Potential role of the hydroxyl carboxylic acid receptor type 2 (HCAR2) in microglia pathophysiology: A possible cross-talk with C-X-C chemokine receptor 1 (CXCR1).

Following insults or injury, microglia cells are activated contributing to the cytotoxic response or by promoting an immune-mediated damage resolution. Microglia cells express HCA2R, a hydroxy carboxylic acid (HCA) receptor, which has been shown to mediate neuroprotective and anti-inflammatory effects. In this study we found that HCAR2 expression levels were increased in cultured rat microglia cells after Lipopolysaccharide (LPS) exposure. In a similar fashion, the treatment with MK 1903, a potent full agonist of HCAR2, increased the receptor protein levels. Moreover, HCAR2 stimulation prevented i) cells viability ii) morphological activation iii) pro/anti-inflammatory mediators production in LPS-treated cells. Likewise, HCAR2 stimulation reduced the proinflammatory mediators mRNA expression induced by neuronal chemokine fractalkine (FKN), a neuronal derived chemokine activating its unique receptor, chemokine receptor 1 (CX3CR1) on microglia surface. Interestingly, electrophysiological recordings in vivo revealed that MK1903 was able to prevent the increase of the nociceptive neurons (NS) firing activity mediated by the spinal FKN application in healthy rats. Collectively, our data demonstrate that HCAR2 is functionally expressed in microglia, by showing its capability to shift microglia toward an anti-inflammatory phenotype. Moreover, we indicated the contribute of HCAR2 in the FKN signaling and suggested a possible HCAR2/CX3CR1 functional interaction. This study paves the way for further investigations aimed at understanding the role HCAR2 as potential target in neuroinflammation-based CNS disorders. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Fractalkine enhances oligodendrocyte regeneration and remyelination in a demyelination mouse model.

Demyelinating disorders of the central nervous system (CNS) occur when myelin and oligodendrocytes are damaged or lost. Remyelination and regeneration of oligodendrocytes can be achieved from endogenous oligodendrocyte precursor cells (OPCs) that reside in the adult CNS tissue. Using a cuprizone mouse model of demyelination, we show that infusion of fractalkine (CX3CL1) into the demyelinated murine brain increases de novo oligodendrocyte formation and enhances remyelination in the corpus callosum and cortical gray matter. This is achieved by increased OPC proliferation in the cortical gray matter as well as OPC differentiation and attenuation of microglia/macrophage activation both in corpus callosum and cortical gray matter. Finally, we show that activated OPCs and microglia/macrophages express fractalkine receptor CX3CR1 in vivo, and that in OPC-microglia co-cultures fractalkine increases in vitro oligodendrocyte differentiation by modulating both OPC and microglia biology. Our results demonstrate a novel pro-regenerative role of fractalkine in a demyelinating mouse model.

Fractalkine/CX3CR1-Dependent Modulation of Synaptic and Network Plasticity in Health and Disease.

CX3CR1 is a G protein-coupled receptor that is expressed exclusively by microglia within the brain parenchyma. The only known physiological CX3CR1 ligand is the chemokine fractalkine (FKN), which is constitutively expressed in neuronal cell membranes and tonically released by them. Through its key role in microglia-neuron communication, the FKN/CX3CR1 axis regulates microglial state, neuronal survival, synaptic plasticity, and a variety of synaptic functions, as well as neuronal excitability via cytokine release modulation, chemotaxis, and phagocytosis. Thus, the absence of CX3CR1 or any failure in the FKN/CX3CR1 axis has been linked to alterations in different brain functions, including changes in synaptic and network plasticity in structures such as the hippocampus, cortex, brainstem, and spinal cord. Since synaptic plasticity is a basic phenomenon in neural circuit integration and adjustment, here, we will review its modulation by the FKN/CX3CR1 axis in diverse brain circuits and its impact on brain function and adaptation in health and disease.

Identification of two theranostic biomarker panels for epithelial ovarian cancer.

BACKGROUND: Epithelial Ovarian cancer (EOC) is the leading cause of death associated with gynecologic tumors. Because the disease is asymptomatic in early-stage, the majority of patients are not diagnosed until late stages, highlighting the need for the development of novel diagnostic biomarkers. Mediators of tumoral microenvironment may affect EOC progression and resistance to treatment. AIM OF THE STUDY: Analysis of serum proteins to identify a panel of theranostic biomarkers for EOC. PATIENTS AND METHODS: Serum levels of 65 analytes were determined in EOC patients, and healthy controls with the ProcartaPlex Human Immune Monitoring 65-Plex Panel. RESULTS: Twenty-one analytes: 7 cytokines (IFN-γ, IL-12p70, IL-13, IL-18 and TSLP), 7 chemokines (Eotaxin, eotaxin-2, IP-10, BLC, I-TAC, SDF-1α, and fractalkine), 2 growth factors (MMP-1, VEGF-α), and 5 soluble receptors (APRIL, CD40L, TWEAK, CD30 and TNFRII; were significantly differentially expressed between the two groups. ROC curves showed that only seven of them (IL-9, TNF-α, Eotaxin, IP-10, BLC, Fractalkine, and Tweak) had AUC values greater than 0.70 and thus had potential clinical utility. Moreover, five cytokines: IFN-γ, IL-1 ß, IL-8, MIP-1ß, and TNF-α are positively associated with patients who developed resistance to taxol-platinum-based chemotherapy (CT). CONCLUSION: This study has revealed a first panel of 7 analytes (IL-9, TNF-α, Eotaxin, IP-10, BLC, Fractalkine and Tweak) that can be used for early detection of EOC and a second panel of five cytokines (IFN-γ, IL-1ß, IL-8, MIP-1ß, TNF-α) that can help clinicians to identify EOC patients who are at higher risk to develop resistance to CT of EOC.

Cytokine expression in rhinovirus- vs. respiratory syncytial virus-induced first wheezing episode and its relation to clinical course.

Rhinovirus (RV) and respiratory syncytial virus (RSV) are common causes of bronchiolitis. Unlike an RSV etiology, an RV etiology is associated with a markedly increased risk of asthma. We investigated the cytokine profiles of RV- and RSV-induced first wheezing episode and their correlation with prognosis. We recruited 52 sole RV- and 11 sole RSV-affected children with a severe first wheezing episode. Peripheral blood mononuclear cells (PBMCs) were isolated during acute illness and 2 weeks later and stimulated in vitro with anti-CD3/anti-CD28. Culture medium samples were analyzed for 56 different cytokines by multiplex ELISA. Recurrences were prospectively followed for 4 years. In adjusted analyses, the cytokine response from PBMCs in the RV group was characterized by decreased expression of interleukin 1 receptor antagonist (IL-1RA), interleukin 1 beta (IL-1ß), and monocyte chemoattractant protein-1 (MCP-1) and increased expression of eosinophil chemotactic protein 2 (eotaxin-2), thymus- and activation-regulated chemokine (TARC), and epithelial-derived neutrophil-activating peptide 78 (ENA-78) in the acute phase and increased expression of fractalkine in the convalescent phase compared to those in the RSV group. An analysis of the change in cytokine expression between study points revealed an increased expression of fractalkine and IL-1ß and decreased expression of I-309 (CCL1) and TARC in the RV group compared to those in the RSV group.. Considering hospitalization time, a significant non-adjusted group × cytokine interaction was observed in the levels of interferon gamma (IFN-γ), macrophage-derived chemokine (MDC), IL-1RA, and vascular endothelial growth factor (VEGF), indicating that a higher expression of cytokine was associated with shorter hospitalization time in the RSV group but not in the RV group. A significant interaction was also found in interleukin 6 (IL-6), but the cytokine response was not associated with hospitalization time in the RSV or RV group. In the RV group, increased expression of I-309 (CCL1) and TARC was associated with fewer relapses within 2 months, and decreased expression of interleukin 13 (IL-13) and increased expression of I-309 (CCL1) were associated with less relapses within 12 months. Differences in cytokine response from PBMCs were observed between RV- and RSV-induced first severe wheezing episode. Our findings also reveal new biomarkers for short- and medium-term prognosis in first-time wheezing children infected with RV or RSV.

CX3CL1 promotes cell sensitivity to ferroptosis and is associated with the tumor microenvironment in clear cell renal cell carcinoma.

BACKGROUND: An increasing number of studies have demonstrated that CX3CL1 is involved in the development of tumors and may thus be considered a new potential therapeutic target for them. However, the function of CX3CL1 in clear cell renal cell carcinoma (ccRCC) remains poorly defined. METHODS: The pan-cancer expression pattern and prognostic value of CX3CL1 were evaluated in this study. Moreover, the relationship of CX3CL1 expression with the tumor microenvironment, especially the tumor immune microenvironment, was analyzed. Our analyses employed public repository data. Additionally, we generated stable CX3CL1-overexpressing 786-O cells to determine the role of CX3CL1 in vitro via cell viability and transwell assays. A xenograft tumor model was used to determine the role of CX3CL1 in vivo. The association between CX3CL1 and ferroptosis sensitivity of tumor cells was assessed using Ferrostatin-1. RESULTS: Our findings indicated the involvement of CX3CL1 in the occurrence and development of ccRCC by acting as a tumor suppressor. We also found that ccRCC patients with high CX3CL1 expression showed better clinical outcomes than those with low CX3CL1 expression. The findings of our epigenetic study suggested that the expression of CX3CL1 in ccRCC is correlated with its DNA methylation level. Furthermore, the CX3CL1 expression level was closely related to the infiltration level of CD8+ T cells into the tumor microenvironment (TME). CX3CL1 showed different predictive values in different immunotherapy cohorts. Finally, CX3CL1 overexpression inhibited tumor cell proliferation and metastasis and promoted tumor ferroptosis sensitivity in ccRCC. CONCLUSIONS: This study revealed the role of CX3CL1 as a tumor suppressor in ccRCC. Our findings indicated that CX3CL1 plays a crucial role in regulating the ccRCC TME and is a potential predictor of immunotherapy outcomes in ccRCC. We also found that CX3CL1 can promote ferroptosis sensitivity in ccRCC cells.

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.

Lessons from the Cerebrospinal Fluid Analysis of HTLV-1-Infected Individuals: Biomarkers of Inflammation for HAM/TSP Development.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease that leads to motor impairment due to a chronic inflammatory process in the central nervous system (CNS). However, the HAM/TSP pathogenesis is not completely clear, and biomarkers to define the disease prognosis are still necessary. Thus, we aimed to identify biomarkers for HAM/TSP and potential mechanisms involved in disease development. To that end, the concentrations of VILIP-1, BDNF, VEGF, ß-NGF, TGF-ß1, fractalkine/CX3CL1, IL-6, IL-18, and TNF-α, and the soluble forms of TREM-1, TREM-2, and RAGE, were assessed using a multiplex bead-based immunoassay in paired cerebrospinal fluid (CSF) and serum samples from HAM/TSP patients (n = 20), asymptomatic HTLV-1 carriers (AC) (n = 13), and HTLV-1-seronegative individuals (n = 9), with the results analyzed according to the speed of HAM/TSP progression. HAM/TSP patients had elevated fractalkine in the serum but not in the CSF, particularly those with low neuroinflammatory activity (CSF/serum ratio of neopterin <1 and of CXCL10 < 2). HAM/TSP patients with normal CSF levels of neurofilament light chain (NfL) showed elevated ß-NGF in serum, and serum BDNF levels were increased in HTLV-1-infected individuals, particularly in HTLV-1 AC. Both HTLV-1 AC and HAM/TSP patients had lower TGF-ß1 levels in CSF compared to uninfected individuals, and HAM/TSP patients with active CNS inflammation showed higher CSF levels of IL-18, which correlated with markers of inflammation, neuronal death, and blood-brain-barrier permeability. Although none of the factors evaluated were associated with the speed of HAM/TSP progression, reduced TGF-ß1 levels in CSF suggest that suppressive responses to control subclinical and/or active neurodegeneration are impaired, while increased CSF IL-18 indicates the involvement of inflammasome-mediated mechanisms in HAM/TSP development.

Increment of CSF fractalkine-positive microvesicles preceded the spatial memory impairment in amyloid beta neurotoxicity.

BACKGROUND: Fractalkine (CX₃CL₁) is a key chemokine, affects neuronal cell communication and involves in Alzheimer's disease pathogenesis. Microvesicles (MVs) participate in neuronal cells' cross-talk in physiological and pathological states. Microvesicles released in cerebrospinal fluid (CSF) may provide a valuable footprint of brain changes. Little information is available regarding the release of fractalkine-positive MVs (CX₃CL₁⁺ -MVs) in the nervous system. METHODS: We induced cognitive impairment by bilateral injection of amyloid-beta (Aß) into the cerebral ventricles. We analyzed the CSF by flow cytometry in two experiments (trained and untrained) to elucidate the presence of CX₃CL₁⁺ -MVs. The hippocampal TNF-α as an inflammatory factor was assessed by immunohistochemistry. RESULTS: The Aß induced spatial memory impairment after two weeks, verified by a decrease in the escape latency in Morris water maze test. It caused an increase in the anxiety-like behaviors demonstrated by a decrease in entries into the open arms of elevated plus maze test. The Aß increased the percent of the positive area for TNF-α staining. Histological evaluation of the hippocampus confirmed the tissue injuries. The CSF levels of CX₃CL₁⁺ -MVs, increased 2 and 7 days after Aß injection. The Aß increased the TNF-α staining and provided an inflammatory context to facilitate the MVs release. The rise of CX₃CL₁⁺ -MVs was transient and subsided after two weeks. Both trained and untrained experiments showed a similar rise pattern of CX₃CL₁⁺ -MVs. CONCLUSION: Increase of fractalkine-positive microvesicles preceded the cognitive impairment, more studies are required to approve the CX₃CL₁⁺ -MVs as a potential biomarker in the early diagnosis of Alzheimer's disease.

CX3CL1 Derived from Bone Marrow Mesenchymal Stem Cells Inhibits Aß 1-42-Induced SH-SY5Y Cell Pathological Damage through TXNIP/NLRP3 Signaling Pathway.

Alzheimer's disease (AD) is the most commonly seen neurodegenerative brain disorder. The paracrine effects of mesenchymal stem cells (MSCs) signify to trigger immunomodulation and neural regeneration. However, the role and mechanism of bone marrow MSC- (BMSC-) derived CX3CL1 in AD remains elusive. In this study, Aß 1-42-intervened SH-SY5Y cells were used for AD cell model construction. pcDNA-ligated CX3CL1 overexpression plasmids were transfected into BMSCs. The levels of soluble and membrane-bound CX3CL1 were detected by ELISA and Western blotting (WB), respectively. The growth, apoptosis, and pathology of AD model cells were evaluated by CCK-8, flow cytometry, immunofluorescence, morphology observation, biochemical examination, and WB. It was found that Aß 1-42 significantly reduced CX3CL1 expression either in soluble or membrane-bound form, cell viability, relative protein expression of synaptic markers, SOD, CAT, and GSH-Px contents, as well as Trx protein expression; in addition, it enhanced the apoptosis rate, the relative expression of cleaved caspase-3, Aß, tau, p-Tau, Iba1, MDA, TXNIP, and NLRP3 in SH-SY5Y cells; however, the above effects were prominently reversed by the coculture of BMSCs. Moreover, overexpression of CX3CL1 in BMSCs observably strengthened the corresponding tendency caused by BMSCs. In conclusion, through the TXNIP/NLRP3 pathway, CX3CL1 derived from BMSCs inhibited pathological damage in Aß 1-42-induced SH-SY5Y.

Role of Innate and Adaptive Cytokines in the Survival of COVID-19 Patients.

SARS-CoV-2 is a new coronavirus characterized by a high infection and transmission capacity. A significant number of patients develop inadequate immune responses that produce massive releases of cytokines that compromise their survival. Soluble factors are clinically and pathologically relevant in COVID-19 survival but remain only partially characterized. The objective of this work was to simultaneously study 62 circulating soluble factors, including innate and adaptive cytokines and their soluble receptors, chemokines and growth and wound-healing/repair factors, in severe COVID-19 patients who survived compared to those with fatal outcomes. Serum samples were obtained from 286 COVID-19 patients and 40 healthy controls. The 62 circulating soluble factors were quantified using a Luminex Milliplex assay. Results. The patients who survived had decreased levels of the following 30 soluble factors of the 62 studied compared to those with fatal outcomes, therefore, these decreases were observed for cytokines and receptors predominantly produced by the innate immune system-IL-1α, IL-1α, IL-18, IL-15, IL-12p40, IL-6, IL-27, IL-1Ra, IL-1RI, IL-1RII, TNFα, TGFα, IL-10, sRAGE, sTNF-RI and sTNF-RII-for the chemokines IL-8, IP-10, MCP-1, MCP-3, MIG and fractalkine; for the growth factors M-CSF and the soluble receptor sIL2Ra; for the cytokines involved in the adaptive immune system IFNγ, IL-17 and sIL-4R; and for the wound-repair factor FGF2. On the other hand, the patients who survived had elevated levels of the soluble factors TNFß, sCD40L, MDC, RANTES, G-CSF, GM-CSF, EGF, PDGFAA and PDGFABBB compared to those who died. Conclusions. Increases in the circulating levels of the sCD40L cytokine; MDC and RANTES chemokines; the G-CSF and GM-CSF growth factors, EGF, PDGFAA and PDGFABBB; and tissue-repair factors are strongly associated with survival. By contrast, large increases in IL-15, IL-6, IL-18, IL-27 and IL-10; the sIL-1RI, sIL1RII and sTNF-RII receptors; the MCP3, IL-8, MIG and IP-10 chemokines; the M-CSF and sIL-2Ra growth factors; and the wound-healing factor FGF2 favor fatal outcomes of the disease.

Dorsal root ganglia CX3CR1 expressing monocytes/macrophages contribute to arthritis pain.

Pain is a persistent symptom of Rheumatoid Arthritis, and the K/BxN serum transfer model recapitulates both association and dissociation between pain and joint inflammation in RA. Furthermore, this model features monocyte/macrophage infiltration in joints and lumbar dorsal root ganglia (DRG), where these immune cells are close to nociceptive neurons. We focussed on CX3CR1-monocyte/macrophage trafficking and show that at peak paw swelling associated with nociception, CX3CR1 deletion altered neither swelling nor macrophage infiltration/phenotype in paws. However, acute nociception and DRG non-classical monocyte numbers were reduced in CX3CR1GFP/GFP (KO) compared to CX3CR1+/GFP (WT). Nociception that persisted despite swelling had resolved was attenuated in KO and correlated with DRG macrophages displaying M2-like phenotype. Still in the DRG, neurons up-regulated neuropeptide CGRP and olcegepant treatment reduced acute swelling, nociception, and leukocyte infiltration in paws and DRG. We delineate in-vitro a signalling pathway showing that CGRP liberates the CX3CR1 ligand fractalkine (FKN) from endothelium, and in bone marrow-derived macrophages, FKN promotes activation of intracellular kinases, polarisation towards M1-like phenotype and release of pro-nociceptive IL-6. These data implicate non-classical CX3CR1-expressing monocyte and macrophage recruitment into the DRG in initiation and maintenance of arthritis pain.

CX3CL1-induced CD16+ monocytes extravasation in myeloperoxidase-ANCA-associated vasculitis correlates with renal damage.

Background: Monocytes are involved in the pathogenesis of ANCA-associated vasculitis (AAV). Monocyte/macrophages are the dominant infiltrating cells in the glomeruli of patients with myeloperoxidase-AAV (MPO-AAV). However, how human monocyte subsets extravasate to the kidney in MPO-AAV with renal damage is unclear. Methods: 30 MPO-AAV patients with renal damage and 22 healthy controls were enrolled in this study. Monocyte subsets and monocyte-related chemokines in the blood and kidneys of MPO-AAV patients were detected. The chemoattractant activity of the CX3CL1-CX3CR1 axis on CD16+ monocytes was observed. The effect of MPO-ANCA on the migration of CD16+ monocytes to human glomerular endothelial cells (HGECs) was detected by flow cytometry and transwell migration assay. Results: Compared with controls, CD16+ monocytes were significantly decreased in the blood and increased in the glomeruli of MPO-AAV patients with renal damage. The level of CX3CL1, but not CCL2, was significantly increased in the plasma of MPO-AAV patients. CX3CL1 co-localized with glomerular endothelial cells in MPO-AAV patients with renal damage. Moreover, we initially found that MPO-ANCA promotes an increase of the chemokine CX3CL1 on HGECs, imposing recruitment on CD16+ monocytes. Finally, the percentage of CD16+ monocytes in the blood was found to be positively correlated with estimated glomerular filtration rate (eGFR) and negatively correlated with urinary protein creatinine ratio in MPO-AAV patients with renal damage. Furthermore, the urinary protein creatinine ratio was positively correlated with the infiltrating of CD14+ and CD16+ cells in the kidneys. Conclusion: Enhanced extravasation of CD16+ monocytes to the kidney via the CX3CL1-CX3CR1 axis may be involved in renal damage in MPO-AAV.

Visualization of macrophage subsets in the development of the fetal human inner ear.

Background: Human inner ear contains macrophages whose functional role in early development is yet unclear. Recent studies describe inner ear macrophages act as effector cells of the innate immune system and are often activated following acoustic trauma or exposure to ototoxic drugs. Few or limited literature describing the role of macrophages during inner ear development and organogenesis. Material and Methods: We performed a study combining immunohistochemistry and immunofluorescence using antibodies against IBA1, CX3CL1, CD168, CD68, CD45 and CollagenIV. Immune staining and quantification was performed on human embryonic inner ear sections from gestational week 09 to 17. Results: The study showed IBA1 and CD45 positive cells in the mesenchymal tissue at GW 09 to GW17. No IBA1 positive macrophages were detected in the sensory epithelium of the cochlea and vestibulum. Fractalkine (CX3CL1) signalling was initiated GW10 and parallel chemotactic attraction and migration of macrophages into the inner ear. Macrophages also migrated into the spiral ganglion, cochlear nerve, and peripheral nerve fibers and tissue-expressing CX3CL1. The mesenchymal tissue at all gestational weeks expressed CD163 and CD68. Conclusion: Expressions of markers for resident and non-resident macrophages (IBA1, CD45, CD68, and CD163) were identified in the human fetal inner ear. We speculate that these cells play a role for the development of human inner ear tissue including shaping of the gracile structures.

The CX3CL1 intracellular domain exhibits neuroprotection via insulin receptor/insulin-like growth factor receptor signaling.

CX3CL1, also known as fractalkine, is best known for its signaling activity through interactions with its cognate receptor CX3CR1. However, its intrinsic function that is independent of interaction with CX3CR1 remains to be fully understood. We demonstrate that the intracellular domain of CX3CL1 (CX3CL1-ICD), generated upon sequential cleavages by α-/ß-secretase and γ-secretase, initiates a back signaling activity, which mediates direct signal transmission to gene expression in the nucleus. To study this, we fused a synthetic peptide derived from CX3CL1-ICD, named Tet34, with a 13-amino acid tetanus sequence at the N terminus to facilitate translocation into neuronal cells. We show that treatment of mouse neuroblastoma Neuro-2A cells with Tet34, but not its scrambled control (Tet34s), induced cell proliferation, as manifested by changes in protein levels of transcription factors and progrowth molecules cyclin D1, PCNA, Sox5, and Cdk2. Further biochemical assays reveal elevation of phosphorylated insulin receptor ß subunit, insulin-like growth factor-1 receptor ß subunit, and insulin receptor substrates as well as activation of proliferation-linked kinase AKT. In addition, transgenic mice overexpressing membrane-anchored C-terminal CX3CL1 also exhibited activation of insulin/insulin-like growth factor-1 receptor signaling. Remarkably, we found that this Tet34 peptide, but not Tet34s, protected against endoplasmic reticulum stress and cellular apoptosis when Neuro-2A cells were challenged with toxic oligomers of ß-amyloid peptide or hydrogen peroxide. Taken together, our results suggest that CX3CL1-ICD may have translational potential for neuroprotection in Alzheimer's disease and for disorders resulting from insulin resistance.

Computational study of the conformational ensemble of CX3C chemokine receptor 1 (CX3CR1) and its interactions with antagonist and agonist ligands.

The CX3C chemokine receptor 1 (CX3CR1), a member of the class A of G Protein-Coupled Receptors (GPCR) superfamily, and its ligand fractalkine constitute an important biochemical axis that influence many cellular pathways involving homeostatic and inflammatory processes. They participate in the activation, chemotaxis and recruitment of multiple immunological cells such as microglia, macrophages and monocytes, and play a critical role in neuroinflammatory conditions such as Alzheimer's disease and multiple sclerosis, in the recovery from central nervous system injuries, in several chronic, peripheral inflammatory entities and in some infective processes including HIV-AIDS. In this work we present the study of the CX3CR1 receptor employing extensive atomistic Molecular Dynamics (MD) simulations with the aim to characterize the conformational ensemble of the receptor in the presence of its antagonist and agonist ligands. We analyzed the receptor conformational changes and described interactions within its key regions and the bounded ligands to identify their notable differences. Finally, we classify the features that would allow the identification of patterns that characterize a functional state to contribute to the understanding of the complexity of the GPCR superfamily.

CX3CL1/CX3CR1 interaction protects against lipotoxicity-induced nonalcoholic steatohepatitis by regulating macrophage migration and M1/M2 status.

BACKGROUND AND OBJECTIVES: Chemokine (C-X3-C motif) ligand 1 (CX3CL1) and its receptor CX3CR1 regulate the migration and activation of immune cells and are involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the mechanism remains elusive. Here, the roles of CX3CL1/CX3CR1 in the macrophage migration and polarization in the livers of NASH mice were investigated. METHODS AND RESULTS: The expression of Cx3cl1 and Cx3cr1 was markedly upregulated in the livers of lipotoxicity-induced NASH mice. CX3CR1 was predominantly expressed by F4/80+ macrophages and to a lesser degree by hepatic stellate cells or endothelial cells in the livers of NASH mice. Flow cytometry analysis revealed that, compared with chow-fed mice, NASH mice exhibited a significant increase in CX3CR1+ expression by liver macrophages (LMs), particularly M1 LMs. CX3CR1 deficiency caused a significant increase in inflammatory monocyte/macrophage infiltration and a shift toward M1 dominant macrophages in the liver, thereby exacerbating the progression of NASH. Moreover, transplantation of Cx3cr1-/- bone marrow was sufficient to cause glucose intolerance, inflammation, and fibrosis in the liver. In addition, deletion of CCL2 in Cx3cr1-/- mice alleviated NASH progression by decreasing macrophage infiltration and inducing a shift toward M2 dominant LMs. Importantly, overexpression of CX3CL1 in vivo protected against hepatic fibrosis in NASH. CONCLUSION: Pharmacological therapy targeting liver CX3CL1/CX3CR1 signaling might be a candidate for the treatment of NASH.

Brain fractalkine-CX3CR1 signalling is anti-obesity system as anorexigenic and anti-inflammatory actions in diet-induced obese mice.

Fractalkine is one of the CX3C chemokine family, and it is widely expressed in the brain including the hypothalamus. In the brain, fractalkine is expressed in neurons and binds to a CX3C chemokine receptor 1 (CX3CR1) in microglia. The hypothalamus regulates energy homeostasis of which dysregulation is associated with obesity. Therefore, we examined whether fractalkine-CX3CR1 signalling involved in regulating food intake and hypothalamic inflammation associated with obesity pathogenesis. In the present study, fractalkine significantly reduced food intake induced by several experimental stimuli and significantly increased brain-derived neurotrophic factor (BDNF) mRNA expression in the hypothalamus. Moreover, tyrosine receptor kinase B (TrkB) antagonist impaired fractalkine-induced anorexigenic actions. In addition, compared with wild-type mice, CX3CR1-deficient mice showed a significant increase in food intake and a significant decrease in BDNF mRNA expression in the hypothalamus. Mice fed a high-fat diet (HFD) for 16 weeks showed hypothalamic inflammation and reduced fractalkine mRNA expression in the hypothalamus. Intracerebroventricular administration of fractalkine significantly suppressed HFD-induced hypothalamic inflammation in mice. HFD intake for 4 weeks caused hypothalamic inflammation in CX3CR1-deficient mice, but not in wild-type mice. These findings suggest that fractalkine-CX3CR1 signalling induces anorexigenic actions via activation of the BDNF-TrkB pathway and suppresses HFD-induced hypothalamic inflammation in mice.

Intestinal fatty acid binding protein (I-FABP) and CXC3L1 evaluation as biomarkers for patients at high-risk for coeliac disease in Johannesburg, South Africa.

Coeliac disease (CD) is an autoimmune disorder and one of the few gastroenteropathies with accurate serological testing. CD serology has decreased accuracy for patients on a gluten-free diet and for monitoring mucosal healing. New ancillary tests would, therefore, be useful. Intestinal Fatty Acid Binding Protein (I-FABP) and CX3CL1 (Fractalkine) are two promising biomarkers for CD but haven't been examined in patients who are at a high-risk for CD such as patients with type one diabetes (TID). This study, therefore, aimed to investigate serum levels of I-FABP and CX3CL1 in a cohort of South African patients with TID at a high-risk of developing CD. The serum I-FABP levels were significantly higher in CD-positive patients compared to CD-negative individuals (p = 0.03). No significant differences in the serum CX3CL1 levels were detected although this may reflect the impact of the comorbid autoimmune diseases had on the serum CX3CL1 levels. In conclusion, this study is the first to assess the levels of these biomarkers in a multiethnic population with comorbid autoimmune disease and determined I-FABP to be the more promising biomarker in such clinical contexts. Future research should focus on a diverse biomarker panel and longitudinal follow-up of patients at a high-risk for CD.

Clinical value of urinary cytokines/chemokines as prognostic markers in patients with crescentic glomerulonephritis.

Crescentic glomerulonephritis (CrGN) usually requires urgent immunosuppressive treatment. However, aggressive immunosuppressive treatment is often difficult because of the patients' medical conditions or comorbidities. Prognostic markers including urinary cytokines/chemokines as noninvasive biomarkers were explored in CrGN patients. This prospective cohort study included 82 patients with biopsy-confirmed CrGN from 2002 to 2015 who were followed up for 5 years. Urine and serum cytokines/chemokines on the day of kidney biopsy were analyzed in 36 patients. The median age was 65 years and 47.6% were male. Baseline estimated glomerular filtration rate (eGFR) and interstitial fibrosis and tubular atrophy (IFTA) scores were identified as significant prognostic factors. Among patients with cytokines/chemokines measurement, increased IL-10 level was identified as an independent predictor of good prognosis, and increased levels of urinary MCP-1 and fractalkine tended to be associated with good prognosis after adjusting for baseline eGFR and IFTA score. However, semiquantitative analysis of intrarenal leukocytes did not show prognostic value predicting renal outcome or correlation with urinary cytokines/chemokines. This study supports the clinical importance of baseline eGFR and IFTA scores and suggests potential usefulness of urinary IL-10, MCP-1, and fractalkine as prognostic markers for predicting renal outcomes in patients with CrGN.