Immunological function of Bombyx Toll9-2 in the silkworm (Bombyx mori) larval midgut: Activation by Escherichia coli/lipopolysaccharide and regulation of growth.

Toll receptors are important regulators of insects' innate immune system which, upon binding of pathogen molecules, activate a conserved signal transduction cascade known as the Toll pathway. RNA interference (RNAi) is a powerful tool to study the function of genes via reverse genetics. However, due to the reported refractory of RNAi efficiency in lepidopteran insects, successful reports of silencing of Toll receptors in the silkworm Bombyx mori have not been reported yet. In this study, a Toll receptor of the silkworm Bombyx Toll9-2 (BmToll9-2) was cloned and its expression and function were analyzed. The results showed that BmToll9-2 contains an ectodomain (ECD) with a signal peptide and nine leucine-rich repeats, a transmembrane helix, and a cytoplasmic region with a Toll/interleukin-1 domain. Phylogenetic analysis indicates that BmToll9-2 clusters with other insect Toll9 receptors and mammalian Toll-like receptor 4. Oral infection of exogenous pathogens showed that the Gram-negative bacterium Escherichia coli and its main cell wall component lipopolysaccharide (LPS), as well as the Gram-positive bacterium Staphylococcus aureus and its main cell wall component peptidoglycan, significantly induce BmToll9-2 expression in vivo. LPS also induced the expression of BmToll9-2 in BmN4 cells in vitro. These observations indicate its role as a sensor in the innate immunity to exogenous pathogens and as a pathogen-associated receptor that is responsive to LPS. RNAi of BmToll9-2 was effective in the midgut and epidermis. RNAi-mediated knock-down of BmToll9-2 reduced the weight and growth of the silkworm. Bacterial challenge following RNAi upregulated the expression of BmToll9-2 and rescued the weight differences of the silkworm, which may be related to its participation in the immune response and the regulation of the microbiota in the midgut lumen of the silkworm larvae.

Toll-9 interacts with Toll-1 to mediate a feedback loop during apoptosis-induced proliferation in Drosophila.

Drosophila Toll-1 and all mammalian Toll-like receptors regulate innate immunity. However, the functions of the remaining eight Toll-related proteins in Drosophila are not fully understood. Here, we show that Drosophila Toll-9 is necessary and sufficient for a special form of compensatory proliferation after apoptotic cell loss (undead apoptosis-induced proliferation [AiP]). Mechanistically, for AiP, Toll-9 interacts with Toll-1 to activate the intracellular Toll-1 pathway for nuclear translocation of the NF-κB-like transcription factor Dorsal, which induces expression of the pro-apoptotic genes reaper and hid. This activity contributes to the feedback amplification loop that operates in undead cells. Given that Toll-9 also functions in loser cells during cell competition, we define a general role of Toll-9 in cellular stress situations leading to the expression of pro-apoptotic genes that trigger apoptosis and apoptosis-induced processes such as AiP. This work identifies conceptual similarities between cell competition and AiP.

Modulation of the transcriptional response of innate immune and RNAi genes upon exposure to dsRNA and LPS in silkmoth-derived Bm5 cells overexpressing BmToll9-1 receptor.

Injection or feeding of dsRNA is commonly used to induce specific gene silencing by RNAi in insects but very little research has been carried out to investigate non-specific effects on gene expression of dsRNA as pathogen-associated molecular pattern (PAMP). This study focuses on the potential role of the BmToll9-1 receptor to modulate the transcriptional response of innate immune and RNAi genes to dsRNA and lipopolysaccharide (LPS), which was used for comparison. To study this role, we took advantage of the silkmoth-derived Bm5 cell line, which does not express BmToll9-1 endogenously, and engineered a transformed cell line that permanently expresses BmToll9-1. Quantitative mRNA expression studies showed that BmToll9-1 can significantly alter the transcriptional response to dsRNA and LPS: (1) BmToll9-1 promotes the transcriptional response of Dicer2, encoding a key component of the RNAi machinery, and, to a lesser extent, that of transcription factors in the Jak-STAT and Toll pathways; and (2) BmToll9-1 represses the transcriptional induction of the IMD and Jak-STAT pathway genes, as well as the antimicrobial peptide (AMP) effector genes, by LPS. Thus, BmToll9-1 was identified as a modulator of innate immune and RNAi machinery gene expression that could be related to its preferential expression in the larval gut, the major barrier of pathogen entry. While BmToll9-1 was found to modulate RNAi-related gene expression, a reporter-based RNAi assay established no evidence for a direct interaction of BmToll9-1 with the intracellular RNAi machinery.