Modeling human diseases as networks simplify complex multi-cellular processes, helps understand patterns in noisy data that humans cannot find, and thereby improves precision in prediction. Using Inflammatory Bowel Disease (IBD) as an example, here we outline an unbiased AI-assisted approach for target identification and validation. A network was built in which clusters of genes are connected by directed edges that highlight asymmetric Boolean relationships. Using machine-learning, a path of continuum states was pinpointed, which most effectively predicted disease outcome. This path was enriched in gene-clusters that maintain the integrity of the gut epithelial barrier. We exploit this insight to prioritize one target, choose appropriate pre-clinical murine models for target validation and design patient-derived organoid models. Potential for treatment efficacy is confirmed in patient-derived organoids using multivariate analyses. This AI-assisted approach identifies a first-in-class gut barrier-protective agent in IBD and predicted Phase-III success of candidate agents.

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 multimodular scaffold, GIV (a.k.a. Girdin), titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress proinflammatory 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 dextran sodium sulfate-induced colitis and sepsis-induced death. The antiinflammatory 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; such dimerization is a prerequisite for proinflammatory signaling. Binding of GIV’s TILL motif to TIR modules inhibits proinflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses.

The gut barrier separates trillions of microbes from the largest immune system in the body; when compromised, a "leaky" gut barrier fuels systemic inflammation, which hastens the progression of chronic diseases. Strategies to detect and repair the leaky gut barrier remain urgent and unmet needs. Recently, a stress-polarity signaling (SPS) pathway has been described in which the metabolic sensor, AMP-kinase acts via its effector, GIV (also known as Girdin) to augment epithelial polarity exclusively under energetic stress and suppresses tumor formation. Using murine and human colon-derived organoids, and enteroid-derived monolayers (EDMs) that are exposed to stressors, we reveal that the SPS-pathway is active in the intestinal epithelium and requires a catalytically active AMP-kinase. Its pharmacologic augmentation resists stress-induced collapse of the epithelium when challenged with microbes or microbial products. In addition, the SPS-pathway is suppressed in the aging gut, and its reactivation in enteroid-derived monolayers reverses aging-associated inflammation and loss of barrier function. It is also silenced during progression of colorectal cancers. These findings reveal the importance of the SPS-pathway in the gut and highlights its therapeutic potential for treating gut barrier dysfunction in aging, cancer, and dysbiosis.

Plasmacytoid dendritic cells (pDC) are type I interferon-producing cells with critical functions in a number of human illnesses; however, their molecular regulation is incompletely understood. Here we show the role of Src family kinases (SFK) in mouse and human pDCs. pDCs express Fyn and Lyn and their activating residues are phosphorylated both before and after Toll-like receptor (TLR) stimulation. Fyn or Lyn genetic ablation as well as treatment with SFK inhibitors ablate pDC (but not conventional DC) responses both in vitro and in vivo. Inhibition of SFK activity not only alters TLR-ligand localization and inhibits downstream signalling events, but, independent of ex-vivo TLR stimulation, also affects constitutive phosphorylation of BCAP, an adaptor protein bridging PI3K and TLR pathways. Our data identify Fyn and Lyn as important factors that promote pDC responses, describe the mechanisms involved and highlight a tonic SFK-mediated signalling that precedes pathogen encounter, raising the possibility that small molecules targeting SFKs could modulate pDC responses in human diseases.

Cellular levels of the versatile second messenger cyclic (c)AMP are regulated by the antagonistic actions of the canonical G protein → adenylyl cyclase pathway that is initiated by G-protein–coupled receptors (GPCRs) and attenuated by phosphodiesterases (PDEs). Dysregulated cAMP signaling drives many diseases; for example, its low levels facilitate numerous sinister properties of cancer cells. Recently, an alternative paradigm for cAMP signaling has emerged in which growth factor–receptor tyrosine kinases (RTKs; e.g., EGFR) access and modulate G proteins via a cytosolic guanine-nucleotide exchange modulator (GEM), GIV/girdin; dysregulation of this pathway is frequently encountered in cancers. In this study, we present a network-based compartmental model for the paradigm of GEM-facilitated cross-talk between RTKs and G proteins and how that impacts cellular cAMP. Our model predicts that cross-talk between GIV, G αs, and G αi proteins dampens ligand-stimulated cAMP dynamics. This prediction was experimentally verified by measuring cAMP levels in cells under different conditions. We further predict that the direct proportionality of cAMP concentration as a function of receptor number and the inverse proportionality of cAMP concentration as a function of PDE concentration are both altered by GIV levels. Taking these results together, our model reveals that GIV acts as a tunable control valve that regulates cAMP flux after growth factor stimulation. For a given stimulus, when GIV levels are high, cAMP levels are low, and vice versa. In doing so, GIV modulates cAMP via mechanisms distinct from the two most often targeted classes of cAMP modulators, GPCRs and PDEs.