key: cord-102886-oo7q05ml
authors: Gomes, Fabio M.; Tyner, Miles D.W.; Barletta, Ana Beatriz F.; Yenkoidiok-Douti, Lampougin; Canepa, Gaspar E.; Molina-Cruz, Alvaro; Barillas-Mury, Carolina
title: “Proliferation of DBLOX Peroxidase-Expressing Oenocytes Maintains Innate Immune Memory in Primed Mosquitoes”
date: 2020-09-10
journal: bioRxiv
DOI: 10.1101/2020.09.09.290312
sha: 
doc_id: 102886
cord_uid: oo7q05ml

Immune priming in Anopheles gambiae mosquitoes following infection with Plasmodium parasites is mediated by the systemic release of a hemocyte differentiation factor (HDF), a complex of lipoxin A4 bound to Evokin, a lipid carrier. HDF increases the proportion of circulating granulocytes and enhances mosquito cellular immunity. We found that Evokin is constitutively produced by hemocytes and fat-body cells, but expression increases in response to infection. Insects synthesize lipoxins, but lack lipoxygenases. Here, we show that the Double Peroxidase (DBLOX) enzyme, present in insects but not in vertebrates, is essential for HDF synthesis. DBLOX is highly expressed in oenocytes in the fat body tissue, and these cells proliferate in response to Plasmodium challenge. We provide direct evidence that modifications mediated by the histone acetyltransferase AgTip60 (AGAP01539) are essential for sustained oenocyte proliferation, HDF synthesis and immune priming. We propose that oenocytes function as a population of “memory” cells that continuously release lipoxin to orchestrate and maintain a broad, systemic and long-lasting state of enhanced immune surveillance.

There is growing evidence that a previous infection can "train" or "prime" the innate immune system, allowing it to respond more effectively to subsequent infections (1-3). This is an ancient response that has been documented in plants (4) , insects (5) and humans (6) . It is not clear whether individual immune cells maintain the training response, or if there are specific "memory keeper" cell populations that orchestrate a persistent re-training of effector immune cells. In Anopheles gambiae, the primary vector of malaria in Africa, Plasmodium infection induces a long-lasting priming response that enhances antiplasmodial immunity (5) . Plasmodium midgut invasion allows direct contact between the gut microbiota and midgut epithelial cells, triggering a burst of prostaglandin E 2 (PGE 2 ) production by midgut cells. The transient systemic release of PGE 2 establishes a long-lasting release of hemocyte differentiation factor (HDF) which, in turn, induces an increase in the proportion of circulating granulocytes, a hallmark of immune priming (7, 8) . HDF is a complex of Evokin, a lipocalin lipid carrier protein, and the lipid mediator Lipoxin A 4 (LXA 4 ) (7). Thus, immune priming can be defined as the enhanced ability of mosquitoes to convert arachidonic acid into LXA 4 that results in a permanent functional state of enhanced immune surveillance (7) . An effective antiplasmodial response requires the coordinated activation of epithelial, cellular, and complement components of the mosquito immune system (9) . PGE 2 attracts hemocytes to the basal surface of the midgut (8), and primed hemocytes release more microvesicles, enhancing complement-mediated ookinete lysis (9) .

In vertebrates, cyclooxygenases (COX) and lipoxygenases (LOX) catalyze the synthesis of prostaglandins and lipoxins, respectively (10) . Although eicosanoids have been detected in mosquitoes and other insects (8, 11, 12) , neither COX nor LOX enzymes are present in insects.

We recently showed that two heme peroxidases, HPX7 and HPX8, are necessary for midgut PGE 2 synthesis and are essential to establish immune priming in response to Plasmodium infection (8) . However, the enzyme(s) mediating LXA 4 synthesis in insects remain unknown. In this manuscript, we identify the Double Peroxidase (DBLOX) enzyme as essential for HDF synthesis, and show that DBLOX is highly expressed in oenocytes, a cell population involved in lipid processing. Furthermore, we discovered that oenocytes proliferate in primed mosquitoes. In vertebrates, monocyte trained immunity is mediated by epigenetic modifications (13, 14) . Here, we identify a histone acetyltransferase (HAT) that is an essential mediator of oenocyte proliferation and mosquito immune priming. mRNA expression of Evokin, an essential component of HDF (7) , was significantly induced in the hemocyte-like immune-responsive A. gambiae Sua 5.1 cell line in response to bacterial challenge (Fig. 1A ) (p=0.0037, Mann-Whitney test). Additionally, antibodies to recombinant Evokin recognized a single band of the expected size (21 kDa) in the fraction containing membrane and insoluble proteins, but not in the cytoplasm (Fig. 1B) . Evokin mRNA expression also increased significantly in hemocytes (p=0.0006, unpaired t-test) and body wall (p= 0.0005, unpaired t-test) of primed mosquitoes 7 days post-infection ( Fig 1C) . In naïve bloodfed females, Evokin is highly expressed in sessile hemocytes associated with the body wall surface ( Fig 1D) . It is also present in oenocytes, with a dotted vesicle-like pattern (Fig 1E) , while in fat body trophocytes a string pattern is observed, suggestive of Evokin clustering in specific membrane regions that may represent lipid rafts ( Fig 1F) . A similar expression pattern was observed in Plasmodium challenged females (Fig. S1 ).

Given the critical role of HPX7 and HPX8 in PGE 2 (Table   S1 ). Both treatments resulted in a prolonged and significant increase in DBLOX expression in the body wall (p=0.0056 and p=0.0059, respectively, Mann-Whitney test) while HPX2 was only induced following PGE 2 injection (p=0.0058, Mann-Whitney test) (Fig. 2B ). HPX2 silencing did not affect priming, as the characteristic increase in granulocytes was observed in response to Plasmodium infection (p=0.0001, Mann Whitney test) (Fig. 2C ). In contrast, DBLOX silencing completely abolished the priming response to infection (Fig. 2D ) and to PGE 2 injection (Fig. S2) .

Furthermore, hemolymph of mosquitoes in which DBLOX was silenced no longer had HDF activity when transferred to naïve mosquitoes (Fig. 2E ). proliferation is long-lasting, as the increased number of clusters and cells per segment were still present 7 days post-feeding (Fig. S4) . Given the essential role of DBLOX in HDF synthesis and immune priming, the high expression of DBLOX in oenocytes, and their sustained proliferation in response to priming, we infer that the documented enhanced ability of primed mosquitoes to synthesize LXA 4 from arachidonic acid (7) is achieved by increasing the number of oenocytes, the cells in the mosquito that are a major site of LXA 4 synthesis.

DBLOX and Evokin mRNA levels remain persistently high in mosquitoes primed through P. berghei challenge or by PGE 2 injection. We explore whether the establishment and persistence of immune priming is mediated by histone acetyltransferases (HAT), enzymes known to catalyze epigenetic chromatin modifications associated with long-lasting changes in transcriptional regulation (15, 16) . We evaluated the effect of silencing each of the ten HATs present in the A. gambiae genome (AgHATs) on immune priming following P. berghei infection (Fig. 4A) . Silencing AGAP000029 resulted in high mortality after blood-feeding and, thus, it could not be further evaluated. Of the other nine AgHATs, only silencing of the homolog of Drosophila Tip60 (AGAP001539 -AgTip60) abolished immune priming (Fig. 4A) . We confirmed that dsLacZ injection had no effect on priming, while the proportion of granulocytes no longer increased when AgTip60 was silenced (Fig. 4B) . Furthermore, hemolymph of primed AgTip60-silenced females no longer had HDF activity when transferred to naïve mosquitoes ( Fig. 4C ). In agreement with our proposed model, the loss of HDF activity following AgTip60 silencing was also associated with a lack of proliferation of DBLOX-expressing oenocytes, as the number of clusters and cells per segment no longer increased following a challenge with P. berghei (Fig. 4D-F) .

DBLOX is a unique enzyme with two duplicated heme peroxidase domains that is present in insects, but not in vertebrates. The first domain has the predicted substrate binding sites but lacks the functional residues present in catalytically active enzymes and has two integrin-binding motifs, typical of peroxinectins (17) . In contrast, the second domain has all the features of a functional heme peroxidase and one integrin-binding motif (17) . DBLOX is essential for LXA 4 synthesis, but the biochemical mechanism of lipoxygenase-independent lipoxin synthesis in insects, and the potential involvement of other enzymes besides DBLOX remain to be explored. We speculate that the systemic burst of PGE 2 when ookinete invasion allows direct contact between the microbiota and gut epithelial cells, or when mosquitoes are injected with PGE 2 , triggers modification mediated by AgTip60 that establish and maintain proliferation of oenocytes. These cells express high levels of DBLOX and their proliferation is essential for HDF synthesis and to maintain the priming response. The function of oenocytes is poorly understood, but they are known to be involved in biosynthesis of cuticular hydrocarbon and pheromones (18) . Taken together, our findings suggest that oenocytes are a major site of lipoxin synthesis.

Immune training of human monocytes involves transcriptional and metabolic reprogramming mediated by epigenetic modifications that allow challenged monocytes to maintain a prolonged state of enhanced immune function (13, 19) . We propose that oenocytes function as "memory keeper" cells that persistently release LXA 4 , which constantly re-trains mosquito hemocytes. Recently, trained immunity in humans induced by vaccination with the anti-tuberculosis bacillus Calmette-Guérin (BCG) vaccine has received much attention in light of the COVID19 pandemic (20) . There is strong epidemiological evidence that BCG vaccination in infants has a broad protective effect and reduces the overall mortality from respiratory infections not related to tuberculosis (21) (22) (23) , and there are ongoing clinical studies to establish if the BCG vaccine also protects from severe COVID19 (24, 25) . Furthermore, there is growing epidemiological evidence of reduced COVID19 mortality (26) and improved outcomes (27) in countries with strong BCG vaccination programs. However, little is known about the mechanism to establish and maintain trained immunity. In A. gambiae, prostaglandins are key to establish the priming response, and lipoxins maintain a broad general state of enhanced immune surveillance that is not pathogen-specific. This begs the question of whether eicosanoids may also be important mediators of trained immunity in humans. 

Innate Immune Memory in Invertebrate Metazoans: A Critical Appraisal

Immune memory in invertebrates

Memory and Specificity in the Insect Immune System: Current Perspectives and Future Challenges

Plant innate immunity: an updated insight into defense mechanism

Hemocyte differentiation mediates innate immune memory in Anopheles gambiae mosquitoes

Therapeutic targeting of trained immunity

A mosquito lipoxin/lipocalin complex mediates innate immune priming in Anopheles gambiae

Activation of mosquito complement antiplasmodial response requires cellular immunity

Synopsis of arachidonic acid metabolism: A review

Eicosanoid-mediated immunity in insects

Prostaglandin E2 modulates the expression of antimicrobial peptides in the fat body and midgut of Anopheles albimanus

Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity

Trained immunity: A program of innate immune memory in health and disease

Histone acetylation: a switch between repressive and permissive chromatin

Transcription: gene control by targeted histone acetylation

Identification, characterization and expression analysis of Anopheles stephensi double peroxidase

The development and functions of oenocytes

Trained immunity: A smart way to enhance innate immune defence

BCG-induced trained immunity: can it offer protection against COVID-19?

Acute lower respiratory tract infections and respiratory syncytial virus in infants in Guinea-Bissau: a beneficial effect of BCG vaccination for girls community based case-control study

Nonspecific (Heterologous) Protection of Neonatal BCG Vaccination Against Hospitalization Due to Respiratory Infection and Sepsis

BCG scar and positive tuberculin reaction associated with reduced child mortality in West Africa. A non-specific beneficial effect of BCG?

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BCG vaccine protection from severe coronavirus disease 2019 (COVID-19)

Significantly Improved COVID-19 Outcomes in Countries with Higher BCG Vaccination Coverage: A Multivariable Analysis

The authors gratefully acknowledge Asher Kantor and Mark Johnson for editorial assistance.

Animal studies were done according to the NIH animal study protocol (ASP) approved by the NIH Animal Care and User Committee (ACUC), with approval ID ASP-LMVR5. Public Health Service Animal Welfare Assurance #A4149-01 guidelines were followed according to the National Institutes of Health Animal (NIH) Office of Animal Care and Use (OACU).