Abstract

MT Tech

Macrophages, literally the big eaters of the immune system, are found in large numbers in nearly all human tissues.  Phagocytosis and degradation of dying cells within tissues, carried out in an immunologically silent manner, is the most common functional activity for these abundant cells.  However, disruption of tissue homeostasis results in changes to any given macrophage’s microenvironment within that tissue and quickly leads to polarization toward one of a huge variety of possible activated macrophage phenotypes.  Infections, wounds, tumor development, smoking tobacco products, and alcohol consumption are just a few examples of homeostasis disruption that can occur that cause macrophages to adopt new activation phenotypes.  Much of the time, macrophages respond appropriately to microenvironmental conditions to return tissues to “normal”.  However, inappropriate responses by macrophages can and do happen.  This justifies research efforts with the goal of therapeutic immunomodulation of macrophages as well as the monocytic cells from which they are derived. 

In this proposal, we will test the hypothesis that Leishmania spp. parasites can be genetically modified to create a novel form of macrophage-activating therapeutics.  Numerous reports have shown that cytokine- and pathogen-associated molecular pattern (PAMP)-expressing protozoan parasites elicit immune responses in mice that minimize pathology yet provide improved responses to challenge with wild-type parasites, suggesting that one use for the transgenic leishmania parasites that we will generate could be used as a live, attenuated leishmania vaccine.  Next, safety considerations such as using nonhuman-parasitic Leishmania spp. or the inclusion of antibiotic sensitivity (commonly referred to as a “suicide gene”) into human parasitic Leishmania spp. are approaches that have been successfully utilized in mouse models suggesting that transgenic parasites vectors could be clinically translated to humans.  In the first specific aim, we will generate transgenic leishmania parasites designed to express cytokines and PAMPs.  By monitoring cytokine transcript levels and microbicidal activity, the new lines will be compared to the parental parasite lines in their respective ability to activate human monocytic cells.  In the second aim, we will use readily-accessible CRISPR tools to knock out cognate receptors in human cells of the cytokines and PAMPs expressed by the transgenic parasites.  We will approach this goal in a two-step manner.  First, using an easy to transfect cell line, we will screen the CRISPR interference constructs for the ability to functionally knock out the receptor.  Subsequently, genes in human monocytic cells will be targeted utilizing a recent breakthrough in efficient lentiviral transduction technology.

Significance of research toward rural and Native American health disparities.  At the most basic level, many of the diseases affecting rural and Native American populations in Montana involve immune responses by monocytic cells such as macrophages.  As leishmania parasites naturally infect monocytic cells, this vector represents an innovative approach for targeted immunomodulatory therapeutics.  Live, attenuated leishmanial vaccine candidates are another potential outcome from the research plan proposed here.  Therefore, progress toward a leishmanial vaccine would benefit millions of Central American and South American rural and indigenous populations

Specific Aims

Statement of problem: Macrophages are cells of the innate immune system that become activated in response to an array of endogenous and exogenous environmental stimuli.  The type of response that these cells have can be either beneficial or detrimental in many disease settings.  Immunotherapy has received much attention and targeting macrophages is gaining support.

Hypothesis: Leishmania parasites can be genetically modified to create a novel form of macrophage-activating therapeutics. In a limited number of studies, leishmania and related parasites that express either cytokines or pathogen-associated molecular patterns (PAMPs) are reported to be immunomodulatory.  These studies have all utilized murine models of leishmaniasis.  Our efforts will be focused on studying human monocytic cell activation in response to attenuated, human Leishmania spp. as well as the nonpathogenic L. tarentolae.

  1. Evaluate the efficacy of transgenic leishmania lines designed to activate macrophages.  Macrophages are a host cell during leishmaniasis.  This situation allows cytokine- and PAMP-expressing leishmania parasites to be a natural vector for targeting human macrophages for immunomodulation, as has been seen in mice.  While generating transgenic leishmania lines for this purpose, we will intentionally be looking to improve the safety profile of these parasites by i) continuously passaging human leishmania parasites in axenic cultures, a known method of reducing virulence, and ii) evaluating whether the nonhuman pathogen, L. tanentolae, is a comparable substitute for various human-specific parasites.
  2. Establish human monocytic cell lines that lack specific cytokine and PAMP receptors.  Efficient transduction of human monocytic cells with lentiviral vectors is a relatively recent advance.  In human monocytic cell lines, we will knock out the cognate receptors of the cytokine- and PAMP-expressing Leishmania spp. parasites generated in specific aim 1.  While working toward this goal, we will become proficient at producing lentiviral vectors and transducing monocytic cell lines so that we may i) move our efforts to utilizing primary human macrophages in subsequent studies and ii) quickly adapt to generating lentiviral vectors designed to create additional monocytic cell lines with useful genetic modifications.

 Significance

Macrophages.  An abundance of macrophages populate nearly every animal tissue [1].  These cells have the opportunity to carry out important roles in countless situations as macrophages are ever-present sentinels.  In fact, macrophages can have a pathogenic or protective role in settings as diverse as arthritis, cancer, cardiovascular disease, diabetes, gastrointestinal health, various infectious diseases, obesity, substance abuse (e.g. liver fibrosis associated with alcohol abuse), and traumatic brain injury [1-6].  Narrowing the scope to a single organ, such as the lungs, emphasizes the importance of a residential population, such as alveolar macrophages, in many settings including allergies, asthma, acute lung injury, acute respiratory distress syndrome, pulmonary infections, occupational exposure to pollutants (e.g. diesel exhaust), COPD, lung cancer, and pulmonary fibrosis [7-14].            

Macrophages can become activated in response to a variety of stimuli within their microenvironment.  The type of macrophage response dictates whether these activated cells will be protective or pathogenic.  Macrophage activation, modeled using either monocytic cell lines or monocyte-derived macrophages (MDMs) and specific activating conditions in vitro, are routinely used to define both gene expression regulation programs and functional attributes, such as microbicidal activity.  Until very recently, transcriptome-based approaches to characterize activated macrophages typically analyzed only two or three macrophage-activating conditions per study.  We have published a microarray-based study that was designed to determine gene expression profiles in human MDMs activated using 33 different conditions [15].  Our results and those from another similarly designed study support a model that is best described as a spectrum [15, 16], rather than the standard model represented linearly with extremes of “M1” and “M2.”

Therapeutics targeting macrophages.  Since macrophages play an influential role in so many disease settings, multiple strategies have been used to therapeutically manipulate these cells.  The receptor for the monocytic cell survival factor M-CSF has been targeted in tumor-associated macrophages (TAMs) using antibodies and small molecule inhibitors [17, 18].  It has been proposed that once reliable macrophage activation markers are established, antibodies could be used to specifically deplete only the macrophages with detrimental activation states [19].  Another successful route of macrophage-based therapies has been the transfer of macrophages, activated ex vivo, back to the donor.  This technique is efficacious in divergent settings, including diabetes, cancer, and Staphylococcus aureus biofilm formation [20-22].

Genetic approaches targeting macrophages are also showing promise.  For example, overexpression of human alpha1-antitrypsin using a lentiviral vector successfully impaired emphysema progression in mice [23].  The same group used lentivirus vectors to control LPS-induced lung injury by RNAi-mediated knock down of the p65 subunit of NF-κB in alveolar macrophages [24].  An impressive range of pathologies have therapeutically targeted macrophages including rodent models of atherosclerosis, acute hepatitis and hepatic injury, arthritis, inflammatory bowel disease, bacterial and viral infections, cancer, and systemic inflammation [25-39].

Leishmaniasis is a parasitic disease that is endemic in 98 countries [40] including many countries in Central and South America were millions of Native Americans live.  The severity of leishmaniasis ranges from asymptomatic to life-threatening depending on host and parasite genetic factors, environmental factors, and the species of leishmania [41].  There are no approved vaccines to protect against leishmaniasis.  Moreover, there is considerable need for the development of novel therapeutic strategies due to expense, toxicity, and emerging resistance to current leishmaniasis treatment regimens [42].

The hypothesis of this proposal is that leishmania parasites can be genetically modified to result in a novel form of macrophage-activating therapeutics.  The macrophage-activating leishmania proposed here would provide candidates for leishmanization-based vaccines as well as novel human macrophage-activating immunotherapeutic.  Thus, the research proposed here is at once applicable to leishmaniasis, but may also be applicable to the wide range of disease states where macrophage activation phenotypes are involved.

Primary Contact

Joel Graff jgraff@mtech.edu