Role of Nimrod receptors in the Drosophila melanogaster cellular immune response

The use of modern molecular biology tools and simple but powerful tractable model organisms such as Drosophila has contributed significantly to our recent advances in the innate immunity field, notably phagocytosis. Since 2001, many phagocytic transmembrane receptors, potential opsonins, and in some cases their ligands, have been discovered. Among them, the Nimrod family, which contains key phagocytic receptors and potential opsonins, deserves special interest as accumulating genetic and biochemical evidence points to their critical role in phagocytosis of both bacteria and apoptotic cells. In this PhD thesis we provided a deeper characterization of two Nimrod transmembrane receptors, Eater and NimC1. In particular, by using single or combined mutations in eater and NimC1, we aimed to further elucidate i) the involvement of each receptor in bacterial phagocytosis and ii) the role of Eater in hemocytes sessility and adhesion. Both receptors have been shown to be specifically expressed in hemocytes, the insect blood cells, and to mediate Gram-positive but not Gram-negative bacteria phagocytosis. In this work we generated a novel NimC1 mutant and demonstrated that the single NimC1 deletion does not affect phagocytosis of bacteria. However, by using the compound NimC1;eater mutant we were able to show that these receptors contribute in a synergistic way to microbes engulfment, but that Eater can bypass the requirement for NimC1. Moreover, we discovered that NimC1, but not Eater, is required for phagocytosis of non-immunogenic particles, namely latex beads and yeast zymosan. Therefore, this work provides evidences of Eater and NimC1 being the main receptors for bacteria phagocytosis, and suggests that those proteins likely play distinct roles in microbial uptake, as tethering and docking receptors. The second part of this thesis re-addresses the role of Eater in hemocyte sessility and adhesion. A previous study showed that this phagocytic receptor is essential to mediate blood cell sessility to the animal integument during larval stages. However, a series of questions about Eater-mediated sessility remains still open. For example, is Eater mediating hemocyte adhesion also in adult flies? And yet, does Eater mediate hemocyte sessility by regulating the expression of cell adhesion genes, therefore functioning as a signalling receptor? By using eater1 mutant adult flies, we showed that, as in larval stages, Eater is required in adult hemocytes to confer adhesion and sessility. Interestingly, we uncovered a potential role of sessility in hemocyte survival during adulthood, since eater1 flies have a decreased number of blood cells. Preliminary transcriptomics analysis led us to hypothesise that this receptor might not work as a signalling molecule, but rather as an adhesion protein providing the initial hemocyte contact to its target surface. In line with Eaterâs role as an adhesion molecule, its over-expression in hemocytes leads to the formation of enlarged cells compared to control. To conclude, this thesis extended our knowledge on the Drosophila cellular immune response, by providing new insights on two Nimrod phagocytic receptors.

The role of the Nimrod proteins in the Drosophila immune system

Bianca Marie Petrignani

The use of tractable model organisms such as Drosophila melanogaster and the powerful genetic tools they offer has contributed significantly to recent advances in comprehension of innate immunity, notably phagocytosis. To date, several phagocytic transmembrane receptors, opsonins, and ligands have been discovered in Drosophila melanogaster. Many of them belong to the Nimrod family, which includes both secreted and transmembrane proteins containing several NIM repeats. Genetic and biochemical studies have revealed the roles of transmembrane Nimrod receptors such as Draper, Simu and Eater in phagocytosis. However, the function of the five secreted Nimrod members, the Nimrod B proteins, remains unstudied. The goals of my thesis were to characterize the function of the secreted Nimrod B proteins using newly generated CRISPR-Cas9 null mutations, focusing on NimB5, NimB4, and NimB1 genes. In this work, we first found that the secreted protein, NimB5, is produced in the fat body upon nutrient scarcity downstream of metabolic sensors and ecdysone signaling. We provide evidence that NimB5 binds to hemocytes to down-regulate their proliferation and adhesion. Blocking this signaling loop results in conditional lethality when larvae are raised on a poor diet due to excessive hemocyte numbers and insufficient energy storage. This pointed to a role of NimB5 in the allocation of resources to blood cells, tailoring the investment in the immune system to metabolic resource availability.In the second part of this thesis, we analyzed the function of NimB4 to reveal its crucial role in clearance of apoptotic cells. NimB4 is expressed by macrophages and glial cells, the two main types of phagocytes in Drosophila. Our study points to a role of NimB4 in phagosome maturation, more specifically in the fusion between the phagosome and lysosomes. We propose that similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis. Finally, in the last part of the thesis, we present preliminary results on the role of NimB1 in the Drosophila immune system. NimB1 is expressed mainly in hemocytes and regulates hemocyte number. Additionally, NimB1 shares several characteristics with NimB4, notably the ability to bind to apoptotic cells. As such, we hypothesize that it could play a similar function in the phagocytosis of apoptotic cells. However, the exact role of NimB1 in phagocytosis is still unclear, and additional work is necessary to understand if NimB1 plays a role in the uptake of apoptotic cells or phagosome maturation. Collectively, this thesis has revealed that the NimB proteins play specific roles in efferocytosis or in blood cell number regulation. Thus, this work extended our knowledge on the Drosophila cellular immune response by providing new insight on the conserved Nimrod family of proteins.

EPFL2022

Two Nimrod receptors, NimC1 and Eater, synergistically contribute to bacterial phagocytosis in Drosophila melanogaster

Andrew Jonathan Bretscher, Jan Paul Dudzic, Bruno Lemaitre, Claudia Melcarne, Elodie Julie Virginie Ramond

Eater and NimC1 are transmembrane receptors of the Drosophila Nimrod family, specifically expressed in haemocytes, the insect blood cells. Previous ex vivo and in vivoRNAi studies have pointed to their role in the phagocytosis of bacteria. Here, we have created a novel NimC1 null mutant to re-evaluate the role of NimC1, alone or in combination with Eater, in the cellular immune response. We show that NimC1 functions as an adhesion molecule ex vivo, but in contrast to Eater it is not required for haemocyte sessility in vivo. Ex vivo phagocytosis assays and electron microscopy experiments confirmed that Eater is the main phagocytic receptor for Gram-positive, but not Gram-negative bacteria, and contributes to microbe tethering to haemocytes. Surprisingly, NimC1 deletion did not impair phagocytosis of bacteria, nor their adhesion to the haemocytes. However, phagocytosis of both types of bacteria was almost abolished in NimC1(1);eater(1) haemocytes. This indicates that both receptors contribute synergistically to the phagocytosis of bacteria, but that Eater can bypass the requirement for NimC1. Finally, we uncovered that NimC1, but not Eater, is essential for uptake of latex beads and zymosan particles. We conclude that Eater and NimC1 are the two main receptors for phagocytosis of bacteria in Drosophila, and that each receptor likely plays distinct roles in microbial uptake.

WILEY2019

Adult Drosophila Lack Hematopoiesis but Rely on a Blood Cell Reservoir at the Respiratory Epithelia to Relay Infection Signals to Surrounding Tissues

Bruno Lemaitre, Elodie Julie Virginie Ramond

The use of adult Drosophila melanogaster as a model for hematopoiesis or organismal immunity has been debated. Addressing this question, we identify an extensive reservoir of blood cells (hemocytes) at the respiratory epithelia (tracheal air sacs) of the thorax and head. Lineage tracing and functional analyses demonstrate that the majority of adult hemocytes are phagocytic macrophages (plasmatocytes) from the embryonic lineage that parallels vertebrate tissue macrophages. Surprisingly, we find no sign of adult hemocyte expansion. Instead, hemocytes play a role in relaying an innate immune response to the blood cell reservoir: through Mid signaling and the Jak/Stat pathway ligand Upd3, hemocytes act as sentinels of bacterial infection, inducing expression of the antimicrobial peptide Drosocin in respiratory epithelia and colocalizing fat body domains. Drosocin expression in turn promotes animal survival after infection. Our work identifies a multisignal relay of organismal humoral immunity, establishing adult Drosophila as model for inter-organ immunity.

CELL PRESS2019

The role of the Nimrod proteins in the Drosophila immune system

Bianca Marie Petrignani

EPFL2022