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Mechanism for malaria: A look into the possible involvement of NK cells, NKT cells,
macrophages and neutrophils
1
Saylor ,
2
Lantz ,
1
Deem
Samantha
Chris
and Tracy
1Department of Biology, Bridgewater College, Bridgewater, Virginia 22812
2Department of Biology, James Madison University, Harrisonburg, Virginia 22801
Abstract
Results
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.
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).
A.
Previous data has shown that IFN-g 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-g, and IP-10 were all significantly higher in WT mice at 4dpi
(Figure 3).
B.
*
.
C.
Figure 3. Cytokines differences in spleen supernatants at 4 days post infection.
Mice were infected with 105 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.
D.
Conclusions
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.
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 105 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.
A.
Macrophages
PMN
Leukocytes
Residential
Macrophages
Inflammatory
Macrophages
Methods
Plasmodium berghei infection (Auclair et al., 2014). Mice
were infected with blood stages of P. berghei by injecting 105
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% NaN3 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).
B.
C.
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.
Literature cited
D.
E.
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
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.
Acknowledgments
Research was made possible through grants to Chris. Lantz (NIH
R15: 1R15AIO94443-01) and Samantha Saylor (Martin
Research Fellowship, Bridgewater College).