Biology subjects

Dotan, I.

Publications and source records attributed to Dotan, I..

9 recordsLinked to original sources

bioRxiv2026

Persistent stromal reprogramming defines incomplete mucosal healing and predicts therapeutic response in ulcerative colitis

Mucosal healing (MH) is the primary therapeutic endpoint in ulcerative colitis (UC), yet frequent relapses suggest it does not reflect complete tissue recovery. To define the basis of this vulnerability, we generated a multimodal atlas of UC integrating single-cell and bulk transcriptomics, Visium HD spatial profiling, and multiplexed imaging across 89 patients. We show that MH represents a distinct biological state marked by persistent stromal remodeling along three axes: emergence of inflammatory fibroblasts, sustained loss of OGN niche-supporting fibroblasts, and expansion of pericytes with matrix-remodeling features and reduced vascular association. Spatial analyses revealed persistent reorganization of mucosal tissue domains despite apparent clinical remission. Across independent cohorts, baseline inflammatory fibroblast and pericyte signatures robustly predicted non-response to anti-TNF therapy. These findings suggest that patients in MH remain in a biologically altered state linked to relapse risk and identify stromal reprogramming as a determinant of disease persistence and therapeutic response in UC.

immunology↗

bioRxiv2024

Development of SYK NanoBRET Cellular Target Engagement Assays for Gain-of-Function Variants

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK. Furthermore, the increased SYK activation resulted in the clinical manifestation of immune dysregulation, organ inflammation, and a predisposition for lymphoma. The knowledge that the SYK GoF variants have enhanced activity was leveraged to develop a SYK NanoBRET cellular target engagement assay in intact live cells with constructs for the SYK GoF variants. Herein, we developed a potent SYK-targeted NanoBRET tracer using a SYK donated chemical probe, MRL-SYKi, that enabled a NanoBRET cellular target engagement assay for SYK GoF variants, SYK(S550Y), SYK(S550F), and SYK(P342T). We determined that ATP-competitive SYK inhibitors bind potently to these SYK variants in intact live cells. Additionally, we demonstrated that MRL-SYKi can effectively reduce the catalytic activity of SYK variants, and the phosphorylation levels of SYK(S550Y) in an epithelial cell line (SW480) stably expressing SYK(S550Y).

cell biology↗

bioRxiv2024

Discordant effects of ex-vivo JAK inhibition on inflammatory responses in colonic compared to ileal mucosa

Background & aimsJanus kinase (JAK) inhibitors modulating JAK-STAT (signal transducers and activators of transcription) signaling pathway, are used for the treatment of patients with inflammatory bowel diseases (IBD). We aimed to identify the molecular effects of JAK inhibition in the human intestinal mucosa, considering the IBD location and phenotype. MethodsColonic and ileal explants from patients with ulcerative colitis (UC), Crohns disease (CD), or non-IBD controls (NC) were treated ex-vivo with the JAK inhibitor, tofacitinib. Phosphorylated STAT (p-STAT) levels were assessed by Western blot and Immunofluorescence. Inflammatory genes expression was assessed with Nanostring nCounter system. Human intestinal organoids were used to assess JAK inhibitors effects on p-STATs and iNOS expression. ResultsExplants were collected from 68 patients (NC=28; UC=20; CD=20). JAK inhibition reduced p-STAT1/3/5 expression in all explants. While p-STAT inhibition rates varied among patients (10%-88%), higher inhibition rates were observed in colonic compared to ileal explants. Significant alterations in 120 of 255 inflammatory genes were observed in colonic explants, while only 30 were observed in ileal NC explants. In colonic explants from UC, significant alterations were observed in 5 genes, including STAT1 and NOS2. Various JAK inhibitors reduced IFN-{gamma}-induced increase in p-STAT1 and iNOS expression in organoids. ConclusionsA site-specific anti-inflammatory effect of JAK inhibition by tofacitinib was noticed, whereby the colon was more robustly affected than the ileum. Ex-vivo response to tofacitinib is individual. JAK inhibition may attenuate inflammation by decreasing iNOS expression. Ex-vivo mucosal platforms may be a valuable resource for studying drug impact and evaluating personalized treatment effects.

immunology↗

bioRxiv2022

Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis

Intestinal epithelial cells (IECs) are the first to encounter luminal microorganisms and actively participate in intestinal immunity. We reported that IECs express the {beta}-glucan receptor Dectin-1, and respond to commensal fungi and {beta}-glucans. In phagocytes, Dectin-1 mediates LC3 associated phagocytosis (LAP) utilizing autophagy components to process extracellular cargo. Dectin-1 can mediate phagocytosis of {beta}-glucan-containing particles by non-phagocytic cells. We aimed to determine whether human IECs phagocytose {beta}-glucan-containing fungal particles via LAP. Zymosan ({beta}-glucan particle) and Heat-killed and UV inactivated C. albicans were phagocytosed by monolayers of human colonic (n=18) and ileal (n=4) organoids and IEC lines. LAP was identified by LC3 and Rubicon recruitment to phagosomes and lysosomal processing of internalized particles was demonstrated by co-localization with lysosomal dyes and LAMP2. Phagocytosis was significantly diminished by blockade of Dectin-1, actin polymerization and NAPDH oxidases. Our results show that human IECs sense luminal fungal particles and internalize them via LAP. This novel mechanism of luminal sampling suggests that IECs may contribute to the maintenance of mucosal tolerance towards commensal fungi.

immunology↗

bioRxiv2021

Escherichia coli strains from patients with inflammatory bowel diseases have disease-specific genomic adaptations

ObjectiveEscherichia coli is over-abundant in the gut microbiome of patients with IBD, yet most studies have focused on the adherent-invasive E. coli pathotype. Here, we aimed to identify IBD-specific or phenotype-specific genomic functions of diverse E. coli lineages. DesignWe investigated E. coli from patients with UC, CD and a pouch and healthy subjects. The majority of E. coli genomes were reconstructed directly from metagenomic samples, including publicly available and newly sequenced fecal metagenomes. Clinical metadata and biomarkers were collected. Functional analysis at the gene and mutation level and genome replication rates of E. coli strains were performed, and correlated with IBD phenotypes and biomarkers. ResultsOverall, 530 E. coli genomes were analysed. A specific E. coli lineage (B2) was more prevalent in UC compared to other IBD phenotypes. Genomic metabolic capacities varied across E. coli lineages and IBD phenotypes. Specifically, sialidases involved in host mucin utilization, were exclusively present in a single lineage and were depleted in patients with a pouch. In contrast, enzymes that hydrolyze inulin were enriched in patients with a pouch. E. coli from patients with UC were twice as likely to encode the genotoxic molecule colibactin than strains from patients with CD or pouch. Strikingly, patients with a pouch showed the highest E. coli growth rates, even in the presence of antibiotics. Fecal calprotectin did not correlate with the relative abundance of E. coli. Finally, we identified multiple IBD-specific loss-of function mutations in E. coli genes encoding for bacterial cell envelope and secretion components. ConclusionThis study presents E. coli as a commensal species better adapted to the overly-active mucosal immune milieu in IBD, that may benefit from intestinal inflammation, rather than causing it. The evidence given here suggests adaptive evolution toward attenuated virulence in some E. coli strains, coupled with a rapid growth despite the presence of antibiotics.

genomics↗

bioRxiv2021

In vivo engineered B cells retain memory and secrete high titers of anti-HIV antibodies in mice

As a potential single-shot HIV therapy, transplanted engineered B cells allow robust secretion of broadly neutralizing antibodies (bNAbs). However, ex vivo engineering of autologous B cells is expensive and requires specialized facilities, while allogeneic B cell therapy necessitates MHC compatibility. Here, we develop in vivo B cell engineering, by injecting two adeno associated viral vectors, one coding for saCas9 and another coding for a bNAb. Following immunizations, we demonstrate memory retention and bNAb secretion at neutralizing titers. We observed minimal CRISPR/Cas9 off-target cleavage, using unbiased CHANGE-Seq analysis, while on-target cleavage at undesired tissues is reduced by expressing saCas9 from a B cell specific promoter. In vivo B cell engineering is thus a safe, potent and scalable method for expressing desired antibodies against HIV and beyond. One sentence summaryB cells can be engineered in vivo to robustly secrete anti-HIV bNAbs in a safe, durable and scalable manner.

immunology↗

bioRxiv2020

B cells engineered to express an anti-HIV antibody allow memory retention, class switch recombination and clonal selection in mice

HIV viremia can be controlled by chronic antiretroviral therapy. As a potentially single-shot alternative, B cells engineered by CRISPR/Cas9 to express anti-HIV broadly neutralizing antibodies (bNAbs) were shown capable of secreting high antibody titers. Here, we demonstrate that, upon immunization of mice, adoptively transferred engineered B cells home to germinal centers (GC) where they predominate over the endogenous response and differentiate into memory and plasma cells while undergoing class switch recombination (CSR). Immunization with a higher affinity antigen increases accumulation in GCs and CSR rates. Boost immunization increases rates of engineered B cells in GCs and antibody secretion, indicating memory retention. Finally, antibody sequences of engineered B cells in the spleen show patterns of clonal selection. B cells may thus be engineered as a living and evolving drug.

immunology↗

bioRxiv2018

Prolonged Antibiotic Treatment Generates a Fluoroquinolone Resistant Gut Microbiome and Collateral Multi-Drug Resistance

One Sentence SummaryThe majority of the gut microbiome develops antibiotic resistance via point-mutations in addition to collateral resistance whereas its density is only moderately decreased following long-term antibiotic treatment.\n\nABSTRACTAntibiotic resistance in bacterial pathogens represents a growing threat to modern medicine. Limitation of lengthy and broad-spectrum antibacterial treatment regimens is generally recommended. Nevertheless, some conditions may require prolonged antibiotic treatment. The effects of such treatments on bacterial communities, specifically their resistome, is yet unknown. Here, we followed a unique cohort of patients with ulcerative colitis who underwent total large bowel resection and the formation of an ileal pouch from their normal small bowel. The majority of these patients tend to develop inflammation of this previously normal small bowel, known as \"pouchitis\". Pouchitis is commonly treated with repeated or prolonged courses of broad-spectrum antibiotics. By using metagenomics of faecal samples obtained longitudinally from patients treated with antibiotics for prolonged periods, we hereby show that the majority of their gut commensal bacteria develop antibiotic resistance by point-mutations. In addition, some bacterial species had acquired multidrug resistance loci with genes that confer resistance to the drug used in the treatment (ciprofloxacin) but co-localized with genes encoding extended-spectrum {beta}-lactamases and other resistance-conferring enzymes. We further show that bacterial density in faecal samples is only modestly reduced despite the long-term antibiotic treatment, thereby questioning the current rationale that antibiotic efficacy in treating pouch inflammation is due to the reduction of bacterial load. This study reveals the impact and dynamics of prolonged antibiotic treatment on human gut microbiomes and provides insights that may guide the development of future IBD therapies. It also provides novel insights into bacterial community recovery after cessation of such prolonged treatment, and highlights the increased risk of spreading mobile antibiotic resistance.

microbiology↗

bioRxiv2018

Gut microbiota density influences host physiology and is shaped by host and microbial factors

To identify factors that regulate gut microbiota density and the impact of varied microbiota density on health, we assayed this fundamental ecosystem property in fecal samples across mammals, human disease, and therapeutic interventions. Physiologic features of the host (carrying capacity) and the fitness of the gut microbiota shape microbiota density. Therapeutic manipulation of microbiota density in mice altered host metabolic and immune homeostasis. In humans, gut microbiota density was reduced in Crohns disease, ulcerative colitis, and ileal pouch-anal anastomosis. The gut microbiota in recurrent Clostridium difficile infection had lower density and reduced fitness that were restored by fecal microbiota transplantation. Understanding the interplay between microbiota and disease in terms of microbiota density, host carrying capacity, and microbiota fitness provide new insights into microbiome structure and microbiome targeted therapeutics.

microbiology↗