BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins.
RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-β activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV.
CONCLUSIONS: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.

Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.

BACKGROUND: Sepsis is one of the most common clinical diseases, which is characterized by a serious and uncontrollable inflammatory response. LPS-induced inflammation is a critical pathological event in sepsis, but the underlying mechanism has not yet been fully elucidated.
METHODS: The animal model was established for two batches. In the first batch of experiments, Adult C57BL/6J mice were randomly divided into control group and LPS (5 mg/kg, i.p.)group . In the second batch of experiments, mice were randomly divided into control group, LPS group, and LPS+VX765(10 mg/kg, i.p., an inhibitor of NLRP3 inflammasome) group. After 24 hours, mice were anesthetized with isoflurane, blood and intestinal tissue were collected for tissue immunohistochemistry, Western blot analysis and ELISA assays.
RESULTS: The C57BL/6J mice injected with LPS for twenty-four hours could exhibit severe inflammatory reaction including an increased IL-1β, IL-18 in serum and activation of NLRP3 inflammasome in intestine. The injection of VX765 could reverse these effects induced by LPS. These results indicated that the increased level of IL-1β and IL-18 in serum induced by LPS is related to the increased intestinal permeability and activation of NLRP3 inflammasome. In the second batch of experiments, results of western blot and immunohistochemistry showed that Slit2 and Robo4 were significant decreased in intestine of LPS group, while the expression of VEGF was significant increased. Meanwhile, the protein level of tight junction protein ZO-1, occludin, and claudin-5 were significantly lower than in control group, which could also be reversed by VX765 injection.
CONCLUSIONS: In this study, we revealed that Slit2-Robo4 signaling pathway and tight junction in intestine may be involved in LPS-induced inflammation in mice, which may account for the molecular mechanism of sepsis.

Background and Objectives: Inflammatory proteins and their prognostic value in patients with carotid artery stenosis (CAS) have not been adequately studied. Herein, we identified CAS-specific biomarkers from a large pool of inflammatory proteins and assessed the ability of these biomarkers to predict adverse events in individuals with CAS. Materials and Methods: Samples of blood were prospectively obtained from 336 individuals (290 with CAS and 46 without CAS). Plasma concentrations of 29 inflammatory proteins were determined at recruitment, and the patients were followed for 24 months. The outcome of interest was a major adverse cardiovascular event (MACE; composite of stroke, myocardial infarction, or death). The differences in plasma protein concentrations between patients with vs. without a 2-year MACE were determined using the independent t-test or Mann-Whitney U test to identify CAS-specific prognostic biomarkers. Kaplan-Meier and Cox proportional hazards analyses with adjustment for baseline demographic and clinical characteristics were performed to assess the prognostic value of differentially expressed inflammatory proteins in predicting a 2-year MACE in patients with CAS. Results: The mean age of the cohort was 68.8 (SD 10.2) years and 39% were female. The plasma concentrations of two inflammatory proteins were significantly higher in individuals with a 2-year MACE relative to those without a 2-year MACE: IL-6 (5.07 (SD 4.66) vs. 3.36 (SD 4.04) pg/mL, p = 0.03) and CD163 (233.825 (SD 230.306) vs. 159.673 (SD 175.669) pg/mL, p = 0.033). Over a follow-up period of 2 years, individuals with elevated levels of IL-6 were more likely to develop MACE (HR 1.269 (95% CI 1.122-1.639), p = 0.042). Similarly, over a 2-year period, patients with high levels of CD163 were more likely to develop MACE (HR 1.413 (95% CI 1.022-1.954), p = 0.036). Conclusions: The plasma levels of inflammatory proteins IL-6 and CD163 are independently associated with adverse outcomes in individuals with CAS. These CAS-specific prognostic biomarkers may assist in the risk stratification of patients at an elevated risk of a MACE and subsequently guide further vascular evaluation, specialist referrals, and aggressive medical/surgical management, thereby improving outcomes for patients with CAS.

The transitory emergence of myeloid-derived suppressor cells (MDSCs) in infants is important for the homeostasis of the immune system in early life. The composition and functional heterogeneity of MDSCs in newborns remain elusive, hampering the understanding of the importance of MDSCs in neonates. In this study, we unraveled the maturation trajectory of polymorphonuclear (PMN)-MDSCs from the peripheral blood of human newborns by performing single-cell RNA sequencing. Results indicated that neonatal PMN-MDSCs differentiated from self-renewal progenitors, antimicrobial PMN-MDSCs, and immunosuppressive PMN-MDSCs to late PMN-MDSCs with reduced antimicrobial capacity. We also established a simple framework to distinguish these distinct stages by CD177 and CXCR2. Importantly, preterm newborns displayed a reduced abundance of classical PMN-MDSCs but increased late PMN-MDSCs, consistent with their higher susceptibility to infections and inflammation. Furthermore, newborn PMN-MDSCs were distinct from those from cancer patients, which displayed minimum expression of genes about antimicrobial capacity. This study indicates that the heterogeneity of PMN-MDSCs is associated with the maturity of human newborns.

Macrophages play essential roles in maintaining tissue homeostasis and immune defence. However, their extensive infiltration into tumours has been linked to adverse outcomes in multiple human cancers. Within the tumour microenvironment (TME), tumour-associated macrophages (TAMs) promote tumour growth and metastasis, making them prime targets for cancer immunotherapy. Recent single-cell analysis suggest that proliferating TAMs accumulate in human cancers, yet their origins and differentiation pathways remain uncertain. Here, we show that a subpopulation of CD163+ TAMs proliferates in situ within the TME of melanoma, lung cancer, and breast cancer. Consistent with their potential role in suppressing anti-tumour activities of T cells, CD163+ TAMs express a range of potent immunosuppressive molecules, including PD-L1, PD-L2, IL-10, and TGF-β. Other phenotypic markers strongly suggested that these cells originate from CD14+ CCR2+ monocytes, a cell population believed to have minimal capacity for proliferation. However, we demonstrate in vitro that certain myelopoietic cytokines commonly available within the TME induce robust proliferation of human monocytes, especially the combination of interleukin 3 (IL-3) and Macrophage Colony-Stimulating Factor 1 (M-CSF). Monocytic cells cultured with these cytokines efficiently modulate T cell proliferation, and their molecular phenotype recapitulates that of CD163+ TAMs. IL-3-driven proliferation of monocytic cells can be completely blocked by IL-4, associated with the induction of CDKN1A, alongside the upregulation of transcription factors linked to dendritic cell function, such as BATF3 and IRF4. Taken together, our work suggests several novel therapeutic routes to reducing immunosuppressive TAMs in human tumours, from blocking chemokine-mediated recruitment of monocytes to blocking their proliferation.

Durable serological memory following vaccination is critically dependent on the production and survival of long-lived plasma cells (LLPCs). Yet, the factors that control LLPC specification and survival remain poorly resolved. Using intravital two-photon imaging, we find that in contrast to most plasma cells (PCs) in the bone marrow (BM), LLPCs are uniquely sessile and organized into clusters that are dependent on APRIL, an important survival factor. Using deep, bulk RNA sequencing, and surface protein flow-based phenotyping, we find that LLPCs express a unique transcriptome and phenotype compared to bulk PCs, fine-tuning expression of key cell surface molecules, CD93, CD81, CXCR4, CD326, CD44, and CD48, important for adhesion and homing. Conditional deletion of Cxcr4 in PCs following immunization leads to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and ultimately accelerated decay of antibody titer. In naïve mice, the endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting LLPC specification is non-random. As mice age, the BM PC compartment becomes enriched in LLPCs, which may outcompete and limit entry of new PCs into the LLPC niche and pool.

Myocardial infarction activates an intense fibro-inflammatory reaction that is essential for cardiac remodeling and heart failure (HF). Bioactive peptide galanin plays a critical role in regulating cardiovascular homeostasis; however, its specific functional relevance in post-infarction fibro-inflammatory reprogramming remains obscure. Here, we show that galanin coordinates the fibro-inflammatory trajectory and mitochondrial integrity in post-infarction reperfusion injury. Aberrant deposition of collagen was associated with a marked increase in CD68-positive macrophage infiltration in cardiac tissue in mice subjected to myocardial ischemia/reperfusion (I/R) for 14 days compared to sham controls. Furthermore, we found that the myocardial expression level of a specific marker of M2 macrophages, CD206, was significantly down-regulated in I/R-challenged mice. In contrast, galanin treatment started during the reperfusion phase blunted the fibro-inflammatory responses and promoted the expression of CD206 in I/R-remodeled hearts. In addition, we found that the anti-apoptotic and anti-hypertrophic effects of galanin were associated with the preservation of mitochondrial integrity and promotion of mitochondrial biogenesis. These findings depict galanin as a key arbitrator of fibro-inflammatory responses to cardiac I/R injury and offer a promising therapeutic trajectory for the treatment of post-infarct cardiovascular complications.

Carbon dioxide (CO2) released by plants can serve as a cue for regulating insect behaviors. Hyphantria cunea is a widely distributed forestry pest that may use CO2 as a cue for foraging and oviposition. However, the molecular mechanism underlying its ability to sense CO2 has not been elucidated. Our initial study showed that CO2 is significantly attractive to H. cunea adults. Subsequently, 44 H. cunea gustatory receptors (GRs) were identified using transcriptome data, and 3 candidate CO2 receptors that are specifically expressed in the labial palps were identified. In vivo electrophysiological assays revealed that the labial palp is the primary organ for CO2 perception in H. cunea, which is similar to findings in other lepidopteran species. By using the Xenopus oocyte expression system, we showed that the HcunGR1 and HcunGR3 co-expressions produced a robust response to CO2, but HcunGR2 had an inhibitory effect on CO2 perception. Finally, immunohistochemical staining revealed sexual dimorphism in the CO2-sensitive labial pit organ glomerulus (LPOG). Taken together, our results clarified the mechanism by which H. cunea sense CO2, laying the foundation for further investigations into the role of CO2 in the rapid spread of H. cunea.

BACKGROUND: Epstein-Barr virus (EBV) is a double-stranded DNA oncogenic virus. Several types of solid tumors, such as nasopharyngeal carcinoma, EBV-associated gastric carcinoma, and lymphoepithelioma-like carcinoma of the lung, have been linked to EBV infection. Currently, several TCR-T-cell therapies for EBV-associated tumors are in clinical trials, but due to the suppressive immune microenvironment of solid tumors, the clinical application of TCR-T-cell therapy for EBV-associated solid tumors is limited. Figuring out the mechanism by which EBV participates in the formation of the tumor immunosuppressive microenvironment will help T cells or TCR-T cells break through the limitation and exert stronger antitumor potential.
METHODS: Flow cytometry was used for analyzing macrophage differentiation phenotypes induced by EBV-infected and EBV-uninfected tumors, as well as the function of T cells co-cultured with these macrophages. Xenograft model in mice was used to explore the effects of M2 macrophages, TCR-T cells, and matrix metalloprotein 9 (MMP9) inhibitors on the growth of EBV-infected tumors.
RESULTS: EBV-positive tumors exhibited an exhaustion profile of T cells, despite the presence of a large T-cell infiltration. EBV-infected tumors recruited a large number of mononuclear macrophages with CCL5 and induced CD163+M2 macrophages polarization through the secretion of CSF1 and the promotion of autocrine IL10 production by mononuclear macrophages. Massive secretion of MMP9 by this group of CD163+M2 macrophages induced by EBV infection was an important factor contributing to T-cell exhaustion and TCR-T-cell therapy resistance in EBV-positive tumors, and the use of MMP9 inhibitors improved the function of T cells cocultured with M2 macrophages. Finally, the combination of an MMP9 inhibitor with TCR-T cells targeting EBV-positive tumors significantly inhibited the growth of xenografts in mice.
CONCLUSIONS: MMP9 inhibitors improve TCR-T cell function suppressed by EBV-induced M2 macrophages. TCR-T-cell therapy combined with MMP9 inhibitors was an effective therapeutic strategy for EBV-positive solid tumors.