Anopheles Imd Pathway Factors and Effectors in Infection Intensity-Dependent Anti-Plasmodium Action

The Anopheles gambiae immune response against Plasmodium falciparum, an etiological agent of human malaria, has been identified as a source of potential anti-Plasmodium genes and mechanisms to be exploited in efforts to control the malaria transmission cycle. One such mechanism is the Imd pathway, a conserved immune signaling pathway that has potent anti-P. falciparum activity. Silencing the expression of caspar, a negative regulator of the Imd pathway, or over-expressing rel2, an Imd pathway-controlled NFkappaB transcription factor, confers a resistant phenotype on A. gambiae mosquitoes that involves an array of immune effector genes. However, unexplored features of this powerful mechanism that may be essential for the implementation of a malaria control strategy still remain. Using RNA interference to singly or dually silence caspar and other components of the Imd pathway, we have identified genes participating in the anti-Plasmodium signaling module regulated by Caspar, each of which represents a potential target to achieve over-activation of the pathway. We also determined that the Imd pathway is most potent against the parasite's ookinete stage, yet also has reasonable activity against early oocysts and lesser activity against late oocysts. We further demonstrated that caspar silencing alone is sufficient to induce a robust anti-P. falciparum response even in the relative absence of resident gut microbiota. Finally, we established the relevance of the Imd pathway components and regulated effectors TEP1, APL1, and LRIM1 in parasite infection intensity-dependent defense, thereby shedding light on the relevance of laboratory versus natural infection intensity models. Our results highlight the physiological considerations that are integral to a thoughtful implementation of Imd pathway manipulation in A. gambiae as part of an effort to limit the malaria transmission cycle, and they reveal a variety of previously unrecognized nuances in the Imd-directed immune response against P. falciparum

Caspar controls resistance to Plasmodium falciparum in diverse anopheline species.

Immune responses mounted by the malaria vector Anopheles gambiae are largely regulated by the Toll and Imd (immune deficiency) pathways via the NF-kappaB transcription factors Rel1 and Rel2, which are controlled by the negative regulators Cactus and Caspar, respectively. Rel1- and Rel2-dependent transcription in A. gambiae has been shown to be particularly critical to the mosquito's ability to manage infection with the rodent malaria parasite Plasmodium berghei. Using RNA interference to deplete the negative regulators of these pathways, we found that Rel2 controls resistance of A. gambiae to the human malaria parasite Plasmodium falciparum, whereas Rel 1 activation reduced infection levels. The universal relevance of this defense system across Anopheles species was established by showing that caspar silencing also prevents the development of P. falciparum in the major malaria vectors of Asia and South America, A. stephensi and A. albimanus, respectively. Parallel studies suggest that while Imd pathway activation is most effective against P. falciparum, the Toll pathway is most efficient against P. berghei, highlighting a significant discrepancy between the human pathogen and its rodent model. High throughput gene expression analyses identified a plethora of genes regulated by the activation of the two Rel factors and revealed that the Toll pathway played a more diverse role in mosquito biology than the Imd pathway, which was more immunity-specific. Further analyses of key anti-Plasmodium factors suggest they may be responsible for the Imd pathway-mediated resistance phenotype. Additionally, we found that the fitness cost caused by Rel2 activation through caspar gene silencing was undetectable in sugar-fed, blood-fed, and P. falciparum-infected female A. gambiae, while activation of the Toll pathway's Rel1 had a major impact. This study describes for the first time a single gene that influences an immune mechanism that is able to abort development of P. falciparum in Anopheline species. Further, this study addresses aspects of the molecular, evolutionary, and physiological consequences of the observed phenotype. These findings have implications for malaria control since broad-spectrum immune activation in diverse anopheline species offers a viable and strategic approach to develop novel malaria control methods worldwide

The JNK Pathway Is a Key Mediator of <i>Anopheles gambiae</i> Antiplasmodial Immunity

<div><p>The innate immune system of <i>Anopheles gambiae</i> mosquitoes limits <i>Plasmodium</i> 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 <i>Hep</i>, <i>JNK</i>, <i>Jun</i> or <i>Fos</i> expression, greatly enhanced <i>Plasmodium</i> infection; while overactivating this cascade, by silencing the suppressor <i>Puckered</i>, 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 <i>Plasmodium</i> infection and regulates expression of two key hemocyte-derived immune effectors (TEP1 and FBN9). Furthermore, the <i>An. gambiae</i> L3–5 strain that has been genetically selected to be refractory (R) to <i>Plasmodium</i> 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 <i>An. gambiae</i> mosquitoes to limit <i>Plasmodium</i> infection and identified several effector genes mediating these responses.</p></div

The JNK Pathway and <i>Plasmodium berghei</i> infection in <i>Anopheles gambiae</i>.

<p>(A) Diagram representing the organization of the JNK signaling cascade based on functional studies from vertebrates and <i>Drosophila</i>. Five <i>An. gambiae</i> orthologs were functionally characterized, including two kinases, hemipterous <i>(hep)</i> and c-Jun N-terminal kinase <i>(jnk)</i>; a phosphatase, <i>puc</i>; and two transcription factors, Jun <i>(jun)</i> and Fos <i>(fos)</i> (B) Basal mRNA expression of putative genes from the JNK pathway in adult females. <i>Hemipterous (Hep)</i>, Jun N-terminal kinase <i>(JNK)</i>, <i>Jun</i> and <i>Fos</i> transcription factors and <i>puckered (puc)</i> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s017" target="_blank">Table S11</a> for gene ID numbers and primer sequences) (C) Midgut expression of members of the JNK pathway in response to <i>Plasmodium</i> infection in three independent experiments. Ratio of expression in infected/control blood-fed mosquitoes of <i>Hep</i>, <i>JNK</i>, <i>Puc</i>, <i>Jun</i> and <i>Fos</i> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s009" target="_blank">Table S3</a>. P-values determined by Student's-T test after log 2 transformation; **, <i>p</i><0.01, ***, <i>p</i><0.001. *, p<0.05; **, p<0.01, ***, p<0.001. (D) Effect of silencing JNK pathway members on <i>P. berghei</i> infection. (E) Effect of silencing the transcription factor <i>jun</i> alone or and co-silencing <i>jun</i> and the negative regulator <i>puc</i> on <i>Plasmodium</i> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s009" target="_blank">Table S3</a>). 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 <i>dsLacZ</i>-injected groups (n = number of midguts).</p

Participation of the JNK Pathway in L3–5 Mosquitoes Refractory (R) responses to <i>Plasmodium berghei</i> Infection.

<p>(A) Basal mRNA expression of genes from the JNK pathway in the midgut and hemocytes of G3 susceptible (S) (gray) and R (blue) mosquitoes. (B) Expression of effector genes regulated by the JNK pathway in S (gray) and R (blue) mosquitoes. Basal mRNA levels of HPx2 and NOX5 in the midgut, and of TEP1 and FBN9 in hemocytes. Graphs represent the expression level in R females, relative to S females, that were adjusted to a value of “1”; for R females samples the bars represent the SEM of three biological replicates from independent experiments (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s010" target="_blank">Table S4</a>). P-values determined by paired Student's-T test after log2 transformation; *, p<0.05, **, p<0.01, ***, <i>p</i><0.001. (C) Effect of silencing JNK (right panel) in the number of melanized and live parasites on individual midguts of R mosquitoes. Red dots indicate the number of parasites on an individual midgut, live (y-axis) and melanized (x-axis). Green horizontal bars indicate median infection intensities. Inset pie graphs represent the percentage of total parasites for each group displaying a live (green) or melanized (black) phenotype; percentage displayed refers to melanized parasites. Graphs represent data from three biological replicates (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s016" target="_blank">Table S10</a>). (n = number of midguts analyzed).</p

Involvement of Gonadal Steroids and Gamma Interferon in Sex Differences in Response to Blood-Stage Malaria Infection

To examine the hormonal and immunological mechanisms that mediate sex differences in susceptibility to malaria infection, intact and gonadectomized (gdx) C57BL/6 mice were inoculated with Plasmodium chabaudi AS-infected erythrocytes, and the responses to infection were monitored. In addition to reduced mortality, intact females recovered from infection-induced weigh loss and anemia faster than intact males. Expression microarrays and real-time reverse transcription-PCR revealed that gonadally intact females exhibited higher expression of interleukin-10 (IL-10), IL-15Rα, IL-12Rβ, Gadd45γ, gamma interferon (IFN-γ), CCL3, CXCL10, CCR5, and several IFN-inducible genes in white blood cells and produced more IFN-γ than did intact males and gdx females, with these differences being most pronounced during peak parasitemia. Intact females also had higher anti-P. chabaudi immunoglobulin G (IgG) and IgG1 responses than either intact males or gdx females. To further examine the effector mechanisms mediating sex differences in response to P. chabaudi infection, responses to infection were compared among male and female wild-type (WT), T-cell-deficient (TCRβδ(−/−)), B-cell-deficient (μMT), combined T- and B-cell-deficient (RAG1), and IFN-γ knockout (IFN-γ(−/−)) mice. Males were 3.5 times more likely to die from malaria infection than females, with these differences being most pronounced among TCRβδ(−/−), μMT, and RAG1 mice. Male mice also exhibited more severe weight loss, anemia, and hypothermia, and higher peak parasitemia than females during infection, with WT, RAG1, TCRβδ(−/−), and μMT mice exhibiting the most pronounced sexual dimorphism. The absence of IFN-γ reduced the sex difference in mortality and was more detrimental to females than males. These data suggest that differential transcription and translation of IFN-γ, that is influenced by estrogens, may mediate sex differences in response to malaria

Caspar-mediated killing of P. falciparum is not dependent on midgut bacteria.

<p>(A) Blue bars represent bacteria colony-forming unit (CFUs) from midguts of mosquitoes undergoing the indicated treatments. Pluses and minuses indicate the presence or absence of antibiotic in GFP or Cpr dsRNA treated group. Each bar represents the average of at least 15 mosquitoes tested, with each mosquito's CFU count determined by averaging counts from three serial dilutions. Bars represent the standard deviation for all mosquitoes in a given treatment group. Cpr, Caspar. (B) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. Pluses and minuses indicate the presence or absence of antibiotic in the GFP or Cpr dsRNA-treated group. Assays represent three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear below each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002737#ppat.1002737.s003" target="_blank">Table S3</a>. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. Cpr, Caspar.</p

Anopheles Imd pathway model.

<p>Components of the Imd pathway explored in this study or others are represented by different colored shapes. Black arrows or lines indicate known interactions or translocations. Gray arrows indicate potential interactions based on <i>D. melanogaster</i> studies. The gray bracketed area indicates the molecules possibly involved in other responses, but not the responses against <i>P. falciparum</i>. Numbers and arrows within colored blocks indicate the -fold change in <i>P. falciparum</i> infection that results when the corresponding pathway member is silenced. The list of genes inside the nucleus portion of the diagram shows those known to be active against <i>Plasmodium</i> and whose expression has been shown by our studies to be REL2-regulated.</p

Some members of the Imd pathway have an effect on P. falciparum infection.

<p>(A–G) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. P-values were derived from Mann-Whitney statistical tests and appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002737#ppat.1002737.s001" target="_blank">Table S1</a>) Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. All assays represent two to three independent biological replicate. Cpr, Caspar. (H) Prevalence of <i>P. falciparum</i> infection following the indicated RNAi treatment.</p