Yaíma L. Lightfoot

Progressive Erosion of β-Cell Function Precedes the Onset of Hyperglycemia in the NOD Mouse Model of Type 1 Diabetes

OBJECTIVE A progressive decline in insulin responses to glucose was noted in individuals before the onset of type 1 diabetes. We determined whether such abnormalities occurred in prediabetic NOD mice—the prototypic model for human type 1 diabetes. RESEARCH DESIGN AND METHODS Morning blood glucose was measured every other day in a cohort of NOD females. Glucose tolerance and insulin secretion were measured longitudinally by intraperitoneal glucose tolerance tests in NOD/ShiLtJ and BALB/cJ mice 6 to 14 weeks of age. Arginine-stimulated insulin secretion and insulin sensitivity were assessed during intraperitoneal arginine or intraperitoneal insulin tolerance tests. RESULTS During prediabetes, NOD females displayed a progressive increase in glucose levels followed by an acute onset of hyperglycemia. First-phase insulin responses (FPIRs) during the intraperitoneal glucose tolerance test (IPGTT) declined before loss of glucose tolerance in NOD. The failure of FPIR could be detected, with a decline in peak insulin secretion during IPGTT. Arginine-stimulated insulin secretion remained unchanged during the study period. The decline in insulin secretion in NOD mice could not be explained by changes in insulin sensitivity. CONCLUSIONS There was an impressive decline in FPIR before changes in glucose tolerance, suggesting that impairment of FPIR is an early in vivo marker of progressive β-cell failure in NOD mice and human type 1 diabetes. We portend that these phenotypes in NOD mice follow a similar pattern to those seen in humans with type 1 diabetes and validate, in a novel way, the importance of this animal model for studies of this disease.

SIGNR3-dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis

Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C‐type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3−/− mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3‐induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD.

Epithelial CaSR deficiency alters intestinal integrity and promotes proinflammatory immune responses

The intestinal epithelium is equipped with sensing receptor mechanisms that interact with luminal microorganisms and nutrients to regulate barrier function and gut immune responses, thereby maintaining intestinal homeostasis. Herein, we clarify the role of the extracellular calcium‐sensing receptor (CaSR) using intestinal epithelium‐specific Casr −/− mice. Epithelial CaSR deficiency diminished intestinal barrier function, altered microbiota composition, and skewed immune responses towards proinflammatory. Consequently, Casr −/− mice were significantly more prone to chemically induced intestinal inflammation resulting in colitis. Accordingly, CaSR represents a potential therapeutic target for autoinflammatory disorders, including inflammatory bowel diseases.

Colonic Immune Stimulation by Targeted Oral Vaccine

Background Currently, sufficient data exist to support the use of lactobacilli as candidates for the development of new oral targeted vaccines. To this end, we have previously shown that Lactobacillus gasseri expressing the protective antigen (PA) component of anthrax toxin genetically fused to a dendritic cell (DC)-binding peptide (DCpep) induced efficacious humoral and T cell-mediated immune responses against Bacillus anthracis Sterne challenge. Methodology/Principal Finding In the present study, we investigated the effects of a dose dependent treatment of mice with L. gasseri expressing the PA-DCpep fusion protein on intestinal and systemic immune responses and confirmed its safety. Treatment of mice with different doses of L. gasseri expressing PA-DCpep stimulated colonic immune responses, resulting in the activation of innate immune cells, including dendritic cells, which induced robust Th1, Th17, CD4+Foxp3+ and CD8+Foxp3+ T cell immune responses. Notably, high doses of L. gasseri expressing PA-DCpep (1012 CFU) were not toxic to the mice. Treatment of mice with L. gasseri expressing PA-DCpep triggered phenotypic maturation and the release of proinflammatory cytokines by dendritic cells and macrophages. Moreover, treatment of mice with L. gasseri expressing PA-DCpep enhanced antibody immune responses, including IgA, IgG1, IgG2b, IgG2c and IgG3. L. gasseri expressing PA-DCpep also increased the gene expression of numerous pattern recognition receptors, including Toll-like receptors, C-type lectin receptors and NOD-like receptors. Conclusion/Significance These findings suggest that L. gasseri expressing PA-DCpep has substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration and may be used as a safe oral vaccine against anthrax challenge.

Targeting aberrant colon cancer-specific DNA methylation with lipoteichoic acid-deficient Lactobacillus acidophilus.

Pathogenic autoinflammatory responses triggered by dysregulated microbial interactions may lead to intestinal disorders and malignancies. Previously, we demonstrated that a lipoteichoic acid (LTA)-deficient Lactobacillus acidophilus strain, NCK2025, ameliorated inflammation-induced colitis, significantly reduced the number of polyps in a colonic polyposis cancer model and restored physiological homeostasis in both cases. Nonetheless, the regulatory signals delivered by NCK2025 to reprogram the gastrointestinal microenvironment, and thus resist colonic cancer progression, remain unknown. Accumulating evidence suggest that epigenetic changes, in the presence and absence of pathogenic inflammation, can result in colorectal cancer (CRC). To test possible epigenetic modifications induced by NCK2025, the expression of epigenetically regulated, CRC-associated genes was measured with and without bacterial treatment. In vivo and in vitro, NCK2025 enhanced the expression of tumor suppressor genes that may regulate CRC development. Therefore, differential epigenetic regulation of CRC-related genes by NCK2025 represents a potential therapy against colitis-associated and sporadic CRC.

Role of the mitochondria in immune-mediated apoptotic death of the human pancreatic β cell line βLox5.

Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA) depleted βLox5 cells, or βLox5 ρ0 cells. βLox5 ρ0 cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ0 cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways.

Tailoring gut immune responses with lipoteichoic acid-deficient Lactobacillus acidophilus

As highlighted by the development of intestinal autoinflammatory disorders when tolerance is lost, homeostatic interactions between gut microbiota, resident immune cells, and the gut epithelium are key in the maintenance of gastrointestinal health. Gut immune responses, whether stimulatory or regulatory, are dictated by the activated dendritic cells (DCs) that first interact with microorganisms and their gene products to then elicit T and B cell responses. Previously, we have demonstrated that treatment with genetically modified Lactobacillus acidophilus is sufficient to tilt the immune balance from proinflammatory to regulatory in experimental models of colitis and colon cancer. Given the significant role of DCs in efficiently orchestrating intestinal immune responses, characterization of the signals induced within these cells by the surface layer molecules, such as lipoteichoic acid (LTA), and proteins of L. acidophilus is critical for future treatment and prevention of gastrointestinal diseases. Here, we discuss the potential regulatory pathways involved in the downregulation of pathogenic inflammation in the gut, and explore questions regarding the immune responses to LTA-deficient L. acidophilus that require future studies.

Colonic Immune Suppression, Barrier Dysfunction, and Dysbiosis by Gastrointestinal Bacillus anthracis Infection

Gastrointestinal (GI) anthrax results from the ingestion of Bacillus anthracis. Herein, we investigated the pathogenesis of GI anthrax in animals orally infected with toxigenic non-encapsulated B. anthracis Sterne strain (pXO1+ pXO2−) spores that resulted in rapid animal death. B. anthracis Sterne induced significant breakdown of intestinal barrier function and led to gut dysbiosis, resulting in systemic dissemination of not only B. anthracis, but also of commensals. Disease progression significantly correlated with the deterioration of innate and T cell functions. Our studies provide critical immunologic and physiologic insights into the pathogenesis of GI anthrax infection, whereupon cleavage of mitogen-activated protein kinases (MAPKs) in immune cells may play a central role in promoting dysfunctional immune responses against this deadly pathogen.

Immune‐mediated β‐cell death in type 1 diabetes: lessons from human β‐cell lines

Eur J Clin Invest 2012; 42 (11): 1244–1251

Oxidative Stress and Beta Cell Dysfunction

Autoimmune Type 1 A Diabetes (T1D) is characterized by dependence on exogenous insulin consequential to the autoimmune attack and destruction of insulin-producing islet beta cells. Pancreatic islet cell inflammation, or insulitis, precedes beta cell death and T1D onset. In the insulitic lesion, innate immune cells produce chemokines and cytokines that recruit and activate adaptive immune cells (Eizirik D et al., Nat Rev Endocrinol 5:219-226, 2009). Locally produced cytokines not only increase immune surveillance of beta cells (Hanafusa T and Imagawa A, Ann NY Acad Sci 1150:297-299, 2008), but also cause beta cell dysfunction and decreased insulin secretion due to the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by the beta cells. This, coupled to the high levels of ROS and RNS secreted by activated macrophages and the low antioxidant capacities of beta cells (Huurman VA, PLoS One 3:e2435, 2008; Schatz D, Pediatr Diabetes 5:72-79, 2004; Verge CF, Diabetes 44:1176-1179, 1995), implicates free radicals as important effectors in T1D pathogenesis (Eizirik D et al., Nat Rev Endocrinol 5:219-226, 2009; Hanafusa T and Imagawa A, Ann NY Acad Sci 1150:297-299, 2008; Eisenbarth GS and Jeffrey J, Arq Bras Endocrinol Metabol 52:146-155, 2008; Pietropaolo M et al., Pediatr Diabetes 6:184-192, 2005).