Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling Angela O. Achieng a,b , Bernard Guyah b , Qiuying Cheng c , John M. Ong'echa d , Collins Ouma a,b , Christophe G. Lambert c , Douglas J. Perkins a,c, ⁎ a University of New Mexico-Kenya Global Health Programs, Kisumu and Siaya, Kenya b Department of Biomedical Sciences and Technology, School of Public Health and Community Development, Maseno University, Maseno, Kenya c University of New Mexico, Center for Global Health, Department of Internal Medicine, NM, USA d Kenya Medical Research Institute, Centre for Global Health Research, Kisumu, Kenya abstract article info Article history: Received 11 February 2019 Received in revised form 18 June 2019 Accepted 20 June 2019 Available online 27 June 2019 Background: Leukocyte-associated immunoglobulin like receptor-1 (LAIR1) is a transmembrane inhibitory recep- tor that influences susceptibility to a myriad of inflammatory diseases. Our recent investigations of severe malar- ial 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 b .0001). The LAIR1 antagonist, sLAIR2, was also elevated in the circulation of children with SMA (1.59 fold-change, P b .0001). There was a positive correlation between sLAIR1 and sLAIR2 (ρ = 0.741, P b .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) availabil- ity. 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 expres- sion, 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 b .050), and inhibited LAIR1 signalling through decreased phosphor- ylation of LAIR1 (P b .0001) and SH2-domain containing phosphatase-1 (SHP-1) (P b .001). This process was as- sociated with NF-κB activation (P b .0001) and enhanced production of IL-6, IL-1β, and TNF-α (all P b .0001). Interpretation: Collectively, these findings demonstrate that SMA is characterized by reduced LAIR1 transmem- brane expression, reduced C1q, and enhanced production of sLAIR1 and sLAIR2, molecular events which can pro- mote 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 dis- ease outcomes. © 2019 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Complement component 1q Leukocyte-associated immunoglobulin like receptor-1 Leukocyte-associated immunoglobulin like receptor-2 Plasmodium falciparum malaria Plasmodium falciparum haemozoin Severe malarial anaemia EBioMedicine 45 (2019) 278–289 Abbreviations: AML, Acute myeloid leukaemia; C1q, Complement component 1q; CLL, Chronic lymphocytic leukaemia; HFRS, haemorrhagic fever with renal syndrome; HIV-1, Human immunodeficiency virus 1; IL-1β, Interleukin 1 beta.; IL-6, Interleukin 6; ITIM, immuno-tyrosine inhibition motifs; LAIR1, Leukocyte-associated immunoglobulin like receptor-1; LAIR2, Leukocyte-associated immunoglobulin like receptor-2; NF-κB, Nuclear factor-kappa beta; PBMCs, Peripheral blood mononuclear cells; P. falciparum, Plasmodium falciparum; PfHz, Plasmodium falciparum haemozoin; PCM, Pigment containing monocytes; PCN, Pigment containing neutrophils; PCR, Polymerase chain reaction; SHP-1, SH2 domain-containing tyrosine phosphatase-1; SHP-2, SH2 domain-containing tyrosine phosphatase-2; SMA, severe malarial anaemia; SLE, Systemic lupus erythematosus; TNF-α, Tumor necrosis factor alpha. ⁎ Corresponding author at: Center for Global Health, Department of Internal Medicine, University of New Mexico Health Sciences Center, MSC10-5550, Albuquerque NM 87131-0001, USA. E-mail address: Dperkins@salud.unm.edu (D.J. Perkins). https://doi.org/10.1016/j.ebiom.2019.06.040 2352-3964/© 2019 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents lists available at ScienceDirect EBioMedicine journal homepage: www.ebiomedicine.com