Insects combat infection through carefully measured cellular (for example, phagocytosis) and humoral (for example, secretion of antimicrobial peptides (AMPs)) innate immune responses. Little is known concerning how these different defense mechanisms are coordinated. Here, we use insect plasmatocytes and hemocyte-like Drosophila S2 cells to characterize mechanisms of immunity that operate in the haemocoel. We demonstrate that a Drosophila cytokine, growth-blocking peptides (GBP), acts through the phospholipase C (PLC)/Ca2+ signalling cascade to mediate the secretion of Pvf, a ligand for platelet-derived growth factor- and vascular endothelial growth factor-receptor (Pvr) homologue. Activated Pvr recruits extracellular signal-regulated protein kinase to inhibit humoral immune responses, while stimulating cell ‘spreading’, an initiating event in cellular immunity. The double-stranded RNA (dsRNA)-targeted knockdown of either Pvf2 or Pvr inhibits GBP-mediated cell spreading and activates AMP expression. Conversely, Pvf2 overexpression enhances cell spreading but inhibits AMP expression. Thus, we describe mechanisms to initiate immune programs that are either humoral or cellular in nature, but not both; such immunophysiological polarization may minimize homeostatic imbalance during infection.

A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed , a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca mobilization in S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by knockdown in three fly model systems: in hemocyte-like S2 cells, knockdown decreases GBP-mediated innate immune responses; in larvae, knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, knockdown increases mortality rate following infection with and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated expression reduces lifespan. Conversely, knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity.

Background Previously, we have shown that the chloride channel ClC-2 modulates intestinal tight junction (TJ) barrier function. The aim of the present study was to investigate the role of ClC-2 in epithelial barrier function and recovery in the event of epithelial injury. Methods We investigated the role of ClC-2 in TJ barrier function in Dextran Sodium Sulfate (DSS)-induced colitis in ClC-2 knockout mice and ClC-2 knockdown intestinal Caco-2 cells. Barrier function was measured electrophysiologically and by transepithelial mannitol fluxes. Select tight junction and associated proteins were western blotted, cytokines were measured using qPCR and human colonic biopsies were examined with immunohistochemistry. Results ClC-2−/− mice had a higher disease activity index, higher histological scores, and increased paracellular permeability compared to wild type (WT) mice when treated with DSS. DSS-treated ClC-2−/− mice had increased claudin-2 expression, greater loss of occludin in the membrane, increased association of occludin with caveolin-1, and significantly increased TNFα and IL-1β mRNA. ClC-2 knockdown in human intestinal Caco-2 cells resulted in a greater loss of epithelial resistance in the event of epithelial injury. The restoration of colonic barrier function after DSS colitis was delayed in ClC-2−/− mice. In human colonic biopsies, the protein and mRNA expression of ClC-2 was found to be reduced in ulcerative colitis (UC) patients. Conclusions ClC-2 plays a critical role in experimental colitis in that its absence increases disease activity, reduces barrier function and recovery, and perturbs tight junctions. Furthermore, ClC-2 expression is markedly reduced in the colon of human patients with ulcerative colitis.