The ZIKV is known for its ability to evade host immune mechanisms, a trait that poses a significant challenge to the immune system. This evasion is achieved through various strategies, including impairing the induction and signaling pathways of the immune response at multiple steps (35). Recent advances in research have shed light on the intricate interplay between mosquito saliva and the host immune response, underscoring the pivotal role of mosquito saliva in shaping the outcomes of arboviral infections (3–5). Our study sought to delineate the immunomodulatory effects of Ae. aegypti salivary glands in the context of ZIKV infection using antigen-presenting T cell models and human PBMCs, juxtaposed against scenarios where saliva is not a contributing factor. The findings presented herein not only deepen our understanding of the immune evasion mechanisms employed by ZIKV but also offer critical insights into the nuanced interactions between Ae. aegypti salivary components and the host immune system. The observed profound influence of Ae. aegypti salivary glands on ZIKV infection dynamics signifies a crucial factor in the intricate web of host-mosquito-pathogen interactions.
The impact of Ae. aegypti SGE on APCs during ZIKV infection aligns with current research exploring the interaction between mosquito saliva and the host immune system. Studies have shown that mosquito saliva can modulate the immune response in various ways (29, 36–38). For example, it's been observed that mosquito saliva has the capacity to alter leukocyte recruitment and cytokine signaling by APCs during arbovirus infections, such as with West Nile Virus (29). This includes effects on dendritic cells and macrophages, which are critical in early arbovirus infection, serving as primary sites of viral replication and key players in orchestrating the immune response to the virus. In the setting of ZIKV infection, we observe that the presence of Ae. aegypti SGE has been shown to increase the production of pro-inflammatory cytokines, such as TNF-α and IL-1β, while simultaneously reducing the levels of IL-12 in dendritic cells. This modulation of cytokine production suggests a critical role for mosquito saliva in influencing the host's immune response, potentially affecting the disease's progression and transmission. The increase in TNF-α and IL-1 modulated by SEG is a strategy that benefits arboviruses, as these cytokines are associated with increased edema, which helps the local retention of viruses and more APCs (DCs and macrophages) infection (39). Our data indicated that this can also be a phenomenon modulated in ZIKV infection. On the other hand the reduction in IL-12 production, an essential cytokine for Th1 antiviral response, further indicates a nuanced dysregulation of APC antiviral signaling by mosquito saliva (36). The IL-12 reduction can downregulate the function of NK and CD8 + T cells, both extremely relevant to controlling ZIKV infection (40).
Our study's outcomes align with previous research demonstrating that Ae. aegypti saliva can suppress innate immune responses, which can facilitate arbovirus infections. The observed alterations in cytokine profiles in DC and macrophages following exposure to Ae. aegypti SGE are consistent with the effects reported in other studies involving arboviral infections and mosquito saliva (29, 36–38). Furthermore, research has revealed that Ae. aegypti SGE can impact macrophage polarization, particularly influencing the M1/M2 macrophage profile, which is crucial for the inflammatory response. This polarization is essential in the host's response to pathogens, with M1 macrophages generally being pro-inflammatory and M2 macrophages being more associated with tissue repair and anti-inflammatory responses (38). Our study unveiled a distinct shift in the Th1/Th2 cytokine polarization in response to Ae. aegypti salivary glands during ZIKV infection. Salivary gland treatment was associated with decreased IFN-γ production and a concurrent increase in IL-4, indicative of a shift towards Th2 polarization. Our results firmly suggest that Ae. aegypti salivary glands drive a shift towards Th2 polarization, as evidenced by the observed changes in this ratio. The modulation of the Th1/Th2 cytokine balance by mosquito saliva has been investigated (41, 42). These studies suggest that mosquito saliva can influence the cytokine milieu of APCs and impact the polarization of T-cell responses (41, 42). Our study aligns with these findings, demonstrating that Ae. aegypti salivary glands can influence the Th1/Th2 cytokine polarization in PBMCs during ZIKV infection(42). The changes in IL-4 and IFN-γ levels suggest mosquito saliva could contribute to the modulation of T cell responses, potentially affecting the outcome of viral infections.
Bridging the findings on Ae. aegypti salivary glands' impact on cytokine polarization and DC profile, it becomes evident that ZIKV exploits these immune alterations to its advantage. The shift towards a Th2 cytokine profile, driven by mosquito saliva, may play a critical role in compromising the traditionally robust antiviral defense mechanisms of DC. This interplay showcases how ZIKV, along with the influence of mosquito saliva, can manipulate key immune cells, turning them from potent defenders into facilitators of viral replication. DC are specialized immune cells known for their unique functions, including recognizing viral threats, presenting foreign antigens to both innate and adaptive immune cells, and initiating immune responses to combat viruses and protect the host. They also possess a remarkable ability to hinder different stages of viral replication by producing a wide range of antiviral molecules, activated in response to type I Interferons (43).Due to these robust characteristics, dendritic cells are traditionally viewed as hostile environments for viral replication, owing to their robust antiviral defense mechanisms. However, ZIKV's ability to infect immune cells, particularly myeloid dendritic cells (mDCs), despite their traditionally robust antiviral mechanisms, showcases a distinct immune escape tactic (44–47). By transforming mDCs into a supportive environment for viral replication, ZIKV effectively circumvents the typical barriers to infection (44–47). Furthermore, our investigation into immune cell populations reveals that mosquito saliva can alter the proportions of dendritic cells. These changes may affect immune surveillance, antigen presentation, and overall immune system function during ZIKV infection. Our findings corroborate the unexpected susceptibility of mDCs to ZIKV infection. Our results align with emerging evidence(44–47) suggesting that ZIKV combined with SGE spread the ability to transform mDCs into a cellular niche conducive to viral propagation, representing a decrease in this population because of death by viral infection and replication.
The link between the manipulation of dendritic cells and T-cell responses by Ae. aegypti SGE in ZIKV infection suggests a coordinated strategy of immune modulation. This strategy not only compromises the dendritic cell's antiviral defenses but also extends to altering T cell dynamics, further indicating the depth and complexity of the immune system manipulation by ZIKV in the presence of SGE. The findings from the investigation into the effects of Ae. aegypti SGE on T cell-mediated immune responses during ZIKV infection are intriguing and align with broader research in the field of immunology. The observed increase in CD4 + T cell frequency and the lack of significant alteration in CD8 + T cells, along with changes in cytokine production, suggest a nuanced modulatory role of SGE in T cell dynamics. It's possible that during ZIKV infection the SGE contributes to the differentiations of CD4 + T cell populations, mostly Th2, since others previously identified the protein SAAG-4 present in SEG that reduces CD4 expression, induces Th2 differentiation and IL-4 production (48).This modulation could potentially affect the immune response's balance, influencing the outcome of ZIKV infection. In particular, the significant reduction in the proliferation of both CD4 + and CD8 + T cell subsets in response to SGE, as well as the impact on granzyme B production in CD8 + T cells, highlights the complex interplay between the mosquito saliva and the host immune system. Previous studies using mouse cells have shown that A. aegypti SGE induces apoptosis in CD4 + and CD8 + T cells, and B cells through a mechanism involving caspase-3 and caspase-8 (25). Aligning with the findings, the results of a previous study indicate that Ae. aegypti SGE induces apoptosis in various lymphocyte subsets, including CD4 + and CD8 + T cells, as well as B cells. This apoptosis mechanism involves caspase-3 and caspase-8 activation, underscoring the intricacy of the immunomodulatory pathways exploited by mosquito saliva. (25). The results suggest that mosquito saliva, through components like LTRIN increases inflammatory cytokines and decreases the Th1 polarization mediated by of antigen-presenting cells and impact T-cell responses(21). Indeed, saliva products from other vectors such as sand flies reduces antigen presenting cells activation and negatively modulate the secretion of Th1 and Th2 cytokines by PBMCs (49).
Additionally, our investigation revealed a link between Ae. aegypti salivary gland components and oxidative stress responses. The alteration of ROS production, lipid peroxidation, and antioxidant levels in PBMCs indicates a multifaceted impact on cellular redox dynamics. The heightened nitric oxide production and the dynamic alterations in lipid peroxidation, GSH levels, and AOPP content suggest that the mosquito's salivary glands play a role in modulating the oxidative stress environment of host cells. To further understand the link between these oxidative stress-related findings and the broader immunomodulatory effects of Ae. aegypti salivary glands, additional studies are warranted to dissect the underlying mechanisms driving these observed changes. Such knowledge could offer promising avenues for therapeutic interventions to mitigate the detrimental effects of ZIKV infection. The impact of mosquito saliva on oxidative stress has been elucidated in studies that highlight the presence of defense-related proteins in mosquito saliva (50, 51). These proteins can decrease and increase the redox state within host cells, depending of the parameters analyzed. Our study extends this understanding by revealing that Ae. aegypti salivary glands have the potential to modulate oxidative stress in PBMCs during ZIKV infection. The elevation of NO levels and changes in GSH content observed in our study align with the findings from these earlier investigations. Therefore, previous study shows that SGE plays a protective role in septic animals, contributing to oxidative and inflammatory balance during sepsis, and this effect seems to be mediated by the control of inflammation and oxidative damage (12). These results underscore mosquito saliva's multifaceted role in impacting cellular oxidative stress and redox signaling pathways.
The SGE from these mosquitoes demonstrated the capacity to potentiate ZIKV infection in vitro, a phenomenon evidenced by the dose-dependent increase cytopathic effect within Vero cells. The dose-dependent relationship observed between the quantity of SGE and the PFU underscores the significant role played by salivary gland factors in influencing ZIKV infection dynamics. Similar to other studies, our results demonstrated that mosquito saliva could enhance viral infection and replication (52–55). The enhancement of ZIKV infection observed in Vero cells and DC treated with Ae. aegypti SGE was also observed by Styer et al. and Wasinpiyamongkol et al., which highlighted the ability of mosquito saliva to augment the infection of West Nile virus and dengue virus (8, 20). Furthermore, Vero cells do not produce type I interferon, an important element of antiviral immunity, which makes the role of SGE more significant in the spread of infection and its relationship with the immunological response. These data suggest a common mechanism by which mosquito saliva might promote the entry and propagation of arboviruses within host cells.
Collectively, the results presented here and insights from previous research emphasize the significance of mosquito saliva in shaping the immune response to arboviral infections. Our findings align with existing literature, illustrating the ability of Ae. aegypti salivary glands to enhance viral infection, manipulate cytokine production, and influence oxidative stress dynamics. These observations strengthen the notion that mosquito saliva is not just a mechanical facilitator of blood feeding but a sophisticated mediator that can tip the balance in favor of viral transmission and disease exacerbation. These findings offer novel insights into the intricate modulation of host immune responses by mosquito salivary gland factors, with potential sources for new therapeutic molecule(s).