Biology subjects

Berkachy, R.

Publications and source records attributed to Berkachy, R..

5 recordsLinked to original sources

bioRxiv2025

Enteric infection priming confers IL-17A dependent protection from chemically-induced Colitis

Background and AimsEnteric infections trigger mucosal immune responses. However, whether such immune imprinting influences susceptibility to sterile inflammatory diseases like colitis remains unclear. The aims of this study were to investigate whether a resolved Citrobacter rodentium (CR) infection in mouse alters host susceptibility to chemically induced colitis and to identify the underlying immune mechanisms. MethodsC57BL/6 mice were infected with wild-type CR or CR {Delta}map{Delta}espF, a mutant lacking tight junction-disrupting effectors. 3 weeks post clearance, mice were subjected to dextran sodium sulfate (DSS)-induced colitis. Disease severity was assessed by weight change, colon length, histopathology, and myeloperoxidase levels. Colonic immune cell populations were characterised by flow cytometry, and cytokine levels were measured from colon explants. Functional roles of IL-17A were evaluated using recombinant cytokine administration and neutralising antibody treatment. ResultsMice that cleared CR infection fared better following DSS treatment compared to uninfected or {Delta}map{Delta}espF-infected mice. This protective phenotype was not directly dependent on microbiota, as confirmed by co-housing experiments. Protected mice displayed elevated numbers of colonic Th1 and Th17 cells and higher levels of IL-17A, IL-22, and IL-2 cytokines. Prophylactic treatment with IL-17A conferred protection in naive mice, whereas IL-17A neutralisation in previously infected mice abrogated the benefit, identifying IL-17A as a key mediator of protection. ConclusionsResolved intestinal infection with CR confers long-term protection against colitis via persistent IL-17A-mediated immune reprogramming. These findings resonate with the "hygiene hypothesis" and highlight how prior microbial exposure can shape mucosal resilience.

microbiology↗

bioRxiv2025

The accessory type III secretion system effectors shape intestinal inflammatory infection outcomes

Injection of effectors via a type III secretion system (T3SS) is an infection strategy shared by various Gram-negative bacterial pathogens, many infecting mucosal surfaces. While individual T3SS effectors are well characterized, their network-level organization and the distinction between core and accessory effectors remain incompletely understood. Here, by systematically dissecting the T3SS effector network of the enteric mouse pathogen Citrobacter rodentium (CR) we identified a subset of 12 accessory effectors that, while dispensable for colonization, significantly alter infection outcomes. A strain lacking the accessory effectors (CRM12) remained virulent in susceptible mouse hosts yet resulted in reduced epithelial barrier damage, inflammation, and immune cell infiltration in resistant mice. Deep proteomic analysis specifically targeting CR-attached colonic epithelial cells revealed that, despite lacking 39% of its effector repertoire, infection with CRM12 results in similar changes to global protein expression as seen in mice infected with the wild-type strain, though key regulators of barrier integrity were differentially expressed. Using a host model with impaired barrier repair, we confirmed that accessory effectors shape infection outcomes without significantly impacting virulence. This study refines the concept of core and accessory effectors, providing a basis for further studies into effector-driven host adaptation.

microbiology↗

bioRxiv2025

IL-18 activates mucosal group 2 innate lymphoid cells following enteric bacterial infection

Group 3 innate lymphoid cells (ILC3s) play a major role in protecting against infection with the enteric mouse pathogen Citrobacter rodentium, used to model infections with enteropathogenic and enterohaemorrhagic Escherichia coli. ILC3s-secreted IL-22, shown to be indispensable for protection against C. rodentium infection, induces secretion of IL-18, antimicrobial peptides and nutritional immunity proteins as well as activation of tissue regeneration processes. While ILC2s have traditionally been associated with immune responses to helminth infection and allergic inflammation via the production of type 2 cytokines (e.g. IL-4, IL-5, IL-9 and IL-13), more recently they have been implicated in protection against Clostridium difficile and Helicobacter pylori infections. Here we show that colonic lamina propria ILC2s proliferate in response to C. rodentium infection and secrete IL-4, IL-5 and IL-13, which are involved in maintenance of the intestinal barrier function, tissue repair and mucus secretion. When stimulated with IL-18, colonic ILC2s from uninfected naive mice secreted type 2 cytokines. Injection of IL-18 binding protein (IL18BP), at 2- and 3-days post C. rodentium infection, blocked activation of ILC2s. These data show that ILC2s are activated in response to infection with an enteric Gram-negative pathogen, where stimulation with IL-18 plays a role in inducing proliferation and secretion of type 2 cytokines. Author SummaryWhile group 3 innate lymphoid cells (ILC3s) play a key role in protecting from bacterial infections, ILC2s are mainly associated with immune responses to helminth infection. Here we investigated if ILC2s are activated in responses to infection with the enteric mouse pathogen Citrobacter rodentium. We show that in infected mice, gut ILC2s expand and secreted type 2 cytokines. ILC2 isolated from uninfected mice were activated by IL-18. Consistently, administration of IL-18 binding protein into C. rodentium-infected mice inhibited ILC2 activation. These findings suggest that gut ILC2s are activated by Gram negative enteric pathogens, which is mediated in part by IL-18.

immunology↗

bioRxiv2024

The exopolysaccharide Poly-N-Acetyl-Glucosamine (PNAG) coats Klebsiella pneumoniae in vivo

The conserved bacterial polysaccharide Poly-N-Acetyl-Glucosamine (PNAG) is a potential broad-spectrum vaccine candidate. While the immunogenicity of PNAG-based vaccine candidates has been established, characterisation of PNAG production across clinically relevant bacteria remains largely unknown. In particular, PNAG production in the Gram-negative pathogen Klebsiella pneumoniae (KP) is not well understood. Here, we demonstrate that PNAG production is prevalent in clinical KP isolates, where it is secreted as extracellular networks during adherent growth conditions. However, during severe KP pulmonary infection, KP PNAG production undergoes a switch to a cell-associated phenotype, coating the bacterial cell surface. By screening a panel of isogenic KP mutants in prominent cell surface components ({Delta}wcaJ,{Delta} rmpADC,{Delta} rfb,{Delta} ompA and{Delta} ompk36), we identified KP capsular polysaccharide as a key determinant underpinning the phenotype. Deleting genes involved in capsule synthesis ({Delta}wcaJ) and regulation ({Delta}rmpADC) resulted in cell-associated PNAG during adherent growth and infection of alveolar epithelial cells in vitro. Taken together, we describe a novel interaction between KP surface polysaccharides and detect for the first time, cell-associated PNAG in KP during lung infection, highlighting PNAG as an attractive KP vaccine antigen. Author summaryThe Gram-negative pathogen Klebsiella pneumoniae (KP) is a leading cause of hospital-associated lung and bloodstream infections worldwide. As KP exhibits resistance to most frontline antibiotics, there is a growing demand for immune-based strategies to treat KP infections. Poly-N-Acetyl-Glucosamine (PNAG) is a surface sugar produced by most clinically relevant bacteria, including KP. However, relatively little is known about PNAG production in KP. Therefore, we set out to characterise PNAG production in KP during in vitro growth and following lung infection in a pulmonary mouse model. During in vitro growth, KP produces extracellular PNAG networks. In contrast, during an in vivo severe lung infection, PNAG is found cell-associated, coating the bacterial surface. We propose that the visible change in KP PNAG between in vitro and in vivo environments is due to crosstalk with capsule, another polysaccharide on the KP surface. Together, this supports PNAG as an attractive KP antigen.

microbiology↗

bioRxiv2022

Temporal profiling of CD4 T-cell activation and differentiation upon SARS-CoV-2 spike protein immunisation

CD4 T-cells require T-cell receptor (TCR) signalling for their activation and differentiation. Foxp3+ regulatory T-cells (Treg) are dependent on TCR signals for their differentiation and suppressive function. However, it is not fully known how TCR signalling controls the differentiation of polyclonal CD4 T-cells upon antigen recognition at the single-cell level in vivo. In this study, using Nr4a3-Tocky (Timer-of-cell-kinetics-and-activity), which analyses temporal changes of antigen-reactive T-cells following TCR signalling, we investigated T-cell response to Spike protein fragments (S1a, S1b, S2a, and S2b) upon immunisation. We show that S1a and S2a induced the differentiation of PD1hiCXCR5+ T follicular helper (Tfh) cells, which is related to CD4 T-cell immunogenicity. In contrast, S1b induced CD25hiGITRhiPD-1int Treg, which intermittently received TCR signalling. Using Foxp3-Tocky, which analyses Foxp3 transcriptional dynamics, the S1b-reactive Treg sustained Foxp3 transcription over time, which is a hallmark of activated Treg. Foxp3 fate-mapping showed that the S1b-reactive Treg were derived not from pre-existing thymic Treg, suggesting Foxp3 induction in non-Treg cells. Thus, the current study reveals temporally dynamic differentiation of CD4 T-cells and Treg upon immunisation in the polyclonal TCR repertoire.

immunology↗