Biology subjects

Santos, A. N.

Publications and source records attributed to Santos, A. N..

4 recordsLinked to original sources

bioRxiv2024

Intestinal Epithelial PTPN2 Limits Pathobiont Colonization by Immune-Directed Antimicrobial Responses

Background and AimsLoss of activity of the inflammatory bowel disease (IBD) susceptibility gene, protein tyrosine phosphatase non-receptor type 2 (PTPN2), is associated with altered microbiome composition in both human subjects and mice. Further, expansion of the bacterial pathobiont, adherent- invasive E. coli (AIEC), is strongly linked to IBD pathogenesis. The mechanism by which intestinal epithelial cells (IEC) maintain equilibrium between commensal microbiota and immune cells to restrict invading pathobionts is poorly understood. Here, we investigated the role of IEC-specific PTPN2 in regulating AIEC colonization. MethodsTamoxifen-inducible, intestinal epithelial cell-specific Ptpn2 knockout mice (Ptpn2{Delta}IEC) and control Ptpn2fl/fl mice were infected with either non-invasive E. coli K12, or fluorescent-tagged mAIEC (mAIECred) for four consecutive days or administered PBS. Subsequently, bacterial colonization in mouse tissues was quantified. mRNA and protein expression were assayed in intestinal epithelial cells (IECs) or whole tissue lysates by PCR and Western blot. Tissue cytokine expression was determined by ELISA. Intestinal barrier function was determined by in vivo administration of 4 kDa FITC-dextran (FD4) or 70kDa Rhodamine-B dextran (RD70) fluorescent probes. Confocal microscopy was used to determine the localization of tight-junction proteins. ResultsPtpn2{Delta}IEC mice exhibited increased mAIECred - but not K12 - bacterial load in the distal colon compared to infected Ptpn2fl/fl mice. The higher susceptibility to mAIECred infection was associated with altered levels of antimicrobial peptide (AMPs). Ileal RNA expression of the alpha-defensin AMPs, Defa5 and Defa6, as well as MMP7, was significantly lower in Ptpn2{Delta}IEC vs. Ptpn2fl/fl mice, after mAIECred but not K12 infection. Further, we observed increased tight junction-regulated permeability determined by elevated in vivo FD4 but not RD70 permeability in Ptpn2{Delta}IEC-K12 mice compared to their respective controls. This effect was further exacerbated in Ptpn2{Delta}IEC mAIEC-infected mice. Further, Ptpn2{Delta}IEC mice displayed lower IL-22, IL-6, IL-17A cytokine expression post mAIEC infection compared to Ptpn2fl/fl controls. Recombinant IL-22 reversed the FD4 permeability defect and reduced bacterial burden in Ptpn2{Delta}IEC mice post mAIEC challenge. ConclusionOur findings highlight that intestinal epithelial PTPN2 is crucial for mucosal immunity and gut homeostasis by promoting anti-bacterial defense mechanisms involving coordinated epithelial-immune responses to restrict pathobiont colonization.

immunology↗

bioRxiv2022

Mapping of the podocin proximity-dependent proteome reveals novels components of the kidney podocyte foot process

The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remains to be elucidated. Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin-BioID fusion. Podocin-BioID localizes to the slit diaphragm and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Gene ontology analysis of 54 proteins specifically enriched in our podocin-BioID sample revealed cell junctions, actin binding, and cytoskeleton organization as top terms. Known foot process components were identified and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and supports the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease.

developmental biology↗

bioRxiv2020

Identification of a Novel Susceptibility Marker for SARS-CoV-2 Infection in Human Subjects and Risk Mitigation with a Clinically Approved JAK Inhibitor in Human/Mouse Cells

Coronavirus disease (COVID-19), caused by SARS-CoV-2, has affected over 65 million individuals and killed over 1.5 million persons (December 8, 2020; www.who.int)1. While fatality rates are higher among the elderly and those with underlying comorbidities2, host factors that promote susceptibility to SARS-CoV-2 infection and severe disease are poorly understood. Although individuals with certain autoimmune/inflammatory disorders show increased susceptibility to viral infections, there is incomplete knowledge of SARS-CoV-2 susceptibility in these diseases.3-7 We report that the autoimmune PTPN2 risk variant rs1893217 promotes expression of the SARS-CoV-2 receptor, ACE2, and increases cellular entry mediated by SARS-CoV-2 spike protein. Elevated ACE2 expression and viral entry were mediated by increased JAK-STAT signalling, and were reversed by the JAK inhibitor, tofacitinib. Collectively, our findings uncover a novel risk biomarker for increased expression of the SARS-CoV-2 receptor and viral entry, and identify a clinically approved therapeutic agent to mitigate this risk.

microbiology↗

bioRxiv2019

The Autoimmune Susceptibility Gene, PTPN2, Restricts Expansion of a Novel Mouse Adherent-Invasive E. coli

Inflammatory bowel diseases (IBD) involve genetic and environmental factors that play major roles in disease pathogenesis. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene increase the risk of IBD and are associated with altered microbiome population dynamics in IBD. Moreover, expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), is strongly implicated in the pathogenesis of IBD as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. The aim of this study was to identify if PTPN2 deficiency disturbs the composition of the intestinal microbiome to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking Ptpn2 we identified increased abundance of a novel adherent-invasive E. coli (AIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages to the IBD associated AIEC, LF82. Furthermore, we confirmed this novel mouse AIEC (mAIEC) caused disease when administered to germ-free and mice lacking segmented-filamentous bacteria (SFB). Moreover, mAIEC infection increased severity of and prevented recovery from dextran-sodium sulfate (DSS)-induced colitis. mAIEC genome sequence analysis showed >90% similarity to LF82. Interestingly, mAIEC contained distinct attachment genes not found in LF82 thereby also demonstrating the novelty of this AIEC. We show here for the first time that an IBD susceptibility gene, PTPN2, plays a key role in modulating the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD.

microbiology↗