Further improvements of the P. falciparum humanized mouse model

Abstract

Background: It has been shown previously that it is possible to obtain growth of Plasmodium falciparum in human erythrocytes grafted in mice lacking adaptive immune responses by controlling, to a certain extent, innate defences with liposomes containing clodronate (clo-lip). However, the reproducibility of those models is limited, with only a proportion of animals supporting longstanding parasitemia, due to strong inflammation induced by P. falciparum. Optimisation of the model is much needed for the study of new anti-malarial drugs, drug combinations, and candidate vaccines.

Materials/methods: We investigated the possibility of improving previous models by employing the intravenous route (IV) for delivery of both human erythrocytes (huRBC) and P. falciparum, instead of the intraperitoneal route (IP), by testing various immunosuppressive drugs that might help to control innate mouse defences, and by exploring the potential benefits of using immunodeficient mice with additional genetic defects, such as those with IL-2Rγ deficiency (NSG mice).

Results: We demonstrate here the role of aging, of inosine and of the IL-2 receptor γ mutation in controlling P. falciparum induced inflammation. IV delivery of huRBC and P. falciparum in clo-lip treated NSG mice led to successful infection in 100% of inoculated mice, rapid rise of parasitemia to high levels (up to 40%), long-lasting parasitemia, and consistent results from mouse-to-mouse. Characteristics were closer to human infection than in previous models, with evidence of synchronisation, partial sequestration, and receptivity to various P. falciparum strains without preliminary adaptation. However, results show that a major IL-12p70 inflammatory response remains prevalent.

Conclusion: The combination of the NSG mouse, clodronate loaded liposomes, and IV delivery of huRBC has produced a reliable and more relevant model that better meets the needs of Malaria research.

Figure 1. Effects of aging and inosine on parasitemia in NOD/SCID mice.

Parasitemia is shown as a percentage of total erythrocytes found in mouse peripheral blood measured on giemsa-stained thin smears. Levels are shown for old mice (>15 weeks) receiving inosine (green) or without inosine (yellow) and young mice (<15 weeks) with inosine (red) or without (blue). P. falciparum 3D7 strain was inoculated on day 0. A 400 µl pellet of huRBC and clo-lip (100 µl) were administered (IV and IP respectively) 3 times a week to all mice. For the inosine groups, inosine (250 mg/kg) was administered IP daily. Results represent the mean ± SEM from 11 different experiments, positive result/success means parasitemia lasting >12 days a) green curve: n = 41, 15 positive, 36,6% success, b) yellow curve; n = 30, 5 positive, 16,6% success; c) red curve; n = 29, 0 positive, 0% success; d) blue curve; n = 10, 1 positive, 10% success.

Figure 2. Effect of the aging process on the production of cytokines in NOD/SCID mice.

Panel (A): levels of various inflammatory mediators (IL-6, MCP-1, TNFα, IFNγ) in supernatants of peritoneal cells (n = 10) and splenocytes (n = 16) from young vs aged mice after stimulation with LPS (1 µg/ml) ; Panel (B) : levels of reactive oxygen intermediates, measured by chemiluminescence, in the presence of PMA (10 µg/ml) produced by peritoneal cells (n = 4) and splenocytes (n = 5) taken from young vs aged mice; Panel (C): in vivo levels of IFNγ in aged mice (with long-lasting parasitemia) (n = 4) compared to young mice (with short-lived parasitemia) (n = 5) as measured in serum samples collected 1, 3, and 6 days post infection with P. falciparum. Cytokine levels were measured by using the CBA mouse inflammatory kit by FACS. Results represent the mean ± SEM from two different experiments.

Figure 3. Effect of inosine and the aging process on production of inflammatory mediators in NOD/SCID mice.

Determination of levels of cytokines in supernatants of peritoneal cells (IL-6, MCP1, TNFα) or splenocytes (IFNγ) collected from NOD/SCID mice and stimulated in vitro with LPS (1 µg/ml). Results from cells of aged mice (>15 weeks) and young mice (<15 weeks) incubated with or without 1 mM inosine are shown. Results represent the mean ± SEM from an experiment performed using 3 mice per group.

Figure 4. Results obtained using the NSG-IV P. falciparum model.

Panel (A): proportion of huRBC (% of total erythrocytes), or “chimerism” seen in peripheral mouse blood, as determined by FACS analysis using an anti-human glycophorin A antibody (average of 18 mice). HuRBC grafting started 10 days before infection with P. falciparum; Panel (B): parasitemia patterns obtained after inoculation with either UPA (red; n = 10), 3D7 (blue; n = 4), or K1 (green; n = 4) P. falciparum strains. Results represent the means ± SEM from 2 different experiments. Panel (C): detailed pattern of P. falciparum growth over time in the NSG-IV model. Arrows indicate points at which >95% of parasites were at ring stage. Parasitemia is expressed as a percentage of total erythrocytes found in mouse peripheral blood. Mice received 750 µl of huRBC delivered IV 3 times a week, along with IP administration of liposomal clodronate (100 µl).

Figure 5. Examples of parasitemia obtained in the NSG-IV model.

Panels (A) and (B) illustrate the typical predominance of ring stage parasites seen at 48 h intervals (see Fig. 4 C and SI-II), with frequent polyparasitism. Such parasite densities are common using UPA parasites in mice having >75% chimerism. Panel (C) fully mature trophozoites seen 24 hours later, as compared to A and B. Single nucleus trophozoites predominate, though some can be mistaken for schizonts due to polyparasitism. However rings and young schizonts are also present in small proportions. Panel (D) illustrates one of the highest parasitemias obtained. Insert: active phagocytosis by a circulating monocyte. Panels (E) and (F) illustrate fully mature schizonts which are only occasionally seen in peripheral blood. Panel (G) stage IV gametocyte, Panel (H) rare occurrence of mature gametocyte (stage V) in peripheral blood.

Figure 6. Residual inflammation present in the IV-NSG model.

Panel A: using the sub-optimal dose of 400 µL huRBC grafting results in a reduction of chimerism (red line) and parasitemia (blue line); Panel B: inflammatory cytokine responses were measured in parallel using the CBA kit. Note the initial rise of IL6, followed by a major increase of IL-12p70, and a modest increase of TNFα and MCP-1 that are temporally associated with anemia, and might play a role in its etiology. Results represent the means ± SEM from 2 experiments (n = 7).