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Thromboxane Mobilizes Insect Blood Cells to Infection Foci.

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 TXB₂ via a Ca²⁺ signal through a PG receptor.

SUBMITTER: Roy MC

PROVIDER: S-EPMC8720849 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The malaria asexual blood-stage antigen PfRipr and its most immunogenic fragment PfRipr5 have recently risen as promising vaccine candidates against this infectious disease. Continued development of high-yielding, scalable production platforms is essential to advance the malaria vaccine research. Insect cells have supplied the production of numerous vaccine antigens in a fast and cost-effective manner; improving this platform further could prove key to its wider use. In this study, insect (Sf9 and High Five) and human (HEK293) cell hosts as well as process-optimizing strategies (new baculovirus construct designs and a culture temperature shift to hypothermic conditions) were employed to improve the production of the malaria asexual blood-stage vaccine candidate PfRipr5. Protein expression was maximized using High Five cells at CCI of 2 × 106 cell/mL and MOI of 0.1 pfu/cell (production yield = 0.49 mg/ml), with high-purity PfRipr5 binding to a conformational anti-PfRipr monoclonal antibody known to hold GIA activity and parasite PfRipr staining capacity. Further improvements in the PfRipr5 expression were achieved by designing novel expression vector sequences and performing a culture temperature shift to hypothermic culture conditions. Addition of one alanine (A) amino acid residue adjacent to the signal peptide cleavage site and a glycine-serine linker (GGSGG) between the PfRipr5 sequence and the purification tag (His6) induced a 2.2-fold increase in the expression of secreted PfRipr5 over using the expression vector with none of these additions. Performing a culture temperature shift from the standard 27–22°C at the time of infection improved the PfRipr5 expression by up to 1.7 fold. Notably, a synergistic effect was attained when combining both strategies, enabling to increase production yield post-purification by 5.2 fold, with similar protein quality (i.e., purity and binding to anti-PfRipr monoclonal antibody). This work highlights the potential of insect cells to produce the PfRipr5 malaria vaccine candidate and the importance of optimizing the expression vector and culture conditions to boost the expression of secreted proteins.

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Thromboxane Mobilizes Insect Blood Cells to Infection Foci.

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