Perivascular neurons instruct 3D vascular lattice formation via neurovascular contact.

The vasculature of the central nervous system is a 3D lattice composed of laminar vascular beds interconnected by penetrating vessels. The mechanisms controlling 3D lattice network formation remain largely unknown. Combining viral labeling, genetic marking, and single-cell profiling in the mouse retina, we discovered a perivascular neuronal subset, annotated as Fam19a4/Nts-positive retinal ganglion cells (Fam19a4/Nts-RGCs), directly contacting the vasculature with perisomatic endfeet. Developmental ablation of Fam19a4/Nts-RGCs led to disoriented growth of penetrating vessels near the ganglion cell layer (GCL), leading to a disorganized 3D vascular lattice. We identified enriched PIEZO2 expression in Fam19a4/Nts-RGCs. Piezo2 loss from all retinal neurons or Fam19a4/Nts-RGCs abolished the direct neurovascular contacts and phenocopied the Fam19a4/Nts-RGC ablation deficits. The defective vascular structure led to reduced capillary perfusion and sensitized the retina to ischemic insults. Furthermore, we uncovered a Piezo2-dependent perivascular granule cell subset for cerebellar vascular patterning, indicating neuronal Piezo2-dependent 3D vascular patterning in the brain.

Segmented Filamentous Bacteria Prevent and Cure Rotavirus Infection.

Rotavirus (RV) encounters intestinal epithelial cells amidst diverse microbiota, opening possibilities of microbes influencing RV infection. Although RV clearance typically requires adaptive immunity, we unintentionally generated RV-resistant immunodeficient mice, which, we hypothesized, reflected select microbes protecting against RV. Accordingly, such RV resistance was transferred by co-housing and fecal transplant. RV-protecting microbiota were interrogated by heat, filtration, and antimicrobial agents, followed by limiting dilution transplant to germ-free mice and microbiome analysis. This approach revealed that segmented filamentous bacteria (SFB) were sufficient to protect mice against RV infection and associated diarrhea. Such protection was independent of previously defined RV-impeding factors, including interferon, IL-17, and IL-22. Colonization of the ileum by SFB induced changes in host gene expression and accelerated epithelial cell turnover. Incubation of RV with SFB-containing feces reduced infectivity in vitro, suggesting direct neutralization of RV. Thus, independent of immune cells, SFB confer protection against certain enteric viral infections and associated diarrheal disease.

Antibiotics-Driven Gut Microbiome Perturbation Alters Immunity to Vaccines in Humans.

Emerging evidence indicates a central role for the microbiome in immunity. However, causal evidence in humans is sparse. Here, we administered broad-spectrum antibiotics to healthy adults prior and subsequent to seasonal influenza vaccination. Despite a 10,000-fold reduction in gut bacterial load and long-lasting diminution in bacterial diversity, antibody responses were not significantly affected. However, in a second trial of subjects with low pre-existing antibody titers, there was significant impairment in H1N1-specific neutralization and binding IgG1 and IgA responses. In addition, in both studies antibiotics treatment resulted in (1) enhanced inflammatory signatures (including AP-1/NR4A expression), observed previously in the elderly, and increased dendritic cell activation; (2) divergent metabolic trajectories, with a 1,000-fold reduction in serum secondary bile acids, which was highly correlated with AP-1/NR4A signaling and inflammasome activation. Multi-omics integration revealed significant associations between bacterial species and metabolic phenotypes, highlighting a key role for the microbiome in modulating human immunity.

Cytonuclear Interactions and Subgenome Dominance Shape the Evolution of Organelle-Targeted Genes in the Brassica Triangle of U.

The interaction and coevolution between nuclear and cytoplasmic genomes are one of the fundamental hallmarks of eukaryotic genome evolution and, 2 billion yr later, are still major contributors to the formation of new species. Although many studies have investigated the role of cytonuclear interactions following allopolyploidization, the relative magnitude of the effect of subgenome dominance versus cytonuclear interaction on genome evolution remains unclear. The Brassica triangle of U features 3 diploid species that together have formed 3 separate allotetraploid species on similar evolutionary timescales, providing an ideal system for understanding the contribution of the cytoplasmic donor to hybrid polyploid. Here, we investigated the evolutionary pattern of organelle-targeted genes in Brassica carinata (BBCC) and 2 varieties of Brassica juncea (AABB) at the whole-genome level, with particular focus on cytonuclear enzyme complexes. We found partial evidence that plastid-targeted genes experience selection to match plastid genomes, but no obvious corresponding signal in mitochondria-targeted genes from these 2 separately formed allopolyploids. Interestingly, selection acting on plastid genomes always reduced the retention rate of plastid-targeted genes encoded by the B subgenome, regardless of whether the Brassica nigra (BB) subgenome was contributed by the paternal or maternal progenitor. More broadly, this study illustrates the distinct selective pressures experienced by plastid- and mitochondria-targeted genes, despite a shared pattern of inheritance and natural history. Our study also highlights an important role for subgenome dominance in allopolyploid genome evolution, even in genes whose function depends on separately inherited molecules.

TLR5-Derived, TIR-Interacting Decoy Peptides to Inhibit TLR Signaling.

TLR5, which is activated by flagellin, plays an important role in initiating immune response to a broad spectrum of motile bacterial pathogens. TLRs induce intracellular signaling via dimerization of their TIR domains followed by adapter recruitment through multiple interactions of receptor and adapter TIRs. Here, a library of cell-permeable decoy peptides derived from the TLR5 TIR was screened for TLR5 signaling inhibition in the HEK-Blue-mTLR5 reporter cell line. The peptide demonstrating the strongest inhibition, 5R667, corresponded to the second helix of the region between the third and fourth ß-strands (helix C″). In addition to the TLR5-induced cytokine expression, 5R667 inhibited cytokine expression elicited by TLR4, TLR2, and TLR9. 5R667 also suppressed the systemic cytokine induction elicited by LPS administration in mice. 5R667 binding specificity was studied by time-resolved fluorescence spectroscopy in a cell-based assay. 5R667 demonstrated a multispecific binding pattern with respect to TIR domains: It bound TIRs of TLR adapters of the MyD88-dependent pathway, Toll/interleukin-1 receptor domain-containing adapter protein/MyD88 adapter-like (TIRAP) and MyD88, and also the TIR of TLR5. TR667, the peptide derived from the TIRAP region, which is structurally homologous to 5R667, demonstrated binding and inhibitory properties similar to that of 5R667. The surface-exposed residues within TIR regions represented by 5R667 and TR667 form motifs, which are nearly 90% conserved in vertebrate evolution and are distinctive of TLR5 and TIRAP TIR domains. Thus, we have identified an evolutionary conserved adapter recruitment motif within TLR5 TIR, the function of which can be inhibited by selective cell-permeable decoy peptides, which can serve as pan-specific TLR inhibitors.

Mechanical strain regulates osteogenesis via Antxr1/LncRNA H19/Wnt/ß-catenin axis.

Alleviating bone loss is an essential way to prevent osteoporotic fractures. Proper exercise improves bone density without the side effects of long-term medications, but the mechanism is unclear. Our study explored the role of Antxr1/LncRNA H19/Wnt/ß-catenin axis in the process of exercise-mediated alleviation of bone loss. Here we discovered that moderate-intensity treadmill exercise alleviates bone loss caused by ovariectomy and ameliorates bone strength accompanied by an increased lncRNA H19 expression. Concomitantly, Antxr1, a mechanosensitive protein was found downregulated by exercise but upregulated by ovariectomy. Interestingly, knockdown expression of Antxr1 increased lncRNA H19 expression and Wnt/ß-catenin signaling pathway in bone marrow mesenchymal stem cells, whereas overexpression of Antxr1 decreased lncRNA H19 expression and Wnt/ß-catenin signaling pathway. Hence, our study demonstrates the regulation of Antxr1/LncRNA H19/Wnt/ß-catenin axis in the process of mechanical strain-induced osteogenic differentiation, which provides further mechanistic insight into the role of mechanical regulation in bone metabolism.

Comprehensive analysis of T-cell receptor repertoires reveals antigen-driven T-cell clusters in patients with Behçet's syndrome.

T lymphocytes are the major components of adaptive immunity in Behçet's syndrome (BS) pathology. However, the precise mechanism of T-cell-induced inflammatory condition remains to be determined. We applied bulk sequencing of the T-cell receptor (TCR) ß chain in peripheral blood samples from 45 patients with BS and 10 healthy donors as controls. TCR repertoires in BS patients displayed more clonality and less diversity than in healthy donors. Male patients exhibited lower diversity metrics of TCR and had a larger proportion in the top 10 clones than females (p = 0.016). There were no TCR clonality differences in other clinical features, such as age, disease duration, organ involvement, disease severity, and activity. By "Grouping of Lymphocyte Interactions by Paratope Hotspots" (GLIPH2) for antigen prediction, we found distinct 2477 clusters of TCR-ß sequences that potentially recognize similar antigens shared between BS patients. We observed clonal T-cell expansion in BS patients. Sexual differences in TCR clonal expansion and public TCR groups deserve further study to reveal the underline T-cell-mediated immunity in BS.

A gain-of-function mutation in BnaIAA13 disrupts vascular tissue and lateral root development in Brassica napus.

Rapeseed (Brassica napus) is an important oilseed crop worldwide. Plant vascular tissues are responsible for material transport and provide mechanical support. The lateral roots (LRs) absorb sufficient water and nutrients. The genetic basis of vascular tissues and LRs development in rapeseed remains unknown. This study characterized an EMS-mutagenized rapeseed mutant, T16, which showed dwarf stature, reduced LRs, and leaf wilting. Scanning electron microscopy observations showed that the internode-cell shortened. Observations of the tissue sections revealed defects in the development of vascular bundles in the stems and petioles. Genetic analysis revealed that the phenotypes of T16 were controlled by a single semi-dominant nuclear gene. Map-based cloning and genetic complementarity confirmed that BnaA03.IAA13 is the functional gene, a G-to-A mutation in second exon changed the glycine at the 79th position to glutamic acid, disrupting the conserved degron motif VGWPP. Transcriptome analysis in roots and stems showed that auxin and cytokinin signaling pathways were disordered in T16. Evolutionary analysis showed that AUXIN/INDOLE-3-ACETIC ACID was conserved during plant evolution. The heterozygote of T16 significantly reduced the plant height while maintaining other agronomic traits. Our findings provide novel insights into the regulatory mechanisms of vascular tissues and LRs development, and provide a new germplasm resource for rapeseed breeding.

High-frequency surface-micromachined optical ultrasound transducer array for 3D micro photoacoustic computed tomography.

This Letter reports a new, to the best of our knowledge, high-frequency surface-micromachined optical ultrasound transducer (HF-SMOUT) array for micro photoacoustic computed tomography (µPACT). An 11 × 11 mm2 2D array of 220 × 220 elements (35 µm in diameter) is designed, fabricated, and characterized. The optical resonance wavelength (ORW) of ≥90% of the elements falls within a 6-nm range. The acoustic center frequency and bandwidth of the elements are ∼14 MHz and ∼18 MHz (129%), respectively. The noise equivalent pressure (NEP) is 161 Pa (or 18 m P a/H z) within a measurement bandwidth of 5-75 MHz. The standard deviation of the ORW drift is 0.45 nm and 0.93 nm within 25°C-55°C, respectively, and during a seven-day continuous water immersion. PACT experiments are conducted to evaluate the imaging performances of the HF-SMOUT array. The spatial resolution is estimated as 90 µm (axial) and 250-750 µm (lateral) within a 10 × 10 mm2 field of view (FoV) and the imaging depth of 16 mm. A 3D PA image of a knotted black hair target is also successfully acquired. These results demonstrate the feasibility of using the HF-SMOUT array for µPACT applications.

Molecular mechanism of allosteric modulation for the cannabinoid receptor CB1.

Given the promising clinical value of allosteric modulators of G protein-coupled-receptors (GPCRs), mechanistic understanding of how these modulators alter GPCR function is of significance. Here, we report the crystallographic and cryo-electron microscopy structures of the cannabinoid receptor CB1 bound to the positive allosteric modulator (PAM) ZCZ011. These structures show that ZCZ011 binds to an extrahelical site in the transmembrane 2 (TM2)-TM3-TM4 surface. Through (un)biased molecular dynamics simulations and mutagenesis experiments, we show that TM2 rearrangement is critical for the propagation of allosteric signals. ZCZ011 exerts a PAM effect by promoting TM2 rearrangement in favor of receptor activation and increasing the population of receptors that adopt an active conformation. In contrast, ORG27569, a negative allosteric modulator (NAM) of CB1, also binds to the TM2-TM3-TM4 surface and exerts a NAM effect by impeding the TM2 rearrangement. Our findings fill a gap in the understanding of CB1 allosteric regulation and could guide the rational design of CB1 allosteric modulators.

Efficacy and Safety of Covered Stents Versus Bare-Metal Stents for Aortoiliac Occlusive Disease: A Systematic Review and Meta-Analysis.

PURPOSE: This study aimed to assess the efficacy and safety outcome of covered stents (CSs), as compared with bare-metal stents (BMSs), for the treatment of patients with aortoiliac occlusive disease (AIOD). MATERIALS AND METHODS: A systematic literature search was conducted in PubMed, Embase, and Cochrane Library up to August 2023 to identify all studies comparing efficacy and safety outcomes of CSs versus BMSs for treating AIOD. Our outcome was primary patency, secondary patency, technical success, ankle-brachial index (ABI) variation, target lesion revascularization (TLR), limb salvage, complications, and long-term survival. Dichotomous outcomes were pooled as relative risks (RR) or hazard ratio with the 95% confidence interval (CI). Continuous outcomes were pooled as weighted mean differences and 95% CI. Model selection was based on the heterogeneity of the included studies. RESULTS: There were 10 studies (2 randomized controlled trials, 8 retrospective cohort studies), comprising 1676 sample size. Compared with BMSs, CSs use was associated with better primary patency of patients with a Trans-Atlantic Inter-Society Consensus II (TASC) D lesion (RR, 1.15, 95% CI, 1.04 to 1.27, p=0.007), TLR (RR, 0.39, 95% CI, 0.27 to 0.56, p<0.001), technical success (RR, 1.01, 95% CI, 1.00 to 1.02, p=0.010), and long-term survival (RR, 1.06, 95% CI, 1.01 to 1.11, p=0.020). There is no difference between CSs and BMSs regarding primary patency of all patients, secondary patency, variation in ABI, limb salvage, and complications. CONCLUSIONS: Compared with BMSs, CSs used in AIOD was associated with more favorable primary patency in patients with TASC D lesions, TLR, technical success rates, and patient long-term survival. These results provide evidence of the advantages of using CSs for AIOD treatment. Future studies focusing on long-term variations in ABI, primary patency of different degrees of calcification, vascular segments, and TASC classification are warranted. CLINICAL IMPACT: Although several studies evaluated the clinical efficacy of CS in the context of AIOD treatment, the significance and consistency of these findings were not determined to date. We found that CS was used in AIOD associated with better technical success rate, long-term patient survival, lower target lesion revascularization, and higher primary patency of patients with a Trans-Atlantic Inter-Society Consensus II D lesion when compared with BMSs. Our study provides evidence supporting the superiority of CSs over BMSs in the treatment of AIOD, and furnishing clinicians with guidance for treatment decisions.

Identification of hub genes and gene modules associated with Behçet's disease by weighted gene co-expression network analysis of neutrophil transcriptome.

OBJECTIVES: Behçet's disease (BD) is a chronic inflammatory condition with recurrent skin lesions, uveitis, and oral and genital ulcers. Neutrophils are important in the pathogenis of BD, but their molecular mechanisms are unclear. METHODS: We performed weighted gene co-expression network analysis on the transcriptome of neutrophils from 10 BD patients and 10 healthy controls to identify hub genes and gene modules associated with BD. RESULTS: We found eight co-expression modules with different biological functions. The turquoise module was involved in response to hydrogen peroxide and reactive oxygen species, the blue module was involved in response to external stimulus and inflammatory response, and the brown module was involved in the type I interferon signaling pathway. We further identified hub genes and transcription factors in each module by using module membership and gene significance. CONCLUSIONS: Our results reveal novel gene modules and hub genes that are associated with neutrophil activation and dysfunction in BD, which could serve as potential biomarkers and therapeutic targets for this disease.

Autophagy mediated degradation of MITA/TBK1/IRF3 by a hnRNP family member attenuates interferon production in fish.

HnRNP A/B belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family and plays an important role in regulating viral protein translation and genome replication. Here, we found that overexpression of hnRNP A/B promoted spring viremia of carp virus (SVCV) and cyprinid herpesvirus 3 (CyHV3) replication. Further, hnRNP A/B was shown to act as a negative regulator of type I interferon (IFN) response. Mechanistically, hnRNP A/B interacted with MITA, TBK1 and IRF3 to initiate their degradation. In addition, hnRNP A/B bound to the kinase domain of TBK1, the C terminal domain of MITA and IAD domain of IRF3, and the RRM1 domain of hnRNP A/B bound to TBK1, RRM2 domain bound to IRF3 and MITA. Our study provides novel insights into the functions of hnRNP A/B in regulating host antiviral response.

A cytokine-like factor 1 homolog acts as a macrophage chemoattractant in grass carp.

Cytokine-like factor 1 (CYTL1) is a small cytokine and has diverse biological functions in mammals. However, whether CYTL1 exists in lower vertebrates is not clear. In this study, we identified cytl homologs in fish and characterized the immune functions in a teleost species, grass carp (Ctenopharyngodon idella). Fish CYTL1 homologs share conserved molecular features with their mammalian counterparts, including 6 cysteine residues in the mature peptide, genomic organization and synteny. Gene expression analysis revealed that cytl1 was constitutively expressed in tissues of grass carp, with the highest expression detected in the heart. Upon infection with Aeromonas hydrophila (A. hydrophila), cytl1 was downregulated in the hindgut, head kidney, skin, and spleen. In the primary head kidney leukocytes (HKLs), stimulation with inactivated A. hydrophila, LPS, poly(I:C), IL-22, IFN-a or IFN-γrel resulted in downregulation of cytl1 expression. Recombinant grass carp CYTL1 protein produced in the HEK293-F cells was potent to induce il-10 expression, but had little effect on the expression of il-1ß and il-6. In vivo experiments revealed that CYTL1 was effective to recruit macrophages to the muscle injected with cytl expression plasmids. Taken together, our results indicate that CYTL1 is a potent chemokine for recruitment of macrophages in fish.

Distinct antiviral activities of IFNφ1 and IFNφ4 in zebrafish.

Interferons (IFNs) are a group of secreted cytokines that play a crucial role in antiviral immunity. Type I IFNs display functional disparities. In teleosts, type I IFNs are categorized into two subgroups containing one or two pairs of disulfide bonds. However, their functional differences have not been fully elucidated. In this study, we comparatively characterized the antiviral activities of zebrafish IFNφ1 and IFNφ4 belonging to the group I type I IFNs. It was found that ifnφ1 and ifnφ4 were differentially modulated during viral infection. Although both IFNφ1 and IFNφ4 activated JAK-STAT signaling pathway via CRFB1/CRFB5 receptor complex, IFNφ4 was less potent in inducing phosphorylation of STAT1a, STAT1b and STAT2 and the expression of antiviral genes than IFNφ1, thereby conferring weaker antiviral resistance of target cells. Taken together, our results provide insights into the functional divergence of type I IFNs in lower vertebrates.

IL-6/STAT3 axis is hijacked by GCRV to facilitate viral replication via suppressing type â IFN signaling.

Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type Ⅰ IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.

Single-cell transcriptome landscape of zebrafish liver reveals hepatocytes and immune cell interactions in understanding nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease in the world. Immunity is the major contributing factor in NAFLD; however, the interaction of immune cells and hepatocytes in disease progression has not been fully elucidated. As a popular species for studying NAFLD, zebrafish, whose liver is a complex immune system mediated by immune cells and non-immune cells in maintaining immune tolerance and homeostasis. Understanding the cellular composition and immune environment of zebrafish liver is of great significance for its application in NAFLD. Here, we established a liver atlas that consists of 10 cell types using single-cell RNA sequencing (scRNA-seq). By examining the heterogeneity of hepatocytes and analyzing the expression of NAFLD-associated genes in the specific cluster, we provide a potential target cell model to study NAFLD. Additionally, our analysis identified two subtypes of distinct resident macrophages with inflammatory and non-inflammatory functions and characterized the successive stepwise development of T cell subclusters in the liver. Importantly, we uncovered the possible regulation of macrophages and T cells on target cells of fatty liver by analyzing the cellular interaction between hepatocytes and immune cells. Our data provide valuable information for an in-depth study of immune cells targeting hepatocytes to regulate the immune balance in NAFLD.

Genome composition in Brassica interspecific hybrids affects chromosome inheritance and viability of progeny.

Interspecific hybridization is widespread in nature and can result in the formation of new hybrid species as well as the transfer of traits between species. However, the fate of newly formed hybrid lineages is relatively understudied. We undertook pairwise crossing between multiple genotypes of three Brassica allotetraploid species Brassica juncea (2n = AABB), Brassica carinata (2n = BBCC), and Brassica napus (2n = AACC) to generate AABC, BBAC, and CCAB interspecific hybrids and investigated chromosome inheritance and fertility in these hybrids and their self-pollinated progeny. Surprisingly, despite the presence of a complete diploid genome in all hybrids, hybrid fertility was very low. AABC and BBAC first generation (F1) hybrids both averaged ~16% pollen viability compared to 3.5% in CCAB hybrids: most CCAB hybrid flowers were male-sterile. AABC and CCAB F1 hybrid plants averaged 5.5 and 0.5 seeds per plant, respectively, and BBAC F1 hybrids ~56 seeds/plant. In the second generation (S1), all confirmed self-pollinated progeny resulting from CCAB hybrids were sterile, producing no self-pollinated seeds. Three AABC S1 hybrids putatively resulting from unreduced gametes produced 3, 14, and 182 seeds each, while other AABC S1 hybrids averaged 1.5 seeds/plant (0-8). BBAC S1 hybrids averaged 44 seeds/plant (range 0-403). We also observed strong bias towards retention rather than loss of the haploid genomes, suggesting that the subgenomes in the Brassica allotetraploids are already highly interdependent, such that loss of one subgenome is detrimental to fertility and viability. Our results suggest that relationships between subgenomes determine hybridization outcomes in these species.

Molecular and Structural Basis of Receptor Binding and Signaling of a Fish Type I IFN with Three Disulfide Bonds.

In mammals, type I IFNs, which commonly contain one or two disulfide bonds, activate the JAK-STAT signaling pathway through binding to the common cell surface receptor formed by IFN-α/ß receptor (IFNAR)1 and IFNAR2 subunits. Although type I IFNs are also known to be essential for antiviral defense in teleost fish, very little is known about mechanisms underlying the recognition of fish type I IFNs by associated receptors. In this study, we demonstrate that a type I IFN of large yellow croaker Larimichthys crocea (LcIFNi), belonging to a new subgroup of fish type I IFNs, triggers antiviral response via the conserved JAK-STAT pathway through stable binding with a heterodimeric receptor comprising subunits LcCRFB5 and LcCRFB2. LcIFNi binds to LcCRFB5 with a much higher affinity than to LcCRFB2. Furthermore, we determined the crystal structure of LcIFNi at a 1.39 Å resolution. The high-resolution structure is, to our knowledge, the first reported structure of a type I IFN with three disulfide bonds, all of which were found to be indispensable for folding and stability of LcIFNi. Using structural analysis, mutagenesis, and biochemical assays, we identified key LcIFNi residues involved in receptor interaction and proposed a structural model of LcIFNi bound to the LcCRFB2-LcCRFB5 receptor. The results show that LcIFNi-LcCRFB2 exhibits a similar binding pattern to human IFN-ω-IFNAR2, whereas the binding pattern of LcIFNi-LcCRFB5 is quite different from that of IFN-ω-IFNAR1. Altogether, our findings reveal the structural basis for receptor interaction and signaling of a type I IFN with three disulfide bonds and provide new insights into the mechanisms underlying type I IFN recognition in teleosts.

Novel Dimeric Architecture of an IFN-γ-Related Cytokine Provides Insights into Subfunctionalization of Type II IFNs in Teleost Fish.

Gene duplication leads to subfunctionalization of paralogs. In mammals, IFN-γ is the sole member of the type II IFN family and binds to a receptor complex consisting of IFN-γR1 and IFN-γR2. In teleost fish, IFN-γ and its receptors have been duplicated due to the teleost-specific whole-genome duplication event. In this study, the functions of an IFN-γ-related (IFN-γrel) cytokine were found to be partially retained relative to IFN-γ in grass carp (Ctenopharyngodon idella [CiIFN-γrel]). CiIFN-γrel upregulated the expression of proinflammatory genes but had lost the ability to activate genes involved in Th1 response. The results suggest that CiIFN-γrel could have been subfunctionalized from CiIFN-γ. Moreover, CiIFN-γrel induced STAT1 phosphorylation via interaction with duplicated homologs of IFN-γR1 (cytokine receptor family B [CRFB] 17 and CRFB13). Strikingly, CiIFN-γrel did not bind to the IFN-γR2 homolog (CRFB6). To gain insight into the subfunctionalization, the crystal structure of CiIFN-γrel was solved at 2.26 Å, revealing that it forms a homodimer that is connected by two pairs of disulfide bonds. Due to the spatial positions of helix A, loop AB, and helix B, CiIFN-γrel displays a unique topology that requires elements from two identical monomers to form a unit that is similar to IFN-γ. Further, mutagenesis analyses identified key residues interacting with CiIFN-γrel receptors and those required for the biological functions. Our study can help understand the subfunctionalization of duplicated IFN-γ paralogs in fish.