Browsing by Subject "Macrophage"

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Determining the Effect of Cell Culture Methods on the Polarization and Phenotype of Macrophages
(2024) Lambrecht, Daniel; Chiu, Maggie; Provenzano, Paolo P.
Macrophages play an important role in the regulation of cancer tumor microenvironments (TME). The specific role they play depends on their polarization, which is divided into two general phenotypes: M1, a pro-inflammatory phenotype, and M2, an anti-inflammatory phenotype known to support tumor growth. As the field of cancer research develops, there is an increased focus on understanding the role of the macrophage in the TME and how it can be manipulated to limit the growth of the tumor. Thus, it is important for researchers who are studying the macrophage’s role in the TME to know the phenotype of the macrophages that they are culturing in their research. The goal of this study was to observe the differentiation and polarization of macrophages during the standard cell culture protocol. We expected the macrophages to be fully differentiated after 7 days of culture with M-CSF and that cell passaging would result in a higher abundance of M1 polarized macrophages in culture. However, we found that macrophages are fully polarized after only 5 days of exposure to M-CSF and that passaging has no significant effect on macrophage polarization. This implies that macrophage differentiation protocols can be shortened with no loss in macrophage yield and that passaging is a suitable cell culture method for macrophages.
Functional role of receptor-interacting protein 140 (RIP140) in adipocyte dysfunctions and inflammatory response in macrophages.
(2012-03) Ho, Ping-Chih
The prevalence of metabolic diseases in modern society, including Type II diabetes mellitus (T2DM), hypertension and cardiovascular diseases, is a major burden on health care systems. Among these diseases, T2DM and its associated complications contribute to the progression of other metabolic diseases such as fatty liver diseases and atherosclerosis. Understanding the initiation and progression of T2DM is critical for developing treatments for T2DM and its associated metabolic disorders. Adipocyte dysfunctions and chronic inflammation have been shown recently to play essential roles in the progression of T2DM. Normally, adipocytes can store energy as triglycerides, fine-tune other metabolic tissues¡¦ lipid and glucose metabolism, and secreted cytokines (adipokines) to modulate immune response. In T2DM or obesity, adipocytes become dysfunctional, with increased lipolysis, an altered adipokine profile, and decreased insulin sensitivity and glucose uptake ability. These changes affect not only the adipocytes themself but also systemic glucose and lipid metabolism. In obese patients and in the high-fat diet (HFD)-fed mouse model, increased inflammatory response in macrophages also contributes to adipocyte dysfunction. The escalated inflammatory response plays pathophysiological roles in various metabolic disorders, including atherosclerosis and arthritis, and increases the incidence of septic shock. However, the underlying mechanisms for initiation of adipocyte dysfunctions and escalation of inflammatory response remain unclear. Receptor-interacting protein 140 (RIP140) is a co-regulator for various transcription factors and nuclear receptors and is expressed mainly in macrophages and metabolic tissues, including adipocytes, hepatocytes and muscle cells. RIP140 affects the progression of T2DM through its nuclear activity as shown by the resistance of knockout mice to diet-induced diabetes and its associated metabolic disorders. In my studies, I found that when I used HFD feeding to induce T2DM, RIP140 could accumulate within the cytoplasm of adipocytes. I further demonstrated that cytoplasmic RIP140 not only interacted with AS160 to impede GLUT4 vesicle trafficking and adiponectin vesicle secretion, but also formed a complex with perilipin A to enhance lipolysis. These findings suggest that HFD feeding can alter RIP140¡¦s cellular distribution, which leads to adipocyte dysfunctions including higher lipolysis, lower glucose uptake, and reduction in adiponectin secretion. I also showed that HFD feeding promoted cytoplasmic accumulation of RIP140 in adipocytes through a PKCϵ-dependent signaling pathway by enhancing intracellular lipid content (as an intrinsic stimulus) and circulating endothelin-1 (as an extrinsic stimulus). Most importantly, administration of a selective ET-1 receptor anatagonist, ambrisentan, reduced HFD-induced cytoplasmic accumulation of RIP140 in adipocytes and further ameliorate hepatic steatosis and insulin sensitivity in vivo. These findings reveal the novel roles of cytoplasmic RIP140 in adipocyte dysfunctions and provide evidence for cytoplasmic RIP140 as a promising target for treatment of T2DM. Recently, RIP140 has also been shown to affect proinflammatory cytokine production by functioning as co-activator for NF-fÛB in macrophages. I showed that HFD feeding up-regulated RIP140 expression by promoting intracellular cholesterol level which led to increased proinflammatory potential in macrophages. In this study, intracellular cholesterol level regulates RIP140 expression by decreasing microRNA-33a, which targeted RIP140 via a conserved region in 3¡¦-UTR of RIP140 mRNA. I further discovered that TLR ligands could trigger RIP140 degradation to resolve inflammation. This RIP140 degradation was modulated by RelA-recruited SCF E3 ligase and Syk-mediated phosphorylation on RIP140. My studies in macrophages demonstrate that RIP140 in macrophages can be modulated by a HFD to affect the systemic inflammatory response and further suggest that defects in RIP140 degradation may cause non-resolving inflammation which is involved in septic shock and various metabolic disorders. Taken together, my studies provide evidence for the novel functions of RIP140 in adipocyte dysfunction and inflammatory response in macrophages and determine the mechanisms by which HFD affect RIP140¡¦s distribution and expression in adipcoytes and macrophages. These findings contribute to our understanding of how HFD causes adipocyte dysfunctions and increase inflammatory response.
The Functional Role of Receptor-Interacting Protein 140 (RIP140) in Innate Immunity and Metabolic Syndrome
(2016-12) Lin, Yi-Wei
Metabolic diseases, such as type II diabetes (T2DM), atherosclerosis and other cardiovascular diseases, are prevalent and are important health issues in the modern world. T2DM contributes to the development of various metabolic diseases. Atherosclerosis is one of the major causes leading to multiple cardiovascular diseases. In order to develop therapeutic strategies, understanding the mechanisms of these metabolic diseases is crucial. It is known that the immune system is highly involved in initiation and progression of metabolic diseases. Macrophages are one of the major leukocytes in innate immunity. Macrophages have two major polarized phenotypes: classical/pro-inflammatory (M1) and alternative/anti-inflammatory (M2). It is widely accepted that M1-M2 switch in macrophage population is essential in disease progression or damage recovery; however, the detailed mechanism of macrophage phenotype switch has not been fully elucidated. In addition, the effect of altering the macrophage phenotype on treating metabolic diseases remains uncertain. Receptor-interacting protein 140 (RIP140) is a co-regulator of numerous nuclear receptors and transcription factors. RIP140 is expressed in various cell types including adipocytes, liver, muscle, heart, neurons, and cells in the monocyte–macrophage lineage. Studies showed that RIP140 expression is positively associated with the progression of metabolic disorders such as obesity, insulin resistance, and glucose intolerance. In addition, studies indicate that RIP140 acts as a co-activator of NFκB to promote macrophage M1 activation and pro-inflammatory responses. My studies further build on this knowledge to uncover the role of RIP140 in the metabolic diseases. First, it was found that RIP140 elevates cholesterol content in macrophages by reducing expression of ABC transporters, which are responsible for cholesterol efflux. The elevated cytosolic cholesterol induces foam cell formation and further enhances progression of atherosclerosis. This study indicated that reducing RIP140 levels effectively ameliorates high-cholesteroldiet-induced atherosclerosis. Second, my study found that reducing RIP140 in macrophages leads to macrophage M2 polarization, resulting in adipose tissue remodeling to brown/beige adipose tissue. This further ameliorates high fat diet-induced T2DM associated metabolic disorders. Moreover, later studies address how RIP140 mediates macrophage M2 activation and M1/M2 switch by its cytosolic function in a wound healing animal model. Final study is to identify a beneficial taxonomic repertoire from macrophage specific RIP140 knockdown (MφRIP140KD) mice. Fecal microbiota transplantation (FMT) from HFD-fed MφRIP140KD to wild type (WT) mice acquired the benefits from donors, which is resistant to development of HFD-induced metabolic diseases. Taken together, this thesis studies elucidate novel functions of RIP140 in polarization and inflammatory responses in macrophages, and identify the benefits of reducing RIP140 expression in macrophages. These findings contribute to our understanding of the relationship between immune and metabolic systems as well as provide a therapeutic target of resolving inflammation and preventing/improving metabolic profiles in T2DM, and atherosclerosis.
Immune Modulation of Adult Neurogenesis during Experimental Herpes Simplex Encephalitis
(2015-05) Rotschafer, Jessica
Herpes Simplex Virus-1 (HSV-1) is the primary cause of sporadic viral encephalitis in the United States. While prompt treatment results in high survival rates, greater than fifty percent of patients develop severe, life-altering neurological deficits subsequent to Herpes Simplex encephalitis (HSE). Inflammatory cues generated during non-HSE viral encephalitis have been shown to alter reparative neurogenesis in humans and mice. However, interactions between induced immune responses and the normal processes of adult neurogenesis remain unexplored during HSE. The present thesis hypothesized that inflammation induced by experimental HSV-1 infection of laboratory mice alters neural stem/progenitor cell (NSC) proliferation. The response of NSCs was dynamic throughout HSV-1 infection of adult BALB/c mice, with a significant increase in the NSC population during acute HSE at 6 d p.i. and a significant decrease by 15 d p.i.. The 6 d p.i. and 15 d p.i. time points coincided with macrophage infiltration and CD8(+) T cell infiltration respectively. At 6 d p.i., infiltrating macrophages were observed to be alternatively activated (M2) whereas macrophages at 15 d p.i. were classically activated (M1 macrophages). Treatment with M2 macrophages resulted in significant increases in the NSC population size both in vivo and in vitro. In vitro, the increase in NSC proliferation was found to be mediated via Wnt5a. In vivo, transplanted M2 macrophages increased the number of NSCs and Wnt5a(+)CD11b(+) macrophages were observed in the brains of 5 d p.i. mice. On the other hand, M1 macrophages activated with interferon-γ did not support NSC proliferation, which suggested that macrophage effects may be modulated by other immune factors present in the infected brain. CD8(+) T cells are the largest population of infiltrating immune cells during chronic HSE and produce robust amounts of IFN-�. The interactions of CD8(+) T cells and NSCs were addressed in vitro and in vivo experiments. Co-culture of activated CD8(+) T cells and NSCs abrogated NSC proliferation in vitro while in vivo depletion of CD8(+) T cells restored the NSC population in infected mice. Further evaluation of the effect of CD8(+) T cells on NSC proliferation was dependent on IFN-� both in vivo and in vitro, as IFN-� receptor 1 (IFN-�R1) knockout mice were unresponsive to CD8(+) T cells in culture and infected IFN-�R1 knockout mice had no reduction in the size of the NSC population. To continue evaluation of interactions of NSC proliferation and IFN-�, alternative models of HSE were assessed in C57BL/6 mice. The kinetics of immune cell and NSC proliferation were similar between BALB/c and C57BL/6 mice, but macrophage and T cell phenotypes were different between the strains. Macrophages infiltrating the brain, at any time point examined, were persistently classically activated with an M1 phenotype, and T cell infiltration was dominated by CD4(+) T cells in C57BL/6 mice. On the other hand, NSC proliferation was significantly decreased when IFN-� was expressed suggesting that the interactions of IFN-� and NSCs were similar in both strains of mice tested. The studies presented in this dissertation are the first to suggest a connection between IFN-� produced by infiltrating T cells and impaired NSC proliferation. The mechanism by which T cells mediate this effect may be direct or through interaction with other cell types in the brain. However, there are implications that NSC proliferation and associated reparative neurogenesis may be impacted in any model of brain damage that exhibits prominent IFN-� expression.
Morphine inhibition of macrophage phagocytosis and bactericidal functions.
(2011-01) Ninkovic, Jana
For centuries, opioids have been implicated in increasing susceptibility to infection, reducing bacterial clearance, and increasing bacterial dissemination. Macrophages as key cells of innate immunity play an essential role in pathogen clearance and antigen presentation. Macrophage phagocytosis is a key mechanism responsible for host defense against bacterial pathogens. Although it is known that opioid addicts are prone to both bacterial and viral infections, the molecular and cellular mechanisms underlying these processes remain to be elucidated. Therefore the goal of this research was to investigate mechanisms of decreased bacterial clearance as a contributing factor in the increased susceptibility to infection in opiate drug abusers. To this end, first set of studies examined the role of morphine on inhibition of key mechanisms involved in Fc-gamma receptor mediated phagocytosis. It was demonstrated that morphine inhibits phagocytosis by inhibiting actin polymerization through a cAMP, PKA and MAPK dependant pathways. By superactivation of adenylyl cyclase morphine increases intracellular cAMP leading to inhibition of actin polymerization. Furthermore, morphine by inhibiting p38 MAPK and ERK 1/2 MAPK causes inhibition of actin polymerization and phagocytosis. By modulating TLR4 receptor function morphine was also able to increase macrophage phagocytosis, indicating that morphine might have a differential effect on internalization of Gram-positive, versus Gram-negative pathogens. These effects were mediated through a MyD88 and p38 MAPK dependant pathways leading to changes in actin polymerization and phagocytosis. In addition to macrophage's ability to internalize pathogens, elimination of internalized pathogen is essential for effective bacterial clearance. We therefore set out to investigate morphine's modulation of macrophage bactericidal mechanisms. We note that morphine inhibits bacterial killing by inhibiting essential mechanisms involved in this process such as formation of reactive oxygen intermediates, reactive nitrogen intermediates, as well as phago-lysosomal fusion. Morphine by inhibiting these essential mechanisms impedes eradication of bacterial infections and leads to detrimental consequences for the host. These series of studies have extended our knowledge in an underrepresented yet clinically significant field of study, however many questions still remain to be addressed and it is crucial to investigate the answers given the prevalence of morphine use today.
Physiological Roles and Regulation of NCOA4 in Macrophages
(2020-09) Guggisberg, Cole
Phagocytosing macrophages are critical to systemic iron homeostasis owing to their capability to recycle iron from senescent RBCs and store iron under systemic distress. NCOA4 has recently been identified as a key regulator of ferritin, mediating its degradation via ferritinophagy. Yet, its function in macrophages remains unclear. The present studies employed a cell culture model of J774 macrophages, to examine the role and regulation of macrophage NCOA4 by iron status, red cell iron recycling, and inflammation. Macrophage NCOA4 is responsive to iron status and inversely related to ferritin abundance. By erythrophagocytosis, ferritin peaks at 12 hours with subsequent decrease at 24 hours which is NCOA4-dependent. Hepcidin activity repressed NCOA4 preventing the turnover of ferritin between 12 and 24 hours in erythrocyte laden macrophages. Macrophages were treated with LPS, which decreased both NCOA4 transcript and protein abundance. Altogether our studies demonstrate an active role of NCOA4-mediated ferritinophagy in macrophage iron homeostasis.
Re-aligning patient prognosis: the role of collagen in establishing an immunosuppressive microenvironment and facilitating cancer cell dissemination in pancreatic ductal adenocarcinoma
(2021-10) Callaway, Mackenzie
Pancreatic ductal adenocarcinoma (PDA) is an aggressive cancer with particularly poor clinical outcomes, in part, because of a dramatically altered stromal environment and striking immune dysfunction. Physical properties within tumors— such as aligned fiber architectures—are fundamental to cancer progression and invasion, and negatively correlate with survival in cancers like those of the breast. However, the influence of aligned architectures in PDA remains unexplored. Here, we elucidate the role extracellular matrix alignment has in establishing an immunosuppressive, metastasis-conducive tumor microenvironment in early, preinvasive PDA, as well as in precursory pancreatic inflammation. Using both mouse and human samples, we demonstrate an inextricable link between collagen, alignment, and 1) immunosuppressive macrophage localization, phenotype, and function (Chapter 2); 2) epithelial cell extrusion and subsequent invasion from intact ductal structures (Chapter 3). The contribution of alignment in both driving macrophage polarization and tumor cell dissemination could be attributed to altered focal adhesion dynamics, as targeting FAK in vivo resulted in a concomitant decrease in aligned collagen architectures, disseminated tumor cells, metastatic burden, and elongated, immunosuppressive macrophages. In Chapter 4, we explore the interplay between macrophages, collagen, and cancer cell extrusion using novel 3D microtissue co-cultures and human biopsies to reveal contributions of macrophages to dissemination in vitro and in vivo. Importantly, we show aligned collagen signatures and immunosuppressive macrophages are abundantly prevalent in pancreatitis, a known risk factor for PDA, suggesting that pancreatic precursory disease may create stromal memory that is later conducive to early immunosuppression and dissemination of PDA. This work highlights the opportunity to utilize FAK inhibitors to target stromal immunity and architectures and supports a model in which collagen architecture drives the early involution of an immunosuppressive, malignant microenvironment. Further, this thesis underscores the importance of targeting stromal matrices in precursor inflammation, limit cancer progression, and “reprogram” stromal immunity.
Role of Innate Immune Macrophages in Pregnancy-Induced Hypertension
(2020-04) Hamm, Cassandra M; Towner, Kendra; Root, Kate; Regal, Jean
Preeclampsia is a pregnancy-specific disease characterized by abnormal arterial remodeling that results in placental insufficiency and placental ischemia. Recent studies have shown a specific association with macrophages and the development of hypertension. Macrophages are large, phagocytic white blood cells that have the ability to attack foreign cells and unhealthy self-cells. The pro-inflammatory cytokines produced by macrophages have been shown to contribute to blood pressure elevation and subsequent tissue damage. Macrophages can further polarize into different subtypes, labelled as M1 and M2 macrophages. In preeclampsia, data suggests that M1 macrophages increase at the maternal-fetal interface. Normally numerous macrophages reside in the peritoneal cavity and can move to different sites throughout the body depending on the circumstances. We hypothesized that placental ischemia results in macrophage movement from the peritoneal cavity to the site of ischemia in the placenta.
The Role of Macrophages in Developmental Programming of Type 2 Diabetes
(2021-03-15) Molin, Alexa M; Root, Kate; Polack, Vonda; Huchthausen, Margaretta; Regal, Jean
Hypertensive disorders are a common pregnancy complication that can increase the risk of developing type 2 diabetes (T2D) in offspring. Preeclampsia, a hypertensive disorder characterized by high blood pressure with new-onset proteinuria is initiated by placental ischemia. Studies from the Regal lab showed placental ischemia induced hypertension in female rat offspring leads to a reduced β-cell area associated with an increase in pancreatic islet macrophages. Therefore, depletion of macrophages may allow beta-cell area to recover and lower the risk of T2D. The goal of this study is to deplete macrophages in the pancreatic islets using Clophosome clodronate injections of postnatal day 13 female rats.
Survival strategies of Mycobacterium avium subsp. paratuberculosis in a variety of microenvironments.
(2012-06) Lamont, Elise A.
Mycobacteria, specifically Mycobacterium avium subsp. paratuberculosis (MAP), are extreme strategists and as a rule live by deception. Mycobacteria represent a group of closely related acid-fast bacilli that encompass a wide-range of host tropisms and diseases. Mycobacteria can be divided into two complexes: the Mycobacterium tuberculosis complex and the Mycobacterium avium complex (MAC). The MAC is comprised of M. avium subsp. avium (M. avium), MAP, M. intracellulare and M. avium subsp. hominissuis (M. hominissuis), all of which share an over 90 percent nucleotide similarity. Despite its genetic similarity, MAC elicits different diseases in both animals and humans including infections of the lung, lymph nodes, bones, skin and gastrointestinal tract. MAP is a unique member of MAC as it infects and establishes itself within the intestine of ruminants and other wildlife. Furthermore, MAP lives in a quiescent state in soil and aquatic environments. Since MAP encounters numerous environments, including those with unfavorable conditions, it has developed several strategies to survive. However, the mechanisms by which MAP survival is achieved remains incompletely understood. The goal of these studies was to determine how MAP may survive and disseminate under unfavorable conditions, which included nutrient starvation and host pressures. We have identified the development of a new MAP morphotype under prolonged nutrient starved conditions. This novel MAP morphotype resembles a spore-like structure and contains dipicolinic acid, which is used to protect DNA located within the core. These novel structures are heat resistant at 70oC and can be enriched for in multiple MAP strains. Furthermore, we describe an unrecognized mechanism by which MAP takes advantage of host responses at the epithelium interface to recruit macrophages to the site of initial infection. MAP is able to safely enter into macrophages and consequently ensures its establishment, survival and dissemination throughout the host. Lastly, we demonstrate the importance of host physiological relevant temperature on successful disease progression. Infection utilizing the temperature of MAP’s natural host, the cow, enhances the speed of infection as well as host and pathogen transcriptomic profiles. Taken together, data generated from these studies will provide the basis for understanding MAP persistence and survival in diverse conditions. The mechanisms by which MAP establishes, disseminates and/or survives difficult conditions may impact new programs to control JD as well as rational vaccine/therapeutic design and the way in which we view other mycobacterioses.

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