Over 34 million people are currently infected by HIV-1, with an estimated 1.7 million AIDS-related deaths in 2011.1 Sub-Saharan Africa (SSA) has been most affected by HIV, and the per-contact risk of HIV transmission is increased 3-fold in studies from SSA compared with high-income countries.2 Malaria is a parasitic infection with a reported 300–500 million infections a year3 that caused an estimated 1.2 million deaths in 2010.4 HIV and malaria overlap in many regions of the world, particularly in SSA. Both infections are holoendemic in Zambia, Zimbabwe, Mozambique, Malawi, and the Central African Republic, countries where the HIV prevalence exceeds 10% and at least 90% of the population is exposed to malaria.5 A striking spatial overlap is also evident in Kenya, where both infections are endemic.6–9
Mounting evidence suggests that HIV and malaria interact at a molecular level.10 HIV infection causes a gradual decline in blood CD4+ T cells and eventual immune dysfunction. In studies from SSA, lower blood CD4+ T-cell levels and more advanced HIV disease stage are both associated with an increased risk of malarial infection and with an increased risk of developing severe malarial disease,11,12 such that HIV has been estimated to cause an additional 3 million malarial cases and 65,000 malarial deaths.13 Conversely, individuals living in areas of high malaria endemicity are twice as likely to be infected with HIV.14 Malaria increases the HIV blood viral load (VL) by almost 10-fold,15 perhaps by inducing immune activation16 and thus enhancing cellular HIV replication.17 Because the blood VL is a strong predictor of sexual transmission risk,18,19 malaria is thought to substantially enhance HIV transmission at a community level. Indeed, based on these data, mathematical models suggest that the interaction between HIV and malaria in coinfected adults from a population of roughly 200,000 in Kisumu, a town in western Kenya, may have been responsible for 8,500 excess HIV infections and 980,000 excess malarial episodes over the last 3 decades.6
Although HIV and malaria are synergistic within coinfected individuals, less is known about the possible effect of malarial infection on HIV susceptibility within a malaria mono-infected person. Relative immune quiescence has been associated with reduced HIV susceptibility,20,21 and HIV acquisition in the recent CAPRISA 004 clinical trial was enhanced by systemic innate immune activation.22 The location of immune activation may be important: nonhuman primate models suggest that activated immune cells within the mucosa are critical for productive HIV infection after a mucosal exposure23 and HIV-exposed, seronegative women demonstrate reduced immune activation in the genital mucosa.24,25 In addition, preliminary findings from a prospective study demonstrate that every 1 log increase in levels of genital proinflammatory cytokines was associated with a 35%–153% increased risk of HIV acquisition.26 Therefore, if malaria were to cause immune activation at mucosal sites of subsequent HIV exposure, then this would be expected to increase HIV susceptibility. However, since malaria is the prototypic systemic (blood) infection, this would require malaria to induce mucosal immune alterations, either through increased mucosal trafficking of HIV target cells or through the immune activation of mucosal cells. Studies of other systemic infections suggest that this may be possible. Masopust et al27 demonstrated that systemic lymphocytic choriomeningitis virus vaccination can transiently upregulate the mucosal homing integrin α4β7 and induce gut T-cell migration. In addition, children infected with malaria have increased shedding of herpes viruses at mucosal sites and malaria treatment reduced herpes viral shedding.28 To the best of our knowledge, however, the mucosal immune impact of systemic malarial infection has not been studied.
Although the effect of malaria on the immunology of the genital mucosa is unknown, increased levels of T-cell immune activation were recently observed in the cervix of young women from Kisumu, Kenya, in the absence of common genital infections.29 Blood samples were not available for malaria diagnostics or immunological characterization in this study, but malaria is holoendemic in Kisumu, and we hypothesized that this might be the cause of increased genital T-cell immune activation.30 To investigate this hypothesis, we have now examined the impact of malaria on mucosal T-cell immunology in a well-established mouse model of malaria. In keeping with our hypothesis, we found that malaria upregulated expression of the mucosal homing integrin α4β7 on blood T cells and increased T-cell immune activation and CCR5 expression in the gut and genital mucosae.
The Animal Care Committee of the University of Toronto approved the animal experimental protocol, and all experiments involving animals were performed in accordance with the guidelines of the University of Toronto, the Canadian Council on Animal Care, and the Animals for Research Act (statutes and regulations of Ontario).
Plasmodium chabaudi chabaudi Strain A Murine Infection
C57BL/6 mice (Charles River, Sherbrooke, Canada) were allowed to acclimatize at the University of Toronto animal facilities for at least 7 days before experiments and kept under pathogen-free conditions with a 12-hour light cycle. Female mice of 8–12 weeks of age were used in all experiments. Five days before animal infection, all mice were treated once with 2 mg of long-lasting Depo-Provera (Depo; dihydroxyprogesterone acetate) by subcutaneous injection to maintain mice in the diestrous stage and minimize hormone-induced cyclical alterations in the female genital mucosa.31 Blood stage P. chabaudi chabaudi strain A (PCCAS malaria) parasites were cultured in the laboratory as previously described32 and were used to inoculate experimental animals by intraperitoneal injection of 1 × 106 parasites. PCCAS malaria parasitemia is known to peak between 8 and 10 days followed by immune clearance within 2–5 days in C57BL/6 mice.33 Therefore, the mice were returned to the pathogen-free housing conditions, and the PCCAS infection was allowed to progress for 10 days. Before the animals were killed, a thick blood smear was collected via tail vein and Giemsa stained to confirm and count parasitemia using protocol R HEMA Stain Set (Thermo Fisher Scientific, Ottawa, Canada).
Sample Collection and Tissue Processing
Immediately after killing (with CO2), blood was sampled from all mice through cardiac puncture into 2 mL microtubes containing 200 μL of acid citrate dextrose to prevent coagulation. Collected blood was then treated with nonfixing lysing solution (BD Pharm Lyse; BD, Mississauga, Canada) as per manufacturer's instructions to lyse red blood cells, and the samples were then stored on ice. In a subset of mice, spleen tissues were also sampled, dispersed using a sterile plunger to release cells, and red blood cells lysed to enrich for lymphocytes. In all animals, mouse cecal (gut), vaginal, and cervical (genital) tissues were then excised and digested to obtain tissue lymphocytes using an established protocol from our previous work.34 Here, tissue sections were sliced open longitudinally, cleaned in 1% fetal bovine serum (FBS) (GIBCO-Life Technologies, Burlington, Canada) in phosphate-buffered saline (PBS) (Gibco), and then cut into ∼1- to 2-cm segments. Mucosal tissues were then washed for 5 minutes in prestripping buffer containing 1 mM dithiothreitol, 5% FBS, and 1 mM EDTA in PBS. Tissues were then transferred into a 50-mL tube containing stripping buffer (PBS, 1% FBS, 5 mM EDTA, and 1 mM dithiothreitol) and were incubated in a heat shaker with agitation at 37°C for 30 minutes. The tissues were then allowed to sediment before the supernatant containing extracted intraepithelial lymphocyte cells was removed, then washed twice in Dulbecco modified Eagle medium (DMEM, Gibco) containing 20% FBS, and stored on ice. Remaining tissues were further minced into ∼4-mm segments using scissors before being digested in a digestion medium containing DMEM, 20% FBS, 2 mg/mL collagenase D (Roche, Mississauga, Canada), and 20 μg/mL DNase (Sigma-Aldrich, Oakville, Canada) in a heat shaker incubator with agitation for 30 minutes at 37°C. The supernatant containing lamina propria lymphocytes (LPL) was then extracted after allowing the tissues to sediment, before fresh digestion medium was used and the digestion repeated for another 30 minutes and the LPL harvested. Both intraepithelial lymphocyte and LPL cells were pooled and washed twice in DMEM and then sequentially passed through a 100-μm and then a 40-μm cell strainer before being placed on ice.34 Iliac lymph nodes (iLN) that drain the genital tract were also excised and placed into culture dishes containing enough 1% FBS in PBS solution to prevent them from drying; a sterile plunger was used to disrupt the lymph node sacs and release viable lymphocytes. Lymph nodes were then collected and rinsed twice in DMEM before being placed on ice. With the exception of blood lymphocytes, all other tissues were pooled into groups of 3 mice each to allow for sufficient cells for flow cytometric analysis.
Flow Cytometry Assays
Cells were stained with aqua live/dead fixable stain (Invitrogen-Life Technologies, Burlington, Canada) to establish viability and with the following surface immune marker antibodies for 30 minutes in 1% FBS in PBS: TCRβ, CD4, CD8, α4β7, CCR9, CCR6, CCR5, CD69, CD38, and MHC-II (eBiosciences, San Diego, CA). Cells were then washed in DMEM before being placed in 2% paraformaldehyde solution in PBS. Fluorescence-activated cell sorting analysis was then performed on the samples using an LSR II flow cytometer (BD) and analyzed using FlowJo software (TreeStar, Ashland, OR).
Mann–Whitney nonparametric tests were performed using Graphpad Prism (GraphPad Software Inc, La Jolla, CA), and P values of <0.05 were considered significant. Tissues were weighed after excision, and the absolute number of cells was reported as cell counts per 1 × 106 cells per 100 g of tissue.
Effect of Murine Malarial Infection on CCR5 Expression and T-Cell Activation in Blood
Fifteen mice were infected with PCCAS malaria for 10 days and matched with uninfected controls. Animals were then killed, and the frequency of T cells expressing CCR5 and immune markers of activation was examined in blood. PCCAS malarial infection was associated with a substantial increase in the frequency of CD4+ T cells expressing CCR5 (Fig. 1A, representative example from 4 experimental replicates). Similarly, there was a significant increase in the CD4+ and CD8+ T-cell expression of CD38, MHC-II, and CD69 (the latter on CD8+ T cells only) in the malaria-infected group compared with malaria-uninfected controls (Figs. 1B–E). A key stage of PCCAS parasite reproductive cycle takes place in the spleen. Therefore, we examined the expression of these immune activation markers here and found that PCCAS-infected mice also had elevated T-cell immune activation at this site (data not shown). Therefore, PCCAS malarial infection induced substantial and consistent systemic T-cell immune activation. These experiments were subsequently repeated 3 times with similar results (data not shown).
Effect of PCCAS Malarial Infection on T-Cell Mucosal Homing Markers
Within the same experimental system, we then examined the expression of mucosal T-cell trafficking receptors in blood lymphocytes, specifically the expression of α4β7, CCR9, and CCR6. Compared with the uninfected controls, PCCAS malaria–infected mice demonstrated dramatic increases in the expression of α4β7 on blood CD4+ and CD8+ T lymphocytes (Figs. 2A, B). However, the expression of CCR9 and CCR6 on T lymphocytes did not differ between study groups, and CCR9+ expression was actually decreased on blood CD8+ T cells in malaria-infected animals (Figs. 2C, D).
The simultaneous co-expression of several susceptibility markers on CD4+ T cells may flag a highly HIV-susceptible cell population.35 We therefore also examined the impact of malaria on the frequency of blood α4β7+ T cells co-expressing CCR5 and other markers of T-cell activation. Malaria-infected mice had a significantly increased frequency of T cells co-expressing α4β7+ together with CCR5 and CD38 (Figs. 3A, B), but not with CD69 or MHC-II (Figs. 3C, D).
Effect of Malarial Infection on Overall T-Cell Numbers in the Gut and Genital Mucosae
The increased expression of α4β7 by activated CD4+ and CD8+ blood T lymphocytes during PCCAS malarial infection would be expected to traffic activated T cells to mucosal sites. Therefore, we next quantified T-cell numbers in the gut and genital mucosae. Malaria was not associated with any difference in the overall number of CD3+, CD4+, or CD8+ T lymphocytes in the gut or genital mucosa (Figs. 4A–C). Furthermore, malaria was not associated with differences in the absolute number of α4β7+ T cells in the gut or genital mucosa (data not shown). Therefore, PCCAS infection did not alter the total number of mucosal T lymphocytes or the expression of α4β7+ by these mucosal T lymphocytes.
Effect of Malaria on Mucosal T-Cell Activation and CCR5 Expression
PCCAS malaria had a profound impact on the expression of CCR5 and immune activation markers by blood T cells. Therefore, we next examined the expression of these markers in the gut tissues (the cecum), the genital mucosa (vagina and cervix), and in the iLN that drain the genital tract (iLN). In the gut, PCCAS-infected animals demonstrated significantly increased T-cell expression of the HIV co-receptor CCR5 and the activation markers CD38 and CD69, although MHC-II expression was similar to uninfected controls (Figs. 5A–D, left column). In the genital mucosa, PCCAS infection was associated with increased T-cell expression of CD38 and increased CCR5 and MHC-II on genital CD4+ T cells, although CD69 expression was unaffected (Figs. 5A–E, middle column). Finally, increased expression of MHC-II and CD69 was apparent on T lymphocytes from the iLN of malaria-infected animals and increased CCR5 and CD38 expression on lymph node CD4+ T cells (Figs. 5A–D, right column). In general, we observed significant and consistent increases in immune activation and CCR5 expression within both the gut and genital mucosal tissues of the PCCAS-infected animals.
The per-contact risk of HIV transmission is 3-fold higher in SSA than in high-income countries.2 Genital immunology plays a key role in susceptibility to HIV,30 and we previously showed that, in the absence of genital infections, young women from western Kenya had a similar overall number of cervical CD4+ T cells compared with women in the United States but that many more cervical CD4+ T cells were activated and expressed the HIV co-receptor CCR5.29 Given the strong geographical overlap between HIV and malaria in Kenya,6–9 we hypothesized that malaria might be one cause of increased genital T-cell activation and enhanced mucosal susceptibility to HIV. We have now tested this hypothesis in a murine model by examining the effects of PCCAS malarial infection on putative markers of HIV susceptibility in the blood and mucosal tissues. PCCAS infection induces nonlethal, noncerebral malaria in C57BL/6 mice and was chosen for this study to represent the disease phenotype in adults living in malaria holoendemic regions with high HIV prevalence, such as western Kenya. First, we demonstrated that malarial infection increased immune activation and expression of the mucosal homing integrin α4β7 in blood, confirming previous in vivo36 and in vitro studies.37,38 In addition, although overall T-cell numbers were not increased in mucosal tissues, malarial infection dramatically increased immune activation and the expression of CCR5 on mucosal (gut and genital) CD4+ T cells.
To the best of our knowledge, this is the first demonstration that malaria, a pure systemic infection, can alter mucosal immunology. However, placental malarial infection in HIV-infected pregnant women has been associated with a 3-fold increase in CCR5 expression on placental cells within the chorionic villus,39 and in some40,41 but not all studies,42 malaria during pregnancy increased the risk of vertical HIV transmission by up to 3-fold; whether the latter was related to fetal immune alterations or to a malaria-induced increase in the maternal VL could not be determined. In our study, malaria induced a dramatic increase in α4β7 T-cell expression in blood, as was reported after systemic vaccination against yellow fever.27 Furthermore, there was a substantially increased frequency of blood T cells co-expressing α4β7 together with the HIV co-receptor CCR5 and the immune activation marker CD38, implying that malaria may be specifically targeting highly HIV-susceptible T cells to mucosal sites. It is not clear why overall mucosal numbers were not increased, but it is very interesting that the increased mucosal T-cell activation without absolute numeric increases so closely reflects our in vivo findings from young women from Kisumu.29 Because α4β7 is downregulated after T-cell mucosal migration, with a switch to αEβ7 in mucosal tissues to facilitate better anchoring of cells,43,44 the lack of increased α4β7 expression by mucosal T cells was not unexpected.
Malaria is an important public health issue, causing up to 500 million infections and an estimated 1.2 million deaths each year.3,4 Evidence from HIV-malaria–coinfected people indicates that this infection may also enhance HIV sexual transmission at a population level, as malaria increases HIV viral levels, which is a known independent risk factor for sexual HIV transmission.15,18,19 Our study suggests that malaria might also cause an increase in HIV susceptibility in HIV-uninfected individuals. However, it will be critical to confirm these murine findings in the human context and (if they are confirmed) to examine whether effective malaria therapy is able to reduce HIV target cell numbers at mucosal sites and hence HIV susceptibility. If this were the case, it would provide a rationale for future clinical trials to evaluate malaria treatment and/or prevention as an HIV prevention tool and would provide further justification for a global effort to eradicate malaria. Nonetheless, our findings in this murine model are provocative and closely parallel our observations of cervical T-cell alterations in young women from western Kenya.29
Some study caveats should be noted. PCCAS malaria in C57BL/6 mice is characterized by an acute phase with peak parasitemia at approximately 10 days postinfection, accompanied by a strong CD4+ T-cell response.33 Based on this, we chose day 10 postinfection as a reference point, and as a proof of concept, we were able to define a clear mucosal immune impact of acute malaria at the time of peak parasitemia. However, the mucosal impact of chronic or repeated malarial infection, the norm in malaria holoendemic areas, will be important to explore in future studies, as will exploration of the effect of natural antimalarial immunity after repeated malarial infections.45 This is particularly relevant because the long duration of genital immune alterations after Herpes simplex virus-type 2 reactivation has been implicated as a reason for the failure of genital herpes suppression to reduce HIV acquisition,46 although the lifelong nature and frequent asymptomatic reactivation of Herpes simplex virus-type 2 make this infection quite distinct to malaria. Peak parasitemia was fairly consistent at 20%–30% ten days after our standardized challenge, and this consistency, together with the relatively small sample size in each of the experimental replicates, means that we were not powered to define the mucosal impact of different parasitemia levels. The use of a murine model that does not sustain HIV infection means that we cannot establish whether the clear malaria-induced mucosal changes that we observed actually cause an increased susceptibility to HIV. The CCR5 expression levels in the mucosal tissues of our mice were lower than that observed in the gut and cervix of some other murine studies.47 Although the reason for this is not known, our mice were kept under pathogen-free conditions before and after PCCAS infection, which may contribute to overall reduced mucosal immune activation. Finally, it is important to note that HIV transmission rates are also extremely high in many areas of southern Africa where malaria is uncommon or absent,48 and so, this infection alone cannot wholly explain the enhanced sexual transmission of HIV in SSA.
In summary, we have used a murine model to demonstrate that malarial infection significantly increases the mucosal expression of immune parameters that would be expected to enhance HIV susceptibility, closely paralleling genital immune findings from young women in a malaria-endemic area. Although our findings suggest a novel mechanism by which malaria may increase HIV transmission in endemic areas, confirmatory human studies are needed.
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