Polymerization of misfolded protein lowers CX3CR1 expression in human PBMCs

The CX3CR1 (chemokine (C-X3-C motif) receptor 1) expression levels on immune cells have significant importance in maintaining tissue homeostasis under physiological and pathological conditions. The factors implicated in the regulation of CX3CR1 and its specific ligand CX3CL1 (fractalkine) expression remain largely unknown. Recent studies provide evidence that host‘s misfolded proteins occurring in the forms of polymers or amyloid fibrils can regulate CX3CR1 expression. Herein, we present a novel example that polymers of human ZZ alpha1-antitrypsin (Z-AAT) protein, resulting from its conformational misfolding due to the Z (Glu342Lys) mutation in SERPINA1 gene, strongly lower CX3CR1 expression in human PBMCs. We also show that extracellular polymers of Z-AAT are internalized by PBMCs, which parallels with increased intracellular levels of CX3CR1 protein. Our findings support the role of extracellular misfolded proteins in CX3CR1 regulation and encourage conducting further studies on this issue.


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The interaction between the chemokine receptors and chemokines, but also other proteins, 49 peptides, lipids, and microbial products, plays a critical role in the recruitment of 50 inflammatory cells into injured/diseased tissues 1 . Many human diseases involve altered 51 surface expression of chemokine receptors, which can lead to a defective cell migration and 52 inappropriate immune response. Most of the human PBMCs express CX3CR1 2 , also known 53 as the G-protein coupled receptor 13 (GPR13) or fractalkine receptor, a mediator of leukocyte 54 migration and adhesion. In the central nervous system, CX3CR1 is largely expressed by 55 microglial cells (brain macrophages) 3 , which play a critical role in the progression of 56 neurodegenerative diseases like Alzheimer's disease. The major role of CX3CR1-expressing 57 cells is to recognize and enter tissue following CX3CL1 (fractalkine or also called 58 neurotactin) gradient, and to crawl or "patrol" in the lumen of blood vessels 4 . Since 59 CX3CR1/CX3CL1 axis is also involved in the synthesis of anti-inflammatory cytokines and 60 has a significant role in cytoskeletal rearrangement, migration, apoptosis and proliferation, its 61 dysregulation is associated with the development of cardiovascular diseases, kidney 62 ischemia-reperfusion injury, cancer, chronic obstructive pulmonary disease, 63 neurodegenerative disorders and others 5, 6, 7 . Some studies indicate that CX3CR1 deficiency 64 contributes to the severity of infectious diseases 8 , and promotes lung pathology in respiratory 65 syncytial virus-infected mice 9 . It is shown that animals with deletion of CX3CR1 have 66 impairments in phagocytosis 10 , which is vital to prevent unwanted inflammation. It is clear 67 that CX3CR1 expressing cells have tissue-specific roles in different pathophysiological 68 conditions. Nevertheless, a comprehensive knowledge on the regulation of CX3CR1 69 expression is still missing. 70 One of the important aspects that needs to be addressed in the chemokine-receptor regulation 71 is the ability of ligands to induce internalization of the receptors and to internalize themselves 72 during this process. Current findings suggest that divergent proteins with a common 73 propensity to form extracellular polymers, independently of amino acid sequence, size, 74 structure, expression level, or function might interact with chemokine receptors and affect 75 their surface expression levels. For example, it has been reported that Alzheimer`s peptide, 76 Aβ, interacts with CX3CR1, and significantly reduces its expression in cultured microglial 77 cells and Alzheimer`s brain 12 . Similarly, highly accumulated extracellular Tau in Alzheimer`s 78 disease, seems to bind to CX3CR1 promoting its internalization and reducing expression in 79 microglial cells 13 . In concordance, we provide novel evidence that polymers of human Z 80 alpha1-antitrypsin (Z-AAT), resulting from its conformational misfolding due to the Z 81 (Glu342Lys) mutation in SERPINA1 gene, strongly lower CX3CR1 expression in human 82 PBMCs. We also show that extracellular oligomers of Z-AAT are internalized by PBMCs, 83 which parallel with increased intracellular CX3CR1 protein levels.

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Inherited α1-antitrypsin deficiency (AATD) is a rare genetic condition caused by mutations in 86 the SERPINA1 gene. Homozygous Z AATD is the most clinically relevant genotype among 87 Caucasians (prevalence is about 1:2000-1:5000) that is characterized by low plasma levels of 88 AAT protein (10-15% compared to the wild type, MM AAT, 1.3-2 g/L) and the presence of 89 intracellular and circulating Z-AAT polymers 14 .

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The intracellular polymers of Z-AAT are known to be harmful for AAT-producing cells 91 whereas in the circulation these polymers are not able to execute the tasks of native AAT-a 92 major inhibitor of neutrophil serine proteases having broad spectrum anti-inflammatory 93 activities. Based on the fact that circulating Z-AAT polymers are dysfunctional and contribute 94 to the risk of developing lung and/or liver pathology 15 (Figure 1-figure supplement 1). 102 To our surprise, the CX3CR1 mRNA expression turned to be much lower in ZZ AATD 103 PBMCs than in PBMCs from non-AATD controls [median (range): 4.1 (2.7-5.5) vs 18.5 (13-104 26.6), p < 0.001] (Figure 1A) independent of individual's age, clinical diagnosis (healthy, 105 lung or liver disease) or augmentation therapy. A previous study has shown that CX3CR1 -/-106 mice have significantly higher plasma levels of CX3CL1 than wild-type mice 18 and that 107 CX3CL1 reveals its biological activity exclusively via an interaction with CX3CR1 1 .

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Taken into account that ZZ AATD individuals, differently from non-AATD, have about 90% 121 lower blood concentration of Z-AAT protein, which may affect cellular microenvironment 23 and the expression of CX3CR1, we thought that there might be a relationship between gene 123 expression of CX3CR1 and Z-AAT plasma levels. However, we could not find any correlation 124 between CX3CR1 mRNA in PBMCs and plasma levels of Z-AAT measured by nephelometry 125 (data not shown). Therefore, we next decided to measure plasma Z-AAT polymers because Z  Most interestingly, however, we found a trend towards an inverse relationship between 147 CX3CR1 mRNA and plasma Z-AAT polymers (r 2 = -0.31, n = 38, p = 0.055) ( Figure 1C). 148 This latter prompted us to investigate whether Z-AAT polymers isolated from ZZ AATD 149 individuals affect CX3CR1 expression when added to healthy donor PBMCs, in vitro. We  As a side note, it has been reported that CD14 ++ monocytes have the lowest expression of 191 CX3CR1 30 . Low and high surface CX3CR1 levels are suggested to delineate two functional 192 subsets of murine blood monocytes: "inflammatory" and "resident monocytes," 31 . This 193 dichotomy appears conserved in humans as CD14 + CD16 − , and CD14 low CD 16+ monocytes 194 resemble "inflammatory" and "resident" monocytes. We previously demonstrated that 195 peripheral blood monocytes from clinically healthy young adults (30-year old) with ZZ 196 AATD have significantly higher mRNA and surface expression of CD14 as compared to age 197 matched MM subjects 32 . We thought that the higher CD14 expression reflects early 198 pathological processes whereas according to our current findings this phenomenon seems to 199 relate with the circulating Z-AAT polymers.

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In summary, we report a novel observation that polymeric Z-AAT lowers the expression of 201 CX3CR1 on PBMCs. We predict that the relationship between the circulating amounts of Z-     We thank the German society Alpha1 Deutschand e.V. and society members for support.