Pro-inflammatory Cytokine Expression of Spleen Dendritic Cells in Mouse Toxoplasmosis

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Abstract

INTRODUCTION

Toxoplasma gondii is an obligate intracellular protozoan parasite infecting felines as its definitive hosts and an important zoonotic pathogen that can cause severe diseases especially in congenitally infected patients and individuals with human immunodeficiency virus (HIV) patients [1,2]. Although T. gondii is well known to infect macrophages primarily, it is capable of invading and replicating within a wide variety of host cells including dendritic cells.

Dendritic cells (DC) have been known as the major sentinels of the strong innate immune cells against infectious organelles [3]. Maturation and activation of DC in response to infection plays a major role in the initiation of innate immunity and the subsequent development of adaptive immunity. In toxoplasmosis, it has been known that mice depleted DC are more susceptible to T. gondii [4]. This phenomenon has been related with the reduced IL-12 production in the infected mice. Although IFN-γ is the major cytokine of resistance to T. gondii [5], other cytokines, such as IL-12, synthesized by DC are also known as crucial triggering cytokines to provoke a series of cell-mediated immunity [4,6]. Therefore, in the present study, we investigated on the changes of pro-inflammatory cytokine expression by splenic dendritic cells in chronic murine toxoplasmosis.

MATERIALS AND METHODS

RT-PCR

RNA was prepared by an RNA easy column (Intron, Seoul, Korea) and primer sets of IL-1α, IL-1β, IL-6, IL-10, and TNF-α were purchased from Bioneer (Cheongwon, Korea) and their primer set is listed in Table 1. RT-PCR reactions using the 1-step RT premix (Intron, Seoul, Korea) were performed according to the manufacturer's instructions; briefly, with an annealing temperature of 55℃ for 35 cycles. Expression results were calculated relative to the expression in β-actin and were performed in triplicate.

Table 1.

Primer sets and corresponding amplicons

RESULTS

FACS analysis of CD8α⁺/CD11c⁺ DC

In Fig. 1, the results of FACS analysis, the number of CD8α⁺ CD11c⁺ DC was significantly increased at week 1 and peaked at week 3 PI, and thereafter the number of CD8α⁺ CD11c⁺ DC decreased.

Fig. 1

FACS analysis of CD8α⁺/CD11c⁺ spleen DC in mouse toxoplasmosis. (A) (a) normal, (b) week 1, (c) week 2, (d) week 3, (e) week 4, and (f) week 6 PI. (B) Number of CD8α⁺/CD11c⁺ splenic DC after T. gondii infection. Note the number of CD8α⁺/CD11c⁺ DC increased significantly at week 1 after T. gondii infection.

Cytokine expression of splenic dendritic cells

As shown in Fig. 2, most pro-inflammatory cytokines, such as IL-1α, IL-6, IL-10, and TNF-α increased significantly at week 1 PI. Especially, IL-1β increased at week 1 and peaked at week 3 PI. Expression of all pro-inflammatory cytokines at week 6 PI was maintained higher than in the pre-infected group.

Fig. 2

RT-PCR findings of pro-inflammatory cytokines in the splenic DC in mouse toxoplasmosis. Note the increased cytokine expressions at week 1 PI. Each graph shows relative densities against β-actin. (a) β-actin, (b) IL-1α (□), (c) IL-1β (░), (d) IL-6 (▒), (e) IL-10 (▓), and (f) TNF-α (■).

Serum IL-10 level

Each cytokine expression of RT-PCR followed similar chronological changes. Therefore, IL-10, which has an important role in DC, was selected and measured its serum levels in mouse toxoplasmosis. The serum level of IL-10 increased significantly at week 1 PI (264.1 pg/ml) than normal (67.4 pg/ml) as in Fig. 3.

Fig. 3

Mouse serum IL-10 levels chronologically in T. gondii infection. The highest IL-10 concentration was measured at week 1 after the infection.

DISCUSSION

DC are well known to be the major sentinels of the innate immune system. Although all DC originate from precursors in the bone marrow, fully matured DC are differentiated into distinct phenotypic subsets. Two major lineages, myeloid and plasmacytoid, are known to develop in mice and humans. Especially in the mouse, myeloid DC are further subdivided into CD8α⁺, CD4⁺, and CDα^-CD4^- subsets [7] and lymphoid DC develop from an early lymphoid progenitor under the influence of IL-3 but independent of GM-CSF [8-10]. Phenotypic characteristics of these lymphoid DC include their expression of CD8α and CD1d in addition to the pan-DC marker CD11c. These subsets show not only specific immunologic functions but also unique repertoires of pattern recognition receptors, therefore, appears to be capable of responding to various microbial stimuli [11]. In our experiment, we performed a FACS analysis that CD8α⁺ CD11c⁺ DC were activated at the early period of week 1 after infection as shown in Fig. 1A and 1B. These results suggested that CD11c⁺ DC in spleen may have important roles both in protective mucosal immunity and propagation of T. gondii tachyzoites transformed from bradyzoites within cysts after oral infection. It is well known that DC could act as a migrating cell. Therefore, DC have been suggested as a means for some parasites to diffuse into the host [12,13]. Also, T. gondii tachyzoites infected DC can offer as shuttle cells for tachyzoites inducing transmigration into the mesenteric lymph node and spleen [14,15].

Recently, CD11c⁺ DC in the spleen are known predominantly to be tachyzoites-infected DC after intraperitoneal inoculation of T. gondii and can transport T. gondii tachyzoites from the intestinal epithelium to the brain [14]. Furthermore, these cells are known to show stronger intracellular associations with a certain type II parasite genotype than type I and contribute the tachyzoites dissemination [16]. In the injection experiments of mice with live tachyzoites or a soluble tachyzoite extract, IL-12p40-producing cells which are consisting entirely of CD11c⁺ DC were observed within a few hr in the T-cell areas of the spleen [6]. In our experiment, staining of the same spleen sections with anti-CD11c monoclonal antibody revealed a massive redistribution of DC in spleen as a consequence of T. gondii infection, with dominant clustering in the T-cell areas (data not shown). In Fig. 1 of FACS scan, CD8α⁺ CD11c⁺ DC is significantly increased at week 1 and peaked at week 3 PI. After then, the number of CD8α⁺ CD11c⁺ DC decreased. Actually, this decreased period is likely to concord with the term of cyst formation in the brain of infected mice, and in chronic infection, possibly DC from blood monocytes are migrating into the inflammatory sites of perivascular and parenchyma in the central nervous system (CNS) [17]. Therefore, these results suggested that CD8α⁺ CD11c⁺ DC might have protective roles in the early phase of murine toxoplasmosis.

In the present study, we compared pro-inflammatory cytokine expression of mouse splenic DC after T. gondii infection. Pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-6, IL-10, and TNF-α showed a significant increase at week 1 PI as shown in Fig. 2. Among them, IL-10, known as an anti-inflammatory cytokine, is produced by a variety of cells, including dendritic cells. The IL-10 signaling cascade is a major pathway involved in the control of pro-inflammatory cytokines, such as IL-12 and TNF-α [18,19]. Although IL-10 secreted from DC does not appear to be a major mechanism responsible for dampening IL-12 production [20], the balance between pro-inflammatory cytokine of IL-12 and down regulatory cytokine of IL-10 is known to be essential to control toxoplasmosis [21]. Actually, IL-10 increased synergistically with the TNF-α expression of splenic dendritic cells at week 1 PI as shown in Fig. 3.

In conclusion, the present data prove the pro-inflammatory cytokine expression of splenic DC in mice and suggested the possible protective roles of DC in the pathogenesis of toxoplasmosis. Therefore, the roles of some other myeloid related DC in toxoplasmosis should be investigated in the future.

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