Research articleGenetic polymorphisms and expression of NLRP3 inflammasome-related genes are associated with Philadelphia chromosome-negative myeloproliferative neoplasms
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, JAK2V617F mutation interconnected with inflammatory microenvironment to promote the development of MPN. Interestingly, inflammatory microenvironment favorably supported the growth of JAK2V617F mutant neoplastic hematopoietic stem cells while inducing apoptosis and cell cycle arrest in JAK2V617F-wild cells. Exposure to proinflammatory cytokines such as TNF-α reduced the colony formation of wild type cells while JAK2V617F-mutant progenitor cells were resistant. Meanwhile, the malignant JAK2V617F 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 JAK2V617F-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.
Section snippets
Patients and controls
In SNP genotyping experiment, clinical information including age, gender, clinical and clinical features were obtained for 269 patients clinically diagnosed with MPN at Qilu Hospital of Shandong University. The diagnosis of MPN corresponded to the 2016 World Health Organization (WHO) classification and diagnostic criteria for MPNs[25]. 291 randomly selected healthy individuals without any history of hematologic malignancies were included as controls during the years 2014 to 2019.
Expression of genes involved in NLRP3 inflammasome signaling were increased in MPN
We analyzed mRNA levels of NLRP3, NF-κB1, CARD8, IL-1β, IL-18, S100A9, HMGB1, and found that NLRP3, NF-κB1, CARD8, IL-1β, IL-18 were expressed at a significantly higher level in BM cells from MPN patients than healthy controls (Fig. 1B, p = 0.0138, 0.0235, 0.0234, 0.0234 and 0.0025, respectively), the mRNA levels were increased by 2.95, 1.63, 1.62, 2.42, and 1.32-fold, respectively. The S100A9 and HMGB1 levels in MPN patients were slightly elevated by 1.32 and 1.05-fold, however, the difference
Discussion
Inflammation plays a crucial role in the initiation and progression of MPN. The inflammatory microenvironment in bone marrow can cause genomic instability and DNA mutations in hematopoietic cells, which contribute to initial hit in the hematopoietic stem cells, or triggered additional mutations for clonal evolution in MPN[26]. Besides, the overactivation of inflammatory signaling pathways, including NF-κB and STAT signaling, resulted in abnormal production of inflammatory cytokines and
Declaration of Competing Interest
The authors declare that there is no conflict of interests regarding the publication of this paper.
Acknowledgments
We would like to thank patients and control individuals for their participation.
Funding information
This research was financed by grants from the Taishan Scholars Program, grants from Key Research and Development Program of Shandong Province (No. 2017GSF218050 and No. 2018GSF118014), and National Natural Science Foundation of China (No. 81600124, No. 91642110 and No. 81873439).
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These authors contributed equally to this work.