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The JNK Pathway Is a Key Mediator of Anopheles gambiae Antiplasmodial Immunity

The JNK Pathway Is a Key Mediator of Anopheles gambiae Antiplasmodial Immunity The innate immune system of Anopheles gambiae mosquitoes limits Plasmodium infection through multiple molecular mechanisms. For example, midgut invasion by the parasite triggers an epithelial nitration response that promotes activation of the complement-like system. We found that suppression of the JNK pathway, by silencing either Hep, JNK, Jun or Fos expression, greatly enhanced Plasmodium infection; while overactivating this cascade, by silencing the suppressor Puckered, had the opposite effect. The JNK pathway limits infection via two coordinated responses. It induces the expression of two enzymes (HPx2 and NOX5) that potentiate midgut epithelial nitration in response to Plasmodium infection and regulates expression of two key hemocyte-derived immune effectors (TEP1 and FBN9). Furthermore, the An. gambiae L3–5 strain that has been genetically selected to be refractory (R) to Plasmodium infection exhibits constitutive overexpression of genes from the JNK pathway, as well as midgut and hemocyte effector genes. Silencing experiments confirmed that this cascade mediates, to a large extent, the drastic parasite elimination phenotype characteristic of this mosquito strain. In sum, these studies revealed the JNK pathway as a key regulator of the ability of An. gambiae mosquitoes to limit Plasmodium infection and identified several effector genes mediating these responses. Citation: Garver LS, de Almeida Oliveira G, Barillas-Mury C (2013) The JNK Pathway Is a Key Mediator of Anopheles gambiae Antiplasmodial Immunity. PLoS Pathog 9(9): e1003622. doi:10.1371/journal.ppat.1003622 Editor: Kirk Deitsch, Weill Medical College of Cornell University, United States of America Received October 3, 2012; Accepted July 31, 2013; Published September 5, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. LSG received funding from the Malaria Infection Biology Research and Training Program, NIAID, NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cbarillas@niaid.nih.gov ´ ´ ´ ¤ Current address: Laboratorio de Entomologia Medica, Instituto Rene Rachou, Fiocruz, Belo Horizonte, Minas Gerais, Brazil. the induction of heme peroxidase 2 (HPX2) and nicotinamide Introduction adenine dinucleotide phosphate (NADPH) oxidase 5 (NOX5) Malaria is a worldwide disease that is highly endemic in Sub- [8,10]. The HPX2/NOX5 system potentiates NO toxicity, Saharan Africa and causes over half a million deaths annually. enhances nitration, and reduces Plasmodium survival. Exposure of The mosquito Anopheles gambiae is a major vector of Plasmodium ookinetes to these chemical reactions as they traverse the midgut falciparum, the parasite responsible for most cases of human malaria cell modifies them and makes them ‘‘visible’’ to the mosquito in Africa. An. gambiae can mount effective antiplasmodial responses complement-like system [8]; however, the immune signaling by activating several signaling cascades involved in immune pathway(s) regulating the midgut epithelial response to infection regulation, such as the Imd, Toll, and STAT pathways [1–4]. have not been identified. Pathway activation leads to the transcription of effector genes that The JNK pathway is a mitogen-activated protein kinase mediate the antiplasmodial mechanism. The thioester-containing (MAPK) pathway that is highly conserved from mammals to protein 1 (TEP1) and the fibrinogen-related protein 9 (FBN9) are insects; however, our understanding of the role of JNK signaling in important components of the mosquito complement-like system insect immunity is limited. Several orthologs of genes that mediate that are produced by hemocytes and secreted into the mosquito JNK signaling in vertebrates have been identified in Drosophila and hemolymph; they bind to the ookinete surface and mediate An. gambiae [11,12]. The Jun-N-terminal kinase (JNK) is a MAP parasite lysis [5,6]. Activation of the Imd and Toll pathways kinase at the core of this signaling cascade that is activated by a decreases ookinete survival as parasites come in contact with the MAPK kinase (hemipterous,in D. melanogaster) (Figure 1A) [11,13– mosquito hemolymph by promoting TEP1-mediated lysis [1,3,7]. 17]. JNK phosphorylates the Jun and Fos transcription factors, In contrast, the STAT pathway targets a later stage of the parasite, giving rise to a Jun/Fos dimer (AP-1 complex) that activates the early oocysts, through a TEP1-independent response [4]. transcription of target genes (reviewed in [18]). JNK signaling is We have recently shown a functional link between midgut modulated by puckered (puc), a phosphatase that suppresses epithelial nitration and another mosquito antiplasmodial response signaling by dephosphorylating JNK. Puckered is part of a that targets the ookinete stage of the parasite, the complement-like negative feedback loop, because transcription of puc is regulated by the JNK pathway [16,19,20]. system [8]. Ookinete invasion results in extensive damage to the invaded cell [9] and induces a two-step epithelial nitration reaction In Drosophila, JNK signaling has been shown to be involved in a in which expression of nitric oxide synthase (NOS) is followed by wide range of biological processes including embryonic develop- PLOS Pathogens | www.plospathogens.org 1 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium Results Author Summary The An. gambiae JNK Pathway Limits Plasmodium berghei The mosquito Anopheles gambiae is a major vector of human malaria, a disease caused by Plasmodium falci- Infection parum parasites that results in more than half a million Five An. gambiae orthologs of genes known to be part of the JNK deaths each year. Several signaling pathways in the pathway signaling cascade in Drosophila have been identified mosquito have been shown to mediate the mosquito including two kinases, hemipterous (hep) and c-Jun N-terminal immune responses to Plasmodium infection. In this kinase (jnk); a phosphatase, puc; and two transcription factors, Jun manuscript we investigated the participation of the Jun- (jun) and Fos (fos) (Figure 1A,) [11]. These five genes are expressed N-terminal kinase (JNK) pathway in mosquito defense in the thoraces, abdomens, midguts, hemocytes, and in undevel- responses. We found that JNK signaling is required for oped ovaries from sugar-fed mosquitoes (Figure 1B, Table S1). Jun mosquito midgut cells to induce expression of two is expressed at low levels in the head, but the mRNAs of the other enzymes, HPx2 and NOX5, that mediate epithelial nitration genes could not be detected in this tissue (Figure 1B, Table S1). A in response to parasite invasion. These reactions modify notable enrichment of hep transcripts in the thorax and of jnk in the the parasites and promote activation of the mosquito ovary was observed (Figure 1B, Table S1). The transcriptional complement-like system that results in parasite lysis. The response of these five genes to infection with P. berghei (rodent JNK pathway also regulates the basal level of expression of malaria parasite) was analyzed in mosquito midguts collected at TEP1 and FBN9, two key components of the complement- different times after feeding on either healthy or P. berghei-infected like system that are produced by hemocytes and secreted mice. A significant increase in jnk, puc, jun and fos expression in into the mosquito hemolymph. Our studies revealed that response to infection was observed between 12–48 hours post JNK signaling plays a key role for mosquitoes to limit infection (hpi). In general, the magnitude and kinetics of the Plasmodium infection, making it an important determinant of malaria transmission to humans. inductions were variable between experiments. Jun at 24 and 48 hpi and JNK at 24 hpi had the most consistent inductions that were significant in three independent experiments (Figure 1C, Table S2). Hep expression changed the least in response to ment, apoptosis, stress response, cell proliferation and differentiation, and immunity [18]. The JNK pathway has a great deal of complexity Plasmodium infection (Figure 1C, Table S2). Only a modest increase was observed in one of the replicates at 12 hpi but, in and is known to receive input from multiple upstream genes, yet to be defined in insects, and from lateral inputs from components of other another, the expression was lower after infection than in the uninfected control. Although activation of the JNK pathway signaling cascades. For example TAK1, a kinase that is part of the Imd pathway, can also activate JNK signaling [21–23]. It is believed involves a cascade of post-translational phosphorylation events, transcription of JNK pathway members has been reported to that this complex organization reflects the broad range of responses that are influenced by JNK signaling. increase upon Plasmodium infection in Anopheles and transcriptional activation of JNK at the mRNA and protein level has also been Many different stimuli are known to activate the JNK pathway, observed in Drosophila midguts in response to bacterial challenge including microbial elicitors. In particular, the participation of [26–28]. JNK protein expression was also induced in the mosquito JNK signaling in antibacterial responses has been well document- midgut in response to Plasmodium infection (Figure S1). This ed in Drosophila. Lipopolysaccharide (LPS) is a key elicitor of JNK indicates that JNK signaling in infected midguts may be enhanced pathway activity in immune-competent cells and flies [13– by increased expression of several components of the cascade. 15,17,22,23], and flies that are deficient in puc (and therefore have an overactive JNK pathway output) display increased We confirmed that JNK silencing (Figure S2) enhances P. berghei infection (Figure 1D), as previously shown [25]. Furthermore, resistance to Gram bacteria [19]. In the An. gambiae 4a3B cell line, JNK signaling was weakly activated by H O , while LPS silencing other genes involved in JNK activation—such as hep, jun, 2 2 and fos (Figure S2)—also enhanced the intensity of infection, elicited a strong response [11]. The response of JNK signaling to increasing the median number of oocysts by 3.8 to 4.9 fold, LPS has also been observed in human dendritic cells and relative to the dsLacZ control (Figure 1, D and E, Table S3) splenocytes [24]. (p,0.001; Kolmogorov-Smirnov [KS] test). As expected, over- We have previously shown that JNK regulates expression of activation of this cascade by silencing puc (Figure S2), a several genes that protect An. gambiae mosquitoes from oxidative phosphatase that normally suppresses JNK signaling, had the damage, such as oxidation resistance 1 (OXR1), catalase, and opposite effect and greatly reduced the intensity (Figure 1D, Table glutathione peroxidase [25]. Silencing of these effector genes S3) (p,0.001; KS test) and the prevalence of infection from 68% increased reactive oxygen species (ROS) levels and reduced to 41% (p,0.005; chi-squared [x ] test). Co-silencing Jun reversed Plasmodium survival. Paradoxically, however, JNK silencing had the antiplasmodial effect of silencing puc (Figure 1E, Table S3) and the opposite effect and enhanced infection, suggesting that— increased the prevalence of infection from 33% to 84% (p,0.001; besides the role in ROS balance—JNK may mediate some x test), indicating that Jun is downstream of puc and confirming antiplasmodial response [25]. In this manuscript, we present a the functional link between these two genes in An. gambiae. detailed functional analysis of several genes that mediate JNK signaling in An. gambiae and identify two key mechanisms by which this cascade mediates antiplasmodial immunity. JNK activation JNK Signaling Activates Midgut Epithelial Nitration induces expression of HPx2 and NOX5, the two enzymes that We have recently shown that the HPx2/NOX5 system mediate epithelial nitration in response to ookinete invasion [8]. In potentiates NO toxicity and mediates nitration of midgut epithelial addition, JNK signaling regulates the basal levels of expression of cells in response to Plasmodium invasion. The potential participa- TEP1 and FBN9, two effector proteins produced by hemocytes tion of JNK signaling in the induction of these two enzymes and that mediate ookinete lysis [5,6]. The participation of JNK epithelial nitration was investigated. A robust increase in HPx2 signaling in the antiplasmodial responses of the A. gambie L3–5 and NOX5 expression was observed in the dsLacZ-injected strain that has been genetically selected to be refractory to control group (Figure 2A) in response to Plasmodium infection, as Plasmodium infection was also investigated. previously shown in uninjected females [8]; however, HPx2 was PLOS Pathogens | www.plospathogens.org 2 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium Figure 1. The JNK Pathway and Plasmodium berghei infection in Anopheles gambiae. (A) Diagram representing the organization of the JNK signaling cascade based on functional studies from vertebrates and Drosophila. Five An. gambiae orthologs were functionally characterized, including two kinases, hemipterous (hep) and c-Jun N-terminal kinase (jnk); a phosphatase, puc; and two transcription factors, Jun (jun) and Fos (fos) (B) Basal mRNA expression of putative genes from the JNK pathway in adult females. Hemipterous (Hep), Jun N-terminal kinase (JNK), Jun and Fos transcription factors and puckered (puc) mRNA levels in different organs of sugar-fed females. Mg, midgut; H, head; Th, thorax; Ab, abdomen; Hc, hemocyte; Ov, ovaries. Expression in different tissues relative to midgut levels, for which the mean was given a value of ‘‘1’’. Error bars indicate SEM of two biological replicates. (see Table S11 for gene ID numbers and primer sequences) (C) Midgut expression of members of the JNK pathway in response to Plasmodium infection in three independent experiments. Ratio of expression in infected/control blood-fed mosquitoes of Hep, JNK, Puc, Jun and Fos mRNA levels in midguts of mosquitoes from 3 independent experiments (green, red and blue bars). Error bars indicate SEM of two technical replicates. The expression analysis in each biological replicate is shown in Table S3. P-values determined by Student’s-T test after log 2 transformation; **, p,0.01, ***, p,0.001. *, p,0.05; **, p,0.01, ***, p,0.001. (D) Effect of silencing JNK pathway members on P. berghei infection. (E) Effect of silencing the transcription factor jun alone or and co-silencing jun and the negative regulator puc on Plasmodium infection. For (D) and (E), the green dots represent oocyst counts from individual midguts and the horizontal red bar indicates the median infection level. Groups were compared using the KS, Mann-Whitney and Kruskal-Wallis tests with Dunn’s post test (see Table S3). The P-values for the Mann-Whitney tests are shown. Graphs represent samples pooled from three biological replicates with comparable (not statistically different) medians in their dsLacZ-injected groups (n = number of midguts). doi:10.1371/journal.ppat.1003622.g001 PLOS Pathogens | www.plospathogens.org 3 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium Figure 2. The JNK Pathway and Midgut Epithelial Nitration. (A) Effect of JNK, Jun or puc silencing on the inducible midgut mRNA expression of HPx2 and NOX5 in response to Plasmodium berghei infection. C, control mosquitoes fed on a healthy mouse (gray bars); I, infected mosquitoes fed on a P. berghei-infected mouse (red bars). Mean expression in infected midguts relative to uninfected blood-fed controls, for which the mean was adjusted to a value of ‘‘1’’; The bars represent the SEM of three biological replicates from independent experiments (see Table S4). P-values determined by paired Student’s-T test after log 2 transformation; **, p,0.01, ***, p,0.001. (B) Effect of silencing JNK, Jun or puc on infection-inducible in vivo midgut nitration. C, control mosquitoes fed on a healthy mouse (gray bars); I, infected mosquitoes fed on a P. berghei-infected mouse (blue bars). Graphs represent one of two biological replicates (see Figure S4 and Table S5); error bars indicate SEM of four technical replicates. P-value determined by Student’s t-test; **, p,0.01, ***, p,0.001. (C and D) Effect of co-silencing hpx2 (C) or nox5 (D) on the phenotype of silencing the negative regulator puc. Green dots represent oocyst counts in individual midguts; horizontal red bar indicates median infection intensity. P-values were determined by Mann-Whitney test; ns, not significant. Graphs represent data from three biological replicates with comparable medians in their dsLacZ-injected groups. n = total number of midguts examined. doi:10.1371/journal.ppat.1003622.g002 no longer induced in infected midguts and NOX5 expression was alone. Co-silencing HPx2 increased the median number of oocysts/ significantly reduced, relative to uninfected controls when JNK midgut from 0 to 17 (p,0.0001; KS test) and the prevalence of infection from 20% to 100% (p,0.0001; x test). Co-silencing was silenced; and expression of both HPx2 and NOX5 is reduced in infected midguts when jun is silenced (Figure 2A, Table S4). The NOX5 and puc (Figure 2D, Table S3) increased the median number of oocysts/midgut from 0 to 10 (p,0.0001; KS test) to the same level transcriptional induction of HPx2 in response to infection was as the dsLacZ control, and the prevalence of infection from 22% to more robust when puc was silenced, but NOX5 induction was no 88% (p,0.0001; x test). This indicates that HPx2 and NOX5 are longer observed (Figure 2A, Table S4). In agreement with the downstream of puc and mediate, to a large extent, the antiplasmodial overall transcriptional responses, when JNK was silenced, in vivo response triggered by the JNK activation. midgut nitration no longer increased in response to Plasmodium infection, was lower than in the uninfected controls when jun was The JNK Pathway Regulates Expression of Hemocyte- silenced, while silencing puc had the opposite effect and enhanced the nitration response. (Figure 2B, Figure S3, Table S5). Derived Antiplasmodial Effectors Furthermore, co-silencing HPx2 (Figure 2C, Table S3) com- Jun expression is induced in mosquito hemocytes 24 hpi with P. pletely rescued the dramatic antiplasmodial effect of silencing puc berghei [27], suggesting that JNK signaling in these cells could also PLOS Pathogens | www.plospathogens.org 4 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium injected groups (see Table S1) n = total number of midguts examined. doi:10.1371/journal.ppat.1003622.g003 be an important component of antiplasmodial immunity. TEP1 and FBN9 are proteins constitutively produced by hemocytes that are secreted into the mosquito hemolymph, bind to the surface of P. berghei ookinetes, and mediate parasite lysis [5,6]. We investigated the hypothesis that these hemocyte-derived proteins are regulated by the JNK pathway and are important effectors of this signaling cascade. Silencing jun or fos significantly reduced TEP1 by 94% and 69%, respectively (p,0.001 and p,0.05; Student’s t-test) and reduced FBN9 expression by 62% and 70%, respectively (Figure 3, A and B, Table S6) (p,0.01 and p,0.001; Student’s t-test) but had no effect on the expression levels of other hemocyte-specific genes such as APL1A or APL1C, and fos silencing actually resulted in a modest increase in LRIM1 expression (Figure S4, Table S6). Conversely, silencing puc significantly increased expression of both TEP1 and FBN9 by 1.86 and 2.6 fold, respectively (Figure 3, A and B, Table S6) (p p,0.01; Student’s t-test). Silencing JNK did not affect the total number of circulating hemocytes or the proportions of granulocytes, oenocytoids, or prohemocytes circu- lating in the mosquito (Figure S5). We have previously shown that induction of HPx2 and NOX5 mediates epithelial nitration and that the activity of these enzymes promotes both TEP1 binding to the ookinete surface and parasite lysis [8]. Participation of TEP1 and FBN9 as final effectors of the JNK antiplasmodial response was explored by co-silencing these genes with puc. Co-silencing TEP1 increased the median number of oocysts/midgut from 1 to 21.5 (p,0.0001; KS test) and the prevalence of infection from 53% to 88% (p,0.02, x test) relative to silencing puc alone (Figure 3C, Table S3). Co-silencing FBN9 had a similar effect, increasing the median number of oocysts/midgut from 0 to 13.5 (p,0.0001; KS test) and the prevalence of infection from 35% to 84% (p,0.0001, x test) (Figure 3D, TableS3). The JNK Pathway Contributes to Parasite Elimination in the An. gambiae Refractory Strain The An. gambiae refractory (R) strain was selected to be refractory to Plasmodium cynomolgi (simian malaria) infection but also eliminates most Plasmodium species, including P. berghei [29]. In this mosquito strain, ookinetes develop and invade the midgut, but they are killed and covered with melanin, a black, insoluble pigment [29]. R females are in a chronic state of oxidative stress that is exacerbated by blood feeding [30], and TEP1 is known to be a critical mediator of P. berghei melanization and killing [5]. We have shown that the JNK pathway regulates expression of two enzymes that mediate midgut epithelial nitration: NOX5, an oxidase that generates ROS, and the heme peroxidase, HPX2. Furthermore, exposure of ookinetes to these nitration reactions as they traverse the midgut epithelial cell promotes TEP1 activation [8]. The hypothesis that the refractory phenotype may be Figure 3. The JNK Pathway and Hemocyte Antiplasmodial mediated, at least in part, by the JNK signaling pathway was Effector Genes. (A and B) Effect of silencing jun, fos,or puc on basal investigated. expression of TEP1 (A) and FBN9 (B) in circulating hemocytes. Mean We first compared the basal level of mRNA expression of the expression level in silenced samples, relative to the dsLacZ-injected genes involved in JNK signaling between the susceptible (S) G3 An. control that was adjusted to a value of ‘‘1’’ and is indicated by the red gambiae and the R strain. The basal midgut expression of all the dotted line. The bars represent the SEM of two biological replicates from independent experiments (see Table S4). P-values determined by genes involved in JNK signaling was higher in the R strain. Student’s-T test after log 2 transformation; *, p,0.05, **, p,0.01, ***, Midgut jnk expression was dramatically higher (4.3 fold), while the p,0.001. (C and D) Effect of co-silencing TEP1 (C) or FBN9 (D) on the overexpression of hep was less prominent (1.5 fold) (Figure 4A, phenotype of silencing negative the regulator puc. Green dots Table S7). The basal expression level of all genes of the JNK represent oocyst counts in individual midguts, and the horizontal red pathway (hep, jnk, puc, jun, and fos) was also significantly higher in bar indicates median infection intensity. P-values were determined by whole body samples of R females, ranging from 2.1 to 4.8 fold Mann-Whitney test; ns, not significant. Graphs represent data from three biological replicates with comparable medians in the dsLacZ- (Figure S6, Table S7). Higher puc expression is indicative of PLOS Pathogens | www.plospathogens.org 5 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium The contribution of the JNK pathway to the refractory phenotype was directly tested by reducing JNK expression via gene silencing. JNK silencing had a dramatic effect, increasing the prevalence of infection from 0 to 70% (Figure 4C) (p,0.0001; x test), and the median number of oocysts from 0 to 6 oocysts/ midgut (Figure 4C, Table S10) (p,0.001; KS test). The total number of parasites (live and melanized) was not significantly different between the dsLacZ control and the JNK-silenced group, indicating that a similar number of ookinetes invaded the midgut and that the difference in infection prevalence was due to ookinete survival once they traversed the midgut. Of the total number of parasites present, 99.6% of parasites were melanized in the dsLacZ group; this decreased to 32% when JNK was silenced (Figure 4C) (p,0.0001, x test). Discussion The immune response of An. gambiae mosquitoes against Plasmodium parasites is mediated by activation of immune-related signal transduction pathways. We carried out a functional characterization of five An. gambiae orthologs of genes known to mediate JNK signaling in Drosophila. Our studies implicate the JNK pathway as an important mediator of two coordinated steps of the mosquito anti-Plasmodium immune response and as a major determinant of the killing mechanism in a highly refractory strain of An. gambiae. JNK signaling triggers the transcriptional activation of HPX2 and NOX5, two key enzymatic effectors of midgut epithelial cells, in response to ookinete invasion. Induction of these two enzymes potentiates nitration and limits Plasmodium survival. This was directly confirmed by the observation that midgut nitration is greatly diminished when JNK signaling is disrupted by silencing JNK or jun. Overactivation of the JNK pathway by silencing puc, greatly increased midgut HPx2 expression and nitration in Figure 4. Participation of the JNK Pathway in L3–5 Mosquitoes response to Plasmodium infection. Interestingly, puc silencing did Refractory (R) responses to Plasmodium berghei Infection. (A) Basal mRNA expression of genes from the JNK pathway in the midgut not induce higher levels of NOX5 expression. This enzyme and hemocytes of G3 susceptible (S) (gray) and R (blue) mosquitoes. (B) generates reactive oxygen species that could be potentially toxic. Expression of effector genes regulated by the JNK pathway in S (gray) Our results suggest that in the absence of puc, there might be and R (blue) mosquitoes. Basal mRNA levels of HPx2 and NOX5 in the alternative mechanisms that limit NOX5 expression,probably to midgut, and of TEP1 and FBN9 in hemocytes. Graphs represent the prevent deleterious effects on the host. Previous studies in a variety expression level in R females, relative to S females, that were adjusted of mammalian cell types have also shown that NOX5 and other to a value of ‘‘1’’; for R females samples the bars represent the SEM of three biological replicates from independent experiments (see Table NADPH oxidases are regulated by the JNK pathway [31–33] and S4). P-values determined by paired Student’s-T test after log2 induction of a nitrogen dioxide-producing heme peroxidase has transformation; *, p,0.05, **, p,0.01, ***, p,0.001. (C) Effect of also been shown to be mediated by the JNK pathway [34]. silencing JNK (right panel) in the number of melanized and live The process of nitration is clearly an essential step in the parasites on individual midguts of R mosquitoes. Red dots indicate the destruction of malaria parasites, evidenced by the significant number of parasites on an individual midgut, live (y-axis) and melanized increase in parasite survival upon silencing either HPx2 or NOX5. (x-axis). Green horizontal bars indicate median infection intensities. Inset pie graphs represent the percentage of total parasites for each It is also a critical outcome of JNK activation, as the considerable group displaying a live (green) or melanized (black) phenotype; resistance conferred by puc silencing is reverted by co-silencing percentage displayed refers to melanized parasites. Graphs represent either of these two enzymes (Figure 2). We therefore propose that data from three biological replicates (see Table S10). (n = number of the JNK pathway is part of an ‘‘alarm system’’ triggered by midguts analyzed). parasite invasion that activates expression of NOX5 and HPx2, doi:10.1371/journal.ppat.1003622.g004 two enzymes that catalyze nitration reactions, that label ookinetes increased JNK activation, because puc expression is transcription- for destruction as they traverse the mosquito midgut. ally regulated by the JNK pathway. In hemocytes, there was no The dramatic reduction in TEP1 and FBN9 mRNA levels in difference in hep, fos and puc expression between the mosquito hemocytes when the JNK pathway was disrupted by silencing jun strains, but jnk and jun levels were also significantly higher in the R or fos appears to be specific, because expression of other hemocyte- strain (Figure 4A, Table S7). Furthermore, expression of effector specific genes involved in the regulation of complement activation genes of the JNK pathway was also higher in the R strain. In the (APL1A, APL1C, and LRIM1) was not reduced. We also midgut, basal HPx2 and NOX5 expression was 2.8 and 3.5 fold confirmed that the differences in expression were not due to times higher, respectively (Figure 4B, Table S8) (p,0.01; paired t- significant changes in the number or type of hemocytes present in test for both); while in hemocytes, TEP1 and FBN9 expression was silenced mosquitoes. The co-silencing experiments with puc 3.2 and 5.9 fold higher in R mosquitoes, respectively (Figure 4B, confirmed that both TEP1 and FBN9 are downstream of JNK. Table S9) (p,0.01; paired t-test for both). This indicates that the basal level of TEP1 and FBN9 expression in PLOS Pathogens | www.plospathogens.org 6 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium mosquito hemocytes is regulated by the JNK pathway and that Mosquito Tissue Dissections both genes are important effectors of the lytic response mediated Approximately 15–20 female mosquitoes 3- to 4 days post- by this cascade. emergence were removed cold anesthetized to immobilize them. Previous studies have shown that the R strain is in a chronic Hemocytes from individual mosquitoes were extracted using the state of oxidative stress that is exacerbated when adult females take method outlined below, Trizol was added and samples were kept a blood meal [30]. Genome-wide transcriptional analysis revealed on ice. Then the head was severed from the thorax, and the thorax higher expression in the R strain of several immune genes, genes from the abdomen using a clean scalpel. The midgut and encoded by the mitochondrial genome, and genes involved in undeveloped ovaries were then pulled from the abdomen using oxido/reductive processes or ROS detoxification relative to S fine forceps. Samples from each tissue were pooled together and females [30]. The R strain also exhibits impaired mitochondrial stored in RNAlater (Ambion, Austin, Texas, USA) in a microfuge state-3 respiration and increased rate of electron leak [35]. NOX5 tube. RNA was extracted, cDNA was generated, and gene is a member of the NAPDH oxidase family and generates expression was quantified using the methods indicated below for superoxide anion, which is quickly converted into hydrogen ‘‘Quantification of gene expression’’ (for tissues except hemocytes) peroxide by superoxide dismutase (reviewed by Bedard and Kraus or ‘‘Hemocyte collection and counting’’ (for hemocytes). [36]). We found that the genes that mediate signaling (hep, JNK, jun, fos, and puc) and key downstream effectors of this pathway in Quantification of Gene Expression the midgut (HPx2 and NOX5) and hemocytes (TEP1 and FBN9) Fifteen to twenty whole female mosquitoes or dissected tissues have increased basal levels of expression. Higher levels of were homogenized in RNAlater (Ambion) and subject to RNA HPx2and NOX5 are expected to accelerate the rate of epithelial extraction using RNAeasy (Qiagen, Los Angeles, California, USA) nitration, and higher hemolymph levels of TEP1 and FBN9 would kits according to the manufacturer’s instructions and first-strand promote parasite lysis. The increase in basal expression of NOX5 cDNA was synthesized using QuantiTect reverse transcriptase may be responsible, at least in part, for the higher constitutive (Qiagen). Gene expression was assessed by SYBR green quanti- levels of systemic ROS that have been observed in the R strain tative real-time PCR (DyNAmo HS; New England Biolabs, [30]. In An. gambiae, ROS levels have been shown to modulate Beverly, Massachusetts, USA) using the CFX96 system (Bio-Rad, immunity to both bacteria and Plasmodium [30,37]. The dramatic Hercules, California, USA). Each sample was assayed using two reduction in melanization and the increase in parasite survival technical replicas and 2–3 biological replicates. The amount of when JNK signaling is disrupted in the R strain confirm the key cDNA template present in each sample was normalized using the role of this pathway in mosquito antiplasmodial immunity. expression An. gambiae ribosomal protein S7 as reference. Fold 2DD We have recently shown that some P. falciparum strains, such as change values were derived using the 2 Ct method. The values NF54, are able to infect the An. gambiae R strain and that silencing were adjusted in each experiment by dividing each of the technical TEP1 did not enhance parasite survival, indicating that the replicates in the control and treatment groups by the mean of the mosquito complement-like system was not activated. In contrast, control group, thus adjusting the control groups to a value of ‘‘1’’. other parasite strains (such as 7G8) were almost completely The statistical analysis was done using the Student’s T-test after eliminated through a TEP1-mediated mechanism [38]. Co- log2 transformation of the mean value of each biological replicate infection experiments with a P. falciparum strain that is melanized for each treatment. Primers used are provided in Table S11; when and one that survives suggest that survival is genetically appropriate, primers were verified against R strain sequences determined by a parasite-autonomous mechanism, because the obtained by Solexa transcriptome sequencing of adult S and R survival (or lack thereof) of one strain does not affect the outcome females (Barillas-Mury Lab, unpublished). The primers used for of the other strain in the same mosquito [38]. Together, this TEP1 expression analysis in Figure 3 (S strain) are located in a indicates that some P. falciparum strains are susceptible to a TEP1- polymorphic region between S and R strains of A. gambiae. For this dependent killing mechanism, while others have the capacity to reason, a different primer set in a conserved region was used for evade it. Given the critical role of TEP1 as an effector of the JNK the TEP1 expression data presented in Figure 4 (comparison pathway, it is likely that P. falciparum strains also differ in their between S and R strains). ability to avoid—or perhaps may even actively suppress— activation of this signaling cascade. Detailed studies on the P. berghei Maintenance and Infection participation of the JNK pathway in mosquito antiplasmodial P. berghei (GFP-CON transgenic 259cl2 strain) parasites from responses to different P. falciparum strains are currently under way frozen stocks were administered intraperitoneally to donor mice. and may shed new insights into immune evasion strategies that When the parasitemias of donor mice reached 10–20%, 20–50 ml promote human malaria transmission. of infected blood was transferred to naı ¨ve mice via intraperitoneal injection. All mice were 3- to 5-week-old BALB/c females. Materials and Methods Parasitemia was assessed by light microscopy inspection of Giemsa-stained thin smears obtained by tail snips. At 2–3 days Ethics Statement post emergence, female mosquitoes were deprived of sucrose Public Health Service Animal Welfare Assurance #A4149-01 solution for 6–12 h, then allowed to feed on anesthetized mice guidelines were followed according to the National Institutes of infected with P. berghei at 3–7% parasitemia and exhibiting 1–3 Health Animal (NIH) Office of Animal Care and Use (OACU). exflagellation events per field, as previously described [40]. Where These studies were done according to the NIH animal study indicated, naıve blood-fed control mosquito groups were fed on protocol (ASP) approved by the NIH Animal Care and User uninfected mice of the same age. All P. berghei-infected mosquitoes Committee (ACUC), with approval ID ASP-LMVR5. and corresponding control mosquitoes were kept at 21uC and 80% humidity. Unless otherwise indicated, P. berghei infection intensities Mosquito Strains and Rearing were quantified 7–9 days post infection (dpi) by epifluorescent An. gambiae G3 and L3–5 mosquitoes were reared at 27uC with microscopy inspection of dissected midguts containing GFP- 80% humidity on a 12-h light/dark cycle. Cotton balls soaked in expressing parasites fixed in 4% paraformaldehyde and mounted 10% sucrose in water were provided as previously described [39]. in Vectashield (Vector Labs, Burlingame, California, USA), PLOS Pathogens | www.plospathogens.org 7 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium enabling manual counting of fluorescent oocysts and/or melanized diluted in PBT (1:3,000) at 4uC. Samples were washed with ookinetes. PBT and 4 were incubated with a secondary alkaline phosphatase- conjugated antibody (1:5,000) diluted in PBT, while the remaining sample was reserved as a background signal control. All samples RNAi Gene-Silencing Assays were incubated with rNPP–r-nitrophenylphosphate (Sigma Al- T7 promoter sequences were introduced at both ends using two drich) and read in a spectrofluorometer plate reader at 405 nm. different strategies. For LacZ, NOX5 and Tep1, cDNA fragments The relative nitration for each experimental treatment was were amplified using the primers given in Table S11 and cloned confirmed in at least two independent experiments. into the pCRII-TOPO vector (Invitrogen, Carlsbad, California, USA) following the manufacturer’s instructions. T7 promoters were introduced by amplifying the cloned insert using the primers: Hemocyte Collection and Counting M13F: 59-GTAAAACGA CGGCCAGT-39 and M13R: 59- Female mosquitoes were cold anesthetized and injected CTCGAGTAATACGACTCACTATAGGGCAGGAAA intrathoracically with a micropipette needle loaded with hemocyte CAGCTATGAC-39, which anneal to the vector as previously perfusion buffer (60% Schneider’s insect medium, 30% citrate, reported [8]. These PCR products were used as templates for 10% FBS). After insertion of the needle into the thorax, a small generating dsRNA as described below. For all other genes, the T7 incision was made in the lower abdomen, and buffer was sequences were included in the gene-specific primers and cDNA dispensed through the needle and collected 2 ml at a time from fragments of about ,300-bp were generated (Table S11). For all the incision using siliconized pipet tips for a total of 10–12 ml. genes, sense and antisense RNAs were synthesized simoulta- Perfusions were then either collected into a siliconized Eppendorf neously from templates and purified using the T7 RNAi Mega- tube for RNA extraction or applied to a disposable hemocytom- script kit (Ambion), eluted in water, and concentrated to 3 mg/ml eter (InCyto, Seoul, South Korea) for counting. For RNA using a Microcon YM-100 filter (Millipore, Bedford, Massachu- extraction, tubes were centrifuged for 30 min at 12,0006g to setts, USA). About 69 nl of this dsRNA preparation was injected pellet the cells; supernatant was removed, and 500 ml Trizol was into the thorax of cold-anesthetized, 2- to 3-day-old female added. RNA was isolated according to phenol/chloroform mosquitoes using a nano-injector (Nanoject; Drummond Scientif- extraction as suggested by Trizol protocol. For counting, cells ic, Broomall, Pennsylvania, USA) fitted with a glass capillary were visualized under light microscope with 406 objective. Cells needle according to previous protocols. dsRNA targeting LacZ contained within the marked grid were separated into three cell was used in each experiment to control for any unspecific effect of types (granulocyte, oenocytoid, prohemocyte) and counted ac- wounding and dsRNA exposure. Efficiency of silencing was cordingly. Population proportions were calculated and total quantified 2–3 days after dsRNA injection by real-time quantita- numbers of cells per mosquito were determined by manufacturer’s tive RT-PCR with the An. gambiae ribosomal S7 gene as the extrapolation. internal control for normalization. Primers for silencing verifica- tion are listed in Table S11, and silencing efficiencies are displayed Statistical Analysis in Figure S2. Fold change differences in gene expression across groups were normalized by log transformation. The statistical analysis of Immunoblotting differences in gene expression was done using the Student’s T-test Midguts were dissected and cleaned of blood meal in cold, after log2 transformation of the mean value of each biological sterile PBS supplemented with 1% levamisole (Sigma-Aldrich, St. replicate from independent experiments. Oocyst distributions were Louis, Missouri, USA). Pools of 5–10 midguts were transferred to a determined not to be normal, and were compared to one another microfuge tube and homogenized in PBS with protease inhibitor, using the Kolmogorov-Smirnov (KS), Mann-Whitney tests and levamisole, and phosphoStop (Roche Applied Science, Madison, Kruskal-Wallis tests with Dunn’s post-test (see Tables S3 and S10). Wisconsin, USA), prepared using NuPAGE buffers and reducing When the median infection levels of the dsLacZ group of two or agent (Invitrogen), and run on NuPAGE Bis-Tris 4–12% gels more biological replicates were not statistically different using the (Invitrogen) according to manufacturer’s instructions. Proteins Mann-Whitney test, the data were merged (See Tables S3 and S10). were then transferred from gels to membranes using the iBlot Oocyst prevalences were compared using x tests. Differences in nitration levels were compared using the Student’s t-test. P-values system (Invitrogen). Membranes were blocked in TBS with 5% milk +0 .05% Tween, washed, and incubated in fresh milk solution represented in figures are given in corresponding figure legends and with primary antibody against JNK (1:2000; Santa Cruz text. All statistical analyses were performed using Prism 5.01 Biotechnology, Santa Cruz, California, USA) overnight. They software (GraphPad Software, La Jolla, California, USA). were then washed and incubated in fresh milk solution with alkaline phosphatase-conjugated secondary antibody against Supporting Information rabbit (1:5000) for 2 h with TBS washings between each step. Figure S1 JNK protein midgut expression in response to Membranes were finally rinsed with TBS and incubated for P. berghei infection. JNK was detected with commercial 30 min (anti-JNK) with Western Blue substrate (Promega Corp., antibodies in Western Blots from midgut homogenates obtained Madison, Wisconsin, USA) to visualize bands. from sugar-fed females (SF), control (C) females fed on a healthy mouse or infected females (I) fed on P. berghei-infected mouse. In vivo Nitration Assays Samples were collected 24 and 48 h after feeding. The size of the Assays were performed according to previously established reference molecular markers is expressed as kDa and is indicated methods [8]. In brief, five midguts were dissected, fixed, and by the dots on the left. washed with PBS, then triturated and incubated in amino triazole (DOCX) (10 mg/ml). Pelleted midgut fragments were incubated with 2 mM levamisole, then blocked with PBT and washed. The pellet Figure S2 Silencing Efficiency in Sugar-Fed Mosquitoes. was subsequently resuspended in 50 ml of PBT, and five replicates Silencing efficiency in sugar-fed mosquitoes after systemic of one-midgut equivalents (10 ml of the 50-ml suspension) were injection of dsRNA for the target gene relative to the expression incubated overnight with anti-nitrotyrosine primary antibody level compared with dsLacZ-injected control mosquitoes. Whole PLOS Pathogens | www.plospathogens.org 8 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium body expression was determined in sugar fed females either 2 days Table S3 Summary of oocyst data for all G3 infections. (HPx2 and NOX5) or 3 days (all other genes) after injection. (DOCX) (Mean 6 SE). Table S4 Quantification of HPx2 and NOX5 expression (DOCX) in the midgut following silencing of JNK pathway Figure S3 Effect of silencing JNK, jun or puc on members. infection-induced in vivo midgut nitration. C, control (DOCX) mosquitoes fed on a healthy mouse (gray bars); I, infected Table S5 Quantification of nitration in the midgut mosquitoes fed on a P. berghei-infected mouse (blue bars). Graphs following silencing of JNK pathway members. represent one of two biological replicates (see Figure 2B and Table (DOCX) S5); error bars indicate SEM of three technical replicates. P-value determined by Student’s t-test; *, p,0.05, **, p,0.01. Table S6 Quantification of effector expression in he- (DOCX) mocytes following silencing of JNK pathway members. (DOCX) Figure S4 Effect of Silencing Jun or Fos on LRIM1, APL1A and APL1C expression. Hemocyte mRNA expression Table S7 Quantification of JNK pathway member of LRIM1, APL1A, and APL1C genes was determined 3 days expression in G3 and L3–5 mosquitoes and tissues. after systemic injection of either dsLacZ, dsJun or dsFos (Mean 6 (DOCX) SEM). (* indicates p,0.05; Student’s t-test) Table S8 Quantification of HPx2 and NOX5 in G3 and (DOCX) L3–5 midguts. Figure S5 Effect of Silencing JNK on hemocyte popula- (DOCX) tions. Effect of silencing JNK on the total number of hemocytes and Table S9 Quantification of HPx2 and NOX5 in G3 and the relative abundance of granulocytes, oenocytoids, and prohemo- L3–5 hemocytes. cytes 4 days after systemic injection of dsLacZ or dsJNK (Mean 6 (DOCX) SEM). No significant differences were observed (Student’s t-test). (DOCX) Table S10 Summary of oocyst data for all L3–5 infections. Figure S6 Relative expression of genes from the JNK (DOCX) pathway in susceptible and refractory An. gambiae mosquitoes. Basal mRNA levels of hep, JNK, jun and fos in Table S11 Primers used for dsRNA templates and susceptible (S, gray) and refractory (R, blue) mosquitoes (Mean 6 silencing validation/real-time PCR. SEM). Graphs represent the expression level in R females, relative (DOCX) to S females, that were adjusted to a value of ‘‘1’’; for R females samples the bars represent the SEM of three biological replicates Acknowledgments (see Table S4). P-values determined by paired Student’s-T test after log2 transformation; *, p,0.05, **, p,0.01,. We thank Alvaro Molina-Cruz for insightful comments and discussions, Andre ´ Laughinghouse and Kevin Lee for insectary support, and Brenda (DOCX) Rae Marshall for editorial assistance. Table S1 Quantification of tissue-specific expression of JNK pathway members. Author Contributions (DOCX) Conceived and designed the experiments: LSG CBM . Performed the Table S2 Time-course quantification of P. berghei- experiments: LSG GdAO . Analyzed the data: LSG. 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Molina-Cruz A, DeJong RJ, Ortega C, Haile A, Abban E, et al. (2012) Some Cell Host Microbe 11: 410–417. strains of Plasmodium falciparum, a human malaria parasite, evade the 27. Baton LA, Robertson A, Warr E, Strand MR, Dimopoulos G (2009) Genome- complement-like system of Anopheles gambiae mosquitoes. Proc Natl Acad wide transcriptomic profiling of Anopheles gambiae hemocytes reveals Sci U S A 109: E1957–1962. pathogen-specific signatures upon bacterial challenge and Plasmodium berghei 39. Benedict MQ (1997) Care and maintenance of anopheline mosquito colonies. infection. BMC Genomics 10: 257. In: Crampton JM, Beard CB, Louis C, editors. The molecular biology of disease 28. Mendes AM, Awono-Ambene PH, Nsango SE, Cohuet A, Fontenille D, et al. (2011) Infection intensity-dependent responses of Anopheles gambiae to the African vectors: A methods manual. London: Champman & Hall. pp. 3–12. malaria parasite Plasmodium falciparum. Infection and Immunity 79: 4708–4715. 40. Billker O, Shaw MK, Margos G, Sinden RE (1997) The roles of temperature, 29. Collins FH, Sakai RK, Vernick KD, Paskewitz S, Seeley DC, et al. (1986) pH and mosquito factors as triggers of male and female gametogenesis of Genetic selection of a Plasmodium-refractory strain of the malaria vector Plasmodium berghei in vitro. Parasitology 115 (Pt 1): 1–7. Anopheles gambiae. Science 234: 607–610. PLOS Pathogens | www.plospathogens.org 10 September 2013 | Volume 9 | Issue 9 | e1003622 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS Pathogens Public Library of Science (PLoS) Journal

The JNK Pathway Is a Key Mediator of Anopheles gambiae Antiplasmodial Immunity

PLoS Pathogens , Volume 9 (9): e1003622 – Sep 5, 2013

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Public Library of Science (PLoS) Journal
Subject
Research Article; Biology; Microbiology; Vector biology; Mosquitoes; Medicine; Infectious diseases; Vectors and hosts; Mosquitoes
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1553-7366
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1553-7374
DOI
10.1371/journal.ppat.1003622
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Abstract

The innate immune system of Anopheles gambiae mosquitoes limits Plasmodium infection through multiple molecular mechanisms. For example, midgut invasion by the parasite triggers an epithelial nitration response that promotes activation of the complement-like system. We found that suppression of the JNK pathway, by silencing either Hep, JNK, Jun or Fos expression, greatly enhanced Plasmodium infection; while overactivating this cascade, by silencing the suppressor Puckered, had the opposite effect. The JNK pathway limits infection via two coordinated responses. It induces the expression of two enzymes (HPx2 and NOX5) that potentiate midgut epithelial nitration in response to Plasmodium infection and regulates expression of two key hemocyte-derived immune effectors (TEP1 and FBN9). Furthermore, the An. gambiae L3–5 strain that has been genetically selected to be refractory (R) to Plasmodium infection exhibits constitutive overexpression of genes from the JNK pathway, as well as midgut and hemocyte effector genes. Silencing experiments confirmed that this cascade mediates, to a large extent, the drastic parasite elimination phenotype characteristic of this mosquito strain. In sum, these studies revealed the JNK pathway as a key regulator of the ability of An. gambiae mosquitoes to limit Plasmodium infection and identified several effector genes mediating these responses. Citation: Garver LS, de Almeida Oliveira G, Barillas-Mury C (2013) The JNK Pathway Is a Key Mediator of Anopheles gambiae Antiplasmodial Immunity. PLoS Pathog 9(9): e1003622. doi:10.1371/journal.ppat.1003622 Editor: Kirk Deitsch, Weill Medical College of Cornell University, United States of America Received October 3, 2012; Accepted July 31, 2013; Published September 5, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by the Intramural Research Program of the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. LSG received funding from the Malaria Infection Biology Research and Training Program, NIAID, NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cbarillas@niaid.nih.gov ´ ´ ´ ¤ Current address: Laboratorio de Entomologia Medica, Instituto Rene Rachou, Fiocruz, Belo Horizonte, Minas Gerais, Brazil. the induction of heme peroxidase 2 (HPX2) and nicotinamide Introduction adenine dinucleotide phosphate (NADPH) oxidase 5 (NOX5) Malaria is a worldwide disease that is highly endemic in Sub- [8,10]. The HPX2/NOX5 system potentiates NO toxicity, Saharan Africa and causes over half a million deaths annually. enhances nitration, and reduces Plasmodium survival. Exposure of The mosquito Anopheles gambiae is a major vector of Plasmodium ookinetes to these chemical reactions as they traverse the midgut falciparum, the parasite responsible for most cases of human malaria cell modifies them and makes them ‘‘visible’’ to the mosquito in Africa. An. gambiae can mount effective antiplasmodial responses complement-like system [8]; however, the immune signaling by activating several signaling cascades involved in immune pathway(s) regulating the midgut epithelial response to infection regulation, such as the Imd, Toll, and STAT pathways [1–4]. have not been identified. Pathway activation leads to the transcription of effector genes that The JNK pathway is a mitogen-activated protein kinase mediate the antiplasmodial mechanism. The thioester-containing (MAPK) pathway that is highly conserved from mammals to protein 1 (TEP1) and the fibrinogen-related protein 9 (FBN9) are insects; however, our understanding of the role of JNK signaling in important components of the mosquito complement-like system insect immunity is limited. Several orthologs of genes that mediate that are produced by hemocytes and secreted into the mosquito JNK signaling in vertebrates have been identified in Drosophila and hemolymph; they bind to the ookinete surface and mediate An. gambiae [11,12]. The Jun-N-terminal kinase (JNK) is a MAP parasite lysis [5,6]. Activation of the Imd and Toll pathways kinase at the core of this signaling cascade that is activated by a decreases ookinete survival as parasites come in contact with the MAPK kinase (hemipterous,in D. melanogaster) (Figure 1A) [11,13– mosquito hemolymph by promoting TEP1-mediated lysis [1,3,7]. 17]. JNK phosphorylates the Jun and Fos transcription factors, In contrast, the STAT pathway targets a later stage of the parasite, giving rise to a Jun/Fos dimer (AP-1 complex) that activates the early oocysts, through a TEP1-independent response [4]. transcription of target genes (reviewed in [18]). JNK signaling is We have recently shown a functional link between midgut modulated by puckered (puc), a phosphatase that suppresses epithelial nitration and another mosquito antiplasmodial response signaling by dephosphorylating JNK. Puckered is part of a that targets the ookinete stage of the parasite, the complement-like negative feedback loop, because transcription of puc is regulated by the JNK pathway [16,19,20]. system [8]. Ookinete invasion results in extensive damage to the invaded cell [9] and induces a two-step epithelial nitration reaction In Drosophila, JNK signaling has been shown to be involved in a in which expression of nitric oxide synthase (NOS) is followed by wide range of biological processes including embryonic develop- PLOS Pathogens | www.plospathogens.org 1 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium Results Author Summary The An. gambiae JNK Pathway Limits Plasmodium berghei The mosquito Anopheles gambiae is a major vector of human malaria, a disease caused by Plasmodium falci- Infection parum parasites that results in more than half a million Five An. gambiae orthologs of genes known to be part of the JNK deaths each year. Several signaling pathways in the pathway signaling cascade in Drosophila have been identified mosquito have been shown to mediate the mosquito including two kinases, hemipterous (hep) and c-Jun N-terminal immune responses to Plasmodium infection. In this kinase (jnk); a phosphatase, puc; and two transcription factors, Jun manuscript we investigated the participation of the Jun- (jun) and Fos (fos) (Figure 1A,) [11]. These five genes are expressed N-terminal kinase (JNK) pathway in mosquito defense in the thoraces, abdomens, midguts, hemocytes, and in undevel- responses. We found that JNK signaling is required for oped ovaries from sugar-fed mosquitoes (Figure 1B, Table S1). Jun mosquito midgut cells to induce expression of two is expressed at low levels in the head, but the mRNAs of the other enzymes, HPx2 and NOX5, that mediate epithelial nitration genes could not be detected in this tissue (Figure 1B, Table S1). A in response to parasite invasion. These reactions modify notable enrichment of hep transcripts in the thorax and of jnk in the the parasites and promote activation of the mosquito ovary was observed (Figure 1B, Table S1). The transcriptional complement-like system that results in parasite lysis. The response of these five genes to infection with P. berghei (rodent JNK pathway also regulates the basal level of expression of malaria parasite) was analyzed in mosquito midguts collected at TEP1 and FBN9, two key components of the complement- different times after feeding on either healthy or P. berghei-infected like system that are produced by hemocytes and secreted mice. A significant increase in jnk, puc, jun and fos expression in into the mosquito hemolymph. Our studies revealed that response to infection was observed between 12–48 hours post JNK signaling plays a key role for mosquitoes to limit infection (hpi). In general, the magnitude and kinetics of the Plasmodium infection, making it an important determinant of malaria transmission to humans. inductions were variable between experiments. Jun at 24 and 48 hpi and JNK at 24 hpi had the most consistent inductions that were significant in three independent experiments (Figure 1C, Table S2). Hep expression changed the least in response to ment, apoptosis, stress response, cell proliferation and differentiation, and immunity [18]. The JNK pathway has a great deal of complexity Plasmodium infection (Figure 1C, Table S2). Only a modest increase was observed in one of the replicates at 12 hpi but, in and is known to receive input from multiple upstream genes, yet to be defined in insects, and from lateral inputs from components of other another, the expression was lower after infection than in the uninfected control. Although activation of the JNK pathway signaling cascades. For example TAK1, a kinase that is part of the Imd pathway, can also activate JNK signaling [21–23]. It is believed involves a cascade of post-translational phosphorylation events, transcription of JNK pathway members has been reported to that this complex organization reflects the broad range of responses that are influenced by JNK signaling. increase upon Plasmodium infection in Anopheles and transcriptional activation of JNK at the mRNA and protein level has also been Many different stimuli are known to activate the JNK pathway, observed in Drosophila midguts in response to bacterial challenge including microbial elicitors. In particular, the participation of [26–28]. JNK protein expression was also induced in the mosquito JNK signaling in antibacterial responses has been well document- midgut in response to Plasmodium infection (Figure S1). This ed in Drosophila. Lipopolysaccharide (LPS) is a key elicitor of JNK indicates that JNK signaling in infected midguts may be enhanced pathway activity in immune-competent cells and flies [13– by increased expression of several components of the cascade. 15,17,22,23], and flies that are deficient in puc (and therefore have an overactive JNK pathway output) display increased We confirmed that JNK silencing (Figure S2) enhances P. berghei infection (Figure 1D), as previously shown [25]. Furthermore, resistance to Gram bacteria [19]. In the An. gambiae 4a3B cell line, JNK signaling was weakly activated by H O , while LPS silencing other genes involved in JNK activation—such as hep, jun, 2 2 and fos (Figure S2)—also enhanced the intensity of infection, elicited a strong response [11]. The response of JNK signaling to increasing the median number of oocysts by 3.8 to 4.9 fold, LPS has also been observed in human dendritic cells and relative to the dsLacZ control (Figure 1, D and E, Table S3) splenocytes [24]. (p,0.001; Kolmogorov-Smirnov [KS] test). As expected, over- We have previously shown that JNK regulates expression of activation of this cascade by silencing puc (Figure S2), a several genes that protect An. gambiae mosquitoes from oxidative phosphatase that normally suppresses JNK signaling, had the damage, such as oxidation resistance 1 (OXR1), catalase, and opposite effect and greatly reduced the intensity (Figure 1D, Table glutathione peroxidase [25]. Silencing of these effector genes S3) (p,0.001; KS test) and the prevalence of infection from 68% increased reactive oxygen species (ROS) levels and reduced to 41% (p,0.005; chi-squared [x ] test). Co-silencing Jun reversed Plasmodium survival. Paradoxically, however, JNK silencing had the antiplasmodial effect of silencing puc (Figure 1E, Table S3) and the opposite effect and enhanced infection, suggesting that— increased the prevalence of infection from 33% to 84% (p,0.001; besides the role in ROS balance—JNK may mediate some x test), indicating that Jun is downstream of puc and confirming antiplasmodial response [25]. In this manuscript, we present a the functional link between these two genes in An. gambiae. detailed functional analysis of several genes that mediate JNK signaling in An. gambiae and identify two key mechanisms by which this cascade mediates antiplasmodial immunity. JNK activation JNK Signaling Activates Midgut Epithelial Nitration induces expression of HPx2 and NOX5, the two enzymes that We have recently shown that the HPx2/NOX5 system mediate epithelial nitration in response to ookinete invasion [8]. In potentiates NO toxicity and mediates nitration of midgut epithelial addition, JNK signaling regulates the basal levels of expression of cells in response to Plasmodium invasion. The potential participa- TEP1 and FBN9, two effector proteins produced by hemocytes tion of JNK signaling in the induction of these two enzymes and that mediate ookinete lysis [5,6]. The participation of JNK epithelial nitration was investigated. A robust increase in HPx2 signaling in the antiplasmodial responses of the A. gambie L3–5 and NOX5 expression was observed in the dsLacZ-injected strain that has been genetically selected to be refractory to control group (Figure 2A) in response to Plasmodium infection, as Plasmodium infection was also investigated. previously shown in uninjected females [8]; however, HPx2 was PLOS Pathogens | www.plospathogens.org 2 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium Figure 1. The JNK Pathway and Plasmodium berghei infection in Anopheles gambiae. (A) Diagram representing the organization of the JNK signaling cascade based on functional studies from vertebrates and Drosophila. Five An. gambiae orthologs were functionally characterized, including two kinases, hemipterous (hep) and c-Jun N-terminal kinase (jnk); a phosphatase, puc; and two transcription factors, Jun (jun) and Fos (fos) (B) Basal mRNA expression of putative genes from the JNK pathway in adult females. Hemipterous (Hep), Jun N-terminal kinase (JNK), Jun and Fos transcription factors and puckered (puc) mRNA levels in different organs of sugar-fed females. Mg, midgut; H, head; Th, thorax; Ab, abdomen; Hc, hemocyte; Ov, ovaries. Expression in different tissues relative to midgut levels, for which the mean was given a value of ‘‘1’’. Error bars indicate SEM of two biological replicates. (see Table S11 for gene ID numbers and primer sequences) (C) Midgut expression of members of the JNK pathway in response to Plasmodium infection in three independent experiments. Ratio of expression in infected/control blood-fed mosquitoes of Hep, JNK, Puc, Jun and Fos mRNA levels in midguts of mosquitoes from 3 independent experiments (green, red and blue bars). Error bars indicate SEM of two technical replicates. The expression analysis in each biological replicate is shown in Table S3. P-values determined by Student’s-T test after log 2 transformation; **, p,0.01, ***, p,0.001. *, p,0.05; **, p,0.01, ***, p,0.001. (D) Effect of silencing JNK pathway members on P. berghei infection. (E) Effect of silencing the transcription factor jun alone or and co-silencing jun and the negative regulator puc on Plasmodium infection. For (D) and (E), the green dots represent oocyst counts from individual midguts and the horizontal red bar indicates the median infection level. Groups were compared using the KS, Mann-Whitney and Kruskal-Wallis tests with Dunn’s post test (see Table S3). The P-values for the Mann-Whitney tests are shown. Graphs represent samples pooled from three biological replicates with comparable (not statistically different) medians in their dsLacZ-injected groups (n = number of midguts). doi:10.1371/journal.ppat.1003622.g001 PLOS Pathogens | www.plospathogens.org 3 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium Figure 2. The JNK Pathway and Midgut Epithelial Nitration. (A) Effect of JNK, Jun or puc silencing on the inducible midgut mRNA expression of HPx2 and NOX5 in response to Plasmodium berghei infection. C, control mosquitoes fed on a healthy mouse (gray bars); I, infected mosquitoes fed on a P. berghei-infected mouse (red bars). Mean expression in infected midguts relative to uninfected blood-fed controls, for which the mean was adjusted to a value of ‘‘1’’; The bars represent the SEM of three biological replicates from independent experiments (see Table S4). P-values determined by paired Student’s-T test after log 2 transformation; **, p,0.01, ***, p,0.001. (B) Effect of silencing JNK, Jun or puc on infection-inducible in vivo midgut nitration. C, control mosquitoes fed on a healthy mouse (gray bars); I, infected mosquitoes fed on a P. berghei-infected mouse (blue bars). Graphs represent one of two biological replicates (see Figure S4 and Table S5); error bars indicate SEM of four technical replicates. P-value determined by Student’s t-test; **, p,0.01, ***, p,0.001. (C and D) Effect of co-silencing hpx2 (C) or nox5 (D) on the phenotype of silencing the negative regulator puc. Green dots represent oocyst counts in individual midguts; horizontal red bar indicates median infection intensity. P-values were determined by Mann-Whitney test; ns, not significant. Graphs represent data from three biological replicates with comparable medians in their dsLacZ-injected groups. n = total number of midguts examined. doi:10.1371/journal.ppat.1003622.g002 no longer induced in infected midguts and NOX5 expression was alone. Co-silencing HPx2 increased the median number of oocysts/ significantly reduced, relative to uninfected controls when JNK midgut from 0 to 17 (p,0.0001; KS test) and the prevalence of infection from 20% to 100% (p,0.0001; x test). Co-silencing was silenced; and expression of both HPx2 and NOX5 is reduced in infected midguts when jun is silenced (Figure 2A, Table S4). The NOX5 and puc (Figure 2D, Table S3) increased the median number of oocysts/midgut from 0 to 10 (p,0.0001; KS test) to the same level transcriptional induction of HPx2 in response to infection was as the dsLacZ control, and the prevalence of infection from 22% to more robust when puc was silenced, but NOX5 induction was no 88% (p,0.0001; x test). This indicates that HPx2 and NOX5 are longer observed (Figure 2A, Table S4). In agreement with the downstream of puc and mediate, to a large extent, the antiplasmodial overall transcriptional responses, when JNK was silenced, in vivo response triggered by the JNK activation. midgut nitration no longer increased in response to Plasmodium infection, was lower than in the uninfected controls when jun was The JNK Pathway Regulates Expression of Hemocyte- silenced, while silencing puc had the opposite effect and enhanced the nitration response. (Figure 2B, Figure S3, Table S5). Derived Antiplasmodial Effectors Furthermore, co-silencing HPx2 (Figure 2C, Table S3) com- Jun expression is induced in mosquito hemocytes 24 hpi with P. pletely rescued the dramatic antiplasmodial effect of silencing puc berghei [27], suggesting that JNK signaling in these cells could also PLOS Pathogens | www.plospathogens.org 4 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium injected groups (see Table S1) n = total number of midguts examined. doi:10.1371/journal.ppat.1003622.g003 be an important component of antiplasmodial immunity. TEP1 and FBN9 are proteins constitutively produced by hemocytes that are secreted into the mosquito hemolymph, bind to the surface of P. berghei ookinetes, and mediate parasite lysis [5,6]. We investigated the hypothesis that these hemocyte-derived proteins are regulated by the JNK pathway and are important effectors of this signaling cascade. Silencing jun or fos significantly reduced TEP1 by 94% and 69%, respectively (p,0.001 and p,0.05; Student’s t-test) and reduced FBN9 expression by 62% and 70%, respectively (Figure 3, A and B, Table S6) (p,0.01 and p,0.001; Student’s t-test) but had no effect on the expression levels of other hemocyte-specific genes such as APL1A or APL1C, and fos silencing actually resulted in a modest increase in LRIM1 expression (Figure S4, Table S6). Conversely, silencing puc significantly increased expression of both TEP1 and FBN9 by 1.86 and 2.6 fold, respectively (Figure 3, A and B, Table S6) (p p,0.01; Student’s t-test). Silencing JNK did not affect the total number of circulating hemocytes or the proportions of granulocytes, oenocytoids, or prohemocytes circu- lating in the mosquito (Figure S5). We have previously shown that induction of HPx2 and NOX5 mediates epithelial nitration and that the activity of these enzymes promotes both TEP1 binding to the ookinete surface and parasite lysis [8]. Participation of TEP1 and FBN9 as final effectors of the JNK antiplasmodial response was explored by co-silencing these genes with puc. Co-silencing TEP1 increased the median number of oocysts/midgut from 1 to 21.5 (p,0.0001; KS test) and the prevalence of infection from 53% to 88% (p,0.02, x test) relative to silencing puc alone (Figure 3C, Table S3). Co-silencing FBN9 had a similar effect, increasing the median number of oocysts/midgut from 0 to 13.5 (p,0.0001; KS test) and the prevalence of infection from 35% to 84% (p,0.0001, x test) (Figure 3D, TableS3). The JNK Pathway Contributes to Parasite Elimination in the An. gambiae Refractory Strain The An. gambiae refractory (R) strain was selected to be refractory to Plasmodium cynomolgi (simian malaria) infection but also eliminates most Plasmodium species, including P. berghei [29]. In this mosquito strain, ookinetes develop and invade the midgut, but they are killed and covered with melanin, a black, insoluble pigment [29]. R females are in a chronic state of oxidative stress that is exacerbated by blood feeding [30], and TEP1 is known to be a critical mediator of P. berghei melanization and killing [5]. We have shown that the JNK pathway regulates expression of two enzymes that mediate midgut epithelial nitration: NOX5, an oxidase that generates ROS, and the heme peroxidase, HPX2. Furthermore, exposure of ookinetes to these nitration reactions as they traverse the midgut epithelial cell promotes TEP1 activation [8]. The hypothesis that the refractory phenotype may be Figure 3. The JNK Pathway and Hemocyte Antiplasmodial mediated, at least in part, by the JNK signaling pathway was Effector Genes. (A and B) Effect of silencing jun, fos,or puc on basal investigated. expression of TEP1 (A) and FBN9 (B) in circulating hemocytes. Mean We first compared the basal level of mRNA expression of the expression level in silenced samples, relative to the dsLacZ-injected genes involved in JNK signaling between the susceptible (S) G3 An. control that was adjusted to a value of ‘‘1’’ and is indicated by the red gambiae and the R strain. The basal midgut expression of all the dotted line. The bars represent the SEM of two biological replicates from independent experiments (see Table S4). P-values determined by genes involved in JNK signaling was higher in the R strain. Student’s-T test after log 2 transformation; *, p,0.05, **, p,0.01, ***, Midgut jnk expression was dramatically higher (4.3 fold), while the p,0.001. (C and D) Effect of co-silencing TEP1 (C) or FBN9 (D) on the overexpression of hep was less prominent (1.5 fold) (Figure 4A, phenotype of silencing negative the regulator puc. Green dots Table S7). The basal expression level of all genes of the JNK represent oocyst counts in individual midguts, and the horizontal red pathway (hep, jnk, puc, jun, and fos) was also significantly higher in bar indicates median infection intensity. P-values were determined by whole body samples of R females, ranging from 2.1 to 4.8 fold Mann-Whitney test; ns, not significant. Graphs represent data from three biological replicates with comparable medians in the dsLacZ- (Figure S6, Table S7). Higher puc expression is indicative of PLOS Pathogens | www.plospathogens.org 5 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium The contribution of the JNK pathway to the refractory phenotype was directly tested by reducing JNK expression via gene silencing. JNK silencing had a dramatic effect, increasing the prevalence of infection from 0 to 70% (Figure 4C) (p,0.0001; x test), and the median number of oocysts from 0 to 6 oocysts/ midgut (Figure 4C, Table S10) (p,0.001; KS test). The total number of parasites (live and melanized) was not significantly different between the dsLacZ control and the JNK-silenced group, indicating that a similar number of ookinetes invaded the midgut and that the difference in infection prevalence was due to ookinete survival once they traversed the midgut. Of the total number of parasites present, 99.6% of parasites were melanized in the dsLacZ group; this decreased to 32% when JNK was silenced (Figure 4C) (p,0.0001, x test). Discussion The immune response of An. gambiae mosquitoes against Plasmodium parasites is mediated by activation of immune-related signal transduction pathways. We carried out a functional characterization of five An. gambiae orthologs of genes known to mediate JNK signaling in Drosophila. Our studies implicate the JNK pathway as an important mediator of two coordinated steps of the mosquito anti-Plasmodium immune response and as a major determinant of the killing mechanism in a highly refractory strain of An. gambiae. JNK signaling triggers the transcriptional activation of HPX2 and NOX5, two key enzymatic effectors of midgut epithelial cells, in response to ookinete invasion. Induction of these two enzymes potentiates nitration and limits Plasmodium survival. This was directly confirmed by the observation that midgut nitration is greatly diminished when JNK signaling is disrupted by silencing JNK or jun. Overactivation of the JNK pathway by silencing puc, greatly increased midgut HPx2 expression and nitration in Figure 4. Participation of the JNK Pathway in L3–5 Mosquitoes response to Plasmodium infection. Interestingly, puc silencing did Refractory (R) responses to Plasmodium berghei Infection. (A) Basal mRNA expression of genes from the JNK pathway in the midgut not induce higher levels of NOX5 expression. This enzyme and hemocytes of G3 susceptible (S) (gray) and R (blue) mosquitoes. (B) generates reactive oxygen species that could be potentially toxic. Expression of effector genes regulated by the JNK pathway in S (gray) Our results suggest that in the absence of puc, there might be and R (blue) mosquitoes. Basal mRNA levels of HPx2 and NOX5 in the alternative mechanisms that limit NOX5 expression,probably to midgut, and of TEP1 and FBN9 in hemocytes. Graphs represent the prevent deleterious effects on the host. Previous studies in a variety expression level in R females, relative to S females, that were adjusted of mammalian cell types have also shown that NOX5 and other to a value of ‘‘1’’; for R females samples the bars represent the SEM of three biological replicates from independent experiments (see Table NADPH oxidases are regulated by the JNK pathway [31–33] and S4). P-values determined by paired Student’s-T test after log2 induction of a nitrogen dioxide-producing heme peroxidase has transformation; *, p,0.05, **, p,0.01, ***, p,0.001. (C) Effect of also been shown to be mediated by the JNK pathway [34]. silencing JNK (right panel) in the number of melanized and live The process of nitration is clearly an essential step in the parasites on individual midguts of R mosquitoes. Red dots indicate the destruction of malaria parasites, evidenced by the significant number of parasites on an individual midgut, live (y-axis) and melanized increase in parasite survival upon silencing either HPx2 or NOX5. (x-axis). Green horizontal bars indicate median infection intensities. Inset pie graphs represent the percentage of total parasites for each It is also a critical outcome of JNK activation, as the considerable group displaying a live (green) or melanized (black) phenotype; resistance conferred by puc silencing is reverted by co-silencing percentage displayed refers to melanized parasites. Graphs represent either of these two enzymes (Figure 2). We therefore propose that data from three biological replicates (see Table S10). (n = number of the JNK pathway is part of an ‘‘alarm system’’ triggered by midguts analyzed). parasite invasion that activates expression of NOX5 and HPx2, doi:10.1371/journal.ppat.1003622.g004 two enzymes that catalyze nitration reactions, that label ookinetes increased JNK activation, because puc expression is transcription- for destruction as they traverse the mosquito midgut. ally regulated by the JNK pathway. In hemocytes, there was no The dramatic reduction in TEP1 and FBN9 mRNA levels in difference in hep, fos and puc expression between the mosquito hemocytes when the JNK pathway was disrupted by silencing jun strains, but jnk and jun levels were also significantly higher in the R or fos appears to be specific, because expression of other hemocyte- strain (Figure 4A, Table S7). Furthermore, expression of effector specific genes involved in the regulation of complement activation genes of the JNK pathway was also higher in the R strain. In the (APL1A, APL1C, and LRIM1) was not reduced. We also midgut, basal HPx2 and NOX5 expression was 2.8 and 3.5 fold confirmed that the differences in expression were not due to times higher, respectively (Figure 4B, Table S8) (p,0.01; paired t- significant changes in the number or type of hemocytes present in test for both); while in hemocytes, TEP1 and FBN9 expression was silenced mosquitoes. The co-silencing experiments with puc 3.2 and 5.9 fold higher in R mosquitoes, respectively (Figure 4B, confirmed that both TEP1 and FBN9 are downstream of JNK. Table S9) (p,0.01; paired t-test for both). This indicates that the basal level of TEP1 and FBN9 expression in PLOS Pathogens | www.plospathogens.org 6 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium mosquito hemocytes is regulated by the JNK pathway and that Mosquito Tissue Dissections both genes are important effectors of the lytic response mediated Approximately 15–20 female mosquitoes 3- to 4 days post- by this cascade. emergence were removed cold anesthetized to immobilize them. Previous studies have shown that the R strain is in a chronic Hemocytes from individual mosquitoes were extracted using the state of oxidative stress that is exacerbated when adult females take method outlined below, Trizol was added and samples were kept a blood meal [30]. Genome-wide transcriptional analysis revealed on ice. Then the head was severed from the thorax, and the thorax higher expression in the R strain of several immune genes, genes from the abdomen using a clean scalpel. The midgut and encoded by the mitochondrial genome, and genes involved in undeveloped ovaries were then pulled from the abdomen using oxido/reductive processes or ROS detoxification relative to S fine forceps. Samples from each tissue were pooled together and females [30]. The R strain also exhibits impaired mitochondrial stored in RNAlater (Ambion, Austin, Texas, USA) in a microfuge state-3 respiration and increased rate of electron leak [35]. NOX5 tube. RNA was extracted, cDNA was generated, and gene is a member of the NAPDH oxidase family and generates expression was quantified using the methods indicated below for superoxide anion, which is quickly converted into hydrogen ‘‘Quantification of gene expression’’ (for tissues except hemocytes) peroxide by superoxide dismutase (reviewed by Bedard and Kraus or ‘‘Hemocyte collection and counting’’ (for hemocytes). [36]). We found that the genes that mediate signaling (hep, JNK, jun, fos, and puc) and key downstream effectors of this pathway in Quantification of Gene Expression the midgut (HPx2 and NOX5) and hemocytes (TEP1 and FBN9) Fifteen to twenty whole female mosquitoes or dissected tissues have increased basal levels of expression. Higher levels of were homogenized in RNAlater (Ambion) and subject to RNA HPx2and NOX5 are expected to accelerate the rate of epithelial extraction using RNAeasy (Qiagen, Los Angeles, California, USA) nitration, and higher hemolymph levels of TEP1 and FBN9 would kits according to the manufacturer’s instructions and first-strand promote parasite lysis. The increase in basal expression of NOX5 cDNA was synthesized using QuantiTect reverse transcriptase may be responsible, at least in part, for the higher constitutive (Qiagen). Gene expression was assessed by SYBR green quanti- levels of systemic ROS that have been observed in the R strain tative real-time PCR (DyNAmo HS; New England Biolabs, [30]. In An. gambiae, ROS levels have been shown to modulate Beverly, Massachusetts, USA) using the CFX96 system (Bio-Rad, immunity to both bacteria and Plasmodium [30,37]. The dramatic Hercules, California, USA). Each sample was assayed using two reduction in melanization and the increase in parasite survival technical replicas and 2–3 biological replicates. The amount of when JNK signaling is disrupted in the R strain confirm the key cDNA template present in each sample was normalized using the role of this pathway in mosquito antiplasmodial immunity. expression An. gambiae ribosomal protein S7 as reference. Fold 2DD We have recently shown that some P. falciparum strains, such as change values were derived using the 2 Ct method. The values NF54, are able to infect the An. gambiae R strain and that silencing were adjusted in each experiment by dividing each of the technical TEP1 did not enhance parasite survival, indicating that the replicates in the control and treatment groups by the mean of the mosquito complement-like system was not activated. In contrast, control group, thus adjusting the control groups to a value of ‘‘1’’. other parasite strains (such as 7G8) were almost completely The statistical analysis was done using the Student’s T-test after eliminated through a TEP1-mediated mechanism [38]. Co- log2 transformation of the mean value of each biological replicate infection experiments with a P. falciparum strain that is melanized for each treatment. Primers used are provided in Table S11; when and one that survives suggest that survival is genetically appropriate, primers were verified against R strain sequences determined by a parasite-autonomous mechanism, because the obtained by Solexa transcriptome sequencing of adult S and R survival (or lack thereof) of one strain does not affect the outcome females (Barillas-Mury Lab, unpublished). The primers used for of the other strain in the same mosquito [38]. Together, this TEP1 expression analysis in Figure 3 (S strain) are located in a indicates that some P. falciparum strains are susceptible to a TEP1- polymorphic region between S and R strains of A. gambiae. For this dependent killing mechanism, while others have the capacity to reason, a different primer set in a conserved region was used for evade it. Given the critical role of TEP1 as an effector of the JNK the TEP1 expression data presented in Figure 4 (comparison pathway, it is likely that P. falciparum strains also differ in their between S and R strains). ability to avoid—or perhaps may even actively suppress— activation of this signaling cascade. Detailed studies on the P. berghei Maintenance and Infection participation of the JNK pathway in mosquito antiplasmodial P. berghei (GFP-CON transgenic 259cl2 strain) parasites from responses to different P. falciparum strains are currently under way frozen stocks were administered intraperitoneally to donor mice. and may shed new insights into immune evasion strategies that When the parasitemias of donor mice reached 10–20%, 20–50 ml promote human malaria transmission. of infected blood was transferred to naı ¨ve mice via intraperitoneal injection. All mice were 3- to 5-week-old BALB/c females. Materials and Methods Parasitemia was assessed by light microscopy inspection of Giemsa-stained thin smears obtained by tail snips. At 2–3 days Ethics Statement post emergence, female mosquitoes were deprived of sucrose Public Health Service Animal Welfare Assurance #A4149-01 solution for 6–12 h, then allowed to feed on anesthetized mice guidelines were followed according to the National Institutes of infected with P. berghei at 3–7% parasitemia and exhibiting 1–3 Health Animal (NIH) Office of Animal Care and Use (OACU). exflagellation events per field, as previously described [40]. Where These studies were done according to the NIH animal study indicated, naıve blood-fed control mosquito groups were fed on protocol (ASP) approved by the NIH Animal Care and User uninfected mice of the same age. All P. berghei-infected mosquitoes Committee (ACUC), with approval ID ASP-LMVR5. and corresponding control mosquitoes were kept at 21uC and 80% humidity. Unless otherwise indicated, P. berghei infection intensities Mosquito Strains and Rearing were quantified 7–9 days post infection (dpi) by epifluorescent An. gambiae G3 and L3–5 mosquitoes were reared at 27uC with microscopy inspection of dissected midguts containing GFP- 80% humidity on a 12-h light/dark cycle. Cotton balls soaked in expressing parasites fixed in 4% paraformaldehyde and mounted 10% sucrose in water were provided as previously described [39]. in Vectashield (Vector Labs, Burlingame, California, USA), PLOS Pathogens | www.plospathogens.org 7 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium enabling manual counting of fluorescent oocysts and/or melanized diluted in PBT (1:3,000) at 4uC. Samples were washed with ookinetes. PBT and 4 were incubated with a secondary alkaline phosphatase- conjugated antibody (1:5,000) diluted in PBT, while the remaining sample was reserved as a background signal control. All samples RNAi Gene-Silencing Assays were incubated with rNPP–r-nitrophenylphosphate (Sigma Al- T7 promoter sequences were introduced at both ends using two drich) and read in a spectrofluorometer plate reader at 405 nm. different strategies. For LacZ, NOX5 and Tep1, cDNA fragments The relative nitration for each experimental treatment was were amplified using the primers given in Table S11 and cloned confirmed in at least two independent experiments. into the pCRII-TOPO vector (Invitrogen, Carlsbad, California, USA) following the manufacturer’s instructions. T7 promoters were introduced by amplifying the cloned insert using the primers: Hemocyte Collection and Counting M13F: 59-GTAAAACGA CGGCCAGT-39 and M13R: 59- Female mosquitoes were cold anesthetized and injected CTCGAGTAATACGACTCACTATAGGGCAGGAAA intrathoracically with a micropipette needle loaded with hemocyte CAGCTATGAC-39, which anneal to the vector as previously perfusion buffer (60% Schneider’s insect medium, 30% citrate, reported [8]. These PCR products were used as templates for 10% FBS). After insertion of the needle into the thorax, a small generating dsRNA as described below. For all other genes, the T7 incision was made in the lower abdomen, and buffer was sequences were included in the gene-specific primers and cDNA dispensed through the needle and collected 2 ml at a time from fragments of about ,300-bp were generated (Table S11). For all the incision using siliconized pipet tips for a total of 10–12 ml. genes, sense and antisense RNAs were synthesized simoulta- Perfusions were then either collected into a siliconized Eppendorf neously from templates and purified using the T7 RNAi Mega- tube for RNA extraction or applied to a disposable hemocytom- script kit (Ambion), eluted in water, and concentrated to 3 mg/ml eter (InCyto, Seoul, South Korea) for counting. For RNA using a Microcon YM-100 filter (Millipore, Bedford, Massachu- extraction, tubes were centrifuged for 30 min at 12,0006g to setts, USA). About 69 nl of this dsRNA preparation was injected pellet the cells; supernatant was removed, and 500 ml Trizol was into the thorax of cold-anesthetized, 2- to 3-day-old female added. RNA was isolated according to phenol/chloroform mosquitoes using a nano-injector (Nanoject; Drummond Scientif- extraction as suggested by Trizol protocol. For counting, cells ic, Broomall, Pennsylvania, USA) fitted with a glass capillary were visualized under light microscope with 406 objective. Cells needle according to previous protocols. dsRNA targeting LacZ contained within the marked grid were separated into three cell was used in each experiment to control for any unspecific effect of types (granulocyte, oenocytoid, prohemocyte) and counted ac- wounding and dsRNA exposure. Efficiency of silencing was cordingly. Population proportions were calculated and total quantified 2–3 days after dsRNA injection by real-time quantita- numbers of cells per mosquito were determined by manufacturer’s tive RT-PCR with the An. gambiae ribosomal S7 gene as the extrapolation. internal control for normalization. Primers for silencing verifica- tion are listed in Table S11, and silencing efficiencies are displayed Statistical Analysis in Figure S2. Fold change differences in gene expression across groups were normalized by log transformation. The statistical analysis of Immunoblotting differences in gene expression was done using the Student’s T-test Midguts were dissected and cleaned of blood meal in cold, after log2 transformation of the mean value of each biological sterile PBS supplemented with 1% levamisole (Sigma-Aldrich, St. replicate from independent experiments. Oocyst distributions were Louis, Missouri, USA). Pools of 5–10 midguts were transferred to a determined not to be normal, and were compared to one another microfuge tube and homogenized in PBS with protease inhibitor, using the Kolmogorov-Smirnov (KS), Mann-Whitney tests and levamisole, and phosphoStop (Roche Applied Science, Madison, Kruskal-Wallis tests with Dunn’s post-test (see Tables S3 and S10). Wisconsin, USA), prepared using NuPAGE buffers and reducing When the median infection levels of the dsLacZ group of two or agent (Invitrogen), and run on NuPAGE Bis-Tris 4–12% gels more biological replicates were not statistically different using the (Invitrogen) according to manufacturer’s instructions. Proteins Mann-Whitney test, the data were merged (See Tables S3 and S10). were then transferred from gels to membranes using the iBlot Oocyst prevalences were compared using x tests. Differences in nitration levels were compared using the Student’s t-test. P-values system (Invitrogen). Membranes were blocked in TBS with 5% milk +0 .05% Tween, washed, and incubated in fresh milk solution represented in figures are given in corresponding figure legends and with primary antibody against JNK (1:2000; Santa Cruz text. All statistical analyses were performed using Prism 5.01 Biotechnology, Santa Cruz, California, USA) overnight. They software (GraphPad Software, La Jolla, California, USA). were then washed and incubated in fresh milk solution with alkaline phosphatase-conjugated secondary antibody against Supporting Information rabbit (1:5000) for 2 h with TBS washings between each step. Figure S1 JNK protein midgut expression in response to Membranes were finally rinsed with TBS and incubated for P. berghei infection. JNK was detected with commercial 30 min (anti-JNK) with Western Blue substrate (Promega Corp., antibodies in Western Blots from midgut homogenates obtained Madison, Wisconsin, USA) to visualize bands. from sugar-fed females (SF), control (C) females fed on a healthy mouse or infected females (I) fed on P. berghei-infected mouse. In vivo Nitration Assays Samples were collected 24 and 48 h after feeding. The size of the Assays were performed according to previously established reference molecular markers is expressed as kDa and is indicated methods [8]. In brief, five midguts were dissected, fixed, and by the dots on the left. washed with PBS, then triturated and incubated in amino triazole (DOCX) (10 mg/ml). Pelleted midgut fragments were incubated with 2 mM levamisole, then blocked with PBT and washed. The pellet Figure S2 Silencing Efficiency in Sugar-Fed Mosquitoes. was subsequently resuspended in 50 ml of PBT, and five replicates Silencing efficiency in sugar-fed mosquitoes after systemic of one-midgut equivalents (10 ml of the 50-ml suspension) were injection of dsRNA for the target gene relative to the expression incubated overnight with anti-nitrotyrosine primary antibody level compared with dsLacZ-injected control mosquitoes. Whole PLOS Pathogens | www.plospathogens.org 8 September 2013 | Volume 9 | Issue 9 | e1003622 JNK Signaling and Mosquito Immunity to Plasmodium body expression was determined in sugar fed females either 2 days Table S3 Summary of oocyst data for all G3 infections. (HPx2 and NOX5) or 3 days (all other genes) after injection. (DOCX) (Mean 6 SE). Table S4 Quantification of HPx2 and NOX5 expression (DOCX) in the midgut following silencing of JNK pathway Figure S3 Effect of silencing JNK, jun or puc on members. infection-induced in vivo midgut nitration. C, control (DOCX) mosquitoes fed on a healthy mouse (gray bars); I, infected Table S5 Quantification of nitration in the midgut mosquitoes fed on a P. berghei-infected mouse (blue bars). Graphs following silencing of JNK pathway members. represent one of two biological replicates (see Figure 2B and Table (DOCX) S5); error bars indicate SEM of three technical replicates. P-value determined by Student’s t-test; *, p,0.05, **, p,0.01. Table S6 Quantification of effector expression in he- (DOCX) mocytes following silencing of JNK pathway members. (DOCX) Figure S4 Effect of Silencing Jun or Fos on LRIM1, APL1A and APL1C expression. Hemocyte mRNA expression Table S7 Quantification of JNK pathway member of LRIM1, APL1A, and APL1C genes was determined 3 days expression in G3 and L3–5 mosquitoes and tissues. after systemic injection of either dsLacZ, dsJun or dsFos (Mean 6 (DOCX) SEM). (* indicates p,0.05; Student’s t-test) Table S8 Quantification of HPx2 and NOX5 in G3 and (DOCX) L3–5 midguts. Figure S5 Effect of Silencing JNK on hemocyte popula- (DOCX) tions. Effect of silencing JNK on the total number of hemocytes and Table S9 Quantification of HPx2 and NOX5 in G3 and the relative abundance of granulocytes, oenocytoids, and prohemo- L3–5 hemocytes. cytes 4 days after systemic injection of dsLacZ or dsJNK (Mean 6 (DOCX) SEM). No significant differences were observed (Student’s t-test). (DOCX) Table S10 Summary of oocyst data for all L3–5 infections. Figure S6 Relative expression of genes from the JNK (DOCX) pathway in susceptible and refractory An. gambiae mosquitoes. Basal mRNA levels of hep, JNK, jun and fos in Table S11 Primers used for dsRNA templates and susceptible (S, gray) and refractory (R, blue) mosquitoes (Mean 6 silencing validation/real-time PCR. SEM). Graphs represent the expression level in R females, relative (DOCX) to S females, that were adjusted to a value of ‘‘1’’; for R females samples the bars represent the SEM of three biological replicates Acknowledgments (see Table S4). P-values determined by paired Student’s-T test after log2 transformation; *, p,0.05, **, p,0.01,. We thank Alvaro Molina-Cruz for insightful comments and discussions, Andre ´ Laughinghouse and Kevin Lee for insectary support, and Brenda (DOCX) Rae Marshall for editorial assistance. Table S1 Quantification of tissue-specific expression of JNK pathway members. Author Contributions (DOCX) Conceived and designed the experiments: LSG CBM . Performed the Table S2 Time-course quantification of P. berghei- experiments: LSG GdAO . Analyzed the data: LSG. 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