1. Results To confirm previously reported results that IL-3 KO mice have a distinct phenotype from WT mice, we infected mice with malaria for four or eight days and then assessed change in weight, hematocrit, and spleen size. Both WT and KO mice had increased weight at 4 dpi but lost weight by 8dpi with KO losing significantly more (Figure 1A,B). Splenomegaly was induced in both mice after 8dpi, but KO mice had spleens that were ~5% larger than WT (Figure 1D). Hematocrit increased over days of infection in WT mice but decreased with days of infection in KO mice (Figure 1C). Methods Plasmodium berghei infection (Auclair et al., 2014). Mice were infected with blood stages of P. berghei by injecting 10 5 parasitized red blood cells (pRBC) intraperitoneally (i.p.) into mice between 8-12 weeks of age. Blood collected from the retroorbital sinus into heparinized tubes was used to determine hematocrit and the cells were used for flow cytometry. Alternatively, plasma was collected for MAGPIX. Flow Cytometry. Heparinized blood and splenocytes were collected and labeled with antibodies to cell surface markers to identify cell subsets. Briefly, cells were washed with FACS staining buffer (1% BSA, 0.15% NaN 3 in PBS). Leukocytes were then incubated for 10 minutes at 4° with a CD16/CD32 Fc blocking antibody, followed by incubation for 20 minutes at 4°C with combination of mAbs. In some experiments, viability was assessed with LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen, Eugene, OR). Cells were fixed with 2% paraformaldehyde and analyzed on a BD Accuri C6 Flow Cytometer (Accuri Cytometers Inc., Ann Arbor, MI). Fluorescence-minus-one (FMO) and isotype controls were used to set gates. Quantification of cytokines (Auclair et al., 2014). Plasma cytokine levels were assayed in duplicate using a 32-analyte multiplex cytokine immunoassay based on xMAP technology (no. MCYTMAG-70K-PX32; Millipore Milliplex,Billerica, MA) on a MAGPIX multiplexing instrument (Luminex Technologies, Austin, TX). Acknowledgments Mechanism for malaria: A look into the possible involvement of NK cells, NKT cells, macrophages and neutrophils Samantha Saylor 1, Chris Lantz 2, and Tracy Deem 1 1 Department of Biology, Bridgewater College, Bridgewater, Virginia 22812 2 Department of Biology, James Madison University, Harrisonburg, Virginia 22801 Literature cited Auclair, Sarah R., et al. (2014). "Interleukin-3-deficient mice have increased resistance to blood-stage Malaria." Infection and Immunity 82: 1-7. Centers for Disease Control and Prevention (2012). Impact of Malaria. http://www.cdc.gov/malaria/malaria_worldwide/impact.html Research was made possible through grants to Chris. Lantz (NIH R15: 1R15AIO94443-01) and Samantha Saylor (Martin Research Fellowship, Bridgewater College ). Figure 1. A) Change in weight at 4 and B) 8 days post infection (dpi). C ) Percent of hematocrit at 4 and 8 days post infection (dpi). D) Average spleen weight at 4 and 8 days post infection (dpi). Mice were weighed and infected with 10 5 parasitized RBCs. After four and eight days of infection, mice were reweighed. Data presented are the mean ± SEM from at least 2 experiments. *p <.05 compared with WT mice. To begin to address possible mechanisms for why IL-3 KO mice survive longer than WT mice, we used flow cytometry to look at macrophages, neutrophils, NK and NKT cells. Abstract Malaria is one of the most severe public health problems worldwide and is a leading cause of death, especially in children and pregnant women in developing countries (CDC, 2012). Murine models of malaria infection have been used to understand malaria pathogenesis. In our laboratory, we previously showed knocking out the hematopoietic growth factor, interleukin-3 (IL-3) during malarial infection with Plasmodium berghei NK65 extended the time of death of male mice, suggesting IL-3 exacerbates disease (Auclair et al.,2014). We hypothesized that the reason WT mice die faster may be due to an overwhelming recruitment of inflammatory cells that results in tissue damage. Therefore, we looked at recruitment of early inflammatory cells, namely NK cells, macrophages, and neutrophils, as well as the expression of inflammatory cytokines. While we saw no differences in the number or percentage of NK cells in the spleen and blood by flow cytometry, there was a significant increase in the number of inflammatory macrophages and neutrophils in WT mice compared to KO mice. Furthermore, there was an increase early in inflammatory cytokines in WT mice. Taken together, these data suggest that WT mice do have an early and possibly an overwhelming inflammatory response. Introduction Malaria is a leading cause of death, especially in children and pregnant women in developing countries (CDC, 2012). Using a murine model, we examined the role of Interleukin-3 (IL-3) during malarial infection. We previously reported Plasmodium berghei NK65-infected IL-3 knock-out (KO) mice survive longer than wild- type (WT) mice for reasons unrelated to hematopoiesis (Auclair et al., 2014). In addition, serum levels of Interferon (IFN)-γ are higher in WT mice early in infection. Therefore, we hypothesized that WT mice die faster than IL-3 KO mice due to a quick and overwhelming inflammatory response mediated by innate immune cells. To test this hypothesis we used flow cytometry to examine different innate immune cells, namely NK and NKT cells, macrophages, and neutrophils, as well as using multiplex cytokine immunoassays to assess cytokine production.. Previous data has shown that IFN-  levels in serum differ between KO and WT mice. To determine if local release of cytokines is different between malaria-infected KO and WT mice, we homogenized spleens, collected supernatants, and analyzed them using Magpix technology. GM-CSF, IFN- , and IP-10 were all significantly higher in WT mice at 4dpi (Figure 3). Figure 3. Cytokines differences in spleen supernatants at 4 days post infection. Mice were infected with 10 5 parasitized RBCs, sacrificed at four days, spleens collected and homogenized, and cytokines measured use multiplex immunoassays. Data presented are the mean ± SEM from at least 2 experiments. *p <.05 compared with KO mice. Figure 2. Difference in PMNs, macrophages, but not NK cells in malaria-infected WT and IL-3 KO mice. Splenocytes were harvested 4dpi, fluorescently-labeled with antibodies to surface markers, and analyzed by flow cyotmetry. A) Gating schematic for identification of cell types. Total B)splenic PMN (Ly-6C - CD11b + ), C) Inflammatory macrophages (Ly-6C ++ CD11b + ), D) Residential macrophages (Ly-6C + CD11b + ), or E) NK cells (B220+CD94+) in WT and KO mice. Data presented are the mean ± SEM from 3 experiments. *p <.05 compared with WT mice. A.A. B.B. C.C. D.D. * A.A. B. C.C. D.D. E.E. Leukocytes Macrophages Residential Macrophages Inflammatory Macrophages PMN Conclusions The experimental model was verified as previously published (Figure 1; Auclair et al., 2014), so studies were continued to determine the mechanism for why KO mice survive longer than WT mice. Since WT mice have higher IFN- γ levels early during infection in both blood (Auclair et al., 2014) and spleen (Figure 3), we hypothesized that these mice are dying from an early, overwhelming inflammatory response. Since innate immunity occurs much earlier than adaptive immunity, we chose to focus on innate immune cells that may contribute to early inflammation. We initially looked at innate immune cells known to secrete IFN-γ, namely NK and NKT cells. Flow cytometry of blood leukocytes and splenocytes showed no difference in the percentages of NK (Figure 2E) and NKT cells (data not shown), suggesting a currently unidentified cell type may be the source of the IFN-γ. In addition to these cells, neutrophils and macrophages also play a role in inflammation. The cytokine GM-CSF which mediates granulocyte recruitment into tissues was higher in the spleen of WT mice early during infection. In addition, IP-10, a cytokine induced by IFN- γ was also higher in spleens of WT mice (Figure 3). In further support of enhanced recruitment of these inflammatory cells, neutrophil percentages were higher in the blood (data not shown) and spleen (WT mice (16%) vs KO (13%)) (Figure 2B). In addition, WT mice had a higher percentage of inflammatory macrophages (47%) compared to KO (40%) (Figure 2C), even though KO mice had a higher residential macrophage percentage (37%) compared to WT (28%) (Figure 2D). Taken together, these data suggest that inflammatory cells are recruited early and at higher levels in WT mice. Future experiments will focus on other possible sources of IFN-γ.