Assessing Immunological Memory in the Solitary Ascidian Ciona robusta

Abstract

The immune defensive mechanisms active in the solitary ascidian Ciona robusta include phagocytic and encapsulating activity, largely brought about by phagocytic cells within the haemocyte population, the presence of complement components, which have been molecularly and functionally identified, and expression of a number of immune-related genes and pathways, identified by genome-based homology with vertebrate counterparts. Since C. robusta only displays highly conserved innate immune mechanisms, being devoid of an adaptive immune system, this organism is an excellent model for studying the features of innate memory, i.e., the capacity of the innate immune system to re-programming its responsiveness to potentially dangerous agents upon repeated exposure. In this study, we have developed an in vivo model for assessing the establishment and molecular/functional features of innate memory, by sequentially exposing C. robusta to a priming stimulus (microbial molecules), followed by a period of resting to return to basal conditions, and a challenge with microbial agents in homologous or cross-stimulation. The endpoints of immune activation were a functional activity (phagocytosis) and the molecular profiles of immune-related gene expression. The results show that exposure of C. robusta to microbial agents induces a reaction that primes animals for developing a different (expectedly more protective) response to subsequent challenges, showing the effective establishment of an immune memory. This immune memory relies on the modulation of a number of different mechanisms, some of which are priming-specific, others that are challenge-specific, and others that are non-specific, i.e., are common to all priming/challenge combinations (e.g., up-regulation of the Tnf and Lbp genes). Memory-dependent expression of the humoral immunity-related gene C3ar inversely correlates with memory-dependent variations of phagocytic rate, suggesting that complement activation and phagocytosis are alternative defensive mechanisms in C. robusta. Conversely, memory-dependent expression of the cellular immunity-related gene Cd36 directly correlates with variations of phagocytic rate, suggesting a direct involvement of this gene in the functional regulation of phagocytosis.

Keywords: Ciona robusta; immune priming; immunological memory; innate immunity; innate memory.

Figure 1

The in vivo model of innate memory in C. robusta. An in vivo model of establishment of innate memory was designed and optimised. This includes treatment of animals at time zero (priming), a period of resting (until day 8) to allow extinction of the response, and a second treatment (challenge). Biological samples were collected at the beginning of the experiment (before priming) for the evaluation of the basal conditions, at day 1 for evaluating the activation due to priming, at the end of the resting period to evaluate the return to basal conditions, and 1 day after challenge to evaluate the changes in the response due to previous priming.

Figure 2

Phagocyte populations in haemocytes of C. robusta exposed in vivo to LPS or LTA. Changes in the percentage of the two different phagocyte populations within haemocytes in response to in vivo exposure of C. robusta to marine solution (MS, control), LPS or LTA alone and in different priming/challenge combinations. Left panel: changes in the percentage of hyaline amoebocytes. Right panel: changes in the percentage of granular amoebocytes. Data are the mean ± SD of five animals.

Figure 3

Innate memory-induced variations in the phagocytic rate of haemocytes of C. robusta exposed in vivo to LPS or LTA. The phagocytic rate (PR), i.e., the percentage of phagocytosing cells within the phagocyte population in haemolymph, was assessed 24 h after primary exposure to LPS or LTA (dark grey columns, no priming), and 24 h after homologous or heterologous challenge of primed animals (light grey columns, with priming). Phagocytosis of two bacterial strains was evaluated, the gram-negative E. coli, and the gram-positive B. cereus. (A,C,E) LPS challenge. (B,D,F) LTA challenge. (A,B) PR of total haemocyte. (C,D) PR of hyaline amoebocytes. (E,F) PR of granular cells. The PR of total haemocytes from control animals receiving MS once or twice (controls) was 17.9 ± 3.7% for E. coli, and 22.4 ± 1.8% for B. cereus; the PR of control hyaline cells was 1.5 ± 1.1 for E. coli and 3.5 ± 0.4 for B. cereus, while the PR of control granular cells was 16.5 ± 1.8 for E. coli and 19.0 ± 0.9 for B. cereus. The mean values ± SD of five animals for each treatment are reported.

Figure 4

Innate memory-induced variations in the phagocytic index of haemocytes of C. robusta exposed in vivo to LPS or LTA. The phagocytic index (PI), i.e., the number of bacteria ingested by single phagocytes in haemolymph, was assessed 24 h after primary exposure to LPS or LTA (dark grey columns, no priming), and 24 h after homologous or heterologous challenge of primed animals (light grey columns, with priming). Phagocytosis of two bacterial strains was evaluated, the gram-negative E. coli, and the gram-positive B. cereus. (A,C,E) LPS challenge. (B,D,F) LTA challenge. (A,B) PI of total haemocyte. (C,D) PI of hyaline amoebocytes. (E,F) PI of granular cells. The PI of total haemocytes from control animals (treated with MS once or twice) was 1.6 ± 0.6 for E. coli, and 2.2 ± 0.4 for B. cereus; the PI of control hyaline cells was 0.9 ± 0.4 for E. coli and 1.5 ± 0.7 for B. cereus, while the PI of control granular cells was 3.0 ± 1.1 for E. coli and 3.4 ± 0.9 for B. cereus. The mean values ± SD of three animals for each treatment are reported.

Figure 5

Innate memory-induced variations in the expression of immune-related genes in the pharynx of C. robusta challenged in vivo with the gram-negative stimulus LPS. LPS-induced expression in the pharynx of genes encoding the immune-related factors C3-1, C3aR, IL-17-2, IL-17R, TNFα, TGFβ, LBP, TLR-2, TLR13, and CD36 was measured in control MS-primed C. robusta animals (upper panel MS/LPS), and in animals that were previously primed with LPS (centre panel LPS/LPS) or LTA (lower panel LTA/LPS). Gene expression is reported relative to expression in control naïve animals. The mean expression values ± SD from three animals for each treatment are reported.

Figure 6

Innate memory-induced variations in the expression of immune-related genes in the pharynx of C. robusta challenged in vivo with the gram-positive stimulus LTA. LTA-induced expression in the pharynx of genes encoding the immune related factors C3-1, C3aR, IL-17-2, IL-17R, TNFα, TGFβ, LBP, TLR-2, TLR13, and CD36 was measured in MS-primed control C. robusta animals (upper panel MS/LTA) and in animals that were previously primed with LTA (centre panel LTA/LTA) or LPS (lower panel LPS/LTA). Gene expression is reported relative to expression in control naïve animals. The mean expression values ± SD from three animals for each treatment are reported.

Figure 7

Expression of immune-related genes in the pharynx of C. robusta primed in vivo with bacterial stimuli. Expression in the pharynx of genes encoding the immune-related factors C3-1, C3aR, IL-17-2, IL-17R, TNFα, TGFβ, LBP, TLR-2, TLR13, and CD36 was measured in C. robusta animals 24 h after in vivo administration of 25 μg of LPS (upper panel LPS) or 25 μg of LTA (lower panel LTA). Gene expression is reported relative to expression in control naïve animals. The mean expression values ± SD from three animals are reported.

Figure 8

Innate memory mechanisms in C. robusta. Schematic representation of the putative mechanisms underlying innate memory in C. robusta, based on the results obtained in this study. Primed animals (with either LPS or LTA; centre), when challenged with LPS experience a general shift towards cellular response (right), while challenge with LTA induces a general shift towards humoral response (left). The shift is evaluated functionally (phagocytosis), in terms of relative number of granular vs. hyaline amoebocytes, and as increased (↑), decreased (↓), or normalised (=) expression of immune-related genes. Two genes, i.e., Tnf and Lbp, are always up-regulated in primed animals independently of the type of challenge.