Terminal tracheal cells of Drosophila are immune privileged to maintain their Foxo-dependent structural plasticity
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eLife Assessment
This is a valuable report of tracheal terminal cells (TTCs) in Drosophila being immune privileged. The authors demonstrated that TTCs lack the expression of membrane-associated peptidoglycan recognition receptor PGRP-LC, which protects these cells from activating immune pathway or JNK-mediated cell death to maintain TTC homeostasis. While genetic experiments using RNAi and overexpression are mostly convincing, the data on the expression of PGRP-LCx and cell death phenotypes following immune activation are currently incomplete. The work will be of interest to researchers in innate immunity across various model systems.
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Abstract
Respiratory organs fulfill several tasks, of which gas exchange is the most important. This function is also true for the Drosophila respiratory organ, the tracheal system, in which the tracheal terminal cells (TTCs), the functional equivalents of the mammalian lung’s alveoli, are the structures where gas exchange occurs. To cope with the plethora of inhaled bacteria, fungi, and viruses, the trachea, like all airway organs, developed a sophisticated innate immune system to protect its large surface area. Bacterial infection of the Drosophila larval tracheal system induced a robust immune response throughout the entire airway epithelium, except for the TTCs that lacked this response. TTCs do not express the membrane-associated peptidoglycan recognition receptor PGRP-LC, which we assume protects these susceptible cells from Immune deficiency (Imd) pathway activation and JNK- mediated cell death. Thus, TTCs can be considered an immune-privileged cell type compared to the rest of the tracheal tissue. Targeted overexpression of PGRP-LCx in these cells led to a significant reduction in branching, cell damage, and ultimately cell death, which depletion of AP-1 or foxo could rescue. We hypothesize that the structural plasticity of TTCs in response to nutritional cues and hypoxia is incompatible with a potent immune function. Both reactions, the structural plasticity, and the immune response, require the transcription factor foxo, and we showed that it is essential for structural plasticity. Thus, the immune-privileged status of TTCs is (presumably) a mechanism that ensures normal TTC function.
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eLife Assessment
This is a valuable report of tracheal terminal cells (TTCs) in Drosophila being immune privileged. The authors demonstrated that TTCs lack the expression of membrane-associated peptidoglycan recognition receptor PGRP-LC, which protects these cells from activating immune pathway or JNK-mediated cell death to maintain TTC homeostasis. While genetic experiments using RNAi and overexpression are mostly convincing, the data on the expression of PGRP-LCx and cell death phenotypes following immune activation are currently incomplete. The work will be of interest to researchers in innate immunity across various model systems.
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Reviewer #1 (Public review):
Summary:
In their manuscript entitled "Terminal tracheal cells of Drosophila are immune privileged to maintain their Foxo-dependent structural plasticity", Bossen and colleagues determine that the terminal cells of the tracheal system differ from other larval tracheal cells in that they do not typically show an Imd-dependent immune response to fungal and viral infections. The authors reach this conclusion based on the expression of a reporter line, Drs-GFP. The authors speculate that this difference may reflect differential expression of an immune pathway component, as tracheal terminal cells (TTCs) do not respond to forced expression of PRGP-LS. The authors then go on to show that, unlike the other cells of the tracheal system, terminal cells do not express PGRP-LC as reported by a GAL4 enhancer trap. …
Reviewer #1 (Public review):
Summary:
In their manuscript entitled "Terminal tracheal cells of Drosophila are immune privileged to maintain their Foxo-dependent structural plasticity", Bossen and colleagues determine that the terminal cells of the tracheal system differ from other larval tracheal cells in that they do not typically show an Imd-dependent immune response to fungal and viral infections. The authors reach this conclusion based on the expression of a reporter line, Drs-GFP. The authors speculate that this difference may reflect differential expression of an immune pathway component, as tracheal terminal cells (TTCs) do not respond to forced expression of PRGP-LS. The authors then go on to show that, unlike the other cells of the tracheal system, terminal cells do not express PGRP-LC as reported by a GAL4 enhancer trap. Forced expression of PGRP-LC in terminal cells resulted in reduced branching, cell damage, and features of the cell death program. These effects could be suppressed by the depletion of AP-1 or Foxo transcription factors. The authors show that Foxo plays a negative role in the branching of TTCs, with ectopic branching occurring upon RNAi (or under hypoxic conditions). The authors speculate that the immune privilege of the TTCs may have evolved to permit Foxo regulation of TTC branching.
Strengths:
The authors provide compelling genetic data.
Weaknesses:
(1) The authors state that after infection 34% of larvae were not GFP+ as defined by the detection of Drs-GFP in dorsal branches. The authors should clarify if these larvae are completely without response to infection, with no Drs-GFP in dorsal trunks and or other tracheal branches. If these larvae are entirely unresponsive, could authors indicate why this might be? Also, at this point in the manuscript, the authors are somewhat misleading regarding TTC expression of Drs-GFP - they should state at this point that there are some TTCs that do express Drs-GFP, and also should address their prior study of Drs-GFP induction which does not claim exclusion of TTC Drs-GFP expression.
(2) The authors describe the terminal cell phenotype as "shrunken" but this implies loss of size or pruning, however, it is not clear whether the defects could equally be due to lack of growth or slower growth.
(3) Figure 1 suggests that GFP+ dorsal branches are not uniform in their expression of Drs-GFP, it seems more patchy. The authors should define the fraction of dorsal branch cells that are Drs-GFP positive. Also, are fusion cells Drs-GFP positive?
(4) Drs-GFP expression is largely absent from terminal cells; however, a still significant # of terminal cells show expression (8%). Authors argue that PRGP-LC expression is absent based on a GAL4 transgenic line. If this line reflects endogenous PRGP-LC expression, should there not be 8% positive TTCs? Or is the 8% Drs-GFP expression independent of the IMD receptor?
(5) Figure 2: the authors state that TTCs are negative even with induced PRGP-LE expression - should there not be at least 8% that are positive?
(6) The authors compare PRGP-LC expression to induction of cell death by expression of reaper and hid. Reaper and Hid had stronger effects and eliminated TTCs. See cleavage of caspase Dpc-1 in PRGP-LC expressing cells. Is caspase cleavage always diagnostic of apoptosis or could the weaker than rpr/hid phenotype imply a different function?
(7) Drs-GFP expression is said to be "completely" absent from tracheal terminal cells when the entire tracheal system is expressing PGRP-LE.
(8) Figure 5, TRE_RFP expression, is not convincing that it is higher or in terminal cells.
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Reviewer #2 (Public review):
Summary:
In this study, Bossen et al. looked at the immune status of the tracheal terminal cells (TTCs) in Drosophila larvae. The authors propose that these cells do show PGFP-LCx expression and, hence, lack immune function. Artificial overexpression of the PGRP-LCx in the TTCs causes these cells to undergo apoptosis.
Strengths:
Only a few groups have tried to look at the immune status of the trachea, though we know that AMPs are expressed there after infection. This exciting study attempts to understand the differences in the tracheal cells that do not produce AMPs upon infection.
Weaknesses:
The reason why the TTCs have some immune privilege still needs to be completely clear. Whether the phenotype is cell autonomous or contributes to the cellular immune system is not evaluated. As we know, crystal cells …
Reviewer #2 (Public review):
Summary:
In this study, Bossen et al. looked at the immune status of the tracheal terminal cells (TTCs) in Drosophila larvae. The authors propose that these cells do show PGFP-LCx expression and, hence, lack immune function. Artificial overexpression of the PGRP-LCx in the TTCs causes these cells to undergo apoptosis.
Strengths:
Only a few groups have tried to look at the immune status of the trachea, though we know that AMPs are expressed there after infection. This exciting study attempts to understand the differences in the tracheal cells that do not produce AMPs upon infection.
Weaknesses:
The reason why the TTCs have some immune privilege still needs to be completely clear. Whether the phenotype is cell autonomous or contributes to the cellular immune system is not evaluated. As we know, crystal cells also maintain oxygen levels in larvae; whether in the absence of terminal trachea, the crystal cells have any role is not explored.
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Reviewer #3 (Public review):
Summary:
The authors report that tracheal terminal cells (TTCs) in Drosophila do not activate innate immunity following bacterial infection. They attribute this to the lack of expression of PGRP-LCx in these cells. Forced activation of the Imd pathway in TTCs leads to cell death and a reduction in tracheal branching. The authors propose a mechanism for cell death induction via pathways involving JNK, AP-1, and foxo. They suggest that the suppression of innate immunity in TTCs may serve to maintain their plasticity, preparing them for responses to hypoxic conditions.
Strengths:
(1) The study addresses the understudied area of immune privilege in innate immunity, providing a potentially important example in Drosophila TTCs.
(2) The molecular characterization of the cell death pathway induced by forced Imd …
Reviewer #3 (Public review):
Summary:
The authors report that tracheal terminal cells (TTCs) in Drosophila do not activate innate immunity following bacterial infection. They attribute this to the lack of expression of PGRP-LCx in these cells. Forced activation of the Imd pathway in TTCs leads to cell death and a reduction in tracheal branching. The authors propose a mechanism for cell death induction via pathways involving JNK, AP-1, and foxo. They suggest that the suppression of innate immunity in TTCs may serve to maintain their plasticity, preparing them for responses to hypoxic conditions.
Strengths:
(1) The study addresses the understudied area of immune privilege in innate immunity, providing a potentially important example in Drosophila TTCs.
(2) The molecular characterization of the cell death pathway induced by forced Imd activation is well-executed and provides solid mechanistic insights.
(3) The authors draw interesting parallels between Drosophila TTCs and mammalian endothelial cells, suggesting broader implications for their findings.
Weaknesses:
(1) The core premise of the study - that TTCs do not activate innate immunity following bacterial infection - relies heavily on a single readout (Drs reporter). Additional markers of immune activation would strengthen this crucial claim.
(2) The evidence for the lack of PGRP-LCx expression in TTCs is based on a single GAL4 reporter line. Given the importance of this observation to the authors' model, validation using alternative methods would be beneficial.
(3) The phenotypes observed upon forced activation of the Imd pathway in TTCs, while intriguing, may be influenced by non-physiological levels of pathway activation. The authors should address this potential caveat and consider examining the effects of more moderate pathway activation.
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