Mechanism of Hip Arthropathy in Ankylosing Spondylitis: Abnormal Myeloperoxidase and Phagosome

BackgroundThe pathogenesis of Ankylosing spondylitis (AS) has not been elucidated, especially involving hip joint disease. The purpose of this study was to analyze the proteome of diseased hip in AS and to identify key protein biomarkers.Material and MethodsWe used label-free quantification combined with liquid chromatography mass spectrometry (LC–MS/MS) to screen for differentially expressed proteins in hip ligament samples between AS and No-AS groups. Key protein was screened by Bioinformatics methods. and verified by in vitro experiments.ResultsThere were 3,755 identified proteins, of which 92.916% were quantified. A total of 193 DEPs (49 upregulated proteins and 144 downregulated proteins) were identified according to P < 0.01 and Log|FC| > 1. DEPs were mainly involved in cell compartment, including the vacuolar lumen, azurophil granule, primary lysosome, etc. The main KEGG pathway included Phagosome, Glycerophospholipid metabolism, Lysine degradation, Pentose phosphate pathway. Myeloperoxidase (MPO) was identified as a key protein involved in Phagosome pathway. The experiment of siRNA interfering with cells further confirmed that the upregulated MPO may promote the inflammatory response of fibroblasts.ConclusionsThe overexpression of MPO may contribute to the autoimmune inflammatory response of AS-affected hip joint through the phagosome pathway.

Mechanism of Hip Arthropathy in Ankylosing Spondylitis: Abnormal Myeloperoxidase

Ankylosing spondylitis (AS) is a chronic inflammation of the joints and spine. Its pathogenesis has not been elucidated, especially involving hip joint disease. The purpose of this study was to analyze the proteome of diseased hip in AS and to identify key protein biomarkers. We used label-free quantification combined with liquid chromatography mass spectrometry (LC–MS/MS) to screen for differentially expressed proteins in hip ligament samples between AS and No-AS groups. Key protein was screened by Bioinformatics methods and verified by in vitro experiments. There were 3755 identified proteins, of which 92.916% were quantified. 193 DEPs (49 up-regulated proteins and 144 down-regulated proteins) were identified according to P < 0.01 and Log|FC| > 1. DEPs were mainly involved in cell compartment, including the vacuolar lumen, azurophil granule,primary lysosome, etc. The main KEGG pathway included Phagosome, Glycerophospholipid metabolism, Lysine degradation, Pentose phosphate pathway. Myeloperoxidase (MPO) was identified as a key protein participated in Phagosome pathway, which induces hip lesions in ankylosing spondylitis. Further experiments confirmed that MPO promoted the inflammatory response of fibroblasts in AS-affected hip joint.

Cathepsin G Is Broadly Expressed By AML and Is an Effective Immunotherapeutic Target Against AML in Vivo

Immunotherapy targeting individual antigens in acute myeloid leukemia (AML) has shown promise. However, in view of leukemia heterogeneity and the loss of tumor antigen expression by AML, it is unlikely that any single antigen will be consistently expressed by all leukemia cells. This highlights the need to identify additional antigens in AML that can be targeted. Azurophil granule proteases have been shown to be effective immunotherapeutic targets. Proteinase 3 and neutrophil elastase, the parent proteins for the HLA-A2 restricted peptide PR1, have been targeted successfully in myeloid leukemia using immunotherapy. We recently discovered the HLA-A2 restricted peptide CG1 (FLLPTGAEA), which is derived from the azurophil granule protease cathepsin G (CG). We showed that CG is highly expressed by AML blasts and leukemia stem cells in a limited number of AML samples and showed that CG1 can be targeted in vitro using CG-specific cytotoxic T lymphocytes (CTL). We sought to determine whether CG1 can be targeted in vivo and to characterize the expression of CG in a large cohort of AML patients. To assess the efficacy of targeting CG1 in vivo, we used a NOD scid gamma (NSG) mouse model engrafted with the human HLA-A2+ AML cell line U937 (U937-A2), which is known to express CG. Mice were injected with U937-A2 (0.5 x 106) cells and on the following day were treated with either CG1-CTL (0.25 x 106), negative control HIV-CTL expanded from the same donor or were left untreated. Mice treated with CG1-CTL demonstrated a significantly greater reduction in U937-A2 in the bone marrow (BM) (8% residual AML; P<0.05; Figure 1A) after 2 weeks compared with mice treated with HIV-CTL (27% residual AML) or that were left untreated (34% residual AML). A similar reduction in AML was seen in the spleens of mice treated with CG1-CTL. Since normal myeloid cells also express CG, to demonstrate the specificity of CG1-CTL for AML, we engrafted NSGS mice with human HLA-A2+ cord blood (CB). After confirming engraftment by detecting human (h) CD45+/HLA-A2+/mouse (m)CD45- cells in peripheral blood, we treated mice with CG1-CTL or HIV-CTL (0.25 x 106). A similar percentage of hCD45+/HLA-A2+/mCD45- cells was detected in the BM of CB-engrafted mice in both CG1-CTL (24% CB) and HIV-CTL (27% CB) after 6 weeks, suggesting the safety of targeting CG1. After demonstrating the efficacy of targeting CG1 in vivo, we then studied CG1 presentation and CG expression in AML patients. To determine CG1 presentation, primary AML blasts, U937-A2 and HLA-A2-trasnduced HL60 (HL60-A2) cell lines were lysed then immunoprecipitated with anti-HLA-A2. Peptides were eluted using standard acid elution methodology and then analyzed by mass spectrometry. CG1 peptide was detected in 6 of 9 primary AML samples and in HL60-A2 and U937-A2 cells. Reverse phase protein analysis (RPPA) was used to study CG protein expression in a large cohort of AML patients and normal donors (ND) to include the following: 511 newly diagnosed patients, 21 ND peripheral blood leukocyte controls, 21 ND CD34+ cell controls and 10 ND BM controls. CG expression in the 511 AML patients samples demonstrated a Gaussian distribution and was higher than CG expression by ND CD34+ cells in 230 samples, equal to normal CD34+ cells in 234 samples and less than normal CD34+ cells in 47 samples. As expected, G-CSF primed ND CD34+ cells had high expression of CG. There were no significant differences in CG expression within the different AML FAB subtypes, although M4 eos and M5b subtypes showed the lowest expression levels. In multivariate Cox model analysis, higher CG expression significantly predicted shorter overall survival (OS) in all patients (P=0.04) (Figure 1B). The association of CG with shorter OS was most significant in patients who had intermediate cytogenetics and FLT3 mutation (P=0.02). Moreover, CG levels were determined in paired samples taken at diagnosis and relapse, and the levels were compared using paired t test and Pearson product-moment correlation analysis. Among 47 cases with paired diagnosis and relapse samples, there was significantly higher CG protein expression in relapse samples (P=0.0001). In summary, we found CG to be an important antigen in AML. CG is associated with worse outcomes in AML patients, however in vivo mouse data show that targeting CG eliminates AML while sparing normal hematopoiesis. Together these data indicate that CG has a promising role as a novel immunotherapeutic target in AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G

Key Points Serine protease inhibitor serpinB1 protects neutrophils by inhibition of their own azurophil granule protease cathepsin G. Granule permeabilization in neutrophils leads to cathepsin G–mediated death upstream and independent of apoptotic caspases.

Serglycin participates in retention of α-defensin in granules during myelopoiesis

The mechanism by which proteins are targeted to neutrophil granules is largely unknown. The intracellular proteoglycan serglycin has been shown to have important functions related to storage of proteins in several types of granules. The possible role of serglycin in the localization of the α-defensin, human neutrophil peptide 1 (HNP-1), a major azurophil granule protein in human neutrophils, was investigated. Murine myeloid cells, stably transfected to express HNP-1, were capable of processing HNP-1, and HNP-1 was found to associate with serglycin in murine and human myeloid cell lines as well as in human bone marow cells. A transgenic mouse expressing HNP-1 in the myeloid compartment was crossed with mice deficient in serglycin or neutrophil elastase to investigate HNP-1 sorting and processing. Neither deficiency affected processing of HNP-1, but the ability to retain fully processed HNP-1 intracellularly was reduced in mice that lack serglycin. Human granulocyte precursors transfected with siRNA against serglycin displayed similar reduced capability to retain fully processed HNP-1, demonstrating a role of serglycin in retaining mature HNP-1 intracellularly, thus preventing potential toxic effects of extracellular HNP-1.

A protease-resistant PML-RARα has increased leukemogenic potential in a murine model of acute promyelocytic leukemia

Previous studies in our laboratory demonstrated that the azurophil granule protease neutrophil elastase (NE) cleaves promyelocytic leukemia–retinoic acid receptor (PML-RAR)α (PR), the fusion protein that initiates acute promyelocytic leukemia (APL). Further, NE deficiency reduces the penetrance of APL in a murine model of this disease. We therefore predicted that NE-mediated PR cleavage might be important for its ability to initiate APL. To test this hypothesis, we generated a mouse expressing NE-resistant PR. These mice developed APL indistinguishable from wild-type PR, but with significantly reduced latency (median leukemia-free survival of 274 days vs 473 days for wild-type PR, P < .001). Resistance to proteolysis may increase the abundance of full-length PR protein in early myeloid cells, and our previous data suggested that noncleaved PR may be less toxic to early myeloid cells. Together, these effects appear to increase the leukemogenicity of NE-resistant PR, contrary to our previous prediction. We conclude that NE deficiency may reduce APL penetrance via indirect mechanisms that are still NE dependent.

Counterregulation of clathrin-mediated endocytosis by the actin and microtubular cytoskeleton in human neutrophils

We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.