimulant CpG on the expression of mRNA biomarkers involved in the immune responses in Atlantic salmon (Salmo salar).

Infectious diseases have significantly impacted Atlantic salmon aquaculture worldwide. Modulating fish immunity with immunostimulant-containing functional feeds could be an effective strategy in mitigating disease problems. Previously, we characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida bacterin on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. A set of miRNA biomarkers of Atlantic salmon head kidney responding to pIC and/or bacterin immune stimulations was identified (Xue et al., 2019) [1]. Herein, we report a complementary qPCR study that investigated the impact of the pIC, bacterin and dietary CpG on the expression of immune-relevant mRNAs (n = 31) using the same samples as in the previous study (Xue et al., 2019) [1]. Twenty-six of these genes were predicted target transcripts of the pIC- and/or bacterin-responsive miRNAs identified in the earlier study. The current data showed that pIC and/or bacterin stimulations significantly modulated the majority of the qPCR-analyzed genes involved in various immune pathways. Some genes responded to both stimulations (e.g. tnfa, il10rb, ifng, irf9, cxcr3, campb) while others appeared to be stimulation specific [e.g. irf3, irf7a, il1r1, mxa, mapk3 (pIC only); clra (bacterin only)]. A. salmonicida bacterin stimulation produced a strong inflammatory response (e.g. higher expression of il1b, il8a and tnfa), while salmon stimulated with pIC showed robust interferon responses (both type I and II). Furthermore, the current data indicated significant down-regulation of immune-relevant transcripts (e.g. tlr9, irf5, il1r1, hsp90ab1, itgb2) by dietary immunostimulant CpG, especially among pre-injection and PBS-injected fish. Together with our prior miRNA study, the present research provided complementary information on Atlantic salmon anti-viral and anti-bacterial immune responses and on how dietary CpG may modulate these responses.

Synthetic Oligodeoxynucleotide CpG Motifs Activate Human Complement through Their Backbone Structure and Induce Complement-Dependent Cytokine Release.

Bacterial and mitochondrial DNA, sharing an evolutionary origin, act as danger-associated molecular patterns in infectious and sterile inflammation. They both contain immunomodulatory CpG motifs. Interactions between CpG motifs and the complement system are sparsely described, and mechanisms of complement activation by CpG remain unclear. Lepirudin-anticoagulated human whole blood and plasma were incubated with increasing concentrations of three classes of synthetic CpGs: CpG-A, -B, and -C oligodeoxynucleotides and their GpC sequence controls. Complement activation products were analyzed by immunoassays. Cytokine levels were determined via 27-plex beads-based immunoassay, and CpG interactions with individual complement proteins were evaluated using magnetic beads coated with CpG-B. In whole blood and plasma, CpG-B and CpG-C (p < 0.05 for both), but not CpG-A (p > 0.8 for all), led to time- and dose-dependent increase of soluble C5b-9, the alternative complement convertase C3bBbP, and the C3 cleavage product C3bc. GpC-A, -B, and -C changed soluble fluid-phase C5b-9, C3bBbP, and C3bc to the same extent as CpG-A, -B, and -C, indicating a DNA backbone-dependent effect. Dose-dependent CpG-B binding was found to C1q (r = 0.83; p = 0.006) and factor H (r = 0.93; p < 0.001). The stimulatory complement effect was partly preserved in C2-deficient plasma and completely preserved in MASP-2-deficient serum. CpG-B increased levels of IL-1ß, IL-2, IL-6, IL-8, MCP-1, and TNF in whole blood, which were completely abolished by inhibition of C5 and C5aR1 (p < 0.05 for all). In conclusion, synthetic analogs of bacterial and mitochondrial DNA activate the complement system via the DNA backbone. We suggest that CpG-B interacts directly with classical and alternative pathway components, resulting in complement-C5aR1-dependent cytokine release.

Targeting c-Myc with decoy oligodeoxynucleotide-loaded polycationic nanoparticles inhibits cell growth and induces apoptosis in cancer stem-like cells (NTERA-2).

BACKGROUND: An increase in cancer stem cell (CSC) populations and their resistance to common treatments could be a result of c-Myc dysregulations in certain cancer cells. In the current study, we investigated anticancer effects of c-Myc decoy ODNs loaded-poly (methacrylic acid-co-diallyl dimethyl ammonium chloride) (PMA-DDA)-coated silica nanoparticles as carriers on cancer-like stem cells (NTERA-2). METHODS AND RESULTS: The physicochemical characteristics of the synthesized nanocomposites (SiO2@PMA-DDA-DEC) were analyzed using FT-IR, DLS, and SEM techniques. UV-Vis spectrophotometer was applied to analyze the release pattern of decoy ODNs from the nanocomposite. Furthermore, uptake, cell viability, apoptosis, and cell cycle assays were used to investigate the anticancer effects of nanocomposites loaded with c-Myc decoy ODNs on NTERA-2 cancer cells. The results of physicochemical analytics demonstrated that SiO2@PMA-DDA-DEC nanocomposites were successfully synthesized. The prepared nanocomposites were taken up by NTERA-2 cells with high efficiency, and could effectively inhibit cell growth and increase apoptosis rate in the treated cells compared to the control group. Moreover, SiO2@PMA-DDA nanocomposites loaded with c-Myc decoy ODNs induced cell cycle arrest at the G0/G1 phase in the treated cells. CONCLUSIONS: The conclusion drawn from this study is that c-Myc decoy ODN-loaded SiO2@PMA-DDA nanocomposites can effectively inhibit cell growth and induce apoptosis in NTERA-2 cancer cells. Moreover, given that a metal core is incorporated into this synthetic nanocomposite, it could potentially be used in conjunction with irradiation as part of a decoy-radiotherapy combinational therapy in future investigations.

SLC15A4 mediates M1-prone metabolic shifts in macrophages and guards immune cells from metabolic stress.

The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR)7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating the mechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage's respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells' ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.

NF-κB Decoy Oligodeoxynucleotide-Loaded Poly Lactic-co-glycolic Acid Nanospheres Facilitate Socket Healing in Orthodontic Tooth Movement.

Orthodontic space closure following tooth extraction is often hindered by alveolar bone deficiency. This study investigates the therapeutic use of nuclear factor-kappa B (NF-κB) decoy oligodeoxynucleotides loaded with polylactic-co-glycolic acid nanospheres (PLGA-NfDs) to mitigate alveolar bone loss during orthodontic tooth movement (OTM) following the bilateral extraction of maxillary first molars in a controlled experiment involving forty rats of OTM model with ethics approved. The decreased tendency of the OTM distance and inclination angle with increased bone volume and improved trabecular bone structure indicated minimized alveolar bone destruction. Reverse transcription-quantitative polymerase chain reaction and histomorphometric analysis demonstrated the suppression of inflammation and bone resorption by downregulating the expression of tartrate-resistant acid phosphatase, tumor necrosis factor-α, interleukin-1ß, cathepsin K, NF-κB p65, and receptor activator of NF-κB ligand while provoking periodontal regeneration by upregulating the expression of alkaline phosphatase, transforming growth factor-ß1, osteopontin, and fibroblast growth factor-2. Importantly, relative gene expression over the maxillary second molar compression side in proximity to the alveolus highlighted the pharmacological effect of intra-socket PLGA-NfD administration, as evidenced by elevated osteocalcin expression, indicative of enhanced osteocytogenesis. These findings emphasize that locally administered PLGA-NfD serves as an effective inflammatory suppressor and yields periodontal regenerative responses following tooth extraction.

TLR Agonists Modify NK Cell Activation and Increase Its Cytotoxicity in Acute Lymphoblastic Leukemia.

Natural killer (NK) cells play a crucial role in innate immunity, particularly in combating infections and tumors. However, in hematological cancers, NK cells often exhibit impaired functions. Therefore, it is very important to activate its endosomal Toll-like receptors (TLRs) as a potential strategy to restore its antitumor activity. We stimulated NK cells from the peripheral blood mononuclear cells from children with acute lymphoblastic leukemia and NK cells isolated, and the NK cells were stimulated with specific TLR ligands (Poly I:C, Imiquimod, R848, and ODN2006) and we evaluated changes in IFN-γ, CD107a, NKG2D, NKp44 expression, Granzyme B secretion, cytokine/chemokine release, and cytotoxic activity. Results revealed that Poly I:C and Imiquimod enhanced the activation of both immunoregulatory and cytotoxic NK cells, increasing IFN-γ, CD107a, NKG2D, and NKp44 expression. R848 activated immunoregulatory NK cells, while ODN2006 boosted CD107a, NKp44, NKG2D, and IFN-γ secretion in cytotoxic NK cells. R848 also increased the secretion of seven cytokines/chemokines. Importantly, R848 and ODN 2006 significantly improved cytotoxicity against leukemic cells. Overall, TLR stimulation enhances NK cell activation, suggesting TLR8 (R848) and TLR9 (ODN 2006) ligands as promising candidates for antitumor immunotherapy.

STAT3 Decoy Oligodeoxynucleotides Suppress Liver Inflammation and Fibrosis in Liver Cancer Cells and a DDC-Induced Liver Injury Mouse Model.

Liver damage caused by various factors results in fibrosis and inflammation, leading to cirrhosis and cancer. Fibrosis results in the accumulation of extracellular matrix components. The role of STAT proteins in mediating liver inflammation and fibrosis has been well documented; however, approved therapies targeting STAT3 inhibition against liver disease are lacking. This study investigated the anti-fibrotic and anti-inflammatory effects of STAT3 decoy oligodeoxynucleotides (ODN) in hepatocytes and liver fibrosis mouse models. STAT3 decoy ODN were delivered into cells using liposomes and hydrodynamic tail vein injection into 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice in which liver injury was induced. STAT3 target gene expression changes were verified using qPCR and Western blotting. Liver tissue fibrosis and bile duct proliferation were assessed in animal experiments using staining techniques, and macrophage and inflammatory cytokine distribution was verified using immunohistochemistry. STAT3 decoy ODN reduced fibrosis and inflammatory factors in liver cancer cell lines and DDC-induced liver injury mouse model. These results suggest that STAT3 decoy ODN may effectively treat liver fibrosis and must be clinically investigated.

Antimetastatic effect of intratumoral Treg antagonists in mice with renal cancer.

The interplay of regulatory T cells (Tregs) within the tumour microenvironment presents a significant challenge in anticancer immunotherapy. This study investigates the potential of Treg blockade to enhance the efficiency of effector T cells. Two distinct treatment cocktails were examined: 3p-hpRNA (5' triphosphate hairpin RNA) combined with unmethylated CpG oligonucleotide (CpG); CpG in combination with OX40 receptor-specific monoclonal antibody (anti-OX40). Treatment efficacy was assessed using a murine model of kidney adenocarcinoma.Renca cells (renal cortical cells with adenocarcinoma) were subcutaneously engrafted in 30 BALB/c mice, then animals were allocated into three treatment groups: Group 1: CpG+anti-OX40, Group 2: CpG+3p-hpRNA, Group 3: untreated control. Treatment efficacy was evaluated based on tumour growth, the occurrence of metastases and overall survival.On day 28 post-implantation, experiments had to be terminated due to tumour progression. Although comparisons of survival times and primary tumour sizes thus became inconsequential, histological examinations provided valuable insights. We observed distinct variations in primary tumour characteristics among the different groups: Groups 1 and 2 displayed demarcations, while Group 3 exhibited diffuse tumours with necrosis. Lung metastases were evident in 70% of untreated mice, whereas none were observed in either of the treated groups.Our findings instil confidence in the potential efficacy of the treatments, thereby laying a solid foundation for future investigations.

The oxidative phosphorylation inhibitor IM156 suppresses B-cell activation by regulating mitochondrial membrane potential and contributes to the mitigation of systemic lupus erythematosus.

Current treatment strategies for autoimmune diseases may not sufficiently control aberrant metabolism in B-cells. To address this concern, we investigated a biguanide derivative, IM156, as a potential regulator for B-cell metabolism in vitro and in vivo on overactive B-cells stimulated by the pro-inflammatory receptor TLR-9 agonist CpG oligodeoxynucleotide, a mimic of viral/bacterial DNA. Using RNA sequencing, we analyzed the B-cell transcriptome expression, identifying the major molecular pathways affected by IM156 in vivo. We also evaluated the anti-inflammatory effects of IM156 in lupus-prone NZB/W F1 mice. CD19+B-cells exhibited higher mitochondrial mass and mitochondrial membrane potential compared to T-cells and were more susceptible to IM156-mediated oxidative phosphorylation inhibition. In vivo, IM156 inhibited mitochondrial oxidative phosphorylation, cell cycle progression, plasmablast differentiation, and activation marker levels in CpG oligodeoxynucleotide-stimulated mouse spleen B-cells. Interestingly, IM156 treatment significantly increased overall survival, reduced glomerulonephritis and inhibited B-cell activation in the NZB/W F1 mice. Thus, our data indicated that IM156 suppressed the mitochondrial membrane potentials of activated B-cells in mice, contributing to the mitigation of lupus activity. Hence, IM156 may represent a therapeutic alternative for autoimmune disease mediated by B-cell hyperactivity.

Immunotherapy for lung cancer combining the oligodeoxynucleotides of TLR9 agonist and TGF-ß2 inhibitor.

Tumor immunotherapies have shown promising antitumor effects, especially immune checkpoint inhibitors (ICIs). However, only 12.46% of the patients benefit from the ICIs, the rest of them shows limited effects on ICIs or even accelerates the tumor progression due to the lack of the immune cell infiltration and activation in the tumor microenvironment (TME). In this study, we administrated a combination of Toll-like receptor 9 (TLR9) agonist CpG ODN and Transforming growth factor-ß2 (TGF-ß2) antisense oligodeoxynucleotide TIO3 to mice intraperitoneally once every other day for a total of four injections, and the first injection was 24 h after LLC cell inoculation. We found that the combination induced the formation of TME toward the enrichment and activation of CD8+ T cells and NK cells, accompanied with a marked decrease of TGF-ß2. The combined therapy also effectively inhibited the tumor growth and prolonged the survival of the mice, even protected the tumor-free mice from the tumor re-challenge. Both of CpG ODN and TIO3 are indispensable, because replacing CpG ODN with TLR9 inhibitor CCT ODN showed no antitumor effect, CpG ODN or TIO3 alone did not lead to ideal antitumor results. This effect was possibly initiated by the activation of dendritic cells at the tumor site. This systemic antitumor immunotherapy with a combination of the two oligonucleotides (an immune stimulant and an immunosuppressive cytokine inhibitor) before the tumor formation may provide a novel strategy for clinical prevention of the postoperative tumor recurrence.

Regulation of reversible conformational change, size switching, and immunomodulation of RNA nanocubes.

In biological systems, conformational changes and allosteric modulation play pivotal roles in regulating biological functions, such as the dynamic change of protein molecules, in response to binding or interacting with other factors such as pH, voltage, salt, light, or ligand. RNA can be manipulated and tuned with a level of simplicity that is characteristic of DNA or polymers, while displaying versatility in structure, diversity in function, and adaptability in a configuration similar to proteins. In the past, the work on the investigation of conformational change mainly focused on protein. The induced-fit and conformational capture in RNA have also been explored, such as in the study of riboswitches. Herein, we report the engineering of three-dimensional RNA nanocubes and demonstrated the operation and regulation for its configuration. We demonstrate the operation of reconfigurable RNA nanocubes whose shapes change precisely and reversibly in response to a specific trigger strand. The shape, size, and conformation can be regulated precisely and reversibly in response to the specific triggering signals. The shape and conformational conversion were observed by cryo-EM and gel electrophoresis, respectively. Harnessing the size, shape, conformation, and self-assembly capabilities of the RNA nanocube can provide a new potential use of this technology as nanocarriers for the treatment of various diseases.

Cytosine-phosphate-guanine oligodeoxynucleotides alleviate radiation-induced kidney injury in cervical cancer by inhibiting DNA damage and oxidative stress through blockade of PARP1/XRCC1 axis.

BACKGROUND: Radiotherapy can cause kidney injury in patients with cervical cancer. This study aims to investigate the possible molecular mechanisms by which CpG-ODNs (Cytosine phosphate guanine-oligodeoxynucleotides) regulate the PARP1 (poly (ADP-ribose) polymerase 1)/XRCC1 (X-ray repair cross-complementing 1) signaling axis and its impact on radiation kidney injury (RKI) in cervical cancer radiotherapy. METHODS: The GSE90627 dataset related to cervical cancer RKI was obtained from the Gene Expression Omnibus (GEO) database. Bioinformatics databases and R software packages were used to analyze the target genes regulated by CpG-ODNs. A mouse model of RKI was established by subjecting C57BL/6JNifdc mice to X-ray irradiation. Serum blood urea nitrogen (BUN) and creatinine levels were measured using an automated biochemical analyzer. Renal tissue morphology was observed through HE staining, while TUNEL staining was performed to detect apoptosis in renal tubular cells. ELISA was conducted to measure levels of oxidative stress-related factors in mouse serum and cell supernatant. An in vitro cell model of RKI was established using X-ray irradiation on HK-2 cells for mechanism validation. RT-qPCR was performed to determine the relative expression of PARP1 mRNA. Cell proliferation activity was assessed using the CCK-8 assay, and Caspase 3 activity was measured in HK-2 cells. Immunofluorescence was used to determine γH2AX expression. RESULTS: Bioinformatics analysis revealed that the downstream targets regulated by CpG-ODNs in cervical cancer RKI were primarily PARP1 and XRCC1. CpG-ODNs may alleviate RKI by inhibiting DNA damage and oxidative stress levels. This resulted in significantly decreased levels of BUN and creatinine in RKI mice, as well as reduced renal tubular and glomerular damage, lower apoptosis rate, decreased DNA damage index (8-OHdG), and increased levels of antioxidant factors associated with oxidative stress (SOD, CAT, GSH, GPx). Among the CpG-ODNs, CpG-ODN2006 had a more pronounced effect. CpG-ODNs mediated the inhibition of PARP1, thereby suppressing DNA damage and oxidative stress response in vitro in HK-2 cells. Additionally, PARP1 promoted the formation of the PARP1 and XRCC1 complex by recruiting XRCC1, which in turn facilitated DNA damage and oxidative stress response in renal tubular cells. Overexpression of either PARP1 or XRCC1 reversed the inhibitory effects of CpG-ODN2006 on DNA damage and oxidative stress in the HK-2 cell model and RKI mouse model. CONCLUSION: CpG-ODNs may mitigate cervical cancer RKI by blocking the activation of the PARP1/XRCC1 signaling axis, inhibiting DNA damage and oxidative stress response in renal tubule epithelial cells.