Diego F. Nino

Necrotizing enterocolitis: new insights into pathogenesis and mechanisms

Necrotizing enterocolitis (NEC) is the most frequent and lethal disease of the gastrointestinal tract of preterm infants. At present, NEC is thought to develop in the premature host in the setting of bacterial colonization, often after administration of non-breast milk feeds, and disease onset is thought to be due in part to a baseline increased reactivity of the premature intestinal mucosa to microbial ligands as compared with the full-term intestinal mucosa. The increased reactivity leads to mucosal destruction and impaired mesenteric perfusion and partly reflects an increased expression of the bacterial receptor Toll-like receptor 4 (TLR4) in the premature gut, as well as other factors that predispose the intestine to a hyper-reactive state in response to colonizing microorganisms. The increased expression of TLR4 in the premature gut reflects a surprising role for this molecule in the regulation of normal intestinal development through its effects on the Notch signalling pathway. This Review will examine the current approach to the diagnosis and treatment of NEC, provide an overview of our current knowledge regarding its molecular underpinnings and highlight advances made within the past decade towards the development of specific preventive and treatment strategies for this devastating disease.

Toll-like receptor 4–mediated lymphocyte influx induces neonatal necrotizing enterocolitis

The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3+CD4+IL-17+ and reduced tolerogenic Foxp3+ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.

Retinoic Acid Improves Incidence and Severity of Necrotizing Enterocolitis by Lymphocyte Balance Restitution and Repopulation of Lgr5+ Intestinal Stem Cells.

ABSTRACT Necrotizing enterocolitis (NEC) is the most devastating gastrointestinal disease of the premature infant. We have recently shown that NEC development occurs after an increase in proinflammatory CD4+Th17 (Th17) cells and reduced anti-inflammatory forkhead box P3+ regulatory T cells (Tregs) to the premature small intestine of mice and humans, which can be experimentally reversed in mice by administration of all-trans retinoic acid (ATRA). We have also shown that NEC is characterized by apoptosis of Lgr5-positive intestinal stem cells (ISCs—Lgr5+ cells) within the crypts of Lieberkühn, which are subsequently essential for intestinal homeostasis. We now hypothesize that the normal lymphocyte balance within the lamina propria of the intestine can be achieved via administration of ATRA which restores mucosal integrity by preventing the loss of ISCs. Using both in vivo and in vitro strategies, we now demonstrate that Th17 recruitment and Treg depletion lead to increased apoptosis within ISC niches, significantly impairing proliferative capacity and mucosal healing. ATRA exerted its protective effects by preventing T cell imbalance, ultimately leading to the protection of the ISC pool preventing the development of NEC in mice. These findings raise the exciting possibility that dietary manipulations could prevent and treat NEC by modulating lymphocyte balance and the ISC pool within the newborn small intestine.

Itraconazole, a commonly used antifungal, inhibits Fcγ receptor-mediated phagocytosis: alteration of Fcγ receptor glycosylation and gene expression.

ABSTRACT Itraconazole (ICZ) is commonly used for the treatment of fungal infections, particularly in immunocompromised patients. In addition, ICZ has been recently found to have antiangiogenic effects and is currently being tested as a new chemotherapeutic agent in several cancer clinical trials. We have previously shown that ICZ impaired complex N-linked glycosylation processing, leading to the accumulation of high-mannose glycoproteins on the surface of macrophages (Møs). This investigation was directed at determining the effects of ICZ on phagocytosis as a major function of Møs. We found a significant decrease in the phagocytosis of opsonized bacterial particles in ICZ-treated murine Møs in comparison with nontreated Møs. Furthermore, the impairment of phagocytosis was associated with a decrease in cell surface expression of Fc&ggr; receptors (Fc&ggr;Rs) as well as alteration of their glycosylation pattern. Concomitantly, a reduction in all three isoforms of the Fc&ggr;R family (i.e., Fcgr1, Fcgr2, and Fcgr3) mRNA levels was observed after incubation with ICZ. The effect of ICZ on phagocytosis and Fc&ggr;R expression was reversed by addition of low-density lipoprotein. These studies indicate that ICZ treatment certainly has a dramatic effect on macrophage function, which could result in a potential impairment of the immune system’;s ability to respond to pathogens and may lead to an elevated incidence of infections.

Deletion of scavenger receptor A gene in mice resulted in protection from septic shock and modulation of TLR4 signaling in isolated peritoneal macrophages

Scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a surface glycoprotein preferentially present in macrophages that plays a primary role in innate immunity. Previous studies have shown that Sra is a modifier gene for the response to bacterial LPS in mice at the level of IL-10 production, in particular. In the present study, we found that Sra(−/−) mice are more resistant to septic shock induced by cecal ligation and puncture than wild-type C57BL/6 J (B6) mice. In addition, Sra(−/−) mice displayed initial elevated high density lipoprotein (HDL) circulating levels. Naïve peritoneal macrophages (PMϕs) were isolated from Sra(−/−) mice to understand the possible protective mechanism. Incubation of these cells with LPS was found to modulate TLR4 signaling, leading to a reduction in IL-10 and IL-6 mRNA levels, but not TNF-α expression, at low concentrations of LPS in comparison with PMϕs isolated from B6 mice. No differences were found in LPS binding between PMϕs derived from Sra(−/−) or B6 mice. The lack of Sra binding to LPS was confirmed after transfection of Chinese hamster ovary (CHO) cells with the Sra gene. The contribution of Sra to the outcome of sepsis may be a combination of changes in TLR4 signaling pathway and elevated levels of HDL in circulation, but also LPS toxicity.

Atropine treatment modifies LPS-induced inflammatory response and increases survival

Abstract.Objective:To explore the effect that Atropine, a competitive antagonist for the muscarinic acetylcholine receptor (mAChR), has on the response to LPS.Subjects:Eight-week-old, male, B6 mice.Treatment:Mice were treated with Atropine prior to, or after LPS challenge.Methods:Survival was monitored and analyzed via Kaplan-Meier analysis using the log-rank test. The effects of atropine on the inflammatory response (TNF-α, IL-6 and IL-10) were monitored at various time intervals following LPS injection in mice that were treated and not treated with atropine.Results:Atropine administration prior to LPS induction of the inflammatory response resulted in reduced TNF-α and elevated IL-10 plasma levels without affecting the production of IL-6. This reduction in TNF-α levels was independent of the increase in IL-10 production. Atropine pretreatment improved the rate of survival from endotoxic shock in mice. The improved survival of mice after endotoxic shock could still be observed when atropine was administered several hours after LPS injection.Conclusion:The administration of atropine after injury may have a beneficial clinical effect.

Sustained expression of lipocalin-2 during polymicrobial sepsis.

Sepsis is a major healthcare problem and a leading cause of death worldwide. There is no dependable diagnosis, and treatment for this condition remains mainly supportive. The etiology of sepsis is related to an overwhelming inflammatory response. In this regard, the antimicrobial protein lipocalin-2 (Lcn2) has been associated with several inflammatory conditions, but its contribution to polymicrobial sepsis is unclear. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP), and Lcn2 mRNA levels and protein expression were measured in liver and lung tissues. We observed that Lcn2 expression was robustly induced in liver and lung of C57BL/6 J (B6) mice, and remained elevated during the stage of innate immune dysfunction observed in sepsis. This response was different in A/J mice, suggesting a contribution of the genetic background, probably due to differences in IL-10 expression between these two mouse strains. Indeed, IL-10 was found to regulate Lcn2 expression in both primary and J774A.1 macrophages. Thus, Lcn2 expression is highly regulated during CLP-induced sepsis, suggesting that this antimicrobial protein could have a role as a potential biomarker for the diagnosis of sepsis.

Pulmonary Epithelial TLR4 Activation Leads to Lung Injury in Neonatal Necrotizing Enterocolitis

We seek to define the mechanisms leading to the development of lung disease in the setting of neonatal necrotizing enterocolitis (NEC), a life-threatening gastrointestinal disease of premature infants characterized by the sudden onset of intestinal necrosis. NEC development in mice requires activation of the LPS receptor TLR4 on the intestinal epithelium, through its effects on modulating epithelial injury and repair. Although NEC-associated lung injury is more severe than the lung injury that occurs in premature infants without NEC, the mechanisms leading to its development remain unknown. In this study, we now show that TLR4 expression in the lung gradually increases during postnatal development, and that mice and humans with NEC-associated lung inflammation express higher levels of pulmonary TLR4 than do age-matched controls. NEC in wild-type newborn mice resulted in significant pulmonary injury that was prevented by deletion of TLR4 from the pulmonary epithelium, indicating a role for pulmonary TLR4 in lung injury development. Mechanistically, intestinal epithelial TLR4 activation induced high-mobility group box 1 release from the intestine, which activated pulmonary epithelial TLR4, leading to the induction of the neutrophil recruiting CXCL5 and the influx of proinflammatory neutrophils to the lung. Strikingly, the aerosolized administration of a novel carbohydrate TLR4 inhibitor prevented CXCL5 upregulation and blocked NEC-induced lung injury in mice. These findings illustrate the critical role of pulmonary TLR4 in the development of NEC-associated lung injury, and they suggest that inhibition of this innate immune receptor in the neonatal lung may prevent this devastating complication of NEC.

Generation of an artificial intestine for the management of short bowel syndrome.

Purpose of reviewThis article discusses the current state of the art in artificial intestine generation in the treatment of short bowel syndrome. Recent findingsShort bowel syndrome defines the condition in which patients lack sufficient intestinal length to allow for adequate absorption of nutrition and fluids, and thus need parenteral support. Advances toward the development of an artificial intestine have improved dramatically since the first attempts in the 1980s, and the last decade has seen significant advances in understanding the intestinal stem cell niche, the growth of complex primary intestinal stem cells in culture, and fabrication of the biomaterials that can support the growth and differentiation of these stem cells. There has also been recent progress in understanding the role of the microbiota and the immune cells on the growth of intestinal cultures on scaffolds in animal models. Despite recent progress, there is much work to be done before the development of a functional artificial intestine for short bowel syndrome is successfully achieved. SummaryContinued concerted efforts by cell biologists, bioengineers, and clinician-scientists will be required for the development of an artificial intestine as a clinical treatment modality for short bowel syndrome.

Acute Acalculous Cholecystitis-Like Phenotype in Scavenger Receptor A Knock-Out Mice

BACKGROUND Sepsis is a major health problem in the United States that affects more than three-quarters of a million people every year. Previous studies have shown that scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a modifier of interleukin 10 (IL-10) expression after injection of bacterial lipopolysaccharide (LPS). Therefore, we investigated the response to sepsis in Sra knock out mice. MATERIALS AND METHODS C57BL/6J (B6) (n = 88) and Sra (-/-) mice (n = 88) were subjected to cecal ligation and puncture (CLP) using 18G or 16G needles, sham operation, or non-operated controls. At the end, mice were autopsied for the determination of abnormalities after the procedure. Cytokine gene expression was examined in lung and liver samples by quantitative RT-PCR (qRT-PCR), and circulating cholesterol levels were also measured. RESULTS Sra (-/-) mice displayed an enlargement of the gallbladder after CLP that was not detected in sham or non-operated mice or in B6 mice (wild-type) after CLP. The enlarged gallbladder resembles a condition of acute acalculous cholecystitis observed in humans. Sra (-/-) mice presented high cholesterol levels in circulation as opposed to wild type B6 mice. Moreover, Sra (-/-) mice exhibited a reduction in IL-10 mRNA levels in lungs compared to wild-type B6 mice after CLP. CONCLUSIONS The development of acute acalculous cholecystitis may be the combination of pre-existing conditions, such as hypercholesterolemia associated with a defect in Sra (Msr1) and a robust inflammation induced by sepsis.