Reactive oxygen species-dependent Toll/NF-κB activation in the Drosophila hematopoietic niche confers resistance to wasp parasitism

Abstract

Hematopoietic stem/progenitor cells in the adult mammalian bone marrow ensure blood cell renewal. Their cellular microenvironment, called 'niche', regulates hematopoiesis both under homeostatic and immune stress conditions. In the Drosophila hematopoietic organ, the lymph gland, the posterior signaling center (PSC) acts as a niche to regulate the hematopoietic response to immune stress such as wasp parasitism. This response relies on the differentiation of lamellocytes, a cryptic cell type, dedicated to pathogen encapsulation and killing. Here, we establish that Toll/NF-κB pathway activation in the PSC in response to wasp parasitism non-cell autonomously induces the lymph gland immune response. Our data further establish a regulatory network where co-activation of Toll/NF-κB and EGFR signaling by ROS levels in the PSC/niche controls lymph gland hematopoiesis under parasitism. Whether a similar regulatory network operates in mammals to control emergency hematopoiesis is an open question.

Keywords: D. melanogaster; Drosophila; Toll/NF-kB pathway; developmental biology; hematopoiesis; parasitism; stem cells.

Figure 1.. Dif-dependent Toll/NF-κB signaling is required for correct timing of lymph gland dispersal in response to wasp parasitism.

(A, B, D, E) Differential interference contrast (DIC) microscopy of lymph gland anterior lobes 30 hr post-parasitism. Lymph glands were classified into three groups (see Materials and methods): Disrupted (*) in wt (wild type, (A) or dl¹ mutant (E); intact in pll²/pll⁷ mutant (B); disrupting in Dif¹ mutant (D). Representative lymph glands are shown. Ring gland, RG; cardiac tube, CT; anterior lobe, AL; posterior lobe, PL. (C, F, G) Quantification of lymph gland disruption (%). The error bars correspond to SEM, ***p<0.001 and ns (not significant) (Pearson’s Chi-squared test). In this and subsequent similar analyses, numbers of lymph glands analyzed are indicated on histograms. (H, I) DIC microscopy of lymph glands in Dif¹ (H) and pll²/pll⁷ (I) mutants 48 hr post-parasitism. Disrupted (*) and disrupting (arrows) lymph glands are shown. (J–M) Perlecan/Trol (white) immunostaining in control (J, L) and Dif¹ anterior lobes (K, M). (N–S) αPS4 (green) immunostaining labels lamellocytes in lymph glands in wt (N, Q), Dif¹ (O, R) and pll²/pll⁷ (P, S) mutants. 20 hr post-parasitism, more than 65% (n = 24) of wt lymph glands displayed lamellocytes, whereas none (n = 24) or less than 15% (n = 18) contained lamellocytes in Dif¹ and pll²/pll⁷ mutants, respectively. 30 hr post-parasitism, Dif¹ and pll²/pll⁷ mutant lymph glands have lamellocytes, whereas most wt lymph glands have disrupted. In this and subsequent figures (unless specified), the scale bar represents 20 µm and nuclei are labeled with TOPRO3 or Draq5.

Figure 1—figure supplement 1.. Dif but not Dorsal is required for proper lymph gland disruption in response to wasp parasitism.

Quantification (%) of lymph gland disruption in (A): +/Df(2L)J4 (control), dl¹/Df(2L)J4 and Dif¹/Df(2L)J4 mutant larvae and (B) in the isogenic strains wt¹¹¹⁸ (control), dl¹ and Dif¹. Error bars correspond to SEM, *p<0.1, **p<0.01, ***p<0.001, ns (not significant) (Pearson’s Chi-squared test).

Figure 2.. Dif, lymph gland lamellocyte differentiation and lymph gland disruption are required for wasp egg neutralization.

(A, B) Brightfield images of control (col>, A) and Dif¹ mutant larvae 48 hr post parasitism (B). The arrow indicates a melanotic capsule. (C) Quantification (%) of wasp egg hatching in control (col>) and Dif¹ mutant. In this and subsequent similar analyses, numbers of (infected) larvae analyzed are indicated on histograms. (D–I) Phalloidin (red) staining of circulating hemocytes in control (hml>, D, E) and Dif¹ mutant (G, H) just prior (D, G) and following (E, H) lymph gland disruption. Arrows indicate discoidal-shaped lamellocytes that are larger than plasmatocytes. (F, I) Quantification (%) of circulating plasmatocytes and lamellocytes in control (F) and Dif¹ mutant (I). Blue, TOPRO3. (J) Quantification of lymph gland disruption (%) in control larvae (dome-MESO>) and when latran is down-regulated in lymph gland progenitors (dome-MESO > lat RNAi). (K) Quantification of wasp egg hatching in control larvae (dome-MESO>) or (antp>) and when latran is down-regulated in lymph gland progenitors (dome-MESO > lat RNAi) or in PSC cells (antp > lat RNAi). Error bars correspond to SEM, ***p<0.001 and ns (not significant) (Pearson’s Chi-squared test).

Figure 2—figure supplement 1.. Toll/NF-κB signaling is required for wasp egg encapsulation.

Quantification (%) of wasp egg hatching in isogenic strains wt¹¹¹⁸ (control), Dif¹, dl¹, Tl^rv1/Tl^r and pll²/pll²¹. Note high level of wasp egg hatching in the wt¹¹¹⁸ iso background. Error bars correspond to SEM, *p<0.1, **p<0.01, ***p<0.001 (Pearson’s Chi-squared test).

Figure 2—figure supplement 2.. At 18°C, lymph gland disruption and the presence of lamellocytes in circulation are delayed.

Circulating hemocytes stained by Phalloidin (red) and TOPRO3 (blue) in control larvae (hml>) 30 hr post-parasitism, prior to lymph gland disruption (A) and 42 hr post-parasitism following lymph gland disruption (B). Arrows indicate lamellocytes. Scale bars represents 0.1 µm.

Figure 3.. Dif-dependent Toll/NF-kB activation in PSC cells.

(A, B) Dif (red) immunostaining of col > mCD8 GFP (col > GFP, green) lymph glands without (A) and 6 hr post-parasitism (B). (A’, A’’, B’, B’’) enlarged views of the PSC. (C, D, F, G, I, J, K, L) LacZ (red) and Col (green) immunostaining of D4–LacZ of wt (C, D), pll²/pll⁷ (F, G), Dif¹ (I, J), and col > Dif RNAi (K, L) lymph glands without (C, F, I, K), and 6 hr post-parasitism (D, G, J, L). (C’, C'’, D’, D'’, F’, F'’, G’, G”, I’, I”, J’ and J”) enlarged views of the PSC. Arrows (K, L) indicate Col-positive PSC cells that do not express D4–LacZ. (E, H) Quantifications of D4-LacZ mean intensity in PSC cells. Error bars represent SDs, *p<0.1 and ns (not significant) (Mann-Whitney nonparametric test).

Figure 3—figure supplement 1.. Dif, Dorsal and Drosomycin-GFP expression in lymph gland cells.

(A and B) Efficient RNAi-mediated downregulation of Dif (red) in PSC cells (green, col > dif RNAi). (C and D) Immunodetection of Dorsal (red) in lymph gland cells under normal conditions (C) and 6 hr post-parasitism (D). Under both conditions, progenitors but not PSC cells express Dorsal. (E and F) Drosomycin-GFP (Drs-GFP, green) is not expressed in lymph gland cells under normal conditions (E) nor 6 hr post-parasitism (F). Nuclei are labeled with Draq5 (blue).

Figure 4.. Toll/NF-kB activation in PSC cells controls lymph gland dispersal, lamellocyte differentiation and wasp egg encapsulation.

(A) Quantification (%) of lymph gland disruption post parasitism in col> (control), col > Dif RNAi, col > Myd88 RNAi and col > pll RNAi lymph glands. (B–C) Representative confocal images of lamellocyte differentiation 20 hr post-parasitism (β Integrin, red) in control lymph glands (col > GFP) (B) and when Toll/NF-kB is downregulated in PSC cells (col > GFP > Myd88 RNAi) (C). At least 60% (n = 44) of lymph glands displayed numerous lamellocytes in the control, whereas less than 15% (n = 41) are observed in col > Myd88 RNAi lymph glands. Note early signs of lymph gland disruption in (B, arrow). (D) Quantification (%) of wasp egg hatching 48 hr post-parasitism in col> (control), col > Myd88 RNAi and col > Dif RNAi lymph glands. Error bars correspond to SEM, ***p<0.001 (Pearson’s Chi-squared test).

Figure 4—figure supplement 1.. Toll/NF-kB activation in PSC cells but not in lymph gland progenitors is required for proper lymph gland disruption in response to wasp parasitism.

Quantifications (%) of lymph gland disruption post-parasitism upon downregulation of Toll/NF-kB signaling in PSC cells (col>, A), (antp>, B) and lymph gland progenitors (dome>, C and D). Error bars correspond to SEM, ***p<0.001, ns (not significant) (Pearson’s Chi-squared test).

Figure 4—figure supplement 2.. Toll/NF-kB signaling is not required in lymph gland progenitors for lamellocyte differentiation.

Representative confocal images of β integrin (red) immunostaining of lamellocytes 20 hr post-parasitism in control lymph gland (dome>) (A) (n = 20) and when Toll/NF-kB is impaired in progenitors (dome > Myd88 RNAi) (B) (n = 12).

Figure 5.. Toll/NF-kB activation depends on SPE expression in PSC cells.

(A–C) Quantification (%) of lymph gland disruption post parasitism in spz^rm7 larvae (A) and when SPE is down-regulated in either PSC cells (col > SPE RNAi) (B) or lymph gland progenitors (dome > SPE RNAi) (C). Error bars correspond to SEM, ***p<0.001 and ns (not significant) (Pearson’s Chi-squared test). (D, E) LacZ (red) and Col immunostaining (green) of D4–LacZ expressing lymph glands 6 hr post-parasitism in wt (col>) (D) and when SPE is down-regulated in the PSC (white arrow) (col > SPE RNAi) (E). (F) Quantifications of D4-lacZ mean intensity in PSC cells 6 hr post-parasitism. Error bars represent SDs. **p<0.01 (Mann-Whitney nonparametric test).

Figure 5—figure supplement 1.. Psh is required for wasp egg neutralization and timely lymph gland disruption.

(A) Quantification (%) of wasp egg hatching in control (col>), modSP¹, psh¹ and double psh¹; modSP¹ mutants. (B) Quantification (%) of lymph gland disruption in control (col>) and psh¹ mutants. Error bars correspond to SEM, ***p<0.001, ns (not significant) (Pearson’s Chi-squared test).

Figure 6.. Wasp-mediated oxidative stress promotes Toll/NF-kB activation in the PSC and lymph gland disruption.

(A, B, E, F) LacZ (red) and Col (green) immunostaining in gstD-lacZ expressing lymph glands in wt context (col>; A, B), when Catalase is expressed in the PSC (col > catalase) (E) and when Toll/NF-kB is inactivated in PSC cells (col > cact > Dif RNAi)(F). (C, D, G, H) Quantifications of gstD-lacZ mean intensity in PSC and progenitor (MZ) cells. Error bars represent SDs. **p<0.01, ****p<0.0001 and ns (not significant) (Mann-Whitney nonparametric test). (I, J) LacZ (red) and Col (green) immunostaining in D4-LacZ expressing lymph glands in wt (col>) (I) and when an intracellular catalase is expressed in the PSC (col > catalase) (J). (K) Quantifications of D4-lacZ mean intensity in PSC cells. **p<0.01 and ns (not significant) (Mann-Whitney nonparametric test). (L–M) Quantifications of lymph gland disruption post parasitism. (N,O) Quantification (%) of wasp egg hatching. Error bars correspond to SEM, **p<0.01, ***p<0.001 (Pearson’s Chi-squared test).

Figure 6—figure supplement 1.. Duox- and Nox-independent generation of ROS in PSC cells 6 hr post-parasitism.

LacZ (green) and Antp (red) immunostaining in gstD-lacZ expressing lymph glands in control larvae (col>) (A), when Duox (col > Duox RNAi) (B) or Nox (col > Nox RNAi) (C) are downregulated in PSC cells, or when the intracellular catalase IRC is expressed in PSC cells (col > IRC) (D). In all contexts, similar levels of gstD-lacZ are observed in PSC cells (arrows). Reduced levels of gstD-lacZ are observed in progenitors of col > Duox RNAi lymph glands (B). Arrows and asterisks indicate the PSC and cardiac cells, respectively, marked with Antp.

Figure 6—figure supplement 2.. ROS are required both in PSC cells and lymph gland progenitors for lamellocyte differentiation.

β integrin (red) immunostaining of control lymph glands (col > GFP, A) or (dome>, C) and when catalase is expressed in the PSC (col > GFP > catalase, B) or in progenitors (dome > catalase, D) 20 hr post-parasitism. Whereas at least 60% (n = 44) of col > GFP lymph glands have numerous lamellocytes less than 10% (n = 45) displayed lamellocytes in col > catalase. Likewise, more than 70% (n = 7) of dome> control lymph glands have lamellocytes, whereas only 13% (n = 15) contained lamellocytes in dome > catalase.

Figure 6—figure supplement 3.. Scavenging ROS in PSC cells or silencing the EGFR pathway in MZ progenitors delays lymph gland disruption upon parasitism.

Differential interference contrast (DIC) microscopy of lymph glands 20 hr (A, B, D, E) and 48 hr (C, F) post parasitism in control (A, D) and when Catalase is expressed in the PSC (col > catalase) (B, C) or when the EGFR pathway is inactivated in lymph gland progenitors (dome-MESO > Egfr^DN) (E, F). Ring gland, RG; cardiac tube, CT; anterior lobe, AL; posterior lobe, PL; disrupted (*) and disrupting (arrow in F) anterior lobes are shown. The scale bar represents 20 µm. All source data are supplied as Excel files.

Figure 7.. Epistatic relationships between EGFR/Erk and Toll/NF-κB signaling.

(A, B) p-ERK (red) immunostaining in col > GFP (PSC, green) larvae. (A’, A”, B’, B”), enlarged views of PSC cells. (C, D) Quantifications of lymph gland disruption post parasitism. (E, F) p-ERK (red) immunostaining in wt (E) and Dif¹ (F) mutant lymph glands. (G) Quantifications (%) of lymph gland disruption post parasitism. Error bars correspond to SEM, ***p<0.001, ns (not significant) (Pearson’s Chi-squared test).

Figure 8.. Proposed gene regulatory network that controls lymph gland rupture upon wasp parasitism.

The PSC is drawn in grey. Wasp parasitism increases ROS in PSC cells that activate Toll/NF-κB and Spitz secretion (sSpi). Toll/NF-kB activation in PSC cells requires SPE in the same cells for Spätzle processing (c-Spz). sSpi non cell-autonomously activates the EGFR pathway in lymph gland progenitors. Both EGFR and Toll/NF-κB activation are required for lymph gland lamellocyte differentiation, lymph gland disruption and wasp egg encapsulation.