Faculty Bibliography

Circulating blood monocytes are a heterogeneous leukocyte population that contributes critical antimicrobial and regulatory functions during systemic and tissue-specific infections. These include patrolling vascular tissue for evidence of microbial invasion, infiltrating peripheral tissues and directly killing microbial invaders, conditioning the inflammatory milieu at sites of microbial tissue invasion, and orchestrating the activation of innate and adaptive immune effector cells. The central focus of this review is the in vivo mechanisms by which monocytes and their derivative cells promote microbial clearance and immune regulation. We include an overview of murine models to examine monocyte functions during microbial challenges and review our understanding of the functional roles of monocytes and their derivative cells in host defense against bacteria, fungi, and parasites.

There is growing interest in treating inflammatory conditions with helminth infection. Recently, Loukas and colleagues have reported promising results from using experimental hookworm infection to reduce gluten sensitivity in celiac disease patients. Analysis of microbiota samples from the trial is contributing to our understanding of the complexity underlying helminth-microbiota-host relationships.

Cellular metabolism is increasingly recognized as a controller of immune cell fate and function. MicroRNA-33 (miR-33) regulates cellular lipid metabolism and represses genes involved in cholesterol efflux, HDL biogenesis, and fatty acid oxidation. Here, we determined that miR-33-mediated disruption of the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Macrophage-specific Mir33 deletion increased oxidative respiration, enhanced spare respiratory capacity, and induced an M2 macrophage polarization-associated gene profile. Furthermore, miR-33-mediated M2 polarization required miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK), but not cholesterol efflux. Notably, miR-33 inhibition increased macrophage expression of the retinoic acid-producing enzyme aldehyde dehydrogenase family 1, subfamily A2 (ALDH1A2) and retinal dehydrogenase activity both in vitro and in a mouse model. Consistent with the ability of retinoic acid to foster inducible Tregs, miR-33-depleted macrophages had an enhanced capacity to induce forkhead box P3 (FOXP3) expression in naive CD4+ T cells. Finally, treatment of hypercholesterolemic mice with miR-33 inhibitors for 8 weeks resulted in accumulation of inflammation-suppressing M2 macrophages and FOXP3+ Tregs in plaques and reduced atherosclerosis progression. Collectively, these results reveal that miR-33 regulates macrophage inflammation and demonstrate that miR-33 antagonism is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and Treg induction.

Gut microbiota plays an important role in mammalian host metabolism and physiological functions. The functions are particularly important in young children where rapid mental and physical developments are taking place. Nevertheless, little is known about the gut microbiome and the factors that contribute to microbial variation in the gut of South East Asian children. Here, we compared the gut bacterial richness and composition of pre-adolescence in Northern Malaysia. Our subjects covered three distinct ethnic groups with relatively narrow range of socioeconomic discrepancy. These included the Malays (n = 24), Chinese (n = 17) and the Orang Asli (indigenous) (n = 20). Our results suggested a strong ethnicity and socioeconomic-linked bacterial diversity. Highest bacterial diversity was detected from the economically deprived indigenous children while the lowest diversity was recorded from the relatively wealthy Chinese children. In addition, predicted functional metagenome profiling suggested an over-representation of pathways pertinent to bacterial colonisation and chemotaxis in the former while the latter exhibited enriched gene pathways related to sugar metabolism.

Cellular metabolism is increasingly recognized to control immune cell fate and functions. miR-33 is a regulator of cellular lipid metabolism that represses genes involved in cholesterol efflux, HDL biogenesis and fatty acid oxidation. We demonstrate that by altering the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation, miR-33 instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Targeted deletion of miR-33 in macrophages increases oxidative respiration, enhances spare respiratory capacity, and induces the expression of genes that define M2 macrophage polarization. We show that these changes are independent of effects on cholesterol efflux, but instead require miR-33 targeting of the energy sensor AMP-activated protein kinase. Notably, inhibition of miR-33 also increases macrophage expression of the retinoic acid-producing enzyme Aldh1a2 and retinal dehydrogenase activity both in vitro and in vivo. Consistent with the ability of retinoic acid to foster inducible regulatory T cells, anti-miR33-treated macrophages have an enhanced capacity to induce FoxP3 expression in naive CD4+ T cells. Finally, treatment of western diet-fed Ldlr-/- mice with miR-33 inhibitors for 8 weeks (conditions that do not alter HDL cholesterol levels) promoted the accumulation of inflammation suppressing M2 macrophages and FoxP3+ T regulatory cells in plaques, and reduced atherosclerosis progression by 40 %. Collectively, these results identify a novel role for miR-33 in the regulation of macrophage inflammation and show that antagonism of miR-33 reduces atherosclerotic inflammation by promoting M2 macrophage polarization and regulatory T cell induction

Much of our understanding of gut-microbial interactions has come from mouse models. Intestinal immunity is complex and a combination of host genetics and environmental factors play a significant role in regulating intestinal immunity. Due to this complexity, no mouse model to date gives a complete and accurate representation of human intestinal diseases, such as inflammatory bowel diseases. However, intestinal tissue from patients undergoing bowel resection reflects a condition of severe disease that has failed treatment, hence a more dynamic perspective of varying inflammatory states in IBD could be obtained through the analyses of pinch biopsy material. Here we describe our protocol for analyzing mucosal pinch biopsies collected predominantly during colonoscopies. We have optimized flow cytometry panels to analyze up to 8 cytokines produced by CD4+ and CD8+ cells, as well as for characterizing nuclear proteins and transcription factors such as Ki67 and Foxp3. Furthermore, we have optimized approaches to analyze the production of cytokines, including TGF-beta from direct ex vivo cultures of pinch biopsies and LPMCs isolated from biopsies. These approaches are part of our workflow to try and understand the role of the gut microbiota in complex and dynamic human intestinal diseases.

In modern societies, diseases that are driven by dysregulated immune responses are increasing at an alarming pace, such as inflammatory bowel diseases and diabetes. There is an urgent need to understand these epidemiological trends, which are likely to be driven by the changing environment of the last few decades. There are complex interactions between human genetic factors and this changing environment that is leading to the increasing prevalence of metabolic and inflammatory diseases. Alterations to human gut bacterial communities (the microbiota) and lowered prevalence of helminth infections are potential environmental factors contributing to immune dysregulation. Helminths have co-evolved with the gut microbiota and their mammalian hosts. This three-way interaction is beginning to be characterized and the knowledge gained may enable the design of new therapeutic strategies to treat metabolic and inflammatory diseases. However, these complex interactions need to be carefully investigated in the context of host genetic backgrounds in order to identify optimal treatment strategies. The complex nature of these interactions raises the possibility that only with highly personalized treatment, with knowledge of individual genetic and microbiota communities, will therapeutic interventions be successful for a majority of the individuals suffering from these complex diseases of immune dysregulation