Thromboxane Mobilizes Insect Blood Cells to Infection Foci
Miltan Chandra Roy1, Kiwoong Nam2, Jaesu Kim3
1Department of Plant Medicals, Andong National University, Andong, South Korea.
Abstract:
Innate immune responses are effective for insect survival to defend against entomopathogens including a fungal pathogen, Metarhizium rileyi, that infects a lepidopteran Spodoptera exigua. In particular, the fungal virulence was attenuated by cellular immune responses, in which the conidia were phagocytosed by hemocytes (insect blood cells) and hyphal growth was inhibited by hemocyte encapsulation. However, the chemokine signal to drive hemocytes to the infection foci was little understood. The hemocyte behaviors appeared to be guided by a Ca2+ signal stimulating cell aggregation to the infection foci. The induction of the Ca2+ signal was significantly inhibited by the cyclooxygenase (COX) inhibitor. Under the inhibitory condition, the addition of thromboxane A2 or B2 (TXA2 or TXB2) among COX products was the most effective to recover the Ca2+ signal and hemocyte aggregation. TXB2 alone induced a microaggregation behavior of hemocytes under in vitro conditions. Indeed, TXB2 titer was significantly increased in the plasma of the infected larvae. The elevated TXB2 level was further supported by the induction of phospholipase A2 (PLA2) activity in the hemocytes and subsequent up-regulation of COX-like peroxinectins (SePOX-F and SePOX-H) in response to the fungal infection. Finally, the expression of a thromboxane synthase (Se-TXAS) gene was highly expressed in the hemocytes. RNA interference (RNAi) of Se-TXAS expression inhibited the Ca2+ signal and hemocyte aggregation around fungal hyphae, which were rescued by the addition of TXB2. Without any ortholog to mammalian thromboxane receptors, a prostaglandin receptor was essential to mediate TXB2 signal to elevate the Ca2+ signal and mediate hemocyte aggregation behavior. Specific inhibitor assays suggest that the downstream signal after binding TXB2 to the receptor follows the Ca2+-induced Ca2+ release pathway from the endoplasmic reticulum of the hemocytes. These results suggest that hemocyte aggregation induced by the fungal infection is triggered by TXB2via a Ca2+ signal through a PG receptor.
Insights
Insect immune cells, hemocytes, aggregate at fungal infection sites, guided by thromboxane B2 (TXB2) signaling. This TXB2 signal, mediated by prostaglandin receptors, triggers a calcium ion (Ca2+) release, crucial for insect defense against pathogens.
Area of Science:
- Insect immunology
- Innate immunity
- Cellular defense mechanisms
Background:
- Innate immune responses are vital for insect survival against entomopathogens like the fungus *Metarhizium rileyi*.
- Cellular immunity, including hemocyte phagocytosis and encapsulation, effectively combats fungal infections in *Spodoptera exigua*.
- The precise signaling pathways guiding hemocytes to infection sites remained largely unknown.
Purpose of the Study:
- To elucidate the chemokine signaling mechanism that directs hemocytes to fungal infection foci.
- To investigate the role of calcium ion (Ca2+) signaling in hemocyte aggregation.
- To identify the specific molecules and pathways involved in mediating insect immune cell aggregation.
Main Methods:
- Investigated Ca2+ signaling in hemocytes using cyclooxygenase (COX) inhibitors and thromboxane A2/B2 (TXA2/TXB2).
- Assessed hemocyte aggregation *in vitro* and *in vivo* following fungal infection.
- Analyzed the expression of phospholipase A2 (PLA2), COX-like peroxinectins (*SePOX-F*, *SePOX-H*), and thromboxane synthase (*Se-TXAS*) genes.
- Utilized RNA interference (RNAi) to study the function of *Se-TXAS* and employed specific inhibitors for receptor and downstream signaling pathway analysis.
Main Results:
- Thromboxane B2 (TXB2) effectively restored Ca2+ signaling and hemocyte aggregation inhibited by COX inhibitors.
- TXB2 induced microaggregation of hemocytes *in vitro*, and its levels increased in infected larvae.
- Elevated TXB2 was linked to induced PLA2 activity and upregulation of *SePOX-F*, *SePOX-H*, and *Se-TXAS* in hemocytes.
- RNAi knockdown of *Se-TXAS* impaired Ca2+ signaling and hemocyte aggregation, which was rescued by TXB2 addition.
- A prostaglandin receptor, not a thromboxane receptor, mediated TXB2 signaling via Ca2+-induced Ca2+ release.
Conclusions:
- Fungal infection triggers TXB2 production in *Spodoptera exigua* hemocytes.
- TXB2 acts as a key chemoattractant, mediating hemocyte aggregation through a Ca2+ signal via a prostaglandin receptor.
- The Ca2+ signal involves Ca2+-induced Ca2+ release from the endoplasmic reticulum, crucial for effective cellular defense against fungal pathogens.
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