In the Reh Lab, our research team focuses on neurodegenerative diseases that affect the retina by causing the death of neurons which result in irreversible blindness. In working towards treatments for retinal diseases we have engineered Muller glia to serve as a source for functional regeneration in the mammalian retina, but not enough to restore lost vision. While this is remarkable progress, limitations to this regeneration strategy still exist in regards to the amount of neurons generated. Recently, we have found that the endogenous immune cell of the retina, the microglia, responds to dead neurons by causing inflammation, which restricts the regenerative capacity of Muller glia. However, microglia are not the only immune cell present during regeneration and we currently do not have an understanding of the diversity of cell types that contribute to this regeneration restricting inflammation. My research project aimed to fill this gap by revealing which immune cells infiltrate the retina after injury and thereby revealing immunomodulation targets to improve retinal regeneration. In order to begin to understand the interaction of the peripheral immune system and the nervous system and how it impacts neurogenesis, I will present on my examination of monocyte and neutrophil invasion after retinal injury. For this study I have processed retinas using cryosectioning, immunohistochemistry, fluorescent antibody labeling, and confocal microscopy. Using this data I visualized and quantified the infiltration dynamics of monocytes and neutrophils in the damaged and regenerating retina. Data from this proposal will determine the types of immune cells and their window of infiltration for cell-type specific immunomodulation strategies to improve the regenerative capacity of the mammalian retina.This understanding of the immune component of regeneration is critical in moving forward to develop therapies for not only retinal diseases such as glaucoma but all neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

Exposure to arsenic (As) through contaminated drinking water is a far-reaching problem in Washington State, as well as the entire United States. There is a demand for novel approaches to arsenic remediation as current technologies prove to be either too expensive or ineffective at lower arsenic concentrations. Periphyton is a community of different organisms, including blue-green algae, bacteria, fungi, plant detritus and animals. In addition to providing dissolved oxygen and soil to its environment, periphyton has been shown to accumulate arsenic at far higher concentrations than the water around it. Our lab aimed to discover if bacteria were in fact responsible for the uptake of arsenic observed in periphyton. So far, we have isolated several species of the Pseudomonas and Janithobacterium genera that are resistant to high levels of arsenic. We further isolated species contained within the periphyton, and then determined that these organisms are metabolizing arsenic. We investigated the genetic basis of the arsenic resistance in our isolates by generating targeted deletions of known arsenic metabolism genes and through a genetic screen of a pool of transposon insertional mutants for gain of arsenic sensitivity. With these identified genes linked to arsenic transformation and resistance of arsenic toxicity, it will then become possible to alter, repress, or enhance these processes. In the future this data may provide utility in arsenic remediation.

The gut microbiome is a central regulator of overall health, and establishing mutually beneficial relationships between the host and resident gut bacteria is important for preventing the later development of pathologies such as ulcerative colitis, metabolic dysregulation, and colon cancer. While microbiota-reactive maternal IgA antibodies in the breastmilk have historically been considered to be a primary regulator of host-microbiota interactions, maternal IgG antibodies have historically been associated with the protection of neonates against pathogens. However, our lab has identified a novel function for microbiota-reactive maternal IgG2b and IgG3 antibodies in preventing dysregulated adaptive immune responses and reinforcing intestinal homeostasis in early life. To investigate how these maternal antibodies function, we created a mouse strain (IgG3^-/-) lacking IgG3 antibodies. Using IgG3^-/- dams, we were able to selectively prevent the transfer of maternal IgG3 antibodies to their pups while maintaining normal transfer of the other antibody isotypes. Compared to the pups born to wild type (i.e. antibody replete) dams, these pups displayed reduced weight gain, increased inflammatory gene expression, dysregulated T cell immunity, and increased susceptibility to intestinal colitis induced by dextran sodium sulfate (DSS). With our data suggesting an integral role for maternal IgG3 antibodies in limiting neonatal immune responses toward gut microbes, my current experiments aim to elucidate the underlying mechanisms by which these antibodies function. Using pups born to C1q deficient and FcγR deficient dams, which lack the ability to activate the complement pathway and FcγRs, respectively, I am aiming to test the roles of these antibody “sensing” pathways in maternal IgG3-mediated suppression of neonatal intestinal immunity. Defining these mechanisms not only gives us a clearer picture of the ways in which neonatal health is regulated; it also advances our ability to manipulate neonatal immunity and early life gut microbiome-targeted treatments to improve human health.

Gene gun technology uses DNA-coated gold microparticle bombardment to achieve DNA vaccine immunogenicity by effective intradermal transfer. This delivery system results in increased uptake of plasmids by cells, potent cellular and humoral immunity compared to other delivery methods like direct needle injection or electroporation. DNA vaccines are expected to exhibit long term stability, ease of manufacturing, low cost, reduce cold chain requirements, and reduce concern for vector immunity from repeated immunizations. Additionally, gene gun DNA vaccine technology allows for multiple antigens to be added in the same immunization. Beyond the advantages above, genetic adjuvants have the potential to further improve the immunogenicity of DNA vaccines. One strategy to improve the immunogenicity of DNA vaccination would be using chemokines like Xcl1 to recruit dendritic cells for induction of CD8+ and CD4+ T-cells. Xcl1 is a ligand that binds to and specifically activates dermal dendritic cells, which presents Xcr1 and efficiently activates T-cell responses. Two ways to introduce Xcl1 as an adjuvant are fusion and cotransfection, the latter of which is the method used in this project. In cotransfection, Xcl1 cytokines induce localized inflammatory responses which recruit dendritic cells. This allows the antigen of interest to target the dendritic cells, leading to the induction of CD8+ T-cells specific to the antigen of interest. I believe that Xcl1 has the potential to induce antigen-specific effector and memory CD8+ T-cells and enhance proliferation of CD4+ and CD8+ T-cells. I gene gun vaccinated mice with and without Xcl1 DNA and assessed the induction of appropriate T-cell responses to determine whether the addition of Xcl1 as an adjuvant enhances the T-cell and antibody response needed to protect against P. yoelii infection challenge. The results of this ongoing work will be presented. This data will inform whether genetic adjuvants have the ability to increase immunogenicity in DNA malaria vaccines.

Kaposi’s Sarcoma (KS) is a highly vascularized tumor, which affects AIDS patients worldwide and remains endemic to sub-Saharan Africa. Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is the etiological agent of KS and its latent infection is involved in tumor formation and the induction of angiogenesis in the spindle cell, a cell of endothelial origin and the main proliferating cell type in a KS tumor. Previous RNA-Seq data obtained by our group showed that osteopontin (opn), a secreted protein known to act as a ligand for integrin receptors that activate signaling cascades that promote angiogenesis, is highly upregulated at the transcript level during KSHV latent infection of endothelial cells. To determine whether opn is required for the activation of angiogenesis in KSHV latently-infected endothelial cells, we used CRISPR-lentiviral constructs to knock out opn and evaluate changes in angiogenic phenotypes upon KSHV infection via cell proliferation, tubule formation, and cell migration assays. Preliminary results reveal a significant reduction in tubule formation in opn knockout KSHV-infected endothelial cells. This finding suggests that opn upregulation in such cells is responsible for the activation of this angiogenic phenotype. Future experimentation will include evaluating how KSHV induces the upregulation of opn by infecting wild-type endothelial cells with mutant viruses lacking certain latency protein genes and evaluating differences in opn transcriptional and translation, as well as evaluating the mechanisms by which opn activates tubule formation in KSHV latently-infected endothelial cells. Identifying  the drivers of angiogenesis in KSHV-infected endothelial cells will aid the characterization of therapeutic targets for KS progression in such cells, given that KS tumors are highly angiogenic from its early stages.

Mycobacterium abscessus (Mabsc) infections occur in more than 5% of patients with cystic fibrosis (CF), increasing over the last decade. Antibiotic treatment for those with the infection can take many months and often are not successful at eradicating the organism, requiring long-term maintenance therapy. Currently, there is not an effective and evidence-based drug regimen for treating Mabsc infections in patients with CF. The conventional recommended treatment consists of 3-4 drugs, which together have an eradication rate as low as 20% in Mabsc subspecies abscessus. The goals of this project were: to collect experimental data to train the INDIGO-MABSC, an in silico model for predicting synergistic and antagonistic antibiotic interactions to treat M. abscessus infections; and to identify possible synergistic interactions between antibiotic combinations. I treated liquid cultures with 40 different antibiotics for 6 hours and then isolated genomic ribonucleic acid (RNA). The RNA was then sequenced to determine gene expression changes which can be used for model construction. Using broth microdilution, I set up checkerboard assays to observe the interactions between two different drugs and assess the combination’s efficiency in inhibiting the growth of Mabsc type strain ATCC19977 qualitatively by eye. I calculated the fractional inhibitory combination for each combination. Preliminary results show putative synergistic interactions between drug pairs Cefoxitin-Rifampin, Imipenem-Cefoxitin, Imipenem-Clarithromycin, and Imipenem-Linezolid. From sequencing results, we saw that Rifampin, Imipenem, Clofazimine, Rifabutin, and Cefoxitin did not induce sufficient gene changes. Future directions include additional checkerboard testing for combinations that were inconclusive due to no minimum inhibitory concentration recorded. Additionally, I will work optimize drug treatment for antibiotics that did not induce enough gene expression changes, and also work to collect RNA from cultures treated with novel drugs and antibiotic combinations that are commercially available.