Drosophila melanogaster harbours a sophisticated cellular immune system to fight invaders. The larval hematopoietic system comprises a circulating and sessile compartment of embryonic origin and the developing larval lymph gland, constituting a hematopietic organ. The circulating and sessile compartment consists primarily of macrophage-like plasmatocytes, plus a small number of crystal cells involved in wound healing and melanization responses to neutralize pathogens. Both cell types differentiate from hemocyte precursors within the lymph gland as well; they are released during pupal stages to serve the adult with immune cells. Finally, lamellocytes represent the third blood cell type in Drosophila. While merely absent in healthy animals, their formation is induced within hours of wasp infestation or wounding (reviewed in: Banerjee et al., 2019; Letourneau et al., 2016; Hultmark and Andó, 2022). In fact, parasitism by endo-parasitoid wasps represents one of the most severe naturally occurring immune challenges to Drosophila, invariably causing death if not defended off properly. Parasitoid wasps deposit their egg into the live Drosophila larva, where it develops into the next wasp generation egressing from the pupa instead of a fly. Hence, wasp infestation is a life-threatening challenge to the Drosophila host, demanding an immediate immune response to overwhelm the parasite. This involves a massive increase in circulating hemocytes, and most energy resources are devoted to fight the invader. Here, lamellocytes play a critical role: they encapsulate the wasp egg and, by expressing Prophenoloxidase, induce a melanization reaction retarding further wasp development (Dudzic et al. 2015; reviewed in: Banerjee et al., 2019; Letourneau et al., 2016; Hultmark and Andó, 2022).

Wasp infestation dramatically remodels the composition of the Drosophila hemocyte population (Cho et al., 2020; Tattikota et al., 2020; reviewed in: Csordás et al., 2021). There is a vast increase in plasmatocytes and intermediate precursors in both hematopoietic compartments, from which lamellocyte differentiate, requiring the combined activity of several pathways, including JAK/STAT, JNK, Toll, Notch, EGFR and INR pathways, which in fact regulate blood cell homeostasis in general (reviewed in Banerjee et al., 2019; Letournau et al., 2018; Csordás et al., 2021). Simultaneous to the massive expansion of lamellocytes, crystal cells derived from the same precursors are significantly reduced (Crozatier et al., 2004; Krzemien et al., 2010; Ferguson and Martinez –Agosto, 2014; Cho et al., 2020; Tattikota et al., 2020; reviewed in Csordás et al., 2021). Crystal cells are generated both in the larval lymph gland as well as by trans-differentiation from plasmatocytes in the sessile compartment. Their formation, differentiation and survival strictly depend on Notch signaling activity (reviewed in: Banerjee et al., 2019; Csordás et al., 2021; Hultmark and Andó, 2022). Lamellocyte and crystal cell lineages are mutually exclusive. Accordingly, while promoting crystal cell fate, Notch activity inhibits differentiation of lamellocytes within the lymph gland, however, is reduced upon wasp infestation (Small et al., 2014). Lamellocyte induction involves the formation and sensing of reactive oxygen species triggered by wasp egg injection (Nappi et al., 1995; Sinenko et al., 2011; Small et al., 2014; Louradour et al., 2017; reviewed in: Banerjee et al., 2019; Csordás et al., 2021). The molecular mechanism underlying the simultaneous crystal cell fate inhibition, however, is less well understood. Obviously, an effective and rapid downregulation of Notch activity is required to fend off parasitic wasps.

The Notch pathway is highly conserved between invertebrates and vertebrates, where it regulates numerous cell fate decisions. Notch signals are transduced by CSL type proteins (abbreviation of human CBF1/RBPJ, Drosophila Suppressor of Hairless [Su(H)], and worm Lag1), that bind the DNA to direct transcriptional activity of Notch target genes by help of recruited cofactors (reviewed in: Giaimo et al., 2021; Kopan and Ilagan, 2009; Siebel and Lendahl, 2017). Hence, CSL is central to Notch pathway activity as no signal transduction could occur in its absence or in the instance of a lack of DNA-binding. Earlier, we observed CSL, i.e. Su(H) phosphorylation at Serine 269 in cultured Drosophila Schneider S2 cells (Nagel et al., 2017). Of note, Schneider S2 cells have hemocyte characteristics (Cherbas et al., 2011; Schneider, 1972; Terriente-Felix et al., 2013). As a consequence of the negative charge conferred by the phosphorylation at Serine 269, Su(H) loses its affinity to the DNA, and without its DNA-binding ability, also its function as a transcriptional regulator (Nagel et al., 2017). Accordingly, a phospho-mimetic Su(H)^S269D mutant behaved like a Su(H) loss of function mutant in all respects. In contrast, the phospho-deficient Su(H)^S269A variant, appeared wild type at the first glance, indicating some tissue-specificity of this regulatory mechanism. In fact, the Su(H)^S269A allele displayed a gain of Notch activity particularly during embryonic and larval hematopoiesis with increased numbers of crystal cells in both hematopoietic compartments (Frankenreiter et al., 2021). Moreover, we found that the general Notch antagonist Hairless is not involved in constraining crystal cell numbers, suggesting that in the context of blood cell homeostasis Notch activity is regulated by the phosphorylation of Su(H) (Maier, 2006; Frankenreiter et al., 2021). As the same set of genes affect blood cell maintenance and differentiation in homeostasis and upon immune challenge (Cho et al., 2020; Tattikota et al., 2020), obviously phospho-mediated downregulation of Notch activity might also occur in response to parasitoid wasp infestation, allowing the formation of encapsulation active lamellocytes at the expense of crystal cells. Briefly, phosphorylation of Su(H) is an elegant mechanism to promptly and transiently curb Notch activity.

In this work, we followed the tempting hypothesis that following wasp infestation, a specific kinase might be activated to phosphorylate Su(H) thereby blocking Notch activity. In this case, the phospho-deficient Su(H)^S269A allele should be immune-compromised, as it cannot respond to phosphorylation, i.e. remaining active even upon infection. Indeed, Su(H)^S269A displayed an increased sensitivity towards parasitoid wasp infestation accompanied by an increase of crystal cells at the expense of lamellocytes. Accordingly, phosphorylation of Su(H) protein was detected in infested wild type larvae but not in the Su(H)^S269A mutant. In a screen for kinases regulating this process, Pkc53E, the homologue of human PKCα, was identified as an important player. In agreement with a role in blood cell homeostasis, a Pkc53E^Δ28 null mutant displayed increased crystal cell numbers. Moreover, genetic and molecular interactions between Su(H) and Pkc53E support the specificity of their relationship. Finally, Pkc53E^Δ28 was impaired in its immune response to wasp infestation as well. Together, these data show that Su(H) is a target of Pkc53E during parasitic wasp infestation, inducing phosphorylation and subsequent downregulation of Notch activity to allow the mass production of lamellocytes required for wasp defense.

The Notch pathway is highly conserved between invertebrates and vertebrates, with regard to both the underlying molecular principles as well as the biological processes it is involved, including hematopoiesis and immune defence. During mammalian hematopoiesis, Notch plays a fundamental role in stem cell maintenance and proliferation as well as in the differentiation of blood cell precursors, notably in T-cell development. Accordingly, aberrant Notch signaling activity has profound consequences for blood cell homeostasis that may result in leukemia (reviewed in: Radtke et al., 2010; Siebel and Lendahl, 2017; Banerjee et al., 2019; Gallenstein et al., 2023). Hence, the principles of the regulation of Notch signaling activity are of great interest.

Notch signals are transduced by CSL-type DNA-binding proteins that are pivotal to Notch pathway activity, as no signal transduction could occur in their absence or in the instance of a lack of DNA-binding. CSL proteins are extremely well conserved. They act as a molecular switch, either activating or repressing Notch target genes, depending on the recruited cofactors. Upon ligand binding, the Notch receptor is cleaved, and the biologically active Notch intracellular domain (NICD) is released, to assemble an activation complex with CSL and further co-factors. Notch pathway repression, however, entails for example the recruitment of co-repressors by CSL; in Drosophila mediated by the binding of Hairless (reviewed in: Maier, 2006; Borggrefe and Oswald, 2009; Kovall and Blacklow, 2010; Bray, 2016; Giaimo et al., 2021).

Studies of Drosophila hematopoiesis have uncovered a pleiotropic role of Notch in all the hematopoietic compartments, where Notch activity needs to be precisely regulated to ensure blood cell homeostasis (reviewed in: Banerjee et al., 2019; Csordás et al., 2021). During blood cell formation, Notch directs the crystal cell lineage. Accordingly, a downregulation of Notch activity causes a lack of crystal cells, whereas a gain of Notch activity results in increased numbers (Duvic et al., 2002; Lebestky et al., 2003; Terriente-Felix et al., 2013; Gosh et al., 2015; Frankenreiter et al., 2021). In our earlier work, we have shown that the regulation of Notch activity during hemocyte differentiation is independent of the general Notch antagonist Hairless. Instead, it relies on the post-translational modification of the Drosophila CSL protein Su(H) (Frankenreiter et al., 2021). Phosphorylation at Ser269 in the beta-trefoil domain of Su(H) impedes DNA-binding activity, and hence the capability of Su(H) to act as a transcriptional regulator in the context of blood cell development, without affecting Su(H) protein expression (Nagel et al., 2017; Frankenreiter et al., 2021). Accordingly, phospho-deficient Su(H)^S269A mutants develop an excess of crystal cells. Now we provide evidence that Su(H) phosphorylation is likewise involved in parasitoid wasp defense, pointing to a dual use of the Notch pathway in blood cell homeostasis as well as in stress response, which is typical for the myeloid system (Banerjee et al., 2019).

The primary cellular immune response of Drosophila to fight parasitoid wasp infestation is an encapsulation of the parasite egg to terminate its further development. Albeit metabolically extremely costly, Drosophila larvae have to remodel their entire hematopoietic system to generate the masses of lamellocytes required for the encapsulation (reviewed in Letournau et al., 2016; Kim-Jo et al., 2019; Csordás et al., 2021; Hultmark & Ando, 2022). There are two major sources for the lamellocytes. One is the transdifferentiation of circulating plasmatocytes, released from the sessile compartment and proliferating upon immune challenge (Márkus et al., 2009; Honti et al., 2010; Stofanko et al., 2010; Vanha-Aho et al., 2015; Anderl et al., 2016). Second is a massive expansion of prohemocytes in the lymph gland followed by a differentiation to lamellocytes and their release due to the premature disintegration of the gland (Lanot et al., 2001, Sorrentino et al., 2002, Louradour et al., 2017; Cho et al., 2020). Wasp infestation hence provokes a biased commitment to the lamellocyte lineage at the expense of crystal cells, as the two lineages are mutually exclusive (Krzemien et al., 2010; Tattikota et al., 2020; Cho et al., 2020).

The processes underlying the Drosophila immune response to wasp parasitism are well-understood, sharing many molecular details with the inflammatory responses of vertebrates (reviewed in Letourneau et al., 2016; Kim-Jo et al., 2019; Banerjee et al., 2019; Kharrat et al., 2022). Interestingly, sterile injury of Drosophila larvae initiates a likewise immune response covering all aspects of wasp-mediated immune challenge (Evans et al., 2022). The epidermal penetration by the wasp ovipositor causes a burst of hydrogen peroxide at the injury site via the activation of the NADPH oxidase DUOX. The oxidative stress then induces a systemic activation of Toll/NF-κB and JNK-signaling within circulating hemocytes as well as within the cells of the posterior signaling center of the lymph gland. Following cytokine release, JAK/STAT and EGFR signalling pathways are activated non-cell autonomously driving the trans/differentiation of pro/hemocytes to lamellocyte fate and lymph gland dispersal (Nappi et al., 1995; Schlenke et al., 2007; Sinenko et al., 2011; Gueguen et al., 2013; Razzell et al., 2013; Louradour et al., 2017; Chakrabarti and Visweswariah, 2020; Evans et al., 2022; reviewed in Letourneau et al., 2016; Banerjee et al., 2019). Whereas the switch to lamellocyte fate is well elaborated, less is known about the processes underlying the simultaneous suppression of crystal cell fate. Obviously, this step requires a downregulation of Notch signalling activity, as the Notch pathway is instrumental to crystal cell fate in all the hematopoietic compartments (Duvic et al., 2002; Lebestky et al., 2003; Schlenke et al., 2007; Small et al., 2014; reviewed in Banerjee et al., 2019). Our work now reveals that a parasitoid wasp attack causes a phosphorylation of Su(H) on Ser269 as means of an efficient, rapid and reversible way to inhibit Notch activity. This idea is consistent with earlier observations, whereby wasp parasitism quenched the expression of a Su(H)-lacZ reporter (Small et al., 2014). Moreover, we provide evidence that Pkc53E is involved in Su(H) phosphorylation during blood cell development as well as during parasitoid wasp-defense in Drosophila. The antagonistic role of Pkc53E in blood cell homeostasis is fully consistent with earlier reports demonstrating that a loss of Pkc53E activity increased the expansion of prohemocytes in a genetic model of myeloid leukemia in Drosophila (Sinenko et al., 2010). Moreover, the involvement of PKCs in wasp defense is not completely unexpected given their well-documented role in the immune responses of mammals and Drosophila alike. For example, Drosophila flies ensure a healthy gut-microbiota homeostasis by modulating DUOX activity as a pathogen-specific defense line (reviewed in Kim and Lee, 2014). DUOX activation and ROS production, induced by gut pathogens was shown to be mediated by Pkc53E and Ca²⁺ via phospholipase Cβ (Ha et al., 2009; Lee et al., 2015). Sterile wounding of the Drosophila embryo triggers an instantaneous Ca²⁺ flash followed by hydrogen peroxide production (Razzel et al., 2013). It is conceivable that the epidermal breach by the wasp’s sting similarly induces a Ca²⁺ flash that may spark Pkc53E activation as a result. However, PKCs may be activated directly by oxidative modification even in the absence of Ca²⁺. Moreover, they may promote endogenous ROS production in a positive feed back loop (reviewed in Cosentino-Gomes et al., 2012), that could support the systemic response in the larval lymph gland.

The role of PKCs in the mammalian hematopoietic and immune systems is well documented. PKCs in this context act redundantly, and presumably, this holds also for Drosophila where several PKCs exist. Upon activation, PKCs may translocate into the nuclear compartment, where they can directly influence gene expression programs, for example by piloting chromatin factors as substrates, pivotal for regulating immune cell differentiation (reviewed in Lim et al., 2015). PKCs, including PKCα, are present in CD34+ long-term hematopoetic stem cells. Moreover, specific roles in both the myeloid and the multilymphoid lineages are known. Notably, some PKC isoforms including PKCα may play a role in Notch-dependent T-cell development. Notch pathway activity is indispensable for T-cell lineage commitment of early thymic progenitors, however, is rapidly downregulated after the β-selection phase. As treatment with phorbol ester upregulates the transcription of TCRα and β chains, a role of PKC in Notch-dependent commitment of αβ T-cells has been proposed. With the expressing of the invariant TCR components, fate commitment of T-cells driven by Notch signaling is completed. Subsequent cell survival and differentiation into CD4+CD8+ double positive cells, however, depends on the formation of a pre-TCR complex and requires PKCα activity (reviewed in Altman and Kong, 2016; Yui and Rothenberg, 2014). During initial T-cell development, Notch1 signaling increases in intensity in the pre-β-selected thymocytes due to an autoregulatory feedback loop directly controlled by E proteins and by Notch1-CSL itself. Following β-selection, as Notch signaling activity becomes dispensable, Notch1 is abruptly downregulated at the transcriptional level. This rapid downregulation is mediated by the inhibition of the E proteins (Yashiro-Ohtani et al., 2009), however, might in addition involve the phosphorylation of CSL by PKCα. Cell fate in more mature thymocytes is then fixed by the silencing of Notch target genes through chromatin modulators (reviewed in Yui and Rothenberg, 2014).

Appropriate silencing of Notch signaling activity in the course of T-cell development is of utmost importance, as prolonged Notch activity during β-selection predisposes the T-cells to leukemic transformation. Phosphorylation of CSL proteins by PKCα kinase offers a way for a rapid and reversible deactivation of Notch signals not only in Drosophila but also in the mammalian system. In fact, the amino acid sequences harboring the respective Serine residue in the CSL beta-trefoil domain are completely conserved between vertebrates and invertebrates (Wilson and Kovall, 2006; Nagel et al., 2017), raising the possibility of a likewise regulatory mechanism in mammalian hematopoiesis and immunity. Indeed, respective phosphorylation of the human CSL protein at the homologous position Ser195 was observed in human embryonic stem cells, where differentiation was induced by phorbol ester treatment (Rigbolt et al., 2011), consistent with an involvement of PKCα in this context as well. In conclusion, our work uncovers an important role for PKC-mediated downregulation of Notch activity via CSL-phosphorylation in blood cell homeostasis and in the immune response to parasitoid wasp infestation in Drosophila. Future work may uncover, whether the same mechanisms apply to mammalian hematopoiesis and immunity as well.

All data are contained within the manuscript.