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

Arefin B, Kucerova L, Krautz R, Kranenburg H, Parvin F, Theopold U - PLoS ONE (2015)

Bottom Line: 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.Finally we show that the nitric oxide donor L-arginine similarly modifies the response against an early stage of tumor development in fly larvae.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

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.

No MeSH data available.


Related in: MedlinePlus

Drosophila larvae lacking hemocytes are not more susceptible to nematode infections.Hemocytes were depleted using two Hid insertion lines (viable-V or lethal-L) and two Hemolectin Gal4 driver lines. To visualize hemocytes, UAS-eGFP combined with hml-Gal4 was employed. Hml-Gal4,UAS-eGFP driven UAS-hid expression in both insertion lines successfully eliminated hemocytes (see S2 and S3 Figs). To maximize Hid expression we also used hml-Gal4 without UAS-eGFP. However, none of the crosses between Gal4 driver lines and Hid responder lines showed significantly increased mortality compared to the positive control (vx.Bc Imd see [13, 18]). The vertical axis shows normalized mortality and the negative control was set to 1. Data presented are means ± SD; t test: * p<0.05; **p<0.01.
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pone.0136593.g002: Drosophila larvae lacking hemocytes are not more susceptible to nematode infections.Hemocytes were depleted using two Hid insertion lines (viable-V or lethal-L) and two Hemolectin Gal4 driver lines. To visualize hemocytes, UAS-eGFP combined with hml-Gal4 was employed. Hml-Gal4,UAS-eGFP driven UAS-hid expression in both insertion lines successfully eliminated hemocytes (see S2 and S3 Figs). To maximize Hid expression we also used hml-Gal4 without UAS-eGFP. However, none of the crosses between Gal4 driver lines and Hid responder lines showed significantly increased mortality compared to the positive control (vx.Bc Imd see [13, 18]). The vertical axis shows normalized mortality and the negative control was set to 1. Data presented are means ± SD; t test: * p<0.05; **p<0.01.

Mentions: To get a better understanding of the role hemocytes play during nematode infections, we depleted them in larvae using a previously established method that relies on expression of the pro-apoptotic protein Hid in combination with the hemocyte-specific Hemolectin driver (hmlDelta-Gal4, in the following abbreviated as hml-Gal4) [5, 6]. We also used a second pro-apoptotic protein (Grim). Initially the strength of two UAS-hid and four UAS-grim lines was tested (see S1 Table for details) and those with the strongest effects chosen for most of our subsequent experiments (UAS-hid (L) and UAS-grim8.1 respectively). After expressing the two Hid lines in hemocytes using 'hml-Gal4,UAS-eGFP' (abbreviated in the following as HFP), almost all hemocytes were GFP-negative, showing that apoptosis had been induced successfully in third instar larvae, adults (S2–S4 Figs) and already at the first larval instar (S5). To our surprise none of the crosses that led to hemocyte depletion showed significant changes in mortality upon nematode infection (Fig 2). To exclude a diluting effect on the transcriptional activator (Gal4) due to dual targeting of UAS-eGFP and UAS-hid we repeated the infection using hml-Gal4 without UAS-eGFP to maximize UAS-hid expression. However, this also did not increase mortality of the Drosophila larvae (Fig 2). Taken together, these observations suggest that counter to our expectation apoptotic hemocyte depletion in larvae did not increase their sensitivity towards nematode infections.


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

Arefin B, Kucerova L, Krautz R, Kranenburg H, Parvin F, Theopold U - PLoS ONE (2015)

Drosophila larvae lacking hemocytes are not more susceptible to nematode infections.Hemocytes were depleted using two Hid insertion lines (viable-V or lethal-L) and two Hemolectin Gal4 driver lines. To visualize hemocytes, UAS-eGFP combined with hml-Gal4 was employed. Hml-Gal4,UAS-eGFP driven UAS-hid expression in both insertion lines successfully eliminated hemocytes (see S2 and S3 Figs). To maximize Hid expression we also used hml-Gal4 without UAS-eGFP. However, none of the crosses between Gal4 driver lines and Hid responder lines showed significantly increased mortality compared to the positive control (vx.Bc Imd see [13, 18]). The vertical axis shows normalized mortality and the negative control was set to 1. Data presented are means ± SD; t test: * p<0.05; **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555835&req=5

pone.0136593.g002: Drosophila larvae lacking hemocytes are not more susceptible to nematode infections.Hemocytes were depleted using two Hid insertion lines (viable-V or lethal-L) and two Hemolectin Gal4 driver lines. To visualize hemocytes, UAS-eGFP combined with hml-Gal4 was employed. Hml-Gal4,UAS-eGFP driven UAS-hid expression in both insertion lines successfully eliminated hemocytes (see S2 and S3 Figs). To maximize Hid expression we also used hml-Gal4 without UAS-eGFP. However, none of the crosses between Gal4 driver lines and Hid responder lines showed significantly increased mortality compared to the positive control (vx.Bc Imd see [13, 18]). The vertical axis shows normalized mortality and the negative control was set to 1. Data presented are means ± SD; t test: * p<0.05; **p<0.01.
Mentions: To get a better understanding of the role hemocytes play during nematode infections, we depleted them in larvae using a previously established method that relies on expression of the pro-apoptotic protein Hid in combination with the hemocyte-specific Hemolectin driver (hmlDelta-Gal4, in the following abbreviated as hml-Gal4) [5, 6]. We also used a second pro-apoptotic protein (Grim). Initially the strength of two UAS-hid and four UAS-grim lines was tested (see S1 Table for details) and those with the strongest effects chosen for most of our subsequent experiments (UAS-hid (L) and UAS-grim8.1 respectively). After expressing the two Hid lines in hemocytes using 'hml-Gal4,UAS-eGFP' (abbreviated in the following as HFP), almost all hemocytes were GFP-negative, showing that apoptosis had been induced successfully in third instar larvae, adults (S2–S4 Figs) and already at the first larval instar (S5). To our surprise none of the crosses that led to hemocyte depletion showed significant changes in mortality upon nematode infection (Fig 2). To exclude a diluting effect on the transcriptional activator (Gal4) due to dual targeting of UAS-eGFP and UAS-hid we repeated the infection using hml-Gal4 without UAS-eGFP to maximize UAS-hid expression. However, this also did not increase mortality of the Drosophila larvae (Fig 2). Taken together, these observations suggest that counter to our expectation apoptotic hemocyte depletion in larvae did not increase their sensitivity towards nematode infections.

Bottom Line: 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.Finally we show that the nitric oxide donor L-arginine similarly modifies the response against an early stage of tumor development in fly larvae.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

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.

No MeSH data available.


Related in: MedlinePlus