Loss of Toll-like receptor 7 alters cytokine production and protects against experimental cerebral malaria

Abstract

Background: Malaria, caused by Plasmodium sp. parasites, is a leading cause of global morbidity and mortality. Cerebral malaria, characterized by neurological symptoms, is a life-threatening complication of malaria affecting over 500,000 young children in Africa every year. Because of the prevalence and severity of cerebral malaria, a better understanding of the underlying molecular mechanisms of its pathology is desirable and could inform future development of therapeutics. This study sought to clarify the role of Toll-like receptors (TLRs) in promoting immunopathology associated with cerebral malaria, with a particular focus on the understudied TLR7.

Methods: Using the Plasmodium berghei ANKA mouse model of experimental cerebral malaria, C57BL/6 mice deficient in various TLRs were infected, and their resistance to cerebral malaria and immune activation through cytokine production were measured.

Results: Loss of TLR7 conferred partial protection against fatal experimental cerebral malaria. Additionally, loss of TLR signalling dysregulated the cytokine profile, resulting in a shift in the cytokine balance towards those with more anti-inflammatory properties.

Conclusion: This work identifies signalling through TLR7 as a source of pathology in experimental cerebral malaria.

Figure 1

Survival of TLR-deficient mice during cerebral malaria. (A) C57BL/6 (B6), Tlr7 ^-/-, Tlr9 ^-/- , Tlr7 ^-/- Tlr9 ^-/- , and Myd88 ^-/- mice were challenged with 10⁶ P. berghei erythrocytes and monitored daily for survival (n = 63 B6, n = 71 Tlr7 ^-/-, n = 45 Tlr9 ^−/−, n = 25 Tlr7 ^-/- Tlr9 ^-/- , n = 38 Myd88 ^−/−). *, p < 0.05 (log rank [Mantel-Cox] test). Data were pooled from six experiments. (B) Infected C57Bl/6, Tlr7 ^−/−, Tlr9 ^-/- , and Myd88 ^−/− mice were monitored for parasitaemia. Right, zoomed-in plot of region contained within gray box on left. (n = 25 B6, n = 25 Tlr7 ^-/-, n = 16 Tlr9 ^−/−; n = 21 Myd88 ^−/−). Data were pooled from three independent experiments.

Figure 2

TLR-dependence of cytokine production during Plasmodium berghei infection. (A, B) Levels of IFNG, IL6, IL4, TNF, MIP1B, and IL10 were measured by cytometric bead array in plasma collected from mice of the indicated genotypes three days (A) or six days (B) after infection with 10⁶ P. berghei-infected erythrocytes. All units are pg/mL. Means with SE are shown. *, p < 0.05 (Mann-Whitney) (n = 30 B6, n = 22 Tlr7 ^-/-, n = 11 Tlr9 ^−/−, n = 3 Tlr7 ^-/- Tlr9 ^-/-, n = 15 Myd88 ^−/−). Data were pooled from three independent experiments.

Figure 3

Association of cytokine ratios with outcomes of experimental cerebral malaria. (A) For each genotype (B6, Tlr7 ^-/-, Tlr9 ^-/-, Tlr7 ^-/- Tlr9 ^-/- , and Myd88 ^-/-) a Pearson’s product moment correlation was used to determine the relationship between the average log₁₀ (plasma cytokine concentration + 1) and the log₁₀ fraction of each genotype that escaped cerebral malaria (survived until day 12). r ² values were calculated from a linear regression of the above. (B) As above, except using ratios of cytokines. A less stringent statistical threshold (α = 0.1) was tested to account for the propagation of error when calculating ratios. Light blue, p < 0.05; dark blue, p < 0.1 (Pearson’s correlation). (C) Significant log₁₀ plasma cytokine ratios from (B) as a function of average survival for each genotype. The linear regression across genotypes is shown with 95% confidence intervals. (D) Significant cytokine ratios from (B) by genotype. Errors represent SE and all statistical comparisons are to B6 mice. *, p < 0.05 (Mann–Whitney). (E) Box plots of significantly correlated, log₁₀ cytokine ratios for all mice, grouped by escape from cerebral malaria (survival past day 12). *, p < 0.05 (Mann–Whitney).