Differential modulation of the cellular and humoral immune responses in Drosophila is mediated by the endosomal ARF1-Asrij axis

Abstract

How multicellular organisms maintain immune homeostasis across various organs and cell types is an outstanding question in immune biology and cell signaling. In Drosophila, blood cells (hemocytes) respond to local and systemic cues to mount an immune response. While endosomal regulation of Drosophila hematopoiesis is reported, the role of endosomal proteins in cellular and humoral immunity is not well-studied. Here we demonstrate a functional role for endosomal proteins in immune homeostasis. We show that the ubiquitous trafficking protein ADP Ribosylation Factor 1 (ARF1) and the hemocyte-specific endosomal regulator Asrij differentially regulate humoral immunity. Asrij and ARF1 play an important role in regulating the cellular immune response by controlling the crystal cell melanization and phenoloxidase activity. ARF1 and Asrij mutants show reduced survival and lifespan upon infection, indicating perturbed immune homeostasis. The ARF1-Asrij axis suppresses the Toll pathway anti-microbial peptides (AMPs) by regulating ubiquitination of the inhibitor Cactus. The Imd pathway is inversely regulated- while ARF1 suppresses AMPs, Asrij is essential for AMP production. Several immune mutants have reduced Asrij expression, suggesting that Asrij co-ordinates with these pathways to regulate the immune response. Our study highlights the role of endosomal proteins in modulating the immune response by maintaining the balance of AMP production. Similar mechanisms can now be tested in mammalian hematopoiesis and immunity.

Figure 1

ARF1 regulates crystal cell- mediated melanization and phenoloxidase activity. (A–F) Photomicrographs showing posterior region of third instar larvae of specific genotypes (A) e33cGal4 (B) UAS arf1rnai (C) UAS arf1rnai/e33cGal4 (D) UAS arf1 (E) UAS arf1/e33cGal4 (F)UAS arf1rnai/UASarf1rnai; UAS arf1/e33cGal4 that were heated at 60 °C for 10 min to visualize the melanization response. (G) Melanized crystal cells were quantified and represented graphically. (H) Graph representing phenoloxidase activity in the hemolymph of the indicated genotypes (detected as absorbance at 490 nm) after conversion of L-3, 4-dihydroxyphenylalanine. Scale Bar: (A–F) 100 μm. Number of larvae analyzed per genotype (n = 10). Error bars show standard error of mean. P-value: ** and *** indicate P < 0.01 and P < 0.001 respectively.

Figure 2

Asrij regulates crystal cell- mediated melanization and phenoloxidase activity. (A–F) Photomicrographs showing posterior region of third instar larvae of specific genotypes (A) w1118 (B) arj9/arj9 (C) e33cGal4 (D) UAS arj (E) UAS arj/e33cGal4 (F) arj9/arj9; UAS arj/e33cGal4 that were heated at 60 °C for 10 min to visualize the melanization response. (G) Melanized crystal cells were quantified and represented graphically. (H) Graph representing phenoloxidase activity in the hemolymph of the indicated genotypes (detected as absorbance at 490 nm) after conversion of L-3, 4-dihydroxyphenylalanine. Scale Bar: (A–F) 100 μm. Number of larvae analyzed per genotype (n = 10). Error bars show standard error of mean. P-value: *, **, *** indicate P < 0.05, P < 0.01 and P < 0.001 respectively.

Figure 3

ARF1 and Asrij negatively regulate Toll pathway- mediated immune response. (A,B) Quantification of Toll pathway-governed antimicrobial peptide expression by qRT-PCR analysis shows that Drosomycin and Metchnikowin are upregulated and Defensin is downregulated in arf1 knockdown (A) and asrij null mutant (B) larvae. (C–F) Quantification for the total percentile of flies expressing the Toll pathway reporters - Drosomycin-GFP and Defensin-GFP in flies with e33cGal4-mediated arf1 knockdown (C,D) or asrij null (E) or e33cGal4-mediated asrij knockdown (F) respectively. (G) Images showing increased colocalization of Cactus and Ubiquitin in arf1 or asrij knockdown circulating larval hemocytes as compared to respective controls, also indicated by adjacent co-localization plots. (H) Images showing higher Dorsal specific signal in the entire hemocyte as well as in DAPI stained region (nucleus) for the asrij null (arj9/arj9) and arf1 knockdown (HmlGal4, UASGFP; UAS arf1rnai) larvae as compared to the respective controls (w1118 and HmlGal4, UASGFP). White dotted line indicates nuclear area under consideration. Arrowheads mark nuclei with higher Dorsal signal (I,J) Quantification of the fluorescence intensity for Dorsal staining in the entire cell as well as in the DAPI stained area of the cell for asrij null (I) and arf1 knockdown hemocytes (n = 10) (J). (K) Model indicating the suggested role of the ARF1-Asrij endocytic axis in regulating the Toll pathway. Error bars indicate standard error of mean. ** indicates P < 0.01 and *** indicates P-value < 0.001. Scale Bar: (G,H) 10 μm.

Figure 4

ARF1 and Asrij differentially regulate the Imd pathway. (A,B) Quantification of Imd pathway-governed antimicrobial peptide expression by qRT-PCR analysis shows that Attacin, Drosocin and Diptericin are highly up-regulated whereas Cecropin levels are unaffected in e33cGal4-mediated ARF1 knockdown flies (A). Cecropin levels are upregulated and Attacin, Drosocin, Diptericin levels are down-regulated in e33cGal4-mediated Asrij knockdown flies (B). (C–J) Quantification of the total percentile of flies expressing the Imd pathway reporters - Attacin-GFP, Cecropin-GFP, Drosocin-GFP and Diptericin-GFP in flies with e33cGal4 mediated ARF1 knockdown (C–F) or asrij knockdown (G–J) respectively. (K) Images showing unchanged intensity of Relish in the nucleus of arj9/arj9 larval fat body but increase in the intensity in arf1 knockdown larval fat body compared to the respective controls. Scale bar 10 µm. (L) Quantification of Relish intensity over the entire area of the tissue in the field of view as well as in the nucleus (DAPI stained area) in arj9/arj9 and arf1 knockdown fat bodies and in the respective controls (n = 10). Error bar represents standard error of mean. ns indicates statistically non-significant difference. * and ** indicates P-value < 0.05 and <0.01 respectively. (M) Model indicating the suggested role of the ARF1-Asrij endocytic axis in regulating the Imd pathway.

Figure 5

ARF1 and Asrij knockdown flies show compromised survival upon infection. (A–D) Survival curves showing that e33cGal4-mediated ARF1 or asrij knockdown flies show a reduced survival ability as compared to its control upon infection with either B. subtilis (A,C) or E. coli (B,D). At least 100 flies were tested per genotype over at least three independent experiments.

Figure 6

Asrij expression is modulated upon infection and in immune mutants. (A,B) qRT-PCR analysis of adult flies showing that asrij mRNA levels are down-regulated 24 and 48 hours post infection (A) and that asrij transcript levels vary among immune mutants (B). (C) Model illustrating loss of immune homeostasis in ARF1-Asrij depleted conditions leading to compromised survival of the flies.