What do mosquitoes inject

Lastly, we saw an increase in the growth factor VEGF at this time point; VEGF is often released in response to endothelium wall breach to stimulate the growth of endothelial cells [ 51 , 52 ].

Since the mosquito bites likely caused limited vascular injury in the mice, it is not unexpected that we observed an increase in serum VEGF in the bitten mice compared to the control mice. To summarize, the serum cytokine concentrations at 6 hours post- bite indicate a dysregulation of inflammatory signaling, a decrease in Th1 and Th2 signaling, an alteration in chemotactic signaling, and an increase in endothelium repair signaling. This cytokine is usually produced in response to viral infection, so its presence after uninfected mosquito bite is unexpected.

We saw an increase in the Th2 cytokine IL-9; this increase contrasts with our observation at 6 hours post-bite where a decrease in IL-9 was coincident with a decrease in the number of total T cells. At 24 hours post-bite, numbers of total T cells in the bitten mice is equivalent to that of the control mice, which may account for the increase in serum concentrations of IL We saw an increase in the chemokines fractalkine and MDC.

An increase in these chemokines in the serum could induce an influx monocytes and other cells that express the appropriate chemokine receptors into the blood [ 56 — 58 ]. We did not see any shifts in monocyte populations in the mice at 24 hours post-bite, but perhaps this shift occurred at a time point that was not analyzed.

As previously mentioned, VEGF is involved in endothelium wall repair; IL-3 is also involved in endothelium wall repair and promotes the production of platelets, which are consumed during an endothelium wall breach [ 59 ]. To summarize, the serum cytokines present at 24 hours post-bite indicate an interferon response, an anti-inflammatory response, a Th2 response, an influx of monocytes, and a continued endothelium repair response.

At 7 days post-bite, we saw a decrease in the pro-inflammatory cytokine sCD40L in the bitten mice compared to the control mice. In addition to stimulating an inflammatory response, this cytokine is also important for immunoglobulin class switching by B cells [ 60 — 62 ]. In the context of a viral infection, decreasing or preventing immunoglobulin class switching could be detrimental for the host. We saw an increase in serum concentrations of the chemokine fractalkine and a decrease in the chemokines IL-8 and MCP Since both fractalkine and MCP-1 attract similar cell types namely monocytes , it is difficult to predict whether changes in concentrations of these two chemokines affect efflux or influx of cells into the blood [ 56 , 57 , 63 ].

While we did not see a significant change in monocyte numbers in the blood, we did see a decrease of monocytes in the bone marrow at this time point. Also, the decrease in IL-8 in the serum did correspond with a decrease in neutrophils the target of IL-8 in the blood and a trend toward an increase in neutrophils in the skin [ 64 ]. We also observed an increase in the growth factors IL-3 and IL IL-7 enhances the growth of early B and T cells and stimulates the proliferation of mature T cells [ 65 , 66 ].

To summarize, at 7 days post-bite, we saw a decrease in pro-inflammatory cytokines, an increase in some but a decrease in other chemokines, and an increase in growth factors. Because hu-NSG mice do not have a human thymus, the T cells present in these mice are not HLA restricted to human cells and may not respond appropriately to signals from other human immune cells in the mice. This could account for the lack of these cytokines in the serum. Lastly, since we only investigated cytokines present in the serum of these mice, our data do not exclude the possibility that these cytokines were produced in the tissues.

Future studies will investigate what cytokines are produced at the site of mosquito bite and at other tissues throughout the body. Mosquitoes deposit numerous salivary proteins into their host's skin while acquiring a blood meal. Past work has shown that mosquito saliva enhances the pathogenicity of dengue, West Nile, and other arbovirus infections.

From these results, we hypothesized that mosquito saliva modulates the host immune system in a way that promotes arbovirus replication and transmission. In this work, we used human PBMCs treated with mosquito saliva and humanized mice bitten by mosquitoes to determine what effects mosquito saliva alone has on the human immune system.

Our results suggest that the human immune response to mosquito saliva is significant and complex: mosquito saliva alters the frequencies of several immune cell populations, in multiple tissues, at several times after blood feeding. Mosquito saliva also affects serum cytokine levels, with the most notable trend being an increase in anti-inflammatory and Th2 cytokines compared to unbitten, control mice, at 7 days post- bite.

The fact that these effects last up to 7 days post-bite is especially interesting, and is of concern in the context of allergic reactions. The long-lasting effects in the humanized bone marrow and skin cells could explain how some of the viruses transmitted by mosquitoes could possibly still be viable in these tissues, or how they could serve as replication reservoirs. Relatively few studies have investigated the effects of mosquito saliva on human cells ex vivo ; fewer have investigated cytokine production from human cells following stimulation with mosquito saliva.

In another ex vivo study, monocyte-derived dendritic cells differentiated from human PBMCs produced higher amounts of the cytokine ILp70 when stimulated with mosquito saliva collected from Ae. In a third study, mosquito saliva from Anopheles stephensi mosquitoes a vector of malaria induced the degranulation of the human mast cell line LAD-2 [ 83 ].

We did not detect a significant decrease in cell populations or an increase in ILp70 production in our ex vivo studies; however, this is most likely due to differences in study design. We used mosquito saliva as opposed to salivary gland extract a heterogeneous mixture of saliva and the cells that make it and monocyte-derived dendritic cells and mast cells are rarely found in the blood and therefore would not be a component of our human PBMCs.

As these cells are rare, and more potent cytokine producers and killers than conventional single positive T cells [ 18 — 22 ], it is interesting to speculate that mosquito saliva may be uniquely capable to induce the differentiation of single positive T cells into this cytotoxic double positive T cell subset. Further studies are needed to examine whether these unique double positive T cells play an important role in host protection from flavivirus infection.

Furthermore, we noticed decreases in total T cells and natural killer cells after mosquito bite; these decreases may point toward a Th2 response. When analyzing the serum cytokine response, we did observe a significant trend toward Th2 cytokine production at 7 days post-bite.

Th2 immune responses occur most often in response to parasitic infections or allergen exposure, and they tend to dampen inflammatory and cytotoxic responses, both of which are needed to clear viral infections. Thus, if mosquito saliva were to trigger a Th2 response in the context of an arbovirus infection, the virus would be able to replicate to higher levels than it would in the presence of a Th1 response.

In most arbovirus infections, higher viral load corresponds to more severe disease. Thus, a saliva-induced Th2 immune response could explain observations in experimental models of arbovirus infections that mosquito saliva enhances pathogenesis. While these cells can kill virus-infected cells and are generally important for clearing viral infections, they have been implicated in increased disease severity of dengue infection in humans [ 85 ]. Nevertheless, the possibility remains that even though mosquito saliva increases some subsets of immune cells typically associated with a Th1 immune response, these cells could be responsible for increased disease severity in humans.

They concluded that mosquito saliva enhances disease by recruiting arbovirus-susceptible cells to the bite site, in their model of Semliki Forest virus infection.

Zeidner et al. In our study, we observed an increase in monocytes and macrophages to the bite site, but only after 7 days post-bite. In the context of arbovirus infection, the migration we observed would be too late to impact the initial replication or dissemination of the virus but could allow for infected cells to return to the skin where they could transmit the virus to new mosquitoes. This is a significant observation that could possibly explain epidemiologic events, where arbovirus transmission is often limited to distinct, small areas of hypertransmission, and where non-viremic hosts could be sources of infection [ 91 — 93 ].

The mouse model we used in this study does not have the same limitations because these mice have been reconstituted with a human immune system, including innate immunity [ 95 ].

However, this model does not have either a full human complement system or a fully functional T cell compartment [ 95 ]. In future studies, we seek to address these limitations by using other types of reconstituted humanized mice e. In addition, we expect to test for the biological significance of these immune cell changes, which might lead to stimulation of infected cells to migrate to important sanctuary tissues e. In the case of arboviruses, many establish infections in brain or bone marrow cells of human patients, leading to specific pathologies such as encephalitis or bone loss and leukopenia and thrombocytopenia [ 97 — 99 ].

It is currently unclear if mosquito saliva contributes to these tissue infections and more severe pathologies. Mosquitoes and the diseases they transmit are of growing public health concern. Often, there are no prophylaxes for these diseases other than mosquito control and no treatments other than palliative care.

Understanding how mosquito saliva interacts with the human immune system not only helps us understand mechanisms of disease pathogenesis but also could provide possibilities for treatments. If we know which mosquito saliva components enhance pathogenesis of diseases, we could create a human vaccine to counteract these effects for multiple arbovirus infections. A similar approach has been used to vaccinate and protect mice against a sandfly saliva protein maxadilan that enhances the infection and progression of Leishmania major [ ].

These approaches have been commercialized and used to interrupt tick transmission of cattle diseases [ ], and we expect that the definition of these factors would help provide the same approaches in humans. Human PBMCs were isolated from two different human donors and stimulated with mosquito saliva, pokeweed mitogen, LPS, or untreated media. Pokeweed mitogen and LPS were used to confirm that the human PBMCs used in these experiments were capable of responding to immunomodulatory compounds.

A Immune cell population changes following stimulation with pokeweed mitogen and LPS. Stimulated human PBMCs were analyzed via flow cytometry five days post stimulation.

Error bars represent 1 standard error of the mean. Supernatants from stimulated human PBMCs were collected daily up to five days post stimulation and analyzed for cytokine concentrations. Data shown are the log10 fold change of each of the stimulated conditions compared to the media stimulated control for the each donor and time point; the darker the color, the higher production of cytokines in treated PBMCs saliva, pokeweed mitogen, or LPS.

Areas with slashes represent samples where no data were collected for that cytokine. These flow charts describe the gating strategies used to analyze flow cytometry data in this study. A This was the gating strategy used for the data presented in Figs 3 and 4.

Grey boxes represent gates that were made during the analysis of all three panels. Blue, red, and yellow boxes represent gates that were made during the analysis of Panels P1, P2, and P3, respectively. Green boxes represent gates that were made during the analysis of both Panels P1 and P3, and orange boxes represent gates that were made during the analysis of both Panels P2 and P3.

B This was the gating strategy used for the data presented in Figs 1 , 5 and 6. Grey boxes represent gates that were made during the analysis of both Panel 1 and Panel 2 data. Red boxes represent gates that were made only during the analysis of Panel 1 data.

Blue boxes represent gates that were made only during the analysis of Panel 2 data. Boxes containing italicized text represent gates that were only used in the analysis of humanized mice samples and not in the analysis of human PBMC samples. There is no evidence of injury or bleeding into the tissues after 3 mosquito bites on each footpad.

The authors would like to thank Mayra Sanabria-Hernandez, Enid Mondragon, Donovan Berens, and Kassandra Peterson for their help in humanized mice production, flow cytometry, and mosquito rearing. We thank Chih-Wei Hsu for stereomicroscope photography. Author summary Mosquito saliva proteins have numerous effects on the immune system, and we describe here the use of mice with a humanized immune system to study the effects of mosquito bites on human cells.

Introduction Approximately , people die of mosquito transmitted diseases each year, including malaria, dengue, West Nile, Zika, and chikungunya fevers [ 1 , 2 ].

Mosquito salivation Mosquito saliva was collected from female Aedes aegypti Rockefeller mosquitoes 4—5 days post-eclosion. Production of humanized mice Humanized mice were engrafted as previously described [ 7 ]. Mosquito biting of Hu-NSG mice Mosquito biting of reconstituted humanized mice was carried out as previously reported [ 7 ], although with uninfected mosquitoes.

Tissue collection and processing Six hours, 24 hours, or 7 days post mosquito bite, mice were humanely euthanized via isoflurane overdose. Flow cytometry Blood, bone marrow, skin, and spleen cells from hu-NSG mice and stimulated human PBMCs were transferred to 96 well plates and incubated with antibodies against extracellular targets Table 1 on ice for 30 minutes.

Download: PPT. Statistical analysis Statistical analysis was performed using Prism v6. Results Mosquito saliva increases the frequency of natural killer T cells in human peripheral blood mononuclear cells Our previous studies demonstrated that mosquito saliva enhances dengue infection in humanized mice [ 7 ].

Fig 1. Mosquito saliva suppresses cytokine production in human PBMC cultures Cytokines are secreted signaling proteins of the innate immune system and are crucial in establishing an effective immune response; thus, we investigated whether mosquito saliva has an effect on cytokines produced by human PBMCs ex vivo.

Fig 2. Mosquito saliva decreases cytokine production in human PBMCs. Preliminary flow cytometry studies After observing alterations in cytokine production and NKT cell frequency in PBMCs treated with mosquito saliva, we expanded our study to investigate the effects of mosquito saliva on the immune system of an entire organism. Fig 3. Preliminary assessment of mosquito saliva on human T cell populations in hu-NSG mice. Fig 4. Preliminary assessment of mosquito saliva on human leukocyte populations in hu-NSG mice.

Mosquito bite impacts lymphocyte and myeloid cells across multiple tissues Because our preliminary flow cytometry study revealed that mosquito bite impacts the immune system of the hu-NSG mice, we performed a similar experiment using different time points 6 hours, 24 hours, and 7 days post-bite to deepen our knowledge of the effects of mosquito saliva on the innate 6 hours, 24 hours and adaptive 7 days immune responses.

Fig 5. Mosquito saliva changes human T cell populations in hu-NSG mice. Fig 6. Mosquito saliva alters human leukocyte populations in hu-NSG mice. Table 2. Changes in T cell populations in bitten hu-NSG mice compared to control mice. Table 3.

Changes in B cells, natural killer cells, and myeloid cells in bitten hu-NSG mice compared to control mice. Mosquito bite alters cytokine levels in hu-NSG mice To further our examination of mosquito bites on the immune system of humanized mice, we investigated whether serum levels of human cytokines changed in mosquito-bitten mice.

Fig 7. Mosquito saliva alters concentrations of select serum cytokines in hu-NSG mice. Discussion Mosquitoes deposit numerous salivary proteins into their host's skin while acquiring a blood meal.

Supporting information. S1 Fig. S2 Fig. Gating strategy for flow cytometry experiments. S3 Fig. Stereomicroscope photographs of a mouse footpad immediately after 3 mosquito bites, and the mosquitoes that bit that humanized mouse 3 per footpad.

S1 Table. List of humanized mice used in these experiments. Acknowledgments The authors would like to thank Mayra Sanabria-Hernandez, Enid Mondragon, Donovan Berens, and Kassandra Peterson for their help in humanized mice production, flow cytometry, and mosquito rearing. References 1. A global brief on vector-borne diseases.

In: Organizaiton WH, editor. Climate change influences on global distributions of dengue and chikungunya virus vectors. The global distribution of the arbovirus vectors Aedes aegypti and Ae. Potentiation of vesicular stomatitis New Jersey virus infection in mice by mosquito saliva.

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To avoid hyperpigmentation , look for creams with vitamin C, E, or niacinamide. The best way to avoid mosquito bites is preparation and prevention. Use insect repellant if you are going to places where mosquitos are. Natural insect repellants are effective, but you may want to use commercial ones if you are traveling to another country. Limiting or avoiding exposure to certain things may help minimize your risk for mosquito bites.

Below is a list of things known to attract mosquitos:. Alcohol intake is also shown to be associated with a higher probability of being bitten. It may also be a good idea to keep some travel-size aloe vera and alcohol wipes handy. Keep reading: 10 natural ingredients that double as mosquito repellants ». Most commercial bug repellents can cause health and environmental problems. Learn about natural repellents that actually work.

While most bug bites cause only mild symptoms, some bug bites can transmit disease. Read on to learn more about symptoms and how to prevent bug bites. Skeeter syndrome is another name for a mosquito bite allergy. Nearly everyone is sensitive to mosquito bites, but the reaction can be serious for….

It can cause a lot of discomfort and may even become a…. Bitten by a mosquito? Here are 13 things you can do from home to alleviate…. Mosquito bites are round, usually puffy, and severely itchy. Learn about which diseases mosquitos can transmit and how to treat and prevent bites. Mosquitoes infect humans with the Zika virus, yellow fever, dengue, malaria, and other diseases.

If you have swelling in just one eye or have found a spider in the area where you were bit, it's safe to assume a spider or similar bug is to blame.

Most spider bites are harmless, and symptoms will go away after a few days. Seek immediate medical care if you suspect the following.

Health Conditions Discover Plan Connect. Medically reviewed by Debra Sullivan, Ph. Why do mosquito bites itch? Scratching mosquito bites. Immediate relief for mosquito bites. Clean the area with rubbing alcohol. Apply honey on the bite. Take an oatmeal bath. Use a cold tea bag.

Make a basil rub. Take over-the-counter antihistamines. Use ointments containing lidocaine or benzocaine. Apply aloe vera.