Apicomplexa: Malaria
3.3 Innate effector mechanisms
3.3.1 Pre-erythrocytic stages
The exact mechanisms governing immune responses targeted against the hep- atic stages of malaria are still unclear. Mouse models of malaria have elucidated some of the immune mechanisms that may be important and, in general, the innate immune response to liver-stage malaria is known to involve the secre- tion of interferon (IFN)-␥from natural killer (NK) cells, NKT cells and␥␦T cells (Figure 3.2). IFN␥promotes the production of interleukin (IL)-12 and IL-18 by localised phagocytes to boost NK cell activation. Effective immune responses against hepatic stages rely on the involvement of cells with a lytic capacity, such as NK cells, to lyse infected hepatocytes, thereby facilitating destruction of the developing LEFs.
3.3.2 Asexual erythrocytic cycle
Many of the symptoms of malaria can be attributed to the inflammatory responses generated from cells of the innate immune system in response to the erythrocytic cycle of malaria. Inflammatory cytokines, such as tumour necrosis factor (TFN), IL-12 and IFN-␥, can be measured in serum around the time that merozoites first burst from hepatocytes before the adaptive immune
Figure 3.2 Features of the immune response mounted against malaria.Recognition of parasite stages by phagocytic cells such as macrophages and dendritic cells induces the production of pro-inflammatory cytokines, which potentiate cytolytic cells such as NK cells,␥␦T cells and CD8+ cytotoxic T cells to produce IFN-␥and target infected hepatocytes with large exoerythrocytic forms. Lysis of infected hepatocytes can prevent the development of merozoites and, in turn, cycling of asexual forms of the parasite in erythrocytes. Effective immune responses against sporozoites and infected hepatocytes is known to be potentiated by phagocytic cells in the draining lymph nodes of the biting site, rather than in the liverper se. Adaptive immune responses leading to antibodies specific for sporozoites are able to neutralise further challenge infections from mosquito bites, providing some protection against reinfection. Immune response against erythrocytic stages is known to occur in the spleen, the main organ filtering the blood stream. This leads to the activation of CD4+ T helper cells, which develop into different phenotypes of CD4+ T cells, depending on the immunological environment at the time of challenge. CD4+ T cells ‘help’ B cells to become activated, proliferate and differentiate in to plasma cells that produce antibodies of different isotypes, including cytophilic antibodies that are highly effective at opsonisation and neutralisation of different parasite stages. The production of IFN-␥from Th1 cells augments the phagocytic capacity of macrophages to clear opsonised parasites from the body.
system becomes activated. Innate immune responses against malaria can help limit the density of the parasite population, but complete elimination of parasitaemia requires more parasite-specific adaptive immune effector mechanisms that take longer to develop.
3.3.2.1 Macrophages/monocytes
Both the number and activation of macrophages is increased during malaria in- fection. Macrophages play a significant role in the clearance of malaria-infected RBCs – particularly splenic macrophages, due to their location in the main lym- phoid organ filtering the bloodstream. The expression of a number of PRRs,
CD36, as well as receptors recognising opsonins, allows macrophages to recog- nise RBC stages of malaria and associated malarial products (Table 3.1). Typi- cal macrophage/monocyte responses to infected RBCs involve the production of pro-inflammatory cytokines such as IL-12 or IL-18 that, in turn, are impor- tant in the activation of other innate immune cell types such as NK cells (see below). Acute phase response cytokines are also produced by macrophages in malaria infection. TNF is secreted in conjunction with IL-1 and IL-6 in response to GPI anchors released at schizogony, causing the cyclical fevers associated with malaria infection.
Macrophages are able to phagocytose infected RBCs in both an antibody- dependent and antibody-independent manner. Antibody-dependent cellular cytotoxicity (ADCC) occurs when macrophages recognise antibody-opsonised infected RBCs or free parasite stages via surface Fc receptors. Antibody- independent phagocytosis by macrophages is thought to be mediated by the scavenger receptor CD36 via recognition of the Pf EMP-1 variant antigen on P.
falciparum and/or platelets adhered on infected RBCs.
3.3.2.2 Granulocytes
The role of granulocytes in malaria infection is not well studied. Molecules re- leased from neutrophil granules, such as myeloperoxidase, can be measured in the serum of patients infected with P. falciparum, demonstrating neutrophil activation in malaria infection. P. falciparum merozoites opsonised by antibody can induce respiratory burst in neutrophils, providing some protection against parasite growth. However, intact infected RBCS are not thought to activate neutrophils.
3.3.2.3 NK cells
The serum of non-immune humans experimentally infected with P. falciparum has been found to contain granzyme A and IFN-␥ before the onset of clini- cal symptoms or detectable circulating parasitaemia, and before activation of CD8+ T cells. This suggests that innate NK cells are activated early after infec- tion. NK cell numbers increase in the circulation of infected children and have a greater capacity for lysis.
Infected RBCs can be recognised by NK cells; incubation of a human NK cell line with P. falciparum-infected RBCs results in rosetting around the NK cells, an interaction that has putatively been linked to the interaction of Pf EMP1 with CD36 expressed on the NK cell surface. Once NK cells have made phys- ical contact with infected RBCs, exposure to the pro-inflammatory cytokines IL-12 and IL-18 from macrophages is required for full activation. Thus, there is cooperation between macrophages/monocytes and NK cells in NK recognition of malaria-infected RBCs.
NK cells are thought to play an important immune-modulatory role in anti- malarial immune responses early after infection, via the secretion of IFN-␥. These cells are the main source of IFN-␥ production when PBMCs from hu- mans are incubated with infected P. falciparum RBCs. Mouse malaria infections suggest that, in the absence of NK cells, IFN-␥levels are decreased in infected animals, and parasites are detectable earlier after infection.
3.3.2.4 ␥␦T cells
Infection with either P. falciparum or P. vivax results in an expansion of ␥␦
T cells that are thought to recognise malaria-derived non-peptidic phospho- antigens. Activation of ␥␦ T cells in malaria infection requires exogenous cytokine stimulation from other cells of the immune system. However, when activated, these cells produce IFN-␥ and perform cytotoxic actions on infected RBCs.
3.3.2.5 Dendritic cells
Myeloid (but not plasmacytoid) DCs are generally believed to play an important role in priming T cells in malaria infection, in turn activating adaptive immune responses against malaria. Uptake of infected RBCs, as in macrophages, can occur via opsonic or non-opsonic routes. Again, non-opsonic uptake of P.
falciparum infected RBC is thought to occur via Pf EMP-1 interactions with CD36. This interaction has been shown to suppress the response of DCs to secondary TLR stimulation, as evidenced by a diminished up-regulation of MHC-II or co-stimulatory molecules CD40, CD80, CD86 in response to lipopolysaccharide (LPS).
In the P. yoelii mouse model of malaria, DCs phagocytose infected RBCs and digest them in acidified mature phagosomes. However, this process does not always appear to result in activation of DCs, which may be dependent on recog- nition of parasite products released at schizogony. Lysed P. yoelii infected RBCs activate DCs via activation of the MyD88, an adaptor protein used by PRRs to activate the transcription of pro-inflammatory cytokines. Some of the products of lysed infected RBCs responsible for this activation are listed in Table 3.1.
In contrast to the P. yoelii model of mouse malaria, intact P. chabaudi-infected RBC can activate mouse DCs, inducing up-regulation of MHC-II and co- stimulatory molecules CD80/CD86. One of the primary splenic DC subsets in the mouse to prime CD4+ T cells towards a Th1 phenotype in P. chabaudi infection are CD8+ DCs. Activation of CD8+ T cells via cross-presented anti- gens on MHC-I in P. berghei infection has also been shown to occur via the CD8+ DC subset. The activation of regulatory DC subsets, such as the CD11clowCD45RBhighDCs in the spleen during P. yoelii infection, may play a role in immunoregulation of malaria infection via the priming and expansion of IL- 10 expressing CD4+ T cells, a T cell subset that is able to reduce immunopathol- ogy in malaria infection.