Carp Il10 Has Anti-Inflammatory Activities on Phagocytes, Promotes Proliferation of Memory T Cells, and Regulates B Cell Differentiation and Antibody Secretion

Abstract

In the current study, we investigated the effects of carp Il10 on phagocytes and lymphocytes. Carp Il10 shares several prototypical inhibitory activities on phagocytes with mammalian IL-10, including deactivation of neutrophils and macrophages, as shown by inhibition of oxygen and nitrogen radical production, as well as reduced expression of proinflammatory genes and mhc genes involved in Ag presentation. Similar to mammalian IL-10, carp Il10 acts through a signaling pathway involving phosphorylation of Stat3, ultimately leading to the early upregulation of socs3 expression. To our knowledge, this is the first study of the effects of Il10 on lymphocytes in fish. Although Il10 did not affect survival and proliferation of T cells from naive animals, it greatly promoted survival and proliferation of T cells in cultures from immunized animals, but only when used in combination with the immunizing Ag. Preliminary gene expression analysis suggests that, under these circumstances, carp Il10 stimulates a subset of CD8+ memory T cells while downregulating CD4+ memory Th1 and Th2 responses. In addition to the regulatory effect on T cells, carp Il10 stimulates proliferation, differentiation, and Ab secretion by IgM+ B cells. Overall, carp Il10 shares several prototypical activities with mammalian IL-10, including downregulation of the inflammatory response of phagocytes, stimulation of proliferation of subsets of memory T lymphocytes, and proliferation, differentiation, and Ab secretion by IgM+ B lymphocytes. To our knowledge, this is the first comprehensive analysis of biological activities of fish Il10 on both phagocytes and lymphocytes showing functional conservation of several properties of Il10.

Cyprinid Herpesvirus 3 Il10 Inhibits Inflammatory Activities of Carp Macrophages and Promotes Proliferation of Igm+ B Cells and Memory T Cells in a Manner Similar to Carp Il10

“…An optimal stimulatory concentration of 0.5 U/ml recombinant cIl10, corresponding to 10 mg/ml recombinant protein, has previously been defined based on titration using a EPC-NF-kB-Luc cell line (9). This method proved unsuitable for the titration of cyhv3Il10 because cyhv3Il10 did not cross-react with the fathead minnow (EPC) cells, for which reason in the present study we show concentrations rather than units.…”

“…To investigate whether cyhv3Il10 would trigger Stat3 phosphorylation, as confirmed previously for cIl10 (9), HKL (5 3 10 6 ) were incubated for 10 min with 10 mg/ml recombinant cyhv3Il10 or with 1:5 diluted cell culture supernatants from CCB cells infected with the above-described wildtype/recombinant/revertant CyHV-3 strains. HKL cell lysates were prepared and the cytoplasmic fraction (20 mg/lane) was assessed for the presence of phospho-Stat3 using a cross-reacting anti-phospho-Stat3 (Tyr 705 ) (3E2) mouse mAb (1:500, Cell Signaling Technology) as previously described (9).…”

“…Briefly, fish were injected with 1 3 10 4 parasites per fish. Two weeks later, when parasitemia reached 1 3 10 6 to 1 3 10 7 parasites/ml blood, fish were injected once with 10 mg/kg melarsoprol (Arsobal), a drug for treatment of infections with human trypanosomes, previously shown to be effective against carp trypanosomes (9,13). All experiments with immunized fish were performed 3 mo after parasite clearance.…”

“…All experiments were performed with the approval of the Animal Experimental Committee of Wageningen University. Fish immunized against the parasite Trypanoplasma borreli were obtained as previously described (9,13). Briefly, fish were injected with 1 3 10 4 parasites per fish.…”

“…Structural analysis indicates that teleost fish, despite low sequence identity for Il10, possess a class II cytokine system surprisingly similar to that of humans (8). In a recent study, we showed that carp Il10 (cIl10) downregulates the inflammatory response of phagocytes, stimulates proliferation of subsets of memory T lymphocytes, and stimulates proliferation, differentiation, as well as Ab secretion by Igm + B lymphocytes (9). cIl10 acts through a conserved signaling pathway involving phosphorylation of Stat3, ultimately leading to an early upregulation of socs3, a cytokine-inducible negative regulator of cytokine signaling.…”