IU Indianapolis ScholarWorks :: Browsing by Author "Brutkiewicz, Randy R."

Browsing by Author "Brutkiewicz, Randy R."

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Alterations in cellular metabolism modulate CD1d-mediated NKT-cell responses
(Oxford, 2016-08) Webb, Tonya J.; Carey, Gregory B.; East, James E.; Sun, Wenji; Bollino, Dominique R.; Kimball, Amy S.; Brutkiewicz, Randy R.; Microbiology and Immunology, School of Medicine
Natural killer T (NKT) cells play a critical role in the host's innate immune response. CD1d-mediated presentation of glycolipid antigens to NKT cells has been established; however, the mechanisms by which NKT cells recognize infected or cancerous cells remain unclear. 5′-AMP activated protein kinase (AMPK) is a master regulator of lipogenic pathways. We hypothesized that activation of AMPK during infection and malignancy could alter the repertoire of antigens presented by CD1d and serve as a danger signal to NKT cells. In this study, we examined the effect of alterations in metabolism on CD1d-mediated antigen presentation to NKT cells and found that an infection with lymphocytic choriomeningitis virus rapidly increased CD1d-mediated antigen presentation. Hypoxia inducible factors (HIF) enhance T-cell effector functions during infection, therefore antigen presenting cells pretreated with pharmacological agents that inhibit glycolysis, induce HIF and activate AMPK were assessed for their ability to induce NKT-cell responses. Pretreatment with 2-deoxyglucose, cobalt chloride, AICAR and metformin significantly enhanced CD1d-mediated NKT-cell activation. In addition, NKT cells preferentially respond to malignant B cells and B-cell lymphomas express HIF-1α. These data suggest that targeting cellular metabolism may serve as a novel means of inducing innate immune responses.
Anthrax Lethal Toxin Impairs CD1d-Mediated Antigen Presentation by Targeting the Extracellular Signal-Related Kinase 1/2 Mitogen-Activated Protein Kinase Pathway
(American Society for Microbiology, 2010-05) Khan, Masood A.; Gallo, Richard M.; Brutkiewicz, Randy R.; Microbiology and Immunology, School of Medicine
Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis and an important means by which this bacterium evades the host's immune system. In this study, we demonstrate that CD1d-expressing cells treated with LT have reduced CD1d-mediated antigen presentation. We earlier showed an important role for the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulation of CD1d-mediated antigen presentation, and we report here that LT impairs antigen presentation by CD1d in an ERK1/2-dependent manner. Similarly, LT and the ERK1/2 pathway-specific inhibitor U0126 caused a decrease in major histocompatibility complex (MHC) class II-mediated antigen presentation. Confocal microscopy analyses revealed altered intracellular distribution of CD1d and LAMP-1 in LT-treated cells, similar to the case for ERK1/2-inhibited cells. These results suggest that Bacillus anthracis has the ability to evade the host's innate immune system by reducing CD1d-mediated antigen presentation through targeting the ERK1/2 pathway.
Brain astrocytes and microglia express functional MR1 molecules that present microbial antigens to mucosal-associated invariant T (MAIT) cells
(Elsevier, 2020-12-15) Priya, Raj; Brutkiewicz, Randy R.; Microbiology and Immunology, School of Medicine
It is unknown whether brain astrocytes and microglia have the capacity to present microbial antigens via the innate immune MR1/MAIT cell axis. We have detected MAIT cells in the normal mouse brain and found that both astrocytes and microglia are MR1+. When we stimulated brain astrocytes and microglia with E. coli, and then co-cultured them with MAIT cells, MR1 surface expression was upregulated and MAIT cells were activated in an antigen-dependent manner. Considering the association of MAIT cells with inflammatory conditions, including those in the CNS, the MR1/MAIT cell axis could be a novel therapeutic target in neuroinflammatory disorders.
Cell Signaling Pathways that Regulate Ag Presentation
(American Association of Immunologists, 2016-10-15) Brutkiewicz, Randy R.; Microbiology and Immunology, School of Medicine
Cell signaling pathways regulate much in the life of a cell: from shuttling cargo through intracellular compartments and onto the cell surface, how it should respond to stress, protecting itself from harm (environmental insults or infections), to ultimately, death by apoptosis. These signaling pathways are important for various aspects of the immune response as well. However, not much is known in terms of the participation of cell signaling pathways in Ag presentation--a necessary first step in the activation of innate and adaptive T cells. In this brief review, I will discuss the known signaling molecules (and pathways) that regulate how Ags are presented to T cells and the mechanism(s) if identified. Studies in this area have important implications in vaccine development and new treatment paradigms against infectious diseases, autoimmunity and cancer.
Characterization of Hepatitis C Virus Infection of Hepatocytes and Astrocytes
(2014) Liu, Ziqing; Yu, Andy; He, Johnny J.; Brutkiewicz, Randy R.; Kao, Cheng C.; Sullivan, William J., Jr.
Approximately 2.8% of the world population is currently infected with hepatitis C virus (HCV). Neutralizing antibodies (nAbs) are often generated in chronic hepatitis C patients yet fail to control the infection. In the first two chapters of this study, we focused on two alternative routes of HCV transmission, which may contribute to HCV’s immune evasion and establishment of chronic infection. HCV was transmitted via a cell-cell contact-mediated (CCCM) route and in the form of exosomes. Formation of HCV infection foci resulted from CCCM HCV transfer and was cell density-dependent. Moreover, CCCM HCV transfer occurred rapidly, involved all four known HCV receptors and intact actin cytoskeleton, and led to productive HCV infection. Furthermore, live cell imaging revealed the temporal and spatial details of the transfer process. Lastly, HCV from HCV-infected hepatocytes and patient plasma occurred in both exosome-free and exosome-associated forms and the exosome-associated HCV remained infectious, even though HCV infection did not significantly alter exosome secretion. In the third chapter, we characterized HCV interaction with astrocytes, one of the putative HCV target cells in the brain. HCV infection causes the central nervous system (CNS) abnormalities in more than 50% of chronically infected subjects but the underlying mechanisms are largely unknown. We showed that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the free virus form or through cell-cell contact. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. In summary, we showed that CCCM HCV transfer and exosome-mediated HCV infection constituted important routes for HCV infection and dissemination and that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction. These findings provide new insights into HCV infection of hepatocytes and astrocytes and shall aid in the development of new and effective strategies for preventing and treating HCV infection.
The Complexity of Microglial Interactions With Innate and Adaptive Immune Cells in Alzheimer’s Disease
(Frontiers Media, 2020-11-19) Wyatt-Johnson, Season K.; Brutkiewicz, Randy R.; Microbiology and Immunology, School of Medicine
In the naïve mouse brain, microglia and astrocytes are the most abundant immune cells; however, there is a complexity of other immune cells present including monocytes, neutrophils, and lymphocytic cells, such as natural killer (NK) cells, T cells, and B cells. In Alzheimer’s disease (AD), there is high inflammation, reactive microglia, and astrocytes, leaky blood–brain barrier, the buildup of amyloid-beta (Aβ) plaques, and neurofibrillary tangles which attract infiltrating peripheral immune cells that are interacting with the resident microglia. Limited studies have analyzed how these infiltrating immune cells contribute to the neuropathology of AD and even fewer have analyzed their interactions with the resident microglia. Understanding the complexity and dynamics of how these immune cells interact in AD will be important for identifying new and novel therapeutic targets. Thus, this review will focus on discussing our current understanding of how macrophages, neutrophils, NK cells, T cells, and B cells, alongside astrocytes, are altered in AD and what this means for the disorder, as well as how these cells are affected relative to the resident microglia.
Control of the temporal development of Alzheimer's disease pathology by the MR1/MAIT cell axis
(BMC, 2023-03-21) Wyatt‑Johnson, Season K.; Kersey, Holly N.; Codocedo, Juan F.; Newell, Kathy L.; Landreth, Gary E.; Lamb, Bruce T.; Oblak, Adrian L.; Brutkiewicz, Randy R.; Microbiology and Immunology, School of Medicine
Background: Neuroinflammation is an important feature of Alzheimer's disease (AD). Understanding which aspects of the immune system are important in AD may lead to new therapeutic approaches. We study the major histocompatibility complex class I-related immune molecule, MR1, which is recognized by an innate-like T cell population called mucosal-associated invariant T (MAIT) cells. Methods: Having found that MR1 gene expression is elevated in the brain tissue of AD patients by mining the Agora database, we sought to examine the role of the MR1/MAIT cell axis in AD pathology. Brain tissue from AD patients and the 5XFAD mouse model of AD were used to analyze MR1 expression through qPCR, immunofluorescence, and flow cytometry. Furthermore, mice deficient in MR1 and MAIT cells were crossed with the 5XFAD mice to produce a model to study how the loss of this innate immune axis alters AD progression. Moreover, 5XFAD mice were also used to study brain-resident MAIT cells over time. Results: In tissue samples from AD patients and 5XFAD mice, MR1 expression was substantially elevated in the microglia surrounding plaques vs. those that are further away (human AD: P < 0.05; 5XFAD: P < 0.001). In 5XFAD mice lacking the MR1/MAIT cell axis, the development of amyloid-beta plaque pathology occurred at a significantly slower rate than in those mice with MR1 and MAIT cells. Furthermore, in brain tissue from 5XFAD mice, there was a temporal increase in MAIT cell numbers (P < 0.01) and their activation state, the latter determined by detecting an upregulation of both CD69 (P < 0.05) and the interleukin-2 receptor alpha chain (P < 0.05) via flow cytometry. Conclusions: Together, these data reveal a previously unknown role for the MR1/MAIT cell innate immune axis in AD pathology and its potential utility as a novel therapeutic target.
Elucidating the Characteristics and Functionality of the Mouse Mucosal-Associated Mucosal Invariant T (MAIT) Cell Receptor
(2023-08) Shrinivasan, Rashmi; Brutkiewicz, Randy R.; Dent, Alexander L.; Tran, Ngoc Tung
Mucosal-associated invariant T cells (MAIT) are a subset of invariant, innate-like T-cells that are abundant in the gut lamina propria, kidney, lungs, and peripheral blood. MAIT cells are stimulated by the recognition of microbial vitamin B-derived metabolites by the MHC class I-like molecule, MR1. Recent studies have implicated MAIT cells in several autoimmune diseases, various cancers, and CNS disorders, making it essential to design animal models that replicate the human disease state. The relatively small population of MAIT cells in mice makes it difficult to isolate and characterize them. The MAIT cell receptor (TCR) is comprised of a Vα7.2-Jα33 rearrangement in humans and TRAV1-TRAJ33 in mice. This project aimed to create a tool to study mouse MAIT cells in detail by generating lentiviral plasmid constructs expressing cDNAs encoding the MAIT cell TCR α and β chains that will be ectopically expressed in TCR-deficient mouse T cells. A bulk TCR analysis of the mouse MR1-restricted MAIT hybridomas 6C2 and 8D12 was performed to confirm variable and joining regions in the TCR α and β chains. This analysis confirmed the proper MAIT cell TCR usage in the MAIT cell hybridomas. As both MAIT cell hybridomas can be stimulated by MR1-presented antigens, we obtained synthetic cDNAs that were generated for the TRAV1-TRAJ33 α chain and TRBV8.2 (TRBV13-2) β chain. These were subcloned into GFP- and mCherry-expressing plasmids and packaged into lentiviruses that will be used for transduction of TCR-deficient mouse T cells. Flow cytometry and ELISAs will ultimately be performed to confirm the functional expression of the MAIT cell TCR. These tools will greatly facilitate the investigation of MAIT cell function in vitro and the ultimate generation of retrogenic mice for the tracking of MAIT cells in vivo.
Elucidating the role of BCL6 in helper T cell activation, proliferation, and differentiation
(2014) Hollister, Kristin N.; Dent, Alexander L.; Brutkiewicz, Randy R.; Harrington, Maureen A.; Kaplan, Mark H.
The transcriptional repressor BCL6 has been shown to be essential for the differentiation of germinal center (GC) B cells and follicular T helper (TFH) cells. The interaction of TFH and GC B cells is necessary for the development of high affinity antibodies specific for an invading pathogen. Germline BCL6-deficient mouse models limit our ability to study BCL6 function in T cells due to the strong inflammatory responses seen in these mice. To overcome this, our lab has developed a new BCL6 conditional knockout (cKO) mouse using the cre/lox system, wherein the zinc finger region of the BCL6 gene is flanked by loxP sites. Mating to a CD4-Cre mouse allowed us to study the effects of BCL6 loss specifically in T cells, without the confounding effects seen in germline knockout models. Using this cKO model, we have reaffirmed the necessity of BCL6 for TFH differentiation, including its role in sustained CXCR5 surface expression, a signature marker for TFH cells. This model also allowed us to recognize the role of BCL6 in promoting the expression of PD-1, another key surface marker for TFH cells. Without BCL6, CD4+ T cells cannot express PD-1 at the high levels seen on TFH cells. Our discovery of DNMT3b as a target for BCL6 suggests BCL6-deficient T cells have increased DNA methyltransferase activity at the PD-1 promoter. This data establishes a novel pathway for explaining how BCL6, a transcriptional repressor, can activate genes. Experiments with the BCL6 cKO model have also established a role for BCL6 in naïve CD4+ T cell activation. Furthermore, we did not observe increased differentiation of other helper T cell subsets, in contrast to what has been reported elsewhere with germline BCL6-deficient models. Unexpectedly, we found decreased T helper type 2 (Th2) cells, whereas mouse models with a germline mutation of BCL6 have increased Th2 cells. These results indicate that BCL6 activity in non-T cells is critical for controlling T cell differentiation. Finally, using an HIV-1 gp120 immunization model, we have, for the first time, shown BCL6-dependent GCs to be limiting for antibody development and affinity maturation in a prime-boost vaccine scheme.
Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses
(Springer Nature, 2021-06-23) Li, Ping; Walsh, Julia R.; Lopez, Kevin; Isidan, Abdulkadir; Zhang, Wenjun; Chen, Angela M.; Goggins, William C.; Higgins, Nancy G.; Liu, Jianyun; Brutkiewicz, Randy R.; Smith, Lester J.; Hara, Hidetaka; Cooper, David K.C.; Ekser, Burcin; Surgery, School of Medicine
Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, β4galNT2, SLA-I α chain, and β2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.

Browsing by Author "Brutkiewicz, Randy R."

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