A short 19 bp dsRNA with 3\'-trinucleotide overhangs acting as immunostimulating RNA (isRNA) demonstrated strong antiproliferative action against cancer cells, immunostimulatory activity through activation of cytokines and Type-I IFN secretion, as well as anti-tumor and anti-metastatic effects in vivo. The aim of this study was to determine the tolerance of chemical modifications (2\'-F, 2\'-OMe, PS, cholesterol, and amino acids) located at different positions within this isRNA to its ability to activate the innate immune system. The obtained duplexes were tested in vivo for their ability to activate the synthesis of interferon-α in mice, and in tumor cell cultures for their ability to inhibit their proliferation. The obtained data show that chemical modifications in the composition of isRNA have different effects on its individual functions, including interferon-inducing and antiproliferative effects. The effect of modifications depends not only on the type of modification but also on its location and the surrounding context of the modifications. This study made it possible to identify leader patterns of modifications that enhance the properties of isRNA: F2/F2 and F2_S/F2 for interferon-inducing activity, as well as F2_S5/F2_S5, F2-NH2/F2-NH2, and Ch-F2/Ch-F2 for antiproliferative action. These modifications can improve the pharmacokinetic and pharmacodynamic properties, as well as increase the specificity of isRNA action to obtain the desired effect.

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BACKGROUND: Hyperactive neutrophil extracellular traps (NETs) formation plays a crucial role in active severe systemic lupus erythematosus (SLE). However, what triggers the imbalance in dysregulated NETs formation in SLE is elusive. Transfer RNA-derived small RNAs (tsRNAs) are novel non-coding RNAs, which participate in various cellular processes. We explore the role of tsRNAs on NETs formation in SLE.
METHODS: We analyzed the levels of NETs DNA and platelet-derived extracellular vesicles (pEVs) from 50 SLE patients and 20 healthy control subjects. The effects of pEVs on NETs formation were evaluated by using immunofluorescence assay and myeloperoxidase-DNA PicoGreen assay. The regulatory mechanism of pEVs on NETs formation and inflammatory cytokines production were investigated using an in vitro cell-based assay.
RESULTS: Increased circulating NETs DNA and pEVs were shown in SLE patients and were associated with disease activity (P < 0.005). We demonstrated that SLE patient-derived immune complexes (ICs) induced platelet activation, followed by pEVs release. ICs-triggered NETs formation was significantly enhanced in the presence of pEVs through Toll-like receptor (TLR) 8 activation. Increased levels of tRF-His-GTG-1 in pEVs and neutrophils of SLE patients were associated with disease activity. tRF-His-GTG-1 interacted with TLR8 to prime p47phox phosphorylation in neutrophils, resulting in reactive oxygen species production and NETs formation. Additionally, tRF-His-GTG-1 modulated NF-κB and IRF7 activation in neutrophils upon TLR8 engagement, resulting IL-1β, IL-8, and interferon-α upregulation, respectively.
CONCLUSIONS: The level of tRF-His-GTG-1 was positively correlated with NETs formation in SLE patients; tRF-His-GTG-1 inhibitor could efficiently suppress ICs-triggered NETs formation/hyperactivation, which may become a potential therapeutic target.
Neutrophils and platelets are key members in the immunopathogenesis of SLE. EVs play a key role in intercellular communication. Abnormal NETs formation promotes vascular complications and organ damage in SLE patients. tsRNA is a novel regulatory small non-coding RNA and participates in diverse pathological processes. Herein, we showed that SLE patient-derived ICs activates platelets directly, followed by intracellular tRF-His-GTG-1 upregulation, which is loaded into pEVs. The pEV-carried tRF-His-GTG-1 could interact with TLR8 in neutrophils, followed by activation of the downstream signaling pathway, including p47phox-NOX2-ROS, which causes NETs enhancement, while IRF7 promotes the expression of IFN-α. The tRF-His-GTG-1 inhibitor could suppress efficiently SLE ICs-induced NETs formation and pEVs primed NETs enhancement. This study offers new molecular machinery to explain the association between the platelets-derived tsRNAs, pEVs, and hyperactive NETs formation in lupus. tRF-His-GTG-1 may serve as a potential therapeutic target and help to advance our understanding of tsRNAs in SLE pathogenesis.

PD-1 blockade therapy has revolutionized melanoma treatment, but still not all patients benefit and pre-treatment identification of those patients is difficult. Increased expression of inflammatory markers such as interleukin (IL)-6 in blood of patients correlates with poor treatment response. We set out to study the effect of inflammatory cytokines on PD-1 blockade in vitro. For this, we studied the effect of IL-6 and type I interferon (IFN) in vitro on human T cells in a mixed leukocyte reaction (MLR) in the absence or presence of PD-1 blockade. While IL-6 reduced IFN-γ secretion by T cells in both the presence and absence of PD-1 blockade, IFN-α specifically reduced the IFN-γ secretion only in the presence of PD-1 blockade. IFN-α reduced T cell proliferation independent of PD-1 blockade and reduced the percentage of cells producing IFN-γ only in the presence of PD-1 blockade. Next we determined the type I IFN score in a cohort of 22 melanoma patients treated with nivolumab. In this cohort, we did not find a correlation between clinical response and type I IFN score, nor between clinical response and IFN-γ secretion in vitro in a MLR in the presence of PD-1 blockade. We conclude that IFN-α reduces the effectiveness of PD-1 blockade in vitro, but that in this cohort, type I IFN score in vivo, nor IFN-γ secretion in vitro in a MLR in the presence of PD-1 blockade correlated to decreased therapy responses in patients.

Autoantibodies to nuclear antigens are hallmarks of systemic lupus erythematosus (SLE) where they contribute to pathogenesis. However, there remains a gap in our knowledge regarding how different isotypes of autoantibodies contribute to this autoimmune disease, including the production of the critical type I interferon (IFN) cytokines by plasmacytoid dendritic cells (pDCs) in response to immune complexes (ICs). We focused on IgA, which is the second-most prevalent isotype in serum and, along with IgG, is deposited in glomeruli in individuals with lupus nephritis. We show that individuals with SLE have serum IgA autoantibodies against most nuclear antigens, correlating with IgG against the same antigen. We investigated whether IgA autoantibodies against a major SLE autoantigen, Smith ribonucleoprotein (Sm/RNP), played a role in IC activation of pDCs. We found that pDCs expressed the IgA-specific Fc receptor, FcαR, and IgA1 autoantibodies synergized with IgG in RNA-containing ICs to generate robust primary blood pDC IFN-α responses in vitro. pDC responses to these ICs required both FcαR and FcγRIIa, showing synergy between these Fc receptors. Sm/RNP IC binding to and internalization by pDCs were greater when ICs contained both IgA1 and IgG. Circulating pDCs from individuals with SLE had higher binding of IgA1-containing ICs and higher expression of FcαR than pDCs from healthy control individuals. Although pDC FcαR expression correlated with the blood IFN-stimulated gene signature in SLE, Toll-like receptor 7 agonists, but not IFN-α, up-regulated pDC FcαR expression in vitro. Together, we show a mechanism by which IgA1 autoantibodies contribute to SLE pathogenesis.

Tumor immunotherapies targeting PD-(L)1 exhibit anti-tumor efficacy in only 10-30% of patients with various cancers. Literature has demonstrated that a \"hot tumor\" which contains high T lymphocytes in the tumor microenvironment exhibits a better response to immunotherapies than a \"cold tumor.\" This study aimed to investigate whether tumor-intrinsic IFNα and CXCL10 determine the recruitment and activation of CD8+ T cells to become \"hot tumor.\" In this study, we found that CXCL10 overexpressed in a variety of tumors including lung, colon, and liver tumors with a correlation with PD-L1. High PD-L1 and CXCL10 are associated with better survival rates in tumor patients receiving immunotherapies. IFNs-downstream transcriptional factor IRF-1 and STAT1 were correlated with PD-L1 and CXCL10 expression. We demonstrated that IRF-1 and STAT1 were both bound with the promoters of PD-L1 and CXCL10, sharing the same signaling pathway and determining IFNs-mediated PD-L1 and CXCL10 expression. In addition, IFNα significantly increased activation marker IFNγ in PBMCs, promoting M1 type monocyte differentiation, CD4+ T, and CD8+ T cell activation. Particularly, we found that CD8+ T lymphocytes abundantly expressed CXCR3, a receptor of CXCL10, by flow cytometry, indicating that tumor-intrinsic CXCL10 potentially recruited CD8+ T in tumor microenvironment. To demonstrate the hypothesis, immunotherapy-sensitive CT26 and immunotherapy-resistant LL/2 were used and we found that CT26 cells exhibited higher IFNα, IFNγ, CXCL10, and PD-L1 levels compared to LL/2, leading to higher IFNγ expression in mouse splenocytes. Moreover, we found that CD8+ T cells were recruited by CXCL10 in vitro, whereas SCH546738, an inhibitor of CXCR3, inhibited T cell migration and splenocytes-mediated anti-tumor effect. We then confirmed that CT26-derived tumor was sensitive to αPD-L1 immunotherapy and LL/2-tumor was resistant, whereas αPD-L1 significantly increased T lymphocyte activation marker CD107a in CT26-derived BALB/c mice. In conclusion, this study revealed that CXCL10 expression is correlated with PD-L1 in tumors, sharing the same signaling pathway and associating with better immunotherapeutic efficacy. Further evidence in the syngeneic tumor models demonstrated that immunotherapy-sensitive CT26 intrinsically exhibited higher IFNα and CXCL10 compared to immunotherapy-resistant LL/2 to recruit and activate CD8+ T cells in the tumor microenvironment, exhibiting \"hot tumor\" characteristic of sensitizing αPD-L1 immunotherapies.

Diseases caused by viruses pose a significant risk to the health of aquatic animals, for which there are presently no efficacious remedies. Interferon (IFN) serving as an antiviral agent, is frequently employed in clinical settings. Due to the unique living conditions of aquatic animals, traditional injection of interferon is cumbersome, time-consuming and labor-intensive. This study aimed to prepare IFN microcapsules through emulsion technique by using resistant starch (RS) and carboxymethyl chitosan (CMCS). Optimization was achieved using the Box-Behnken design (BBD) response surface technique, followed by the creation of microcapsules through emulsification. With RS at a concentration of 1.27 %, a water‑oxygen ratio of 3.3:7.4, CaCl2 at 13.67 %, CMCS at 1.04 %, the rate of encapsulation can escalate to 80.92 %. Rainbow trout infected with Infectious hematopoietic necrosis virus (IHNV) and common carp infected with Spring vireemia (SVCV) exhibited a relative survival rate (RPS) of 65 % and 60 % after treated with IFN microcapsules, respectively. Moreover, the microcapsules effectively reduced the serum AST levels and enhanced the expression of IFNα, IRF3, ISG15, MX1, PKR and Viperin in IHNV-infected rainbow trout and SVCV-infected carp. In conclusion, this integrated IFN microcapsule showed potential as an antiviral agent for treatment of viral diseases in aquaculture.

Human respiratory viruses are the most prevalent cause of disease in humans, with the highly infectious RSV being the leading cause of infant bronchiolitis and viral pneumonia. Responses to type I IFNs are the primary defense against viral infection. However, RSV proteins have been shown to antagonize type I IFN-mediated antiviral innate immunity, specifically dampening intracellular IFN signaling. Respiratory epithelial cells are the main target for RSV infection. In this study, we found RSV-NS1 interfered with the IFN-α JAK/STAT signaling pathway of epithelial cells. RSV-NS1 expression significantly enhanced IFN-α-mediated phosphorylation of STAT1, but not pSTAT2; and neither STAT1 nor STAT2 total protein levels were affected by RSV-NS1. However, expression of RSV-NS1 significantly reduced ISRE and GAS promoter activity and anti-viral IRG expression. Further mechanistic studies demonstrated RSV-NS1 bound STAT1, with protein modeling indicating a possible interaction site between STAT1 and RSV-NS1. Nuclear translocation of STAT1 was reduced in the presence of RSV-NS1. Additionally, STAT1\'s interaction with the nuclear transport adapter protein, KPNA1, was also reduced, suggesting a mechanism by which RSV blocks STAT1 nuclear translocation. Indeed, reducing STAT1\'s access to the nucleus may explain RSV\'s suppression of IFN JAK/STAT promoter activation and antiviral gene induction. Taken together these results describe a novel mechanism by which RSV controls antiviral IFN-α JAK/STAT responses, which enhances our understanding of RSV\'s respiratory disease progression.

Herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) are widespread human pathogens that establish chronic latent infections leading to recurrent episodes. Current treatments are limited, necessitating the development of novel antiviral strategies. This study aimed to assess the antiviral efficacy of novel topical formulations containing interferon alpha-2b (IFN α-2b) against HSV-1 and HSV-2. The formulations, Oftalmoferon® forte (eye drops) and Interferon Vaginal Tablets, demonstrated potent antiviral effects against HSV-1 and HSV-2 in Vero cells, respectively, with concentration-dependent inhibition of viral replication. Subsequently, their efficacy was tested in animal models: HSV-1 keratitis in the rabbit eye model and HSV-2 genital herpes in mice. Oftalmoferon® forte effectively treated HSV-1 keratitis, reducing clinical symptoms and ulcerations compared to virus control. Interferon Vaginal Tablets showed promising results in controlling HSV-2 genital herpes in mice, improving survival rates, reducing clinical signs, weight loss and viral replication. The novel IFN α-2b formulations exhibited significant antiviral activity against HSV infections in cell culture and animal models. These findings suggest the potential of these formulations as alternative treatments for HSV infections, particularly in cases resistant to current therapies. Further studies are warranted to optimize treatment regimens and assess clinical efficacy in humans.

Interferon-alpha (IFN-α) is a first-line drug for treating chronic hepatitis B (CHB). Guanylate-binding protein 1 (GBP1) is one of the interferon-stimulating factors, which participates in the innate immunity of the host and plays an antiviral and antibacterial role. In this study, we explored how GBP1 is involved in IFN-α antiviral activity against HBV. Before being gathered, HepG2-NTCP and HepG2 2.15 cells were transfected with the wild-type hGBP1 plasmid or si-GBP1, respectively, and followed by stimulation with Peg-IFNα-2b. We systematically explored the role of GBP1 in regulating HBV infection in cell models. Additionally, we also examined GBP1 levels in CHB patients. GBP1 activity increased, and its half-life was prolonged after HBV infection. Overexpression of GBP1 inhibited the production of HBsAg and HBeAg, as well as HBs protein and HBV total RNA levels, whereas silencing of GBP1 inhibited its ability to block viral infections. Interestingly, overexpressing GBP1 co-treatment with Peg-IFNα-2b further increased the antiviral effect of IFN-α, while GBP1 silencing co-treatment with Peg-IFNα-2b partly restored its inhibitory effect on HBV. Mechanistically, GBP1 mediates the anti-HBV response of Peg-IFNα-2b by targeting HBs. Analysis of clinical samples revealed that GBP1 was elevated in CHB patients and increased with Peg-IFNα-2b treatment, while GBP1 showed good stability in the interferon response group. Our study demonstrates that GBP1 inhibits HBV replication and promotes HBsAg clearance. It is possible to achieve antiviral effects through the regulation of IFN-α induced immune responses in response to HBV.