Mosquitoes have been a bane to human health for centuries, with different species carrying the pathogens causing malaria, filaria, dengue and other debilitating or even potentially deadly diseases.
A new preprint study looks at the immunomodulatory effects of mosquito bites, using Aedes aegypti as the model species and humans as the host.
Viral infections transmitted by mosquitoes have been the focus of much research worldwide. Arboviruses such as the dengue, yellow fever, Zika and chikungunya viruses are of especial importance in terms of their reach, clinical severity and mortality or teratogenicity. All of these are transmitted by the Aedes species of mosquito, found in abundance in tropical and subtropical regions.
At present, climate change and the spread of urban centers have enlarged the habitat of these species and also increased the size of the population at risk from these mosquitoes. The mechanism of transmission is via the bite. The infected host first transfers the virus to the mosquito when the latter pierces the skin to obtain a blood meal. The virus then replicates in the insect’s midgut, spreads beyond these cells and reaches the salivary glands.
Afterwards, the mosquito bites another potential host to transmit the virus through its saliva. The saliva of these blood-sucking creatures contains many bioactive ingredients, including some which prevent clot formation, and others that promote the growth of new blood vessels. In addition, some have immunomodulatory functions.
Over time, the exposure to mosquito saliva, with the repeated bites of these insects, lead to immunomodulation of the antiviral response occurring in the skin and nearby tissues. The result is a disruption of the inflammatory pathways, increasing the recruitment of other immune cells to the site of the bite and enhanced autophagy. Neutrophils also infiltrate the inflamed site.
Potential consequences include the elevated levels of viral particles in the blood, or a delay in their appearance, following mosquito bites as the route of inoculation, compared to other routes such as needles or non-vector inoculation. Again, the levels of viral nucleic acid were higher in mouse models when both mosquito saliva and Semliki Forest virus were inoculated.
Such animals showed higher levels of virus genetic material in the skin, indicating greater viral replication; earlier spread to the brain; and increased mortality; vs animals that were inoculated only with the virus. The current preprint, available on the bioRxiv server, aimed to examine the effects of mosquito bites on the immune response in humans in an area where these vectors are abundant, in contrast to regions where mosquito bites are occasional or rare.
The study included 30 healthy individuals in an area of Cambodia where A. aegypti are prevalent. The researchers compared the immune responses before and after the bites, using skin biopsies, in an attempt to identify the important genes and cell types that participate in these phenomena at various time points following the bite.
The results showed that the skin underwent significant clinical changes after mosquito bites occurred, mainly redness and swelling at the site. The average size of the wheal was ~5 mm at 15 minutes, going down to half by 48 hours. At this point, immune cells were recruited to the skin.
At the same time, several genes showed up-regulation and down-regulation, with the pattern varying depending on the durationof time since the bite, but not differing much between individuals. The changes peaked at 48 hours, with over 470 genes showing increased expression and ~30 being downregulated. Overall, >700 genes were upregulated.
The changes related to these gene changes were mostly to do with inflammatory changes like neutrophil degranulation and gamma-interferon signaling, as well as interleukin (IL)-4 and IL-13 signaling, at an early stage. Beginning four hours post-bite, these continued until 48 hours.
At 4 hours, extracellular matrix was breaking down, while at later stages, at 48 hours, adaptive immune responses were present. Neutrophil recruitment early in the reaction was followed by type 1 and type 2 T helper cell (Th1 and Th2 cells) and regulatory pathways.
The top genes to be upregulated early on included KRT6C, CXCL8, TNIP3, IL-20 and IL-1B. These have to do with keratinization, neutrophil recruitment, inhibition of NF-kappa-B activation, keratinocyte proliferation and differentiation, and inflammation, respectively. All were increased by 50-80 times.
At this point, M2 macrophages and dendritic cells (DCs) were increased, while natural killer (NK) cells were decreased, signaling the innate immune response.
At 48 hours, the top upregulated genes were KRT6C, DEFB4A, GZMB, TCL1A and CCL18, representing keratinization, antimicrobial peptide, granzyme B, T and B cell proliferation and Th2 cell responses, respectively. At this point, T cells in the skin underwent change, with more activated or exhausted CD8+ T cells being observed. In addition, CD4+ T cells were polarized towards a Th2/Th17 profile.
The saliva of this mosquito appeared to dampen the inflammatory cytokine response in the skin.
The results of this study cover the gross impact of the different factors operating with a mosquito bite, including the exposure to human saliva as well as the inoculation of mosquito and human skin microbiota into the puncture site. The local reaction, comprising a wheal and flare, was observed 15 minutes after the bite.
Most changes were seen after this point, however. The observed vasodilation, fluid build-up and neutrophil infiltrate could be the result of histamine release from cutaneous mast cells activated by the mosquito bite. Over time, the extracellular matrix broke down, and immune cells flooded in, observable by 4 hours.
Along with genes regulating these processes, other inflammation-associated genes were upregulated at 48 hours. The inflammation could be promoted by the anti-clotting proteins in mosquito saliva, which could also disrupt vascular function and promote infection by the arbovirus. The increased movement of white cells could also present more susceptible cells to the site of viral inoculation.
This could also promote viral replication, especially as these cells allow arboviruses to enter and establish infection, even as they serve as the first line of defense. The strong influence on neutrophil influx at the bite site is seen early and is traceable to salivary proteins NeSt1 and AgBr1, which induce an increase in many chemokines that attract neutrophils and induce angiogenesis.
With the shift from innate to adaptive immunity, a T cell response emerged, observable from 4 hours post-bite. The upregulation of the Th2 pathway indicates an allergic inflammatory response to the bite. However, this was also regulated by the CD4+ T cells, thus avoiding excessive inflammation and skin damage, especially with repeated exposure to the bites.
A special finding here was of the upregulation of the DC antigen-2 CLEC4C at 48 hours, probably leading to inhibition of the NF-κB pathway, and thus blocking TRAIL-mediated cytotoxic activity. This might distinguish individuals who are repeatedly bitten from those who are rarely exposed.
According to the authors, “Skin immune tolerance to constant mosquito exposure may also explain the reducing effect of SGE on cytokine production.”
The high level of DEFB4A expression also points to the role played by this antimicrobial peptide in inflammation following the mosquito bite, mediating the recruitment of memory T cells, neutrophils, mast cells and immature DCs.
These newly identified genes, cell types, and processes of human cutaneous immunity can be leveraged in the development of novel therapeutics and vector-targeted vaccine candidates. Such new pathogen-agnostic strategies have shown promising results in recent years from vector saliva-based vaccinations to topical antiviral applications post-bite.”, explained the researchers.
BioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.