Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: 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 anaemia (SMA) pathogenesis in Kenyan children discovered that novel LAIR1 genetic variants which were associated with decreased LAIR1 transcripts enhanced the longitudinal risk of SMA and all-cause mortality.

Methods: To characterize the molecular mechanism(s) responsible for altered LAIR1 signalling in severe malaria, we determined LAIR1 transcripts and protein, sLAIR1, sLAIR2, and complement component 1q (C1q) in children with malarial anaemia, followed by a series of in vitro experiments investigating the LAIR1 signalling cascade.

Findings: Kenyan children with SMA had elevated circulating levels of soluble LAIR1 (sLAIR1) relative to non-SMA (1.69-fold P < .0001). The LAIR1 antagonist, sLAIR2, was also elevated in the circulation of children with SMA (1.59 fold-change, P < .0001). There was a positive correlation between sLAIR1 and sLAIR2 (ρ = 0.741, P < .0001). Conversely, circulating levels of complement component 1q (C1q), a LAIR1 natural ligand, were lower in SMA (-1.21-fold P = .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 haemozoin (PfHz) by monocytes (P = .028), we explored the relationship between acquisition of intraleukocytic PfHz, LAIR1 expression, and subsequent impacts on leukocyte signalling in cultured PBMCs from malaria-naïve 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 < .050), and inhibited LAIR1 signalling through decreased phosphorylation of LAIR1 (P < .0001) and SH2-domain containing phosphatase-1 (SHP-1) (P < .001). This process was associated with NF-κB activation (P < .0001) and enhanced production of IL-6, IL-1β, and TNF-α (all P < .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.

Keywords: Complement component 1q; Leukocyte-associated immunoglobulin like receptor-1; Leukocyte-associated immunoglobulin like receptor-2; Plasmodium falciparum haemozoin; Plasmodium falciparum malaria; Severe malarial anaemia.

Fig. 1

Relationship between sLAIR1, sLAIR2, and C1q serum levels in children with malaria. Differences between circulating sLAIR1 (ng/mL), sLAIR2 (ng/mL), and C1q (ng/mL) in children with non-SMA and SMA were determined using Student t-test and are presented as mean ± SEM. Relationships between sLAIR1, sLAIR2, C1q, and haemoglobin levels were determined by Spearman's correlation test. (a) Normalised sLAIR1 levels in children with non-SMA and SMA. Children with SMA had elevated sLAIR1 levels relative to non-SMA. (b) Normalised sLAIR1 vs. haemoglobin concentrations in children with non-SMA and SMA. Circulating sLAIR1 levels were inversely correlated with haemoglobin levels. (c) Normalised sLAIR1 levels in children with non-SMA and SMA. sLAIR2 serum levels were elevated in children with SMA relative to non-SMA. (d) Normalised sLAIR2 vs. haemoglobin concentrations in children with non-SMA and SMA. Normalised sLAIR2 vs. haemoglobin. sLAIR2 levels were inversely correlated with haemoglobin concentrations. (e) Normalised sLAIR1 vs. normalised sLAIR2 in children with non-SMA and SMA. Circulating sLAIR1 and sLAIR2 levels were positively correlated. (f) Circulating C1q levels in children with non-SMA and SMA. Circulating C1q levels were lower in children with SMA.

Fig. 2

Relationship between LAIR1 expression and intraleukocytic haemozoin. (a) Pairwise comparisons of LAIR1 transcript levels between children with and without pigment-containing monocytes (PCM) and pigment-containing neutrophils (PCN) in the non-SMA and SMA groups were determined using Mann-Whitney U test. Data are presented as box-plots, where the box represents the interquartile range, the line through the box is the median, and whiskers show the 10th and 90th percentiles. LAIR1 transcript levels were lower in children with PCM compared to those without, while LAIR1 transcript levels were comparable between absence/presence of PCN. (b) Temporal kinetics of LAIR1 transcript levels in response to PfHz treatment of PBMC from malaria-naïve donors (n = 6, measured in triplicate). Pairwise comparisons were determined using Student t-test. Significant (P < .05) differences in transcript levels between no treatment and PfHz treatment (10 μg/mL) groups represented by *at 0.5, 2, and 4 h time points. Data represent average of individuals (n = 3) with each condition performed in triplicate (error bars represent SEM). (c) Immunoblot analysis of non-treated (baseline) and PfHz-treated (10 μg/mL) PBMC lysates for 12, 24, and 48 h. (d) Densitometric analysis of normalised cellular LAIR1 protein production presented as mean ± SEM. Across group comparisons analysed using ANOVA. Pairwise comparisons analysed using Student t-test. Data represent average of individuals (n = 6) with each condition performed in triplicate (error bars represent SEM). LAIR1 protein levels were lower in PfHz-treated PBMC lysates compared to no treatment at 12, 24, and 24 h.

Fig. 3

Effect of intraleukocytic PfHz on LAIR1 and SHP-1 phosphorylation. Temporal kinetics of the signalling pathway in response to the different treatment conditions was determined in PBMCs from malaria-naïve donors (n = 6, measured in triplicate). (a) Human Phospho-immunoreceptor array results showing LAIR1 and SHP-1 phosphorylation upon no treatment (baseline), C1q (25 μg/mL), PfHz (10 μg/mL), and PfHz + C1q. Phosphorylation (spots) in the different conditions with control represented by blue boxes, LAIR1 by red boxes, and SHP-1 by green boxes. (b) Densitometric analysis of human phosphor-immunoreceptor array data. Data presented as (mean ± SEM). Across group comparisons analysed using ANOVA. *indicates significant differences (P < .05) determined by Student t-test in pLAIR1 and pSHP-1 compared to C1q treatment. Phagocytosis of PfHz resulted in a reduction of pLAIR1 relative to C1q (alone) treatment. Similarly, ingestion of PfHz also caused a marked decrease in pSHP-1 levels relative to C1q treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Effect of haemozoin on NF-κB activation and cytokine production. Measurements ndwere determined in PBMCs from malaria-naïve donors (n = 6, measured in triplicate). Data presented as mean ± SEM. Across group and pairwise comparisons determined using Student t-test and Anova analyses, respectively. (a) Comparisons of phosphorylated NF-κB immediately after stimulation of PBMCs with no treatment (NT), C1q (25 μg/mL), PfHz (10 μg/mL), and PfHz + C1q. NF-κB phosphorylation levels were elevated in the PfHz and PfHz + C1q treatment groups relative to no treatment (denoted by *P < .05). (b) IL-1β levels (pg/mL) were elevated in culture supernatants from PBMCs treated with PfHz and PfHz + C1q. (c) IL-6 levels (pg/mL) were elevated in culture supernatants from PBMCs treated with PfHz and PfHz + C1q. (d) TNF-α levels (pg/mL) were elevated in culture supernatants from PBMCs treated with PfHz and PfHz + C1q.

Fig. 5

LAIR1 signalling pathway in malaria. The LAIR1 pathway is activated by attachment of collagen and collagenous ligands (C1q) to LAIR1 extracellular surface. This results in phosphorylation of intracellular LAIR1 ITIM tyrosine residues by Src family kinases. Phosphorylated ITIMs serve as docking sites for recruitment of SHP-1 and SHP-2 phosphatases. SHP-1 and SHP-2 become localized to phosphorylated ITIMs through their regulatory SH2 domains, subsequently inducing their phosphatase activity. Activated SHP-1 has been shown to block activation and nuclear translocation of nuclear factor nuclear factor-kappa beta (NF-κB) through de-phosphorylation of inhibitor of kappa-beta kinase complex (IKK). SHP-1 phosphatase activity also inhibits activation and translocation of IRFs from the cytoplasm to the nucleus by preventing TANK-binding kinase 1 (TANK-1) phosphorylation of interferon regulatory factors (IRFs). These events subsequently block transcription of inflammatory mediator encoding genes with response elements for IRFs and NF-κB. SHP-2 inhibits activation of IRF 8 and blocks expression of phagocyte NADPH oxidase (gp91^PHOX). LAIR1 inhibitory signalling is regulated by soluble LAIR1 and LAIR2 (soluble homolog of LAIR1) through competition for collagenous ligands. Children with SMA had increased circulating levels of sLAIR1 and sLAIR2 indicative of enhanced receptor shedding. C1q was reduced in children with SMA, thereby, limiting ligand availability. Phagocytosis of PfHz antagonizes LAIR1 signalling through down-regulation of LAIR1 ITIM and SHP-1 phosphorylation, but does not alter SHP-2 phosphorylation. Leukocytic ingestion of PfHz also decreases LAIR1 transcripts and protein. These events result in NF-κB activation and the consequent production of pro-inflammatory mediators that enhance the pathogenesis of SMA. Red arrows represent ligand-receptor interaction, black solid arrows represent activation, and dashed black arrows represent blockade. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)