Pseudorabies virus (PRV), characterized by latent infection, lifelong viral shedding, and high mortality rates, has inflicted substantial economic losses on the swine industry. While vaccination remains the most cost-effective control strategy, emerging virulent variants with enhanced immune evasion capabilities have compromised conventional vaccines, manifesting as short-lived protection, suboptimal efficacy, and risks of latency reactivation. To overcome rapid antigen clearance and undefined immunomodulatory mechanisms in traditional emulsion vaccines, we engineered three nanoemulsions with distinct architectures and sizes: an oil-in-water nanovaccine (O/W nanovaccine, 163.1 ± 3.84 nm), a water-in-oil-in-water nanovaccine (W/O/W nanovaccine, 32 ± 4.04 nm), and a Pickering emulsion nanovaccine (326.07 ± 9.19 nm). The immunogenicity and biocompatibility of nanoemulsion formulations are systematically evaluated through in vitro cellular models, followed by comprehensive in vivo investigations in murine/porcine models to elucidate immune mechanisms, protective efficacy, and challenge resistance. All formulations demonstrated immunostimulatory potential with distinct functional advantages. O/W nanovaccine exhibited superior antigen-presenting cell uptake efficiency and sustained cytokine induction. W/O/W nanovaccine showed maximal dendritic cell activation and high-titer neutralizing antibodies. Pickering emulsion nanovaccine enhanced specific antibody titers. In addition, mechanistic studies revealed that nanoscale lymphatic targeting of O/W nanovaccine and W/O/W nanovaccine leveraged immune cell size preferences for polyfunctional cytokine release. Multi-layered design of W/O/W nanovaccine enabled compartmentalized antigen delivery, induced CD8+ T cell response, and synergistically enhanced cross-presentation to elicit coordinated humoral and cellular immunity. Particulate-stabilized interface of Pickering emulsion nanovaccine enhanced humoral immunity via DC-mediated IFN-γ hyper-secretion and CD4+ T cell differentiation. Furthermore, all nanovaccines demonstrated higher protective efficacy compared to commercial vaccines in animal challenge models infected with PRV, O/W nanovaccine achieved 100% survival in mice while exhibiting the lowest viral shedding in pigs. This study establishes a transformative prevention paradigm against PRV through nanovaccine engineering, providing critical insights for developing next-generation veterinary vaccine platforms.

Pelvic organ prolapse (POP) is a common and distressing condition affecting women, particularly those with a history of vaginal delivery. The impact of extracellular vesicles derived from adipose-derived mesenchymal stem cells (ADSC-EVs) on pelvic floor tissue injury remains unclear. Due to their short half-life and rapid clearance in vivo, ADSC-EVs lose efficacy quickly. To address this, an injectable tetra-PEG hydrogel to encapsulate ADSC-EVs (PEG@EVs) is developed. The hydrogel is formed by tetra-PEG-NH2 and tetra-PEG-NHS through an ammonolysis reaction, leading to the formation of amide bonds within seconds. Vaginal wall tissue from POP patients shows disruption in the extracellular matrix, lipid peroxidation, and inflammation. In vitro, ADSC-EVs significantly reduce H₂O₂-induced oxidative stress, lipid oxidation, and apoptosis, while enhancing the expression of Nrf2 and its downstream targets-CAT, NQO1, HO-1, and SOD2. ADSC-EVs also upregulate GPX4 and SLC7A11, reducing mitochondrial damage and mitigating ferroptosis. The Nrf2 inhibitor ML385 reverses these protective effects. In a rat model of childbirth injury, PEG@EVs treatment promotes Nrf2 nuclear translocation, induces the M1-to-M2 macrophage conversion, reduces inflammation, and stimulates collagen deposition, thereby accelerating vaginal wall repair. The findings of this study may serve as a foundation for early targeted intervention in POP, representing a promising therapeutic approach.

Background Pulmonary arterial hypertension (PAH) is one of the most severe complications and the leading cause of death in patients with systemic lupus erythematosus (SLE). Pathogenic mechanisms leading to SLE-PAH are still not fully understood. In idiopathic PAH, the dysfunction of the bone morphogenetic protein (BMP) pathway was found to be involved in pulmonary artery remodeling [1]. In SLE-PAH, our previous study identified serum autoantibodies targeting BMP receptors (BMPR) as the potential biomarker, including anti-BMPR2, BMPR1A, and Activin Receptor-Like Kinase type 1 (ALK1) antibodies [2]. However, the clinical and pathological roles of the above autoantibodies targeting BMP signaling in SLE-PAH remain uncertain. Objectives To investigate the clinical and pathological roles of autoantibodies targeting BMPR2, BMPR1A, and ALK1 in SLE-PAH. Methods Patients of SLE-PAH confirmed by right heart catheterization were enrolled and serum levels of autoantibodies targeting BMPR2, BMPR1A, and ALK1 were measured by ELISA. Patients with SLE-PAH were subtyped by cluster analysis. Survival and treatment goal achievement were further compared between different clusters. The Id1 and phospho-SMAD1/5 (p-SMAD1/5) levels were examined in human pulmonary artery endothelial cells (hPAEC) under the induction of BMP9 combined with anti-BMPR2, anti-ALK1, and anti-BMPR1A neutralizing antibodies (5 μg/mL). The effect of anti-ALK1 antibodies on the extent of monolayer permeability and apoptosis of hPAEC was further examined. Results 60 SLE-PAH patients were enrolled and cluster analysis revealed two distinct clusters according to the positivity of autoantibodies targeting BMP signaling and clinical manifestations. Cluster 1 was a “low-antibody and high-disease activity” cluster while cluster 2 was a “high-antibody and low-disease activity” cluster. Patients in cluster 1 showed a higher proportion of nephropathy (76.9%) and SLE activity, however a low positivity rate of autoantibodies targeting BMP signaling. Patients in cluster 2 were characterized by a higher rate of anti-BMPR2 antibodies (82.4%), anti-ALK antibodies (70.6%), and lower SLE activity. Prognostic analysis showed that the proportion of patients who reached the treatment target was relatively higher in cluster 2. Mechanism study showed that the p-SMAD1/5 level and the Id1expression were decreased, indicating the suppression of BMP signaling in the presence of anti-ALK1 antibodies. Functional studies showed that anti-ALK1 antibodies increased the monolayer permeability of hPAEC. The late-stage apoptosis of hPAECs was also induced by anti-ALK1 antibodies. Conclusion The serum positivity of autoantibodies targeting BMP signaling has clinical potential in dividing SLE-PAH patients into two distinct clusters. The anti-ALK1 antibodies can downregulate BMP signaling and mediate great permeability and apoptosis in hPAECs, which may be involved in the pathogenesis of SLE-PAH. References [1]Dewachter L, et.al. Eur Respir J 34 (5):1100-1110.[2]Xing Y, et.al. FASEB J 35 (12):e22044. Acknowledgements We thank CSTAR co-authors as following for assistance with cases collections. Disclosure of Interests None Declared.Table 1The clinical features of the patients in cluster 1 and cluster 2.Cluster 1 (N=26)Cluster 2 (N=34)PAnti-BMPR2 antibodies5(19.2)28(82.4)<0.001Anti-BMPR1A antibodies8(30.2)4(1.8)0.068Anti-ALK antibodies5(19.2)24(70.6)<0.001Arthritis12(46.2)25(73.5)0.031Nephropathy20(76.9)1(2.9)<0.001SLEDAI5.46±4.282.62±2.100.001WHO III/IV17(65.4)14(41.2)0.063Figure 1(A). Kaplan-Meier analysis of the prognosis of cluster1 and cluster2. (B) The level of Id1 expression under the induction of anti-BMPR2, anti-ALK1, and anti-BMPR1A antibodies in the presence or absence of BMP9. The ID1 (C) and p-Smad 1/5/8 (D) measured by western blotting under the induction of anti-ALK antibodies, BMP9, or anti-ALK antibodies+ BMP9. The fluorescence of the receiver plate well solution (E) and the percentage of late-stage apoptotic cells (E) under the induction of anti-ALK neutralizing antibodies.

BackgroundPulmonary arterial hypertension (PAH) is one of the most important complications that seriously threatens the prognosis of patients with systemic lupus erythematosus (SLE), with complicated and unclear pathogenesis.ObjectivesBased on genomic studies and functional experiments, we aim to investigate candidate biomarkers and targeted therapy for the early diagnosis and timely treatment of SLE-PAH patients.Methods:1) In order to screen susceptible genes of SLE-PAH, a number of 150 peripheral blood from SLE-PAH patients were subject to whole-exome sequencing (WES), and genome-wide association study (GWAS) was performed by comparing with 934 healthy controls.2) The transcriptional expression levels on peripheral blood of SLE-PAH patients were examined by RT-qPCR to further evaluate the possible pathogenesis of the above screened genes.3) Intervention experiments on human pulmonary artery endothelial cells (hPAEC) were performed to figure out the potential pathogenesis of the selected gene in vitro. RNA-seq and gene ontology were applied to identify the downstream pathways.4) Established by pristane injection and hypoxia induction, SLE-PAH mice model was used to evaluate the pathogenicity and therapeutic value of selected gene. Pulmonary arterial pressure (PAP) was measured by right heart catheterization after tail-intravenous injection of therapeutic vectors.Results:1) The tumor necrosis factor receptor-associated factor 5 (TRAF5) was identified as a susceptible gene of SLE-PAH based on WES and GWAS.2) The significant reductions of TRAF5 on transcriptional level in peripheral blood of SLE-PAH patients were identified, indicating clinical diagnosis values.3) Knockdown of TRAF5 significantly increased early apoptosis of hPAEC and triggered the pathogenesis of PAH through distinct pathways.4) SLE-PAH mouse model was successfully established since they showed lupus phenotype and the mean PAPs were measured as over 40mmHg. Tail-intravenous injection of TRAF5-overexpression vector attenuated PAH.ConclusionLack of TRAF5 triggers the pathogenesis of PAH in SLE patients through inducing hPAEC abnormality. It is a susceptible gene of SLE-PAH and could be a candidate marker for diagnosis and therapy for SLE-PAH patients.Figure 1.A) Genomic and protein simulation structure TRAF5 (a susceptible gene of SLE-PAH). Red dots represent mutation sites that screened from SLE-PAH patients. P.G468R mutation causes dysfunction of protein. B) TRAF5 mRNA expression levels in PBMC of healthy-controls, SLE-PAH and SLE-nPAH patients. *p<0.05. C) shTRAF5 transfected human PAEC. The transfection efficiency reaches 80% (The percentage of EGFP positive cells in all cells of bright field) when MOI=20. All three knockdown vectors of shTRAF5 showed significantly down regulation of TRAF5. *p<0.05, **p<0.01. D) FACS was performed to detect early apoptotic cells labeled with Annexin V-APC (Apoptosis Detection Kit). The group of shTRAF5 showed significantly increased apoptosis compared with groups of control/shScramble. *p<0.05 E) Wound healing experiments were performed in different groups, and the distances of scratches at the same area in each group were measured at the time point of 0h, 6h, 24h, 48h. Abnormal migration was observed in shTRAF5 transfected PAECs.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

The human testis can be infected by a large number of RNA and DNA viruses. While various RNA virus infections may induce orchitis and impair testicular functions, DNA virus infection rarely affects the testis. Mechanisms underlying the differential effects of RNA and DNA viral infections on the testis remain unclear. In the current study, we therefore examined the effects of viral RNA and DNA sensor signaling pathways on mouse Sertoli cells (SC) and Leydig cells (LC). The local injection of viral RNA analogue polyinosinic-polycytidylic acid [poly(I:C)] into the testis markedly disrupted spermatogenesis, whereas the injection of the herpes simplex virus (HSV) DNA analogue HSV60 did not affect spermatogenesis. Poly(I:C) dramatically induced the expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 in SC and LC through Toll-like receptor 3 and interferon β promoter stimulator 1 signaling pathways, impairing the integrity of the blood-testis barrier and testosterone synthesis. Poly(I:C)-induced TNF-α production thus plays a critical role in the impairment of cell functions. In contrast, HSV60 predominantly induced the expression of type 1 interferons and antiviral proteins via the DNA sensor signaling pathway, which did not affect testicular cell functions. Accordingly, the Zika virus induced high levels of TNF-α in SC and LC and impaired their respective cellular functions, whereas Herpes simplex virus type 2 principally induced antiviral responses and did not impair such functions. These results provide insights into the mechanisms by which RNA viral infections impair testicular functions.

The abnormal expression of sialic acids (SAs) on cells and tissues is closely related to various pathophysiological states. Here we applied phenylboronic acid (PBA) functionalized graphitic carbon nitride fluorescent quantum dots (PCQDs) with sizes from 3 to 5 nm in efficient and selective labeling SAs on the surface of living cells and tissues. With abundant PBA in their structure, the water soluble PCQDs showed the relative SA level on the cell surface via selectively and efficiently staining different cell lines in 30 min and revealed that M1 macrophages may express more SAs on their surfaces compared with M0 and M2. The distinct demarcation of cancerous and para-noncancerous areas on cancer tissue sections was showed by PCQDs staining. PCQDs with their high selectivity, stable photoluminescence, low cost, and nontoxicity can be an ideal SA fluorescent probe for living cells and tissues.

Macrophages, the important cells of immune system, have exhibited distinct gene phenotypes with diverse functions in different microenvironments. In the present study, macrophages RAW264.7 (M0 macrophages) and lipopolysaccharide (LPS) plus interferon gamma (INF-γ)-treated M0 macrophages (M1 macrophages) were cultured in different lung cell-derived culture supernatants (CSs) as imitative tumor microenvironments. The lipids (mainly from cell membrane) of intact macrophages were in situ detected by matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry. Approximately 300 of small molecules were observed in negative ion mode. Partial least square-discriminant analysis (PLS-DA) suggested that two types of the macrophages have different membrane lipid phenotypes. Changes in the levels of phosphatidylethanolamine PE(16:1/18:0), PE(18:1/18:0), PE(36:2), PE-Cer(d36:1), and PE(P-16:0/18:1) were closely associated with membrane phenotypes of macrophages. The heatmap also revealed that directional induction to classically activated macrophages (M1 macrophages) in vitro had greater impact on the membrane lipid phenotypes of macrophages than different lung cell-derived CSs. The results are consistent with the data obtained by biological technologies.

Viral infections of the ovary may perturb ovarian functions. However, the mechanisms underlying innate immune responses in the ovary are poorly understood. The present study demonstrates that cytosolic viral DNA sensor signaling initiates the innate immune response in mouse ovarian granulosa cells and affects endocrine function. The cytosolic DNA sensors p204 and cGAS and their common signaling adaptor stimulator of interferon (IFN) genes (STING) were constitutively expressed in granulosa cells. Transfection with VACV70, a synthetic vaccinia virus (VACV) DNA analog, induced the expression of type I interferons (IFNA/B) and major inflammatory cytokines (TNFA and IL6) through IRF3 and NF-κB activation respectively. Moreover, several IFN-inducible antiviral proteins, including 2',5'-oligoadenylate synthetase, IFN-stimulating gene 15 and Mx GTPase 1, were also induced by VACV70 transfection. The innate immune responses in granulosa cells were significantly reduced by the transfection of specific small-interfering RNAs targeting p204, cGas or Sting Notably, the VACV70-triggered innate immune responses affected steroidogenesis in vivo and in vitro The data presented in this study describe the mechanism underlying ovarian immune responses to viral infection.

Uropathogenic Escherichia coli (UPEC) may cause epididymitis and impair male fertility. The mechanisms underlying the innate immune responses to UPEC infection in the epididymis are not fully understood. This study showed that UPEC induced innate immune responses in mouse epididymal epithelial cells (EECs) through the activation of Toll-like receptor 4 (TLR4) and TLR5. Infection with UPEC significantly induced the expression of proinflammatory cytokines, including tumor necrosis factor alpha, interleukin 6, and monocyte chemoattractant protein 1, in EECs through the activation of nuclear factor kappa B. Moreover, UPEC induced the production of type 1 interferons by EECs through the activation of interferon regulatory factor 3. The UPEC-induced innate immune responses were significantly reduced in the EECs of Tlr4 or Tlr5 knockout mice. The innate immune responses were further reduced in Tlr4 and Tlr5 double-knockout EECs. Furthermore, we demonstrated that TLR4 and TLR5 cooperatively initiated the epididymal innate immune responses to UPEC infection in vivo. The results provide novel insights into the mechanisms underlying the epididymal innate immune responses to UPEC infection.

Mumps virus (MuV) infection may lead to oophoritis and perturb ovarian function. However, the mechanisms underlying the activation of innate immune responses to MuV infection in the ovary have not been investigated. This study showed that Toll-like receptor 2 (TLR2) and retinoic acid-inducible gene I (RIG-I) cooperatively initiate innate immune responses to MuV infection in mouse ovarian granulosa cells. Ovarian granulosa cells infected with MuV significantly produced pro-inflammatory cytokines and chemokines, including interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), monocyte chemotactic protein 1 (MCP-1), and type 1 interferons (IFN-α and IFN-β). Knockdown of RIG-I significantly decreased MuV-induced cytokine expression. TLR2 deficiency reduced the expression of IL-1β, TNF-α, and MCP-1 but did not affect the expression of IFN-α and IFN-β in granulosa cells after infection with MuV. Intraperitoneal injection of MuV induced the ovarian innate immune responses in vivo, which suppressed estradiol synthesis and induced granulosa cell apoptosis. The results provide novel insights into the mechanisms underlying MuV-induced innate immune responses in the mouse ovary.

Mumps virus (MuV) infection frequently causes orchitis and impairs male fertility. However, the mechanisms underlying the innate immune responses to MuV infection in the testis have yet to be investigated. This study showed that MuV induced innate immune responses in mouse Sertoli and Leydig cells through TLR2 and retinoic acid-inducible gene I (RIG-I) signaling, which result in the production of proinflammatory cytokines and chemokines, including TNF-α, IL-6, MCP-1, CXCL10, and type 1 interferons (IFN-α and IFN-β). By contrast, MuV did not induce the cytokine production in male germ cells. In response to MuV infection, Sertoli cells produced higher levels of proinflammatory cytokines and chemokines but lower levels of type 1 IFNs than Leydig cells did. The MuV-induced cytokine production by Sertoli and Leydig cells was significantly reduced by the knockout of TLR2 or the knockdown of RIG-I signaling. The local injection of MuV into the testis triggered the testicular innate immune responses in vivo. Moreover, MuV infection suppressed testosterone synthesis by Leydig cells. This is the first study examining the innate immune responses to MuV infection in testicular cells. The results provide novel insights into the mechanisms underlying the MuV-induced innate immune responses in the testis.

Uropathogenic Escherichia coli (UPEC) may cause epididymitis and impair male fertility. The mechanisms underlying the innate immune responses to UPEC infection in the epididymis are not fully understood. This study showed that UPEC induced innate immune responses in mouse epididymal epithelial cells (EECs) through the activation of Toll-like receptor 4 (TLR4) and TLR5. Infection with UPEC significantly induced the expression of proinflammatory cytokines, including tumor necrosis factor alpha, interleukin 6, and monocyte chemoattractant protein 1, in EECs through the activation of nuclear factor kappa B. Moreover, UPEC induced the production of type 1 interferons by EECs through the activation of interferon regulatory factor 3. The UPEC-induced innate immune responses were significantly reduced in the EECs of Tlr4 or Tlr5 knockout mice. The innate immune responses were further reduced in Tlr4 and Tlr5 double-knockout EECs. Furthermore, we demonstrated that TLR4 and TLR5 cooperatively initiated the epididymal innate immune responses to UPEC infection in vivo. The results provide novel insights into the mechanisms underlying the epididymal innate immune responses to UPEC infection.

ABSTRACT Viral infections may perturb ovarian functions and female fertility. Mechanisms underlying viral perturbation of ovarian functions are incompletely understood. This study found that intraperitoneal injection of polyinosinic–polycytidylic acid [poly (I:C)] in female mice inhibits estradiol synthesis and induces ovarian granulosa cell apoptosis. Poly (I:C) is a synthetic viral double-stranded RNA analog, which induces innate antiviral responses mimicking a viral infection through activation of pattern recognition receptors, including toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated gene 5. Injection of poly (I:C) significantly induced granulosa cell apoptosis in antral follicles and reduced antral follicle numbers. These effects were significantly diminished in Tlr3 knockout or tumor necrosis factor-alpha (Tnfa) knockout mice. We demonstrated that poly (I:C) induced TNFA production at a relatively high level in wild-type mice compared with that in Tlr3 knockout mice. Notably, TNFA neutralizing antibody significantly reduced poly (I:C)-induced ovarian dysfunction. In vitro assays confirmed that TNFA inhibits estradiol synthesis and induces granulosa cell apoptosis. Results provide novel insights into the mechanisms by which a mimicked viral infection perturbs ovarian functions in mice.

Viral infections of the epididymis may impair male fertility and spread sexually transmitted pathogens. The innate antiviral immune responses in the epididymis have yet to be intensively investigated. This study found that mouse epididymal epithelial cells (EECs) constitutively express several viral sensors, including TLR3, retinoic acid-inducible gene I, and DNA-dependent activator of IFN regulatory factors. Other DNA sensors, including p204 and cGMP-AMP synthase, can be induced by transfection of synthetic HSV genomic DNA (HSV60). TLR3 and retinoic acid-inducible gene I in EECs can be activated by their common agonist, polyinosinic-polycytidylic acid [poly(I:C)]. The signaling pathway of DNA sensors can be initiated by HSV60. Both poly(I:C) and HSV60 induced the expression of type 1 IFNs and various antiviral proteins, including IFN-stimulated gene 15, 2',5'-oligoadenylate synthetase, and myxovirus resistance 1. Poly(I:C), but not HSV60, also dramatically induced the expression of major proinflammatory cytokines, including TNF-α and MCP-1, in EECs. In vivo assay confirmed that the local injection of poly(I:C) or HSV60 induced the innate antiviral responses in EECs. This study provided novel insights into the mechanisms underlying the innate antiviral responses in the mouse epididymis.

Viral infections may perturb ovarian functions and female fertility. Mechanisms underlying viral perturbation of ovarian functions are incompletely understood. This study found that intraperitoneal injection of polyinosinic-polycytidylic acid [poly (I:C)] in female mice inhibits estradiol synthesis and induces ovarian granulosa cell apoptosis. Poly (I:C) is a synthetic viral double-stranded RNA analog, which induces innate antiviral responses mimicking a viral infection through activation of pattern recognition receptors, including toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated gene 5. Injection of poly (I:C) significantly induced granulosa cell apoptosis in antral follicles and reduced antral follicle numbers. These effects were significantly diminished in Tlr3 knockout or tumor necrosis factor-alpha (Tnfa) knockout mice. We demonstrated that poly (I:C) induced TNFA production at a relatively high level in wild-type mice compared with that in Tlr3 knockout mice. Notably, TNFA neutralizing antibody significantly reduced poly (I:C)-induced ovarian dysfunction. In vitro assays confirmed that TNFA inhibits estradiol synthesis and induces granulosa cell apoptosis. Results provide novel insights into the mechanisms by which a mimicked viral infection perturbs ovarian functions in mice.