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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

Induction of apoptosis in plasmatocytes and crystal cells triggers lamellocyte differentiation.Hemocyte preparations from 3rd instar larvae were analyzed under the epi-fluorescence microscope. (A-I) Both Hid- and Grim-expressing samples showed massive lamellocyte differentiation (F and I) whereas control samples showed none (C). In addition when apoptotic cell bodies were included in the counts, an increase in counts was observed (using DAPI staining) in Hid- and Grim-expressing samples (D and G) compared to controls (A). (J, K) Some GFP-positive hemocytes were still detectable after Hid (J) or Grim (K) expression (the scale bars correspond to 50 μm). (L) Quantification of total cell (including apoptotic bodies) and lamellocyte numbers (hemocyte counts were determined within a defined area, see Material and Methods for details). Significantly higher numbers of total cells/cell fragments and lamellocytes were detected in Hid or Grim samples compared to controls. (M, N) Quantification of lamellocyte numbers per larva at 25° (M) and 29°C (N). Black arrow indicates lamellocytes (F and I), and green arrows indicate GFP-positive live hemocytes (J and K). HFP: hml-Gal4,UAS-eGFP. Data represent means ± SD; t test: **p<0.01, *** p<0.001.
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pone.0136593.g003: Induction of apoptosis in plasmatocytes and crystal cells triggers lamellocyte differentiation.Hemocyte preparations from 3rd instar larvae were analyzed under the epi-fluorescence microscope. (A-I) Both Hid- and Grim-expressing samples showed massive lamellocyte differentiation (F and I) whereas control samples showed none (C). In addition when apoptotic cell bodies were included in the counts, an increase in counts was observed (using DAPI staining) in Hid- and Grim-expressing samples (D and G) compared to controls (A). (J, K) Some GFP-positive hemocytes were still detectable after Hid (J) or Grim (K) expression (the scale bars correspond to 50 μm). (L) Quantification of total cell (including apoptotic bodies) and lamellocyte numbers (hemocyte counts were determined within a defined area, see Material and Methods for details). Significantly higher numbers of total cells/cell fragments and lamellocytes were detected in Hid or Grim samples compared to controls. (M, N) Quantification of lamellocyte numbers per larva at 25° (M) and 29°C (N). Black arrow indicates lamellocytes (F and I), and green arrows indicate GFP-positive live hemocytes (J and K). HFP: hml-Gal4,UAS-eGFP. Data represent means ± SD; t test: **p<0.01, *** p<0.001.

Mentions: To further investigate the effects of apoptosis in hemocytes, we examined hemocyte preparations from non-infected larvae where apoptosis had been induced. When we bled mid 3rd to late 3rd instar larvae after hemocyte-specific expression of Hid or Grim, GFP-positive hemocytes were drastically reduced in both lines (Fig 3E and 3H) compared to hml-Gal4,UAS-eGFP/+ control crosses.


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)

Induction of apoptosis in plasmatocytes and crystal cells triggers lamellocyte differentiation.Hemocyte preparations from 3rd instar larvae were analyzed under the epi-fluorescence microscope. (A-I) Both Hid- and Grim-expressing samples showed massive lamellocyte differentiation (F and I) whereas control samples showed none (C). In addition when apoptotic cell bodies were included in the counts, an increase in counts was observed (using DAPI staining) in Hid- and Grim-expressing samples (D and G) compared to controls (A). (J, K) Some GFP-positive hemocytes were still detectable after Hid (J) or Grim (K) expression (the scale bars correspond to 50 μm). (L) Quantification of total cell (including apoptotic bodies) and lamellocyte numbers (hemocyte counts were determined within a defined area, see Material and Methods for details). Significantly higher numbers of total cells/cell fragments and lamellocytes were detected in Hid or Grim samples compared to controls. (M, N) Quantification of lamellocyte numbers per larva at 25° (M) and 29°C (N). Black arrow indicates lamellocytes (F and I), and green arrows indicate GFP-positive live hemocytes (J and K). HFP: hml-Gal4,UAS-eGFP. Data represent means ± SD; t test: **p<0.01, *** p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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pone.0136593.g003: Induction of apoptosis in plasmatocytes and crystal cells triggers lamellocyte differentiation.Hemocyte preparations from 3rd instar larvae were analyzed under the epi-fluorescence microscope. (A-I) Both Hid- and Grim-expressing samples showed massive lamellocyte differentiation (F and I) whereas control samples showed none (C). In addition when apoptotic cell bodies were included in the counts, an increase in counts was observed (using DAPI staining) in Hid- and Grim-expressing samples (D and G) compared to controls (A). (J, K) Some GFP-positive hemocytes were still detectable after Hid (J) or Grim (K) expression (the scale bars correspond to 50 μm). (L) Quantification of total cell (including apoptotic bodies) and lamellocyte numbers (hemocyte counts were determined within a defined area, see Material and Methods for details). Significantly higher numbers of total cells/cell fragments and lamellocytes were detected in Hid or Grim samples compared to controls. (M, N) Quantification of lamellocyte numbers per larva at 25° (M) and 29°C (N). Black arrow indicates lamellocytes (F and I), and green arrows indicate GFP-positive live hemocytes (J and K). HFP: hml-Gal4,UAS-eGFP. Data represent means ± SD; t test: **p<0.01, *** p<0.001.
Mentions: To further investigate the effects of apoptosis in hemocytes, we examined hemocyte preparations from non-infected larvae where apoptosis had been induced. When we bled mid 3rd to late 3rd instar larvae after hemocyte-specific expression of Hid or Grim, GFP-positive hemocytes were drastically reduced in both lines (Fig 3E and 3H) compared to hml-Gal4,UAS-eGFP/+ control crosses.

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