Reduced circulating dendritic cells in acute Plasmodium knowlesi and Plasmodium falciparum malaria despite elevated plasma Flt3 ligand levels

Human blood dendritic cells (DCs) are a heterogeneous population of antigen presenting cells, comprising several subsets with distinct phenotypes and functions [1]. Peripheral blood DCs can be divided into myeloid DCs (mDCs or classical DCs) and plasmacytoid DCs (pDCs), and are identified by their surface expression of CD11c and CD123, respectively [2]. There are three phenotypically distinct subpopulations of mDCs based on expression of CD1c (BDCA1⁺, DC2), CD16⁺ (FcγRIII) and CD141⁺ (BDCA3⁺, DC1) [1, 3]. DCs are key activators of the adaptive immune response to pathogens [4], including the malaria causing parasite Plasmodium [5]. Previous studies have shown that during experimental blood-stage Plasmodium infection [6‐10], and in adults with uncomplicated Plasmodium falciparum and Plasmodium vivax malaria [11‐13] numbers of all DC subsets are reduced in the periphery. The effect of the zoonotic parasite Plasmodium knowlesi on circulating DC subsets in human malaria is yet to be determined.

DC homeostasis is in part regulated by the haematopoietic cytokine FMS-like tyrosine kinase 3 ligand (Flt3L) [14‐16]. A role for Flt3L in DC development was uncovered when mice and humans were administered Flt3L and DC numbers subsequently expanded [14, 17]. Further, bone marrow progenitors treated with FLT3L, preferentially support the development of mature DCs in vitro [18]. In murine malaria, Flt3L preferentially stimulates the expansion of CD8α⁺ CD103⁺ DCs which activate CD8⁺ T cells [19]. In humanized mice, Flt3L is associated with CD141⁺ DC expansion, and the maintenance and expansion of other DC subsets including CD1c⁺ DCs and pDC [15].

In children with severe P. falciparum malaria, Plasmodium-induced Flt3L is reported to selectively stimulate the expansion of blood CD141⁺ DCs, but not other DC subsets [19]. Increased Flt3L is observed in adults with uncomplicated P. falciparum malaria [20], yet associations with DCs have not been investigated in adults. Here, plasma Flt3L and circulating DC numbers were quantified in adults experimentally infected with P. falciparum, and adults with clinical P. falciparum or P. knowlesi malaria to better understand the impact of a Plasmodium infection on circulating DC numbers and their association with plasma Flt3L levels.

The relationship between DCs and Flt3L during malaria is incompletely understood. Here we show that similar to CD1c⁺ DCs and pDC subsets, circulating CD141⁺ DCs decline in numbers during experimental P. falciparum infection. Despite this decline, there was no detectable perturbation of plasma Flt3L in a first sub-microscopic infection in adults. Furthermore, the previously reported loss of pDC, CD1c⁺ DC and CD141⁺ DC subsets was confirmed in adults with acute falciparum malaria; and for the first time a significant decline in pDC, CD1c⁺ DC and CD141⁺ DC subsets in acute uncomplicated knowlesi malaria is reported. Previous reports show that plasma Flt3L is elevated in falciparum malaria, and here, for the first time elevated plasma Flt3L in patients with knowlesi malaria is reported, suggesting a pan Plasmodium effect of increased Flt3L in malaria. However, there was no association between plasma Flt3L and CD141⁺ DCs, in either adults with uncomplicated malaria or submicroscopic infection. These data may imply that other haematopoetic factors such as GM-CSF, M-CSF and/or IL-4 may be involved individually or in combination with Flt3L for the maintenance of circulating DC [24] and highlight the complicated relationship between DC subsets and Flt3L during malaria.

Early dysfunction of DCs during experimental malaria has been reported previously [6, 10]. Here, CD141⁺ DCs are also lost from peripheral circulation during experimental P. falciparum infection. Loss of CD141⁺ DCs during experimental P. vivax infection in healthy adult volunteers [25], in clinical P. vivax [12, 13] and now also clinical knowlesi malaria suggests a clear pan Plasmodium effect on CD141⁺ DCs in adults with subpatent or clinical malaria. Indeed, all DC subsets are reduced in both falciparum and knowlesi malaria. Changes in peripheral DC numbers during malaria may be explained by DC migration [26], DC apoptosis [13] or the failure to re-populate DCs from the bone marrow [27]. Increased expression of apoptotic markers annexin V [10] and caspase-3 [6] on DC subsets has been shown, suggesting, early apoptosis may contribute to premature loss of DCs. Future studies are required to establish whether the marked loss of CD141⁺ DCs from the circulation during Plasmodium infection is due to migration to organs such as the spleen and to identify haematopoietic factors that may be impaired by Plasmodium infection.

The plasma Flt3L increase observed in acute, uncomplicated falciparum or knowlesi malaria, are consistent with previous studies that found plasma Flt3L levels to be increased in clinical disease [19, 20]. In children, Flt3L levels are significantly higher in severe malaria patients compared to uncomplicated malaria patients [19], however, Flt3L concentrations reported in uncomplicated malaria in children are lower than those recorded in adults in this study and by others [20]. In adults with uncomplicated malaria, higher Flt3L levels are positively correlated with parasitaemia [20] yet, here there was no correlation with parasitaemia during clinical malaria. A parasite density threshold may trigger Flt3L release, via sensing of uric acid crystal accumulation by mast cells as demonstrated in Plasmodium chabaudi infection [19, 30]. In mice, release of Flt3L by mast cells was reported to drive the expansion of splenic CD8α⁺ DCs [19]. Only one study has assessed mast cell function in human malaria, with increased activation of mast cells in patients with severe malaria when compared to uncomplicated malaria patients [29]. In contrast to clinical malaria, no increase in Flt3L during experimental infection was observed. These data suggest a parasite threshold and/or symptomatic disease are associated with Flt3L elevation in malaria.

In the current study, no association between plasma Flt3L and CD141⁺ DC, CD1c⁺ DC or pDC peripheral cell numbers, in adults with clinical malaria, nor subpatent infection. Rather, despite increased Flt3L in clinical malaria, circulating CD141⁺ DCs and other DC subsets were significantly reduced. Further, Flt3L plasma levels and DC subset numbers were comparable between convalescent patients and healthy controls. However, the recovery of DC subsets was not correlated with Flt3L. These observations may be explained by the role of other haematopoietic cytokines including GM-CSF, M-CSF and IL-4 in DC development [30, 31]. In mice, the lack of Flt3L is compensated by increased production of M-CSF and IL-4, which results in continued expansion of DCs from progenitor cells [24]. Paradoxically, DC numbers significantly expand from progenitor cells when Flt3L receptor is deleted, suggesting multiple cytokines can induce DC development [24]. Future studies are required to assess M-CSF and other DC generating cytokines longitudinally in clinical malaria cohorts and the associations of these factors with DC subsets. The lack of an observed correlation between Flt3L and parasitaemia or DC subsets during clinical malaria could also be impacted by the limited longitudinal sampling within our cohorts and restricted sample numbers.

In summary, similar to classical CD1c⁺ DCs (6), and pDCs (7) CD141⁺ DCs are reduced during experimental P. falciparum infection. Similarly, this study shows that pDC, CD1c⁺ DC and CD141⁺ DCs subsets are significantly reduced during clinical falciparum and for the first time, also in knowlesi malaria. These data suggest that Flt3L released in adults with uncomplicated malaria may not be sufficient to expand or restore circulating DC subsets during acute infection. Rather, multiple signals, not just Flt3L, may be required to expand CD141⁺ DCs and other DC subsets [30, 31].