Factors contained within mosquito saliva enhance transmission of mosquito-borne viruses to the mammalian host. In this issue, Wang et al (2024) identify AaNRP as a crucial pro-viral factor in Aedes aegypti mosquito saliva that promotes recruitment of Zika and dengue virus-susceptible myeloid cells

Barningham, L.D.

Keskek Turk, Y.

McKimmie, C.S.

White Rose Research Online

News & ViewsSensing the danger in mosquito spitYonca Keskek Turk, Liam D Barningham & Clive S McKimmie ✉Factors contained within mosquito salivaenhance transmission of mosquito-borneviruses to the mammalian host. In thisissue, Wang et al (2024) identify AaNRP asa crucial pro-viral factor in Aedes aegyptimosquito saliva that promotes recruitmentof Zika and dengue virus-susceptiblemyeloid cells.See also: Z Wang et al (February 2024)Mosquito-borne viruses representan increasing threat to humanhealth. The incidence of infectionwith these viruses, such as the Zika virus(ZIKV), dengue virus (DENV), and chikun-gunya virus (CHIKV), have significantlyincreased, infecting many millions eachyear. Although infection by these viruses isoften asymptomatic or mild, many infec-tions can result in severe disease or death.ZIKV is particularly noteworthy in itsability to cause severe defects in thedeveloping nervous system of gestatingembryos in pregnant mothers. The ongoingclimate crisis combined with increasedglobalization of trade and transport isconspiring to put more people at risk ofinfection, and yet we have few vaccines, andno specific antiviral medicines are licensedfor their treatment (Weaver et al, 2018).There are several challenges faced by thosedeveloping virus specific anti-virals. Thisincludes the tendency of these viruses toemerge and re-emerge unpredictably andthat each outbreak may be driven by agenetically distinct virus and/or serotype.Consequently, the development of a pan-viral medicine effective against multiplemosquito-borne virus species infectionswould be highly advantageous.Anyone who has been bitten by amosquito knows that this results in anitchy, inflamed swelling. It is into thisunique inflammatory niche that mosquitoestransmit virus as they search for a bloodmeal. This is important for defining theseverity of infection, as the host response tomosquito bites, and the saliva deposited,significantly enhances infection with theseviruses (Pingen et al, 2016, 2017). This is acrucial stage of infection, as bitten skin iswhere the virus must replicate to enableefficient dissemination to the blood andremote tissues. An exciting concept that hasgained ground in recent years is thetherapeutic targeting of mosquito salivacomponents, or the bite itself, as a putativepan-viral therapy (Marin-Lopez et al, 2021;Bryden et al, 2020).Mosquito saliva contains a complexmixture of factors, some of which haveevolved to enable highly efficient bloodfeeding. Intriguingly, a number of thesefactors also enhance the infection of thevertebrate host with virus. These factorsinclude sialokinin (Lefteri et al, 2022;Martin-Martin et al, 2022) which actswithin seconds to permeabilise blood vesselsand promote fluid and leukocyte entry;NeSt1 which activates neutrophils (Hastingset al, 2019); AaVA1 which activates autop-hagy (Sun et al, 2020); and LTRIN thatsuppresses lymphotoxin-beta function (Jinet al, 2018). These findings have helpeddefine which factors in mosquito saliva areresponsible for inducing the rapid androbust influx of leukocytes that occursfollowing mosquito biting. Some of thesecells are susceptible to infection and repli-cate virus to higher quantities than wouldotherwise occur if virus was deposited inuninflamed, non-bitten skin (Pingen et al,2017). However, most mosquito salivaryfactors remain poorly characterized, andtheir role in modulating host susceptibilityto virus infection not studied. In a recentpublication, Wang et al (2024) report apartial screen of mosquito salivary factorsleading to the identification of the novel,pro-inflammatory salivary factor AaNRP.Fascinatingly, AaNRP directly activatesinnate immune sensors on skin residentmacrophages, thus activating aninflammatory program that recruits leuko-cytes into the skin. These newly recruitedcells provide new cellular fodder for viralreplication and thereby enhance infection(as summarized in Fig. 1).To begin with, Wang et al use a non-biased screening-based approach to assessthe ability of 25 different salivary proteins torecruit neutrophils into the skin. Neutro-phils are rapidly recruited during mosquitobiting and serve to coordinate localizedinflammatory response that recruits virus-permissive cells such as dendritic cells,monocytes, and macrophages. Injectionwith three of these salivary proteins, parti-cularly AaNRP, induced similar responsesin the skin to a mosquito bite and resultedin leukocyte recruitment. Similarly, deple-tion of AaNRP expression in mosquitosalivary glands limited the ability of mos-quito biting to induce inflammatoryresponses in skin. The entry of neutrophilsappeared crucial, as in their absence AaNRPdid not induce inflammation.Next, they assessed how AaNRP couldact to recruit neutrophils. Firstly, theydiscounted the ability of AaNRP to bechemotactic through use of transwell assays.Surprising, they also found mast cells, whichare required for many immune responses toallergens and other immunogens, were notsignificantly activated by mosquito biting.Instead, skin bitten by AaNRP-sufficientmosquitoes, or injected with purifiedAaNRP, activated skin resident macro-phages directly to initiate a chemokinecentric program that recruited neutrophilsand other innate immune leukocytes. Spe-cifically, they used single-cell sequencing todefine high expression of neutrophil-attracting chemokines CXCL1-3 in macro-phages. Depletion of macrophages limitedthe ability of skin to recruit neutrophilsfollowing biting, suggesting an essential rolein this process. To define the molecularmechanism by which AaNRP activatedmacrophages, they assessed whether theVirus Host Interaction Team, School of Medicine, University of Leeds, Leeds, UK. ✉E-mail: c.s.mckimmie@leeds.ac.ukhttps://doi.org/10.1038/s44318-024-00073-w |1234567890();,:© The Author(s) The EMBO Journal 1Downloaded from https://www.embopress.org on April 4, 2024 from IP 94.8.8.34.well-described canonical activator of che-mokine expression, MyD88/NF-κB signal-ing, was necessary. Interestingly, luciferase-based assays showed strong induction of thecanonical MyD88-dependent pathway byAaNRP, an effect that was reversed by aMyD88-specific inhibitor. To define whichreceptor could be involved in activatingMyD88, they performed immunoprecipita-tion assays to show direct binding ofAaNRP specifically to TLR1 and TLR4, butnot other TLRs. TLR1/4 inhibitors alsoprevented AaNRP-mediated expression ofchemokine, while a knockdown of TLR1and TLR4 aborted NF-κB signaling byAaNRP. Together this demonstrates forthe first time that a mosquito-derivedsalivary factor is a potent activator ofmammalian TLR signaling in skin macro-phages, and that this initiates a chemokine-associated inflammatory response thatrecruits leukocytes.To define whether AaNRP activation ofTLRs modulated host susceptibility to virusinfection, they next inoculated mice withZIKV in the presence or absence of AaNRP.Following ZIKV infection, AaNRP robustlyrecruited virus-permissive myeloid cells,some of which some became infected andreleased new infectious virus, resulting inmore efficient virus dissemination andworse infection in a MyD88 pathway-dependent manner. Similar results werefound following infection with the denguevirus. Impressively, they also showed thatZIKV-infected mosquitoes, when allowed tobite mice, were less able to induce high titerviremia and disease in mice, if depletedfor AaNRP.Although AaNRP is unlikely to haveevolved for the specific purpose of activatingTLR signaling in mammalian skin, thesedata nonetheless show it is unambiguouslyinflammatory and that this host responsesignificantly enhances infection by the virus.This is particularly intriguing as mosquitoesthat lacked AaNRP had normal skin prob-ing times and the ability to engorge withblood. As such, it does pose the questionwhy mosquitoes would have evolved toexpress AaNRP at all, as the ability toenhance virus infection in vertebrates wouldnot have asserted any evolutionary pressurein this respect.In the final chapter of this interestingstory, the authors ask whether therapeutictargeting of AaNRP-induced signaling canlimiting the efficient establishment of virusin the skin following bite by an infectedmosquito. Such an approach is not unpre-cedented, as mice vaccinated against salivafactors NeSt1 and AgBR1 (Marin-Lopezet al, 2021) exhibit some protection frommosquito-transmitted ZIKV infection.Here, Wang et al show that it is possibleto limit AaNRP-mediated enhancement ofZIKV infection through use of the well-described MyD88-NF-κB pathway inhibitorresveratrol. This interesting compound isfound naturally, e.g., in red grapes. Here,administration of resveratrol to mice lim-ited chemokine expression and leukocyterecruitment to AaNRP injection and mos-quito biting. In doing so, this afforded asubstantial degree of protection from ZIKVinfection, as observed through decreasedvirus quantities, ability to disseminate andcause disease.Together these data help solidify theconcept that host responses to mosquitosaliva that promotes rapid and robustleukocyte entry to bitten skin are centralfor its pro-viral capacity. A key step in thismechanism has now been defined with theidentification of AaNRP as a crucial pro-viral factor in Aedes aegypti mosquito saliva.This species of mosquito is responsible fortransmitting most arbovirus infectionsworldwide and constitutes a new target thatcoud limit the development of diseasecaused by a wide number of these geneti-cally diverse viruses. This proof-of-conceptstudy uses resveratrol to limit AaNRPfunction, which, if extrapolated for limitingthe development of disease in humans,would most likely require prophylactic androutine dosing. As such, careful considera-tion, and further pre-clinical testing, wouldbe needed to define the potential efficiencyand side effects of such putative AaNRPinhibitors before devising trials in humans.Although seemingly controversial, such aprophylactic approach is common in othercontexts, such as the use of aspirin for thoseat high risk of heart disease. Similarly,outbreaks of mosquito-borne disease arehighly seasonal. Those individuals, such asthe immunosuppressed that are at higherrisk during these periods, may benefit fromsuch a strategy; only time and furtherstudies will tell if such an approach iseffective and safe.ReferencesBryden SR, Pingen M, Lefteri DA, Miltenburg J,Delang L, Jacobs S, Abdelnabi R, Neyts J,Pondeville E, Major J et al (2020) Pan-viralprotection against arboviruses by activating skinmacrophages at the inoculation site. Sci TranslMed 12:eaax2421Hastings AK, Uraki R, Gaitsch H, Dhaliwal K, StanleyS, Sproch H, Williamson E, MacNeil T, Marin-Lopez A, Hwang J et al (2019) Aedes aegyptiNeSt1 protein enhances ZIka virus pathogenesisby activating neutrophils. J Virol 93:e00395–19Jin L, Guo X, Shen C, Hao X, Sun P, Li P, Xu T, Hu C,Rose O, Zhou H et al (2018) Salivary factor LTRINfrom Aedes aegypti facilitates the transmission ofZika virus by interfering with the lymphotoxin-βreceptor. Nat Immunol 19:342–353Lefteri DA, Bryden SR, Pingen M, Terry S, McCaffertyA, Beswick EF, Georgiev G, Van der Laan M,Mastrullo V, Campagnolo P et al (2022)Mosquito saliva enhances virus infection throughsialokinin-dependent vascular leakage. Proc NatlAcad Sci USA 119:e2114309119Figure 1. A simplified cartoon depicting the findings from Wang et al.Mosquito saliva contains multiple proteins including AaNRP, which is deposited into the dermis during biting.AaNRP activates TLR1 and TLR4-MyD88-NF-KB signaling in skin resident macrophages, resulting in theexpression of leukocyte-recruiting chemokines. If virus, such as ZIKV, is present, some of these infiltratingmyeloid cells become infected, release new infectious virus and make outcome of infection substantially worse.Selective inhibition of the MyD88 pathway by resveratrol inhibited AaNRP function. Created withBiorender.com.The EMBO Journal Yonca Keskek Turk et al2 The EMBO Journal © The Author(s)Downloaded from https://www.embopress.org on April 4, 2024 from IP 94.8.8.34.Marin-Lopez A, Wang Y, Jiang J, Ledizet M, Fikrig E(2021) AgBR1 and NeSt1 antisera protect micefrom Aedes aegypti-borne Zika infection. Vaccine39:1675–1679Martin-Martin I, Valenzuela Leon PC, Amo L,Shrivastava G, Iniguez E, Aryan A, Brooks S, KojinBB, Williams AE, Bolland S et al (2022) Aedesaegypti sialokinin facilitates mosquito bloodfeeding and modulates host immunity andvascular biology. Cell Rep. 39:110648Pingen M, Bryden SR, Pondeville E, Schnettler E, KohlA, Merits A, Fazakerley JK, Graham GJ,McKimmie CS (2016) Host inflammatoryresponse to mosquito bites enhances the severityof arbovirus infection. Immunity 44:1455–69Pingen M, Schmid MA, Harris E, McKimmie CS(2017) Mosquito biting modulates skin responseto virus infection. Trends Parasitol 33:645–657Sun P, Nie K, Zhu Y, Liu Y, Wu P, Liu Z, Du S, Fan H,Chen C-H, Zhang R et al (2020) A mosquitosalivary protein promotes flavivirus transmissionby activation of autophagy. Nat Commun 11:260Wang Z, Nie K, Liang Y, Niu J, Yu X, Zhang O, Liu L,Shi X, Wang Y, Feng X et al (2024) A mosquitosalivary protein-driven influx of myeloid cellsfacilitates flavivirus transmission. EMBOJ https://doi.org/10.1038/s44318-024-00056-xWeaver SC, Charlier C, Vasilakis N, Lecuit M (2018)Zika, chikungunya, and other emerging vector-borne viral diseases. Annu Rev Med 69:395–408Disclosure and competing interestsstatementThe authors declare no competing interests.Open Access This article is licensed under aCreative Commons Attribution 4.0 InternationalLicense, which permits use, sharing, adaptation,distribution and reproduction in any medium orformat, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link tothe Creative Commons licence, and indicate ifchanges were made. The images or other third partymaterial in this article are included in the article’sCreative Commons licence, unless indicatedotherwise in a credit line to thematerial. If material isnot included in the article’s Creative Commonslicence and your intended use is not permitted bystatutory regulation or exceeds the permitted use,you will need to obtain permission directly from thecopyright holder. To view a copy of this licence, visithttp://creativecommons.org/licenses/by/4.0/.Creative Commons Public Domain Dedicationwaiver http://creativecommons.org/publicdo-main/zero/1.0/ applies to the data associated withthis article, unless otherwise stated in a credit line tothe data, but does not extend to the graphical orcreative elements of illustrations, charts, or figures.This waiver removes legal barriers to the re-use andmining of research data. According to standardscholarly practice, it is recommended to provideappropriate citation and attribution whenevertechnically possible.© The Author(s) 2024Yonca Keskek Turk et al The EMBO Journal© The Author(s) The EMBO Journal 3Downloaded from https://www.embopress.org on April 4, 2024 from IP 94.8.8.34.

Sensing the danger in mosquito spit

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Sensing the danger in mosquito spit

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