In Vivo MMP Functions Figure 10. Detection of MMP-like collagenolytic activity on the surface of stimulated Tribolium hemocytes. Typical granular hemocytes from Tribolium (A,C) exhibited MMP-like enzymatic activity localized at their surface by hydrolyzing DQTM collagen type IV resulting in green fluorescence (B) and of quenched, fluorogenic MMP-1/MMP-9 peptidic substrate resulting in bright blue fluorescence (D), respectively. Scale bar, 10 mm. doi:10.1371/journal.pone.0004751.g010 Innate immunity Besides developmental functions, we provide evidence that at least MMP-1 exerts pleitrophic functions by additionally mediating proper innate immune defense against pathogens because MMP-1 (RNAi) beetles were more susceptible to the entomopathogenic fungus B. bassiana. We further determined elevated levels of MMP activity as well as gene expression of MMP-1, TIMP, and RECK in stimulated immune-competent hemocytes. This is in agreement with recent observations that activated human immune cells express high levels of MMPs [43] and that immune-competent hemocytes from the mollusk Crassostrea virginica exhibit increased levels of MMP activities [44] as well as induced gene expression of TIMP [45], in response to immune-challenge. In insect hemocytes, MMPs may be required for tissue transmigration to reach the site of infection or to activate cytokines or antimicrobial peptides, similar to what has been recently shown for mammalian lymphocytes [1–6], whereas TIMP and RECK may have regulatory functions. It is also noteworthy, that we have recently described the ability of the insect innate immune system to sense collagen IV fragments that represent endogenous danger signals during infection [46,47]. Accruing collagen IV fragments by increasing hemocyte-derived MMP activity, therefore, may serve as a potentiating factor of innate immune responses against pathogens according to the proposed danger model by Matzinger [48]. Since insect MMP-1s are most closely related to mammalian MMP-19 and MMP-28, we postulate that some functions may have been conserved between insects and mammals. Indeed, it has recently been shown that MMP-28 expression is induced during dermal wound healing [49] and that MMP-19 has indispensable functions in cutaneous homeostasis by modulating epidermal proliferation [50,51], cutaneous immune responses [27] as well as skin tumorigenesis [52,53]. Furthermore, human MMP-19 associates with the surface of human monocytes and macrophages [54] and impacts the maturation and response of mammalian T-cells that are involved in both innate and adaptive immunity [27]. Thus, insect model organisms lacking adaptive immune system may help to elucidate direct role of MMPs in innate specific immune responses. Moreover, we have recently determined septic injury-inducible MMP in the planarian Schmidtea mediterranea [55] belonging to the Lophotrochozoa, suggesting that MMPs exhibit evolutionarily conserved functions in innate immunity of many diverse animals. In sum, results from Tribolium MMPs shed new light on MMP roles in insect development and immunity in particular and on evolutionarily conserved in vivo roles of MMPs in Metaozoa in general. Materials and Methods Animals, RNAi and infection experiments Wild-type Tribolium castaneum strain San Bernardino and strain Pig-19 expressing EGFP in muscle tissues [29] beetles, kindly provided by Gregor Bucher (Georg August University, Goettin- PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4751