An interspecies translation model implicates integrin signaling in infliximab-resistant inflammatory bowel disease.

Anti-tumor necrosis factor (anti-TNF) therapy resistance is a major clinical challenge in inflammatory bowel disease (IBD), due, in part, to insufficient understanding of disease-site, protein-level mechanisms. Although proteomics data from IBD mouse models exist, data and phenotype discrepancies contribute to confounding translation from preclinical animal models of disease to clinical cohorts. We developed an approach called translatable components regression (TransComp-R) to overcome interspecies and trans-omic discrepancies between mouse models and human subjects. TransComp-R combines mouse proteomic data with patient pretreatment transcriptomic data to identify molecular features discernable in the mouse data that are predictive of patient response to therapy. Interrogating the TransComp-R models revealed activated integrin pathway signaling in patients with anti-TNF-resistant colonic Crohn's disease (cCD) and ulcerative colitis (UC). As a step toward validation, we performed single-cell RNA sequencing (scRNA-seq) on biopsies from a patient with cCD and analyzed publicly available immune cell proteomics data to characterize the immune and intestinal cell types contributing to anti-TNF resistance. We found that ITGA1 was expressed in T cells and that interactions between these cells and intestinal cell types were associated with resistance to anti-TNF therapy. We experimentally showed that the α1 integrin subunit mediated the effectiveness of anti-TNF therapy in human immune cells. Thus, TransComp-R identified an integrin signaling mechanism with potential therapeutic implications for overcoming anti-TNF therapy resistance. We suggest that TransComp-R is a generalizable framework for addressing species, molecular, and phenotypic discrepancies between model systems and patients to translationally deliver relevant biological insights.

Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI).

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 µL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.

The Role of Substance P in Pulmonary Clearance of Bacteria in Comparative Injury Models.

Neural input to the immune system can alter its ability to clear pathogens effectively. Patients suffering mild traumatic brain injury (mTBI) have shown reduced rates of pneumonia and a murine model replicated these findings, with better overall survival of TBI mice compared with sham-injured mice. To further investigate the mechanism of improved host response in TBI mice, this study developed and characterized a mild tail trauma model of similar severity to mild TBI. Both mild tail trauma and TBI induced similar systemic changes that normalized within 48 hours, including release of substance P. Examination of tissues showed that injuries are limited to the target tissue (ie, tail in tail trauma, brain in mTBI). Pneumonia challenge showed that mild TBI mice showed improved immune responses, characterized by the following: i) increased survival, ii) increased pulmonary neutrophil recruitment, iii) increased bacterial clearance, and iv) increased phagocytic cell killing of bacteria compared with tail trauma. Administration of a neurokinin-1-receptor antagonist to block substance P signaling eliminated the improved survival of mTBI mice. Neurokinin-1-receptor antagonism did not alter pneumonia mortality in tail trauma mice. These data show that immune benefits of trauma are specific to mTBI and that tail trauma is an appropriate control for future studies aimed at elucidating the mechanisms of improved innate immune responses in mTBI mice.

Acute-Phase Deaths from Murine Polymicrobial Sepsis Are Characterized by Innate Immune Suppression Rather Than Exhaustion.

Sepsis, a leading cause of death in the United States, has poorly understood mechanisms of mortality. To address this, our model of cecal ligation and puncture (CLP) induced sepsis stratifies mice as predicted to Live (Live-P) or Die (Die-P) based on plasma IL-6. Six hours post-CLP, both Live-P and Die-P groups have equivalent peritoneal bacterial colony forming units and recruitment of phagocytes. By 24 h, however, Die-P mice have increased bacterial burden, despite increased neutrophil recruitment, suggesting Die-P phagocytes have impaired bacterial killing. Peritoneal cells were used to study multiple bactericidal processes: bacterial killing, reactive oxygen species (ROS) generation, and phagocytosis. Total phagocytosis and intraphagosomal processes were determined with triple-labeled Escherichia coli, covalently labeled with ROS- and pH-sensitive probes, and an ROS/pH-insensitive probe for normalization. Although similar proportions of Live-P and Die-P phagocytes responded to exogenous stimuli, Die-P phagocytes showed marked deficits in all parameters measured, thus suggesting immunosuppression rather than exhaustion. This contradicts the prevailing sepsis paradigm that acute-phase sepsis deaths (<5 d) result from excessive inflammation, whereas chronic-phase deaths (>5 d) are characterized by insufficient inflammation and immunosuppression. These data suggest that suppression of cellular innate immunity in sepsis occurs within the first 6 h.

Location, location, location: cytokine concentrations are dependent on blood sampling site.

OBJECTIVE: Considerable breakthroughs in the field of sepsis have been made using animal models. Sepsis exhibits a wide array of derangements that may be evaluated in the blood, including the release of proinflammatory and anti-inflammatory cytokines. The Shock journal adheres to the ARRIVE guidelines regarding reporting in vivo results to allow reproducibility of data findings. It is generally assumed that blood cytokine concentrations collected from typical sampling sites will be similar, but there are no data validating that this is true. The main purpose of the present study was to determine if the location of blood sampling results in cytokine concentration differences following inflammatory insults. METHODS: Two different models of acute inflammation were studied. Adult, female ICR (Institute of Cancer Research) mice were injected with Escherichia coli lipopolysaccharide (n = 28) or subjected to cecal ligation and puncture (n = 16). They were killed at early time points following these inflammatory challenges for the collection of blood from the facial vein, retro-orbital sinus, and heart. Additional samples were collected in EDTA and heparin. Plasma cytokines from the same mouse were collected from each sampling site and evaluated by enzyme-linked immunosorbent assay. Clinical chemical parameters including plasma blood urea nitrogen and total protein were also analyzed. RESULTS: Regardless of model, time of collection, or cytokine measured, cytokine values from heart blood were higher than facial vein values from the same mouse. Interleukin (IL-6) collected from the heart relative to the facial vein demonstrated elevated concentrations following injection of lipopolysaccharide. In a similar manner, higher concentrations of IL-6, macrophage inflammatory protein 2, IL-10, and IL-1 receptor antagonist were found in cardiac puncture samples compared with other sampling sites 24 h after sepsis induced by cecal ligation and puncture. Similar differences were not seen when comparing blood urea nitrogen and total protein values from the two different sites. Using plasma IL-6 collected from the heart would incorrectly stratify predicted-to-live mice into the predicted-to-die category. Therefore, a simple linear regression model was developed to correctly restratify mice to their predicted fate. These data demonstrate that proinflammatory and anti-inflammatory cytokine concentrations are dramatically elevated when drawn centrally from the heart compared with collection from peripheral locations such as the facial vein. It is critical for publications to document the sampling location when evaluating plasma cytokines and attempting to compare studies.

Early murine polymicrobial sepsis predominantly causes renal injury.

Multiple organ failure in sepsis substantially increases mortality. This study examined if there was greater hepatic, pancreatic, splenic, or renal injury in mice that would die during sepsis induced by cecal ligation and puncture (CLP) compared with that of those that would survive. Mice were stratified into groups predicted to die (Die-P) or predicted to live (Live-P) in the first 5 days after CLP based on plasma interleukin 6 levels. Groups were sacrificed to harvest organs for histology. Separate animals were followed for survival with daily blood sampling to examine renal function. No significant histological evidence of organ injury was observed in either the Live-P or Die-P mice. Minimal hepatic injury occurred as plasma aspartate transaminase demonstrated less than a 2-fold increase over normal in both groups. In addition, pancreatic injury was minimal as there was also less than a 2-fold increase in plasma amylase levels. In contrast, blood urea nitrogen levels were nearly five times higher within 24 h in Die-P mice compared with those of mice predicted to live. Mice with blood urea nitrogen levels higher than 44 mg/dL had a 17.6 higher relative risk of dying (95% confidence interval, 4.5-69.4). Cystatin C, a more specific kidney function biomarker, was also elevated at 24 h after CLP. When the cystatin C levels were analyzed relative to the hours before death, rather than hours after CLP, they were also significantly increased in mice Dead by day 5 compared with those Alive after day 5. We conclude that limited liver, pancreas, and spleen injury develops during murine CLP-induced sepsis while significant kidney injury is present. The renal injury becomes worse closer to death.

Detection and quantification of cytokines and other biomarkers.

Accurate measurement of cytokine concentrations is a powerful and essential approach to the study of inflammation. The enzyme-linked immunosorbent assay (ELISA) is a simple, low-cost analytical tool that provides both the specificity and sensitivity required for the study of cytokines in vitro or in vivo. This communication describes a systematic approach to develop an indirect sandwich ELISA to detect and quantify cytokines, or other biomarkers, with accuracy and precision. Also detailed is the use of sequential ELISA assays to analyze multiple cytokines from samples with limited volumes. Finally, the concept of a multiplex ELISA is discussed with considerations given to cost and additional time required for development.