Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila Immune System and Leads to a Pro-Inflammatory State

Abstract

Apart from their role in cellular immunity via phagocytosis and encapsulation, Drosophila hemocytes release soluble factors such as antimicrobial peptides, and cytokines to induce humoral responses. In addition, they participate in coagulation and wounding, and in development. To assess their role during infection with entomopathogenic nematodes, we depleted plasmatocytes and crystal cells, the two classes of hemocytes present in naïve larvae by expressing proapoptotic proteins in order to produce hemocyte-free (Hml-apo, originally called Hemoless) larvae. Surprisingly, we found that Hml-apo larvae are still resistant to nematode infections. When further elucidating the immune status of Hml-apo larvae, we observe a shift in immune effector pathways including massive lamellocyte differentiation and induction of Toll- as well as repression of imd signaling. This leads to a pro-inflammatory state, characterized by the appearance of melanotic nodules in the hemolymph and to strong developmental defects including pupal lethality and leg defects in escapers. Further analysis suggests that most of the phenotypes we observe in Hml-apo larvae are alleviated by administration of antibiotics and by changing the food source indicating that they are mediated through the microbiota. Biochemical evidence identifies nitric oxide as a key phylogenetically conserved regulator in this process. Finally we show that the nitric oxide donor L-arginine similarly modifies the response against an early stage of tumor development in fly larvae.

Nitric Oxide Mediates Insect Cellular Immunity via Phospholipase A2 Activation

“…melanogaster, a strain resistant to the endoparasitoid wasp, Leptoplina boulardi, produces a significant increase in NO concentrations from differentiated lamellocytes [47], and the elevated NO level mediates hemocyte encapsulation of the parasitoid eggs [16]. NO also acts in humoral immunity by inducing genes encoding AMPs in insects.…”

Extraction of Hemocytes from <em>Drosophila melanogaster</em> Larvae for Microbial Infection and Analysis

“…As melanization is a rapid process, these cells would be excluded from the pathogen infection experiments. Additionally, the anti-coagulant phenylthiourea can be added to DHIM to inhibit the phenoloxidase-activating system and melanization during wounding 9 . Nevertheless, as melanization and apoptosis are innate immune responses of Drosophila , their levels can be quantified following hemocyte extraction and stimulation using the methods described here.…”

“…Hemocytes have been used for morphological observations 5,6,7 , morphometric analysis 2,8 , phagocytosis analysis 2,3 , qRT-PCR 2,9 , immunoprecipitation 10,11 , immunofluorescent analysis 10,12 , immunostaining 13 , immunoblotting 3,10,11 and immunohistochemistry 9,14 . Although Drosophila S2 cells are also available for various in vitro experiments, immortalization and potential pre-existing viral infection change their behavior 15,16 .…”

Macrophages and cellular immunity in Drosophila melanogaster

“…Surprisingly, for the most part macrophages may not be essential during pupariation and adulthood. Animals with genetically ablated hemocytes are viable [174, 175], although developmental defects have been found at low penetrance [174, 176]. …”

“…This suggests that cellular immunity may have additional functions over the course of infection, not just in the short-term. However, alternative scenarios are possible, as it was recently shown that hemocyte ablation leads to a shift in inflammation status, with an upregulation of Toll signaling and downregulation of the Imd pathway [176]. Interestingly, adult macrophage responses may also be linked to environmental conditions.…”