Impaired phagocytosis in macrophages from patients affected by lysinuric protein intolerance

doi:10.1016/j.ymgme.2012.01.008

Molecular Genetics and Metabolism

Volume 105, Issue 4, April 2012, Pages 585-589

https://doi.org/10.1016/j.ymgme.2012.01.008 Get rights and content

Abstract

Lysinuric Protein Intolerance (LPI, MIM 222700) is a recessive aminoaciduria caused by defective cationic amino acid transport in epithelial cells of intestine and kidney. SLC7A7, the gene mutated in LPI, codifies for the y + LAT1 subunit of system y⁺L amino acid transporter. LPI patients frequently display severe complications, such as pulmonary disease, haematological abnormalities and disorders of the immune response. The transport defect may explain only a part of the clinical aspects of the disease, while the mechanisms linking the genetic defect to the clinical features of the patients remain thus far obscure. The aim of the study is to investigate the consequences of SLC7A7 mutations on specific macrophage functions, so as to evaluate if a macrophage dysfunction may have a role in the development of pulmonary and immunological complications of LPI.

The results presented 1) confirm previous data obtained in one LPI patient, demonstrating that arginine influx through system y⁺L is markedly compromised in LPI macrophages; 2) demonstrate that also system y⁺L-mediated arginine efflux is significantly lower in LPI macrophages than in normal cells and 3) demonstrate that the phagocytic activity of LPI macrophages is severely impaired.

In conclusion, SLC7A7/y + LAT1 mutations lead to a defective phenotype of macrophages, supporting the pathogenetic role of these cells in the development of LPI-associated complications.

Highlights

► Arginine influx/efflux through system y⁺L is compromised in LPI macrophages. ► LPI macrophages display a significant impairment of the phagocytic activity. ► An altered macrophage phenotype may underlie the pathogenesis of LPI complications.

Introduction

Lysinuric Protein Intolerance (LPI, MIM 222700) is an autosomic, recessive aminoaciduria caused by defective cationic amino acid (CAA; l-arginine, l-lysine, l-ornithine) transport at the basolateral membrane of epithelial cells of intestine and kidney [1]. SLC7A7, the gene mutated in LPI [2], [3], codifies for the y + LAT1 subunit of system y⁺L transporter. This system is a member of the large group of heterodimeric amino acid transporters and is accounted for by a light subunit (y + LAT1 or y + LAT2) and a glycoprotein (4F2hc/CD98hc) that is necessary for the correct expression of the transporter in the plasma membrane [4]. System y⁺L selectively transports CAA in the absence of sodium, while it requires the cation to interact with neutral amino acids (leucine, glutamine). Operatively, it works as an antiport coupling the efflux of CAA to the influx of neutral amino acids and sodium [4].

Because of the transport defect, LPI patients have high renal clearance and low intestinal absorption of CAA and, as a consequence, their CAA plasma levels are usually low [5]. The disease is characterized by nausea and vomiting after protein ingestion, failure to thrive, post-prandial hyperammonemia, hepato- and spleno-megaly [5]. The patients can also exhibit one or more symptoms related to pulmonary, renal, hematologic, musculoskeletal, and neurological involvement. An extensive clinical variability, even observed for the same genotype, is a typical feature of LPI patients [2], [6]. Most of the clinical findings of LPI may be related to the metabolic abnormality originating from altered absorption and reabsorption of cationic amino acids. However, nutritional imbalance of cationic amino acids cannot explain the complex multiorgan involvement of LPI, especially the complications affecting lung and immune and hematologic systems.

Respiratory involvement is the most threatening complication of LPI. Patients are, indeed, highly predisposed to develop interstitial lung disease and/or a secondary form of Pulmonary Alveolar Proteinosis (PAP), a rare disorder in which alveolar spaces of the lungs are excessively filled with lipoproteinaceous material (surfactant) and alveolar macrophages (AM) appear foamy and lipid-filled because of the impaired surfactant clearance [7]. Moreover, LPI subjects also display hematological abnormalities and disorders of the immune response including anemia, thrombocytopenia, leukopenia, systemic autoimmune diseases (lupus erythematosus) and increased susceptibility to hemophagocytic lymphohistiocytosis (HLH), a syndrome characterized by fever, hepatosplenomegalia, hemophagocytosis in bone marrow and increased levels of serum LDH and ferritin. HLH hallmark is the excessive activation and proliferation of T lymphocytes and macrophages with massive hypersecretion of proinflammatory cytokines such as IFNγ, soluble IL2-R, IL6, TNFα [8], [9].

The pathophysiological mechanisms involved in these complications are still unclear. The involvement of the mononuclear phagocyte system appears crucial, but the elucidation of the initiating events and the subsequent cellular and molecular processes which lead to the development of pulmonary and immunological complications still remain obscure. In our previous study describing the case of a young man affected by LPI [10], we demonstrated that the activity of system y⁺L was impaired in monocytes and alveolar macrophages but not in fibroblasts from the patient. However, the differentiation of patient's monocytes to macrophages upon exposure to GM-CSF appeared comparable to that of cells from healthy subjects.

Here, we have extended the study to other LPI subjects and assessed the involvement of SLC7A7 mutations in specific macrophage functions. In particular, given the pivotal role of macrophages in both host defense and surfactant homeostasis, we have investigated if LPI macrophages exhibit alterations in the phagocytic activity.

Section snippets

Subjects

Three LPI patients and five normal healthy donors were recruited for this study after subscription of an informed consent. The clinical aspects and the mutations of the patients are summarized in Table 1. The clinical history of LPI₁ has been already described [10], [11], [12]. Patient LPI₂ presents with a very mild phenotype with normal physical and intellectual development and aversion for protein-rich foods. The initial clinical history of patient LPI₃ has also been already reported [13]; at

Activity of system y⁺L in LPI monocytes and macrophages

In our previous study we demonstrated that the activity of system y⁺L was significantly reduced in monocytes and macrophages isolated from a patient affected by LPI, here indicated as LPI₁ [10]. In the present study, we measured the discriminated influx of arginine in monocytes and in monocyte-derived macrophages (MDM) obtained from 2 other LPI patients (LPI₂ and LPI₃). Five healthy subjects were used as control (Fig. 1). The activity of system y⁺L, predominant in cells from normal subjects,

Discussion

In this study we have analyzed the phenotypic consequences of SLC7A7 defect in macrophages, so as to validate the hypothesis of a role of macrophage dysfunction in the pathogenesis of pulmonary and immunological complications of LPI.

We demonstrate here for the first time that the impairment of phagocytosis is a common feature of LPI macrophages. Defects in neutrophil functions (adhesion, phagocytosis, oxygen radical production, microbial killing) and an impaired phagocytosis by alveolar

Acknowledgments

This work was partially supported by CLIMB, Children Living with Inherited Metabolic Diseases, Crewe, UK, by project EuPAPnet, granted by E-Rare call 2008 and by MICINN (SAF 2009-12606-C02-02) to Virginia Nunes. The Association “La Vita è un Dono” supports the clinical fellowship of Dr. Diego Martinelli.

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The decreased factor V level cannot be explained by hepatic dysfunction as the other coagulation factors were normal. The remaining hematological findings were similar to the patients reported in the literature [9,10,15,16,18–23]. Growth hormone deficiency is secondary to arginine depletion in LPI [2].
We report a 9-year-old boy with lysinuric protein intolerance (LPI). He had developmental delay, short stature, failure to thrive, high-protein food aversion, hypothyroidism, growth hormone deficiency, features of hemophagocytic lymphohistiocytosis (HLH), decreased bone mineral density and multiple thoracic spine compression fractures on X-ray. LPI was suspected, but urine amino acid profile and normal orotic acid did not suggest biochemical diagnosis of LPI. Targeted next generation sequencing panel for HLH (including SLC7A7) was organized. Due to elevated glutamine in plasma amino acid analysis, a metabolic consultation was initiated and his asymptomatic post-prandial ammonia was 295 μmol/L. We then suspected n-acetylglutamate synthase or carbamoyl-phosphate synthase I deficiency due to marked hyperammonemia, elevated glutamine level, normal orotic acid, and normalization of ammonia at 2 h of carglumic acid (200 mg/kg/d). His targeted next generation sequencing panel for HLH revealed homozygous pathogenic variant in SLC7A7 ((NM_001126106.2): c.726G>A (p.Trp242*)) and confirmed the diagnosis of LPI. We emphasize the importance of genetic investigations in the diagnosis of LPI.
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Herpes simplex encephalitis (HSE) is a widely accepted risk factor for anti N-methyl-d-aspartate receptor (NMDAR) encephalitis. Association of inherited metabolic disease has never been reported in a patient with HSE and anti-NMDAR encephalitis. Herein, we report a case of pediatric HSE complicated by development of anti-NMDAR encephalitis; this patient showed subsequent recurrent, unexplained episodes of encephalopathy associated with hyperammonemia. The patient was diagnosed with lysinuric protein intolerance (LPI), a rare inborn metabolic disorder. Although it would be difficult to make conclusions regarding the casual link of HSE and anti-NMDAR encephalitis with LPI from a single case, there have been many reports that autoimmune diseases and immunologic abnormalities are frequently associated with LPI. Thus, we speculate that LPI may contribute to the development of anti-NMDAR encephalitis following HSE.
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The most surprising finding in this observation is the evolution towards monotypic deposits, which is not evocative of standard lupus nephritis evolution. Unlike monocyte/macrophage dysfunction in LPI, little is known regarding B lymphocytes in LPI [31,32]. Ogier de Baulny et al reported pneumococcal invasive arthritis despite vaccination [6].
Lysinuric protein intolerance (LPI) is a rare autosomal recessive disease caused by mutations in the SLC7A7 gene encoding the light subunit of a cationic amino acid transporter. Symptoms mimic primary urea cycle defects but dysimmune symptoms are also described. Renal involvement in LPI was first described in the 1980s. In 2007, it appeared that it could concern as much as 75% of LPI patients and could lead to end-stage renal disease. The most common feature is proximal tubular dysfunction and nephrocalcinosis but glomerular lesions are also reported. However, very little is known regarding histological lesions associated with LPI. We gathered every kidney biopsy of LPI-proven patients in our highly specialized pediatric and adult institution. Clinical, biological, and histological information was analyzed. Five LPI patients underwent kidney biopsy in our institution between 1986 and 2015. Clinically, 4/5 presented with proximal tubular dysfunction and 3/5 with nephrotic range proteinuria. Histology showed unspecific tubulointerstitial lesions and nephrocalcinosis in 3/5 biopsies and marked peritubular capillaritis in one child. Glomerular lesions were heterogeneous: lupus-like–full house membranoproliferative glomerulonephritis (MPGN) in one child evolved towards monotypic IgG1κ MPGN sensitive to immunomodulators. One patient presented with glomerular non-AA non-AL amyloidosis. Renal biopsy is particularly relevant in LPI presenting with glomerular symptoms for which variable histological lesions can be responsible, implying specific treatment and follow-up.
Dysfunction in macrophage toll-like receptor signaling caused by an inborn error of cationic amino acid transport
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y+LAT1 is encoded by SLC7A7, the gene which is mutated in lysinuric protein intolerance (LPI [MIM 222700]) (Torrents et al., 1999; Borsani et al., 1999). LPI is an autosomal recessive disorder in which the transport of lysine, arginine and ornithine is defective in the basolateral membrane of epithelial cells in the small intestine and proximal kidney tubules (Näntö-Salonen et al., 2012), and in the plasma membrane of monocytes and macrophages (Barilli et al., 2010, 2012). Although a large number of causative mutations in the SLC7A7 gene have been described in patients with LPI, all the Finnish patients share the same LPIFin point mutation IVS6AS, A-T, -2, c.895-2A > T (Torrents et al., 1999; Borsani et al., 1999).
Amino acids, especially arginine, are vital for the well-being and activity of immune cells, and disruption of amino acid balance may weaken immunity and predispose to infectious and autoimmune diseases. We present here a model of an inborn aminoaciduria, lysinuric protein intolerance (LPI), in which a single mutation in y⁺LAT1 cationic amino acid transporter gene SLC7A7 leads to a multisystem disease characterized by immunological complications, life-threatening pulmonary alveolar proteinosis and nephropathy. Macrophages are suggested to play a central role in LPI in the development of these severe secondary symptoms. We thus studied the effect of the Finnish y⁺LAT1 mutation on monocyte-derived macrophages where toll-like receptors (TLRs) act as the key molecules in innate immune response against external pathogens. The function of LPI patient and control macrophage TLR signaling was examined by stimulating the TLR2/1, TLR4 and TLR9 pathways with their associated pathogen-associated molecular patterns. Downregulation in expression of TLR9, IRF7, IRF3 and IFNB1 and in secretion of IFN-α was detected, suggesting an impaired response to TLR9 stimulation. In addition, secretion of TNF-α, IL-12 and IL-1RA by TLR2/1 stimulation and IL-12 and IL-1RA by TLR4 stimulation was increased in the LPI patients. LPI macrophages secreted significantly less nitric oxide than control macrophages, whereas plasma concentrations of inflammatory chemokines CXCL8, CXCL9 and CXCL10 were elevated in the LPI patients. In conclusion, our results strengthen the relevance of macrophages in the pathogenesis of LPI and, furthermore, suggest that cationic amino acid transport plays an important role in the regulation of innate immune responses.
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¹: Contributed equally.

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Impaired phagocytosis in macrophages from patients affected by lysinuric protein intolerance

Abstract

Highlights

Introduction

Section snippets

Subjects

Activity of system y+L in LPI monocytes and macrophages

Discussion

Acknowledgments

Curr. Opin. Genet. Dev.

J. Biol. Chem.

Am. J. Med.

Am. J. Hum. Genet.

J. Pediatr.

FEBS Lett.

Immunity

Blood

Curr. Opin. Immunol.

SLC7A7, encoding a putative permease-related protein, is mutated in patients with lysinuric protein intolerance

Nat. Genet.

CATs and HATs: the SLC7 family of amino acid transporters

Pflugers. Arch.

Pulmonary alveolar proteinosis

N. Engl. J. Med.

Activity of system y⁺L in LPI monocytes and macrophages