Molecular Basis of Reduced LAIR1 Expression in Childhood Severe Malarial Anemia: Implications for Leukocyte Inhibitory Signalling

Abstract

Background: Leukocyte-associated immunoglobulin like receptor-1 (LAIR1) is a transmembrane inhibitory receptor that influences susceptibility to a myriad of inflammatory diseases. Our recent investigations of severe malarial anemia (SMA) pathogenesis in Kenyan children discovered that novel LAIR1 genetic variants associated with decreased LAIR1 enhanced the longitudinal risk of SMA and all-cause mortality. Methods: To further characterize LAIR1 expression and identify novel molecular mechanisms, at least in part, responsible for reduced LAIR1 in severe malaria, we conducted a series of experiments in samples from children with malaria, followed by in vitro investigations driven by the in vivo findings. Findings: Kenyan children with SMA had elevated circulating levels of soluble LAIR1 (sLAIR1) relative to non-SMA (1.69-fold P<0.0001). The LAIR1 antagonist, sLAIR2, was also elevated in the circulation of children with SMA (1.59 fold-change, P<0.0001). There was a positive correlation between sLAIR1 and sLAIR2 (ρ= 0.741, P<0.0001). Conversely, circulating levels of complement component 1q (C1q), a LAIR1 natural ligand, were lower in SMA (-1.21-fold P=0.048). These in vivo findings suggest that reduced membrane-bound LAIR1 expression in SMA is associated with elevated production of sLAIR1, sLAIR2 (antagonist), and limited C1q (agonist) availability. Since reduced LAIR1 transcripts in SMA were associated with increased acquisition of hemozoin (PfHz) by monocytes (P=0.028), we explored the relationship between acquisition of intraleukocytic PfHz, LAIR1 expression, and subsequent impacts on leukocyte signalling in cultured PBMCs from malaria-naive donors stimulated with physiological concentrations of PfHz (10 µg/mL). Phagocytosis of PfHz reduced LAIR1 transcript and protein expression in a time-dependent manner (P<0.050), and inhibited LAIR1 signalling through decreased phosphorylation of LAIR1 (P<0.0001) and SH2-domain containing phosphatase-1 (SHP-1) (P<0.001). This process was associated with NF-κB activation (P<0.0001) and enhanced production of IL-6, IL-1β, and TNF-α (all P<0.0001). Interpretation: Collectively, these findings demonstrate that SMA is characterized by reduced LAIR1 transmembrane expression, reduced C1q, and enhanced production of sLAIR1 and sLAIR2, molecular events which can promote enhanced production of cytokines that contribute to the pathogenesis of SMA. These investigations are important for discovering immune checkpoints that could be future targets of immunotherapy to improve disease outcomes. Funding: The work was supported by National Institutes of Health (NIH) Research Grants R01AI130473, R01AI51305 and D43TW05884 (DJP), and the University of New Mexico Health Sciences Clinical and Translational Science Center (UL1TR001449). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The study was approved by the ethical and scientific review committees at the, the University of New Mexico and the Kenya Medical Research Institute.