Biology subjects

Pranadinata, R.

Publications and source records attributed to Pranadinata, R..

4 recordsLinked to original sources

bioRxiv2023

A Living Organoid Biobank of Crohn's Disease Patients Reveals Molecular Subtypes for Personalized Therapeutics

ABSTRACT (Structured)Crohns disease (CD) is a complex, clinically heterogeneous disease of multifactorial origin; there is no perfect pre-clinical model, little insight into the basis for such heterogeneity, and still no cure. To address these unmet needs, we sought to explore the translational potential of adult stem cell-derived organoids that not only retain their tissue identity, but also their genetic and epigenetic disease-driving traits. We prospectively created a biobank of CD patient-derived organoid cultures (PDOs) using biopsied tissues from colons of 34 consecutive subjects representing all clinical subtypes (Montreal Classification B1-B3 and perianal disease). PDOs were generated also from healthy subjects. Comparative gene expression analyses enabled benchmarking of PDOs as tools for modeling the colonic epithelium in active disease and revealed that despite the clinical heterogeneity there are two major molecular subtypes: immune-deficient infectious-CD [IDICD] and stress and senescence-induced fibrostenotic-CD [S2FCD]. The transcriptome, genome and phenome show a surprising degree of internal consistency within each molecular subtype. The spectrum of morphometric, phenotypic, and functional changes within the "living biobank" reveals distinct differences between the molecular subtypes. These insights enabled drug screens that reversed subtype-specific phenotypes, e.g., impaired microbial clearance in IDICD was reversed using agonists for nuclear receptors, and senescence in S2FCD was rectified using senotherapeutics, but not vice versa. Phenotyped-genotyped CD-PDOs may fill the gap between basic biology and patient trials by enabling pre-clinical Phase 0 human trials for personalized therapeutics. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/532245v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1edcea0org.highwire.dtl.DTLVardef@198a3c8org.highwire.dtl.DTLVardef@28d394org.highwire.dtl.DTLVardef@5a9dbb_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefThis work creates a prospectively biobanked phenotyped-genotyped Crohns disease patient-derived organoids (CD-PDOs) as platforms for molecular subtyping of disease and for ushering personalized therapeutics. HIGHLIGHTSO_LIProspectively biobanked CD-organoids recapitulate the disease epithelium in patients C_LIO_LIThe phenome-transcriptome-genome of CD-organoids converge on two molecular subtypes C_LIO_LIOne subtype shows impaired microbial clearance, another increased cellular senescence C_LIO_LIPhenotyped-genotyped PDOs are then used for integrative and personalized therapeutics C_LI

immunology↗

bioRxiv2021

The interaction of enteric bacterial effectors with the host engulfment pathway control innate immune responses

BackgroundHost engulfment protein ELMO1 generates intestinal inflammation following internalization of enteric bacteria. In Shigella, bacterial effector IpgB1 interacts with ELMO1 and promotes bacterial invasion. IpgB1 belongs to the WxxxE effector family, a motif found in several effector of enteric pathogens. Here, we have studied the role of WxxxE effectors, with emphasis on Salmonella SifA and whether it interacts with ELMO1 to regulate inflammation. MethodologyIn-silico-analysis of WxxxE effectors was performed using BLAST search and Clustal W program. The interaction of ELMO1 with SifA was assessed by GST pulldown assay and co-immunoprecipitation. ELMO1 knock-out mice, and ELMO1-depleted murine macrophage J774 cell lines were challenged with WT and SifA mutant Salmonella. Bacterial effectors containing the WxxxE motif were transfected in WT and ELMO1-depleted J774 cells to assess the inflammatory cytokines. ResultsELMO1 generates differential pro-inflammatory cytokines between pathogenic and non-pathogenic bacteria. WxxxE motif is present in pathogens and in the TIR domain of host proteins. The C-terminal part of ELMO1 interacts with SifA where WxxxE motif is important for interaction. ELMO1-SifA interaction affects the bacterial colonization, dissemination, and inflammatory cytokines in vivo. Moreover, ELMO1-SifA interaction increases TNF- and IL-6 production from the macrophage cell line and is associated with enhanced Rac1 activity. ELMO1 also interacts with WxxxE effectors IpgB1, IpgB2, and Map, and induces inflammation after challenge with microbe or microbial ligand. ConclusionELMO1 generates a differential response through interaction with the WxxxE motif which is absent in commensals. ELMO1-WxxxE interaction plays a role in bacterial pathogenesis and induction of inflammatory response. HighlightsO_LIELMO1 generates a differential immune response between enteric pathogens and commensals. C_LIO_LIEnteric bacterial effectors containing WxxxE signature motif interact with ELMO1. C_LIO_LIThe WxxxE effector of Salmonella SifA interacts with the C-terminal part of ELMO1. C_LIO_LIELMO1-SifA interaction increases the inflammatory response in vivo and in vitro. C_LI

microbiology↗

bioRxiv2020

TLR4 signaling and macrophage inflammatory responses are dampened by GIV/Girdin

Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multi-modular scaffold, GIV (a.k.a Girdin) titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (LPS, a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress pro-inflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene depletion studies confirmed that the presence of GIV ameliorates DSS-induced colitis and sepsis-induced death. The anti-inflammatory actions of GIV are mediated via its C-terminally located TIR-like BB-loop (TILL)-motif which binds the cytoplasmic TIR-modules of TLR4 in a manner that precludes receptor dimerization; the latter is a pre-requisite for pro-inflammatory signaling. Binding of GIVs TILL motif to other TIR modules inhibits pro-inflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses. SignificanceTo ensure immunity, and yet limit pathology, inflammatory responses must be confined within the proverbial Goldilocks zone. TLR4 is the prototypical sensor that orchestrates inflammatory responses through a series of well characterized downstream cascades. How TLR4 signals are confined remain incompletely understood. Using trans-scale approaches ranging from disease modeling in live animals, through cell-based interventional studies, to structure-guided biochemical studies that offer an atomic-level resolution, this study unravels the existence of a brake within the TLR4 signaling cascade, i.e., GIV; the latter is a prototypical member of an emerging class of scaffold proteins. By showing that GIV uses conserved mechanisms to impact multi-TLR signaling, this work unravels a multi-scale point of convergence of immune signaling of broader impact beyond TLR4.

immunology↗

bioRxiv2020

E-cigarettes compromise the gut barrier and trigger gut inflammation

E-cigarette and vaping device use continue to rise, particularly in adolescents and young adults, but the safety of inhaling the multitude of chemicals within e-cigarette aerosols has been questioned. While several studies have evaluated vaping effects on the lungs and heart; effects on the gastrointestinal tract remain unknown. Using established murine models of acute (1 week) and chronic (3 month) daily e-cigarette aerosol inhalation, both with nicotine-containing and vehicle control e-liquids, murine colon transcriptomics and organoid co-culture models, we assessed the effects of e-cigarette use on the gut barrier and mucosal health. Histologic analyses revealed that chronic exposure to nicotine-free e-cigarette aerosols induced mucosal inflammation. Transcriptome analyses revealed that chronic, but not acute, nicotine-free e-cigarette use significantly reduced expression of tight junction markers, including occluding, and drove expression of pro-inflammatory cytokines. Exposure of murine and human enteroid-derived monolayers (EDMs) to nicotine-free e-cigarette aerosols alone, or in co-culture with invasive E. coli, confirmed that repetitive exposure was sufficient to recapitulate the key findings observed in vivo, i.e., barrier-disruption, downregulation of occludin, inflammation, and an accentuated risk of and response to bacterial infection. These data highlight an unexpected harmful effect of e-cigarette use on the gut barrier and pinpoint non-nicotine chemical components common across >90% of e-cigarette e-liquids as the source of harm. Given the ever-expanding importance of the integrity of the gut barrier for host fitness, and impact of gut mucosal inflammation on a multitude of chronic diseases, these findings are broadly relevant to medicine and public health. SIGNIFICANCEThe safety of electronic cigarettes has been questioned amidst emerging evidence that they may derail our immune system and increase our susceptibility to infections. Despite these insights, their impact on the most critical entity that separates trillions of microbes from the largest immune system in our body, i.e., the gut barrier, remains unexplored. Using a combination of mouse models, gut transcriptomics, and murine and human gut-derived organoids, here we show that chronic exposure to aerosols of electronic-cigarette disrupts the gut barrier, increases its susceptibility to bacterial infections and triggers inflammation. Given the importance of the gut barrier in the maintenance of immune homeostasis, these findings provide valuable insights into the potential long-term harmful effects of electronic cigarettes on health.

immunology↗