Individuality in the Immune Repertoire and Induced Response of the Sponge Halichondria panicea

Abstract

The animal immune system mediates host-microbe interactions from the host perspective. Pattern recognition receptors (PRRs) and the downstream signaling cascades they induce are a central part of animal innate immunity. These molecular immune mechanisms are still not fully understood, particularly in terms of baseline immunity vs induced specific responses regulated upon microbial signals. Early-divergent phyla like sponges (Porifera) can help to identify the evolutionarily conserved mechanisms of immune signaling. We characterized both the expressed immune gene repertoire and the induced response to lipopolysaccharides (LPS) in Halichondria panicea, a promising model for sponge symbioses. We exposed sponges under controlled experimental conditions to bacterial LPS and performed RNA-seq on samples taken 1h and 6h after exposure. H. panicea possesses a diverse array of putative PRRs. While part of those PRRs was constitutively expressed in all analyzed sponges, the majority was expressed individual-specific and regardless of LPS treatment or timepoint. The induced immune response by LPS involved differential regulation of genes related to signaling and recognition, more specifically GTPases and post-translational regulation mechanisms like ubiquitination and phosphorylation. We have discovered individuality in both the immune receptor repertoire and the response to LPS, which may translate into holobiont fitness and susceptibility to stress. The three different layers of immune gene control observed in this study, - namely constitutive expression, individual-specific expression, and induced genes -, draw a complex picture of the innate immune gene regulation in H. panicea. Most likely this reflects synergistic interactions among the different components of immunity in their role to control and respond to a stable microbiome, seawater bacteria, and potential pathogens.

Keywords: LPS; Porifera; RNA-seq; early-diverging metazoa; gene expression; holobiont; host-microbe interaction; innate immunity.

Figure 1

The repertoire of pattern recognition receptors in Halichondria panicea. The PRR families GPCRs, SRCRs, CTLD genes, NLRs and TIR-domain receptors were identified based on conserved Pfam domains. Identification numbers of representative transcripts are shown next to protein models, and bold IDs indicate constitutive expression (i.e. present in each analyzed sponge). The number of additional transcripts with the same protein domain architecture is given behind the representative ID. TIR, Toll/interleukine-1 receptor; CARD, caspase recruitment domain; LEM, in nuclear membrane associated proteins; IG, immunoglobulin; NHL, repeat; LRR, leucine-rich repeat; WD40, repeat of 40 amino acids typically terminating in Trp-Asp; DUF4062, conserved domain of unknown function; CTL, C-type lectin; FN3, fibronectin III; Kazal, part of serine protease inhibitors; EGF, epidermal growth factor; EGF-calcium-binding, epidermal growth factor calcium-binding; EGF-LAM, Laminin-type epidermal growth factor-like; LU, Ly-6 antigen/uPA receptor-like; TSP, Thrombospondini domain; peptidase28; VWA, von Willebrand factor; SR, scavenger receptor cysteine-rich; ZP, zona pelludica; FZ, frizzled domain; Pkinase, phosphate kinase; TyrKc, tyrosine-specific kinase; GPS, G-protein-coupled receptor proteolytic site; HormR, present in hormone receptors; LDLa, low density lipoprotein receptor class A repeat; SO, somatomedin B-like; LamG, laminin G; CA, cadherin repeat; TM, transmembrane region; 7TM, 7 helix transmembrane domain. Other domains: DEATH, NACHT, MAM, WSC, Sushi, NIDO, IgGFc-binding, Calx-beta, Lysyl-oxidase, GAIN. The Figure was created with BioRender.com.

Figure 2

Expression patterns of PRRs. (A) 38% of the identified PRRs were constitutively expressed (present in all samples), 62% were expressed individual specific (PRRs present in only some samples). (B) Expression levels of constitutively expressed PRRs. Each row represents one gene and each column one sponge sample. The color gradient indicates the expression level (TMM normalized TPM). (C) Presence/absence of individual-specific PRRs. Each row represents one gene and each column one sponge sample. Note the clustering of individuals independent of the timepoint (LPS01 was only sampled at 1h and not at 6h). PRR, pattern recognition receptor; GPCR, G-Protein coupled receptor; CTLD, C-type lectin like domain; NLR, nucleotide-binding domain and leucine-rich repeat containing receptors; SRCR, scavenger receptor cysteine-rich; TIR, Toll/interleukin-1 receptor.

Figure 3

(A) Number of DEGs comparing LPS treated versus control sponges and the overlap between the two time points after LPS exposure, 1 h and 6 h after treatment. Arrows indicate up- and downregulation in comparison to control treatment. (B) Proportion of consistent and variable DEGs (with consistent/variable expression patterns across replicate samples, see Methods). Total DEGs 1 h: 225, total DEGs 6 h: 257 (genes defined as differentially expressed with FDR p-value < 0.005 and log2|FC|≥2).

Figure 4

Differentially expressed genes at 1 h and 6 h after LPS exposure. The heatmap shows the TMM-normalized relative expression per DEG (rows) for control and LPS treated samples (columns). DEGs are divided according to expression pattern in consistent and variable expressed (see Methods). Genes were defined as differentially expressed with FDR p-value < 0.005 and log2|FC|≥2.

Figure 5

Annotated differentially expressed genes at 1 h and 6 h after LPS exposure separated into functional categories based on their annotation.

Figure 6

COG association network analysis from DEGs at 1 h and 6 h after LPS exposure identified from closest annotated proteins in Amphimedon queenslandica. Created with STRING. Edges represent protein-protein associations coded by color according to the type of evidence for the shown interaction.