Biology subjects

Hosur, V.

Publications and source records attributed to Hosur, V..

5 recordsLinked to original sources

bioRxiv2026

Redundant γc cytokines license IL-1-driven neutrophil inflammation through MEK/ERK convergence

Interleukin-1 (IL-1) is a central driver of autoinflammatory disease, yet IL-1 blockade often provides incomplete benefit in complex, neutrophil-driven conditions. Here we identify a licensing circuit in which common {gamma}-chain ({gamma}c) cytokines provide a redundant signal required for maximal IL-1-driven neutrophil inflammation. IL-1 and {gamma}c cytokines synergize to drive inflammatory cytokine production exceeding either stimulus alone, and these signals engage the MEK/ERK pathway, an effect substantially suppressed by pharmacological MEK inhibition. We validated this circuit in vivo in a mouse model of IL-1-driven neutrophil-dominant autoinflammation. Ablation of the shared {gamma}c receptor markedly prolonged survival and attenuated pathology, whereas deletion of individual {gamma}c cytokine pathways had no major effect--demonstrating in vivo necessity and functional redundancy. Analysis of public phospho-proteomic and transcriptomic datasets confirms MEK/ERK as a conserved neutrophil response to diverse inflammatory stimuli and coordinated IL-1, {gamma}c, and MEK/ERK activation in neutrophils from patients with systemic juvenile idiopathic arthritis (sJIA) and in lesional skin from hidradenitis suppurativa. Together, these findings define a signaling architecture in which redundant {gamma}c inputs enhance MEK/ERK-dependent inflammatory output, identify the {gamma}c receptor as an in vivo disease-modifying node, and position MEK/ERK as a mechanistically grounded therapeutic target. eTOC SummaryLorenzo et al. show that common {gamma}-chain ({gamma}c) cytokines provide redundant licensing signals that amplify IL-1-driven neutrophil inflammation through MEK/ERK convergence. Blocking any single {gamma}c cytokine fails to suppress disease, but ablating the shared {gamma}c receptor or inhibiting MEK/ERK markedly attenuates pathology, identifying these nodes as therapeutic targets in autoinflammatory disease.

immunology↗

bioRxiv2024

Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice

The Cre-Lox recombination system is a powerful tool in mouse genetics, offering spatial-temporal control over gene expression and facilitating the large-scale generation of conditional knockout mice. Its versatility also extends to other research models, such as rats, pigs, and zebrafish. However, the Cre-Lox technology presents a set of challenges that includes high costs, a time-intensive process, and the occurrence of unpredictable recombination events, which can lead to unexpected phenotypic outcomes. To better understand factors affecting recombination, we embarked on a systematic and genome-wide analysis of Cre-mediated recombination in mice. To ensure uniformity and reproducibility, we generated 11 novel strains with conditional alleles at the ROSA26 locus, utilizing a single inbred mouse strain background, C57BL/6J. We examined several factors influencing Cre-recombination, including the inter-loxP distance, mutant loxP sites, the zygosity of the conditional alleles, chromosomal location, and the age of the breeders. We discovered that the selection of the Cre-driver strain profoundly impacts recombination efficiency. We also found that successful and complete recombination is best achieved when loxP sites are spaced between 1 to 4 kb apart, with mutant loxP sites facilitating recombination at distances of 1 to 3 kb. Furthermore, we demonstrate that complete recombination does not occur at an inter-loxP distance of [≥] 15 kb with wildtype loxP sites, nor at a distance of [≥] 7 kb with mutant lox71/66 sites. Interestingly, the age of the Cre-driver mouse at the time of breeding emerged as a critical factor in recombination efficiency, with best results observed between 8 and 20 weeks old. Moreover, crossing heterozygous floxed alleles with the Cre-driver strain resulted in more efficient recombination than using homozygous floxed alleles. Lastly, maintaining an inter-loxP distance of 4 kb or less ensures efficient recombination of the conditional allele, regardless of the chromosomal location. While CRISPR/Cas has revolutionized genome editing in mice, Cre-Lox technology remains a cornerstone for the generation of sophisticated alleles and for precise control of gene expression in mice. The knowledge gained here will enable investigators to select a Cre-Lox approach that is most efficient for their desired outcome in the generation of both germline and non-germline mouse models of human disease, thereby reducing time and cost of Cre-Lox technology-mediated genome modification.

genetics↗

bioRxiv2021

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

Efficient, targeted integration of large DNA constructs represent a significant hurdle in genetic engineering for the development of mouse models of human disease and synthetic biology research. To address this, we developed a system for efficient and precise, targeted single-copy integration of large transgenes directly into the zygote using multiple mouse genetic backgrounds. Conventional approaches, such as random transgenesis, CRISPR/Cas9-mediated homology-directed repair (HDR), lentivirus-based insertion, or DNA transposases all have significant limitations. Our strategy uses in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) to efficiently generate precise single-copy integrations of transgenes. This is achieved using a transgene "landing pad" composed of dual heterologous Bxb1 attachment (att) sites in cis, pre-positioned in the Gt(ROSA)26Sor safe harbor locus. Successful RMCE is achieved in att carrier zygotes using donor DNA carrying cognate attachment sites flanking the desired donor transgene microinjected along with Bxb1-integrase mRNA. This approach routinely achieves perfect vector-free integration of donor constructs at efficiencies as high as 43% and has generated transgenic animals containing inserts up to [~]43kb. Furthermore, when coupled with a nanopore-based Cas9-targeted sequencing (nCATS) approach, complete verification of the precise insertion sequence can be achieved. As a proof-of-concept we describe the creation and characterization of C57BL/6J and NSG Krt18-ACE2 transgenic mouse models for SARS-CoV2 research with verified heterozygous N1 animals available for experimental use in [~]4 months. In addition, we created a diverse series of mouse backgrounds carrying a single att site version of the landing pad allele in C57BL/6J, NSG, B6(Cg)-Tyrc-2J/J, FVB/NJ, PWK/PhJ, 129S1/SvImJ, A/J, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, and DBA/2J for rapid transgene insertion. Combined, this system enables predictable, rapid creation of precisely targeted transgenic animals across multiple genetic backgrounds, simplifying characterization, speeding expansion and use.

genetics↗

bioRxiv2020

Genes adapt to outsmart gene targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation

Gene disruption in mouse embryonic stem cells or zygotes is a conventional genetics approach to identify gene function in vivo. However, because different gene-disruption strategies use different mechanisms to disrupt genes, the strategies can result in diverse phenotypes in the resulting mouse model. To determine whether different gene-disruption strategies affect the phenotype of resulting mutant mice, we characterized Rhbdf1 mouse mutant strains generated by three commonly used strategies--definitive-null, targeted knockout (KO)-first, and CRISPR/Cas9. We find that Rhbdf1 responds differently to distinct KO strategies, for example, by skipping exons and reinitiating translation to potentially yield gain-of-function alleles rather than the expected null or severe hypomorphic alleles. Our analysis also revealed that at least 4% of mice generated using the KO-first strategy show conflicting phenotypes, suggesting that exon skipping is a widespread phenomenon occurring across the genome. Additionally, our study emphasizes that at least 35% of mouse and 45% of human protein-coding genes could be predisposed to targeted KO-first- and CRISPR/Cas9-mediated unexpected translation. Our findings have significant implications for the application of genome editing in both basic research and clinical practice.

genetics↗

bioRxiv2017

ADAM17 is the Principal Ectodomain Sheddase of the EGF-Receptor Ligand Amphiregulin

The epidermal growth factor (EGF)-receptor ligand amphiregulin (AREG) is a potent growth factor implicated in proliferative skin diseases and in primary and metastatic epithelial cancers. AREG in vitro, synthesized as a pro-peptide, requires conversion to an active peptide by metalloproteases by a process known as ectodomain shedding. Although, (Adam17) a disintegrin and metalloprotease 17 is implicated in ectodomain shedding of AREG, it remains to be established in vivo whether ADAM17 contributes to AREG shedding. In the present study, using a curly bare (Rhbdf2cub) mouse model that shows loss-of-hair, enlarged sebaceous glands, and rapid cutaneous wound-healing phenotypes mediated by enhanced Areg mRNA and protein levels, we sought to identify the principal ectodomain sheddase of AREG. To this end, we generated Rhbdf2cub mice lacking ADAM17 specifically in the skin and examined the above phenotypes of Rhbdf2cub mice. We find that ADAM17 deficiency in the skin of Rhbdf2cub mice restores a full hair coat, prevents sebaceous-gland enlargement, and impairs the rapid wound-healing phenotype observed in Rhbdf2cub mice. Furthermore, in vitro, stimulated shedding of AREG is abolished in Rhbdf2cub mouse embryonic keratinocytes lacking ADAM17. Thus, our data demonstrate that ADAM17 is the major ectodomain sheddase of AREG.

genetics↗