Detection of PD-L1-Expressing Myeloid Cell Clusters in the Hyaluronan-Enriched Stroma in Tumor Tissue and Tumor-Draining Lymph Nodes.

Expression of the transmembrane protein PD-L1 is frequently upregulated in cancer. Because PD-L1-expressing cells can induce apoptosis or anergy of T lymphocytes through binding to the PD1 receptor, the PD-L1-mediated inhibition of activated PD1+ T cells is considered a major pathway for tumor immune escape. However, the mechanisms that regulate the expression of PD-L1 in the tumor microenvironment are not fully understood. Analysis of organotypic tumor tissue slice cultures, obtained from mice with implanted syngeneic tumors (MBT2 bladder tumors in C3H mice, Renca kidney, and CT26 colon tumors in BALB/c mice), as well as from patients with cancer, revealed that tumor-associated hyaluronan (HA) supports the development of immunosuppressive PD-L1+ macrophages. Using genetically modified tumor cells, we identified epithelial tumor cells and cancer-associated mesenchymal fibroblast-like cells as a major source of HA in the tumor microenvironment. These HA-producing tumor cells, and particularly the vimentin-positive fibroblast-like cells of bone marrow origin, directly interact with tumor-recruited myeloid cells to form large stromal congregates/clusters that are highly enriched for both HA and PD-L1. Furthermore, similar cell clusters composed of HA-producing fibroblast-like cells and PD-L1+ macrophages were detected in tumor-draining, but not in distant, lymph nodes. Collectively, our findings indicate that the formation of multiple large HA-enriched stromal clusters that support the development of PD-L1-expressing APCs in the tumor microenvironment and draining lymph nodes could contribute to the immune escape and resistance to immunotherapy in cancer.

Immunotherapy for stone disease.

PURPOSE OF REVIEW: In addition to traditional risk factors such as low urine volume or hypercalciuria, emerging data suggest that calcium oxalate (CaOx), one of the most common mineral complexes in the urine, elicits a strong immunologic response. This review highlights those studies and projects how future therapies may be directed for kidney stone prevention. RECENT FINDINGS: Over the last 2 years, several groups have studied the response of the immune system to CaOx crystals using cell culture and animal models. Dominguez et al. found that CaOx crystals were recognized by monocytes through an lipopolysaccharide-mediated mechanism, leading to M1 'inflammatory' macrophage phenotype. Patel et al. proposed excessive oxalate-mediated reactive oxygen species within macrophage mitochondria may impair their ability to properly clear stones. Two other groups developed mouse models (an androgen receptor knock-out and an overexpression of Sirtuin 3 protein) and demonstrated increased renal anti-inflammatory macrophage differentiation and decreased CaOx deposition in experimental compared with controls. Anders et al. fed hyperoxaluric mice 1,3-butanediol, which blocks an inflammatory form of cell death called NLRP3 inflammasome and found less intrarenal oxidative damage and higher anti-inflammatory renal infiltrates in experimentals. Finally, monocytes exposed to CaOx crystals followed by hydroxyapatite had reduced inflammatory cytokine and chemokine production compared with those without hydroxyapatite, suggesting that Randall's plaque may play a role in dampening M1-mediatiated CaOx inflammation. SUMMARY: By modulating the immune response, immunotherapy could provide the means to prevent stone recurrences in certain individuals. The promotion of M2 over M1 macrophages and inhibition of inflammation could prevent the cascade that leads to CaOx nucleation. Future therapies may target the ability of macrophages to degrade CaOx crystals to prevent stones.

Calcium Oxalate Stone Fragment and Crystal Phagocytosis by Human Macrophages.

PURPOSE: In murine and human hyperoxaluric conditions macrophages can be seen surrounding renal calcium oxalate crystal deposits. We hypothesized that macrophages have a role in degrading and destroying these deposits. We investigated the inflammatory response and phagocytic mechanisms when macrophages were exposed to human kidney stones and inorganic crystals. MATERIALS AND METHODS: Human monocytes were differentiated into resting, fully differentiated macrophages by treatment with recombinant human macrophage colony-stimulating factor (M-CSF) or GM-CSF (granulocyte M-CSF) for 6 days. After confirming phenotype by flow cytometry the macrophages were exposed for 20 hours to fragments of sterile human calcium oxalate stones or calcium oxalate crystals. Crystal uptake was determined, and supernatant cytokine and chemokine profiles were analyzed using antibody arrays. Quantitative reverse transcriptase-polymerase chain reaction was done to validate mRNA profile expression. RESULTS: Under direct vision fluorescence microscopy activated human macrophages were noted to surround stone fragments and synthesized crystals, and destroy them in a step-by-step process that involved clathrin mediated endocytosis and phagocytosis. An inflammatory cascade was released by macrophages, including the chemokines chemokine ligand (CCL)2, CCL3, interleukin (IL)-1 receptor antagonist (IL-1ra), complement component C5/C5a and IL-8. Response patterns to stone and crystal material depended on macrophage phenotype and activation status. CONCLUSIONS: In our in vitro study macrophages differentiated with M-CSF showed greater ability to phagocytize crystal deposits than those treated with GM-CSF. Following clathrin mediated endocytosis macrophages released a number of cytokines that are crucial for the inflammatory immune response. This suggests that tissue macrophages have an important role in preventing kidney stone disease by removing and digesting interstitial renal crystal deposits.

Regulation of TLR2-mediated tolerance and cross-tolerance through IRAK4 modulation by miR-132 and miR-212.

Innate immune response is the first defense against pathogens via recognition by various conserved pattern recognition receptors, such as TLRs, to initiate a rapid and strong cytokine alarm. TLR signaling-mediated cytokine production must be properly regulated to prevent pathological conditions deriving from overproduction of cytokines. In this study, the role of specific microRNAs in TLR-signaling pathway was investigated to reveal the cross-interaction and -regulation in the MyD88 pathway. In peptidoglycan (PGN)/TLR2-stimulated THP-1 monocytes, PBMCs, and primary macrophages showed rapid and dramatic miR-132 and miR-212 (miR-132/-212) upregulation. This newly identified response appeared earlier in time than the characteristic miR-146a response in LPS-TLR4 stimulation. The rapid induction of miR-132/-212 was transcription factor CREB dependent, and the sustained expression of miR-132/-212 was responsible for inducing tolerance to subsequent PGN challenge. Cross-tolerance was observed by TLR5 ligand flagellin and heat-killed or live bacteria resulting from miR-132/-212 upregulation. Mechanistically, IRAK4 was identified and validated as a target of miR-132/-212 by luciferase reporter assay and seed-sequence mutagenesis of the reporter. Transfection of miR-132 or miR-212 alone mimicked PGN tolerance in monocytes, whereas transfected specific miRNA inhibitors tampered the tolerance effect. During bacterial infection, PGN-mediated TLR2 signaling induces miR-132/-212 to downregulate IRAK4, an early component in the MyD88-dependent pathway, whereas LPS/TLR4-induced miR-146a downregulates downstream components of the same MyD88-dependent pathway. The identification of miR-132/-212 and miR-146a together to prevent damaging consequences from the overproduction of proinflammatory cytokines by targeting a common signaling pathway is significant and will provide insights into future design and development of therapeutics.

Positive correlation of STAT1 and miR-146a with anemia in patients with systemic lupus erythematosus.

PURPOSE: Anemia is one of the most common hematological manifestations in SLE patients, occurring in about 50% of active cases. STAT1 is a critical signaling molecule required for the production of type-1 interferon (I-IFN), CCL2, and CXCL10, all of which are upregulated in SLE. Overexpression of STAT1 has been described to be involved in anemia in animal models. The aim of this study is to analyze how these components are involved in SLE-associated anemia. METHODS: Blood samples were collected from 39 healthy donors and 101 SLE patients fulfilling ACR criteria. Samples were collected from a total of 180 visits (58 patients had 2 or more visits) of which 52 visits included a diagnosis of anemia. Healthy donors had only single visit. Total RNA, isolated from leukocytes, was analyzed by Taqman qPCR. Relative expression levels of I-IFN signature genes, chemokines, and miR-146a were determined by the ΔΔCT method. Results were correlated with clinical data and analyzed by the Wilcoxon/Kruskal-Wallis test and Fisher's exact test. RESULTS: Significant increases in IFN score (p < 0.0001), STAT1 (p < 0.0001), miR-146a (p < 0.0005), CCL2 (p = 0.0047), and CXCL10 (p = 0.017), as well as a significant decrease in pri-miR-146a (p = 0.0002), were detected in the anemic SLE patient visits (n = 52) compared to non-anemic SLE visits (n = 128). Regardless of disease activity, lupus nephritis, or race, anemic SLE patients displayed significantly elevated levels of STAT1 and miR-146a compared to non-anemic SLE patients. CONCLUSIONS: STAT1 and miR-146a may be upregulated during inflammation and via proinflammatory cytokines and chemokines in SLE. Prolonged upregulation of STAT1 and miR-146a appears to play an important role in anemia in SLE patients.

Expression and secretion of glycosylated barley oxalate oxidase in Pichia pastoris.

Oxalate oxidase is an enzyme that degrades oxalate and is used in commercial urinary assays to measure oxalate levels. The objective of this study was to establish an enhanced expression system for secretion and purification of oxalate oxidase using Pichia pastoris. A codon optimized synthetic oxalate oxidase gene derived from Hordeum vulgare (barley) was generated and cloned into the pPICZα expression vector downstream of the N-terminal alpha factor secretion signal peptide sequence and used for expression in P. pastoris X-33 strain. A novel chimeric signal peptide consisting of the pre-OST1 sequence fused to pro-αpp8 containing several amino acid substitutions was also generated to enhance secretion. Active enzyme was purified to greater than 90% purity using Q-Sepharose anion exchange chromatography. The purified oxalate oxidase enzyme had an estimated Km value of 256µM, and activity was determined to be 10U/mg. We have developed an enhanced oxalate oxidase expression system and method for purification.

Deregulated hyaluronan metabolism in the tumor microenvironment drives cancer inflammation and tumor-associated immune suppression.

Hyaluronan (HA) is known to be a prominent component of the extracellular matrix in tumors, and many solid cancers are characterized by aberrant HA metabolism resulting in increased production in tumor tissue. HA has been implicated in regulating a variety of cellular functions in tumor cells and tumor-associated stromal cells, suggesting that altered HA metabolism can influence tumor growth and malignancy at multiple levels. Importantly, increased HA production in cancer is associated with enhanced HA degradation due to high levels of expression and activity of hyaluronidases (Hyal). Understanding the complex molecular and cellular mechanisms involved in abnormal HA metabolism and catabolism in solid cancers could have important implications for the design of future cancer therapeutic approaches. It appears that extensive crosstalk between immune cells and HA-enriched stroma contributes to tumor growth and progression in several ways. Specifically, the interaction of tumor-recruited Hyal2-expressing myeloid-derived suppressor cells (MDSCs) of bone marrow origin with HA-producing cancer-associated fibroblasts and epithelial tumor cells results in enhanced HA degradation and accumulation of small pro-inflammatory HA fragments, which further drives cancer-related inflammation. In addition, hyaluronan-enriched stroma supports the transition of tumor-recruited Hyal2+MDSCs to the PD-L1+ tumor-associated macrophages leading to the formation of an immunosuppressive and tolerogenic tumor microenvironment. In this review, we aim to discuss the contribution of tumor-associated HA to cancer inflammation, angiogenesis, and tumor-associated immune suppression. We also highlight the recent findings related to the enhanced HA degradation in the tumor microenvironment.

High Levels of PD-L1+ and Hyal2+ Myeloid-derived Suppressor Cells in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) patients frequently have increased number of immunosuppressive myeloid cells in circulation. High number of myeloid-derived suppressor cells (MDSCs) in the blood are associated with immune suppression as well as with cancer-related inflammation which drives the mobilization of myeloid cells to tumor tissue. Here, we show that peripheral blood from a previously untreated RCC patient has increased the number of monocytic CD33+CD11b+ MDSCs, which also co-expressed PD-L1 and membrane-bound enzyme hyaluronidase 2 (Hyal2). PD-L1 expression is associated with immune suppression, whereas expression of Hyal2 is associated with inflammation, because Hyal2+ myeloid cells can degrade the extracellular hyaluronan (HA), leading to the accumulation of pro-inflammatory HA fragments with low molecular weight. These findings implicate the potential involvement of monocytic MDSCs in both tumor-associated immune suppression and cancer-related inflammation. Analysis of organotypic tumor-tissue slice cultures prepared from cancer tissue of the same patient revealed the significant presence of PD-L1+ HLA-DR+ macrophage-like or dendritic cell-like antigen-presenting cells in tumor stroma. Interestingly, stroma-associated PD-L1+ cells frequently have intracellular hyaluronan. Collectively, data presented in this study suggest that the interplay between tumor-recruited myeloid cells and stromal HA may contribute to the inflammation and immune tolerance in kidney cancer.

Randall's plaque and calcium oxalate stone formation: role for immunity and inflammation.

Idiopathic calcium oxalate (CaOx) stones often develop attached to Randall's plaque present on kidney papillary surfaces. Similar to the plaques formed during vascular calcification, Randall's plaques consist of calcium phosphate crystals mixed with an organic matrix that is rich in proteins, such as inter-α-trypsin inhibitor, as well as lipids, and includes membrane-bound vesicles or exosomes, collagen fibres and other components of the extracellular matrix. Kidney tissue surrounding Randall's plaques is associated with the presence of classically activated, pro-inflammatory macrophages (also termed M1) and downregulation of alternatively activated, anti-inflammatory macrophages (also termed M2). In animal models, crystal deposition in the kidneys has been associated with the production of reactive oxygen species, inflammasome activation and increased expression of molecules implicated in the inflammatory cascade, including osteopontin, matrix Gla protein and fetuin A (also known as α2-HS-glycoprotein). Many of these molecules, including osteopontin and matrix Gla protein, are well known inhibitors of vascular calcification. We propose that conditions of urine supersaturation promote kidney damage by inducing the production of reactive oxygen species and oxidative stress, and that the ensuing inflammatory immune response promotes Randall's plaque initiation and calcium stone formation.

Hyal2 Expression in Tumor-Associated Myeloid Cells Mediates Cancer-Related Inflammation in Bladder Cancer.

The increased presence of myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in tumor tissue has been extensively reported. However, their role in the regulation of hyaluronan (HA) metabolism in the tumor microenvironment has not been established. Here we describe a novel function of tumor-associated myeloid cells related to the enhanced breakdown of extracellular HA in human bladder cancer tissue, leading to the accumulation of small HA fragments with molecular weight (MW) <20 kDa. Increased fragmentation of extracellular HA and accumulation of low molecular weight HA (LMW-HA) in tumor tissue was associated with elevated production of multiple inflammatory cytokines, chemokines, and angiogenic factors. The fragmentation of HA by myeloid cells was mediated by the membrane-bound enzyme hyaluronidase 2 (Hyal2). Increased numbers of Hyal2+CD11b+ myeloid cells were detected in the tumor tissue as well as in the peripheral blood of patients with bladder cancer. Coexpression of CD33 suggested that these cells belong to monocytic myeloid-derived suppressor cells. The HA-degrading function of Hyal2-expressing MDSCs could be enhanced by exposure to tumor-conditioned medium, and IL1ß was identified as one of the factors involved in the stimulation of Hyal2 activity. CD44-mediated signaling played an important role in the regulation of HA-degrading activity of Hyal2-expressing myeloid cells, as the engagement of CD44 receptor with specific mAb triggered translocation of Hyal2 enzyme to the cellular surface and stimulated secretion of IL1ß. Taken together, this work identifies Hyal2-expressing tumor-associated myeloid cells as key players in the accumulation of LMW-HA in the tumor microenvironment and cancer-related inflammation and angiogenesis. SIGNIFICANCE: This study identifies Hyal2-expressing tumor-associated myeloid cells of monocyte-macrophage lineage as contributors to hyaluronan degradation in bladder cancer tissue, leading to accumulation of inflammatory and proangiogenic low molecular weight hyaluronan fragments.

Formation of GW/P bodies as marker for microRNA-mediated regulation of innate immune signaling in THP-1 cells.

GW bodies (GWB or P bodies) are cytoplasmic foci thought to result from microRNA (miRNA) regulation of messenger RNA (mRNA) targets and subsequent mRNA degradation. The purpose of this study is to examine the effects of lipopolysaccharide (LPS) stimulation of human monocytes on GWB formation, miRNA induction, miRNA target regulation and downstream cytokine and chemokine expression. In response to LPS stimulation, the number of GWB consistently increased by twofold at 8 h after stimulation and this increase was abolished when the miRNA-effector proteins Rck/p54 or argonaute 2 were depleted. As the level of miR-146a increased from 19-fold up to 100-fold during LPS stimulation, the transfection of a miR-146a mimic into THP-1 cells was examined to determine whether miR-146a alone can induce similar changes in GWB. The results showed transfected miR-146a could produce a comparable increase in the number of GWB and this was accompanied by a reduction in major cytokines/chemokines induced by LPS. These data show that the increase in size and number of GWB may serve as a biomarker for miRNA-mediated gene regulation, and miR-146a has a significant role in the regulation of LPS-induced cytokine production in THP-1 cells.

Calcium Oxalate Differentiates Human Monocytes Into Inflammatory M1 Macrophages.

Purpose: A number of hyperoxaluric states have been associated with calcium oxalate (CaOx) deposits in the kidneys. In animal models of stone disease, these crystals interact with circulating monocytes that have migrated into the kidney as part of innate immunity. Similarly, macrophages surround CaOx crystals in kidneys of patients excreting high levels of oxalate. We investigate the effect of this exposure and subsequent human immunological response in vitro. Materials and methods: Primary human monocytes were collected from healthy donors and exposed to CaOx, potassium oxalate, and zinc oxalate (ZnOx). Cytokine production was measured with a multiplex ELISA. Quantitative reverse transcription-polymerase chain reaction was done to validate the mRNA profile expression. M1 macrophage phenotype was confirmed with immunofluorescence microscopy. Results: Both primary monocytes and THP-1 cells, a human monocytic cell line, respond strongly to CaOx crystals in a dose-dependent manner producing TNF-α, IL-1ß, IL-8, and IL-10 transcripts. Exposure to CaOx followed by 1 h with LPS had an additive effect for cytokine production compared to LPS alone, however, LPS followed by CaOx led to significant decrease in cytokine production. Supernatants taken from monocytes were previously exposed to CaOx crystals enhance M2 macrophage crystal phagocytosis. CaOx, but not potassium or ZnOx, promotes monocyte differentiation into inflammatory M1-like macrophages. Conclusion: In our in vitro experiment, human monocytes were activated by CaOx and produced inflammatory cytokines. Monocytes recognized CaOx crystals through a specific mechanism that can enhance or decrease the innate immune response to LPS. CaOx promoted M1 macrophage development. These results suggest that monocytes have an important role promoting CaOx-induced inflammation.

Elevated signal transducers and activators of transcription 1 correlates with increased C-C motif chemokine ligand 2 and C-X-C motif chemokine 10 levels in peripheral blood of patients with systemic lupus erythematosus.

INTRODUCTION: The present study examines the levels of recently reported biomarkers, adenosine deaminase acting on RNA (ADAR), C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine 10 (CXCL10), signal transducers and activators of transcription 1 (STAT1), and miR-146a in systemic lupus erythematosus (SLE) patients over multiple visits. METHODS: Peripheral blood leukocytes were collected from 65 healthy donors and 103 SLE patients, 60 of whom had samples from 2 or more visits. Total RNA was isolated and analyzed for the expression of mRNA and microRNA using Taqman real time PCR assays. Relative expression of I-IFN signature genes, chemokines, and miR-146a were determined by the ΔΔCT method. Results were correlated with clinical data and analyzed by Wilcoxon/Kruskal-Wallis test and Fisher's exact test. RESULTS: Levels of ADAR, CCL2, CXCL10, and STAT1 in SLE were significantly elevated compared with the healthy controls (P <0.0001). ADAR, CCL2, and CXCL10 showed significant correlation with IFN score in both healthy donors (P <0.0033) and SLE patients (P <0.0001). In SLE patients, miR-146a level was not significantly different from healthy controls nor correlated to the IFN score. Two STAT1 populations were identified: a low STAT1 and a high STAT1 group. High STAT1 patient visits displayed higher (P ≤0.0020) levels of CCL2 and CXCL10 than the low STAT1 patient visits. STAT1 levels correlated with IFN score in low STAT1 group but not in high STAT1 group. More importantly, high STAT1 levels appeared as an enhancer of CCL2 and CXCL10 as indicated by the significantly stronger correlation of CCL2 and CXCL10 with IFN score in high STAT1 patient visits relative to low STAT1 patient visits. CONCLUSION: Our data indicate a novel role for STAT1 in the pathogenesis of SLE as an expression enhancer of CCL2 and CXCL10 in SLE patients with high levels of STAT1. Future study is needed to examine the exact role of STAT1 in the etiology of SLE.

Reduced levels of CCL2 and CXCL10 in systemic lupus erythematosus patients under treatment with prednisone, mycophenolate mofetil, or hydroxychloroquine, except in a high STAT1 subset.

INTRODUCTION: Our recent data showed that signal transducers and activators of transcription 1 (STAT1), adenosine deaminase acting on RNA (ADAR), C-C motif chemokine ligand 2 (CCL2), and C-X-C motif chemokine 10 (CXCL10) were significantly elevated in a systemic lupus erythematosus (SLE) cohort compared to healthy donors. High and low STAT1 subsets were identified in SLE patient visits. The present study analyzed the correlation of common treatments used in SLE with the levels of these biomarkers. METHODS: Peripheral blood leukocytes were collected from 65 healthy donors and 103 SLE patients, of whom 60 had samples from two or more visits. Total RNA was isolated and analyzed for the expression of mRNA and microRNA using Taqman real-time polymerase chain reaction (PCR) assays. Relative expression of interferon signature genes, CCL2, and CXCL10 were determined by the ΔΔCT method. Results were correlated with therapy using prednisone, mycophenolate mofetil, and hydroxychloroquine and analyzed by Wilcoxon/Kruskal-Wallis test and Fisher's exact test. RESULTS: CCL2 and CXCL10 were significantly higher in untreated patients compared to treated patients, however, in high STAT1 patient visits there is no significant difference between treated and untreated patients' visits. When comparing linear regression fits of interferon (IFN) score with CCL2 and CXCL10, untreated patients and high STAT1 patients displayed significantly higher slopes compared to treated patients. There was no significant difference between the slopes of high STAT1 and untreated patients indicating that CCL2 and CXCL10 were correlated with type-I IFN in high STAT1 patients similar to that in untreated patients. CCL2 and CXCL10 levels in the high STAT1 subset remained high in treated patient visits compared to those of the low STAT1 subset. CONCLUSIONS: Among the biomarkers analyzed, only CCL2 and CXCL10 showed significantly reduced levels in treated compared to untreated SLE patients. STAT1, CCL2, and CXCL10 are potentially useful indicators of therapeutic action in SLE patients. Further work is needed to determine whether high STAT1 levels convey resistance to therapies commonly used to treat SLE and whether STAT1 inhibitors may have therapeutic implication for these patients.

MicroRNAs in systemic rheumatic diseases.

MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNAs about 21 nucleotides in length. miRNAs have been shown to regulate gene expression and thus influence a wide range of physiological and pathological processes. Moreover, they are detected in a variety of sources, including tissues, serum, and other body fluids, such as saliva. The role of miRNAs is evident in various malignant and nonmalignant diseases, and there is accumulating evidence also for an important role of miRNAs in systemic rheumatic diseases. Abnormal expression of miRNAs has been reported in autoimmune diseases, mainly in systemic lupus erythematosus and rheumatoid arthritis. miRNAs can be aberrantly expressed even in the different stages of disease progression, allowing miRNAs to be important biomarkers, to help understand the pathogenesis of the disease, and to monitor disease activity and effects of treatment. Different groups have demonstrated a link between miRNA expression and disease activity, as in the case of renal flares in lupus patients. Moreover, miRNAs are emerging as potential targets for new therapeutic strategies of autoimmune disorders. Taken together, recent data demonstrate that miRNAs can influence mechanisms involved in the pathogenesis, relapse, and specific organ involvement of autoimmune diseases. The ultimate goal is the identification of a miRNA target or targets that could be manipulated through specific therapies, aiming at activation or inhibition of specific miRNAs responsible for the development of disease.