CXCL6 promotes the progression of NAFLD through regulation of PPARα.

An increasing number of studies have shown that Nonalcoholic fatty liver disease (NAFLD) is strongly associated with obesity, insulin resistance, dyslipidemia, hypertension and metabolic syndrome, but its specific pathogenesis remains unclear. By analyzing GEO database, we found CXCL6 was upregulated in liver tissues of patients with NAFLD. We also confirmed with qPCR that CXCL6 is highly expressed in serum of patients with NAFLD. To identify the underlying impact of CXCL6 on NAFLD, we established animal and cell models of NAFLD. Similarly, we confirmed by qPCR and Western blot that CXCL6 was upregulated in the NAFLD model in vitro and vivo. After transfecting NAFLD cells with siRNA targeting CXCL6 (si-CXCL6), a series of functional experiments were carried out, and these data indicated that the inhibition of CXCL6 reduced intracellular lipid deposition, decreased AST, ALT and TG level, facilitate cell proliferation and suppress their apoptosis. Furthermore, western blot and qPCR analyses displayed that the suppression of CXCL6 could raise the PPARα expression, but PPAR α inhibitor, GW6471 could partially counteract this effect. What's more, Oil Red O staining, biochemical analyzer and TG detection kit revealed that GW6471 could reverse the inhibitory effect of si-CXCL6 on NAFLD. In summary, we provide convincing evidence that CXCL6 is markedly elevated in NAFLD, and the CXCL6/PPARα regulatory network mediates disease progression of NAFLD.

Increased expression of CXCL6 in secretory cells drives fibroblast collagen synthesis and is associated with increased mortality in idiopathic pulmonary fibrosis.

RATIONALE: Recent data suggest that the localisation of airway epithelial cells in the distal lung in idiopathic pulmonary fibrosis (IPF) may drive pathology. We set out to discover whether chemokines expressed in these ectopic airway epithelial cells may contribute to the pathogenesis of IPF. METHODS: We analysed whole lung and single-cell transcriptomic data obtained from patients with IPF. In addition, we measured chemokine levels in blood, bronchoalveolar lavage (BAL) of IPF patients and air-liquid interface cultures. We employed ex vivo donor and IPF lung fibroblasts and an animal model of pulmonary fibrosis to test the effects of chemokine signalling on fibroblast function. RESULTS: By analysis of whole-lung transcriptomics, protein and BAL, we discovered that CXCL6 (a member of the interleukin-8 family) was increased in patients with IPF. Elevated CXCL6 levels in the BAL of two cohorts of patients with IPF were associated with poor survival (hazard ratio of death or progression 1.89, 95% CI 1.16-3.08; n=179, p=0.01). By immunostaining and single-cell RNA sequencing, CXCL6 was detected in secretory cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased collagen I levels in donor and IPF fibroblasts 4.4-fold and 1.7-fold, respectively. Both silencing of and chemical inhibition of CXCR1/2 blocked the effects of CXCL6 on collagen, while overexpression of CXCR2 increased collagen I levels 4.5-fold in IPF fibroblasts. CONCLUSIONS: CXCL6 is expressed in ectopic airway epithelial cells. Elevated levels of CXCL6 are associated with IPF mortality. CXCL6-driven collagen synthesis represents a functional consequence of ectopic localisation of airway epithelial cells in IPF.

CXCL6: A potential therapeutic target for inflammation and cancer.

Chemokines were originally defined as cytokines that affect the movement of immune cells. In recent years, due to the increasing importance of immune cells in the tumor microenvironment (TME), the role of chemokines has changed from a single "chemotactic agent" to a key factor that can regulate TME and affect the tumor phenotype. CXCL6, also known as granulocyte chemoattractant protein-2 (GCP-2), can recruit neutrophils to complete non-specific immunity in the process of inflammation. Cancer-related genes and interleukin family can promote the abnormal secretion of CXCL6, which promotes tumor growth, metastasis, epithelial mesenchymal transformation (EMT) and angiogenesis in the TME. CXCL6 also has a role in promoting fibrosis and tissue damage repair. In this review, we focus on the regulatory network affecting CXCL6 expression, its role in the progress of inflammation and how it affects tumorigenesis and progression based on the TME, in an attempt to provide a potential target for the treatment of diseases such as inflammation and cancer.

Basic fibroblast growth factor promotes meniscus regeneration through the cultivation of synovial mesenchymal stem cells via the CXCL6-CXCR2 pathway.

OBJECTIVE: To investigate the efficacy of basic fibroblast growth factor (bFGF) in promoting meniscus regeneration by cultivating synovial mesenchymal stem cells (SMSCs) and to validate the underlying mechanisms. METHODS: Human SMSCs were collected from patients with osteoarthritis. Eight-week-old nude rats underwent hemi-meniscectomy, and SMSCs in pellet form, either with or without bFGF (1.0 × 106 cells per pellet), were implanted at the site of meniscus defects. Rats were divided into the control (no transplantation), FGF (-) (pellet without bFGF), and FGF (+) (pellet with bFGF) groups. Different examinations, including assessment of the regenerated meniscus area, histological scoring of the regenerated meniscus and cartilage, meniscus indentation test, and immunohistochemistry analysis, were performed at 4 and 8 weeks after surgery. RESULTS: Transplanted SMSCs adhered to the regenerative meniscus. Compared with the control group, the FGF (+) group had larger regenerated meniscus areas, superior histological scores of the meniscus and cartilage, and better meniscus mechanical properties. RNA sequencing of SMSCs revealed that the gene expression of chemokines that bind to CXCR2 was upregulated by bFGF. Furthermore, conditioned medium derived from SMSCs cultivated with bFGF exhibited enhanced cell migration, proliferation, and chondrogenic differentiation, which were specifically inhibited by CXCR2 or CXCL6 inhibitors. CONCLUSION: SMSCs cultured with bFGF promoted the expression of CXCL6. This mechanism may enhance cell migration, proliferation, and chondrogenic differentiation, thereby resulting in superior meniscus regeneration and cartilage preservation.

CXCL1 and CXCL6 Are Potential Predictors for HCC Response to TACE.

Distinct immune patterns of hepatocellular carcinoma (HCC) may have prognostic implications in the response to transarterial chemoembolization (TACE). Thus, we aimed to exploratively analyze tumor tissue of HCC patients who do or do not respond to TACE, and to identify novel prognostic biomarkers predictive of response to TACE. We retrospectively included 15 HCC patients who had three consecutive TACE between January 2019 and November 2019. Eight patients had a response while seven patients had no response to TACE. All patients had measurable disease according to mRECIST. Corresponding tumor tissue samples were processed for differential expression profiling using NanoString nCounter® PanCancer immune profiling panel. Immune-related pathways were broadly upregulated in TACE responders. The top differentially regulated genes were the upregulated CXCL1 (log2fc 4.98, Benjamini-Hochberg (BH)-p < 0.001), CXCL6 (log2fc 4.43, BH-p = 0.016) and the downregulated MME (log2fc -4.33, BH-p 0.001). CD8/T-regs was highly increased in responders, whereas the relative number of T-regs to tumor-infiltrating lymphocytes (TIL) was highly decreased. We preliminary identified CXCL1 and CXCL6 as candidate genes that might have the potential to serve as therapeutically relevant biomarkers in HCC patients. This might pave the way to improve patient selection for TACE in HCC patients beyond expert consensus.

circ_0006089 Facilitates Gastric Cancer Progression via Decoying miR- 515-5p and Up-regulating CXCL6.

BACKGROUND: Gastric cancer (GC) is the most common cancer globally. Recent research has suggested that circular RNAs (circRNAs) play crucial roles in GC tumorigenesis and progression. The present study is performed to clarify the possible mechanism of circRNA has_circ_0006089 (circ_0006089) in GC. METHODS: The differentially expressed circRNAs were screened out by analyzing the dataset GSE83- 521. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect circ_0006089, miR-515-5p and CXCL6 expression levels in GC tissues and cell lines. CCK-8, BrdU and Transwell assays were adopted to examine the biological function of circ_0006089 in GC cells. The interaction between miR-515-5p and circ_0006089, as well as between CXCL6 and miR-515-5p, was confirmed through bioinformatics, RNA immunoprecipitation (RIP) assay, dual-luciferase reporter gene assay and RNA pull-down assay. RESULTS: Circ_0006089 was significantly upregulated in GC tissues and cells, and miR-515-5p was remarkably downregulated. After knocking down circ_0006089 or overexpressing miR-515-5p, the growth, migration and invasion of GC cells were markedly reduced. In terms of mechanism, miR-515- 5p was verified to be the target of circ_0006089, and CXCL6 was validated as miR-515-5p's downstream target gene. Inhibiting miR-515-5p reversed the inhibitory effect knocking down circ_0006089 had on GC cell proliferation, migration and invasion. CONCLUSION: Circ_0006089 facilitates the malignant biological behaviors of GC cells via the miR-515- 5p/CXCL6 axis. Circ_0006089 can probably act as one of the important biomarkers and therapeutic targets in GC treatment strategies.

Disease modification and symptom relief in osteoarthritis using a mutated GCP-2/CXCL6 chemokine.

We showed that the chemokine receptor C-X-C Motif Chemokine Receptor 2 (CXCR2) is essential for cartilage homeostasis. Here, we reveal that the CXCR2 ligand granulocyte chemotactic protein 2 (GCP-2) was expressed, during embryonic development, within the prospective permanent articular cartilage, but not in the epiphyseal cartilage destined to be replaced by bone. GCP-2 expression was retained in adult articular cartilage. GCP-2 loss-of-function inhibited extracellular matrix production. GCP-2 treatment promoted chondrogenesis in vitro and in human cartilage organoids implanted in nude mice in vivo. To exploit the chondrogenic activity of GCP-2, we disrupted its chemotactic activity, by mutagenizing a glycosaminoglycan binding sequence, which we hypothesized to be required for the formation of a GCP-2 haptotactic gradient on endothelia. This mutated version (GCP-2-T) had reduced capacity to induce transendothelial migration in vitro and in vivo, without affecting downstream receptor signaling through AKT, and chondrogenic activity. Intra-articular adenoviral overexpression of GCP-2-T, but not wild-type GCP-2, reduced pain and cartilage loss in instability-induced osteoarthritis in mice. We suggest that GCP-2-T may be used for disease modification in osteoarthritis.

Pilot study of peripheral blood chemokines as biomarkers for atrial fibrillation-related thromboembolism and bleeding in elderly patients.

Background: The scoring systems currently used to identify the potential for thrombosis and bleeding events in high-risk atrial fibrillation patients have certain limitations. The aim of this pilot study was to identify inflammatory chemokines with potential utility as sensitive biomarkers for the risk of thrombosis and bleeding in elderly patients with non-valvular atrial fibrillation. Methods: From January 1, 2014, to December 31, 2017, 200 consecutive elderly patients with atrial fibrillation (average age: 87.6 ± 7.7 years) were enrolled and followed up for 2 years to observe thromboembolic (arterial and venous) and bleeding events. Serum was collected upon enrollment, and the baseline levels of 27 chemokines were analyzed. During the 2-year follow-up, 12 patients were lost to follow-up. Among the 188 patients, there were 32 cases (17.0%) of AF-related thrombosis, 36 cases (19.1%) of arterial thrombosis, and 35 cases (18.6%) of major bleeding events. Results: Among 188 patients, 30 patients without clinical events (control group), 23 with arterial thrombosis, 15 with atrial fibrillation-related venous thromboembolism, and 12 with major bleeding were selected and randomly matched to compare chemokine levels. The baseline levels of interleukin-6, interleukin-10, vascular cell adhesion molecule-1, chemokine C-C-motif ligand, B-lymphocyte chemoattractant 1, interleukin-4, E-selectin, fractalkine, C-X-C motif chemokine 12, and granulocyte chemotactic protein 2 were found to differ statistically among the four groups (p < 0.05). Compared with that in the control group, the level of interleukin-4 in patients with atrial fibrillation-related thrombosis, arterial thrombosis, or major bleeding increased by 53-fold (0.53 vs. 0.01 pg/ml), 17-fold (0.17 vs. 0.01 pg/ml), and 19-fold (0.19 vs. 0.01 pg/ml), respectively. Compared with that in the control group, the level of interleukin-6 in patients with arterial thrombosis increased by six-fold (39.78 vs. 4.98 pg/ml). Conclusions: Among elderly patients with atrial fibrillation at high risk of thromboembolism and bleeding, the baseline levels of interleukin-6, interleukin-4, and E-selectin were significantly increased in those that experienced thrombosis and bleeding events during the 2-year follow-up, indicating that these chemokines may serve as potential biomarkers for an increased risk of thrombosis and bleeding in this population. Clinical trial registration number: ChiCTR-OCH-13003479.

Allicin May Promote Reversal of T-Cell Dysfunction in Periodontitis via the PD-1 Pathway.

We evaluated the role of allicin in periodontitis using an in silico and in vitro design. An in silico docking analysis was performed to assess the plausible interactions between allicin and PD-L1. The cytokine profile of gingival crevicular fluid (GCF) samples obtained from periodontitis patients was estimated by cytometric bead array. CD3+ lymphocytes isolated from the peripheral blood were sorted and characterized using immunomagnetic techniques. Cultured and expanded lymphocytes were treated with the GCF samples to induce T-cell exhaustion. Optimum concentrations of allicin were added to exhausted lymphocytes to compare the expression of TIM-3 and LAG-3 gene expression at baseline and post-treatment. Allicin was found to bind to the PD-L1 molecule as revealed by the in-silico experiment, which is possibly an inhibitory interaction although not proven. GCF from periodontitis patients had significantly higher concentrations of TNF-α, CCL2, IL-6, IFN-γ, and CXCL8 than controls. GCF treatment of CD3+ lymphocytes from the periodontitis patients significantly increased expression of T-cell exhaustion markers TIM-3 and LAG-3. Allicin administration with GCF treatment resulted in significant lowering of the expression of exhaustion markers. Allicin may exert an immunostimulatory role and reverse immune-destructive mechanisms such as T-cell exhaustion.

Chemokine Profiles Are Affected in Serum of Patients with Acute Rejection of Kidney Allograft.

Kidney allograft transplantation improved the prognosis and quality of life of patients with end-stage renal diseases but the occurrence of acute rejection represents a limitation of the final outcome. Noninvasive biomarkers are needed as well as further advancements in the understanding of immune mechanisms of reaction to the allograft. Our study of 138 patients focused on one-year monitoring of serum concentrations of 12 chemokines regulating the recruitment of different immune cells into transplanted allograft and on in vitro regulation of the same chemokines release by interactions of renal proximal epithelial cells with monocyte/macrophage cell line stimulated with TNF alpha. In a group of 44 patients with acute rejection, higher serum pretransplant levels of CXCL1, CXCL5, CXCL6, CCL2, CCL21, and particularly CXCL10 and CX3CL1(both p < 0.001) were found suggesting their higher proinflammatory status as compared to subjects with the uncomplicated outcome. In samples collected at the day of biopsy positive for acute rejection, chemokines CXCL9 and CXCL11 attracting preferentially Th1 lymphocytes were found to be upregulated. In our in vitro model with TNF alpha induction, renal proximal epithelial cells seemed to be a more potent source of chemokines attracting neutrophils as compared to monocyte/macrophage cell line but the coculture of these cells potentiated release of neutrophilic chemokines CXCL5 and CXCL6. Similar augmentation of chemokine production was found also in the case of CCL2. On the other hand, adding of monocytes/macrophages to a culture of renal epithelial cells suppressed the release of CXCL10 and CXCL11 attracting T lymphocytes. We assume from our data that in kidney allograft transplantation, chemokines attracting neutrophils, T lymphocytes, and monocytes are induced simultaneously and measurement some of them in combination might be used as biomarkers of acute rejection. Mutual cell-cell interactions of immune cells with renal parenchyma seem to be important for fine regulation of chemokine release.

Cancer-Associated Fibroblast-Mediated Cellular Crosstalk Supports Hepatocellular Carcinoma Progression.

BACKGROUND AND AIMS: Cancer-associated fibroblasts (CAFs) are key players in multicellular, stromal-dependent alterations leading to HCC pathogenesis. However, the intricate crosstalk between CAFs and other components in the tumor microenvironment (TME) remains unclear. This study aimed to investigate the cellular crosstalk among CAFs, tumor cells, and tumor-associated neutrophils (TANs) during different stages of HCC pathogenesis. APPROACH AND RESULTS: In the HCC-TME, CAF-derived cardiotrophin-like cytokine factor 1 (CLCF1) increased chemokine (C-X-C motif) ligand 6 (CXCL6) and TGF-ß secretion in tumor cells, which subsequently promoted tumor cell stemness in an autocrine manner and TAN infiltration and polarization in a paracrine manner. Moreover, CXCL6 and TGF-ß secreted by HCC cells activated extracellular signal-regulated kinase (ERK) 1/2 signaling of CAFs to produce more CLCF1, thus forming a positive feedback loop to accelerate HCC progression. Inhibition of ERK1/2 or CLCF1/ciliary neurotrophic factor receptor signaling efficiently impaired CLCF1-mediated crosstalk among CAFs, tumor cells, and TANs both in vitro and in vivo. In clinical samples, up-regulation of the CLCF1-CXCL6/TGF-ß axis exhibited a marked correlation with increased cancer stem cells, "N2"-polarized TANs, tumor stage, and poor prognosis. CONCLUSIONS: This study reveals a cytokine-mediated cellular crosstalk and clinical network involving the CLCF1-CXCL6/TGF-ß axis, which regulates the positive feedback loop among CAFs, tumor stemness, and TANs, HCC progression, and patient prognosis. These results may support the CLCF1 cascade as a potential prognostic biomarker and suggest that selective blockade of CLCF1/ciliary neurotrophic factor receptor or ERK1/2 signaling could provide an effective therapeutic target for patients with HCC.

CXCL6 fuels the growth and metastases of esophageal squamous cell carcinoma cells both in vitro and in vivo through upregulation of PD-L1 via activation of STAT3 pathway.

CXCL6, contraction of C-X-C motif chemokine ligand 6, whose biological roles have been rarely described in esophageal squamous cell carcinoma (ESCC). To understand the clinicopathological and biological roles played by CXCL6 in the growth and metastasis of ESCC, immunohistochemistry was used to detect the expression of CXCL6 in ESCC tissues, totaling 105 cases; and the correlation was statistically analyzed between CXCL6 expression and clinicopathological parameters. The role mediated in migration and invasion was evaluated using wound-healing and Transwell assays. MTT and flow cytometry were used to assay the proliferative variation. In vivo, tail vein injection model was established in nude mice xenografted with human ESCC cell lines whose CXCL6 were artificially manipulated. It was found that relative to normal control, CXCL6 was profoundly higher in ESCC; upregulated CXCL6 only significantly correlated with differentiation degree. In vitro, CXCL6 was found to promote the proliferation, migration, and invasion of ESCC cells; which was fully corroborated by nude mice experiment that CXCL6 can promote the growth and metastases of ESCC cells in vivo. Mechanistically, CXCL6 was discovered to be capable of promoting epithelial-mesenchymal transition and upregulating PD-L1 expression through activation of the STAT3 pathway. Collectively, all the data we showed here demonstrate that CXCL6 can enhance the growth and metastases of ESCC cells both in vivo and in vitro.

Potential Repressive Impact of microRNA-20a on Renal Tubular Damage in Diabetic Kidney Disease by Targeting C-X-C Motif Chemokine Ligand 6.

BACKGROUND AND AIMS: C-X-C Motif Chemokine Ligand 6 (CXCL6) is an important chemokine. We attempt in this investigation to explore its role and possible mechanism in diabetic kidney disease (DKD). METHODS: By intergrating GEO data, CXCL6 expression in DKD patients and normal controls was exhibited. miRWalk website and luciferase reporter assay were used to predict and verify the upstream miRNA of CXCL6. CCK-8 assay and flow cytometry were performed to detect proliferation and apoptosis capacities. The levels of inflammatory key factors (TNF-α, IL-6 and IL-8) were measured using ELISA analysis. Expression of CXCL6, miR-20a, and JAK/STAT3 pathway-related markers were detected by qRT-PCR or western blot assays. RESULTS: CXCL6 was increased in DKD. miR-20a was identified as an upstream regulatory miRNA of CXCL6, and its expression was decreased in DKD and HG-treated HK-2 cells. miR-20a overexpression facilitated the proliferation of HG-treated HK-2 cells, whereas miR-20a depletion exhibited the opposite phenomenon. The levels of TNF-α, IL-6 and IL-8 were increased by HG treatment in HK-2 cells. CXCL6 antagonized the promoting impacts of miR-20a mimics on HG-exposed HK-2 cell proliferation. The suppressive effect of miR-20a overexpression on apoptosis and inflammatory response of HG-induced HK-2 cell was rescued by CXCL6 enhancement. The protein expression of p-JAK and p-STAT3 were reduced by miR-20a mimic while facilitated by CXCL6 overexpression in HG-stimulated HK-2 cells. CONCLUSION: These consequences hinted that miR-20a might exert a repressive impact on DKD, possibly through targeting CXCL6 and mediating JAK/STAT3 pathway, which offer new targets for DKD treatment.

CXCL6 regulates cell permeability, proliferation, and apoptosis after ischemia-reperfusion injury by modulating Sirt3 expression via AKT/FOXO3a activation.

Chemokine (C-X-C motif) ligand 6 (CXCL6), a member of the CXC chemokine family, reportedly mediates several processes such as inflammation, immunoreaction, cell growth, and metastasis through interaction with the chemokine receptors CXCR1 and CXCR2 in humans; further, CXCR1 and CXCR2 can promote repair and regeneration of organs or tissues after ischemia-reperfusion injury (IRI). In this study, we found that HIF-1α, CXCL6, and CXCR2 expression levels were elevated in human brain microvascular endothelial cells (HBMECs) after IRI, whereas silent information regulator of transcription (Sirt) 3 expression level had reduced. HIF-1α inhibition in an IRI model potently promoted HBMEC proliferation, accompanied by increased Sirt3 and decreased CXCL6/CXCR2 expression levels. CXCL6 knockdown in the IRI model significantly decreased HBMEC permeability and promoted HBMEC proliferation, concurrent with a decrease in apoptosis; it also increased Sirt3 expression levels and decreased CXCL6/CXCR2 protein and phosphorylated AKT (p-AKT) and class O of forkhead box (FOXO) 3a (p-FOXO3a) levels. In addition, CXCL6-induced HBMEC permeability and inhibition of HBMEC proliferation were counteracted by Sirt3 overexpression, and the AKT inhibitor LY294002 counteracted the effect of CXCL6 recombinant proteins on Sirt3, p-AKT, and p-FOXO3a expressions. These results suggest that CXCL6 and Sirt3 are downstream of HIF-1α and that CXCL6 regulatesHBMEC permeability, proliferation, and apoptosis after IRI by modulating Sirt3 expression via AKT/FOXO3a activation.

Hypoxia-CXCL6 axis affects arteriolar niche remodeling in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant clonal disease derived from hematopoietic stem/progenitor cell. Leukemia blasts cause extensive hypoxia of bone marrow (BM), which lead to disorder and remodeling of BM niche, thereby becoming "leukemic niche" to support the development and drug-resistance of AML as well as the maintenance of normal hematopoietic stem cells. In this study, the biological characteristics (such as self-renewal, apoptosis, migration, autocrine) and function (vascularization) of mesenchymal stem cells (MSCs) and human umbilical artery endothelial cells (HUAECs) that make up BM arteriolar niche in simulated hypoxia AML context were investigated. It was found that moderate hypoxia enhanced the viability of the arteriolar niche cells, but severe hypoxia of AML BM resulted in the damage of arteriolar niche cells and the disorder of vascular cytokines C-X-C motif chemokine ligand 6 (CXCL6). The dynamic changes of CXCL6 in the system as well as its anti-apoptotic and promoting angiogenic effects suggested that CXCL6 played an important role in the remodeling of BM arteriolar niche in AML. Taking advantage of CXCL6 can save the damaged MSCs and HUAECs, which is the hope of rescuing arteriolar niche. It is suggested that CXCL6 may be an assistant strategy for microenvironment targeted therapy of AML.

Formalin-fixed paraffin-embedded renal biopsy tissues: an underexploited biospecimen resource for gene expression profiling in IgA nephropathy.

Primary IgA nephropathy (IgAN) diagnosis is based on IgA-dominant glomerular deposits and histological scoring is done on formalin-fixed paraffin embedded tissue (FFPE) sections using the Oxford classification. Our aim was to use this underexploited resource to extract RNA and identify genes that characterize active (endocapillary-extracapillary proliferations) and chronic (tubulo-interstitial) renal lesions in total renal cortex. RNA was extracted from archival FFPE renal biopsies of 52 IgAN patients, 22 non-IgAN and normal renal tissue of 7 kidney living donors (KLD) as controls. Genome-wide gene expression profiles were obtained and biomarker identification was carried out comparing gene expression signatures a subset of IgAN patients with active (N = 8), and chronic (N = 12) renal lesions versus non-IgAN and KLD. Bioinformatic analysis identified transcripts for active (DEFA4, TNFAIP6, FAR2) and chronic (LTB, CXCL6, ITGAX) renal lesions that were validated by RT-PCR and IHC. Finally, two of them (TNFAIP6 for active and CXCL6 for chronic) were confirmed in the urine of an independent cohort of IgAN patients compared with non-IgAN patients and controls. We have integrated transcriptomics with histomorphological scores, identified specific gene expression changes using the invaluable repository of archival renal biopsies and discovered two urinary biomarkers that may be used for specific clinical decision making.

Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case.

The serum amyloid A (SAA) gene family is highly conserved and encodes acute phase proteins that are upregulated in response to inflammatory triggers. Over the years, a considerable amount of literature has been published attributing a wide range of biological effects to SAAs such as leukocyte recruitment, cytokine and chemokine expression and induction of matrix metalloproteinases. Furthermore, SAAs have also been linked to protumorigenic, proatherogenic and anti-inflammatory effects. Here, we investigated the biological effects conveyed by murine SAA3 (mu rSAA3) recombinantly expressed in Escherichia coli. We observed the upregulation of a number of chemokines including CCL2, CCL3, CXCL1, CXCL2, CXCL6 or CXCL8 following stimulation of monocytic, fibroblastoid and peritoneal cells with mu rSAA3. Furthermore, this SAA variant displayed potent in vivo recruitment of neutrophils through the activation of TLR4. However, a major problem associated with proteins derived from recombinant expression in bacteria is potential contamination with various bacterial products, such as lipopolysaccharide, lipoproteins and formylated peptides. This is of particular relevance in the case of SAA as there currently exists a discrepancy in biological activity between SAA derived from recombinant expression and that of an endogenous source, i.e. inflammatory plasma. Therefore, we subjected commercial recombinant mu rSAA3 to purification to homogeneity via reversed-phase high-performance liquid chromatography (RP-HPLC) and re-assessed its biological potential. RP-HPLC-purified mu rSAA3 did not induce chemokines and lacked in vivo neutrophil chemotactic activity, but retained the capacity to synergize with CXCL8 in the activation of neutrophils. In conclusion, experimental results obtained when using proteins recombinantly expressed in bacteria should always be interpreted with care.