Elsevier

Immunobiology

Volume 218, Issue 2, February 2013, Pages 263-271
Immunobiology

NK cells and conventional dendritic cells engage in reciprocal activation for the induction of inflammatory responses during Plasmodium berghei ANKA infection

https://doi.org/10.1016/j.imbio.2012.05.018Get rights and content

Abstract

Cerebral malaria (CM) is the most severe syndrome associated with Plasmodium falciparum infections. Experimental evidence suggests that disease results from the sequestration of parasitized-red blood cells (pRBCs) together with inflammatory leukocytes within brain capillaries. We have previously shown that NK cells stimulate migration of CXCR3+ T cells to the brain of Plasmodium berghei ANKA-infected mice. Here we investigated whether interactions between NK cells and dendritic cells (DCs) are required for the induction of T cell responses involved in disease. For that, NK cell-depleted and control mice were infected with transgenic parasites expressing model T cell epitopes. T cells from TCR transgenic mice specific for those epitopes were adoptively transferred and proliferation was determined. NK cell depletion significantly reduced CD8+ but not CD4+ DC-mediated T cell priming. Lack of NK cells did not compromise CD8+ T cell responses in IL-12−/− mice, suggesting that NK cells stimulate IL-12 output by DCs required for optimal T cell priming. The contribution of DCs to NK cell function was also investigated. DC depletion and genetic deletion of IL-12 dramatically reduced NK cell-mediated IFN-γ responses to malaria. Thus NK cells and DCs engage in reciprocal activation for the induction of inflammatory responses involved in severe malaria.

Introduction

Plasmodium falciparum malaria causes around 500 million clinical cases each year worldwide. The blood stage of the parasite is largely responsible for the induction of disease (Miller et al. 2002). The fatalities are associated with a spectrum of disease syndromes including respiratory distress, metabolic acidosis, hypoglycemia, renal failure, pulmonary edema and cerebral involvement (White and Ho 1992). It is widely accepted that high parasite densities are a major determinant of disease development. To avoid clearance in the spleen, mature forms of blood-stage malaria adhere to vascular endothelial cells. This parasite sequestration induces obstructions in the blood flow resulting in hypoxia and hemorrhages (Miller et al. 2002) that are associated with the development of organ-specific syndromes such as CM and placental malaria. A large body of literature indicates that in addition to parasite sequestration, inflammatory responses also contribute to severe disease. High TNF-α levels (Molyneux et al. 1993) as well as increased production of IFN-γ and IL-1β (Pongponratn et al. 2003) have been associated with disease severity in human malaria cases. Inflammatory chemokines including MIP-1α, MIP-1β (Ochiel et al. 2005) as well as the CXCR3 chemokine IFN-γ-inducible protein 10 (IP-10) (Armah et al., 2007, Jain et al., 2008) have been found to be associated with increased risk of CM associated mortality.

The P. berghei ANKA infection of mice is a model of severe malaria. This rodent infection has many features in common with human disease and is thus a good model for certain aspects of clinical malaria (Brian De Souza and Riley, 2002, Miller et al., 2002, Schofield and Grau, 2005). Like in human malaria, pro-inflammatory cytokines and chemokines such as TNF-α (Grau et al. 1987), LT-α (Engwerda et al. 2002), IFN-γ (Grau et al. 1989) and IP-10 (Nie et al. 2009) have been shown to contribute to severe malaria in mice. Experimental evidence suggests that these inflammatory factors contribute to disease by up-regulating the expression of adhesion molecules involved in the binding of pRBC to the vascular endothelium and by stimulating recruitment of inflammatory leukocytes to the site of parasite sequestration.

Several leukocyte cell lineages have been shown to participate in the development of experimental CM. Depletion studies as well as experimental infection of mice deficient in CD4+ (Yanez et al. 1996) and CD8+ T (Belnoue et al., 2002, Nitcheu et al., 2003) cells demonstrated that these populations contribute to the induction of severe disease. Both CD4+ and CD8+ T cells have been found sequestered in brain blood vessels of malaria-infected mice (Belnoue et al., 2002, Nitcheu et al., 2003). Moreover, the vast majority of inflammatory T cells recruited to the brain during rodent malaria appear to migrate via a CXCR3-IP-10-dependent mechanism (Campanella et al., 2008, Miu et al., 2008, Nie et al., 2009). CD8+ T cells, which are substantially more abundant than CD4+ T cells within brain intravascular infiltrates, have been found to mediate cerebral disease in a perforin-dependent manner (Nitcheu et al. 2003). Furthermore, recent studies revealed that CD8+ T cells recruited to the brain during infection are parasite-specific (Lundie et al. 2008). DCs appear to be essential for priming of T cell responses involved in the development of experimental CM (Dewalick et al. 2007). Amongst these cells, CD8α+ conventional DCs were shown to be the main subset involved in the cross-presentation of parasite-expressed antigens to naïve CD8+ T cells (Lundie et al. 2008).

In addition to T cells, NK cells have been also shown to mediate inflammatory responses during malaria. These cells readily secrete IFN-γ in response to malaria parasites in humans (Artavanis-Tsakonas and Riley, 2002, Baratin et al., 2005) and mice (Ing and Stevenson 2009). Moreover, NK cells appear to contribute to the induction of experimental CM as depletion of this cell lineage with specific antibodies protects mice from P. berghei ANKA-mediated severe disease (Hansen et al. 2007). NK cells were shown to stimulate the recruitment of CXCR3+ T cells to the brain of malaria-infected mice. IFN-γ secretion by NK cells appears to be required to stimulate T cell recruitment, since adoptive transfer of NK cell-depleted recipients with wild-type but not IFN-γ−/− NK cells significantly reconstitutes the capacity of T cells to migrate to the brain of malaria-infected mice (Hansen et al. 2007).

The precise mechanism by which NK cells stimulate T cell function during malaria remains elusive. Emerging evidence demonstrated several interactions between NK cells and DCs, which result in reciprocal activation and appear to be important for the development of effective immune responses (Andoniou et al., 2005, Marcenaro et al., 2005, Vitale et al., 2005). Whereas DCs have been shown to activate NK cells via IFN-α, IL-12 and IL-15-dependent mechanisms, NK cells promote DC maturation via Nkp30 engagement and the through the release of TNF-α and IFN-γ(Vitale et al. 2005). NK cell-DC interactions during malaria infection have not been extensively investigated. In vitro studies indicated that monocytes and myeloid DCs are required for IFN-γ production by human NK cells in response to P. falciparum (Baratin et al., 2005, Newman et al., 2006). NK cells were shown to stimulate DC maturation and IL-12 production during P. chabaudi-AS infection in mice. These interactions appeared to stimulate in vitro proliferation and IFN-γ output by anti-CD3-stimulated CD4+ T cells (Ing and Stevenson 2009). The relevance of NK cell-DC interactions in the context of severe malaria induction remains elusive. Recent studies in cancer models (Geldhof et al., 2002, Adam et al., 2005) revealed that NK cells stimulate DCs to produce IL-12 for the induction of CD8+ T cell responses via an IFN-γ-dependent mechanism that appears to be independent of CD4+ T cell help (Mocikat et al. 2003). To determine if a similar process is responsible for the induction of T cell responses involved in the induction of severe malaria we investigated if NK cells influence the capacity of DCs to prime naive T cells during infection. We found that NK cells stimulate the DC-mediated priming of CD8+ T cells in response to P. berghei ANKA. Moreover, our results also revealed a reciprocal contribution of DCs and IL-12 for NK cell-mediated IFN-γ responses to malaria in vivo.

Section snippets

Mice and infections

Eight to 12 week-old C57BL/6 or IL-12p40−/− (F8 generation) mice were used. Groups of 6–10 mice were injected i.p. with 1 × 106 P. berghei ANKA pRBC or transgenic P. berghei ANKA expressing MHC-I and MHC-II-restricted chicken OVA epitopes (OVA257–264, H-2Kb restricted; OVA323–339 I-Ab and IAd restricted) fused to GFP (PbTG) or GFP alone (PbG) (Lundie et al. 2008). Transgenic parasite lines express a selection cassette encoding a mutated form of the dihydrofolate reductase synthase gene of

NK cells contribute to the DC-mediated priming of parasite-specific CD8+ T cells during P. berghei ANKA infection

Conventional DCs are the main antigen presenting cell population required for the induction of T cell responses involved in experimental CM (Dewalick et al. 2007). To determine whether NK cells modulate the capacity of DCs to prime naïve T cells during infection, C57BL/6 mice were treated with anti-Asialo GM1 antibody to deplete NK cells. Since it has been shown that in addition to NK cells, anti-Asialo GM1 might bind to activated but not naïve T cells (Charley et al. 1988), in previous work we

Discussion

Two contributing factors have been identified as the main determinants of severe malaria: parasite sequestration and pro-inflammatory responses. In experimental animal models of severe malaria, parasite sequestration in the brain and the subsequent recruitment of CXCR3+CD8+ T cells have been found to be responsible for CM induction (Belnoue et al., 2002, Nitcheu et al., 2003, Nie et al., 2009). Previous work revealed that DCs are able to cross-present antigen and prime naïve CD8+ T cells during

Acknowledgments

This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council IRIISS and Project Grant 575538.

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