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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

Hml-apo flies show a defective leg phenotype, which can be rescued by blocking apoptosis, pharmacological inhibition of NOS and antibiotic treatment.(A) Control adults (HFP/+) where legs were normal. (B-C’) Both Hid- and Grim-expressing adults showed defective legs ranging from shortened leg segments (mild phenotype, B and C) to the complete absence of a leg (strong phenotype, B’ and C’). In both cases, phenotypes were most pronounced for the 3rd leg pair. Arrows indicate the defective leg phenotype (B, B’, C and C’). (D) Inhibiting apoptosis by co-expressing UAS-grim28.2 with UAS-p35 rescued the defective leg phenotype. (E-G’’’) shows isolated legs including (E) normal control adult legs (E, HFP/+) and defective legs in both Hid and Grim lines (F, G, and G’) in non-treated conditions. (E”) Feeding L-NAME (a pharmacological inhibitor of NOS) to 3rd instar larvae, rescued the leg defects in both Hid and Grim adult flies (F” and G’’’) while feeding D-NAME (inactive isomer of NAME) did not (F’ and G”). (H) Quantification of the defective leg phenotype in apoptotic adults, and after rescue with antibiotic treatment and upon co-expressing UAS-grim28.2 with UAS-p35. Defective legs were found in both Hid and Grim lines. Hid lines showed a stronger phenotype (such as a complete absence of legs, bracket 1) and a higher frequency of defective legs than Grim adults. Using a different food source (DIM) rescued the defective leg phenotype in Hid- but not Grim-expressing flies (bracket 2) and the same was true for antibiotics treatment (bracket 3). Blocking apoptosis using UAS-p35 also rescued the defective leg phenotype (right part). (I) Quantification of defective leg penetrance after treatment (D-NAME and L-NAME) compared to non-treated flies. Data presented are means ± SD; t test: * p<0.05; **p<0.01 (n = 81, 120 and 62 for controls, 189, 114 and 62 for Hid-expressing flies and 206, 120 and 72 for Grim-expressing flies).
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pone.0136593.g008: Hml-apo flies show a defective leg phenotype, which can be rescued by blocking apoptosis, pharmacological inhibition of NOS and antibiotic treatment.(A) Control adults (HFP/+) where legs were normal. (B-C’) Both Hid- and Grim-expressing adults showed defective legs ranging from shortened leg segments (mild phenotype, B and C) to the complete absence of a leg (strong phenotype, B’ and C’). In both cases, phenotypes were most pronounced for the 3rd leg pair. Arrows indicate the defective leg phenotype (B, B’, C and C’). (D) Inhibiting apoptosis by co-expressing UAS-grim28.2 with UAS-p35 rescued the defective leg phenotype. (E-G’’’) shows isolated legs including (E) normal control adult legs (E, HFP/+) and defective legs in both Hid and Grim lines (F, G, and G’) in non-treated conditions. (E”) Feeding L-NAME (a pharmacological inhibitor of NOS) to 3rd instar larvae, rescued the leg defects in both Hid and Grim adult flies (F” and G’’’) while feeding D-NAME (inactive isomer of NAME) did not (F’ and G”). (H) Quantification of the defective leg phenotype in apoptotic adults, and after rescue with antibiotic treatment and upon co-expressing UAS-grim28.2 with UAS-p35. Defective legs were found in both Hid and Grim lines. Hid lines showed a stronger phenotype (such as a complete absence of legs, bracket 1) and a higher frequency of defective legs than Grim adults. Using a different food source (DIM) rescued the defective leg phenotype in Hid- but not Grim-expressing flies (bracket 2) and the same was true for antibiotics treatment (bracket 3). Blocking apoptosis using UAS-p35 also rescued the defective leg phenotype (right part). (I) Quantification of defective leg penetrance after treatment (D-NAME and L-NAME) compared to non-treated flies. Data presented are means ± SD; t test: * p<0.05; **p<0.01 (n = 81, 120 and 62 for controls, 189, 114 and 62 for Hid-expressing flies and 206, 120 and 72 for Grim-expressing flies).

Mentions: When examining adults after eclosion, we observed defects that particularly affected the third pair of legs. Either tarsal segments were missing or in extreme cases the whole leg was affected (Fig 8B, 8B’, 8C and 8C’). These defective leg phenotypes could also be rescued either by co-expressing p35, by feeding antibiotics or using DIM. The antibiotic treatment suggests that the microbiota contributes even to this phenotype either directly or via its effects on hemocytes. Interestingly, defective legs had been observed upon ectopic expression of mouse nitric oxide synthase [24]. This prompted us to test whether NO might similarly contribute to the leg defects in our system and indeed feeding a pharmacological inhibitor of NOS (L-NAME) but not its less active enantiomer D-NAME rescued the leg defects (Fig 8F, 8G and 8I). Potential sources for NO include the gut epithelium and lamellocytes [25, 26]. Due to its developmental effects when fed to larvae, the influences of L-NAME on eclosure rates could not be tested with sufficient stringency [27]. Instead when we applied the nitric oxide donor L-arginine to control flies, lamellocyte levels increased although neither melanization nor the leg defects could be further enhanced (S6 Fig). Conversely, feeding L-NAME (but not D-NAME) to Hid-expressing larvae reduced lamellocyte counts (S7 Fig). Together this means that NO promotes lamellocyte differentiation and that lamellocytes may in turn further enhance the NO concentration [26]. This positive feedback might explain the substantial increase in lamellocytes counts we observe (Fig 3, see Discussion for further details).


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)

Hml-apo flies show a defective leg phenotype, which can be rescued by blocking apoptosis, pharmacological inhibition of NOS and antibiotic treatment.(A) Control adults (HFP/+) where legs were normal. (B-C’) Both Hid- and Grim-expressing adults showed defective legs ranging from shortened leg segments (mild phenotype, B and C) to the complete absence of a leg (strong phenotype, B’ and C’). In both cases, phenotypes were most pronounced for the 3rd leg pair. Arrows indicate the defective leg phenotype (B, B’, C and C’). (D) Inhibiting apoptosis by co-expressing UAS-grim28.2 with UAS-p35 rescued the defective leg phenotype. (E-G’’’) shows isolated legs including (E) normal control adult legs (E, HFP/+) and defective legs in both Hid and Grim lines (F, G, and G’) in non-treated conditions. (E”) Feeding L-NAME (a pharmacological inhibitor of NOS) to 3rd instar larvae, rescued the leg defects in both Hid and Grim adult flies (F” and G’’’) while feeding D-NAME (inactive isomer of NAME) did not (F’ and G”). (H) Quantification of the defective leg phenotype in apoptotic adults, and after rescue with antibiotic treatment and upon co-expressing UAS-grim28.2 with UAS-p35. Defective legs were found in both Hid and Grim lines. Hid lines showed a stronger phenotype (such as a complete absence of legs, bracket 1) and a higher frequency of defective legs than Grim adults. Using a different food source (DIM) rescued the defective leg phenotype in Hid- but not Grim-expressing flies (bracket 2) and the same was true for antibiotics treatment (bracket 3). Blocking apoptosis using UAS-p35 also rescued the defective leg phenotype (right part). (I) Quantification of defective leg penetrance after treatment (D-NAME and L-NAME) compared to non-treated flies. Data presented are means ± SD; t test: * p<0.05; **p<0.01 (n = 81, 120 and 62 for controls, 189, 114 and 62 for Hid-expressing flies and 206, 120 and 72 for Grim-expressing flies).
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Related In: Results  -  Collection

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pone.0136593.g008: Hml-apo flies show a defective leg phenotype, which can be rescued by blocking apoptosis, pharmacological inhibition of NOS and antibiotic treatment.(A) Control adults (HFP/+) where legs were normal. (B-C’) Both Hid- and Grim-expressing adults showed defective legs ranging from shortened leg segments (mild phenotype, B and C) to the complete absence of a leg (strong phenotype, B’ and C’). In both cases, phenotypes were most pronounced for the 3rd leg pair. Arrows indicate the defective leg phenotype (B, B’, C and C’). (D) Inhibiting apoptosis by co-expressing UAS-grim28.2 with UAS-p35 rescued the defective leg phenotype. (E-G’’’) shows isolated legs including (E) normal control adult legs (E, HFP/+) and defective legs in both Hid and Grim lines (F, G, and G’) in non-treated conditions. (E”) Feeding L-NAME (a pharmacological inhibitor of NOS) to 3rd instar larvae, rescued the leg defects in both Hid and Grim adult flies (F” and G’’’) while feeding D-NAME (inactive isomer of NAME) did not (F’ and G”). (H) Quantification of the defective leg phenotype in apoptotic adults, and after rescue with antibiotic treatment and upon co-expressing UAS-grim28.2 with UAS-p35. Defective legs were found in both Hid and Grim lines. Hid lines showed a stronger phenotype (such as a complete absence of legs, bracket 1) and a higher frequency of defective legs than Grim adults. Using a different food source (DIM) rescued the defective leg phenotype in Hid- but not Grim-expressing flies (bracket 2) and the same was true for antibiotics treatment (bracket 3). Blocking apoptosis using UAS-p35 also rescued the defective leg phenotype (right part). (I) Quantification of defective leg penetrance after treatment (D-NAME and L-NAME) compared to non-treated flies. Data presented are means ± SD; t test: * p<0.05; **p<0.01 (n = 81, 120 and 62 for controls, 189, 114 and 62 for Hid-expressing flies and 206, 120 and 72 for Grim-expressing flies).
Mentions: When examining adults after eclosion, we observed defects that particularly affected the third pair of legs. Either tarsal segments were missing or in extreme cases the whole leg was affected (Fig 8B, 8B’, 8C and 8C’). These defective leg phenotypes could also be rescued either by co-expressing p35, by feeding antibiotics or using DIM. The antibiotic treatment suggests that the microbiota contributes even to this phenotype either directly or via its effects on hemocytes. Interestingly, defective legs had been observed upon ectopic expression of mouse nitric oxide synthase [24]. This prompted us to test whether NO might similarly contribute to the leg defects in our system and indeed feeding a pharmacological inhibitor of NOS (L-NAME) but not its less active enantiomer D-NAME rescued the leg defects (Fig 8F, 8G and 8I). Potential sources for NO include the gut epithelium and lamellocytes [25, 26]. Due to its developmental effects when fed to larvae, the influences of L-NAME on eclosure rates could not be tested with sufficient stringency [27]. Instead when we applied the nitric oxide donor L-arginine to control flies, lamellocyte levels increased although neither melanization nor the leg defects could be further enhanced (S6 Fig). Conversely, feeding L-NAME (but not D-NAME) to Hid-expressing larvae reduced lamellocyte counts (S7 Fig). Together this means that NO promotes lamellocyte differentiation and that lamellocytes may in turn further enhance the NO concentration [26]. This positive feedback might explain the substantial increase in lamellocytes counts we observe (Fig 3, see Discussion for further details).

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