Insights into the Complement System of Tunicates: C3a/C5aR of the Colonial Ascidian Botryllus schlosseri

Abstract

As an evolutionary ancient component of the metazoan immune defense toolkit, the complement system can modulate cells and humoral responses of both innate and (in jawed vertebrates) adaptive immunity. All the three known complement-activation pathways converge on the cleavage of C3 to C3a and C3b. The anaphylatoxin C3a behaves as a chemokine in inflammatory responses, whereas C3b exerts an opsonic role and, ultimately, can activate the lytic pathway. C3aR, one of the mammalian receptors for C3a, is a member of the G-protein-coupled receptor family sharing seven transmembrane alpha helixes. C3aR can act as a chemokine and recruit neutrophils, triggering degranulation and respiratory burst, which initiates an inflammatory reaction. Mining the transcriptome of the colonial ascidian Botryllus schlosseri, we identified a transcript showing homology with both mammalian C3aR and C5aR. The gene (bsc3/c5ar) is actively transcribed in morula cells, the circulating immunocyte triggering the inflammatory reactions in response to the recognition of nonself. Its transcription is modulated during the recurrent cycles of asexual reproduction known as blastogenetic cycles. Moreover, the treatment of hemocytes with C3aR agonist, induces a significant increase in the transcription of BsC3, revealing the presence of an autocrine feedback system able to modulate the expression of C3 in order to obtain a rapid clearance of potentially dangerous nonself cells or particles. The obtained results support the previously proposed role of complement as one of the main humoral components of the immune response in tunicates and stress the importance of morula cells in botryllid ascidian innate immunity.

Keywords: Botryllus; C3a/C5aR; complement; tunicates.

Figure 1

Schematic organization of BsC3a/C5aR gene. Exons are represented by boxes and introns by lines. Numbers refer to the length of the nucleotide sequence.

Figure 2

Evolutionary relationships among deuterostome anaphylatoxin receptors obtained with the maximum likelihood (ML) method. Similar topologies were obtained with neighbor joining (NJ) and unweighted pair group method with arithmetic mean (UPGMA). Bootstrap confidence values are indicated at the left of each branch. Sequence accession numbers are reported in Table S2.

Figure 3

Schematic domain organization of BsC3a/C5aR protein. Numbers refer to the length of the amino acid sequence. IL: intracellular loop; EL: extracellular loop; CT: cytoplasmic tail; TM: transmembrane domain.

Figure 4

(A): level of bsc3a/c5ar transcription product in three phases of the colonial blastogenetic cycle as assessed by qRT PCR. EC: early cycle; MC: mid cycle; TO: takeover. (B): transcription of bsc3a/c5ar and bsc3 in colonies previously injected with C3aR agonist (dark grey bars) as compared to controls (light grey bars). Asterisks mark significant differences with respect to EC or the control (**: p < 0.01; ***: p < 0.001).

Figure 5

ISH with riboprobes for bsc3a/c5ar on hemocyte monolayers. (A): sense riboprobe; (B): antisense riboprobe. Scale bar: 10 µm.

Figure 6

Fraction of hemocytes labeled by the riboprobes for bsc3a/c5ar at MC and TO. Asterisks indicate significant differences with respect to the control (***: p < 0.001).