Phospholipase D (PLD) is an enzyme that catalyzes the conversion of membrane phosphatidylcholine to choline and phosphatidic acid (PA; a second messenger). PLD is expressed in nearly all types of leukocytes and has been associated with phagocytosis, degranulation, microbial killing, and leukocyte maturation. With the application of recently developed molecular tools (i.e., expression vectors, silencing RNA, and specific antibodies), the demonstration of a key role for PLD in those and related cellular actions has contributed to a better awareness of its importance. A case in point is the recent findings that RNA interference-mediated depletion of PLD results in impaired leukocyte adhesion and chemotaxis toward a gradient of chemokines, implying that PLD is necessary for leukocyte movement. We forecast that based on results such as those, leukocytes may prove to be useful tools to unravel still-unresolved mechanistic issues in the complex biology of PLD. Three such issues are considered here: first, whether the cellular actions of PLD are mediated entirely by PA (the product of its enzymatic reaction) or whether PLD by itself interacts with other protein signaling molecules; second, the current difficulty of defining a “PA consensus site” in the various intracellular protein targets of PA; and third, the resolution of specific PLD location (upstream or downstream) in a particular effector signaling cascade. There are reasons to expect that leukocytes and their leukemic cell line counterparts will continue yielding invaluable information to cell biologists to resolve standing molecular and functional issues concerning PLD.

Bovine Viral Diarrhea Virus (BVDV) fetal infections occur in two forms; persistent infection (PI) or transient infection (TI), depending on what stage of gestation the fetus is infected. Examination of lymphoid organs from both PI and TI fetuses reveals drastically different fetal responses, dependent upon the developmental stage of the fetal immune system. Total RNA was extracted from the thymuses and spleens of uninfected control, PI, and TI fetuses collected on day 190 of gestation to test the hypothesis that BVDV infection impairs the innate and adaptive immune response in the fetal thymus and spleen of both infection types. Transcripts of genes representing the innate immune response and adaptive immune response genes were assayed by Reverse Transcription quatitative PCR (RT-qPCR) (2^−ΔΔCq; fold change). Genes of the innate immune response, interferon (IFN) inducible genes, antigen presentation to lymphocytes, and activation of B cells were downregulated in day 190 fetal PI thymuses compared to controls. In contrast, innate immune response genes were upregulated in TI fetal thymuses compared to controls and tended to be upregulated in TI fetal spleens. Genes associated with the innate immune system were not different in PI fetal spleens; however, adaptive immune system genes were downregulated, indicating that PI fetal BVDV infection has profound inhibitory effects on the expression of genes involved in the innate and adaptive immune response. The downregulation of these genes in lymphocytes and antigen-presenting cells in the developing thymus and spleen may explain the incomplete clearance of BVDV and the persistence of the virus in PI animals while the upregulation of the TI innate immune response indicates a more mature immune system, able to clear the virus.

We report the molecular mechanisms that underlie chemotaxis of macrophages and cell migration of fibroblasts, cells that are essential during the body's innate immune response and during wound repair, respectively. Silencing of phospholipase D1 (PLD1) and PLD2 reduced cell migration (both chemokinesis and chemotaxis) by approximately 60% and >80%, respectively; this migration was restored by cell transfection with PLD2 constructs refractory to small interfering RNA (siRNA). Cells overexpressing active phospholipase D1 (PLD1) but, mostly, active PLD2 exhibited cell migration capabilities that were elevated over those elicited by chemoattractants alone. The mechanism for this enhancement is complex. It involves two pathways: one that is dependent on the activity of the lipase (and signals through its product, phosphatidic acid [PA]) and another that involves protein-protein interactions. The first is evidenced by partial abrogation of chemotaxis with lipase activity-defective constructs (PLD2-K758R) and by n-butanol treatment of cells. The second is evidenced by PLD association with the growth factor receptor-bound protein 2 (Grb2) through residue Y(169), located within a Src homology 2 (SH2) consensus site. The association Grb2-PLD2 could be visualized by fluorescence microscopy in RAW/LR5 macrophages concentrated in actin-rich membrane ruffles, making possible that Grb2 serves as a docking or intermediary protein. The Grb2/PLD2-mediated chemotaxis process also depends on Grb2's ability to recognize other motility proteins, like the Wiskott-Aldrich syndrome protein (WASP). Cell transfection with WASP, PLD2, and Grb2 constructs yields the highest levels of cell migration response, particularly in a macrophage cell line (RAW/LR5) and only modestly in the fibroblast cell line COS-7. Further, RAW/LR5 macrophages utilize for cell migration an additional pathway that involves S6 kinase (S6K) through PLD2-Y(296), known to be phosphorylated by epidermal growth factor receptor (EGFR) kinase. Thus, both fibroblasts and macrophages use activity-dependent and activity-independent signaling mechanisms. However, highly mobile cells like macrophages use all signaling machinery available to them to accomplish their required function in rapid immune response, which sets them apart from fibroblasts, cells normally nonmobile that are only briefly involved in wound healing.

Animal models are critical to the advancement of our knowledge of infectious disease pathogenesis, diagnostics, therapeutics, and prevention strategies. The use of animal models requires thoughtful consideration for their well-being, as infections can significantly impact the general health of an animal and impair their welfare. Application of the 3Rs—replacement, refinement, and reduction—to animal models using biohazardous agents can improve the scientific merit and animal welfare. Replacement of animal models can use in vitro techniques such as cell culture systems, mathematical models, and engineered tissues or invertebrate animal hosts such as amoeba, worms, fruit flies, and cockroaches. Refinements can use a variety of techniques to more closely monitor the course of disease. These include the use of biomarkers, body temperature, behavioral observations, and clinical scoring systems. Reduction is possible using advanced technologies such as in vivo telemetry and imaging, allowing longitudinal assessment of animals during the course of disease. While there is no single method to universally replace, refine, or reduce animal models, the alternatives and techniques discussed are broadly applicable and they should be considered when infectious disease animal models are developed.

Bovine viral diarrhea virus (BVDV) continues to cost the cattle industry millions of dollars each year despite control measures. The primary reservoirs for BVDV are persistently infected (PI) animals, which are infected in utero and shed the virus throughout their lifetime. The difficulty in controlling the virus stems from a limited understanding of transplacental transmission and fetal development of immunotolerance. In this study, pregnant BVDV naïve heifers were inoculated with BVDV on day 75 of gestation and fetal spleens were collected on gestational days 82, 97, 190, and 245. Microarray analysis on splenic RNA from days 82 and 97 revealed an increase in signaling for the innate immune system and antigen presentation to T cells in day 97 PI fetuses compared to controls. RT-qPCR on select targets validated the microarray revealing a downregulation of type I interferons and lymphocyte markers in day 190 PI fetuses compared to controls. Protein was visualized using western blot and tissue sections were analyzed with H&E staining and immunohistochemistry. Data collected indicates that fetal immunotolerance to BVDV developed between days 97 and 190, with mass attenuation of the immune system on day 190 of gestation. Furthermore, lymphocyte transcripts were initially unchanged then downregulated, suggesting that immunotolerance to the virus stems from a blockage in lymphocyte activation and hence an inability to clear the virus. The identification of lymphocyte derived immunotolerance will aid in the development of preventative and viral control measures to implement before or during pregnancy. BVDV is an infectious RNA virus which is a major cause of production losses in cattle due to its contribution to bovine respiratory disease in feedlot animals and reproductive losses in cow-calf (breeding) herds. Persistently infected (PI), immunotolerant animals are the main source of BVDV infections in cattle. The generation of PI animals by vertical transmission of BVDV during early pregnancy has been known for over 50 years; however, the mechanisms responsible for the immunotolerance to and persistence of BVDV in PI animals have not been elucidated [1-3]. This in vivo study provides not only a unique perspective on the development of immunotolerance to BVDV in PI fetuses, but contributes to our understanding the development of the bovine fetal immune system.