Wolbachia surface protein induces innate immune responses in mosquito cells
© Pinto et al; licensee BioMed Central Ltd. 2012
Published: 18 January 2012
Wolbachiaendosymbiotic bacteria are capable of inducing chronic upregulation of insect immune genes in some situations and this phenotype may influence the transmission of important insect-borne pathogens. However the molecules involved in these interactions have not been characterized.
Here we show that recombinant Wolbachia Surface Protein (WSP) stimulates increased transcription of immune genes in mosquito cells derived from the mosquito Anopheles gambiae, which is naturally uninfected with Wolbachia; at least two of the upregulated genes, TEP1 and APL1, are known to be important in Plasmodium killing in this species. When cells from Aedes albopictus, which is naturally Wolbachia-infected, were challenged with WSP lower levels of upregulation were observed than for the An. gambiaecells.
We have found that WSP is a strong immune elicitor in a naturally Wolbachia-uninfected mosquito species (Anopheles gambiae) while a milder elicitor in a naturally-infected species (Aedes albopictus). Since the WSP of a mosquito non-native (nematode) Wolbachia strain was used, these data suggest that there is a generalized tolerance to WSP in Ae. albopictus.
Wolbachia pipientis is a maternally inherited endosymbiotic bacterium that infects a wide range of nematodes and arthropods. It is responsible for the induction of several forms of reproductive manipulation in its arthropod hosts, all of which favour infected females at the expense of their uninfected counterparts. Cytoplasmic incompatibility, classically seen in its unidirectional form in crosses between uninfected females and infected males where there is high embryo mortality, provides a powerful insect population invasion capacity. Recently, the presence of Wolbachia has been associated with the inhibition of viral [1–5] filarial nematode  and Plasmodium [3, 7] pathogens. In addition, Wolbachia is capable of inducing the production of anti-oxidant enzymes and reactive oxygen species (ROS) , innate immune effectors [6, 7, 9] as well as increasing haemocyte densities . However the molecular nature of the interactions between this symbiotic bacterium and the insect immune system are not well characterized. If Wolbachia is to be used optimally in applied strategies to disrupt pathogen transmission in mosquitoes and other pest insects, it is important to gain a better understanding of what Wolbachia molecules are involved in eliciting insect immune responses, and whether responses to these molecules differ between naturally Wolbachia-infected and uninfected hosts.
Wolbachia and its products have been shown to evoke strong innate immune responses in mammals and are very important in establishing and augmenting inflammatory pathogenesis of the diseases caused by filarial nematodes [11–13]. In particular the Wolbachia Surface Protein (WSP) has been shown to elicit innate immune induction via TLR2 and TLR4 activation in both humans and mice  and to inhibit apoptosis in neutrophils through inhibition of caspase-3 activity .
In this study we investigated whether WSP can also induce innate immune responses in insects, using mosquito cell lines originating from both naturally Wolbachia-uninfected and Wolbachia-infected mosquito species. An additional aim was to identify PAMPs (pathogen associated molecular patterns) that can elicit strong immune responses in mosquitoes, which could be useful for novel disease control strategies; thus in order to avoid the complications of possible strain-host co-adaptations, we have initially used WSP derived from a nematode Wolbachia rather than from an insect-derived Wolbachiastrain.
WSP is a strong innate immune response elicitor in An. gambiae cells.
WSP is a mild innate immune response elicitor in Ae. albopictuscells
We next examined whether WSP has the same capacity to elicit an immune response in a species that naturally harbours Wolbachia. Uninfected Ae. albopictus Aa23 cells  were challenged with WSP and transcription level of immunity genes monitored as for the An gambiae cell line. All genes tested showed elevation in mRNA levels with increased WSP concentration up to 5μg/ml (Fig1B), but these were less pronounced when compared to the 4a3A cell line. Statistically significant upregulation was seen only for CEC and TEP when 5μg/ml WSP was used (p<0.05, Fig1B).
Only early phase induction is seen after WSP challenge in both cell lines
The Ae. albopictuscells are capable of mounting a strong immune response
Obtaining a better understanding of Wolbachia-host immune interactions in insects is particularly important at the current time given the recently described effects of Wolbachia in inhibiting the development or dissemination of several very important mosquito-borne human pathogens. This study shows that, as previously observed using mammalian cells, the Wolbachia WSP protein is a potent innate immune elicitor in insects. The responses between the two mosquito cell lines to WSP challenge are mechanistically similar: 1) they are dosage dependent, increasing with increasing amounts of WSP up to 5μg/ml; 2) peak induction is seen at 5μg/ml, while higher concentrations sometimes reduce the mRNA levels; and 3) the immune gene transcription was at a maximum at 3h post challenge (early phase induction) and do not show late phase induction. The major difference is the level of upregulation between the two species: detected peak induction of 3 to 5-fold in the naturally Wolbachia-uninfected cell line compared to just 2-fold induction in the naturally infected one. Tolerance effects due to previous natural Wolbachia exposure have been described  and seem likely to be contributing to the differences observed between these cell lines in their response to WSP. The control experiments also show that Aa23T can show strong induction of immune gene transcription and can effectively clear a bacterial infection. Thus the differences seen between WSP-associated immune induction between these cell lines are not due to impaired immune responses in Aa23T.
In this experimental set-up the WSP protein will be extracellular, and although Wolbachia itself is mostly located within intracellular vacuoles in insects, bacterial protein will be released into the hemocele, for example through regular cellular turnover / apoptosis. This mirrors the situation in humans where WSP elicits antibody responses in lymphatic filariasis patients despite Wolbachia itself being located inside vacuoles within the filarial nematodes . In the insect hemocele WSP has the potential to elicit innate immune responses from hemocyte immune cells, and the same applies in these cell lines.
Further studies of insect immune responses to WSP may include the examination of levels of immune response to intracellular WSP, using transformation / transfection studies (although these will not exactly replicate the intra-vacuole localization of Wolbachia itself). Furthermore, the possibility of different levels of immune response to WSP derived from various insect Wolbachia strains can be examined, particularly in the case of the Ae. albopictus cells which are derived from a naturally Wolbachia-infected species and could thus show varying degrees of tolerance to different WSP molecules. These basic biology questions are also relevant to the important applied aim of identifying potent PAMPs that might be incorporated in transgenic strategies to ‘prime’ the mosquito immune system, and thus impair pathogen transmission. The Dirofilaria Wolbachia-derived WSP used here appears to hold potential in this respect, since it induces the upregulation of genes (particularly TEP1 and APL1) that are directly involved in Plasmodium killing in Anopheles mosquitoes.
Similarly to mammals, the major surface protein of the endosymbiotic bacteria Wolbachia (WSP) can induce strong innate immune responses in insects at the transcriptomic level. Antimicrobial peptides as well as important immune effector genes are up-regulated when recombinant WSP is used to challenge mosquito cell lines. Interestingly the response between a naturally-uninfected mosquito and a naturally -infected mosquito is qualitatively similar but quantitatively distinct. The Wolbachia naïve host is capable of mounting a very strong upregulation to WSP as opposed to the Wolbachia cleared host suggesting that tolerance effects due to previous Wolbachiaexposure may be contributing to this particular phenotype.
Two cell lines were used: 4a3A derived from the naturally Wolbachia-uninfected mosquito species Anopheles gambiae  and Aa23 from the naturally Wolbachia-infected mosquito species Aedes albopictus . wAlbB-strain infection present in Aa23 was cured via Tetracycline treatment (100μg/ml) for 5 days. Wolbachiaabsence after drug treatment was confirmed using PCR and the derived cell line was subsequently called Aa23T. Cell lines were maintained at 27 °C and grown in Schneider medium (Promo Cell) supplemented with 10% heat-inactivated FCS, 1% penicillin-streptomycin (Gibco).
WSP and bacterial cell challenges
Prior to cell challenges, cultures were re-suspended in growth medium and counted using a heamocytometer. For all experiments, approximately 2million cells were seeded per well in 6-well plates. Varying concentrations (0.5-10μg/ml) of stringently purified endotoxin-free recombinant WSP, obtained from the nematode Dirofilaria immitis [14, 19], were used to challenge the cells. Proteinase k-treated WSP (pkWSP) [14, 19] was used at a concentration of 5μg/ml.
Logarithmic phase cultures of E. coli and E. faecalis were washed three times in PBS and re-suspended in Hank-balanced salt solution (Sigma) at OD (A600 nm) of 0.4 prior to heat inactivation at 80 C. For challenge, 30 μl of a 1:1 mixture of heat killed E. coli and E. faecalis were used per well. Logarithmic phase cultures of E. coli K12 TETr strain (NEB) were washed and re-suspended in PBS to a final OD (A600 nm) of 0.05. For challenge, 25 μl of the bacterial culture was added to 3hr conditioned cell culture or 3hr incubated Schneider medium (cell-free). Cell medium was collected at 3 and 9hr post E. coliaddition, plated in serial dilutions onto LB-TET agar plates and the next day the number of CFUs was determined.
RNA isolation, cDNA synthesis and real-time quantitative reverse transcription PCR (qRT-PCR)
Primers used in qRT-PCR
Ae albopictus !
DEF (D) *
CEC (A1), TEP (20), PGRP (SP1) and CLIP (B37) ref 
This study was supported by the Wellcome Trust (grant number 079059) and by MIUR-PRIN 2009. MM received support from EU-COST Action FA0701 "Arthropod Symbiosis: from fundamental research to pest and disease management”.
The authors thank M. Blagrove for sharing primer sequences prior to publication.
This article has been published as part of BMC Microbiology Volume 11 Supplement 1, 2012: Arthropod symbioses: from fundamental studies to pest and disease mangement. The full contents of the supplement are available online at http://0-www.biomedcentral.com.brum.beds.ac.uk/1471-2180/12?issue=S1.
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