DOI: 10.5061/dryad.9zw3r22kw
title: Lipidomic data of macrophages isolated from adult fruit flies (Drosophila melanogaster) 24 hours post-infection

description: The immune response is an energy-demanding process that must be
 coordinated with systemic metabolic changes redirecting nutrients from
 stores to the immune system. Although this interplay is fundamental for
 the function of the immune system, the underlying mechanisms remain
 elusive.  Our data show that the pro-inflammatory polarization of
 Drosophila macrophages is coupled to the production of the insulin
 antagonist ImpL2 through the activity of the transcription factor HIF1α.
 ImpL2 production, reflecting nutritional demands of activated macrophages,
 subsequently impairs insulin signaling in the fat body, thereby triggering
 FOXO-driven mobilization of lipoproteins. This metabolic adaptation is
 fundamental for the function of the immune system and an individual’s
 resistance to infection. We demonstrated that analogically to Drosophila,
 mammalian immune-activated macrophages produce ImpL2 homolog IGFBP7 in a
 HIF1α-dependent manner and that enhanced IGFBP7 production by these cells
 induces mobilization of lipoproteins from hepatocytes. Hence, the
 production of ImpL2/IGFBP7 by macrophages represents an evolutionarily
 conserved mechanism by which macrophages alleviate insulin signaling in
 the central metabolic organ to secure nutrients necessary for their
 function upon bacterial infection.
description: Fat bodies from six flies and one hundred thousand isolated
 macrophages were obtained for each analyzed group. Tissue lipid fraction
 was extracted by 500 ul of cold chloroform: methanol solution (v/v; 2:1).
 The samples were then homogenized by a Tissue Lyser II (Qiagen, Prague,
 Czech Republic) at 50 Hz, -18°C for 5 min and kept further in an
 ultrasonic bath (0°C, 5 min). Further, the mixture was centrifuged at
 10,000 RPM at 4°C for 10 min followed by the removal of the supernatant.
 The extraction step was repeated under the same conditions. The lower
 layer of pooled supernatant was evaporated to dryness under a gentle
 stream of Argon. The dry total lipid extract was re-dissolved in 50 µl of
 chloroform: methanol solution (v/v; 2:1) and directly measured using
 previously described methods. Briefly, high-performance liquid
 chromatography (Accela 600 pump, Accela AS autosampler) combined with mass
 spectrometry LTQ-XL (all Thermo Fisher Scientific, San Jose, CA, USA) were
 used. The chromatographic conditions were as follows: Injection volume 5
 µl; column Gemini 3 µM C18 HPLC column (150 × 2 mm ID, Phenomenex,
 Torrance, CA, USA) at 35°C; the mobile phase (A) 5 mM ammonium acetate in
 methanol with ammonia (0.025%), (B) water and (C) isopropanol: MeOH (8:2);
 gradient change of A:B:C as follows: 0 min: 92:8:0, 7 min: 97:3:0, 12 min:
 100:0:0, 19 min: 93:0:7, 20-29 min: 90:0:10, 40–45 min: 40:0:60, 48 min:
 100:0:0, and 50–65 min: 92:8:0 with flow rate 200 µl/min. The mass
 spectrometry condition: positive (3kV) and negative (-2,5kV) ion detection
 mode; capillary temperature 200°C. Eluted ions were detected with full
 scan mode from 200 to 1000 Da with the collisionally induced MS2
 fragmentation (NCE 35). Data were acquired and processed by means of
 XCalibur 4.0 software (Thermo Fisher). The corrected areas under
 individual analytical peaks were expressed in percentages assuming that
 the total area of all detected is 100%. Lipidomics data were subsequently
 analyzed in the online platform LipidSuite (https://suite.lipidr.org/). Data were inputted by the K-Neares Neighbours method (KNN), and normalization was performed by the PQN algorithm. Subsequently, data were explored by PCA and OPLS-DA methods. Differential analysis of lipidomic data was done by univariate analysis and visualized in Volcano plots.
description: # Lipidomic data of macrophages isolated from adult fruit flies
 (Drosophila melanogaster) 24 hours post-infection
 [https://doi.org/10.5061/dryad.9zw3r22kw](https://doi.org/10.5061/dryad.9zw3r22kw) Title of Dataset: Lipidomic data of macrophages isolated from adult fruit flies (Drosophila melanogaster) 24 hours post-infection. 2\. Author Information A. Principal Investigator Contact Information Name: Bajgar Adam Institution: University of South Bohemia Address: Branisovska 31 Email: bajgaradam@seznam.cz 3\. Associated publication: Macrophage\-derived insulin antagonist ImpL2 induces lipoprotein mobilization upon bacterial infection 4\. Abstract: The immune response is an energy\-demanding process that must be coordinated with systemic metabolic changes redirecting nutrients from stores to the immune system\. Although this interplay is fundamental for the function of the immune system\, the underlying mechanisms remain elusive\. Our data show that the pro\-inflammatory polarization of *Drosophila* macrophages is coupled to the production of the insulin antagonist *ImpL2* through the activity of the transcription factor HIF1α. *ImpL2* production, reflecting nutritional demands of activated macrophages, subsequently impairs insulin signaling in the fat body, thereby triggering FOXO-driven mobilization of lipoproteins. This metabolic adaptation is fundamental for the function of the immune system and an individual’s resistance to infection.We demonstrated that analogically to *Drosophila*, mammalian immune-activated macrophages produce *ImpL2* homolog IGFBP7 in a HIF1α-dependent manner and that enhanced IGFBP7 production by these cells induces mobilization of lipoproteins from hepatocytes. Hence, the production of *ImpL2*/IGFBP7 by macrophages represents an evolutionarily conserved mechanism by which macrophages alleviate insulin signaling in the central metabolic organ to secure nutrients necessary for their function upon bacterial infection. 5\. Data were generated by metabolomic service on Biology Center of Czech Academy of Sciences\. For procedural details please refer to : https://bclab\.eu/contact\-us 6\. Data were collected from croquemort GFP flies infected by injection of 20thousan of Streptococcus pneumoniae in 2020\. Tissue lipid fraction was extracted by 500 ul of cold chloroform: methanol solution \(v/v; 2:1\)\. The samples were then homogenized by a Tissue Lyser II \(Qiagen\, Prague\, Czech Republic\) at 50 Hz\, \-18¯C for 5 min and kept further in an ultrasonic bath \(0¯C\, 5 min\)\. Further\, the mixture was centrifuged at 10\,000 RPM at 4¯C for 10 min followed by the removal of the supernatant\. The extraction step was repeated under the same conditions\. The lower layer of pooled supernatant was evaporated to dryness under a gentle stream of Argon\. The dry total lipid extract was re\-dissolved in 50 Êl of chloroform: methanol solution \(v/v; 2:1\) and directly measured using previously described methods49\. Briefly\, high\-performance liquid chromatography \(Accela 600 pump\, Accela AS autosampler\) combined with mass spectrometry LTQ\-XL \(all Thermo Fisher Scientific\, San Jose\, CA\, USA\) were used\. The chromatographic conditions were as follows: Injection volume 5 Êl; column Gemini 3 ÊM C18 HPLC column \(150 ? 2 mm ID\, Phenomenex\, Torrance\, CA\, USA\) at 35¯C; the mobile phase \(A\) 5 mM ammonium acetate in methanol with ammonia \(0\.025%\)\, \(B\) water and \(C\) isopropanol: MeOH \(8:2\); gradient change of A:B:C as follows: 0 min: 92:8:0\, 7 min: 97:3:0\, 12 min: 100:0:0\, 19 min: 93:0:7\, 20\-29 min: 90:0:10\, 40\-45 min: 40:0:60\, 48 min: 100:0:0\, and 50\-65 min: 92:8:0 with flow rate 200 Êl/min\. The mass spectrometry condition: positive \(3kV\) and negative \(\-2\,5kV\) ion detection mode; capillary temperature 200¯C\. Eluted ions were detected with full scan mode from 200 to 1000 Da with the collisionally induced MS2 fragmentation \(NCE 35\)\. Data were acquired and processed by means of XCalibur 4\.0 software \(Thermo Fisher\)\. The corrected areas under individual analytical peaks were expressed in percentages assuming that the total area of all detected is 100%\. Lipidomics data were subsequently analyzed in the online platform LipidSuite \(https://suite\.lipidr\.org/\)53\. Data were inputted by the K\-Neares Neighbours method \(KNN\)\, and normalization was performed by the PQN algorithm\. Subsequently\, data were explored by PCA and OPLS\-DA methods\. Differential analysis of lipidomic data was done by univariate analysis and visualized in Volcano plots\. Individual data files contain raw data representing obtained peak area values for analyzed lipid species. Data were used as relative proportion values of lipids making alltogether 100%. Identified lipids and particular lipid sclasses were analyzed by using online available app LipidSuite. * PB\_Bajgar - these samples represent standards * Hem\_Buff - indicates samples of Drosophila macrophages obtained from control flies injected with 50nl pf PBS * Hem\_SP - indicates samples of Drosophila macrophages obtained from experimental flies injected with 20 thousand of *Streptococcus* *pneumoniae* * L3W - indicates control macrophages obtained from wandering larvae of third instar * WP - indicates samples obtained from fies in developmental stage of white prepupa * V0 - indicates macrophage obtained from frwshly emerged flies * V48 - indicates samples obtained from flies 48 hours post emergence; All samples were analyzed for positive and negative(NESI) charge List of analyzed samples: * PB\_Bajgar\_50ul\_1\_210805\_01; * PB\_Bajgar\_500ul\_2\_210805\_01; * VZ\_Hem\_Buf\_33\_210805\_01; * VZ\_Hem\_Buf\_34\_210805\_01; * VZ\_Hem\_Buf\_35\_210805\_01; * VZ\_Hem\_Buf\_36\_210805\_01; * VZ\_Hem\_Buf\_37\_210805\_01; * VZ\_Hem\_SP\_28\_210805\_01; * VZ\_Hem\_SP\_29\_210805\_01; * VZ\_Hem\_SP\_30\_210805\_01; * VZ\_Hem\_SP\_31\_210805\_01; * VZ\_Hem\_SP\_32\_210805\_01; * VZ\_MFS\_L3W\_1\_210805\_01; * VZ\_MFS\_L3W\_2\_210805\_01; * VZ\_MFS\_L3W\_3\_210805\_01; * VZ\_MFS\_L3W\_4\_210805\_01; * VZ\_MFS\_L3W\_5\_210805\_01; * VZ\_MFS\_V0\_6\_210805\_01; * VZ\_MFS\_V0\_7\_210805\_01; * VZ\_MFS\_V0\_8\_210805\_01; * VZ\_MFS\_V0\_9\_210805\_01; * VZ\_MFS\_V0\_10\_210805\_01; * VZ\_MFS\_V48\_11\_210805\_01; * VZ\_MFS\_V48\_12\_210805\_01; * VZ\_MFS\_V48\_13\_210805\_01; * VZ\_MFS\_V48\_14\_210805\_01; * VZ\_MFS\_V48\_15\_210805\_01; * VZ\_V0\_20\_210805\_01; * VZ\_V0\_21\_210805\_01; * VZ\_V0\_22\_210805\_01; * VZ\_V0\_23\_210805\_01; * VZ\_V48\_24\_210805\_01; * VZ\_V48\_25\_210805\_01; * VZ\_V48\_26\_210805\_01; * VZ\_V48\_27\_210805\_01; * VZ\_WP\_16\_210805\_01; * VZ\_WP\_17\_210805\_01; * VZ\_WP\_18\_210805\_01; * VZ\_WP\_19\_210805\_01; * PB\_Bajgar\_50ul\_1\_NESI\_210805\_01; * PB\_Bajgar\_500ul\_2\_NESI\_210805\_01; * VZ\_Hem\_Buf\_33\_NESI\_210805\_01; * VZ\_Hem\_Buf\_34\_NESI\_210805\_01; * VZ\_Hem\_Buf\_35\_NESI\_210805\_01; * VZ\_Hem\_Buf\_36\_NESI\_210805\_01; * VZ\_Hem\_Buf\_37\_NESI\_210805\_01; * VZ\_Hem\_SP\_28\_NESI\_210805\_01; * VZ\_Hem\_SP\_29\_NESI\_210805\_01; * VZ\_Hem\_SP\_30\_NESI\_210805\_01; * VZ\_Hem\_SP\_31\_NESI\_210805\_01; * VZ\_Hem\_SP\_32\_NESI\_210805\_01; * VZ\_MFS\_L3W\_1\_NESI\_210805\_01; * VZ\_MFS\_L3W\_2\_NESI\_210805\_01; * VZ\_MFS\_L3W\_3\_NESI\_210805\_01; * VZ\_MFS\_L3W\_4\_NESI\_210805\_01; * VZ\_MFS\_L3W\_5\_NESI\_210805\_01; * VZ\_MFS\_V0\_6\_NESI\_210805\_01; * VZ\_MFS\_V0\_7\_NESI\_210805\_01; * VZ\_MFS\_V0\_8\_NESI\_210805\_01; * VZ\_MFS\_V0\_9\_NESI\_210805\_01; * VZ\_MFS\_V0\_10\_NESI\_210805\_01; * VZ\_MFS\_V48\_11\_NESI\_210805\_01; * VZ\_MFS\_V48\_12\_NESI\_210805\_01; * VZ\_MFS\_V48\_13\_NESI\_210805\_01; * VZ\_MFS\_V48\_14\_NESI\_210805\_01; * VZ\_MFS\_V48\_15\_NESI\_210805\_01; * VZ\_V0\_20\_NESI\_210805\_01; * VZ\_V0\_21\_NESI\_210805\_01; * VZ\_V0\_22\_NESI\_210805\_01; * VZ\_V0\_23\_NESI\_210805\_01; * VZ\_V48\_24\_NESI\_210805\_01; * VZ\_V48\_25\_NESI\_210805\_01; * VZ\_V48\_26\_NESI\_210805\_01; * VZ\_V48\_27\_NESI\_210805\_01; * VZ\_WP\_16\_NESI\_210805\_01; * VZ\_WP\_17\_NESI\_210805\_01; * VZ\_WP\_18\_NESI\_210805\_01; * VZ\_WP\_19\_NESI\_NESI\_210805\_01;