A short receptor downregulates JAK/STAT signalling to control the Drosophila cellular immune response

Abstract

The posterior signalling centre (PSC), a small group of specialised cells, controls hemocyte (blood cell) homeostasis in the Drosophila larval hematopoietic organ, the lymph gland. This role of the PSC is very reminiscent of the "niche," the micro-environment of hematopoietic stem cells in vertebrates. We have recently shown that the PSC acts in a non-cell-autonomous manner to maintain janus tyrosine kinase/signal transducers and activators of transcription (JAK/STAT) signalling in hematopoietic progenitors (prohemocytes), thereby preserving the multipotent character necessary for their differentiation into lamellocytes, a cryptic and dedicated immune cell type required to fight specific immune threats such as wasp parasitism. In this report, on the basis of a knock out generated by homologous recombination, we show that a short type I cytokine-related receptor CG14225/Latran is required for switching off JAK/STAT signalling in prohemocytes. This is a prerequisite to massive differentiation of lamellocytes upon wasp parasitisation. In vivo and cell culture assays indicate that Latran forms heteromers with Domeless, the Drosophila type I cytokine signalling receptor related to mammalian GP130, and antagonises Domeless activity in a dose-dependent manner. Our analysis further shows that a primary immune response to wasp parasitism is a strong decrease in cytokine mRNA levels in the lymph gland, followed by an increase in the latran/domeless ratio. We propose that this sequence of events culminates in the complete inhibition of residual JAK/STAT signalling by Latran. JAK/STAT activity has been associated with several human diseases including leukaemia while knock-out studies in mice point to a central role of this pathway in hematopoiesis and regulation of immune functions. The specific function of Drosophila Latran is, to our knowledge, the first in vivo example of a role for a nonsignalling receptor in controlling a dedicated immune response, and thus raises the question of whether short, nonsignalling receptors also control specific aspects of vertebrate cellular immunity.

Figure 1. Latran: homology with JAK/STAT receptors and expression in the LG.

(A) Schematic of the D. melanogaster dome-lat genomic region. The ORFs and untranslated 5′ and 3′ regions are indicated by black and grey boxes, respectively. Positions of the PG125/domeGal4 P-element insertion and the lat genomic fragment that was deleted to generate a null allele are indicated by an arrowhead and a red bar, respectively. (B) Schematic alignment of the Lat, Dome, and human GP130 proteins. The green box corresponds to the CBM and the blue box to a conserved region between Lat and Dome LDHR; the percentage of sequence identity is given. Fibronectin III (Fn III) motifs are indicated in red, the signal peptide in yellow. TM indicates the transmembrane domain. The intracytoplasmic regions are in grey, the position of the STAT binding site in orange. (C–E). Overlapping expression of lat (ISH red) (C), dome>GFP (GFP, light green) and Col (Ab, bright green, arrows) (D), and triple overlap (E) in LGs of a third instar larva. Nuclei are highlighted in blue (TOPRO-3); anterior is to the left. Scale bar, 80 µm. (F) Schematic of an anterior lobe of the LG.

Figure 2. lat mutant LGs do not properly respond to wasp parasitisation.

(A–C) DAPI staining of circulating hemocytes from (A) wt, (B) lat mutant, and (C) lat mutant larvae expressing lat in the LG (srp>lat). 48 h after parasitisation, lamellocytes are found in large numbers in the hemolymph of wt larvae (A, insert) but lacking in lat mutants. This lack-of-lamellocyte phenotype is rescued by expressing lat in the LG (C, arrows). (D–I) Prohemocytes, marked by dome-MESO expression (green) are not maintained in wt LGs following wasp parasitisation (D and E); this is paralleled by massive lamellocyte differentiation (integrin α chain [α-PS4], red) (E, insert). In lat mutant larvae, dome-MESO remains expressed after parasitisation (F, G); few lamellocytes differentiate (G, insert). (H–J) Stat-GFP (green) subcellular localisation in wt (H and I) and lat mutant LG (J), either without (H) or 4–6 h after parasitism (I and J). srp-Gal4 was used to drive UAS-Stat-GFP in the LG (srp>Stat-GFP). Stat-GFP localisation is nuclear in nonparasitised wt and parasitised lat LG, whereas it is both cytoplasmic and nuclear in parasitised wt LG. The scattered distribution of GFP-labelled cells is due to nonuniform activation of the srp-Gal4 driver in the LG . Nuclei (TOPRO-3) are in blue. Scale bars, 50 µm (A); 80 µm (D).

Figure 3. Lat acts as a dominant-negative JAK/STAT receptor.

(A–D) dome-Gal4 driven expression of lat in the MZ switches off JAK/STAT signalling, as monitored by dome-MESO expression (LacZ, green). proPO (red, A and B) and P1 stainings (red, C and D) indicate differentiating crystal cells and plasmatocytes, respectively. (E) Drosophila S2-NP cells were transfected with 10×STAT92E-luciferase, Act-Renilla, and Act-upd, and various amounts of Act-dome and Act-lat plasmids (indicated in nanograms). Luciferase assays were performed 4 d later and the reporter activity normalised as the ratio of firefly luciferase/Renilla. The results are from three independent experiments. Vertical bars correspond to SD. (F–G) Immunodetection of Dome-V5 (green) and HA-Lat (red) in Drosophila S2-NP cells, in conditions where the JAK/STAT pathway is either on (F) or off (G) (MM and Figure 3E). HA-Lat and Dome-V5 colocalise in trafficking cytoplasmic vesicles in both conditions. (G) Shows a phase-contrast view of a transfected cell. Scale bars, 80 µm (A); 8 µm (G).

Figure 4. Lat forms heterodimers with Dome in the LG.

(A) Immunoprecipitation of the Lat/Dome protein complex. Lysates from either control S2-NP cells or cells transfected with pAc-DomeV5 and pAc-HALat were immunoprecipitated with either anti-V5 or anti-HA antibodies as indicated above each lane and subjected to Western blot analysis. Both conditions indicate the formation of Dome/Lat protein complexes. (B, D–E) Formation of Dome homodimers, Lat homodimers, and Lat/Dome heterodimers in (B) da-Gal4xUAS-DomeΔα/UAS-DomeΔω, (D) da-Gal4; UAS-LatΔα/UAS-LatΔω, and (E) da-Gal4; UAS-LatΔα/UAS-DomeΔω embryos, respectively, as visualised by X-Gal staining (blue). In stage 13 embryos, strong staining is observed in the salivary glands (black arrow) and the hindgut (black arrowhead). (C) da-Gal4; UAS-LatΔα ubiquitous expression of LatΔα detected by LacZ immunostaining. (F) Formation of Dome/Lat heterodimers in the MZ of the LG. Scale bars, 40 µm (F).

Figure 5. Downregulation of upd3 and dome transcripts in response to wasp infestation.

Quantitative analysis of dome, lat, and upd3 relative to rp49 transcripts is given in either (A) wt or (B) lat mutant LGs. A significant decrease of dome (p = 5×10⁻⁵) and increase of lat (p = 1.5×10⁻¹⁶) transcripts is observed 4 h after wasp egg-laying. The level of upd3 transcripts drops to almost undetectable. This decrease of upd3 mRNA is also observed in lat mutant LGs (B). (C) αPS4 (lamellocytes, black) immunostaining of wt, lat mutant, and lat,srp>dsRNA-hop LGs 30 h following wasp infestation. Massive lamellocyte differentiation is observed in lat;srp>dsRNA-hop mutant LGs similar to wt. (D) dome-Gal4> upd3dsRNA-induced degradation of upd3 transcripts in the LG leads to a significant decrease of dome (p = 1×10⁻⁸) transcripts whereas the level of lat (p = 0.2) is not affected. rp49 and rpL17 mRNAs were used as internal controls (unpublished data). Three independent experiments were performed, vertical bars correspond to SD.

Figure 6. upd3 is expressed and required to maintain JAK/STAT signalling in prohemocytes.

(A) upd3 expression (white and red, left and right panels, respectively) overlaps that of dome > GFP (green, middle and right panels) in LGs of third instar larvae. (B) upd3 (red, top panel) is also expressed in the PSC (pcol>GFP, green, middle panel), overlay at the bottom. (C and D) dome-Gal4 driven expression of upd3 dsRNA leads to loss of dome-MESO expression (LacZ, green) in the MZ. proPO staining (red) indicates differentiating crystal cells. Nuclei (TOPRO-3) are in blue. Scale bars, 40 µm (A); 60 µm (B).

Figure 7. Model for lat function in Drosophila larval hematopoiesis.

During normal development (left panel), PSC cells (orange) act, in a non–cell-autonomous manner (arrow) to maintain JAK/STAT signalling activity and preserve a pool of multipotent prohemocytes in the MZ (green shades). The PSC signal overrides lat function in the MZ (grey shades). In response to parasitisation (middle panel), there is a decrease of upd3 and dome and increase of lat transcripts, which ultimately lead to an increased lat/dome ratio. The PSC signal is short-circuited. As a result, JAK/STAT signalling is switched off, thus licensing prohemocytes to differentiate into lamellocytes. Lat activity is strictly required in the LG for this switch. In the absence of lat (right panel), residual upd3 levels maintain JAK/STAT activity, therefore preserving a pool of prohemocytes (grey shades). Upon wasp parasitisation some differentiating hemocytes become lamellocytes, however, indicating that lat is not required for this differentiation program per se. Arrows indicate activation, vertical bars repression.