Received: 20 June 2017
Revised: 15 October 2017
Accepted: 26 November 2017
DOI: 10.1111/cmi.12819
RESEARCH ARTICLE
Interferon regulatory factor 1 is essential for pathogenic CD8+ T cell migration and retention in the brain during experimental cerebral malaria | Carla Claser1 | Teck Hui Teo1 | Shanshan W. Howland1 | Sin Yee Gun1,2 1,2* | 1,3 | 1 1,2 | Laurent Rénia Chek Meng Poh Rebecca Ren Ying Chye Lisa F.P. Ng 1
Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR), Singapore
2
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
3
Department of Biological Science, National University of Singapore, Singapore
Correspondence Laurent Rénia, Singapore Immunology Network, Agency for Science, Technology and Research (ASTAR), Immunos, 8A Biomedical Grove, #03–15, Singapore 138648, Singapore. Email:
[email protected]‐star.edu.sg Present Address School of Public Health, Hong Kong University, Hong Kong SAR
Abstract Host immune response has a key role in controlling the progression of malaria infection. In the well‐established murine model of experimental cerebral malaria (ECM) with Plasmodium berghei ANKA infection, proinflammatory Th1 and CD8+ T cell response are essential for disease development. Interferon regulatory factor 1 (IRF1) is a transcription factor that promotes Th1 responses, and its absence was previously shown to protect from ECM death. Yet the exact mechanism of protection remains unknown. Here we demonstrated that IRF1‐deficient mice (IRF1 knockout) were protected from ECM death despite displaying early neurological signs. Resistance to ECM death was a result of reduced parasite sequestration and pathogenic CD8+ T cells in the brain. Further analysis revealed that IRF1 deficiency suppress interferon‐γ production and delayed CD8+ T cell proliferation. CXCR3 expression was found to be decreased in pathogenic CD8+ T cells, which limited their migration to the brain. In addition, reduced expression of
*
adhesion molecules by brain endothelial cells hampered leucocyte retention in the brain. Taken together, these factors limited sequestration of pathogenic CD8+ T cells and consequently its ability to induce extensive damage to the blood–brain barrier. KEY W ORDS
immunopathogenesis, IRF1, malaria, migration, Plasmodium berghei, T cells
1
|
I N T RO D U CT I O N
interferon‐γ (IFNγ) and lymphotoxin‐α (Hunt & Grau, 2003). Although these proinflammatory cytokines play an important role in the parasite
Innate and adaptive host immune response have been shown to play
clearance, their overwhelming production through immune cells stimu-
an important role in controlling the progression of malaria infection
lated by malaria parasite contribute to the CM pathogenesis. Unfortu-
(Artavanis‐Tsakonas, Tongren, & Riley, 2003). Plasmodium berghei
nately, the exact mechanism by which the immune system triggers the
ANKA (PbA) infection in C57BL/6 mice is the classical murine model
systemic inflammation and immune cells trafficking during malaria
of severe cerebral malaria (CM) associated with systemic disease
infection has yet to be fully uncovered.
syndromes and neurological symptoms (Rest, 1982). Susceptible mice
Interferon regulatory factor 1 (IRF1) is a transcription factor that
(e.g., C57BL/6) succumb to infection within 6–10 days (de Souza &
plays a central role in Th1 response. It is expressed in many cell types
Riley, 2002, Lou, Lucas, & Grau, 2001) as a result of dysregulated
and respond to cytokine such as IFN‐alpha/beta, IL‐12 and IFNγ, a
inflammation mediated by proinflammatory cytokines, such as
strong IRF1 inducer (Fujita, Kimura, Miyamoto, Barsoumian, &
Abbreviations: PbAluc, Plasmodium berghei ANKA tagged luciferase; CM, cerebral malaria; ECM, experimental cerebral malaria; dpi, days of post infection; WT, wild type; IRF1KO, IRF1 knockout; BBB, blood–brain barrier; BSL, brain‐sequestered leucocytes; iRBC, infected red blood cells; GrB, granzyme B This work is supported by core grants to the Singapore Immunology Network from Agency for Science, Technology and Research (A*STAR). Sin Yee Gun and Chek Meng Poh were supported by a postgraduate scholarship from Yong Loo Lin School of Medicine, National University of Singapore (Singapore). The funders played no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript.
Cellular Microbiology. 2018;20:e12819. https://doi.org/10.1111/cmi.12819
wileyonlinelibrary.com/journal/cmi
© 2017 John Wiley & Sons Ltd
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Taniguchi, 1989; Kimura et al., 1996; Miyamoto et al., 1988). IRF1 is
neurotoxin that gains access to the brain parenchyma in the presence
involved in many immunological functions, such as development of
of minute BBB damage (Hermsen, Mommers, van de Wiel, Sauerwein,
CD8+ T cells (Matsuyama et al., 1993) and modulation of balance
& Eling, 1998; Howland et al., 2013). To detect if minute damage in the
between Th1 and Th2 response (Kroger, Koster, Schroeder, Hauser,
BBB of IRF1KO mice could be responsible for the neurological symp-
& Mueller, 2002), and as a consequence, it is likely to affect suscepti-
toms observed, we injected folic acid intravenously on 7 dpi. Folic acid
bility to infectious disease. It was first reported in two West African
injection led to convulsion and death in all infected WT and IRF1KO
ethnic groups (Mangano et al., 2008) and in a Brazilian Amazonian
mice (n = 5 per group), indicating BBB damage in both groups of mice.
population (da Silva Santos et al., 2012) that a common genetic poly-
From the results of both assays, we concluded that the BBB is dam-
morphism at the IRF1 locus is associated with control of peripheral
aged in IRF1KO mice, but the extent of damage is less than WT mice
Plasmodium falciparum parasite load. However, no association with
and is insufficient to lead to ECM death.
severe anaemia and CM was detected when assessed in three other African populations (Mangano et al., 2009). Notwithstanding, animal studies have repeatedly supported the role of IRF1 in experimental
2.2
|
IRF1 controls parasite biomass
cerebral malaria (ECM) development because IRF1 knockout (IRF1KO)
Previously, two contradictory reports stated that IRF1 deficiency leads
mice were protected from ECM (Berghout et al., 2013; Senaldi et al.,
to either an increase or a decrease of parasitaemia upon infection
1999; Tan, Feng, Asano, & Kara, 1999). Because the exact implication
(Senaldi et al., 1999; Tan et al., 1999). Here, we showed that PbAluc‐
of IRF1 in ECM pathogenesis was not elucidated, in this present study,
infected IRF1KO mice displayed significantly higher parasitaemia than
we have dissected the immune response regulated by IRF1 during
WT mice throughout infection (Figure 2a). A similar effect was also
malaria infection to understand its contribution to the ECM pathology.
observed in PbA BdS infection where infected IRF1KO mice displayed significantly higher parasitaemia on 7 and 8 dpi than infected WT mice (Figure S2c). In contrast, IRF1 F1 mice displayed similar parasitaemia as
2
|
RESULTS
WT mice (Figure 2d). These demonstrated that IRF1 is involved in the control of peripheral parasite growth and the presence of a single allele
2.1 | PbA‐infected IRF1KO mice is partially protected from ECM death
is sufficient to perform this role. Because peripheral parasitaemia was higher in IRF1KO mice, we next tested if this could influence total parasite biomass and parasite
IRF1KO and WT mice were infected intraperitoneally with 106 PbAluc
sequestration in the tissues. Sequestration of PbAluc iRBC, particularly
iRBC. Although all infected WT mice died of ECM by 9 dpi, only one
in the brain, has been associated with ECM development (Claser et al.,
out of 20 IRF1KO mice died within the same ECM time window
2011). Using in vivo bioluminescence imaging, we assessed total para-
(Figure 1a). Although only 5% of IRF1KO mice died of ECM, approxi-
site accumulated in deep organs during PbAluc infection. Significantly
mately 40% displayed neurological signs between 6 and 9 dpi, which
lower bioluminescence signals were detected in the whole body
is still statistically lower on the basis of chi‐square analysis (Table 1).
(Figure 2b) and head (Figure 2c) of IRF1KO mice as compared to WT
IRF1KO mice that did not die of ECM succumbed to hyperparasitaemia
mice on 5–7 dpi and 7 dpi, respectively. Because dynamic in vivo imag-
and died in the later part of the infection (Figure 1a). To show that this
ing takes into account both circulating parasites and those sequestered
observation is not limited to a single strain of PbA, we infected IRF1KO
in the tissues, we performed ex vivo imaging to quantify iRBC accumu-
mice with another strain, PbA BdS. Once again, protection from ECM
lation in perfused and isolated organs on 7 dpi. Significantly lower
death was observed in IRF1KO mice, whereby all PbA BdS‐infected
bioluminescence signals were observed in the brain (Figure 2d), lungs,
WT mice succumbed to ECM, whereas none of the PbA BdS‐infected
heart, and spleen (Figure S3) of IRF1KO mice as compared to WT mice.
IRF1KO mice died from ECM (Figure S2a). It was reported that the
Taken together with the higher parasitaemia in IRF1KO mice, these
immune system of IRF1 F1 and IRF1KO mice responded to Leishmania
results demonstrated that absence of IRF1 curtails parasite accumula-
infection in a gene dose‐dependent manner (Lohoff et al., 1997). To
tion in tissues.
investigate if ECM outcome is also affected by the number of functional IRF1 gene, we infected IRF1 F1 with PbAluc. Presence of only one functional IRF1 allele had no effect on ECM susceptibility because IRF1 F1 developed ECM like the WT mice (Figure S2b).
2.3 | IRF1 does not influence antigen cross‐ presentation by brain microvessels
Neurological complications in ECM stems from haemorrhagic
Activated brain endothelial cells are capable of taking up parasite
damage in the brain (Nacer et al., 2012; Nacer et al., 2014) and plasma
sequestered in the brain for cross‐presentation of parasite‐derived epi-
leakage in the interstitial tissue as a result of BBB damage (Neill &
topes to parasite‐specific CD8+ T cells during ECM (Howland et al.,
Hunt, 1995). We first performed histopathological examination of
2013, Howland, Poh, & Renia, 2015). In addition, cross‐presentation
the brain tissues and observed that infected IRF1KO mice had signifi-
is directly dependent on the level of parasite sequestration in the brain
cantly fewer haemorrhages than WT mice (Figure 1b). We next per-
(Howland et al., 2013). Because infected IRF1KO mice had significantly
formed Evans Blue extravasation assay to quantify the extent of
less parasite sequestered in the brain and it was also reported that
plasma leakage in the interstitial tissue in the brain (Neill & Hunt,
IRF1KO mice exhibited defects in antigen processing machinery
1995). Significantly less vascular leakage was observed in infected
(Penninger & Mak, 1998) and MHC I expression (Hobart, Ramassar,
IRF1KO as compared to WT mice (Figure 1c). Folic acid is a lethal
Goes, Urmson, & Halloran, 1996; Hobart, Ramassar, Goes, Urmson, &
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FIGURE 1
IRF1 deficiency largely prevented experimental cerebral malaria death with minor damage in the brain. (a) Survival of WT (n = 10) and IRF1KO (n = 20) throughout infection with Plasmodium berghei ANKA tagged luciferase. The shaded area in the survival curve represents days when WT mice displayed neurological symptoms. #p < .05, log‐rank test. Data were pooled from two independent experiments. (b) Enumeration of cerebellar haemorrhages and representative histopathology images of haematoxylin and eosin‐stained brain sections of naive WT (n = 2), infected WT (n = 5), naive IRF1KO (n = 2), and infected IRF1KO (n = 5) on 7 dpi. Data are expressed as mean ± SD. Haematoxylin and eosin sections at 5× magnifications and inset at 20× magnifications. Scale bar, 100 μm. (c) Extent of Evans Blue dye leakage in the brain tissue of naive WT (n = 4), infected WT (n = 6), naive IRF1KO (n = 4), and infected IRF1KO (n = 6) on 7 dpi. Data are expressed as mean ± SEM. *p < .05, **p < .01, ***p < .001, Mann–Whitney test. IRF1 = interferon regulatory factor 1; WT = wild type; IRF1KO = IRF1 knockout; dpi = days of post infection
TABLE 1 Comparing ECM susceptibility in PbAluc‐infected WT and IRF1KO mice using Fisher's exact test
ECM NCM
WT (n = 10)
IRF1KO (n = 20)
n
n
10 0
% 100 0
40 60
degree of cross‐presentation by brain endothelial cells is not reduced.
p‐value
% 8 20
reduced parasite sequestration in the brain of IRF1KO mice, the
.0001
Note. ECM = experimental cerebral malaria; PbAluc = Plasmodium berghei ANKA tagged luciferase; WT = wild type; IRF1KO = interferon regulatory factor 1 knockout; NCM = non‐cerebral malaria.
2.4 | IRF1 is essential for parasite‐specific CD8+ T cell sequestration in the brain An afflux of leucocytes to the brain at the time of neurological manifestation is a hallmark of ECM (Belnoue et al., 2002). Therefore, we next determined the number of leucocytes sequestered in the brain on 7 dpi, when WT mice manifested neurological symptoms. Leucocytes
Halloran, 1997; Penninger & Mak, 1998), we investigated whether
were isolated from the brain of perfused mice and characterized by
absence of IRF1 affected malaria antigen presentation by brain endo-
flow cytometry. First, we observed no significant difference in the total
thelial cells. Using an ex vivo brain microvessel cross‐presentation
leucocyte number in the brains of infected WT and IRF1KO mice on
assay (Howland et al., 2013), we quantified the presentation of Pb1,
7 dpi (Figure 3a). Both total CD4+ and CD8+ T cell numbers were sig-
a dominant parasite CD8 epitope. The extent of Pb1 presentation
nificantly reduced in PbAluc‐infected IRF1KO mice (Figure 3b and 3c).
was similar between WT and IRF1KO mice (Figure 2e). Hence, despite
Pb1‐specific CD8+ T cells were also significantly lower (less than 10%
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FIGURE 2 Peripheral parasitaemia, parasite sequestration, and malaria antigen presentation by brain endothelial cells in infected IRF1KO mice. (a) Parasitaemia (%) of infected WT (n = 5) and IRF1KO (n = 5) mice. For parasitaemia, data were log‐transformed for comparison between groups for each day and expressed as mean ± SD. *p < .05, **p < .01, ***p < .001, unpaired Student's t test. Dynamic in vivo quantification of parasite sequestrations in the (b) whole body and (c) head of infected WT (n = 5) and IRF1KO (n = 5) mice throughout the course of infection starting from 5 dpi. (d) Ex vivo imaging of brain isolated from infected WT (n = 4) and IRF1KO (n = 5) mice on 7 dpi. Data are expressed as mean ± SD. **p < .01, ***p < .001, unpaired Student's t test. (e) Antigen cross‐presentation by brain endothelial cells of naive WT (n = 5), infected WT (n = 10), naive IRF1KO (n = 4), and infected IRF1KO (n = 9) mice on 8 dpi when WT controls displayed experimental cerebral malaria signs in this experiment. Data are expressed as mean ± SD. *p < .05, Mann–Whitney test. IRF1KO = interferon regulatory factor 1 knockout; WT = wild type; dpi = days of post infection
of the Pb1‐specific CD8+ T cells detected in WT mice) in the brains of
2013; Piva et al., 2012). Naive mice lacking IRF1 display severe
IRF1 KO mice than in those of WT mice (Figure 3d). In addition, there
splenic T cell deficiency (Matsuyama et al., 1993; White et al.,
was also a reduction in the number of brain‐sequestered CD8+ T cells
1996). Confirming this observation, we also found that the number
expressing GrB or IFNγ, two molecules critical for pathogenicity of
of leucocytes in naive and infected mice was lower in IRF1KO than
CD8+ T cells during ECM (Amani et al., 2000; Grau et al., 1989; Haque
in WT mice (Figure S4a and S4b). On 4 dpi, the total number of
et al., 2011; Figure 3e and 3f). In essence, these results demonstrated a
CD4+ and CD8+ T cells increased in the spleen of IRF1KO mice but
fundamental defect in T cell sequestration in the brain of IRF1KO mice.
not to the level observed in the spleen of infected WT mice
This suggested that absence of IRF1 either crippled cell migration to
(Figure 4a and 4b). Significantly fewer Pb1‐specific CD8+ T cells were
the brain or impaired retention of these leucocytes in the brain.
also found in the spleen of infected IRF1KO mice than that of WT mice (Figure 4c). T cell activation is a prerequisite for clonal expansion and migra-
2.5 | Lack of IRF1 impairs parasite‐specific CD8+ T cell activation after priming
tion (Masopust & Schenkel, 2013; Pennock et al., 2013). When TCR
CD8+ Clec9a dendritic cells present in the spleen are involved in the
cules such as CD69 (Maino, Suni, & Ruitenberg, 1995; Simms & Ellis,
priming and activation of T cells in PbA model (Guermonprez et al.,
1996) and CD11a (Bose et al., 2013). Significantly fewer total activated
activation occurs, it induces the upregulation of expression of mole-
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FIGURE 3 Fewer brain‐sequestered leucocytes in IRF1KO than WT on 7 dpi. (a) Total leucocyte, (b) CD4+ T cell, and (c) CD8+ T cell numbers sequestered in the brain of naive WT (n = 5), infected WT (n = 4), naive IRF1KO (n = 5), and infected IRF1KO (n = 8) mice. Number of (d) Pb1‐ specific CD8+ T cells in the brain of infected WT (n = 4) and infected IRF1KO (n = 8) mice. Number of (e) GrB + CD8+ T cells and (f) IFNγ‐secreting CD8+ T cells sequestered in the brain of naive WT (n = 8), infected WT (n = 8), naive IRF1KO (n = 10), and infected IRF1KO (n = 11) mice. Mice were perfused intracardially before brains were isolated. Data are representative of two independent experiments and expressed as mean ± SD. *p < .05, **p < .01, ***p < .001, unpaired Student's t test. IRF1KO = interferon regulatory factor 1 knockout; WT = wild type; GrB = granzyme B; IFNγ = interferon‐γ; dpi = days of post infection
CD11ahighCD69+ CD4+ and CD8+ T cells were found in the spleen of
numbers of GrB‐secreting Pb1‐specific CD8+ T cells (Figure S5a and
infected IRF1KO mice than in its WT counterparts on 4 dpi (Figure 4d
S5b). These cells expressed similar levels of GrB, as determined by
and 4e). Likewise, the number of activated parasite‐specific CD8+ T
mean fluorescence intensity on 7 dpi (Figure S5c). In addition, para-
cells was lower in the spleen of infected IRF1KO mice compared to
site‐specific CD8+ T cells from IRF1KO mice were able to efficiently
that of WT mice (Figure 4f).
kill Pb1‐pulsed splenocytes in vivo and ex vivo with the same effi-
Next, a similar analysis was performed on 7 dpi. To our surprise,
ciency (Figure S5d and S5e).
despite the lower number of leucocytes (Figure S4b) and total CD4+
Taken together, we showed that T cell expansion was delayed and
(Figure 5a) and CD8+ T cells (Figure 5b), the number of parasite‐specific
activation was partial in the IRF1KO mice, with no effect on the func-
CD8+ T cells was similar in both WT and IRF1KO mice (Figure 5c). How-
tional cytotoxic capacity in the parasite‐specific CD8+ T cells.
ever, the number of total activated CD11ahighCD69+ CD8+ T cells and parasite‐specific CD11ahighCD69+ CD8+ T cells was still lower on 7 dpi than that of WT mice (Figure 5e and 5f). No difference in the number of CD11ahighCD69+ CD4+ T cells were found in infected WT and IRF1KO
2.6 | Alteration of migratory capacity of parasite‐ specific CD8+ T cells in IRF1KO mice
mice (Figure 5d). We further tested if the lack of T cells activation in IRF1KO mice
Because we showed that IRF1 deficiency results in a suboptimal
alters the GrB expression and cytotoxic capacity of parasite‐specific
activation of parasite‐specific CD8+ T cells, we hypothesised that
CD8+ T cells. Surprisingly, both groups of infected mice had similar
these cells may not express chemokine receptor necessary for T cell
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FIGURE 4
Infected IRF1KO mice have less activated splenicT cells than WT mice on 4 dpi. Numbers of (a) CD4+ T cells and (b) CD8+ T cells in naive WT (n = 6), infected WT (n = 4), naive IRF1KO (n = 3), and infected IRF1KO (n = 5) mice. Number of (c) Pb1‐specific CD8+ T cells in infected WT (n = 4) and infected IRF1KO (n = 5) mice. Numbers of (d) CD4+ T cells and (e) CD8+ T cells expressing the CD11a and CD69 activation markers in the spleen of naive WT (n = 6), infected WT (n = 4), naive IRF1KO (n = 3), and infected IRF1KO (n = 4) mice. Number of (f) Pb1‐specific CD8+ T cells expressing CD11a and CD69 activation markers in the spleen of infected WT (n = 4) and infected IRF1KO (n = 4) mice. Data are expressed as mean ± SD. *p < .05, **p < .01, ***p < .001, unpaired Student's t test. Mann–Whitney test employed for analysis of activated Pb1‐specific CD8+ T cells because data did not follow a normal distribution. IRF1KO = interferon regulatory factor 1 knockout; WT = wild type; dpi = days of post infection
migration to the brain. CXCR3 (Campanella et al., 2008; Van den
chamber assay by seeding in the upper compartment of a transwell
Steen et al., 2008) and CCR5 (Belnoue et al., 2003; Nitcheu et al.,
the exact same number of CD8+ T cells isolated from infected WT
2003) have been implicated in the migration of pathogenic CD8+ T
and IRF1KO mice on 7 dpi. Both activated (LFA1high) CD8+ T cells
cells to the brain during ECM. We focused on CXCR3 because we
and activated parasite‐specific CD8+ T cells from IRF1KO mice had
observed that CCR5KO mice are fully susceptible to ECM when
lower chemotaxis index as compared to cells from WT mice in
infected with PbAluc (data not shown). Expression of CXCR3 was
response to CXCL9 and CXCL10 (Figure 6e and 6f). This clearly
analysed on splenic CD8+ and Pb1‐specific CD8+ T cells by flow
demonstrated that absence of IRF1 in CD8+ T cells crippled cell
cytometry. Reduction in the number of CXCR3‐expressing CD8+ T
migration.
cells and Pb1‐specific CD8+ T cells was observed in the spleen of infected IRF1KO mice compared to that of infected WT mice (Figure 6a and 6b). Furthermore, mean fluorescence intensity
2.7
|
Lack of IRF1 alters IFNγ production by T cells
measurement showed lower levels of CXCR3 expressed on the
IFNγ is the main soluble mediator driving the pathogenesis of ECM, as
surface of these cells (Figure 6c and 6d). Thus, we showed here that
IFNγ or IFNγ receptor‐deficient mice are protected from ECM death
IRF1 deficiency led to a lower expression of CXCR3 on total CD8+ T
(Amani et al., 2000; Grau et al., 1989). IFNγ secreted by T cells and
cells as well as parasite‐specific CD8+ T cells. To confirm that CD8+ T
in particular CD4+ T cells (Villegas‐Mendez et al., 2012) is essential
cells suffer from migratory deficiency, we performed a chemotaxis
for activation of brain endothelial cells and cross‐presentation
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FIGURE 5 Infected IRF1KO mice have similar number of splenic Pb1‐specific CD8+ T cells as infected WT mice on 7 dpi despite less T cell activation. (a) CD4+ T cell and (b) CD8+ T cell numbers in naive WT (n = 5), infected WT (n = 5), naive IRF1KO (n = 5), and infected IRF1KO (n = 8) mice. Number of (c) Pb1‐specific CD8+ T cells in infected WT (n = 5) and infected IRF1KO (n = 8) mice. Data are representative of two independent experiments. Numbers of (d) CD4+ T cells and (e) CD8+ T cells expressing the CD11a and CD69 activation markers in the spleen of naive WT (n = 5), infected WT (n = 5), naive IRF1KO (n = 3), and infected IRF1KO (n = 4) mice. Number of (f) Pb1‐specific CD8+ T cells expressing the CD11a and CD69 activation markers in the spleen of infected WT (n = 5) and infected IRF1KO (n = 4) mice. Data are expressed as mean ± SD. *p < .05, **p < .01, ***p < .001, unpaired Student's t test. IRF1KO = interferon regulatory factor 1 knockout; WT = wild type; dpi = days of post infection
(Howland, Poh, et al., 2015), sequestration of infected erythrocyte in the brain, and, more importantly, for the migration of CD4+ and
2.8 | Reduced expression of adhesion molecules by brain endothelial cells from IRF1KO mice
CD8+ T cells to the brain during ECM (Belnoue et al., 2008). Therefore, we sought to investigate whether the lack of T cell activation in
Adhesion molecules LFA‐1 and very late antigen‐4 are expressed by
IRF1KO mice is associated with a decrease in IFNγ level. Serum IFNγ
pathogenic CD8+ T cells during ECM, and these molecules interact with
levels in infected IRF1KO mice were significantly lower than those in
ICAM‐1 and VCAM‐1 on the surface of activated brain endothelial cells,
WT mice (Figure 7a). This was associated with a reduced number of
respectively. These interactions facilitate the retention of effector
IFNγ‐producing total CD4+ and CD8+ T cells in the spleen on 7 dpi
CD8+ T cells in the brain intravascularly, leading to brain vascular leak-
(Figure 7b and 7c). However, no difference in the number of
age and ECM death (Swanson II et al., 2016). Because IFNγ stimulates
IFNγ‐producing Pb1‐specific CD8+ T cells was observed between
activation of brain endothelial cells (Howland, Poh, et al., 2015; Weiser,
both infected groups (Figure 7d).
Miu, Ball, & Hunt, 2007) and IRF1KO mice produced less IFNγ, we
Because we observed a difference in circulating IFNγ, we investi-
hypothesised that endothelial cells may be partially activated and
gated whether the serum levels of two IFNγ‐inducible chemokines,
expressed less ICAM‐1 and VCAM‐1. A lower expression of ICAM‐1
CXCL9 and CXCL10, which bind to CXCR3 (Muller, Carter, Hofer, &
and VCAM‐1 on brain endothelial cells of IRF1KO mice as compared
Campbell, 2010) and are involved in ECM (Campanella et al., 2008),
to WT mice was observed (Figure 8a and 8b).
were modified. Both infected WT and IRF1KO mice had similar levels of serum CXCL9 and CXCL10 on 7 dpi (Figure 7e and 7f).
Next, to determine if the reduction of adhesion molecules would lead to a reduction in CD8+ T cell retention, we transferred splenic
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FIGURE 6
CD8+ and parasite‐specific CD8+ T cells of IRF1KO mice displayed migratory defect as compared to cells from WT mice. Number of (a) CD8+ T cells expressing CXCR3 in naive WT (n = 3), infected WT (n = 9), naive IRF1KO (n = 5), and infected IRF1KO (n = 6) mice. Number of (b) Pb1‐specific CD8+ expressing CXCR3 in infected WT (n = 9) and infected IRF1KO (n = 6) mice. CXCR3 expression, as measured by MFI, on (c) CD8+ and (d) Pb1‐specific CD8+ T cells in infected WT and infected IRF1KO. Chemotaxis index of (e) activated CD8+ T cells and (f) Pb1‐specific CD8+ T cells isolated from infected WT (n = 10) and IRF1KO (n = 7) mice in response to CXCL9 and CXCL10. Data are expressed as mean ± SEM. **p < .01, ***p < .001, unpaired Student's t test. IRF1KO = interferon regulatory factor 1 knockout; WT = wild type; MFI = mean fluorescence intensity
CD8+ T cells from infected CD45.1 mice into infected CD45.2 WT
CD8+ T cells in the brain. We observed that IRF1KO mice displayed
(which have normal levels of CXCR3 expression and are fully activated)
defects in both trafficking of parasite‐specific CD8+ T cells to the brain
and IRF1KO recipient mice and assessed the numbers of CD45.1+
as well as retention of leucocytes in the brain. Migratory deficiency of
CD8+ T cells in the brain of recipient mice 2 days post transfer. We
parasite‐specific CD8+ T cells was a direct result of downregulated
high
high
Pb1‐
CXCR3 expression, possibly due to delayed proliferation and subopti-
specific CD8+ T cells was lower in IRF1‐KO than in WT mice
mal activation of these cells in the spleen of IRF1KO mice. On the
(Figure 8c). This supported the notion that the brain endothelial cells
other hand, lower retention of leucocytes in the brain was most likely
of IRF1KO mice are incapable of retaining leucocytes.
an outcome of reduced expression of adhesion molecules by brain
observed that the number of total LFA1
cells and LFA‐1
endothelial cells. IRF1KO mice displayed delayed T cell proliferation because naive
3
|
DISCUSSION
IRF1KO mice have a deficit in T cell numbers and it remained low by 4 dpi (Figure 4a–c), but this did not impede parasite‐specific CD8+ T
Previous studies using knockout mice have reported a role for IRF1 in
cells in the spleen of IRF1KO mice from reaching similar levels as in
ECM but did not identify the mechanism responsible for protection
WT mice on 7 dpi (Figure 5c). Such phenomenon was previously
(Berghout et al., 2013; Senaldi et al., 1999; Tan et al., 1999). Here,
reported in West Nile virus infection where the pool of West Nile
we have confirmed that absence of IRF1 protects mice from PbA‐
virus‐specific cytolytic CD8+ T cells expanded in IRF1KO mice despite
induced ECM death and have further revealed that protection from
less CD8+ T cells than that of WT mice (Brien et al., 2011). Priming of
ECM death is a consequence of reduced sequestration of pathogenic
PbA‐specific CD8+ T cells are driven by signals from Clec9A+ CD8a+
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FIGURE 7
Absence of IRF1 lowers IFNγ production in infected IRF1KO mice on 7 dpi. (a) Concentration of IFNγ in the serum of naive WT (n = 4), infected WT (n = 5), naive IRF1KO (n = 3), and infected IRF1KO (n = 8) mice. The dotted line represents the lower limit of detection for the ELISA kit. IFNγ‐producing. Data are expressed as mean ± SEM. Number of IFNγ‐secreting (b) CD4+ T cells and (c) CD8+ T cells in the spleen of naive WT (n = 6), infected WT (n = 9), naive IRF1KO (n = 9), and infected IRF1KO (n = 12) mice. Number of IFNγ‐secreting (d) Pb1‐specific CD8+ T cells in the spleen of infected WT (n = 9) and infected IRF1KO (n = 12) mice. Data are representative of two independent experiments. Numbers of (e) CD4+ T cells and (f) CD8+ T cells expressing T‐bet in the spleen of naive WT (n = 5), infected WT (n = 4), naive IRF1KO (n = 3), and infected IRF1KO (n = 4) mice. Concentration of (g) CXCL9 and (h) CXCL10 in the serum of WT (n = 11) and IRF1KO (n = 14) mice on 7 dpi. Data are expressed as mean ± SD. *p < .05, **p < .01, ***p < .001, unpaired Student's t test. IRF1KO = interferon regulatory factor 1 knockout; WT = wild type; IFNγ = interferon‐γ; dpi = days of post infection
dendritic cells through MHC molecules presenting malaria epitopes
essential for full maturation of these cells during ECM (Ball et al.,
(Signal 1) and costimulation molecules (Signal 2; Li et al., 1999).
2013). On the other hand, IFNγ is required to stimulate CD8+ T cells
Reduced activation of pathogen‐specific CD8+ T cell response in
to expand and mature in many systems (Sercan, Stoycheva,
IRF1KO mice is not due to defect in expression in costimulatory
Hammerling, Arnold, & Schuler, 2010). In addition, low level of IFNγ
molecules, such as CD28, because it was shown that stimulation with
production by CD8+ T cells after engagement of antigen and
plate‐bound anti‐CD3 and anti‐CD28 allows IRF1KO cells to prolifer-
costimulation leads to autocrine IFNγ signals, which further drives
ate more rapidly than WT cells (Brien et al., 2011). The more likely
effector IFNγ production (Curtsinger, Agarwal, Lins, & Mescher,
explanation is that cytokines, such as IL‐12, type I interferon, or IFNγ
2012). We have seen here that IFNγ are reduced in CD8+ T cells in
that provide a third signal for clonal expansion and effector function,
infected IRF1KO mice and is likely to lead to the observed activation
are not secreted to adequate levels. During infection, IRF1 binds to
deficit.
IFN‐stimulated regulatory element of several IFN‐inducible genes
Endothelial activation is a feature of ECM pathogenesis with
including IFN‐alpha/beta and IL‐12 (Fujita et al., 1989; Kimura et al.,
expression of adhesion molecules being responsible for sequestration
1996; Miyamoto et al., 1988), thereby generating a positive feedback
of leucocytes (Swanson et al., 2016). Here, IRF1KO mice exhibited less
loop. IL‐12 is dispensable for ECM to occur (Claser et al., 2011;
leucocyte sequestration in the brain due to downregulated ICAM‐1
Fauconnier et al., 2012) hence eliminating its role in CD8+ T cell prim-
and VCAM‐1 expressions on the brain endothelial cells (Figure 8a
ing and maturation. This is also the case for type I IFN, because it was
and 8b). VCAM‐1 expression was reported to be upregulated as early
shown that expression of type I IFN receptor on CD8+ T cells is not
as 4 dpi, whereas ICAM‐1 expression was only intensely observed on
10 of 15
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ET AL.
FIGURE 8 Absence of IRF1 mitigates leucocyte retention in the brain due to lower expression of adhesion molecules by cerebral endothelial cells. Representative histogram (left) from naive and infected WT and IRF1KO mice show the expression of (a) ICAM‐1 and (b) VCAM‐1 on live CD45‐CD31+ endothelial cells. The bar graph (right) depicts the mean fluorescent intensity of (a) ICAM‐1 and (b) VCAM‐1 on brain endothelial cells from naive WT (n = 5), infected WT (n = 7), naive IRF1KO (n = 5), and infected IRF1KO (n = 10) mice. (c) Design of the in vivo migration assay. Normalised ratio of activated CD8+ T cells and Pb1‐specific CD8+ T cells collected from the brain of infected WT (n = 5) and infected IRF1KO (n = 6) mice. Data are expressed as mean ± SD. *p < .05, **p < .01, ***p < .001, unpaired Student's t test. IRF1 = interferon regulatory factor 1; WT = wild type; IRF1KO = IRF1 knockout; ICAM‐ 1 = intracellular adhesion molecule 1; VCAM‐ 1 = vascular cell adhesion molecule 1; MFI = mean fluorescence intensity; dpi = days of post infection
6 dpi (Bauer, van der Heyde, Sun, Specian, & Granger, 2002). Direct
sufficient for optimal epitope cross‐presentation. This also implies that
effect of IRF1 on VCAM‐1 expression has been previously reported
endothelial cells can respond to minimal local stimuli very efficiently.
(Lechleitner, Gille, Johnson, & Petzelbauer, 1998; Neish et al., 1995;
It is intriguing to note that IRF1KO mice displayed neurological
Sun et al., 2012), whereas ICAM‐1 expression is shown to be indepen-
signs during the ECM time window, which were associated with
dent of IRF1 activity (Sun et al., 2012). Expression of ICAM‐1 and
minute ruptures in the BBB. This suggests that even a small number
VCAM‐1 during ECM is induced primarily by IFNγ (Amani et al.,
of pathogenic T cells can cause damage to the BBB as shown previ-
2000; Bauer et al., 2002; Swanson et al., 2016). We observed that
ously (Howland et al., 2013; Poh, Howland, Grotenbreg, & Rénia,
the level of circulating IFNγ was reduced, and thus, it was logical that
2014) and a threshold needs to be attained to induce ECM death. At
ICAM‐1 and VCAM‐1 expressions were affected by the lower level
later time points, IRF1KO mice stopped showing any neurological signs
of IFNγ in IRF1KO mice. Hence, we proposed that during early ECM
and survived the ECM window suggesting that they might have recov-
development, IRF1 directly stimulates VCAM‐1 expression on brain
ered from these injuries. Further studies are needed to uncover the
endothelial; at later time points, IRF1‐induced IFNγ upregulates
recovery mechanisms involved, and this may lead to the discovery of
ICAM‐1 expression and perpetuates VCAM‐1 expression in the brain.
novel therapeutic approaches.
IRF1 was previously demonstrated to be involved in the control of
In conclusion, we have established that IRF1 deficiency conferred
parasitaemia (Berghout et al., 2013; Senaldi et al., 1999; Tan et al.,
ECM protection. An association study of 18 single‐nucleotide
1999). Control in parasite load is likely through the action of IFNγ, as
polymorphisms among three African populations did not reveal signif-
previously reported (Amani et al., 2000; Claser et al., 2011). Our find-
icant correlation with severe malaria or subphenotypes such as CM
ings also supported this as we observed higher parasitaemia (Figure 2
and severe anaemia (Mangano et al., 2009). However, the extent of
a) and lower level IFNγ (Figure 7a) in infected IRF1KO mice
IRF1 expression in these single‐nucleotide polymorphisms were not
(Figure 6). IFNγ activation and parasites sequestration are essential
known. Given that IRF1 F1 mice are still susceptible to ECM, such
for successful endothelial PbA cross‐presentation (Howland, Poh,
alterations in IRF1 expression are unlikely to reverse the outcome of
et al., 2015). Surprisingly, reduced parasite sequestration in the brain
CM. Alleles that drastically lower IRF1 expression or differential
and lower circulating IFNγ levels did not affect antigen presentation
binding to promoter elements of genes regulated by IRF1 might influ-
in the brain. This suggests that these levels in IRF1KO mice are
ence some aspect of the immune responses and hence result in varied
GUN
11 of 15
ET AL.
forms of malaria severity. Further studies integrating a deeper
performed to obtain 5‐μm‐thick brain sections that were stained with
immunophenotyping and severity phenotyping are definitely needed
haematoxylin and eosin. Slides were acquired on Metafer4
to identify these interactions.
(MetaSystems), and haemorrhages were manually counted.
4
E X P E R I M E N T A L P R O C E DU RE S
|
4.5
|
Folic acid challenge
Mice were injected intravenously with 5 mg of folic acid (25 mg/ml in
4.1
|
PBS adjusted to pH 7.0) twice at 1 hr apart and monitored (Howland
Mice
et al., 2013). Mice with a compromised BBB convulsed and died within Six‐ to eight‐week‐old wild‐type (WT) CD45.1 and CD45.2 C57BL/6
90 min post injection. Survivors were euthanised the following day.
mice and IRF1KO mice (The Jackson Laboratory) were used. IRF1 heterozygous mice (IRF1 F1) mice were generated by crossing IRF1KO with WT C57BL/6 mice. All mice were kept under specific pathogen‐
4.6
free conditions in Biomedical Resource Centre, Singapore. All proce-
Accumulation of iRBC in the whole body and head of mice infected
dures were approved by the Institutional Animal Care and Use
with PbAluc was assessed using an imaging system (IVIS, Xenogen,
Committee of the Agency for Science, Technology and Research
Alameda, CA, USA) as described previously (Claser et al., 2011). Each
(Biopolis, Singapore; Institutional Animal Care and Use Committee
anaesthetised mice was subcutaneously injected with 0.5 mg of
#110630 and #140968) in agreement with the regulations of the
D‐luciferin (D‐luciferin potassium salt, Caliper Life Sciences, dissolved
National Advisory Committee for Laboratory Animal Research of
in PBS), which was allowed to circulate for 2 min prior to image acqui-
Singapore and the Agri‐Food and Veterinary Authority.
sition. For in vivo analysis, whole body and head imaging were
|
Bioluminescence imaging
acquired with the mice in ventral and dorsal positions, respectively.
4.2
|
Parasite infection and pathology
Images were taken under the following settings: medium binning factor, exposure time of 5–60 s and 21.7 cm (whole body) or 4 cm (head)
Transgenic P. berghei ANKA 231cl11 expressing luciferase and green
field of vision. For ex vivo experiments, 7 dpi mice were subcutane-
fluorescent protein (referred here as PbAluc) was obtained from Dr
ously administered with D‐luciferin for 3 min prior to intracardiac per-
Christian Engwerda (QIMR, Brisbane, Australia) (Amante et al., 2007;
fusion. Isolated organs were placed in a Petri dish and imaged with
Franke‐Fayard et al., 2005). PbA clone BdS (Belnoue et al., 2008;
medium binning factor, exposure time of 10–60 s and 10 cm field of
Hearn, Rayment, Landon, Katz, & de Souza, 2000) was also used in
vision. For background subtraction, uninfected mice injected with
selected experiments. Both parasites were passaged in C57BL/6J
luciferin were imaged. Data acquired were analysed using Living Imag-
mice, and blood was harvested for parasite stock when mice displayed
ing 3.0 software where the region of interest was determined. Data
neurological signs. These stocks were frozen in liquid nitrogen (107
were expressed as average radiance (photons/s/cm2/sr).
infected red blood cells [iRBC]/ml in Alsever's solution). Mice were infected with 106 iRBC intraperitoneally and monitored for neurological signs associated with ECM, such as abnormal posturing, paralysis, ataxia, convulsion, and/or coma. Mice that did not develop ECM died
4.7 | Ex vivo brain microvessels cross‐presentation assay
in the later stage of infection with hyperparasitaemia and anaemia.
Anaesthetised mice were terminally bled on 8 dpi when WT mice
Parasitaemia levels were monitored daily from 3 days of post infection
displayed neurological signs in this experiment. The assay has been
(dpi), and every 2–3 days from 12 dpi onwards, by flow cytometry
performed as previously described protocol (Howland et al., 2013,
method using Hoechst and ethidium dyes (Malleret et al., 2011).
Howland, Gun, Claser, Poh, & Renia, 2015). Briefly, brain microvessel fragments isolated by dextran gradient centrifugation and retention
4.3
|
Evans Blue extravasation assay
on a cell strainer were digested with collagenase. They were incubated in a 96‐well filter plate overnight with a reporter cell line (LR‐BSL8.4a)
Assessment of blood–brain barrier (BBB) permeability was performed
that expresses LacZ upon recognising the malaria‐specific Pb1 epitope
through the Evans Blue extravasation assay. Briefly, Evans Blue dye
complexed with H2‐Db (Howland et al., 2013). Cells were stained with
(1% w/v in 0.9% NaCl; Sigma Aldrich) was intravenously administered
X‐gal the following day. Number of blue spots were counted on a CTL
and allowed to circulate for 1 hr, after which anaesthetised mice were
ImmunoSpot Analyzer.
exsanguinated. Whole brains were quickly removed, weighed, and suspended in N,N‐dimethylformamide (Sigma) for 2–3 days. Dye in the extracts was quantified by absorbance at 620 nm. All data were expressed as OD620
nm
per gram of tissue weight.
4.8
|
Mononuclear cell isolation
On 4 or 7 dpi, mice were anaesthetised and intracardially perfused with PBS to isolate brain and spleen. Organs were placed in Rosewell Park
4.4
|
Histology
Memorial Institute (RPMI) medium‐1640, supplemented with 10% heat‐inactivated fetal bovine serum and 100 U/ml penicillin/streptomycin
Infected mice were sacrificed on 7 dpi. Mice were perfused with PBS
(Gibco, Life Technologies). Brains and spleens were mashed through
followed by 4% formaldehyde (VWR). Isolated brain was immersed in
100‐ and 40‐μm cell strainers (BD, Bioscience, San Jose, CA, USA),
4% formaldehyde and embedded in paraffin. Sagittal sectioning was
respectively. For isolation of brain‐sequestered leucocytes (BSL),
12 of 15
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ET AL.
samples were digested with 0.5 mg/ml CollagenaseType IV (Worthington
0.5 μM Carboxyfluorescein Succinimidyl Ester (CFSE) (Sigma) for
Biochemical, Lakewood, NJ, USA), 10 μg/ml DNase I (Roche Applied
10 min. The other portion remained unpulsed and was stained with
Science) in 10 ml PBS for 30 min at room temperature. After filtering
5 μM CFSE. Equal number of washed pulsed (low CFSE) and unpulsed
through a 40‐μm cell strainer, samples were centrifuged at 500 rpm
cells (high CFSE; 107 each) were injected intravenously into naive and
for 1 min to remove large debris. The supernatant was layered over a
infected mice on 6 dpi. Twenty hours later, recipient mice were
30% Percoll gradient and spun down at 3,000 rpm for 10 min. Pellet
euthanised to harvest for splenocytes to analyse the CFSE‐labelled
obtained was treated with ammonium–chloride–potassium lysis buffer
cells. Capacity of in vivo killing was assessed using the following for-
and washed with RPMI medium. Spleen cells were also treated with
mula:
lysis buffer. After washing, cells were counted using a haemocytometer.
(
Brain endothelial cells were isolated as described above (see Section
"
1−
4.7). Mice were perfused, and their brains were processed to obtain
CFSEhigh
!
CFSElow
= naive
CFSEhigh
!
CFSElow
#) ×100
infected
brain microvessel fragments. Cells were kept at 4 °C before staining. For ex vivo CTL assay, splenocytes from naive donor mice were pulsed with 10 μg/ml Pb1 peptide and then stained with 5 μM CFSE.
4.9
Mononuclear cell phenotyping
|
On the other hand, CD8+ T cells were isolated from the spleen of
Leucocytes were stained with the following: Fixable Aqua dead cell
infected mice on 7 dpi using mouse CD8a+ T cell isolation kit (Miltenyi
stain kit (405 nm excitation, Life Technologies), anti‐mouse CD45.2
Biotec
conjugated with PerCP‐Cy5.5 (clone 145‐2C11, eBioscience), anti‐
manufacturer's instructions. The percentage of Pb1‐specific CD8+ T
mouse CD3e conjugated with Pe‐Cy7 (clone 145‐2C11, Life
cells was next assessed by flow cytometry in order to calculate for
Technologies), anti‐mouse CD4 conjugated with Pacific Blue (clone
number of Pb1‐specific CD8+ T cells in each sample. Next, 105 target
RM4–5, Biolegend), anti‐mouse CD8a conjugated with Brilliant Violet
cells (CFSE + Pb1‐pulsed splenocytes) were seeded with different
GmbH,
Bergisch
Gladbach,
Germany)
following
the
605 (clone 53–6.7, Biolegend) and SQLLNAKYL‐H‐2D (Pb1) tetramer
number of effector cells (Pb1‐specific CD8+ T cells) and tested in
conjugated with Phycoerythrin (PE) or Allophycocyanin (APC)
triplicates at different effector–target ratio. After 4 hr, cells were
b
(Howland et al., 2013). To assess the activation status of T cells, cells
harvested and stained with 4,6‐diamidino‐2‐phenylindole before
were stained with anti‐mouse CD11a conjugated with FITC (Clone
analysis with flow cytometry to assess cytolytic activity in each sample.
M17/4, eBioscience) and anti‐mouse CD69 conjugated with PE (Clone H1.2F3, BD Pharmingen) antibodies. CXCR3 expression was determined using anti‐mouse CXCR3 conjugated with PerCP Cy5.5 antibodies (Clone CXCR3–173, eBioscience). For intracellular staining, BSL and splenocytes were incubated with 10 μg/ml Brefeldin A (eBioscience) in 1 ml RPMI medium at 37 °C for 3 hr before extracellular staining. Samples were then fixed in 2% formaldehyde and kept overnight at 4 °C. The following day, cells were permeabilised with 0.5% w/v Saponin (Sigma Aldrich) and stained with anti‐mouse IFNγ conjugated with PerCP Cy5.5 (Clone XMG1.2, eBioscience) and anti‐mouse granzyme B (GrB) conjugated with Pe‐Cy7 (Clone NGZB, eBioscience) antibodies on ice. Brain endothelial cells were stained with Fixable Aqua dead cell stain kit, anti‐mouse CD45 conjugated to APC (clone 30F11, Miltenyi Biotec), anti‐mouse CD31 conjugated with Pe‐Cy7 (Clone 390, Biolegend), anti‐mouse intercellular adhesion molecule 1 (ICAM‐1) conjugated with FITC (clone YN1/1.7.4, Biolegend), anti‐mouse vascular
4.11
|
Transwell migration assay
CD8+ T cells were purified from the spleen of infected mice using mouse CD8α+ T cell isolation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). For migration studies, 0.6 × 106 cells were seeded in Transwell inserts (Corning Costar, Acton, MA, USA) that were placed in 24‐well plate containing either 200 ng/ml mouse recombinant IP‐10 (CXCL10) or MIG (CXCL9; both from Peprotech, Rocky Hill, NJ, USA). After 3 hr, cells in the lower chamber were collected, stained with anti‐mouse CD8a conjugated with Brilliant Violet 605 (clone 53‐6.7, Biolegend), Pb1 tetramer conjugated with PE and anti‐mouse lymphocyte function‐associated antigen 1 (LFA‐1) conjugated with PerCP‐Cy5.5 (clone H155–78, Biolegend) and counted using flow cytometry. Chemotaxis index was calculated by normalising the number of cells migrating in response to chemokines by the number of cells migrating in wells with medium alone.
cell adhesion molecule 1 (VCAM‐1) conjugated with Pe (clone 429, Biolegend) and anti‐mouse MHC II (I‐A/I‐E) conjugated with eFluor 450 (clone M5/114.15.2, eBioscience). Thereafter, cells were washed
4.12
|
In vivo migration assay
and resuspended in FACS buffer. Samples were acquired with BD
At 6 dpi, total splenic CD8+ T cells were isolated from infected CD45.1
LSRFortessa cell analyser and subsequently analysed using FlowJo
donor mice through negative selection with a CD8α+ T cells isolation
software (Tree Star). Cell populations in brain and spleen were identified
kit (Miltenyi Biotec). 5 × 106 cells in 200 μl of PBS was injected
and defined as described in Figure S1. Number of cells in each leucocyte
intravenously into PbA‐infected C57BL/6 and IRF1KO recipient mice
population were calculated from total cell count from each organ.
at 5dpi. Profile of the numbers of total, LFA‐1high and Pb1‐specific CD8+ T cells in donor cells was performed using flow cytometry by
4.10
|
Cytotoxicity T lymphocyte assay
staining a portion of these cells with anti‐mouse CD45, anti‐mouse CD3, anti‐mouse CD4, anti‐mouse CD8, anti‐mouse LFA‐1 antibodies,
For in vivo CTL assay, splenocytes from naive donor mice were divided
and Pb1 tetramer. Two days later, brains were extracted from the
into two fractions of 4 × 107 cells/ml. One portion was pulsed with
recipient mice and processed similar to mononuclear cell isolation.
10 μg/ml Pb1 peptide for 1 hr at 37 °C followed by staining with
Cells were stained with Fixable Aqua dead cell stain kit, followed by
GUN
13 of 15
ET AL.
the same panel of antibodies as before for flow cytometry acquisition. Migration capacity of the donor cells towards the brain was determined by the ratio of donor cells obtained from the recipient brain divided by the initial number of donor cells transferred.
4.13
|
Measurement of cytokine
IFNγ, CXCL9, and CXCL10 levels were measured in duplicates by ELISA using commercially available kits (R&D Systems). Serum samples were collected from naive and infected mice on 7 dpi and tested at a dilution of 1/5. The limit of detection was 9.4 pg/ml for IFNγ, 31.25 pg/ml for CXCL9, and 62.5 pg/ml for CXCL10.
4.14
|
Statistical analysis
Survival was assessed using log‐rank Mantel–Cox test. All data were subjected to normality test. If results follow a normal distribution (based on Shapiro–Wilk normality test assayed on the minimum eight samples), Student's t test was employed; otherwise, Mann–Whitney was used. Data were compared pair wise because there were two naive controls (WT and IRF1KO mice) in each dataset. Chi‐square analysis was used to compare data for ECM‐symptomatic and nonsymptomatic mice. Values obtained from parasitaemia, imaging studies, and brain microvessel assays were log‐transformed for normalisation and difference assessed by Student's t test. ACKNOWLEDGEMEN TS We would like to thank SIgN Flow Cytometry Core for their technical excellence. We also express gratitude to SIgN Mouse core, especially Florida Toh, for assistance in breeding and providing the mutant mice in this study. This work was supported by an intramural grant from Singapore's Agency for Science, Technology and Research (A*STAR; L. R.). G. S. Y. and P. C. M. were supported by a postgraduate scholarship from the Yong Loo Lin School of Medicine, National University of Singapore (Singapore). CONF LICT S OF INT ER E ST The authors have no financial conflicts of interest. ORCID Sin Yee Gun
http://orcid.org/0000-0001-6158-6293
Carla Claser
http://orcid.org/0000-0002-5395-9632
Teck Hui Teo
http://orcid.org/0000-0001-9858-9412
Shanshan W. Howland Lisa F.P. Ng
http://orcid.org/0000-0003-0349-1557
http://orcid.org/0000-0003-4071-5222
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SUPPOR TI NG INF ORMATI ON Additional Supporting Information may be found online in the supporting information tab for this article.
How to cite this article: Gun SY, Claser C, Teo TH, et al. Interferon regulatory factor 1 is essential for pathogenic CD8+ T cell migration and retention in the brain during experimental cerebral
malaria.
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https://doi.org/10.1111/cmi.12819
2018;20:e12819.
