Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors

Nature Immunology volume 17, pages 1150–1158 (2016)Cite this article

Subjects

Abstract

The innate immune system needs to distinguish between harmful and innocuous stimuli to adapt its activation to the level of threat. How Drosophila mounts differential immune responses to dead and live Gram-negative bacteria using the single peptidoglycan receptor PGRP-LC is unknown. Here we describe rPGRP-LC, an alternative splice variant of PGRP-LC that selectively dampens immune response activation in response to dead bacteria. rPGRP-LC-deficient flies cannot resolve immune activation after Gram-negative infection and die prematurely. The alternative exon in the encoding gene, here called rPGRP-LC, encodes an adaptor module that targets rPGRP-LC to membrane microdomains and interacts with the negative regulator Pirk and the ubiquitin ligase DIAP2. We find that rPGRP-LC-mediated resolution of an efficient immune response requires degradation of activating and regulatory receptors via endosomal ESCRT sorting. We propose that rPGRP-LC selectively responds to peptidoglycans from dead bacteria to tailor the immune response to the level of threat.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The PGRP-LC locus encodes cytosolic tail variant isoforms with regulatory potential.
Figure 2: rLC is a ligand-specific regulator of IMD pathway activation.
Figure 3: The rLC regulatory isoform resolves IMD pathway activation after infection.
Figure 4: rLC interacts with the negative regulator Pirk.
Figure 5: Endocytic mechanisms modulate IMD pathway kinetics and rLC localization.
Figure 6: rLC and ESCRT synergize to degrade activating PGRP-LC and resolve IMD pathway activation.

Similar content being viewed by others

References

  1. Ausubel, F.M. Are innate immune signaling pathways in plants and animals conserved? Nat. Immunol. 6, 973–979 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Janeway, C.A. Jr. The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol. Today 13, 11–16 (1992).

    Article  CAS  PubMed  Google Scholar 

  3. Medzhitov, R. & Janeway, C.A. Jr. Decoding the patterns of self and nonself by the innate immune system. Science 296, 298–300 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Blander, J.M. & Sander, L.E. Beyond pattern recognition: five immune checkpoints for scaling the microbial threat. Nat. Rev. Immunol. 12, 215–225 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Vance, R.E., Isberg, R.R. & Portnoy, D.A. Patterns of pathogenesis: discrimination of pathogenic and nonpathogenic microbes by the innate immune system. Cell Host Microbe 6, 10–21 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dworkin, J. The medium is the message: interspecies and interkingdom signaling by peptidoglycan and related bacterial glycans. Annu. Rev. Microbiol. 68, 137–154 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Park, J.T. & Uehara, T. How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan). Microbiol. Mol. Biol. Rev. 72, 211–227 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kleino, A. & Silverman, N. The Drosophila IMD pathway in the activation of the humoral immune response. Dev. Comp. Immunol. 42, 25–35 (2014).

    Article  CAS  PubMed  Google Scholar 

  9. Kaneko, T. et al. Monomeric and polymeric gram-negative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway. Immunity 20, 637–649 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Leulier, F. et al. The Drosophila immune system detects bacteria through specific peptidoglycan recognition. Nat. Immunol. 4, 478–484 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Stenbak, C.R. et al. Peptidoglycan molecular requirements allowing detection by the Drosophila immune deficiency pathway. J. Immunol. 173, 7339–7348 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Choe, K.M., Lee, H. & Anderson, K.V. Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor. Proc. Natl. Acad. Sci. USA 102, 1122–1126 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Neyen, C., Poidevin, M., Roussel, A. & Lemaitre, B. Tissue- and ligand-specific sensing of gram-negative infection in drosophila by PGRP-LC isoforms and PGRP-LE. J. Immunol. 189, 1886–1897 (2012).

    Article  CAS  PubMed  Google Scholar 

  14. Werner, T., Borge-Renberg, K., Mellroth, P., Steiner, H. & Hultmark, D. Functional diversity of the Drosophila PGRP-LC gene cluster in the response to lipopolysaccharide and peptidoglycan. J. Biol. Chem. 278, 26319–26322 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Mellroth, P. et al. Ligand-induced dimerization of Drosophila peptidoglycan recognition proteins in vitro. Proc. Natl. Acad. Sci. USA 102, 6455–6460 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zaidman-Rémy, A. et al. The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 24, 463–473 (2006).

    Article  PubMed  CAS  Google Scholar 

  17. Aggarwal, K. et al. Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway. PLoS Pathog. 4, e1000120 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Basbous, N. et al. The Drosophila peptidoglycan-recognition protein LF interacts with peptidoglycan-recognition protein LC to downregulate the Imd pathway. EMBO Rep. 12, 327–333 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Costechareyre, D. et al. Tissue-specific regulation of Drosophila NF-kB pathway activation by peptidoglycan recognition protein SC. J. Innate Immun. 8, 67–80 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Kleino, A. et al. Pirk is a negative regulator of the Drosophila Imd pathway. J. Immunol. 180, 5413–5422 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Maillet, F., Bischoff, V., Vignal, C., Hoffmann, J. & Royet, J. The Drosophila peptidoglycan recognition protein PGRP-LF blocks PGRP-LC and IMD/JNK pathway activation. Cell Host Microbe 3, 293–303 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Paredes, J.C., Welchman, D.P., Poidevin, M. & Lemaitre, B. Negative regulation by amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from innocuous infection. Immunity 35, 770–779 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Lhocine, N. et al. PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling. Cell Host Microbe 4, 147–158 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Choe, K.M., Werner, T., Stöven, S., Hultmark, D. & Anderson, K.V. Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 296, 359–362 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Gottar, M. et al. The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 416, 640–644 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Clark, A.G. et al. Evolution of genes and genomes on the Drosophila phylogeny. Nature 450, 203–218 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. DiNitto, J.P., Cronin, T.C. & Lambright, D.G. Membrane recognition and targeting by lipid-binding domains. Sci. STKE 2003, re16 (2003).

    Article  PubMed  Google Scholar 

  28. Gozani, O. et al. The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor. Cell 114, 99–111 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Kaneko, T. et al. PGRP-LC and PGRP-LE have essential yet distinct functions in the Drosophila immune response to monomeric DAP-type peptidoglycan. Nat. Immunol. 7, 715–723 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Bosco-Drayon, V. et al. Peptidoglycan sensing by the receptor PGRP-LE in the Drosophila gut induces immune responses to infectious bacteria and tolerance to microbiota. Cell Host Microbe 12, 153–165 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Perrin, J. et al. The nonaspanins TM9SF2 and TM9SF4 regulate the plasma membrane localization and signalling activity of the peptidoglycan recognition protein PGRP-LC in Drosophila. J. Innate Immun. 7, 37–26 (2014).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  32. Libert, S., Chao, Y., Chu, X. & Pletcher, S.D. Trade-offs between longevity and pathogen resistance in Drosophila melanogaster are mediated by NF-κB signaling. Aging Cell 5, 533–543 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Micheau, O. & Tschopp, J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114, 181–190 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Schneider-Brachert, W. et al. Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles. Immunity 21, 415–428 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. Compagnon, J., Gervais, L., Roman, M.S., Chamot-Boeuf, S. & Guichet, A. Interplay between Rab5 and PtdIns(4,5)P2 controls early endocytosis in the Drosophila germline. J. Cell Sci. 122, 25–35 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2, 107–117 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Rusten, T.E. et al. Fab1 phosphatidylinositol 3-phosphate 5-kinase controls trafficking but not silencing of endocytosed receptors. Mol. Biol. Cell 17, 3989–4001 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Katzmann, D.J., Odorizzi, G. & Emr, S.D. Receptor downregulation and multivesicular-body sorting. Nat. Rev. Mol. Cell Biol. 3, 893–905 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Zeigerer, A. et al. Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature 485, 465–470 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Katzmann, D.J., Babst, M. & Emr, S.D. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106, 145–155 (2001).

    Article  CAS  PubMed  Google Scholar 

  41. Guruharsha, K.G. et al. Drosophila protein interaction map (DPiM): a paradigm for metazoan protein complex interactions. Fly (Austin) 6, 246–253 (2012).

    Article  CAS  Google Scholar 

  42. Leulier, F., Lhocine, N., Lemaitre, B. & Meier, P. The Drosophila inhibitor of apoptosis protein DIAP2 functions in innate immunity and is essential to resist gram-negative bacterial infection. Mol. Cell. Biol. 26, 7821–7831 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Meinander, A. et al. Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling. EMBO J. 31, 2770–2783 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Valanne, S., Kleino, A., Myllymäki, H., Vuoristo, J. & Rämet, M. Iap2 is required for a sustained response in the Drosophila Imd pathway. Dev. Comp. Immunol. 31, 991–1001 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Maminń´ska, A. et al. ESCRT proteins restrict constitutive NF-κB signaling by trafficking cytokine receptors. Sci. Signal. 9, ra8 (2016).

    Google Scholar 

  46. Nakamura, N. et al. Endosomes are specialized platforms for bacterial sensing and NOD2 signalling. Nature 509, 240–244 (2014).

    Article  CAS  PubMed  Google Scholar 

  47. Takehana, A. et al. Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity. EMBO J. 23, 4690–4700 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Brubaker, S.W., Bonham, K.S., Zanoni, I. & Kagan, J.C. Innate immune pattern recognition: a cell biological perspective. Annu. Rev. Immunol. 33, 257–290 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Kagan, J.C. & Medzhitov, R. Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling. Cell 125, 943–955 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Marek, L.R. & Kagan, J.C. Phosphoinositide binding by the Toll adaptor dMyD88 controls antibacterial responses in Drosophila. Immunity 36, 612–622 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Neyen, C., Bretscher, A.J., Binggeli, O. & Lemaitre, B. Methods to study Drosophila immunity. Methods 68, 116–128 (2014).

    Article  CAS  PubMed  Google Scholar 

  52. Lim, J.H. et al. Structural basis for preferential recognition of diaminopimelic acid-type peptidoglycan by a subset of peptidoglycan recognition proteins. J. Biol. Chem. 281, 8286–8295 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  54. Stöven, S., Ando, I., Kadalayil, L., Engström, Y. & Hultmark, D. Activation of the Drosophila NF-kappaB factor Relish by rapid endoproteolytic cleavage. EMBO Rep. 1, 347–352 (2000).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was funded by the National Research Fund Luxembourg (AFR08/037) (C.N.) and UK National Health Service funding to the National Institute for Health Research Biomedical Research Centre (P.M.). We thank members of B.L.'s lab for discussions, D. Mengin-Lecreulx (Centre National de la Recherche Scientifique, Université de Paris-Sud) for PGN and TCT, S. Boy, J. Rybniker (Ecole Polytechnique Fédérale de Lausanne) and A. Kleino (University of Massachusetts) for sharing advice and plasmids, C. Day, M. Miaczynska and N. Silverman for insightful comments, K. Hofmann and A. Kajava for help with RHIM analysis, M. Gonzalez-Gaitán (University of Geneva), H. Krämer (University of Texas Southwestern Medical Center), A.H. Tang (Eastern Virginia Medical School), and the Bloomington Drosophila Stock Center (BDSC), the Transgenic RNAi Project (TRiP) and the Vienna Drosophila Resource Centre (VDRC) for fly stocks.

Author information

Author notes

  1. Pascal Meier and Bruno Lemaitre: These authors jointly directed this work.

Authors and Affiliations

  1. Global Health Institute, Swiss Federal Institute of Technology, Lausanne, Switzerland

    Claudine Neyen, Fanny Schüpfer & Bruno Lemaitre

  2. The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, London, UK

    Christopher Runchel & Pascal Meier

Authors
  1. Claudine Neyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher Runchel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fanny Schüpfer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pascal Meier
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bruno Lemaitre
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.N. conceived the project, carried out experiments and wrote the manuscript. F.S. assisted with experiments. C.R. provided additional experimental data. P.M. and B.L. advised on experimental design and commented on the manuscript.

Corresponding authors

Correspondence to Claudine Neyen or Bruno Lemaitre.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The PGRP-LC locus encodes a subset of isoforms containing an alternative first exon.

(a) Clustal Omega multiple sequence alignment of 5’ and 3’ regions of exon 5 in 11 sequenced Drosophila species indicate a conserved Kozak sequence upstream of the potential translation start site, and a splice donor site in 3’.

(b) RT-qPCR amplification of rLC isoforms from DNase-treated total RNA from the white1118 strain, reverse-transcribed using oligo dT primers, shows that exon 5 encoding the rLC cytosolic domain is spliced to all ectodomain-encoding exons (x, a and y isoforms). See also Fig. 1c for details of PGRP-LC locus architecture. Sequenced products were blasted against the UCSC Genome Browser D. melanogaster Apr. 2006 (BDGP R5/dm3) Assembly. Note that FlyBase lists only the rLC isoform containing the x ectodomain, referred to as PGRP-LC-RE.

(c) PCR products as sequenced in b.

Supplementary Figure 2 Predicted protein domains in the cytosolic tail of rLC.

(a) The rLC cytosolic tail contains a PHD motif according to motif prediction using Phyre2 (not shown) and InterProScan (hits and E-values shown) 55, 56, 57.

(b) Phylogram of Drosophila proteins with known function containing the RING/FYVE/PHD domain predicted for rLC, built using ClustalW and protein sequences corresponding to the RING/FYVE/PHD (IPR013083) domain of each protein. Grey area highlights phosphoinositide-binding membrane adaptors. rLC clusters with this group, suggesting lipid binding potential.

(c) Helical wheel diagram of the conserved residues 76 to 91, built using HeliQuest 58, indicating amphipathic properties. Positively charged residues (orange), negatively charged residues (blue), aliphatic residues (green).

(d) The conserved α-helical domain (residues 76 to 911) is predicted to have a strong propensity for forming coiled-coils (prediction method: Coils 59).

Supplementary Figure 3 Physiological and immunological consequences of rLC loss of function.

(a) eGFP mRNA expression (representing LC transcripts from P[acman] constructs only) shows that deletion of exon 5 from the engineered P[acman]-GFP -LC locus does not affect PGRP-LC expression in cis. Tested on unchallenged flies (initial insertion lines, carrying a wild-type PGRP-LC locus on chromosome 3, plus one genomic copy of P[acman]-GFP-LC at the attP landing site 51C on chromosome 2).

(b) Quantification of adults hatching per female per 24h egg lay shows that reproductive output is not significantly affected in rLC loss-of-function flies (resc(LCΔex5)) compared to WT or resc(LCwt) flies. Each dot represents counts from one fly vial.

(c) Quantification of Diptericin (Dpt) mRNA expression (relative to ribosomal (RpL32) mRNA) in flies infected with Erwinia carotovora carotovora strain 15 (Ecc15) shows that rLCx is necessary and sufficient for resolution of IMD activation, and that rLCx-mediated regulation is dose-dependent. Efficiency of regulation correlated with genomic copy number of rLC (compare efficiency of rescue constructs in the ΔLCE12 background, which has zero genomic rLC copies, versus rescue in the ΔLCird7(1) background, which has two genomic rLC copies). See also Fig. 2d, e for a visual explanation of genotypes.

(d) Quantification of Dpt mRNA expression in dissected tissues of unchallenged flies shows that loss of rLC has no impact on PGRP-LE-dependent basal IMD activation by commensals in the midgut of conventionally reared flies, but leads to increased immune activation in whole guts where PGRP-LC contributes to sensing (see also Fig. 1d for receptor expression levels). Loss of rLC did not cause any significant changes in baseline IMD activation in the fat body of conventionally reared but uninfected flies.

(e) Quantification of Dpt mRNA expression over time in in flies with (resc(LCwt)) or without (resc(LCΔex5)) rLC or controls (WT = w1118 and ΔLCE12) infected with heat-killed E.coli (see also Fig. 3b).

(f) Survival of rLC overexpressing flies (c564-Gal4 > UAS-PGRP-LCx) compared to controls (c564-Gal4) infected with Ecc15.

NS P > 0.05, * P < 0.05, **P < 0.01, *** P < 0.001 (Student’s t test (a), one-way ANOVA, Tukey’s post hoc test (b, c), two-way ANOVA, Bonferroni post hoc test (d, e); in f, P = 0.0002 (log-rank test). Data are pooled from three (b, c, d, e) or four (a, f) independent experiments and represent mean ± s.e.m (a, b, c, d, e).

Supplementary Figure 4 rLC interacts with the negative regulator Pirk.

(a) Quantification of Dpt mRNA expression over time in flies overexpressing rLC (c564-Gal4 > UAS-rLCx), Pirk (c564-Gal4 > UAS-Pirk) or both in the fat body after challenge with heat-killed Ecc15. Control, driver c564-Gal4 (see Fig. 4d).

(b) Quantification of Dpt mRNA expression over time in flies with genomic Pirk deficiency and overexpressing rLC in the fat body (driver: w;c564-Gal4) after challenge with heat-killed Ecc15 (see Fig. 4e). *** P < 0.001 (one-way ANOVA, Tukey’s post hoc test (a, b). Data are pooled from three independent experiments and represent mean + s.e.m.

Supplementary Figure 5 rLCx overexpression constructs for subcellular localization.

(a) Schematic representation of all UAS constructs generated in this study (left panel). Quantification of Dpt mRNA (at 8h after infection, left) or rLCx mRNA (fold induction, right) in flies overexpressing rLCx constructs (driver: c564-Gal4) and challenged with heat-killed Ecc15 (WT control: c564-Gal4 crossed to w1118). Unfortunately, any large truncation of the rLC cytosolic tail will leave a short cytosolic stump coupled to a peptidoglycan-binding PGRP ectodomain, strongly resembling PGRP-LF. It is likely that overexpression of such constructs will still regulate IMD activation, albeit by receptor competition mechanistically resembling regulation through PGRP-LF rather than through rLC-specific (or PHD-domain specific) mechanisms. An adequate but time-intensive approach would be to replace wild-type rLC with deletion mutants at its genomic locus.

(b) Confocal imaging of overexpressed (driver: c564-Gal4) FYVE-GFP (left, single z slice through cell body) and PLCδ-PH-GFP (right, single z slices close to cell surface and through cell body) shows localization of FYVE marker to intracellular vesicles and of PLCδ-PH marker to plasma membranes. Scale bars, 20 μm.

(c) Confocal imaging of overexpressed GFP-rLCx in fixed and DAPI-stained hemocytes from hml-Gal4>UAS-GFP-rLCx larvae shows GFP localizes to plasma membrane (solid arrowheads), to cell-cell contact points (open arrowheads), and to filopodia (arrow). Image is an average intensity z-projection of 10 consecutive z-slices. Scale bar 10 μm.

(d) Quantification of immunoblot in Fig. 5f shows that GFP-rLCx accumulates when the endocytic machinery is impaired.

(e) and (f) Localization of overexpressed GFP-rLCx (driver: c564-Gal4) in tsg101 knock-down (e) and Rab5 knock-down (f) fat bodies. Sum projection of 3 consecutive apical slices; scale bar 10 μm (compare to Fig. 5g).

(g) Quantification of fat body cell size for indicated genotypes shows that only Rab5 knock-down significantly affects cell size. To correct for this, vesicle numbers were normalized to respective cell area in Fig. 5j.

(h) Quantification of Dpt mRNA expression 8h after infection with heat-killed Ecc15 in controls (c564-Gal4) or in flies overexpressing rLCx (c564-Gal4 > UAS-rLCx) in the presence or absence of RNAi against indicated endocytic components. WT control, w;c564-Gal4 x w1118. Fab1 is a PI3P-5 kinase, Vps28 and Tsg101 are components of the ESCRT machinery, UbcD10 is the unique Drosophila E1 ligase involved in protein ubiquitination. * P < 0.05, **P < 0.01, *** P < 0.001 (one-way ANOVA, Dunnett’s post hoc test (g), two-way ANOVA, Bonferroni post hoc test (h). Data are pooled from three (a, h, except for Rab5 RNAi which caused significant developmental lethality) or n (indicated above graph (g)) independent experiments and represent mean + s.e.m. (a, g, h).

Supplementary Figure 6 Proposed model for rLCx-mediated resolution of IMD pathway activation.

(a) During Gram-negative infection, TCT released from live bacteria efficiently activates the IMD pathway by engaging PGRP-LCx-LCa homodimers (as opposed to PGRP-LC-rLC heterodimers). Since ligand binding depends on the ectodomains alone, homodimers and heterodimers of activating and regulatory isoforms are equally likely to assemble. IMD recruitment requires dimerization of activating receptors, therefore neither rLC-rLC homodimers nor rLC-LC heterodimers can activate the pathway. In this sense, rLC acts similarly to PGRP-LF with regards to TCT: it can form ligand-bound dimers with LC but cannot signal 18. IMD pathway activation triggers transcriptional induction of antimicrobial peptide genes and the production of negative regulators of the pathway, such as rLC. (b) Once bacteria are killed, the balance of available ligands shifts from TCT monomers to PGN polymers, which can recruit both activating PGRP-LC and regulatory rPGRP-LC isoforms in one complex. rPGRP-LC promotes ESCRT-mediated clearance of signalling complexes. Components of the ESCRT machinery capture signalling receptors, trafficking them into multivesicular bodies, from where the cytosolic tails can no longer interact with signalling adaptors. Receptors are eventually degraded to resolve IMD pathway activation. Note that the intracellular negative regulator Pirk (not shown) contributes to regulation in both scenarios, regardless of the type of ligand.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–4 (PDF 1741 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Neyen, C., Runchel, C., Schüpfer, F. et al. The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors. Nat Immunol 17, 1150–1158 (2016). https://doi.org/10.1038/ni.3536

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3536

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing