AeMOPE-1, a Novel Salivary Peptide From Aedes aegypti, Selectively Modulates Activation of Murine Macrophages and Ameliorates Experimental Colitis.

The sialotranscriptomes of Aedes aegypti revealed a transcript overexpressed in female salivary glands that codes a mature 7.8 kDa peptide. The peptide, specific to the Aedes genus, has a unique sequence, presents a putative secretory nature and its function is unknown. Here, we confirmed that the peptide is highly expressed in the salivary glands of female mosquitoes when compared to the salivary glands of males, and its secretion in mosquito saliva is able to sensitize the vertebrate host by inducing the production of specific antibodies. The synthetic version of the peptide downmodulated nitric oxide production by activated peritoneal murine macrophages. The fractionation of a Ae. aegypti salivary preparation revealed that the fractions containing the naturally secreted peptide reproduced the nitric oxide downmodulation. The synthetic peptide also selectively interfered with cytokine production by murine macrophages, inhibiting the production of IL-6, IL-12p40 and CCL2 without affecting TNF-α or IL-10 production. Likewise, intracellular proteins associated with macrophage activation were also distinctively modulated: while iNOS and NF-κB p65 expression were diminished, IκBα and p38 MAPK expression did not change in the presence of the peptide. The anti-inflammatory properties of the synthetic peptide were tested in vivo on a dextran sulfate sodium-induced colitis model. The therapeutic administration of the Ae. aegypti peptide reduced the leukocytosis, macrophage activity and nitric oxide levels in the gut, as well as the expression of cytokines associated with the disease, resulting in amelioration of its clinical signs. Given its biological properties in vitro and in vivo, the molecule was termed Aedes-specific MOdulatory PEptide (AeMOPE-1). Thus, AeMOPE-1 is a novel mosquito-derived immunobiologic with potential to treat immune-mediated disorders.

AeMOPE-1: a novel salivary peptide from Aedes aegypti, selectively modulates activation of murine macrophages and ameliorates experimental Colitis

The sialotranscriptomes of Aedes aegypti revealed a transcript overexpressed in female salivary glands that codes a mature 7.8 kDa peptide. The peptide, specific to the Aedes genus, has a unique sequence, presents a putative secretory nature and its function is unknown. Here, we confirmed that the peptide is highly expressed in the salivary glands of female mosquitoes when compared to the salivary glands of males, and its secretion in mosquito saliva is able to sensitize the vertebrate host by inducing the production of specific antibodies. The synthetic version of the peptide downmodulated nitric oxide production by activated peritoneal murine macrophages. The fractionation of a Ae. aegypti salivary preparation revealed that the fractions containing the naturally secreted peptide reproduced the nitric oxide downmodulation. The synthetic peptide also selectively interfered with cytokine production by murine macrophages, inhibiting the production of IL-6, IL-12p40 and CCL2 without affecting TNF-α or IL-10 production. Likewise, intracellular proteins associated with macrophage activation were also distinctively modulated: while iNOS and NF-κB p65 expression were diminished, IκBα and p38 MAPK expression did not change in the presence of the peptide. The anti-inflammatory properties of the synthetic peptide were tested in vivo on a dextran sulfate sodium-induced colitis model. The therapeutic administration of the Ae. aegypti peptide reduced the leukocytosis, macrophage activity and nitric oxide levels in the gut, as well as the expression of cytokines associated with the disease, resulting in amelioration of its clinical signs. Given its biological properties in vitro and in vivo, the molecule was termed Aedes-specific MOdulatory PEptide (AeMOPE-1). Thus, AeMOPE-1 is a novel mosquito-derived immunobiologic with potential to treat immune-mediated disorders.

Evaluation of inflammatory skin infiltrate following Aedes aegypti bites in sensitized and non-sensitized mice reveals saliva-dependent and immune-dependent phenotypes.

During probing and blood feeding, haematophagous mosquitoes inoculate a mixture of salivary molecules into their vertebrate hosts' skin. In addition to the anti-haemostatic and immunomodulatory activities, mosquito saliva also triggers acute inflammatory reactions, especially in sensitized hosts. Here, we characterize the oedema and the cellular infiltrate following Aedes aegypti mosquito bites in the skin of sensitized and non-sensitized BALB/c mice by flow cytometry. Ae. aegypti bites induced an increased oedema in the ears of both non-sensitized and salivary gland extract- (SGE-)sensitized mice, peaking at 6 hr and 24 hr after exposure, respectively. The quantification of the total cell number in the ears revealed that the cellular recruitment was more robust in SGE-sensitized mice than in non-sensitized mice, and the histological evaluation confirmed these findings. The immunophenotyping performed by flow cytometry revealed that mosquito bites were able to produce complex changes in cell populations present in the ears of non-sensitized and SGE-sensitized mice. When compared with steady-state ears, the leucocyte populations significantly recruited to the skin after mosquito bites in non-sensitized and sensitized mice were eosinophils, neutrophils, monocytes, inflammatory monocytes, mast cells, B-cells and CD4+ T-cells, each one with its specific kinetics. The changes in the absolute number of cells suggested two cell recruitment profiles: (i) a saliva-dependent migration; and (ii) a migration dependent on the immune status of the host. These findings suggest that mosquito bites influence the skin microenvironment by inducing differential cell migration, which is dependent on the degree of host sensitization to salivary molecules.

Aedes aegypti saliva impairs M1-associated proinflammatory phenotype without promoting or affecting M2 polarization of murine macrophages.

BACKGROUND: During the feeding process, the mouthparts of hematophagous mosquitoes break the skin barrier and probe the host tissue to find the blood. The saliva inoculated in this microenvironment modulates host hemostasis, inflammation and adaptive immune responses. However, the mechanisms involved in these biological activities remain poorly understood and few studies explored the potential roles of mosquito saliva on the individual cellular components of the immune system. Here, we report the immunomodulatory activities of Aedes aegypti salivary cocktail on murine peritoneal macrophages. RESULTS: The salivary gland extract (SGE) of Ae. aegypti inhibited the production of nitric oxide and inflammatory cytokines such as interleukin-6 (IL-6) and IL-12, as well as the expression of inducible nitric oxide synthase and NF-κB by murine macrophages stimulated by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). The spare respiratory capacity, the phagocytic and microbicidal activities of these macrophages were also reduced by Ae. aegypti SGE. These phenotypic changes are consistent with SGE suppressing the proinflammatory program of M1 macrophages. On the other hand, Ae. aegypti SGE did not influence M2-associated markers (urea production, arginase-1 and mannose receptor-1 expression), either in macrophages alternatively activated by IL-4 or in those classically activated by LPS plus IFN-γ. In addition, Ae. aegypti SGE did not display any cytokine-binding activity, nor did it affect macrophage viability, thus excluding supposed experimental artifacts. CONCLUSIONS: Given the importance of macrophages in a number of biological processes, our findings help to enlighten how vector saliva modulates vertebrate host immunity.

Exposure to Aedes aegypti Bites Induces a Mixed-Type Allergic Response following Salivary Antigens Challenge in Mice.

Classical studies have shown that Aedes aegypti salivary secretion is responsible for the sensitization to mosquito bites and many of the components present in saliva are immunogenic and capable of inducing an intense immune response. Therefore, we have characterized a murine model of adjuvant-free systemic allergy induced by natural exposure to mosquito bites. BALB/c mice were sensitized by exposure to A. aegypti mosquito bites and intranasally challenged with phosphate-buffered saline only or the mosquito's salivary gland extract (SGE). Blood, bronchoalveolar lavage (BAL) and lung were collected and evaluated for cellularity, histopathological analyses, cytokines and antibody determination. Respiratory pattern was analyzed by Penh measurements and tracheal segments were obtained to study in vitro reactivity to methacholine. BAL recovered from sensitized mice following challenge with SGE showed an increased number of eosinophils and Th2 cytokines such as IL-4, IL-5 and IL-13. Peribronchoalveolar eosinophil infiltration, mucus and collagen were also observed in lung parenchyma of sensitized mice, suggesting the development of a typical Th2 response. However, the antibody profile in serum of these mice evidenced a mixed-type response with presence of both, IgG1/IgE (Th2-related) and IgG2a (Th1-related) isotypes. In addition, changes in breathing pattern and tracheal reactivity to methacholine were not found. Taken together, our results show that A. aegypti bites trigger an atypical allergic reaction, with some classical cellular and soluble Th2 components in the lung, but also systemic Th1 and Th2 antibody isotypes and no change in either the respiratory pattern or the trachea responsiveness to agonist.

The dual effect of paradoxical sleep deprivation on murine immune functions.

We aimed to evaluate the effect of paradoxical sleep deprivation on the cellular migration during inflammation, the peritoneal macrophage phenotype and the infectious stimulus outcomes. A/J mice were inoculated with thioglycollate and exposed to paradoxical sleep deprivation. Sleep-deprived animals presented decreased cell migration compared to controls. Nitric oxide production was reduced in macrophages from sleep-deprived mice compared to controls. Cell surface analysis showed that sleep deprivation reduced F4/80(+)/CD80(low) peritoneal cell population induced by thioglycollate injection. Sleep-deprived mice were not more susceptible to infection than control mice. Our findings challenge the general perception that sleep loss always increases infection susceptibility.

Transcriptome sequencing and developmental regulation of gene expression in Anopheles aquasalis.

BACKGROUND: Anopheles aquasalis is a major malaria vector in coastal areas of South and Central America where it breeds preferentially in brackish water. This species is very susceptible to Plasmodium vivax and it has been already incriminated as responsible vector in malaria outbreaks. There has been no high-throughput investigation into the sequencing of An. aquasalis genes, transcripts and proteins despite its epidemiological relevance. Here we describe the sequencing, assembly and annotation of the An. aquasalis transcriptome. METHODOLOGY/PRINCIPAL FINDINGS: A total of 419 thousand cDNA sequence reads, encompassing 164 million nucleotides, were assembled in 7544 contigs of ≥ 2 sequences, and 1999 singletons. The majority of the An. aquasalis transcripts encode proteins with their closest counterparts in another neotropical malaria vector, An. darlingi. Several analyses in different protein databases were used to annotate and predict the putative functions of the deduced An. aquasalis proteins. Larval and adult-specific transcripts were represented by 121 and 424 contig sequences, respectively. Fifty-one transcripts were only detected in blood-fed females. The data also reveal a list of transcripts up- or down-regulated in adult females after a blood meal. Transcripts associated with immunity, signaling networks and blood feeding and digestion are discussed. CONCLUSIONS/SIGNIFICANCE: This study represents the first large-scale effort to sequence the transcriptome of An. aquasalis. It provides valuable information that will facilitate studies on the biology of this species and may lead to novel strategies to reduce malaria transmission on the South American continent. The An. aquasalis transcriptome is accessible at http://exon.niaid.nih.gov/transcriptome/An_aquasalis/Anaquexcel.xlsx.

Effects of Aedes aegypti salivary components on dendritic cell and lymphocyte biology.

BACKGROUND: Saliva is a key element of interaction between hematophagous mosquitoes and their vertebrate hosts. In addition to allowing a successful blood meal by neutralizing or delaying hemostatic responses, the salivary cocktail is also able to modulate the effector mechanisms of host immune responses facilitating, in turn, the transmission of several types of microorganisms. Understanding how the mosquito uses its salivary components to circumvent host immunity might help to clarify the mechanisms of transmission of such pathogens and disease establishment. METHODS: Flow cytometry was used to evaluate if increasing concentrations of A. aegypti salivary gland extract (SGE) affects bone marrow-derived DC differentiation and maturation. Lymphocyte proliferation in the presence of SGE was estimated by a colorimetric assay. Western blot and Annexin V staining assays were used to assess apoptosis in these cells. Naïve and memory cells from mosquito-bite exposed mice or OVA-immunized mice and their respective controls were analyzed by flow cytometry. RESULTS: Concentration-response curves were employed to evaluate A. aegypti SGE effects on DC and lymphocyte biology. DCs differentiation from bone marrow precursors, their maturation and function were not directly affected by A. aegypti SGE (concentrations ranging from 2.5 to 40 µg/mL). On the other hand, lymphocytes were very sensitive to the salivary components and died in the presence of A. aegypti SGE, even at concentrations as low as 0.1 µg/mL. In addition, A. aegypti SGE was shown to induce apoptosis in all lymphocyte populations evaluated (CD4+ and CD8+ T cells, and B cells) through a mechanism involving caspase-3 and caspase-8, but not Bim. By using different approaches to generate memory cells, we were able to verify that these cells are resistant to SGE effects. CONCLUSION: Our results show that lymphocytes, and not DCs, are the primary target of A. aegypti salivary components. In the presence of A. aegypti SGE, naïve lymphocyte populations die by apoptosis in a caspase-3- and caspase-8-dependent pathway, while memory cells are selectively more resistant to its effects. The present work contributes to elucidate the activities of A. aegypti salivary molecules on the antigen presenting cell-lymphocyte axis and in the biology of these cells.