Mechanisms of Immune Activation and Suppression by Parasitic Wasps of \u3cem\u3eDrosophila\u3c/em\u3e

Drosophila melanogaster has served as an excellent model organism to study the molecular processes of innate immunity. Flies essentially lack adaptive immunity and the innate immune system is often divided into the humoral and cellular responses (Lemaitre and Hoffmann 2007). The humoral arm involves the production of antimicrobial peptides, secreted from the fat body, to combat bacterial and fungal infections. The cellular response involves the production of hemocytes (blood cells: crystal cells, plasmatocytes, and lamellocytes) in the larval lymph gland, in the sessile pools, and in circulation (Gold and Bruckner 2014). Microbial pathogens are phagocytosed by plasmatocytes whereas larger parasites such as parasitic wasp eggs are neutralized by egg encapsulation, principally by lamellocytes. The innate immune response is vital for survival against the abundant pathogens and parasites in their natural habitats. The range of microbial as well as Hymenoptera species that attack their Diptera hosts is vast. These pathogens and parasitoids have evolved strategies to either evade or suppress host immune responses (Keebaugh 2013). This thesis contains two chapters. In Chapter 1, we focused on the mechanisms underlying host defense in response to specialist wasps of D. melanogaster, Leptopilina boulardi. Chapter 1 is already published (Small et al. 2014) and I shared first authorship with Dr. Small. Previous experiments demonstrated that Notch (N) signaling is essential for crystal cell specification and differentiation (Duvic et al. 2002; Lebestky et al. 2003), and also promotes lamellocyte differentiation (Duvic et al. 2002). The N ligand, Serrate is expressed in the posterior signaling center (PSC), a non-hematopoietic cell population, also called the niche. Through direct contact, the PSC activates N signaling in the developing hematopoietic cells and instructs them to become crystal cells (Lebestky et al. 2003). L. boulardi infection promotes lamellocyte but inhibits crystal cell differentiation (Krzemien et al. 2010). ROS production is also activated in the PSC upon wasp infection (Sinenko et al. 2011). In Chapter 1, we demonstrate a second function for N signaling: L. boulardi parasitization inactivates N signaling in the developing lymph gland lobes; reduction of N signaling correlates with lamellocyte differentiation. We also demonstrate an unexpected link between N signaling and ROS in restricting differentiation of hematopoietic progenitors (Small et al. 2014). In chapter 2, we focused on strategies that the generalist parasitic wasp L. heterotoma employs to actively suppress the hosts’ immune responses. Building on previous work that showed that L. boulardi infection activates NF-κB signaling in the PSC (Gueguen et al. 2013), we examined changes in the PSC and hematopoietic progenitors after L. heterotoma infection and found reduction in gene expression in the PSC, presence of VLPs around (but not within) PSC cells, and significant reduction in the progenitor population. Consistent with previous results (Chiu 2002), this reduction correlates with Caspase activation in plasmatocytes and lysis of lamellocytes, within the lymph gland and circulating hemocyte populations. These responses are mediated by virus-like particles (VLPs) produced in the L. heterotoma venom. L. heterotoma VLPs have 4-8 spikes and the spike-to-spike distance is roughly 300 nm (Rizki and Rizki 1990). A mouse polyclonal antibody against VLPs was generated previously in our lab and immuno-electron microscopy (EM) experiments localized this protein’s origin to secretory cells of the venom gland (Chiu et al. 2006). The p40 protein is also present in large amounts in the lumen of the venom gland where VLPs undergo biogenesis and assembly (Morales et al. 2005)(Chiu et al. 2006). VLPs are ultimately deposited into the host hemocoel during the egg laying process (Chiu et al. 2006). Immuno-EM of purified mature VLPs localizes p40 to the VLP spike surface and spike termini. p40 is also present in plasmatocytes and lamellocytes of host cells. Proteomic analyses of L. heterotoma VLPs reveal more than 150 proteins, some of which are not expressed in L. boulardi (Govind lab, unpub. results). In Chapter 2, we show (1) differential effects of L. boulardi (lamellocyte differentiation, activation of gene expression in the PSC) and L. heterotoma (cell death, repression of gene expression in PSC) on lymph gland homeostasis; (2) the subcellular localization of p40 (punctate and vesicular in plasmatocytes, nuclear in lamellocytes); (3) Rab5-dependent entry into plasmatocytes but not in lamellocytes; (4) an immune function for the PSC. Molecular characterization of p40 revealed a protein with signal sequence, a central helical domain, and C-terminal transmembrane domain. The central helical domain share structural similarity with proteins of the SipD/IpaD family, normally present on tips of Gram negative bacterial type three secretion system needles. Incubation of bacterial extracts with live lamellocytes resulted in alteration in cell morphology. We hypothesize a direct role for p40 in mediating VLP entry into lamellocytes. These studies constitute the first detailed investigation of any VLP protein and begin to uncover mechanisms of active immune suppression by VLPs. They also contribute to our understanding of the biotic nature of VLPs

Differential Transcriptional Regulation of meis1 by Gfi1b and Its Co-Factors LSD1 and CoREST

Gfi1b (growth factor independence 1b) is a zinc finger transcription factor essential for development of the erythroid and megakaryocytic lineages. To elucidate the mechanism underlying Gfi1b function, potential downstream transcriptional targets were identified by chromatin immunoprecipitation and expression profiling approaches. The combination of these approaches revealed the oncogene meis1, which encodes a homeobox protein, as a direct and prominent target of Gfi1b. Examination of the meis1 promoter sequence revealed multiple Gfi1/1b consensus binding motifs. Distinct regions of the promoter were occupied by Gfi1b and its cofactors LSD1 and CoREST/Rcor1, in erythroid cells but not in the closely related megakaryocyte lineage. Accordingly, Meis1 was significantly upregulated in LSD1 inhibited erythroid cells, but not in megakaryocytes. This lineage specific upregulation in Meis1 expression was accompanied by a parallel increase in di-methyl histone3 lysine4 levels in the Meis1 promoter in LSD1 inhibited, erythroid cells. Meis1 was also substantially upregulated in gfi1b2/2 fetal liver cells along with its transcriptional partners Pbx1 and several Hox messages. Elevated Meis1 message levels persisted in gfi1b mutant fetal liver cells differentiated along the erythroid lineage, relative to wild type. However, cells differentiated along the megakaryocytic lineage, exhibited no difference in Meis1 levels between controls and mutants. Transfection experiments further demonstrated specific repression of meis1 promoter driven reporters by wild type Gfi1b but neither by a SNAG domain mutant nor by a DNA binding deficient one, thus confirming direct functional regulation of this promoter by the Gfi1b transcriptional complex. Overall, our results demonstrate direct yet differential regulation of meis1 transcription by Gfi1b in distinct hematopoietic lineages thus revealing it to be a common, albeit lineage specific, target of both Gfi1b and its paralog Gfi1

A parasitoid wasp of Drosophila employs preemptive and reactive strategies to deplete its host's blood cells.

The wasps Leptopilina heterotoma parasitize and ingest their Drosophila hosts. They produce extracellular vesicles (EVs) in the venom that are packed with proteins, some of which perform immune suppressive functions. EV interactions with blood cells of host larvae are linked to hematopoietic depletion, immune suppression, and parasite success. But how EVs disperse within the host, enter and kill hematopoietic cells is not well understood. Using an antibody marker for L. heterotoma EVs, we show that these parasite-derived structures are readily distributed within the hosts' hemolymphatic system. EVs converge around the tightly clustered cells of the posterior signaling center (PSC) of the larval lymph gland, a small hematopoietic organ in Drosophila. The PSC serves as a source of developmental signals in naïve animals. In wasp-infected animals, the PSC directs the differentiation of lymph gland progenitors into lamellocytes. These lamellocytes are needed to encapsulate the wasp egg and block parasite development. We found that L. heterotoma infection disassembles the PSC and PSC cells disperse into the disintegrating lymph gland lobes. Genetically manipulated PSC-less lymph glands remain non-responsive and largely intact in the face of L. heterotoma infection. We also show that the larval lymph gland progenitors use the endocytic machinery to internalize EVs. Once inside, L. heterotoma EVs damage the Rab7- and LAMP-positive late endocytic and phagolysosomal compartments. Rab5 maintains hematopoietic and immune quiescence as Rab5 knockdown results in hematopoietic over-proliferation and ectopic lamellocyte differentiation. Thus, both aspects of anti-parasite immunity, i.e., (a) phagocytosis of the wasp's immune-suppressive EVs, and (b) progenitor differentiation for wasp egg encapsulation reside in the lymph gland. These results help explain why the lymph gland is specifically and precisely targeted for destruction. The parasite's simultaneous and multipronged approach to block cellular immunity not only eliminates blood cells, but also tactically blocks the genetic programming needed for supplementary hematopoietic differentiation necessary for host success. In addition to its known functions in hematopoiesis, our results highlight a previously unrecognized phagocytic role of the lymph gland in cellular immunity. EV-mediated virulence strategies described for L. heterotoma are likely to be shared by other parasitoid wasps; their understanding can improve the design and development of novel therapeutics and biopesticides as well as help protect biodiversity

Impact of Phosphoproteomics in the Era of Precision Medicine for Prostate Cancer

Prostate cancer is the most common malignancy in men in the United States. While androgen deprivation therapy results in tumor responses initially, there is relapse and progression to metastatic castration-resistant prostate cancer. Currently, all prostate cancer patients receive essentially the same treatment, and there is a need for clinically applicable technologies to provide predictive biomarkers toward personalized therapies. Genomic analyses of tumors are used for clinical applications, but with a paucity of obvious driver mutations in metastatic castration-resistant prostate cancer, other applications, such as phosphoproteomics, may complement this approach. Immunohistochemistry and reverse phase protein arrays are limited by the availability of reliable antibodies and evaluates a preselected number of targets. Mass spectrometry-based phosphoproteomics has been used to profile tumors consisting of thousands of phosphopeptides from individual patients after surgical resection or at autopsy. However, this approach is time consuming, and while a large number of candidate phosphopeptides are obtained for evaluation, limitations are reduced reproducibility, sensitivity, and precision. Targeted mass spectrometry can help eliminate these limitations and is more cost effective and less time consuming making it a practical platform for future clinical testing. In this review, we discuss the use of phosphoproteomics in prostate cancer and other clinical cancer tissues for target identification, hypothesis testing, and possible patient stratification. We highlight the majority of studies that have used phosphoproteomics in prostate cancer tissues and cell lines and propose ways forward to apply this approach in basic and clinical research. Overall, the implementation of phosphoproteomics via targeted mass spectrometry has tremendous potential to aid in the development of more rational, personalized therapies that will result in increased survival and quality of life enhancement in patients suffering from metastatic castration-resistant prostate cancer

Differential Transcriptional Regulation of <em>meis1</em> by Gfi1b and Its Co-Factors LSD1 and CoREST

<div><p>Gfi1b (growth factor independence 1b) is a zinc finger transcription factor essential for development of the erythroid and megakaryocytic lineages. To elucidate the mechanism underlying Gfi1b function, potential downstream transcriptional targets were identified by chromatin immunoprecipitation and expression profiling approaches. The combination of these approaches revealed the oncogene <em>meis1</em>, which encodes a homeobox protein, as a direct and prominent target of Gfi1b. Examination of the <em>meis1</em> promoter sequence revealed multiple Gfi1/1b consensus binding motifs. Distinct regions of the promoter were occupied by Gfi1b and its cofactors LSD1 and CoREST/Rcor1, in erythroid cells but not in the closely related megakaryocyte lineage. Accordingly, Meis1 was significantly upregulated in LSD1 inhibited erythroid cells, but not in megakaryocytes. This lineage specific upregulation in Meis1 expression was accompanied by a parallel increase in di-methyl histone3 lysine4 levels in the Meis1 promoter in LSD1 inhibited, erythroid cells. Meis1 was also substantially upregulated in <em>gfi1b−/−</em> fetal liver cells along with its transcriptional partners Pbx1 and several Hox messages. Elevated Meis1 message levels persisted in <em>gfi1b</em> mutant fetal liver cells differentiated along the erythroid lineage, relative to wild type. However, cells differentiated along the megakaryocytic lineage, exhibited no difference in Meis1 levels between controls and mutants. Transfection experiments further demonstrated specific repression of <em>meis1</em> promoter driven reporters by wild type Gfi1b but neither by a SNAG domain mutant nor by a DNA binding deficient one, thus confirming direct functional regulation of this promoter by the Gfi1b transcriptional complex. Overall, our results demonstrate direct yet differential regulation of <em>meis1</em> transcription by Gfi1b in distinct hematopoietic lineages thus revealing it to be a common, albeit lineage specific, target of both Gfi1b and its paralog Gfi1.</p> </div

Regulation of the isolated <i>meis1</i> promoter by exogenous Gfi1b.

<p>Luciferase reporter based promoter assays in HEK-293T cells. 1 µg of reporter plasmid along with the indicated amount of expression plasmid and 50 ng of β-galactosidase expression vector was transfected into ∼10⁶ cells and assayed for luciferase activity. The 0.5 kb <i>gfi1b</i> core promoter was used as a positive control. The <i>meis1L</i> promoter consisted of 2.7 kb of promoter sequence from −2.1 kb to +0.65 kb (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053666#pone-0053666-g002" target="_blank">Figure 2A</a>) and the <i>meis1S</i> consisted of 1.25 kb of promoter sequence from −0.60 kb to +0.65 kb relative to the tss of <i>meis1</i>. P2A-represents the P2A-Gfi1b SNAG domain mutant; Gfi1b-del5+6-represents the Gfi1b deletion mutant lacking zinc fingers 5 and 6. For each promoter set, values shown are relative to that obtained in the absence of the corresponding expression vector, following normalization of all luciferase values for β-galactosidase levels. The average (solid bars) and standard deviations (error bars) from 3 independent experiments is shown.</p

Microarray profiling of Gfi1b and Meis1 expression in erythroid (MEL) cells upon LSD1 inhibition.

<p>Increase in mRNA levels of Gfi1b and Meis1 (fold increase) in LSD1 depleted MEL cells relative to controls.</p

Di-methyl H3–K4 levels in <i>meis1</i> promoter chromatin.

<p>Chromatin immunoprecipitation (ChIP) analysis of the <i>gfi1b</i> promoter and two <i>meis1</i> promoter segments as indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053666#pone-0053666-g002" target="_blank">Figure 2A</a> in erythroid and megakaryocytic lineages. Relative enrichment of the indicated promoter sequences for di-methyl H3–K4 in control (scrambled) versus LSD1 knocked down cells was calculated relative to that for immunoglobulin switch µ (Sµ) sequences in MEL (A) and L8057 (B) cells respectively. Results shown are the average (solid bars) and standard deviations (error bars) of three independent experiments.</p

Message levels of Gfi1b and Meis1 in erythroid and megakaryocytic cells.

<p>Quantitative PCR (qPCR) of relative Gfi1b and Meis1 mRNA levels (normalized for HPRT) in (A) MEL (erythroid) and (B) L8057 (megakaryocytic) cells transduced with empty vector (mIR-PIG) or LSD1 shRNA (LSD1 k/d). Average of three experiments is shown, error bars represent standard deviation. C. Steady state protein levels of Gfi1b, LSD1 and CoREST relative to β-actin in HEK-293T, MEL and L8057 cells.</p