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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

Lamellocyte differentiation in the lymph gland of Hml-apo larvae.Dissected fixed lymph glands from 3rd instar larvae were stained with DAPI and the early lamellocyte-specific antibody L1. (B and G) Control (HFP/+) lymph glands show the presence of some plasmatocytes and/or crystal cells (B) whereas no GFP signal was observed in Hid-expressing lymph gland (G), which confirms effective elimination through apoptosis. Strong and extensive L1 staining was observed in the lymph gland of Hid-expressing larvae (H) whereas no L1 staining was found in the control (C). HFP: hml-Gal4,UAS-eGFP. The scale bars represent 50 μm.
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pone.0136593.g004: Lamellocyte differentiation in the lymph gland of Hml-apo larvae.Dissected fixed lymph glands from 3rd instar larvae were stained with DAPI and the early lamellocyte-specific antibody L1. (B and G) Control (HFP/+) lymph glands show the presence of some plasmatocytes and/or crystal cells (B) whereas no GFP signal was observed in Hid-expressing lymph gland (G), which confirms effective elimination through apoptosis. Strong and extensive L1 staining was observed in the lymph gland of Hid-expressing larvae (H) whereas no L1 staining was found in the control (C). HFP: hml-Gal4,UAS-eGFP. The scale bars represent 50 μm.

Mentions: Unexpectedly, we found massive differentiation of lamellocytes in both Grim- and Hid-expressing larvae (Fig 3F and 3I). Of note hml-Gal4,UAS-eGFP drives GFP transgene expression only in plasmatocytes and crystal cells and not in lamellocytes, which therefore are not targeted by expression of proapoptotic genes in our set-up [19]. Lamellocytes were already detected in the second and early third larval instar at a stage when the lymph gland still displayed a GFP signal indicating that depletion of gland-derived hemocytes had not occurred yet (S5) and is therefore likely due to the depletion of embryonic hemocytes. At the late third instar only few GFP-positive cells were left in both apoptotic lines (Fig 3J and 3K). When apoptotic cell bodies were included, total counts were found significantly increased in both Hid and Grim over-expressing larvae compared to control crosses (Fig 3L). This indicates that prior to their apoptotic death, the pool of plasmatocytes might have expanded. Alternatively, although we only included large DAPI-positive fragments in the counts, the fragmentation of apoptotic cells may lead to an overestimation of cell numbers. Increased lamellocyte titers were found both at 25°C and further increased at 29°C, the optimal temperature for the Gal4 system (Fig 3M and 3N). To detect lamellocyte differentiation in the lymph gland, we stained the glands using an early lamellocyte-specific antibody (L1) (Fig 4). Lymph glands from Hid over-expressing larvae showed substantial accumulation of L1 staining compared to the controls, suggesting that lamellocyte differentiation had occurred. In conclusion, these data suggest that plasmatocyte depletion under these conditions had been largely effective but appears to have induced a differentiation of lamellocytes. These findings confirm previous observations where lamellocyte-like cells had been reported after depletion of plasmatocytes with the hml-Gal4 driver [6].


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)

Lamellocyte differentiation in the lymph gland of Hml-apo larvae.Dissected fixed lymph glands from 3rd instar larvae were stained with DAPI and the early lamellocyte-specific antibody L1. (B and G) Control (HFP/+) lymph glands show the presence of some plasmatocytes and/or crystal cells (B) whereas no GFP signal was observed in Hid-expressing lymph gland (G), which confirms effective elimination through apoptosis. Strong and extensive L1 staining was observed in the lymph gland of Hid-expressing larvae (H) whereas no L1 staining was found in the control (C). HFP: hml-Gal4,UAS-eGFP. The scale bars represent 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0136593.g004: Lamellocyte differentiation in the lymph gland of Hml-apo larvae.Dissected fixed lymph glands from 3rd instar larvae were stained with DAPI and the early lamellocyte-specific antibody L1. (B and G) Control (HFP/+) lymph glands show the presence of some plasmatocytes and/or crystal cells (B) whereas no GFP signal was observed in Hid-expressing lymph gland (G), which confirms effective elimination through apoptosis. Strong and extensive L1 staining was observed in the lymph gland of Hid-expressing larvae (H) whereas no L1 staining was found in the control (C). HFP: hml-Gal4,UAS-eGFP. The scale bars represent 50 μm.
Mentions: Unexpectedly, we found massive differentiation of lamellocytes in both Grim- and Hid-expressing larvae (Fig 3F and 3I). Of note hml-Gal4,UAS-eGFP drives GFP transgene expression only in plasmatocytes and crystal cells and not in lamellocytes, which therefore are not targeted by expression of proapoptotic genes in our set-up [19]. Lamellocytes were already detected in the second and early third larval instar at a stage when the lymph gland still displayed a GFP signal indicating that depletion of gland-derived hemocytes had not occurred yet (S5) and is therefore likely due to the depletion of embryonic hemocytes. At the late third instar only few GFP-positive cells were left in both apoptotic lines (Fig 3J and 3K). When apoptotic cell bodies were included, total counts were found significantly increased in both Hid and Grim over-expressing larvae compared to control crosses (Fig 3L). This indicates that prior to their apoptotic death, the pool of plasmatocytes might have expanded. Alternatively, although we only included large DAPI-positive fragments in the counts, the fragmentation of apoptotic cells may lead to an overestimation of cell numbers. Increased lamellocyte titers were found both at 25°C and further increased at 29°C, the optimal temperature for the Gal4 system (Fig 3M and 3N). To detect lamellocyte differentiation in the lymph gland, we stained the glands using an early lamellocyte-specific antibody (L1) (Fig 4). Lymph glands from Hid over-expressing larvae showed substantial accumulation of L1 staining compared to the controls, suggesting that lamellocyte differentiation had occurred. In conclusion, these data suggest that plasmatocyte depletion under these conditions had been largely effective but appears to have induced a differentiation of lamellocytes. These findings confirm previous observations where lamellocyte-like cells had been reported after depletion of plasmatocytes with the hml-Gal4 driver [6].

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