The mammalian intestine houses a complex microbial community, which influences normal epithelial growth and development, and is integral to the repair of damaged intestinal mucosa1–3. Restitution of injured mucosa involves the recruitment of immune cells, epithelial migration and proliferation4,5. Although microenvironmental alterations have been described in wound healing6, a role for extrinsic influences, such as members of the microbiota, has not been reported. Here, we show that a distinct subpopulation of the normal mucosal-associated gut microbiota expands and preferentially colonizes sites of damaged murine mucosa in response to local environmental cues. Our results demonstrate that formyl peptide receptor 1 (FPR1) and neutrophilic NADPH oxidase (NOX2) are required for the rapid depletion of microenvironmental oxygen and compensatory responses, resulting in a dramatic enrichment of an anaerobic bacterial consortium. Furthermore, the dominant member of this wound-mucosa-associated microbiota, Akkermansia muciniphila (an anaerobic, mucinophilic gut symbiont7,8), stimulated proliferation and migration of enterocytes adjacent to the colonic wounds in a process involving FPR1 and intestinal epithelial-cell-specific NOX1-dependent redox signalling. These findings thus demonstrate how wound microenvironments induce the rapid emergence of ‘probiont’ species that contribute to enhanced repair of mucosal wounds. Such microorganisms could be exploited as potential therapeutics.

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of hACE-2 binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using vaccine and delta variant viral strains showed strong correlation with cell-based pseudovirus and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta variant resistance to neutralization in samples with paired vaccine and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.

The human enteric flora plays a significant role in intestinal health and disease. Populations of enteric bacteria can inhibit the NF-κB pathway by blockade of IκB-α ubiquitination, a process catalyzed by the E3-SCFβ-TrCP ubiquitin ligase. The activity of this ubiquitin ligase is regulated via covalent modification of the Cullin-1 subunit by the ubiquitin-like protein NEDD8. We previously reported that interaction of viable commensal bacteria with mammalian intestinal epithelial cells resulted in a rapid and reversible generation of reactive oxygen species (ROS) that modulated neddylation of Cullin-1 and resulted in suppressive effects on the NF-κB pathway. Herein, we demonstrate that butyrate and other short chain fatty acids supplemented to model human intestinal epithelia in vitro and human tissue ex vivo results in loss of neddylated Cul-1 and show that physiological concentrations of butyrate modulate the ubiquitination and degradation of a target of the E3-SCFβ-TrCP ubiquitin ligase, the NF-κB inhibitor IκB-α. Mechanistically, we show that physiological concentrations of butyrate induces reactive oxygen species that transiently alters the intracellular redox balance and results in inactivation of the NEDD8-conjugating enzyme Ubc12 in a manner similar to effects mediated by viable bacteria. Because the normal flora produces significant amounts of butyrate and other short chain fatty acids, these data provide a functional link between a natural product of the intestinal normal flora and important epithelial inflammatory and proliferative signaling pathways.

The enteric pathogen Salmonella typhimurium secretes the preformed AvrA effector protein into host cells. This acetyltransferase has been shown to modulate mammalian intestinal immune and survival responses by inhibition of JNK MAPK. To study the role of this effector in natural enteric infection, we used a mouse model to compare wild type Salmonella typhimurium to an isogenic AvrA null Salmonella mutant. Salmonella lacking AvrA induced increased intestinal inflammation, more intense systemic cytokine responses, and increased apoptosis in epithelial cells. Increased apoptosis was also observed in extra epithelial macrophages. AvrA null infected mice consistently showed higher bacterial burden within mucosal lymphoid tissues, spleen and liver by 5 days post infection, which indicated a more severe clinical course. To study the molecular mechanisms involved, recombinant adenoviruses expressing AvrA or mutant AvrA proteins were constructed, which showed appropriate expression and mediated the expected inhibition of JNK signaling. Cultured epithelial cells and macrophages transduced with AvrA expressing adenovirus were protected from apoptosis induced by exogenous stimuli. In conclusion, the results demonstrated that Salmonella AvrA modulates survival of infected macrophages likely via JNK suppression, and prevents macrophage death and rapid bacterial dissemination. AvrA suppression of apoptosis in infected macrophages may allow for establishment of a stable intracellular niche typical of intracellular pathogens.

Bacterial enteric pathogens have evolved efficient mechanisms to suppress mammalian inflammatory and immunoregulatory pathways. By exploiting the evolutionary relationship between the gut and pathogenic bacteria, we have developed a potential mucosal therapeutic. Our findings suggest that engineered preparations of the Salmonella acetyltransferase, AvrA, suppress acute inflammatory responses such as those observed in inflammatory bowel disease (IBD). We created 125 nm diameter cross-linked protein nanoparticles directly from AvrA and carrier protein to deliver AvrA in the absence of Salmonella. AvrA nanoparticles are internalized in vitro and in vivo into barrier epithelial and lamina propria monocytic cells. AvrA nanoparticles inhibit inflammatory signaling and confer cytoprotection in vitro, and in murine colitis models, we observe decreased clinical and histological indices of inflammation. Thus, we have combined naturally evolved immunomodulatory proteins with modern bioengineering to produce AvrA nanoparticles, a potential treatment for IBD.

Flagellin, the primary structural component of bacterial flagella, is recognized by Toll-like receptor 5 (TLR5) present on the basolateral surface of intestinal epithelial cells. Utilizing biochemical assays of proinflammatory signaling pathways and mRNA expression profiling, we found that purified flagellin could recapitulate the human epithelial cell proinflammatory responses activated by flagellated pathogenic bacteria. Flagellin-induced proinflammatory activation showed similar kinetics and gene specificity as that induced by the classical endogenous proinflammatory cytokine TNF-α, although both responses were more rapid than that elicited by viable flagellated bacteria. Flagellin, like TNF-α, activated a number of antiapoptotic mediators, and pretreatment of epithelial cells with this bacterial protein could protect cells from subsequent bacterially mediated apoptotic challenge. However, when NF-κB-mediated or phosphatidylinositol 3-kinase/Akt proinflammatory signaling was blocked, flagellin could induce programmed cell death. Consistently, we demonstrate that flagellin and viable flagellate Salmonella induces both the extrinsic and intrinsic caspase activation pathways, with the extrinsic pathway (caspase 8) activated by purified flagellin in a TLR5-dependant fashion. We conclude that interaction of flagellin with epithelial cells induces caspase activation in parallel with proinflammatory responses. Such intertwining of proinflammatory and apoptotic signaling mediated by bacterial products suggests roles for host programmed cell death in the pathogenesis of enteric infections. Copyright © 2006 the American Physiological Society.

The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin-mediated degradation of important signaling intermediates, including β-catenin and the NF-κB inhibitor IκB-α. Ubiquitination of these proteins as well as others is catalyzed by the SCFβTrCP ubiquitin ligase, which itself requires regulated modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8-conjugating enzyme, resulting in complete but transient loss of cullin-1 neddylation and consequent effects on NF-κB and β-catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin–proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.

Activation of the PI3K-mTOR pathway via HER2: HER3-mediated signaling in HER2+ breast cancers pose one of the major threats towards the success of trastuzumab. First, trastuzumab cannot perturb survival/proliferative signals following HER2: HER3 heterodimerization in HER2+ tumor cells. Second, trastuzumab treatment has been reported to cause drug-mediated resistance in over 50% of HER2+ breast cancers. We have reported that treatment with an anti-angiogenic drug imparted a significant anti-tumor advantage when combined with trastuzumab plus pertuzumab in the trastuzumab-resistant model of HER2+ breast cancers (PMID: 23959459). The very fact as revealed by our study that an inclusion of anti-angiogenic drug conferred a significant anti-tumor advantage when combined with dual anti-HER2 therapy clearly indicated a critical and indispensable role of angiogenesis in these tumors. Hence, we hypothesized that BEZ235 a dual PI3K/mTOR inhibitor will have an effect on the tumor as well as the angiogenic stromal compartments. In vitro and in vivo efficacy of BEZ235 was determined in HER2+ trastuzumab-sensitive, trastuzumab-resistant and HER2 amplified/PIK3CA mutated cell lines. BEZ235 alone and in combination with trastuzumab was tested on the tumor as well as stromal compartments. AKT-mTOR signal was suppressed following BEZ235 treatment in a concentration and time-dependent manner. AnnexinV, cl-CASPASE3, SURVIVIN and p-FOXO1 indicated that BEZ235-induced cell death occurred predominantly via an apoptotic pathway. Heregulin-induced HIF1a synthesis was also significantly decreased. Oncoprint data (cBioPortal) representing PAM50 Her2 enriched tumors (TCGA, Nature 2012) and Her2-positive breast tumors (TCGA, cell 2015) showed 91.4% genetic alterations and 79.2% genetic alterations in a set of four genes comprised of PIK3CA, ERBB2, VEGFA and HIF1alpha. The co-occurrence of HIF1alpha with VEGFA in PAM50 Her2 enriched tumors (TCGA, Nature 2012) and the co-occurrence of HIF1alpha with VEGFA pair as well as HIF1alpha with PIK3CA pair in Her2-positive breast tumors (TCGA, cell 2015) were found statistically significant. In xenograft models, BEZ235 blocked tumor growth and decreased Ki67, CD31, p-AKT, p-S6RP, p-4EBP1 IHC-expressions. These decreases were more pronounced when BEZ235 was combined with trastuzumab in HER2+/trastuzumab-sensitive, trastuzumab-resistant and HER2+/PIK3CA mutated models. We demonstrated that combined targeting of HER2 and the PI3K-AKT-mTOR pathway is superior to HER2-directed therapy alone. Mechanistically the inhibition of tumor-induced angiogenesis by BEZ235 caused by the down-regulation of PI3K-mTOR-HIF1alpha signaling irrespective of the trastuzumab-sensitivity status of HER2+ breast cancers proving evidence for the first time that the inhibition of angiogenesis is an important component of the anti-tumor efficacy of BEZ235 in HER2 defined breast cancers.

Inflammatory bowel disease (IBD) results from aberrant immune stimulation against a dysbiotic mucosal but relatively preserved luminal microbiota and preferentially affects males in early onset disease. However, factors contributing to sex-specific risk and the pattern of dysbiosis are largely unexplored. Core 1 β3GalT-specific molecular chaperone (Cosmc), which encodes an X-linked chaperone important for glycocalyx formation, was recently identified as an IBD risk factor by genome-wide association study. We deleted Cosmc in mouse intestinal epithelial cells (IECs) and found marked reduction of microbiota diversity in progression from the proximal to the distal gut mucosa, but not in the overlying lumen, as seen in IBD. This loss of diversity coincided with local emergence of a proinflammatory pathobiont and distal gut restricted pathology. Mechanistically, we found that Cosmc regulates host genes, bacterial ligands, and nutrient availability to control microbiota biogeography. Loss of one Cosmc allele in males (IEC-Cosmc-/y) resulted in a compromised mucus layer, spontaneous microbe-dependent inflammation, and enhanced experimental colitis; however, females with loss of one allele and mosaic deletion of Cosmc in 50% of crypts (IEC-Cosmc+/-) were protected from spontaneous inflammation and partially protected from experimental colitis, likely due to lateral migration of normal mucin glycocalyx from WT cells over KO crypts. These studies functionally validate Cosmc as an IBD risk factor and implicate it in regulating the spatial pattern of dysbiosis and sex bias in IBD.

Intestinal homeostasis is regulated in-part by reactive oxygen species (ROS) that are generated in the colonic mucosa following contact with certain lactobacilli. Mechanistically, ROS can modulate protein function through the oxidation of cysteine residues within proteins. Recent advances in cysteine labeling by the Isotope Coded Affinity Tags (ICATs) technique has facilitated the identification of cysteine thiol modifications in response to stimuli. Here, we used ICATs to map the redox protein network oxidized upon initial contact of the colonic mucosa with Lactobacillus rhamnosus GG (LGG). We detected significant LGG-specific redox changes in over 450 proteins, many of which are implicated to function in cellular processes such as endosomal trafficking, epithelial cell junctions, barrier integrity, and cytoskeleton maintenance and formation. We particularly noted the LGG-specific oxidation of Rac1, which is a pleiotropic regulator of many cellular processes. Together, these data reveal new insights into lactobacilli-induced and redox-dependent networks involved in intestinal homeostasis.