Matrix Metalloproteinase Expression Varies Throughout Development and During the Innate Immune Response of Tobacco Hornworm. Manduca sexta

Abstract

Matrix metalloproteinases (MI'v1Ps) are a family of conserved, proteolytic enzymes. MMPs can degrade most extracellular matrix proteins. As such, they are key enzymes in tissue remodeling processes. such as molting and metamorphosis in insects. The presence of MMP in tracheae of 4th and 5th instar Manduca scxra (M sexta) larvae was investigated using zymograms and Western blots, and the expression was found to decrease at the end of the instar. Failure to completely inhibit gelatinolytic activity in a zymogram indicates the existence of various other proteases that arc involved in the molting process. However, delay in the appearance of the dorsal blood vessel along with a reduction in the body mass upon inhibition of MMP supports our hypothesis that MMPs are required for molting. MMPs are also known to be up-regulated in many disease states, where they may cause tissue damage. Their presence may be beneficial, as their inhibition may result in worsening of symptoms or increased mortality. We tested the hypothesis that MMPs play a role in innate immunity in the tobacco hornworm caterpillar. Jf. sex/a. Larvae infected with E. coli DH5a had significantly more circulating hemocytes (insect blood cells) when compared to the larvae v,:hich received an MMP inhibitor. suggesting that MMPs may be important for hernocyte recruitment from the hematopoietic organs such as the lymph gland and hematopoictic cells present near the v.'ing imaginal discs. The expression of MMP was increased by 50% in larvae that had been infected with E. coli DH5a. However, this response was abrogated upon inhibition of MMPs. reducing expression tocontrol levels. The majority of the circulating hemocytes were granulocytes. a cell type that phagocytoses foreign bodies. including pathogens. We used flow cytometry to assess phagocytic ability. Hemocytes in the presence of the MMP inhibitor were less abk to phagocytose sephadex beads. supporting the hypothesis that this MMP contributes to the cellular innate immune response. Our results suggest that MMP expression is high at the beginning of the in star while it reduces as the caterpillar reaches molting. during which other proteases like chitinases and molting fluid proteinases play a more prominent role. Our results also suggest that M. sex/a larvae have a reduced immunity to bacterial infections when MMPs are inhibited. Collectively, inhibition of MMPs during the molting process indicates that the larvae. by down regulating the expression of MMPs. prevent the elicitation of immune responses that is generally seen during the larval pupal metamorphosis.