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Subpopulation of Macrophage-Like Plasmatocytes Attenuates Systemic Growth via JAK/STAT in the Drosophila Fat Body

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Subpopulation of Macrophage-Like Plasmatocytes Attenuates Systemic Growth via JAK/STAT in the Drosophila Fat Body

Mingyu Shin et al. Front Immunol.

Abstract

Drosophila hemocytes, like those of mammals, are given rise from two distinctive phases during both the embryonic and larval hematopoiesis. Embryonically derived hemocytes, mostly composed of macrophage-like plasmatocytes, are largely identified by genetic markers. However, the cellular diversity and distinct functions of possible subpopulations within plasmatocytes have not been explored in Drosophila larvae. Here, we show that larval plasmatocytes exhibit differential expressions of Hemolectin (Hml) and Peroxidasin (Pxn) during development. Moreover, removal of plasmatocytes by overexpressing pro-apoptotic genes, hid and reaper in Hml-positive plasmatocytes, feeding high sucrose diet, or wasp infestation results in increased circulating hemocytes that are Hml-negative. Interestingly these Hml-negative plasmatocytes retain Pxn expression, and animals expressing Hml-negative and Pxn-positive subtype largely attenuate growth and abrogate metabolism. Furthermore, elevated levels of a cytokine, unpaired 3, are detected when Hml-positive hemocytes are ablated, which in turn activates JAK/STAT activity in several tissues including the fat body. Finally, we observed that insulin signaling is inhibited in this background, which can be recovered by concurrent loss of upd3. Overall, this study highlights heterogeneity in Drosophila plasmatocytes and a functional plasticity of each subtype, which reaffirms extension of their role beyond immunity into metabolic regulation for cooperatively maintaining internal homeostatic balance.

Keywords: Drosophila melanogaster; Hemolectin; JAK/STAT; Peroxidasin; insulin signaling; plasmatocytes; upd3.

Figures

Figure 1
Figure 1
Distribution of total hemocytes during larval development. (A,B) Absolute numbers of hemocytes per mm2 at 72, 96, and 120 h after egg laying (AEL). Total (blue), Hml+Pxn+ (yellow), Pxn+Hml (magenta), and Hml+Pxn (green) hemocytes are increased over development in circulating (A) and total hemocytes (B). Total hemocytes are counted after vortexing larvae, therefore, indicating sessile and circulating populations (54). Error bars indicate S.D. (C–E) Circulating hemocytes bled at 72 (C), 96 (D), and 120 h AEL (E). Subtypes of plamatocytes are visualized by Hml (green), Pxn (magenta), and DAPI (blue) (HmlΔ-Gal4 UAS-EGFP; Pxn-LexA LexAop-mCherry). Co-localization of Hml and Pxn is indicated in white. Scale bar, 40 μm. (F–H) Pie chart quantitation of circulating plasmatocytes at 72 (F, related to C), 96 (G, related to D), and 120 h AEL (H, related E). Hml+Pxn+ (white), Pxn+Hml (magenta), and Hml+Pxn (green). (I–K) Circulating and sessile hemocytes bled at 72 (I), 96 (J), and 120 h AEL (K). Subtypes of plamatocytes are visualized by Hml (green), Pxn (magenta), and DAPI (blue) (HmlΔ-Gal4 UAS-EGFP; Pxn-LexA LexAop-mCherry). Co-localization of Hml and Pxn is indicated in white. Scale bar, 40 μm. (L–N) Pie chart quantitation of circulating and sessile plasmatocytes at 72 (L, related to I), 96 (M, related to J), and 120 h AEL (N, related K). Hml+Pxn+ (white), Pxn+Hml (magenta), and Hml+Pxn (green).
Figure 2
Figure 2
Ablation of Hml+ hemocytes increases the number of circulating and sessile hemocytes, and induces upd3 expression. (A,B) Expression of Pxn (magenta) in HmlΔ-Gal4 UAS-EGFP, Pxn-LexA LexAop-mCherry background. Compared to hemocytes sampled from wild type (A), the genetic ablation of Hml+ cells increases the number of Pxn+ (magenta) cells (HmlΔ-Gal4 UAS-EGFP UAS-hid,rpr, Pxn-LexA LexAop-mCherry) (B). Hml (green), Pxn (magenta), and DAPI (blue). Scale bar, 40 μm. (C) Quantitation of circulating and sessile hemocytes in genetic backgrounds used in (A,B). Graphs indicate median plots of DAPI positive cells per mm2 in each genotype. Highest and lowest bars indicate maximum and minimum values, respectively. Controls (green) (Pxn-LexA LexAop-mCherry or UAS-hid,rpr; Pxn-LexA LexAop-mCherry), Hml ablated background (pink) (HmlΔ-Gal4 UAS-EGFP UAS-hidrpr; Pxn-LexA LexAop-mCherry). Statistical significance was determined by t-test. ***p < 0.001; not-significant, ns. (D) Pie chart shows the proportion of Hml+Pxn+ (white), Hml+Pxn (green), or Pxn+Hml (magenta) in circulating and sessile plasmatocytes of Hml+ablated background (HmlΔ-Gal4 UAS-EGFP UAS-hidrpr; Pxn-LexA LexAop-mCherry). Quantitation of (B). (E,F) mRNA levels of signaling molecules and antimicrobial peptides in Hml+ablated background (HmlΔ-Gal4 UAS-hid,rpr). RT-qPCR analysis of genes related to active immunity normalized by rp49 using whole larvae (E). upd3 expression in relevant organs (F). upd3 expression is highly increased in whole larvae (E), and hemocytes show identical increase in upd3 (F). Statistical analyses were performed using two-way ANOVA in whole larvae and organs. ***p < 0.001; ****p < 0.0001; not significant, ns. (G,G') upd3 (magenta; upd3-LexA LexAop-mRFP) is co-localized with Pxn (green) in total hemocytes. Inset in magnified view. DAPI is blue. Scale bars, 40 μm. (H,H',I,I') STAT92E::edGFP (green) expression is increased in the fat body upon loss of Hml+ hemocytes. (H,H') is control (Hml-Gal4; STAT92E::edGFP) and (I,I') is Hml+ ablated background (Hml-Gal4 UAS-hid, rpr; STAT92E::edGFP). Scale bars, 80 μm. (J,K) Socs36E is increased in the fat body when Hml+ hemocytes are genetically ablated or upd3 is ectopically expressed in Pxn+ cells. mRNA expression of Socs36E is increased in the fat body extracted from HmlΔ-Gal4 UAS-hid, rpr (J) or from Pxn-gal4 UAS-upd3 (K). Graph indicates RT-qPCR analyses of Socs36E in in the fat body. Error bar in graph is S.D. Statistical significance was determined by using t-test. ***p < 0.001.
Figure 3
Figure 3
Attenuated insulin signaling in the fat body upon loss of Hml+ hemocytes. (A–C) Membrane localization of tGPH, a reporter for PI3K activity, is reduced by ablating Hml+ hemocytes. Fat body isolated from wild types exhibits membrane-expression of tGPH (green) (A), whereas this pattern disappears in Hml-Gal4 UAS-hid,rpr background (B). Bottom images indicate magnified Z-stacks of corresponding images. Quantitation of tGPH expression in the fat body membrane (C). DAPI, blue. Scale bar, 40 μm. Statistical significance was determined by t-test. ****p < 0.0001. (D–F) Expression of dFOXO upon genetic ablation of Hml+ hemocytes. Cytosolic and low expressions of dFOXO in the wild-type fat body (D,D'). The level and nuclear localization of dFOXO is enhanced in Hml-Gal4 UAS-hid,rpr background (E,E'). DAPI (blue) and dFOXO (red) are overlaid in (D,E), and dFOXO (red) alone is shown in (D',E'). Nuclear dFOXO levels are quantitated in (F). Statistical significance was determined by t-test. ****p < 0.0001. Scale bar, 40 μm. (G,H) mRNA expression of 4EBP is increased in both HmlΔ-Gal4 UAS-EGFP UAS-hidrpr (G) and Pxn-Gal4 UAS-upd3 (H) backgrounds in the fat body. Error bar in graph is S.D. Statistical significance was determined by t-test. ***p < 0.001. (I–K) Expression of lipid droplets in the fat body. Compared to wild types (I,I') (HmlΔ-Gal4 UAS-EGFP), the size of lipid droplets is decreased when Hml+ hemocytes are ablated (J,J') (Hml-Gal4 UAS-hid, rpr). Quantitation of BODIPY diameter in (I',J') (K). Highest and lowest bars indicate maximum and minimum values, respectively. Statistical significance was determined by t-test. ****p < 0.0001. Scale bar, 40 μm. (L) Biochemical measurement of triacyl glyceride (TAG) levels in the fat body normalized by protein contents. The level of TAG is decreased upon loss of Hml+ hemocytes (Hml-Gal4 UAS-hid, rpr). Highest and lowest bars indicate maximum and minimum values, respectively. Statistical significance was determined by t-test. *p < 0.05.
Figure 4
Figure 4
upd2Δupd3Δ mutants rescue the phenotypes caused by genetic ablation of Hml+ hemocytes. (A–E) The number of total hemocytes is decreased in upd2Δupd3Δ; Hml-Gal4 UAS-hid background. Compared to wild types (A), overexpression of hid in Hml+ hemocytes (Hml-Gal4 UAS-hid) induces the number of Pxn+ (magenta) and/or DAPI+ (blue) cells (B). This phenotype is recovered by combining upd2Δupd3Δ in the Hml-Gal4 UAS-hid background (C,D). There is no significant difference between HmlΔ-Gal4 UAS-EGFP controls (A) and upd2Δupd3Δ (C) or upd2Δupd3Δ; Hml-Gal4 UAS-hid (D). Controls (A,C) express Hml (green), Pxn (magenta), and DAPI (blue); therefore, are indicated in white. Quantitation of each genotype is shown in (E). Graphs indicate median plots of DAPI positive cells per mm2 in each genotype. Highest and lowest bars indicate maximum and minimum values, respectively. Statistical significance was determined by t-test. *p < 0.05; not significant, ns. Scale bar, 40 μm. (F,G) Increased levels of Socs36E or 4EBP are reverted by loss of upd2 and upd3. Increased levels of Socs36E (F) or 4EBP (G) upon loss of Hml+ hemocytes in the fat body are recovered in upd2Δupd3Δ; Hml-Gal4 UAS-hid genetic background. Rescue is indicated in red. Statistical analyses were performed using two-way ANOVA. *p < 0.05; ****p < 0.0001; not significant, ns. (H–K) Nuclear location and levels of dFOXO in the fat body are rescued by loss of upd2 and upd3. In controls, low levels of dFOXO (red) are located in the cytoplasm and nucleus (H,H'). Overexpression of hid in Hml+ hemocytes further induces the nuclear expression of dFOXO (red) in the fat body (I,I'). Genetic combination of upd2Δupd3Δ and Hml-Gal4 UAS-hid reduces the nuclear expression of dFOXO (red) (J,J'). Quantitation of dFOXO intensity shown in (H–J') (K). Highest and lowest bars indicate maximum and minimum values, respectively. Statistical significance was determined by t-test. ****p < 0.0001. Scale bar, 40 μm.
Figure 5
Figure 5
Working model of this study. We identified heterogenic expression of larval plasmatocytes based on two genetic markers, Hemolectin (Hml) and Peroxidasin (Pxn). There are Hml+ Pxn+ (yellow), Hml+ Pxn (green), and Pxn+ Hml (pink) plamatocytes which show definitive distributions across 72, 96, 120 h AEL developmental time points. This suggests plasmatocyte proportions are constantly maintained during development. Pxn+ hemocyte (pink) is increased when Hml+ hemocytes are ablated. Also, total remaining hemocytes emit a cytokine, upd3 (red dot). upd3 increases JAK/STAT signaling in the fat body which possibly inhibits insulin receptor signaling, and consequently affects growth and metabolism.

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