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cGMP signaling pathway that modulates NF-κB activation in innate immune responses

Summary The nuclear factor-kappa B (NF-κB) pathway is an evolutionarily conserved signaling pathway that plays a central role in immune responses and inflammation. Here, we show that Drosophila NF-κB signaling is activated via a pathway in parallel with the Toll receptor by receptor-type guanylate cyclase, Gyc76C. Gyc76C produces cyclic guanosine monophosphate (cGMP) and modulates NF-κB signaling through the downstream Tollreceptor components dMyd88, Pelle, Tube, and Dif/Dorsal (NF-κB). The cGMP signaling pathway comprises a membrane-localized cGMP-dependent protein kinase (cGK) called DG2 and protein phosphatase 2A (PP2A) and is crucial for host survival against Gram-positive bacterial infections in Drosophila. A membrane-bound cGK, PRKG2, also modulates NF-κB activation via PP2A in human cells, indicating that modulation of NF-κB activation in innate immunity by the cGMP signaling pathway is evolutionarily conserved.

Guanylate cyclase (GC) converts guanosine triphosphate (GTP) into cGMP, which acts as a second messenger to regulate a wide range of physiologic processes (Feil and Kemp-Harper, 2006). GCs consist of soluble (Montfort et al., 2017) and receptor-type (r)GCs (Kuhn, 2016) and are regulated by diverse extracellular ligands, such as peptide hormones, bacterial toxins, and free radicals, as well as by calcium and adenine nucleotides. Once GCs are activated, cGMP accumulates in the cytoplasm and initiates signaling cascades via downstream effectors, such as cGMP-dependent protein kinase (cGK), cGMP-regulated phosphodiesterases (PDE), and cyclic nucleotide-gated ion channels (Cng) (Tsai and Kass, 2009). cGMP signaling, which is evolutionarily conserved, is involved in renal homeostasis (Davies, 2006), behavior (Morton, 2011;Reaume and Sokolowski, 2009), development (Patel et al., 2012), and stress tolerance (Davies et al., 2014) in mammals and Drosophila. rGCs also function in inflammation and immune cell differentiation and regulation (Zhang et al., 2015). Despite the widespread roles of cGMP and rGCs, however, it is unclear whether rGC-dependent signaling and NF-kB signaling interact in the context of immune regulation.
Here, we report the existence of a cGMP-dependent signaling pathway that modulates NF-kB activation in Drosophila immune responses. This pathway was discovered through the identification of Gyc76C, an rGC that functions as an immune receptor. This Gyc76C-induced cGMP signaling pathway is mediated by the membrane-localized cGK called DG2 and by protein phosphatase 2A (PP2A). Our findings also show that Gyc76C and dg2 (foraging) are crucial for host survival against Gram-positive bacterial infections in Drosophila. We also show that the membrane-bound cGK PRKG2 modulates NF-kB activation via PP2A in human cells as well, indicating an evolutionarily conserved role for this cGMP signaling pathway in modulating NF-kB activation in innate immunity.

RESULTS
A receptor guanylate cyclase, Gyc76C, activates NF-kB signaling (the Toll pathway) in parallel with the Toll receptor Our previously reported genome-wide gain-of-function genetic screen based on modular misexpression using GAL4/UAS in Drosophila was used to identify immune-response activating genes Takehana et al., 2002;Yano et al., 2008). Here, we expanded this earlier screen with 11,761 additional lines and identified 8 lines that constitutively express two AMP reporter genes, Drosomycin (Drs)-GFP and Diptericin (Dpt)-lacZ, which monitor Toll and IMD pathway activation, respectively (Jung et al., 2001), in a GAL4-dependent manner. The GAL4/UAS system revealed the upregulated expression of CG3221, CG3216, and CG10543, which lie adjacent to a single UAS vector insertion in one of these eight lines, GS 2198 (Figures S1A and S1B). These three genes encode dim g-tubulin 3, a putative rGC, and a putative zinc finger nucleic-acid-binding factor, respectively. We investigated the role of CG3216 in immune responses, as CG3216 encodes a putative receptor with an extracellular ligand-binding domain, a transmembrane domain, as well as intracellular kinase homology and guanylate cyclase domains; it also has amino acid sequence similarity with mammalian rGCs that produce cGMP, such as the atrial natriuretic peptide receptor (Davies, 2006).
Overexpression of CG3216 in the fat body using the c564-GAL4 driver and in epithelial tissues such as the trachea using the NP2610-GAL4 driver resulted in the constitutive expression of Drs-GFP and Dpt-lacZ in the larval fat body and epithelia, respectively ( Figure S1C). Quantitative RT-PCR analyses revealed that CG3216 overexpression induces strong Drs expression and weak Dpt expression compared with the overexpression of positive regulators of the iScience Article (Lemaitre et al., 1996;Weber et al., 2003) and the IMD pathway (PGRP-LC, an IMD pathway receptor) (Gottar et al., 2002), indicating that CG3216 overexpression preferentially activates the Toll pathway (Figure S1D). The rGC gene family in Drosophila includes CG3216, Gyc76C, Gyc32E, and CG31183 (FlyBase data; http://flybase.org). Our analysis of Drosophila rGC expression in wild-type larvae revealed little endogenous expression of CG3216 ( Figure S1E) but high ( Figure S1E), preferential expression of Gyc76C in immune-related tissues such as the fat body, hemocytes, and Malpighian (renal) tubules (Figure S1F, and FlyAtlas data; http://flyatlas.org/). Gyc76C also more effectively induced Drs expression than CG3216 ( Figure S1G). Gyc76C overexpression also induced expression of Drs-GFP in larvae (Figure S1H). Gyc76C has known roles in neuronal pathfinding and development (Ayoob et al., 2004;Chak and Kolodkin, 2014) in muscle (Patel et al., 2012), epithelia (Myat and Patel, 2016), and wing (Schleede and Blair, 2015); salt stress response (Overend et al., 2012); and gut immunocompetence (Bou Sleiman et al., 2015). The present findings indicate that Gyc76C also has a role in the immune response.
Because Gyc76C overexpression preferentially induces the expression of Drs relative to Dpt ( Figure 1A), we examined the relationship between Gyc76C and Toll pathway components by epistatic analysis in larvae. The Gyc76C-dependent induction of Drs was not affected by mutations of Toll, spz, or imd but was significantly suppressed by mutations of downstream components of the Toll pathway, Tube, Pelle, dMyd88, and Dif/Dorsal (NF-kB factors in the Toll pathway) ( Figure 1B). In reciprocal experiments, processed Spz-dependent induction of Drs was not affected by a hypomorphic mutation caused by a P-element insertion in the first intron of Gyc76C (gyc76C KG03723 ) (Ayoob et al., 2004) ( Figure 1B). The imd-, PGRP-LC-, and PGRP-LEdependent induction of Dpt and Drs was not affected in the presence of gyc76C KG03723 ( Figure S2A). Together, these findings indicate that Gyc76C induces Drs expression via downstream Toll receptor components in a Toll-receptor-independent manner. Consistent with these results, co-expression of processed Spz and Gyc76C led to a strong synergistic induction of Drs ( Figure 1C). Epistasis in adults was not analyzed because Gyc76C expression in the fat body, as driven by c564-GAL4 ( Figure S2B), and in the fat body and hemocytes, as driven by Cg-GAL4 (data not shown), failed to induce Drs when using a temperature-sensitive GAL80 that avoids the pupal lethality caused by Gyc76C expression. Consistent with these results, induction of Gyc76C mRNA expression and cGMP production by Gyc76C overexpression induced by c564-GAL4 was low in adults in comparison with larvae ( Figures S2C and S2D). The production of cGMP is required for Gyc76C-dependent induction of Drs, as described below.
All rGC family members have a highly conserved cyclase domain that converts GTP to cGMP (Davies, 2006). Gyc76C overexpression induces Drs expression and cGMP production, and both constitutive Drs expression and cGMP production were severely reduced by a Gyc76C point mutation at amino acid 945 (Gyc76C D945A ) that disrupts cyclase activity (Ayoob et al., 2004) ( Figure 1D). Gyc76C-dependent Drs induction was also severely reduced by the co-expression of Drosophila PDE-5/6, a cGMP-specific PDE that catalyzes the hydrolysis of cGMP to inactive 5 0 nucleotides (Day et al., 2005) ( Figure 1E). As expected, PDE5/6 abolished the Gyc76C-induced increase in cGMP but did not affect the forced expression of Gyc76C driven by c564-GAL4 ( Figures 1E and S2E). cGMP production was not induced by Toll pathway activation via processed Spz and activated Toll (Toll 10B ) or by IMD pathway activation via imd and PGRP-LC ( Figure S2F). Consistently, the expression of PDE5/6, which reduces cGMP levels, did not suppress the processed Spz-dependent induction of Drs or the PGRP-LC-dependent induction of Dpt ( Figure S2G). Together, these findings demonstrate that a cGMP-mediated signaling pathway leads to AMP induction via downstream Toll receptor components that function in parallel with the Toll receptor.
Gyc76C mediates ModSP-dependent and psh-dependent NF-kB signaling activation in cooperation with the Toll receptor As previously reported (Buchon et al., 2009), Drs is induced by the overexpression of ModSP, an upstream regulator of the Toll receptor ( Figure 2A). The ModSP-dependent induction of Drs was stronger than that induced in response to the expression of Gyc76C, Toll 10B , or processed Spz ( Figure 2A) but was suppressed by mutations of gyc76C KG03723 and Toll 1-RXA/r632 ( Figure 2B). Thus, the ModSP-dependent induction of Drs requires Gyc76C. This requirement was confirmed by a transheterozygote of gyc76C KG03723 and Df(3L) Exel9061 ( Figure 2B). Forced expression of ModSP via c564-GAL4 was not affected by these mutations (Figure S2H). These findings indicate that both Gyc76C and the Toll receptor are required for ModSP-dependent induction of Drs, consistent with the synergistic induction of Drs by both cGMP pathway and Toll receptor activation resulting from the co-expression of Gyc76C and processed Spz ( Figure 1C). GNBP-1 acts as a recognition protein that activates ModSP. GNBP-1-dependent induction of Drs was also suppressed ll

OPEN ACCESS
iScience 24, 103473, December 17, 2021 3 iScience Article by the gyc76C KG03723 mutation, providing further support that Gyc76C mediates ModSP-dependent NF-kB activation ( Figure 2C). To investigate whether Gyc76C ligand activity is induced in larvae by ModSP overexpression, we used the Drs reporter (Drs-luciferase) in Drosophila DL1 cells in which Gyc76C is expressed ( Figure S2J). The Drs expression was induced by treating DL1 cells with an acid (0.1% trifluoroacetic acid in H 2 O) extract of the homogenate of larvae expressing lacZ ( Figure S2I). The Drs-inducing activity in the larval extract was increased by ModSP overexpression, which was reduced by RNAi targeting Gyc76C in DL1 cells ( Figure S2I). Consistent with the partial reduction of Drs-inducing activity by Gyc76C RNAi, Gyc76C mRNA iScience Article expression was partially reduced to 50% in DL1 cells by Gyc76C RNAi ( Figure S2J). These results suggest that the Gyc76C ligand exists in larvae overexpressing ModSP. The Gyc76C ligand activity was both acid-(0.1% trifluoroacetic acid) and heat-(100 C, 1 min) stable and was retained on the ultrafiltration membrane with a molecular weight cut-off of 10,000 (Millipore, Amicon Ultra-4 10K), suggesting that the Gyc76C ligand is an acid-and heat-stable molecule with a molecular weight greater than 10 kD (Table S1). The Toll pathway is activated by some bacterial and fungal proteases through Psh, independent of ModSP. As previously reported (Jang et al., 2006), Drs is induced by overexpression of Psh ( Figure 2D). The Pshdependent induction of Drs was suppressed by the gyc76C KG03723 mutation ( Figure 2D), suggesting that Gyc76C also mediates Psh-dependent NF-kB activation.
cGMP levels were also increased by ModSP overexpression ( Figure 2E), a response that was suppressed by the gyc76C KG03723 , but not the Toll 1-RXA/r632 , mutation ( Figure 2E). Thus, Gyc76C is required for the ModSPdependent increase in cGMP. This requirement was confirmed by analyzing the transheterozygote of gyc76C KG03723 and Df(3L)Exel9061 ( Figure 2E). Drs was induced by Toll 10B expression and processed Spz expression ( Figure 2A), whereas cGMP was not ( Figures 2F and S2F). These results indicate that NF-kB signaling activation is induced by the independent activation of Gyc76C and the Toll receptor through the forced expression of ModSP. GNBP-1-dependent and Psh-dependent Drs induction was reduced by co-expression with PDE-5/6, which reduces cGMP levels, confirming that cGMP production is required for GNBP-1-dependent and Psh-dependent Drs induction ( Figures 2C and 2D).

Roles of the cGMP pathway and Gyc76C in resistance to Gram-positive bacterial infections
We next performed infection experiments to evaluate the role of Gyc76C and cGMP signaling in host defense against various pathogens. Because the Gyc76C null mutant is lethal (Ayoob et al., 2004), we used hypomorphic Gyc76C mutant flies, gyc76C KG03723 , in which endogenous Gyc76C mRNA expression is reduced to 35% than in yw control flies ( Figure S3A). In contrast to wild-type (Oregon-R) and w 1118 flies, gyc76C KG03723 flies were susceptible to Gram-positive bacterial infections (Staphylococcus saprophyticus, Enterococcus faecalis, and Staphylococcus aureus), but not to Gram-negative bacterial infections (Erwinia carotovora carotovora 15 [Ecc15] and Escherichia coli), indicating an important role for Gyc76C in self-defense against Gram-positive bacteria ( Figure 3A). This finding is consistent with the activation of Gyc76c by ModSP, which itself is activated by the recognition of Gram-positive bacteria (Buchon et al., 2009). Notably, in revertant flies in which the P element was precisely excised from the gyc76C KG03723 genome, the S. saprophyticus-susceptible phenotype was restored, ruling out the possibility of genetic background effects ( Figure 3A). The gyc76C KG03723 flies accumulated significantly higher Gram-positive bacterial loads ( Figure S3B), but not Gram-negative bacterial loads ( Figure S3C), in their hemolymph relative to control yw flies. Susceptibility to S. saprophyticus infection was also induced by Cg-GAL4-mediated RNAi knockdown of Gyc76C in the fat body and hemocytes but not in lacZ-expressing control flies ( Figures 3B and  S3A). Susceptibility to Gram-positive bacterial infection was also more severe in flies in which both Gyc76C and Toll were knocked down by RNAi, relative to the susceptibility induced by RNAi-mediated targeting of Toll alone ( Figure 3B). Expression of PDE5/6 by Cg-GAL4 also induced susceptibility to Grampositive bacterial infections, suggesting an important role for cGMP signaling in self-defense against Gram-positive bacteria ( Figure 3C). A role for Gyc76C and cGMP signaling in host defense against Gram-positive bacteria was confirmed by larval infection experiments. In contrast to yw larvae, gyc76C KG03723 larvae were susceptible to S. saprophyticus but not to Ecc15 infection ( Figure S4A). iScience Article Expression of Gyc76C RNAi and of PDE5/6 by c564-GAL4 also induced larval susceptibility to S. saprophyticus infections ( Figures S4B and S4C). Susceptibility of gyc76C KG03723 larvae to S. saprophyticus was completely rescued by c564-GAL4-dependent expression of Gyc76C, but not Gyc76C D945A , which failed to produce cGMP ( Figures S4D and 1D).
Interestingly, as previously reported (Lemaitre et al., 1996), the spz rm7 mutant was susceptible to natural fungal infection (Beauveria bassiana), whereas the gyc76C KG03723 mutant was not, indicating a distinct role for Spz/Toll and Gyc76C in antifungal defense in Drosophila ( Figure 3A). In addition to the natural infection experiments, we investigated the role of Gyc76C in host defense against fungal infection by injection in the same manner as for bacterial infection. Double mutant adults and larvae of modSP and iScience Article psh were susceptible to Candida glabrata injection, whereas gyc76C KG03723 mutant adults and larvae were not, indicating that Gyc76C-mediated NF-kB activation does not have a significant role in host defense against fungi, unlike its role in host defense against Gram-positive bacteria ( Figures S4E and S4F).

Gyc76C is involved in inducing numerous genes against Gram-positive bacterial infections
To investigate the effect of the Gyc76C mutation on AMP induction, we compared AMP induction in gyc76C KG03723 and Gyc76C-revertant flies following Gram-positive and Gram-negative bacterial infections.
Drs induction in response to S. saprophyticus infection was partially reduced in gyc76C KG03723 flies relative to that in spz rm7 flies, whereas the induction of Defensin (Def), an AMP that acts on Gram-positive bacteria, was reduced in both gyc76C KG03723 and spz rm7 flies following S. saprophyticus infection (Figure 4). The induction of Dpt, Attacin, and Drosocin was also reduced in gyc76C KG03723 flies after S. saprophyticus infection ( Figure 4). Induction of Def in response to Ecc15 infection was also reduced in gyc76C KG03723 , but, compared with Relish E20 , significant induction of the tested AMPs was observed in gyc76C KG03723 in response to Ecc15 infection ( Figure 4). DNA microarray analysis also revealed that the induction of 139 other genes was reduced in gyc76C KG03723 flies compared with controls following S. saprophyticus infection, including 9 genes that encode innate immune-related factors, mainly AMPs: 19 serine protease genes; 8 metallopeptidase genes; 5 polysaccharide-binding protein genes; and 4 metal-ion-binding protein genes ( Figures S5A, S5B, and Table S1, for details see http://www.pharm.tohoku.ac.jp/seimei/m_array/ index.htm). Many of these genes were upregulated by Gyc76C overexpression in larvae, although they were suppressed in response to S. saprophyticus infection in spz rm7 flies (Table S1). DNA microarray analysis of gyc76C KG03723 larvae also confirmed that Gyc76C is involved in the induction of numerous genes, including innate immune-related genes, serine protease genes, and polysaccharide-binding protein genes, in response to Gram-positive bacterial infections in larvae (Table S1 and Figure S5C). Many of these genes were upregulated by Gyc76C overexpression in larvae and were suppressed in response to S. saprophyticus infection in Dif/Dorsal (Drosophila NF-kB) mutant larvae and in psh;modSP double mutant larvae (Table S1). Together, these findings demonstrate that Gyc76C is involved in the induction of numerous genes in response to Gram-positive bacterial infections.
The cGMP signaling pathway is mediated by the membrane-localized cGMP-dependent protein kinase DG2 and protein phosphatase 2A cGK and Cng are candidate downstream mediators of cGMP signaling (Davies, 2006). We therefore investigated the effect of their loss on Gyc76C-dependent induction of Drs by RNAi-mediated knockdown of the Drosophila cGKs dg2 (foraging), dg1 (Pkg21D), and CG4839 (Pkg-like) and two Drosophila Cngs, Cng and Cng-like. When dg2 was knocked down in the fat body via c564-GAL4, Gyc76C-dependent induction of Drs was inhibited ( Figure 5A). In contrast, RNAi-mediated knockdown of Cng, Cng-like, and CG4839 in the fat body did not inhibit Gyc76C-dependent induction of Drs, whereas dg1 RNAi induced embryonic lethality (data not shown). These results indicate that DG2, a membrane-localized cGK (Davies, 2006), mediates cGMP signaling, thus activating immune responses mediated by Gyc76C. RNAi-mediated knockdown of dg2 by Cg-GAL4 in flies and by c564-GAL4 in larvae induced susceptibility to S. saprophyticus infection but not to Ecc15 infection ( Figures 5B and S4B). Susceptibility to S. saprophyticus infection was also induced by adult-stage-specific expression of dg2 RNAi via c564-GAL4 using a temperature-sensitive GAL80, indicating that the S. saprophyticus-susceptible phenotype was not caused by developmental defects in the fat body ( Figure S4G). These findings indicate that immune responses mediated by DG2 are crucial for self-defense against Gram-positive bacteria in Drosophila flies and larvae.
Consistent with the synergistic induction of Drs by co-overexpression of Gyc76C and processed Spz in larvae ( Figure 1C), Drs was synergistically induced by co-overexpression of one of two isoforms of dg2 (MacPherson et al., 2004), P1 or P2, and processed Spz in larvae ( Figure 5C). Drs and a Drs reporter gene (Drs-luciferase) were similarly synergistically induced by co-overexpression in Drosophila DL1 cells of dg2 and dMyd88 and that of dg2 and an active Toll receptor (delta leucine-rich repeat) ( Figures 5D  and 5E). Drs-luciferase activation by the co-expression of dg2 and dMyd88 in DL1 cells was enhanced by Gyc76C, indicating that DG2 cooperates with Gyc76C to enhance dMyd88-dependent Dif/Dorsal iScience Article (Drosophila NF-kB) activation ( Figure 5F). The enhanced activation of dMyd88-dependent Dif/Dorsal by dg2 was abolished by point mutations in DG2 at amino acid 555 or 573 that disrupt cGMP-dependent kinase activity ( Figure 5G), indicating that this DG2 kinase activity is required to enhance dMyd88-dependent Dif/Dorsal activation. Consistent with functional interactions of dMyd88 and DG2, co-immune precipitation results revealed that dMyd88 forms a complex with DG2 in DL1 cells ( Figure 5H).
dMyd88 contains a consensus sequence for phosphorylation by cGK (RRXS/T) (Wong et al., 2012). Enhanced dMyd88-dependent Dif/Dorsal activation by DG2 was not reduced by a point mutation in this consensus sequence at amino acid 461 (S461A; Figure S6A). Moreover, a point mutation at amino acid 461 to glutamic acid (S461E) did not enhance dMyd88-dependent Dif/Dorsal activation ( Figure S6A). These findings indicate that dMyd88 consensus sequence phosphorylation does not contribute to the dMyd88dependent enhanced activation of Dif/Dorsal by DG2. In addition to dMyd88, Pelle-dependent Dif/Dorsal activation was also enhanced by DG2 ( Figure 5I). The dMyd88-dependent enhanced activation of Dif/Dorsal by DG2 was suppressed by RNAi knockdown of dMyd88, tube, and pelle ( Figure S6B). On the other hand, enhanced Pelle-dependent activation of Dif/Dorsal by DG2 was suppressed by pelle RNAi, but not by dMyd88 and tube RNAi, indicating that dMyd88 and tube are not required for DG2-induced enhancement of Pelle-dependent Dif/Dorsal activation ( Figure S6C). Pelle does not contain a consensus sequence for cGK phosphorylation. These findings suggest that additional components exist between DG2 and Pelle in cGMP signaling.
To identify the genes involved in the modulation of NF-kB activation by DG2, we performed a genomewide RNAi screen in DL1 cultured cells. The results revealed that the enhanced activation of dMyd88dependent Dif/Dorsal by Gyc76C and DG2 was suppressed by RNAi-mediated knockdown of pelle and Dif ( Figure S6D). We compared the results of this screen with those of a similar RNAi screen of dMyd88dependent Dif/Dorsal activation (Kanoh et al., 2015) and identified microtubule star (mts, Drosophila PP2A catalytic subunit), Pp2A-29B (Drosophila PP2A regulatory subunit A), twins (tws, Drosophila PP2A regulatory subunit B), and Pp4-19C (Drosophila protein phosphatase 4 catalytic subunit), in addition to 22 other genes ( Figures S6E and S6F). We investigated the effects of knocking down the Drosophila PP2A subunits tws, mts, and Pp2A-29B on the Gyc76C-dependent induction of Drs in larvae. The findings demonstrated that, similarly to when dg2 was knocked down in the fat body by c564-GAL4 using a temperature-sensitive GAL80, loss of the Drosophila PP2A subunits inhibited the Gyc76C-dependent induction of Drs ( Figure 5J). These findings indicate that both DG2 and PP2A modulate NF-kB activation by the cGMP pathway in Drosophila.

cGMP signaling modulation of NF-kB activation is conserved
Seven mouse and human rGC homologs, including the atrial natriuretic peptide receptor, are reported, but only one membrane-bound cGK (cGK II) is reported (Davies, 2006). Expression of the human cGK II, PRKG2, enhanced the MyD88-dependent activation of NF-kB signaling in human HEK293 cells, as well as Drosophila DL1 cells ( Figure 6A). This enhanced MyD88-dependent NF-kB activation by PRKG2 was reduced by a PRKG2 point mutation at amino acid 576 that disrupts its cGMP-dependent kinase activity ( Figure 6B). Thus, cGMP-dependent kinase activity of PRKG2 is required to enhance MyD88-dependent NF-kB activation in human cells, as it is in Drosophila. Deletion of the cGMP-binding domain of PRKG2 reduced this enhanced MyD88-dependent NF-kB activation by PRKG2 ( Figure 6B). IRAK1 is the human iScience Article homolog of Pelle (Daigneault et al., 2013). We found that IRAK1-dependent activation of NF-kB was enhanced by PRKG2 in human cells as well as in Drosophila, whereas TRIF (another adaptor protein for TLR signaling)-dependent NF-kB activation was not ( Figure 6C). The enhanced IRAK1-dependent NF-kB activation by PRKG2 was suppressed by siRNA-mediated targeting IRAK1 and the PP2A regulatory subunit B beta (PPP2-R2B, Twins homolog) but not by targeting MyD88, indicating that PP2A is required to enhance the IRAK1-dependent activation of NF-kB by PRKG2, as in Drosophila ( Figure 6D). The siRNA-mediated silencing of MyD88 reduced the enhanced MyD88-dependent NF-kB activation induced by PRKG2 (Figure S6G). Okadaic acid, a PP2A inhibitor, inhibited both the enhanced IRAK1-dependent and MyD88dependent NF-kB activation by PRKG2 in HEK293 cells ( Figure S6H), suggesting that the phosphatase iScience Article activity of PP2A is required for the cGMP-signaling-mediated modulation of NF-kB signaling. We also investigated IRAK1 modification ( Figure 6E), as it undergoes phosphorylation and ubiquitination when activated (Tun-Kyi et al., 2011). PRKG2 expression increased modified IRAK1, including phosphorylation levels at serine 376, which is essential for IRAK1 activation ( Figure 6F). In contrast, MyD88 expression was not affected by PRKG2 ( Figure 6E). This enhanced modification of IRAK1 by PRKG2 was inhibited by siRNAmediated targeting of PPP2-R2B ( Figure 6G). These findings suggest that, in addition to conservation of the Toll and TLR pathways in innate immunity, modulation of NF-kB activation by a membrane-bound cGK and a protein phosphatase, PP2A, in the cGMP signaling pathway is also evolutionarily conserved in insects and mammals.

DISCUSSION
In the present study, we set out to address immune response modulation by cGMP signaling and receptor guanylate cyclases. We show that NF-kB (Toll pathway) signaling in the Drosophila immune response is regulated by the activation of both Toll-receptor-dependent signaling and Gyc76C-dependent cGMP signaling.
Our findings indicate the presence of a dual receptor system that allows for modulation of the intensity of the Drosophila immune response depending on the situation; activating both receptors induces a strong immune response. Further, Gyc76C is activated by ModSP and Psh, members of the serine protease cascade that cleaves the Spz precursor, suggesting that, as the Toll receptor ligand, the Gyc76C ligand is also produced endogenously by ModSP and Psh activation. A peptide, NPLP1-VQQ, is a reported ligand of Gyc76C in response to salt stress (Overend et al., 2012). NPLP1-VQQ, however, is not likely to be a ligand for Gyc76C in response to Gram-positive bacterial infections, because a null mutant of Nplp1 encoding the NPLP1-VQQ precursor Nplp EY11089 is not susceptible to Gram-positive bacterial infections, in contrast to the gyc76C mutant (data not shown). Additional studies are needed to identify and analyze the Gyc76C ligand and/or its precursor to elucidate how NF-kB signaling and Drosophila immunity are modulated by the dual receptor system we identified here.
The present study demonstrated that, in parallel with the Toll receptor, Gyc76C mediates ModSP-dependent and Psh-dependent NF-kB activation. ModSP and Psh are activated by both Gram-positive bacteria and fungi. gyc76C KG03723 mutant adults and larvae, however, are resistant to fungal infection ( Figures 3A,  S4E, and S4F), in contrast to Gram-positive bacterial infections ( Figures 3A and S4A). modSP;psh double mutants are susceptible to fungal infection under the same conditions ( Figures S4E and S4F). Therefore, Psh-and ModSP-dependent NF-kB activation through the Toll receptor is sufficient for host defense against fungi. On the other hand, cGMP-pathway-mediated NF-kB modulation through Gyc76C has a crucial role in host defense against Gram-positive bacteria. The enhanced activation of NF-kB signaling by both receptors, the Toll receptor and Gyc76C, could be required for host defense against Gram-positive bacteria with more rapid proliferation than fungi. We recently reported that Gyc76C mediates hemocyte proliferation in a distinct manner from the cGMP pathway (Iwashita et al., 2020). A small GTPase, Ras85D, mediates Gyc76C-dependent hemocyte proliferation, and the loss-of-function of Ras85D causes host susceptibility against Gram-positive bacterial infection (Iwashita et al., 2020). Therefore, the Gyc76Cdependent cellular response may also be involved in host defense against Gram-positive bacteria.
Our results also demonstrate that NF-kB signaling activation is modulated by a membrane-bound cGK and PP2A in fly and human cells. cGMP levels are increased in Drosophila by the overexpression of ModSP as well as the overexpression of Gyc76C ( Figures 1D and 2E), whereas no Gyc76C-dependent increase in cGMP is detected in Drosophila following Gram-positive bacterial infections (data not shown). Gyc76C is a membrane receptor that converts GTP to cGMP, and DG2 is a membrane-localized cGMP-dependent protein kinase. Moreover, DG2 physically associates with dMyd88 to form a signaling complex with Tube and Pelle after binding to the intracellular Toll/IL-1 receptor homologous domain of the activated Toll receptor. It is possible that cGMP is produced by Gyc76C at a specific membrane region under physiologic conditions, which could initiate the physical and functional interactions of DG2 with the dMyd88-dependent signaling complex. A higher cGMP level is not induced by bacterial infections, whereas it is induced by overexpression of Gyc76C and ModSP. We speculate that cGMP is rapidly degraded by PDEs in vivo, and therefore, no increase is observed in cGMP during infection. On the other hand, in the case of overexpression, cGMP is continuously produced, and its increase is detected. In mammals, activation of the dMyd88-dependent signaling complex induces IRAK1 phosphorylation and ubiquitination ll OPEN ACCESS iScience 24, 103473, December 17, 2021 13 iScience Article (Tun-Kyi et al., 2011). Our study demonstrates that IRAK1 modifications, including phosphorylation at serine 376, which is essential for IRAK1 activation, are enhanced by the expression of PRKG2, a membrane-bound cGK, in human cells. The enhanced modification of IRAK1 is inhibited by PP2A knockdown, which inhibits the PRKG2-dependent enhancement of NF-kB activation. Therefore, the complex regulation of the phosphorylation and de-phosphorylation by two protein kinases, IRAK4 and IKAK1, in NF-kB signaling; a protein kinase, PRKG2; and a protein phosphatase, PP2A, in cGMP signaling may be involved in immune response modulation. Gyc76C is reported to be required for activation of the IMD pathway, another NF-kB pathway in Drosophila, in adult Malpighian tubules in response to salt stress (Overend et al., 2012). The present study demonstrated that Gyc76C activates the Toll pathway in larvae. Together these findings indicate that Gyc76C-mediated signaling modulates 2 NF-kB pathways, the IMD and Toll pathways, in a context-dependent manner in Drosophila. Further detailed studies are required to clarify how the cGMP signaling pathway modulates NF-kB signaling.
Our findings also suggest that the modulation of NF-kB activation by the cGMP signaling pathway in innate immune responses is evolutionarily conserved. For example, our findings show that a membrane-bound cGK, PRKG2, and PP2A are involved in modulating NF-kB activation in human cells, as in Drosophila. Other similarities between animals and plants in the regulation of immune responses include AtPepR1, an Arabidopsis receptor that bears an extracellular leucine-rich repeat, similar to that of the Toll receptor, and an intracellular guanylyl cyclase domain, similar to Gyc76C, with guanylate cyclase activity that produces cGMP (Qi et al., 2010). AtPepR1 is reported to be involved in the expression of pathogen-defense genes through cyclic-nucleotide-gated ion channel (Cng) 2, a cGMP-activated Ca 2+ channel. The findings of Qi et al. (2010) together with those of the present study suggest that an extracellular leucine-rich repeat and intracellular guanylyl cyclase are involved in mediating innate immune responses in both animals and plants.

Limitations of study
This study demonstrated that NF-kB signaling in the Drosophila immune response is regulated by the activation of both Toll-receptor-dependent and Gyc76C-dependent cGMP signaling. Although we demonstrated that cGMP signaling is required for host defense against Gram-positive bacterial infections, the physiological conditions under which NF-kB signaling is modulated by cGMP signaling and the mechanisms by which it is controlled remain unclear. Future studies are needed to identify the ligands for Gyc76C and to clarify the physiological conditions under which Gyc76C is activated by the ligands.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Materials availability
This study did not generate new unique reagents.

Data and code availability
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

DNA constructs
To generate UAS-CG3216, a PCR fragment was amplified with the following primers: forward 5'-CCGGAATTCATGCATCTGCTTGGGATCAGCATC -3' and reverse 5'-TTGCGGCCGCTACTTTCCCA TGCCGGCGA -3'. The resulting fragment was subcloned into the EcoRI/NotI sites of a pUAST vector. In the case of UAS-Gyc76C-RNAi, the 333-bp PCR fragment (corresponding to nt 2395 -2727 of GenBank accession number NM_079441) was subcloned into a pWIZ RNAi vector. To generate the in vitro expression vector under the control of a metallothionine promoter in Drosophila cells, full-length Gyc76C, dg2 P2, dMyd88, and Toll lacking residues 152-800 (designated TollDLRR) were subcloned into a pMT vector (Invitrogen). For immunoprecipitation, dMyd88 with a V5 epitope tag (as a C-terminal fusion) and dg2 with a HA epitope tag (inserted between Gly15 and Gly16 in a putative linker region) were expressed. The cDNA clone of dMyd88 (LD20892) was obtained through the Drosophila Genomic Resource Center (Indiana University). The human MyD88 construct, ELAM-NF-kB luciferase reporter, and pRL-TK were provided by Dr. T.
Cell culture, luciferase reporter assay, and RNAi DL1 cells (provided by the Drosophila RNAi Screening Center) were maintained at 25 C in Schneider's Drosophila medium (Invitrogen) containing 10% (v/v) heat-inactivated fetal bovine serum. The scheme of the luciferase assay in Drosophila cells was described previously (Sun et al., 2002). To monitor the Toll pathway activity, 4.0x10 4 cells/well in a 96-well plate were transfected with 40 ng of pGL3-Drosomycin-Luciferase (Drs-Luc) reporter vector and 10 ng of an Actin5C-lacZ transfection control vector together with the indicated in vitro expression vectors using Effectene (Qiagen) following the manufacturer's recommendations. At 12 to 16 h after transfection, expression was induced by adding 500 mM CuSO4 and incubated at 28 C for 2 d. DL1 cells were then lysed in Glo Lysis Buffer (Promega). Luciferase activity was measured with the One-Glo Luciferase Assay System (Promega) in a luminometer (SpectraMax L-TYA, Molecular Devices). b-Galactosidase (b-gal) activity was measured with b-gal assay buffer (100 mM HEPES pH 7.3/150 mM NaCl/10 mM MgCl 2 /0.5 mg/ml chlorophenol red-b-D-galactopyranoside; Roche), and the values of the absorbance of free chlorophenol red at 595 nm were used to normalize the variability in transfection efficiency. For RNAi and the dual-glo assay, DL1 cells (1.0x10 5 ) were plated in 96-well plates and transfected with 100 ng dsRNA, 40 ng pGL3-Drosomycin-Firefly luciferase reporter vector, and 10 ng pAc5.1-Renilla luciferase transfection control vector together with the indicated in vitro expression vectors using the Effectene transfection reagent (Qiagen). At 24 h after transfection, expression was induced by the addition of CuSO4 at a final concentration of 1.0 mM and cells were incubated at 28 C for another 24 h. Luciferase activity was measured with the Dual-glo Luciferase Assay System (Promega) using the Spectra-Max L-TYA luminometer (Molecular Devices). HEK293 cells were maintained at 37 C in 5% CO 2 . To monitor NF-kB activation, 20,000 cells/well in 96-well plates were transfected with 20 ng of ELAM-NF-kB luciferase and 20 ng of pRL-TK together with the expression vectors using Lipofectamine LTX (Invitrogen). After 48 h of transfection, relative luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega).

Assay for Gyc76C ligand activity
Fifty larvae overexpressing lacZ or ModSP were homogenized with 500 mL of 0.1% (v/v) trifluoroacetic acid (Wako) in H 2 O using a tissue homogenizer (Bertin Technologies, Precellys 24). The resulting homogenate was centrifuged for 10 min at 20,000g. The clear supernatant was evaporated in a Spin Driver (TAITEC, VC-96R) and solved in phosphate-buffered saline (PBS) as an acid extract. DL1 cells transfected with a Drs-Firefly luciferase reporter vector and an Actin5C-Renilla luciferase transfection control vector were incubated with the acid extract for 3 h. The luciferase reporter assay was carried out as described above. Heat treatment was performed at 100 C for 1 min. The ultrafiltration membrane with a molecular weight cut-off of 10,000 (Millipore, Amicon Ultra-4 10K) was used to estimate the molecular weight of the Gyc76 ligand. One arbitrary unit (AU) of the Gyc76C ligand activity was defined as the amount that showed 0.5 relative luciferase activity (firefly/renilla), calculated using a calibration curve created using the same assay.

Total RNA isolation and real-time PCR
Total RNAs were isolated from each genotype of 20 flies with Trizol reagent (GIBCO/BRL). Total RNA (1 mg) was used for cDNA synthesis with ReverTraAce reverse transcriptase (Toyobo) and oligo(dT) 15 primer (Promega). Using the first-strand cDNA (0.5 ml), real-time PCR was performed using a LightCycler (Roche Diagnostics). Rp49 was used as the internal control. The primers used for real-time PCR were as follows (F=forward, R=reverse): Rp49 The data are represented as mean with standard deviation. Statistical analyses between 2 groups were performed using the independent Student's t-test. Log-rank tests were performed to estimate the significance of differences in overall survival assays. P<0.05 was considered to indicate statistical significance.