Genetic polymorphisms and expression of NLRP3 inflammasome-related genes are associated with Philadelphia chromosome-negative myeloproliferative neoplasms

Abstract

Inflammation plays a crucial role in the initiation, progression and prognosis of Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), which could be clinically subdivided into polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Nucleotide binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasomes affect inflammatory diseases and carcinomas by excessive production of cytokines. To investigate a possible association of NLRP3 inflammasome signaling with MPN, we investigated the expression of selected inflammasome-related genes from bone marrow cells of 67 MPN patients as well as gene polymorphisms in NLRP3 (rs35829419), NF-κB1 (rs28362491), CARD8 (rs2043211), IL-1β (rs16944), and IL-18 (rs1946518). It showed that inflammasome-related genes (NLRP3, NF-κB1, CARD8, IL-1β, and IL-18) were highly expressed in BM cells from MPN patients and the increased expression was associated with JAK2^V617F mutation, white blood cell counts and splenomegaly. Analysis of genetic polymorphisms in 269 MPN patients and 291 healthy controls demonstrated that NF-κB1 (rs28362491) was associated with MPN and increased expression of NF-κB1, NLRP3 and IL-1β. This research provided novel biomarkers and potential targets for MPN.

Introduction

Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are characterized by excessive production of terminally differentiated myeloid cells in peripheral blood and bone marrow. Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are the classical subtypes of MPN characterized by an increased number of erythrocytes, platelets and bone marrow fibrosis, respectively [1]. Targeting JAK-STAT signaling has gained great benefits in the treatment of MPN. However, current therapeutic regimens are still beyond satisfaction in the challenge of completely reversing the progress of fibrosis or reducing the mutant allele burden [2]. It is necessary to investigate a complex system of pathogenesis beyond JAK-STAT that drives the initiation and development of MPN.

Recent studies have revealed the crucial role of inflammation in MPN. Inflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) are commonly increased in MPN [3]. Besides, patients with inflammatory or autoimmune diseases, including Crohn’s disease and primary immune thrombocytopenia (ITP), suffer a higher risk of MPN [4]. Moreover, as the most frequent driver mutation in MPN, JAK2^V617F mutation interconnected with inflammatory microenvironment to promote the development of MPN. Interestingly, inflammatory microenvironment favorably supported the growth of JAK2^V617F mutant neoplastic hematopoietic stem cells while inducing apoptosis and cell cycle arrest in JAK2^V617F-wild cells. Exposure to proinflammatory cytokines such as TNF-α reduced the colony formation of wild type cells while JAK2^V617F-mutant progenitor cells were resistant. Meanwhile, the malignant JAK2^V617F mutant cells contribute the inflammatory microenvironment by secreting cytokines, as well as activated the bystander immune cells [5], [6], suggesting that MPN cells alter the immediate local environment to promote their own growth while inhibiting the growth of the normal counterparts[7]. On the contrary, in a murine model of JAK2^V617F-positive MPN, absence of TNF-α limited clonal expansion and attenuated disease progression [8].

The Nod-like receptor protein 3 (NLRP3) inflammasome, as the most extensively studied inflammasome, is a large protein complex typically comprises of Nod-like receptor 3, the adapter protein apoptosis-associated speck-like protein containing caspase recruitment domain-containing protein (CARD), and Caspase-1 [9]. Activation of NLRP3 inflammasomes results from the recognition of pathogen-associated molecular patterns (PAMPs), such as cell wall proteoglycans, pore-forming toxins and pathogen RNA and DNA [10]. Meanwhile, several damage-associated molecular patterns (DAMPs) have been identified, such as external adenosine 5′-triphosphate (ATP), host DNA, synthetic nanoparticles and biological particulate matter [11]. Upon activation, NLRP3 inflammasome assembles and subsequently recruits procaspase-1. Oligomerization of pro-caspase-1 proteins induces autoproteolytic cleavage into active caspase-1, thus cleaves the precursor cytokines pro-interleukin-1 beta (pro-IL-1β) and pro-interleukin-18 (pro-IL-18) into biologically active cytokines IL-1β and IL-18. Notably, the activation of nuclear factor-kappa B (NF-κB) is required to regulate the expression of precursor IL-1β [12]. In addition to interleukins, activation of the inflammasome induce the release of several DAMPs including S100 calcium-binding protein A9 (S100A9) and high molecular group box 1 (HMGB1), which are important activators of the complement cascade (ComC) in the mannan binding lectin (MBL)-dependent pathway.[13]. Our previously published studies have revealed that activated NLRP3 inflammasome signaling is correlated with hematological diseases, such as acute lymphoblastic leukemia (ALL) [14] and immune thrombocytopenia (ITP) [15]. However, it is still unknown whether and how NLRP3 inflammasome contributes to the pathogenesis and clinical features of MPN.

With the development of high-throughput techniques, the research of genetic variations including copy number variants (CNVs), indels (deletions or insertions), structural variants and single nucleotide polymorphisms (SNPs) has boosted in aided diagnosis and treatment of diseases [16]. Single nucleotide polymorphisms (SNPs) of the NLRP3 inflammasome-related genes, such as the Q705k polymorphism in NLRP3, is a gain-of- function alteration that increases the susceptibility in Crohn’s disease [17] and causes poor prognosis celiac diseases [18]. NF-κB-94 ATTG ins/del polymorphism contributes to poor prognosis of colorectal cancer [19], higher susceptibility of squamous cell carcinoma [20] and gastric cancer [21] via impropriate activation of NF-κB signaling. Our research group have demonstrated previously that GT genotype of IL-18 (rs1946518) served as a reverse prognosis factor for acute myeloid leukemia (AML). Besides, the distribution of IL-1β (rs16944) polymorphisms was statistically different in AML patients of different cytogenetics risk subgroups [22]. Our group also proved that CARD8 (rs2043211) AT genotype contributed to the susceptibility of multiple myeloma (MM) [23] and increases the prognosis risk of chronic myeloid leukemia (CML)[24].

To date, no data has been demonstrated in the association of NLRP3 inflammasome-related genes and MPN. In the present case-control study, we analyzed the expression of selected inflammasome-related genes (NLRP3, NF-κB1, CARD8, IL-1β, and IL-18) from bone marrow cells of MPN patients as well as gene polymorphisms in NLRP3 (rs35829419), NF-kB1 (rs28362491), CARD8 (rs2043211), IL-1β (rs16944), and IL-18 (rs1946518), and investigated their roles in susceptibility and clinical significance of MPN.