Reference

Elimination of plasmatocytes by targeted apoptosis reveals their role in multiple aspects of the Drosophila immune response.

Charroux B Royet J
Proceedings of the National Academy of Sciences of the United States of America. 2009.

Abstract

Drosophila hemocytes have strong phagocytic capacities and produce antimicrobial peptides (AMPs). However, the precise role of blood cells during immune responses and developmental processes has only been studied using indirect means. To overcome this limitation, we generated plasmatocyte-depleted flies by specifically overexpressing the proapoptotic protein Hid into plasmatocytes. Unexpectedly, these plasmatocyte-depleted animals have a normal larval and pupal development and do not exhibit any obvious defect after birth. Remarkably, plasmatocyte-depleted adults show a strong susceptibility to infections by various microorganisms, although activation of systemic AMP gene transcription via the Toll and immune deficiency (IMD) pathways is wild-type. Our data show that this susceptibility, which correlates with overproliferation of bacteria, is likely due to the absence of phagocytosis. We also demonstrate that during larval stages, plasmatocytes play an essential role in mediating AMP production by the fat body after oral bacterial infection. Finally, we show that plasmatocytes are involved in immune surveillance during pupal development, because they prevent bacterial infection that causes pupal lethality.

Main claims

  1. Genetic ablation of hemocytes reveals no role of hemocytes in systemic activation of antimicrobial peptides in adult flies, but shows that hemocytes contribute to combatting infection through other processes including phagocytosis. [v1]
    (Defaye et al., 2009) similarly show that hemocytes are dispensable for systemic activation of AMPs in response to septic infection in adult flies, while many studies show that phagocytosis is an important component of defense against various microbial infections (e.g. (Avet-Rochex et al., 2007; Kocks et al., 2005)).

    Last change on 2022-08-15 15:32 by Anonymous

Major claims

  1. Plasmatocytes are largely dispensable for third-instar larval and pupal development. [v1]
    (Defaye et al., 2009; Shia et al., 2009) initially confirmed that plasmatocytes were dispensable for larval and pupal development (although note that plasmatocytes are essential at the embryonic stage). However, (Stephenson et al., 2022) recently found that a stronger hml driver that ablates more hemocytes reveals an essential role of hemocyte during metamorphosis, indicating that some remaining hemocytes are necessary for this process.

    Last change on 2022-08-15 15:32 by Anonymous

    1. Plasmatocyte-depleted individuals (hml>hid) reached the pupal stage with the same timing as wild-type siblings. [v1]

      Use of a stronger hml driver reveals strong lethality in hemoless pupae (Stephenson et al., 2022).

      Last change on 2022-08-15 15:36 by Anonymous

    2. Larval lethality of mutations used to study hemocytes such as domino, l(3)hem or psidin suggests they affect other tissues; in contrast to these mutants, hml>hid larvae and flies do not produce melanotic tumors or other obvious defects [v1]

      To be assessed

      Last change on 2022-08-15 15:37 by Anonymous

  2. Activation of systemic AMP gene transcription via the Toll and Imd pathways in adult flies following septic infection does not require plasmatocytes. [v1]
    Supported by results of (Defaye et al., 2009).

    Last change on 2022-08-15 15:32 by Anonymous

    1. Induction and kinetics of Drosomycin, Diptericin, Attacin-A, and Defensin in response to Gram-positive and -negative bacteria in hml>hid flies was similar to wild-type flies [v1]

      To be assessed

      Last change on 2022-08-15 15:37 by Anonymous

    2. Hml>hid, spzrm7 flies had higher susceptibility than either of these modifications alone, showing that phagocytosis and AMP production act independently but in concert to fight infection [v1]

      To be assessed

      Last change on 2022-08-15 15:37 by Anonymous

  3. Plasmatocyte-depleted adults are susceptible to infections by various microorganisms due to overproliferation of bacteria, likely due to the absence of phagocytosis [v1]
    This largely supported by results of (Defaye et al., 2009). These authors showed that phagoless flies had increased susceptibility to septic infection with Ecc15, E. coli, E. faecalis, and S. aureus, but not several other bacteria. (Kocks et al., 2005) similarly found normal AMP expression (Diptericin only) but reduced survival of eater mutant flies to natural S. marcescens infection and increased bacterial loads. Experiments performed for ReproSci show that null mutations of NimC1 or eater, or a phagocytosis-deficient NimC1/eater double mutant, produce several pathogen-specific survival effects (see comment). However, these results are not consistent with a model where loss of phagocytosis causes strong susceptibility to E. faecalis, P. entomophila or C. albicans septic infection, suggesting that RNAi against eater and NimC1 does not have equivalent effects to mutation of these same genes, or to total loss of hemocytes.

    Last change on 2022-08-15 15:32 by Anonymous

    1. Hml>hid flies were susceptible to septic infection with E. faecalis, P. entomophila, and C. albicans, and had faster and higher bacterial growth than controls. [v1]

      To be assessed

      Last change on 2022-08-15 15:38 by Anonymous

    2. In vivo RNAi silencing of eater or NimC1 genes affecting phagocytosis produced similar phenotypes to hml>hid flies, suggesting that the survival phenotype is mainly due to a lack of phagocytosis [v1]

      Experiments performed for ReproSci show that loss of NimC1 and eater does have pathogen-specific effects on survival to various infections, but the phenotype of eater and NimC1 is likely exaggerated here due to use of RNAi and is not equivalent to loss of hemocytes (see comment). (Bretscher et al., 2015) found that larvae carrying the eater1 null mutation (a line that also carried a mutation of NimC1, see Kocks et al., 2020) had reduced phagocytosis of Gram-positive bacteria (S. aureus, M. luteus, S. epidermidis) but not of Gram-negative bacteria (E. coli, S. marcescens); these authors did not assess survival to bacteria in adults. (Melcarne et al., 2019) further showed using the NimC11 mutation that single mutants for NimC1 are not defective in phagocytosis, and that only Eater;NimC1 double mutants have impaired phagocytosis of Gram-negative bacteria, zymosan and latex beads, while eater single mutants are defective only for Gram-positive bacterial phagocytosis. (Hashimoto et al., 2009) found that draper and eater mutant flies had reduced phagocytosis of S. aureus, and that draper mutants had reduced survival to S. aureus (survival of eater flies not assessed). (Christofi and Apidianakis, 2013) found reduced survival of eater mutant flies to septic infection with P. aeruginosa. (Defaye et al., 2009) found that eater mutant or RNAi depleted flies had increased susceptibility to S. aureus similar to phagoless flies. (Nehme et al., 2011) similarly found increased susceptibility of eater mutant flies to E. faecalis and S. aureus, but not M. luteus.

      Last change on 2022-08-15 15:38 by Anonymous

  4. Plasmatocytes are involved in immune surveillance during pupal development, and prevent bacterial infection causing pupal lethality [v1]
    Lethality of phagoless animals at the pupal stage was also observed by (Defaye et al., 2009). (Arefin et al., 2015) also found high pupal lethality in hml>hid and hml>grim larvae that was rescued by antibiotic treatment, but assigned this effect to reduced Imd signaling in these larvae (reduced Dpt expression) rather than a loss of phagocytic activity. However, note that the suppression of the Imd response in these larvae was very minor (~2-4x fold decrease in Dpt and CecA1), and controls are not shown. Supported by results of (Shia et al., 2009) who found that larval lethality associated with ablation of post-embryonic hemocytes is rescued by raising larvae in axenic conditions. Consistent with studies showing that other immune programs are also needed to control bacteria during metamorphosis (Nunes et al., 2021)

    Last change on 2022-08-15 15:33 by Anonymous

    1. 45% of hml>hid larvae emerged as adults; the rate of emergence was normal under axenic conditions, showing that uncontrolled bacterial infection during the pupal stage resulted in lethality. [v1]

      To be assessed

      Last change on 2022-08-15 15:38 by Anonymous

Minor claims

  1. Larval plasmatocytes are required for AMP production by the fat body following natural infection. [v1]
    Supported by results of (Basset et al., 2000; Foley and O’Farrell, 2003).

    Last change on 2022-08-15 15:33 by Anonymous

    1. A Dpt-cherry reporter and RT-PCR showed that systemic (fat body) immune response was reduced in hml>hid larvae in response to oral infection with Ecc15. [v1]

      To be assessed

      Last change on 2022-08-15 15:39 by Anonymous

    2. A systemic immune response in 1(3)hem larvae shows that a very few hemocytes are sufficient to activate a response in the fat body. [v1]

      Using l(3)hem larvae and quantification of a lac-Z reporter, (Basset et al., 2000) found that a loss of hemocytes virtually eliminated systemic induction upon natural (Ecc15) infection, but a reduced and patchy expression of Dpt was still observed (24h post infection).

      Last change on 2022-08-15 15:39 by Anonymous

    3. Dpt-cherry expression in domino larvae is reduced but not absent, patchy and restricted to the anterior and posterior lobes of the fat body [v1]

      This patchy but reduced expression of Dpt was observed in l(3)hem larvae by (Basset et al., 2000).

      Last change on 2022-08-15 15:40 by Anonymous

  2. Phagocytosis is not essential for activation of AMP expression in the fat body upon oral infection of larvae. [v1]
    The evidence given by (Charroux and Royet, 2009) is not strong support for this claim as Gram-negative bacteria (Ecc15) was used, and neither eater nor NimC1 mutants (which were used as phagocytosis-deficient lines) have significantly impaired phagocytosis of Gram-negative bacteria (Melcarne et al., 2019). Although (Melcarne et al., 2019) showed that AMP (Dpt, Drs) induction was normal in NimC1 and NimC1;eater double mutant larvae in response to septic infection, they did not assess induction following natural infection. Experiments performed for the reproducibility project show that systemic AMP expression in larvae naturally infected with the Gram-negative bacterium Ecc15 is impaired by loss of eater, but not NimC1. As phagocytosis of Gram-negative bacteria is not impaired in the eater mutant, we hypothesize that reduced AMP expression is not due to reduced phagocytosis in these mutants, but rather another factor such as impaired hemocyte sessility (see comment). This claim is difficult to effectively test, as methods of arresting phagocytosis typically have pleiotropic effects.

    Last change on 2022-08-15 15:33 by Anonymous

    1. Eater or NimC1-deficient larvae with reduced phagocytic capacity had a normal systemic response to oral infection with Ecc15, showing that either reduced phagocytosis is sufficient to activate the systemic response, or that phagocytosis is not a major contributor in transduction of the signal by hemocytes from gut to fat body. [v1]

      To be assessed

      Last change on 2022-08-15 15:40 by Anonymous

  3. Plasmatocytes are not required for local AMP activation in epithelia [v1]
    (Tzou et al., 2000) also showed using domino larvae and natural infection with Ecc15 that loss of hemocytes did not reduce AMP reporter activation in epithelia. Epithelia are in direct contact with pathogens and express components of signaling pathways (e.g. (Buchon et al., 2013; Wagner et al., 2008)) and thus it is logical that they respond directly to stimuli and do not require motile cells as a transducer. Although (Sanchez Bosch et al., 2019) found that expression of Drosocin in the tracheal epithelium (and fat body) of the adult fly was severely reduced by ablation of hemocytes, this Drosocin expression was due to hemocytes colonizing the respiratory epithelium rather than reduced AMP expression in the epithelia itself.

    Last change on 2022-08-15 15:33 by Anonymous

    1. Dpt-cherry expression was normal in gut epithelia of naturally Ecc15-infected hml>hid larvae [v1]

      To be assessed

      Last change on 2022-08-15 15:40 by Anonymous

  4. A population of plasmatocytes is observed in the proventriculus of third instar larvae [v1]
    Confirmed by results of (Zaidman-Rémy et al., 2012)

    Last change on 2022-08-15 15:33 by Anonymous

  5. A population of Hml positive hemocytes is found in the anterior part of the larval midgut. [v1]
    (LaJeunesse et al., 2010) confirms a population of hml-expressing cells in this region, but these cells are enteroendocrine cells expressing the Hml marker and not hemocytes.

    Last change on 2022-08-18 13:17 by Anonymous

Methods

  1. Plasmatocyte-depleted flies were generated by specifically overexpressing the proapoptotic protein Hid in plasmatocytes. More than 96% of circulating larval hemocytes were eliminated by hml>hid; embryonic plasmatocytes were not ablated. Adults had very few (5%) plasmatocytes. Hemoless. [v1]

    Last change on 2022-08-15 15:34 by Anonymous

Additional context

Not annotated yet

Additional files

Not annotated yet

References

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    Last change on 2023-01-05 10:38 by Anonymous