The mechanisms of crosstalk between haemolysis, coagulation and innate immunity are evolutionarily conserved from the invertebrate haemocyanin to the vertebrate haemoglobin (Hb). In vertebrates, extracellular Hb resulting from haemolytic infections binds bacterial lipopolysaccharide (LPS) to unleash the antimicrobial redox activity of Hb. Because bacterial invasion also upregulates tissue factor (TF), the vertebrate coagulation initiator, we asked whether there may be functional interplay between the redox activity of Hb and the procoagulant activity of TF. Using real-time PCR, TF-specific ELISA, flow cytometry and TF activity assay, we found that Hb upregulated the expression of functional TF in macrophages. ELISA, flow cytometry and immunofluorescence microscopy showed binding between Hb and TF, in isolation and in situ. Bioinformatic analysis of Hb and TF protein sequences showed co-evolution across species, suggesting that Hbβ binds TF. Empirically, TF suppressed the LPS-induced activation of Hb redox activity. Furthermore, Hb desensitised TF to the effects of antioxidants like glutathione or serum. This bi-directional regulation between Hb and TF constitutes a novel link between coagulation and innate immunity. In addition, induction of TF by Hb is a potentially central mechanism for haemolysis to trigger coagulation.

Serine protease cascades boost immune responses while maintaining homeostasis. These crucial actions are intricately regulated by cognate serine protease inhibitors. However, the mechanism underlying such a dynamic immunomodulation during acute phase infection remains obscure, particularly where the pathogen’s serine protease adds a new challenge to the host. Here, we found that infection of horseshoe crab, Carcinoscorpius rotundicauda, induced reciprocal profiles of CrSPI (serine protease inhibitor) and CrFurin (serine protease) with respect to their transcription and protein activities. Using recombinant rCrSPI, we explored its inhibitory activity against various microbial proteases and found it most efficacious against a model serine protease, subtilisin A. rCrSPI inhibited subtilisin at Ki 10^–9M with a molar ratio of 1 rCrSPI:2 subtilisin. The rCrSPI also inhibited plasma CrFurin, suppressed subtilisin-mediated activation of prophenoloxidase (PPO) and interacted with complement C3. Taken together, CrSPI acts as a key immunomodulatory ‘on-off’ switch in a 2-way regulation of serine protease microbial subtilisin and host serine proteases (CrFurin and CrC3), thereby controlling immune responses involving the complements and the PPO-mediated antimicrobial activities, while maintaining homeostasis.

Background: Redox activity of hemoglobin (Hb) is augmented by lipopolysaccharide (LPS) to boost immune defense. Results: Computational analysis of Hb identified LPS-binding hot spots, which were further defined via peptide-based binding assays and confirmed by mutagenesis of Hb subunits. Conclusion: Regions of Hb that interact with LPS have been delineated. Significance: Knowledge of LPS-binding residues on Hb may be exploited for designing antimicrobial peptides.

Hemolysis releases hemoglobin (Hb), a prooxidant, into circulation. While the heme iron is a nutrient for the invading pathogens, it releases ROS, which is both microbicidal and cytotoxic, making it a double-edged sword. Previously, we found a two-pass detoxification mechanism involving the endocytosis of Hb into monocytes in collaboration with vascular endothelial cells to overcome oxidative damage. This prompted us to examine the effect of Hb priming on host cell viability and intracellular bacterial clearance during a hemolytic infection. Here, we demonstrate that Hb-primed macrophages harbor a higher intracellular bacterial load but with suppressed apoptosis. p-ERK and p-p38 MAPK were significantly downregulated, with concomitant impairment of Bax and downstream caspases. The Hb-primed cells harboring intracellular bacteria upregulated anti-inflammatory IL-10 and downregulated proinflammatory TNF-α, which further enhanced the infectivity of the neighboring cells. Our findings suggest that opportunistic intracellular pathogens exploit the Hb-scavenging machinery of the host to silently persist within the circulating phagocytes by suppressing apoptosis while escaping immune surveillance.