Terminal tracheal immune reaction to natural infection measured via AMP reporter response.

GFP reporter larvae were infected with P. carotovorum for 24 h, and GFP fluorescence in the terminal structures of the tracheal system was monitored. The Images were taken in the DIC and GFP channels. Scale, 50 µm. (A-E) Dorsal tracheal structures of non-infected larvae. (A’-E’) Dorsal tracheal structures of infected larvae. (F) Dorsal view of the tracheal system, showing the dorsal trunks branching into the dorsal branch (DB), fusion cells (FC) and the dorsal TTCs. The quantification explanation is shown in magnification emphasized with colors. Presence of fluorescence in DB/FC counts as a GFP positive larva (light green, GFP+). Presence of fluorescence in the TTCs in GFP+ animals is indicated by dark green color (GFP TTCs). (G) Proportion of animals with tracheal fluorescence following the mentioned quantification procedure. Scale bar, 50 µm. Drs = Drosomycin, Def = Defensin, Att = Attacin, Mtk = Metchnikowin, Dpt = Diptericin.

Tracheal terminal cells (TTCs) show very rare Drs expression upon natural infection.

Drs-GFP larvae were infected with P. carotovorum for 24 h, and GFP fluorescence in the TTCs of the tracheal system was monitored. Images were taken in the DIC (A, C-G) and GFP channels (A’, C’-F’). (A) Dorsal TTCs without fluorescence. (B) Visceral TTCs without fluorescence. (C) Percentage of larvae showing GFP fluorescence in the DB and TTCs. (D–G) TTCs with expression of Drs-GFP. White arrows indicate shortened TTC branches (G). The black arrows mark a melanization site and the arrowhead marks a translucent branch without air filling (G). Dashed lines represent the proximal end of the TTCs. Scale bar, 50 µm.

TTCs do not express the Imd receptor PGRP-LCx.

(A–D) The secreted Imd pathway receptor PGRP-LE is expressed in the main parts of the tracheal system (ppk4>PGRP-LE (GFP-Drs)). The arrows indicate TTCs not expressing GFP. The dashed lines represent the proximal end of the TTC. (A–C) An activated immune response in the larvae is visualized by expression of GFP-tagged Drosomycin (Drs). (D, E) Detailed TTCs were observed in fillet preparations (D) and in the dissected intestine (E) in both the DIC (D, E) and GFP channels (D’, E’). (F) Example TTC of infected PGRP-LE animals with fluorescence. (G) Percentage of PGRP-LEOE animals with tracheal GFP expression. (H, I) Expression of GFP under the control of a PGRP-LCx promoter (PGRP-LCx-Gal4 > UAS-GFP) revealed a lack of promoter activity, and expression of GFP, in TTCs on the cuticle (H, H’) and intestine (I, I’), which is in contrast to the rest of the tracheal system. (J, K) The TTCs in the tracheal system are visualized by GFP expression in the cuticle (J) and intestine (K) of wild-type larvae (btl-Gal4; UAS-GFP). (L) Percentage of PGRP-LCx animals with tracheal GFP expression. Scale bar, 50 µm. Dashed lines represent the proximal end of the TTCs. Dt = dorsal trunk.

Expression of PGRP-LCx by TTCs leads to size reduction and loss of functionality.

(A, B) Dorsal TTCs in the control (A) and DSRF-driven overexpression of PGRP-LCx in TTCs (B). (C, D) Measurement and quantification of the number (C) and length (D) of branches (n=22–45). Data are presented as the mean ± SD. (E) The hypoxia sensitivity assay was conducted with control and PGRP-LCx-expressing 3rd instar larvae under hypoxic conditions (2–3 % O2, n = 11–14). Data are presented as the means ± SEM. Statistical significance was tested using Mann-Whitney-U test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (F) Culture vials containing control, hid;rpr, and Tak1 larvae (DSRF > hid;rpr/Tak1) at 4 days post-oviposition. (G, H) Transmission light microscopy of dissected intestines from 3rd instar larvae (F, G) or 2nd instar larvae (H) with the connected TTCs of control (G) showing expression of hid;rpr (H) or PGRP-LCx (I). (J, K) Dissected intestines from control larvae (J) or larvae expressing PGRP-LCx in TTCs (K) were stained with an antibody specific for cleaved Drosophila Dcp-1 (purple). (I’, J’) and then counterstained to detect GFP (green). (I’’, J’’) Merged channels. Scale bars, 50 μm.

JNK signaling is associated with impaired TTC branching.

(A) Schematic showing how the PGRP-LC-activated Imd signaling pathway is subdivided into the NF-κB (Relish) and JNK (hep = JNKK, bsk = JNK) pathways. (B–D) Relish (B, Rel68), as well as constitutively active hep (C, hepCA) and bsk (D, bskOE), were expressed in TTCs (DSRF >). (E, F) Measurement and quantification of the number (E) and length (F) of branches in control (w1118) versus PGRP-LCx-expressing TTCs (n=22–45). Data are presented as the mean ± SD. Statistical significance was evaluated using Mann-Whitney-U test, ** p < 0.01, **** p < 0.0001, ns = not significant. (G, H) Dissected intestines from control (DSRF > w1118) and PGRP-LCx-expressing flies (DSRF > PGRP-LCx), in which the TTCs were stained to detect Relish (Rel, G) and pJNK (purple, H). TTCs were counterstained with GFP (green). Arrows mark the TTC nucleus. Merged channels are shown. Scale bars, 50 µm.

The TTC phenotype induced by PGRP-LCx is dependent on the transcription factors kay and foxo.

(A) Schematic illustration of the JNK signaling pathway downstream of Tak1, which includes Ets21C, kay, and Jra (AP1). (B–E) DSRF-driven PGRP-LCxOE in TTCs was combined with the dominant-negative form of Tak1, bsk (B), and kay (C), or with RNAi targeting foxo (D) or Ets21C (E). (F, G) Measurement and quantification of the number (F) and length (G) of branches (n=16–45). (H–K) Measurement and quantification of the number (H) and length (I) of GFP expressing branches in control (w1118) and PGRP-LCx-expressing cells (n=7–45). TTCs overexpressing foxo (J, foxoOE), and kay and Jra (K, kayOE + JraOE). (L, M). TRE-RFP expression in control (L) and PGRP-LCx-expressing TTCs (M). (N) Foxo promoter activity in TTCs (foxo-Gal4 > UAS-GFP). Data are expressed as the mean ± SD. Statistical significance was evaluated using Mann-Whitney-U test, * p < 0.05, *** p < 0.001, **** p < 0.0001, ns = not significant. The color of the asterisk indicates the corresponding comparison. Dashed lines represent the mean control value. Scale bar, 50 µm.

Targeted reduction of foxo expression in TTCs leads to hyperbranching.

(A, B) Representative tracheal branching in control (A) and DSRF-driven foxoRNAi(B) cells under normoxic conditions. (C, D) Representative images showing tracheal branching in control (C) and foxORNAi(D) cells under hypoxic conditions. (E) Quantification of branches in control and DSRF > foxORNAi TTCs under normoxic (21%) and hypoxic (5%) conditions. Scale bar, 50 µm. n=21, Data are presented as the mean ± SD. Statistical significance was evaluated using Mann-Whitney-U t-test, * p < 0.05, **** p < 0.0001, ns = not significant.

Schematic summarizing the JNK-mediated immune/stress response in the trachea and TTCs.

(A) Tracheal infection leads to an immune response involving expression of antimicrobial peptides such as Drosomycin (Drs, green). In most cases, the immune response is restricted to the tracheal trunks and the TTCs are unaffected (bold arrow). In rare cases, TTCs express Drs, resulting in an impaired phenotype (dashed arrow). (B) Imd signaling in the main tracheal trunks is induced by peptidoglycan recognition receptors (PGRP)-LC and -LE. Downstream, the signaling branches into a Relish (Rel) and a JNK signaling pathway. Activation of the pathways mediates airway remodeling (13). However, activation in TTCs is avoided by the absence of PGRP-LC, even though all other JNK signaling pathway components are present. The pathway can be activated by external stressors, resulting in AP-1-mediated cell death. The transcription factor foxo, which is component not only of the JNK signaling pathway but also of the insulin signaling pathway, plays a role in TTC homeostasis and their ability to branch.

Drs expression in infected larvae.

GFP reporter larvae were infected with P. carotovorum for 24 h, and GFP fluorescence was monitored. Drs expression was visible in different larval structures like hemocytes and fat body (A-D). Scale bar, 50 µm. Drs = Drosomycin, Def = Defensin, Mtk = Metchnikowin, Dpt = Diptericin.

PGRP-LCx mediated epithelial thickening of the dorsal trunks.

PGRP-LCx expression in the tracheal system was induced for 24 h in 3rd instar larvae. Epithelial thickness of control larvae (A, btl-Gal4, UAS-GFP; tub-Gal80ts > w1118) were compared to PGRP-LCxOE larvae with PGRP-LCx on the third chromosome (III, B, btl-Gal4, UAS-GFP; tub-Gal80ts > UAS-PGRP-LCx) and on the second chromosome (II, C). Epithelial thickness was quantified (D). Scale bar, 50 µm. Data are presented as the mean ± SD. Statistical significance was evaluated using unpaired t-test, **** p < 0.0001.

Increase in apoptotic signalling in PGRP-LC overexpression.

The influence of PGRP-LC overexpression on apoptotic cells was investigated using -Dcp1 staining. For the analysis, the 8th tracheal metamere of the controls (A; ppk4-Gal4 > w1118 (ControlD), w1118 > UAS-PGRP-LCx (ControlR)) and PGRP-LEOE (B; ppk4-Gal4 > UAS-PGRP-LCx) were used. (C) Quantification was performed by measuring the fluorescence intensity and calculating the CTCF (corrected total cell fluorescence) of ControlD, ControlR and PGRP-LEOE. The statistical significance was evaluated using the Mann-Whitney test. ****= p < 0.0001, ***= p < 0.001, ns = not significant. Scale bar, 50 µm.

Overexpression of PGRP-LE and infection increased hypoxia sensitivity.

The influence of PGRP-LEOE and bacterial infection on hypoxia sensitivity was analyzed based on the escape response of 3rd instar larvae exposed to 2.5 % OLJ. Control: w1118 > UAS-PGRP-LE (Drs-GFP), PGRP-LE overexpression: Btl-Gal4;Gal80 > UAS-PGRP-LE (Drs-GFP). Six independent replicates, each with 10 larvae of the control (grey), infected control (black), PGRP-LEOE (pink) and infected PGRP-LEOE (burgundy) were used for quantification. Values are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA. **= p < 0.01, *= p < 0.1.