Resident cardiac macrophages mediate adaptive myocardial remodeling.

Cardiac macrophages represent a heterogeneous cell population with distinct origins, dynamics, and functions. Recent studies have revealed that C-C Chemokine Receptor 2 positive (CCR2+) macrophages derived from infiltrating monocytes regulate myocardial inflammation and heart failure pathogenesis. Comparatively little is known about the functions of tissue resident (CCR2-) macrophages. Herein, we identified an essential role for CCR2- macrophages in the chronically failing heart. Depletion of CCR2- macrophages in mice with dilated cardiomyopathy accelerated mortality and impaired ventricular remodeling and coronary angiogenesis, adaptive changes necessary to maintain cardiac output in the setting of reduced cardiac contractility. Mechanistically, CCR2- macrophages interacted with neighboring cardiomyocytes via focal adhesion complexes and were activated in response to mechanical stretch through a transient receptor potential vanilloid 4 (TRPV4)-dependent pathway that controlled growth factor expression. These findings establish a role for tissue-resident macrophages in adaptive cardiac remodeling and implicate mechanical sensing in cardiac macrophage activation.

CCR2-dependent placental migration of inflammatory monocytes suppresses abnormal pregnancies caused by Toxoplasma gondii infection.

Toxoplasma gondii (T. gongii) is a zoonotic protozoan parasite that causes congenital toxoplasmosis, including fetal death, abortion, stillbirth, morphological abnormalities, and premature birth. Primary T. gondii infection in pregnant women results in congenital toxoplasmosis. C-C chemokine receptor (CCR) 2 is reportedly a critical host defense factor against T. gondii infection. However, details of the role of CCR2 in the host immune response to T. gondii in congenital toxoplasmosis remain unclear. Here, we infected pregnant CCR2-deficient mice with T. gondii, resulting in stillbirth, embryonic resorption, fetal morphological abnormalities, and preterm delivery at significantly higher rates than those in pregnant wild-type mice. Consistent with the severity of abnormal pregnancy, a large area of placental hemorrhage and a large number of T. gondii infections around the hemorrhagic area were observed in the placentas of CCR2-deficient mice. In addition, the accumulation of inflammatory monocytes in the placenta was reduced in CCR2-deficient mice during infection. We further confirmed that the adoptive transfer of inflammatory monocytes collected from wild-type mice into T. gondii-infected pregnant CCR2-deficient mice effectively suppressed placental damage and abnormal pregnancy. Collectively, CCR2 contributes to pregnancy maintenance by regulating the migration of inflammatory monocytes into the placenta of T. gondii-infected pregnant mice.

Targeting CCL2-CCR2 signaling pathway alleviates macrophage dysfunction in COPD via PI3K-AKT axis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) remains a leading cause of morbidity and mortality worldwide, characterized by persistent respiratory symptoms and airflow limitation. The involvement of C-C motif chemokine ligand 2 (CCL2) in COPD pathogenesis, particularly in macrophage regulation and activation, is poorly understood despite its recognized role in chronic inflammation. Our study aims to elucidate the regulatory role and molecular mechanisms of CCL2 in the pathogenesis of COPD, providing new insights for therapeutic strategies. METHODS: This study focused on the CCL2-CCR2 signaling pathway, exploring its role in COPD pathogenesis using both Ccl2 knockout (KO) mice and pharmacological inhibitors. To dissect the underlying mechanisms, we employed various in vitro and in vivo methods to analyze the secretion patterns and pathogenic effects of CCL2 and its downstream molecular signaling through the CCL2-CCR2 axis. RESULTS: Elevated Ccl2 expression was confirmed in the lungs of COPD mice and was associated with enhanced recruitment and activation of macrophages. Deletion of Ccl2 in knockout mice, as well as treatment with a Ccr2 inhibitor, resulted in protection against CS- and LPS-induced alveolar injury and airway remodeling. Mechanistically, CCL2 was predominantly secreted by bronchial epithelial cells in a process dependent on STAT1 phosphorylation and acted through the CCR2 receptor on macrophages. This interaction activated the PI3K-AKT signaling pathway, which was pivotal for macrophage activation and the secretion of inflammatory cytokines, further influencing the progression of COPD. CONCLUSIONS: The study highlighted the crucial role of CCL2 in mediating inflammatory responses and remodeling in COPD. It enhanced our understanding of COPD's molecular mechanisms, particularly how CCL2's interaction with the CCR2 activates critical signaling pathways. Targeting the CCL2-CCR2 axis emerged as a promising strategy to alleviate COPD pathology.

C-C Chemokine Receptor 7 Promotes T-Cell Acute Lymphoblastic Leukemia Invasion of the Central Nervous System via ß2-Integrins.

C-C Chemokine Receptor 7 (CCR7) mediates T-cell acute lymphoblastic leukemia (T-ALL) invasion of the central nervous system (CNS) mediated by chemotactic migration to C-C chemokine ligand 19 (CCL19). To determine if a CCL19 antagonist, CCL198-83, could inhibit CCR7-induced chemotaxis and signaling via CCL19 but not CCL21, we used transwell migration and Ca2+ mobilization signaling assays. We found that in response to CCL19, human T-ALL cells employ ß2 integrins to invade human brain microvascular endothelial cell monolayers. In vivo, using an inducible mouse model of T-ALL, we found that we were able to increase the survival of the mice treated with CCL198-83 when compared to non-treated controls. Overall, our results describe a targetable cell surface receptor, CCR7, which can be inhibited to prevent ß2-integrin-mediated T-ALL invasion of the CNS and potentially provides a platform for the pharmacological inhibition of T-ALL cell entry into the CNS.

Sulfotyrosine residues: Interaction specificity determinants for extracellular protein-protein interactions.

Tyrosine sulfation, a post-translational modification, can determine and often enhance protein-protein interaction specificity. Sulfotyrosyl residues (sTyrs) are formed by the enzyme tyrosyl-protein sulfotransferase during protein maturation in the Golgi apparatus and most often occur singly or as a cluster within a six-residue span. With both negative charge and aromatic character, sTyr facilitates numerous atomic contacts as visualized in binding interface structural models, thus there is no discernible binding site consensus. Found exclusively in secreted proteins, in this review, we discuss the four broad sequence contexts in which sTyr has been observed: first, a solitary sTyr has been shown to be critical for diverse high-affinity interactions, such as between peptide hormones and their receptors, in both plants and animals. Second, sTyr clusters within structurally flexible anionic segments are essential for a variety of cellular processes, including coreceptor binding to the HIV-1 envelope spike protein during virus entry, chemokine interactions with receptors, and leukocyte rolling cell adhesion. Third, a subcategory of sTyr clusters is found in conserved acidic sequences termed hirudin-like motifs that enable proteins to interact with thrombin; consequently, many proven and potential therapeutic proteins derived from blood-consuming invertebrates depend on sTyrs for their activity. Finally, several proteins that interact with collagen or similar proteins contain one or more sTyrs within an acidic residue array. Refined methods to direct sTyr incorporation in peptides synthesized both in vitro and in vivo, together with continued advances in mass spectrometry and affinity detection, promise to accelerate discoveries of sTyr occurrence and function.

CCL5/CCR5-mediated peripheral inflammation exacerbates blood‒brain barrier disruption after intracerebral hemorrhage in mice.

BACKGROUND: Owing to metabolic disequilibrium and immune suppression, intracerebral hemorrhage (ICH) patients are prone to infections; according to a recent global analysis of stroke cases, approximately 10 million new-onset ICH patients had experienced concurrent infection. However, the intrinsic mechanisms underlying the effects of infection related peripheral inflammation after ICH remain unclear. METHODS: Lipopolysaccharide (LPS) was intraperitoneally injected into ICH model mice to induce peripheral inflammation. Neurobehavioral deficits, blood‒brain barrier (BBB) disruption, and the expression of CCR5, JAK2, STAT3, and MMP9 were evaluated after treatment with recombinant CCL5 (rCCL5) (a CCR5 ligand), maraviroc (MVC) (an FDA-approved selective CCR5 antagonist), or JAK2 CRISPR plasmids. RESULTS: Our study revealed that severe peripheral inflammation increased CCL5/CCR5 axis activation in multiple inflammatory cell types, including microglia, astrocytes, and monocytes, and aggravated BBB disruption and neurobehavioral dysfunction after ICH, possibly in part through the JAK2/STAT3 signaling pathway. CONCLUSIONS: CCR5 might be a potential target for the clinical treatment of infection-induced exacerbation of BBB disruption following ICH.

Effects of let-7a microRNA and C-C chemokine receptor type 7 expression on cellular function and prognosis in esophageal squamous cell carcinoma.

BACKGROUND: C-C chemokine receptor type 7 (CCR7) participates in chemotactic and metastatic responses in various cancers, including in esophageal squamous cell carcinoma (ESCC). The microRNA (miRNA) let-7a suppresses migration and invasion of various types of cancer cells by downregulating CCR7 expression. METHODS: The expression levels of CCR7 and let-7a were measured in the cell lines, tumor, and peritumoral tissues of ESCC patients. KYSE cell lines were transfected with synthetic let-7a miRNA and a let-7a miRNA inhibitor, and their CCR7 expression levels as well as invasive ability were evaluated. A highly invasive cell line was established via an invasion assay, and CCR7 expression level along with let-7a level was subsequently evaluated. Cancer cells overexpressing CCR7 were injected subcutaneously into mice, and the animals were monitored for tumor growth along with lymph node metastasis. RESULTS: A negative correlation between CCR7 and let-7a expression was observed in the ESCC cell lines as well as in tissue samples from patients. Synthetic let-7a decreased CCR7 expression level, while the let-7a inhibitor increased it. In vitro, the established highly invasive cancer cells with high and low levels of CCR7 and let-7a expression, respectively, exhibited a greater invasive ability than the wild-type cell line. The cells were associated with tumor growth and lymph node metastasis in mice. Patients in the high-CCR7/low-let-7a group had the worst prognosis, with a five-year recurrence free survival (5-RFS) rate of 37.5%, followed by the high-CCR7/high-let-7a (5-RFS: 60.0%) and low-CCR7 (5-RFS: 85.7%; p = 0.038) groups. CONCLUSIONS: The expression of CCR7 was downregulated by let-7a miRNA in esophageal cancer cells. The decrease in let-7a expression level led to the increased expression level of CCR7 in ESCC cells, consequently increasing their invasive ability and malignancy and resulting in a worse prognosis for ESCC patients. TRIAL REGISTRATION: Retrospectively registered.

Exacerbation of non-steroidal anti-inflammatory drug-induced enteropathy in C-C chemokine receptor type 7-deficient mice.

BACKGROUND AND AIM: Non-steroidal anti-inflammatory drugs (NSAIDs) induce intestinal enteropathy and the pathophysiology is related to immune-mediated mechanisms. We aimed to investigate the role of C-C chemokine receptor type 7 (CCR7) which regulates immune cell migration in NSAID-induced enteropathy. METHODS: Injury of the small intestine was evaluated 24 h after the subcutaneous injection of indomethacin in CCR7-deficient (Ccr7-/- ) and wild-type (WT) mice. The cellular profile and cytokine production in intestinal cells were analyzed. Indomethacin-induced enteropathy was evaluated in mice adoptively transferred with CD103+ dendritic cells (DCs) from Ccr7-/- or WT mice. RESULTS: Indomethacin induced more severe intestinal injury in Ccr7-/- mice than in WT mice. The major inflammatory cytokines were not increased and the proportion of regulatory T cells following indomethacin injection was not decreased in Ccr7-/- mice compared with WT mice. The expression of interleukin (IL)-22 binding protein (IL-22BP), which inhibits IL-22 activity, was significantly higher in CD103+ DCs from Ccr7-/- mice than those from WT mice. Mice adoptively transferred with CD103+ DCs isolated from Ccr7-/- mice exhibited more severe intestinal injury following indomethacin injection compared with those adoptively transferred with CD103+ DCs of WT mice. Ccr7-/- mice injected with indomethacin showed a significant reduction in regenerating islet-derived 1 (Reg1) mRNA expression, which is regulated by IL-22, in intestinal epithelial cells. CONCLUSIONS: C-C chemokine receptor type 7 deficiency exacerbated NSAID-induced enteropathy in association with an altered phenotype of CD103+ DCs that produces IL-22BP. CCR7 contributes to protect the small intestine from NSAID-induced mucosal injury.

CCR5 as a prognostic biomarker correlated with immune infiltrates in head and neck squamous cell carcinoma by bioinformatic study.

BACKGROUND: C-C chemokine receptor 5 (CCR5) has recently been recognized as an underlying therapeutic target for various malignancies. However, the association of CCR5 with prognosis in the head and neck squamous cell carcinoma (HNSC) patients and tumor-infiltrating lymphocytes (TILs) is unclear. METHODS: In the current experiment, methods such as the Tumor Immune Estimation Resource Analysis (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, and Kaplan-Meier plotter Analysis were used to comprehensively evaluate the expression of CCR5 in human various malignancies and the clinical prognosis in HNSC patients. Subsequently, we used the TIMER database and the TISIDB platform to investigate the correlation between CCR5 expression levels and immune cell infiltration in the HNSC tumor microenvironment. Furthermore, immunomodulatory and chemokine profiling were performed using the TISIDB platform to analyse the correlation between CCR5 expression levels and immunomodulation in HNSC patients. RESULTS: We found that CCR5 expression in HNSC tumor tissues was significantly upregulated than in normal tissues. In HNSC, patients with high CCR5 expression levels had worse overall survival (OS, HR = 0.59, p = 0.00015) and worse recurrence-free survival (RFS, HR = 3.27, p = 0.00098). Upregulation of CCR5 expression is closely associated with immunomodulators, chemokines, and infiltrating levels of CD4+ T cells, neutrophils, macrophages, and myeloid dendritic cells. Furthermore, upregulated CCR5 was significantly associated with different immune markers in the immune cell subsets of HNSC. CONCLUSIONS: High expression of CCR5 plays an important prognostic role in HNSC patients and may serve as a prognostic biomarker correlated with immune infiltration, and further studies are still needed to investigate therapeutic targeting HNSC patients in the future.

Cellular Electrical Impedance as a Method to Decipher CCR7 Signalling and Biased Agonism.

The human C-C chemokine receptor type 7 (CCR7) has two endogenous ligands, C-C chemokine ligand 19 (CCL19) and CCL21, displaying biased agonism reflected by a pronounced difference in the level of ß-arrestin recruitment. Detecting this preferential activation generally requires the use of separate, pathway-specific label-based assays. In this study, we evaluated an alternative methodology to study CCR7 signalling. Cellular electrical impedance (CEI) is a label-free technology which yields a readout that reflects an integrated cellular response to ligand stimulation. CCR7-expressing HEK293 cells were stimulated with CCL19 or CCL21, which induced distinct impedance profiles with an apparent bias during the desensitisation phase of the response. This discrepancy was mainly modulated by differential ß-arrestin recruitment, which shaped the impedance profile but did not seem to contribute to it directly. Pathway deconvolution revealed that Gαi-mediated signalling contributed most to the impedance profile, but Gαq- and Gα12/13-mediated pathways were also involved. To corroborate these results, label-based pathway-specific assays were performed. While CCL19 more potently induced ß-arrestin2 recruitment and receptor internalisation than CCL21, both chemokines showed a similar level of Gαi protein activation. Altogether, these findings indicate that CEI is a powerful method to analyse receptor signalling and biased agonism.

Phosphoproteomic characterization of the signaling network resulting from activation of the chemokine receptor CCR2.

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein-coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function.

Effects of a CCR2 antagonist on macrophages and Toll-like receptor 9 expression in a mouse model of diabetic nephropathy.

The pathogenesis of diabetic nephropathy (DN) is related to macrophage (Mφ) recruitment to the kidneys, tumor necrosis factor-α (TNF-α) production, and oxidative stress. Toll-like receptor 9 (TLR9) activation is reportedly involved in systemic inflammation, and it exacerbates this condition in metabolic syndrome. Therefore, we hypothesized that TLR9 plays a role in the pathogenesis of DN. Two subsets of kidney Mφs in DN model (db/db) mice were analyzed using flow cytometry to evaluate their distribution and TLR9 expression and function. Mice were administered the CCR2 antagonist INCB3344 for 8 wk; changes in Mφ distribution and function and its therapeutic effects on DN pathology were examined. Bone marrow-derived CD11bhigh (BM-Mφ) and tissue-resident CD11blow Mφs (Res-Mφ) were identified in the mouse kidneys. As DN progressed, the BM-Mφ number, TLR9 expression, and TNF-α production increased significantly. In Res-Mφs, reactive oxygen species (ROS) production and phagocytic activity were enhanced. INCB3344 decreased albuminuria, serum creatinine level, BM-Mφ abundance, TLR9 expression, and TNF-α production by BM-Mφs and ROS production by Res-Mφs. Both increased activation of BM-Mφ via TLR9 and TNF-α production and increased ROS production by Res-Mφs were involved in DN progression. Thus, inactivating Mφs and their TLR9 expression by INCB3344 is a potential therapeutic strategy for DN.NEW & NOTEWORTHY We classified kidney macrophages (Mφs) into bone marrow-derived Mφs (BM-Mφs) expressing high CD11b and tissue-specific resident Mφ (Res-Mφs) expressing low CD11b. In diabetic nephropathy (DN) model mice, Toll-like receptor 9 (TLR9) expression and TNF-α production via TLR9 activation in BM-Mφs and ROS production in Res-Mφs were enhanced. Furthermore, CCR2 antagonist suppressed the kidney infiltration of BM-Mφs and their function and the ROS production by Res-Mφs, with concomitant TLR9 suppression. Our study presents a new therapeutic strategy for DN.

Recombinant CCL17-dependent CCR4 activation alleviates neuroinflammation and neuronal apoptosis through the PI3K/AKT/Foxo1 signaling pathway after ICH in mice.

BACKGROUND: Intracerebral hemorrhage (ICH), a devastating subtype of stroke, is associated with high mortality and morbidity. Neuroinflammation is an important factor leading to ICH-induced neurological injuries. C-C Chemokine Receptor 4 (CCR4) plays an important role in enhancing hematoma clearance after ICH. However, it is unclear whether CCR4 activation can ameliorate neuroinflammation and apoptosis of neurons following ICH. The aim of the present study was to examine the effects of recombinant CCL17 (rCCL17)-dependent CCR4 activation on neuroinflammation and neuronal apoptosis in an intrastriatal autologous blood injection ICH model, and to determine whether the PI3K/AKT/Foxo1 signaling pathway was involved. METHODS: Two hundred twenty-six adult (8-week-old) male CD1 mice were randomly assigned to sham and ICH surgery groups. An intrastriatal autologous blood injection ICH model was used. rCCL17, a CCR4 ligand, was delivered by intranasal administration at 1 h, 3 h, and 6 h post-ICH. CCL17 antibody was administrated by intraventricular injection at 1 h post-ICH. C021, a specific inhibitor of CCR4 and GDC0068, an AKT inhibitor were delivered intraperitoneally 1 h prior to ICH induction. Brain edema, neurobehavioral assessments, western blotting, Fluoro-Jade C staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunofluorescence staining were conducted. RESULTS: Endogenous expression of CCL17 and CCR4 were increased following ICH, peaking at 5 days post-induction. CCR4 was found to co-localize with microglia, neurons, and astrocytes. rCCL17 treatment decreased brain water content, attenuated short- and long-term neurological deficits, deceased activation of microglia/macrophages and infiltration of neutrophils, and inhibited neuronal apoptosis in the perihematomal region post-ICH. Moreover, rCCL17 treatment post-ICH significantly increased the expression of CCR4, PI3K, phosphorylated AKT, and Bcl-2, while Foxo1, IL-1ß, TNF-α, and Bax expression were decreased. The neuroprotective effects of rCCL17 were reversed with the administration of C021 or GDC0068. CONCLUSIONS: rCCL17-dependent CCR4 activation ameliorated neurological deficits, reduced brain edema, and ameliorated neuroinflammation and neuronal apoptosis, at least in part, through the PI3K/AKT/Foxo1 signaling pathway after ICH. Thus, activation of CCR4 may provide a promising therapeutic approach for the early management of ICH.

Antifibrogenic effects of C-C chemokine receptor type 2 antagonist in a bleomycin-induced scleroderma model.

There have been several studies on the role of the monocyte chemotactic protein-1/C-C chemokine receptor type 2 (CCR2) signalling pathway in fibrotic diseases, which identified the blockade of this pathway as a potential therapeutic target for treating fibrosis. We examined the efficacy of CCR2 antagonist (RS-504393) in a mouse model of scleroderma induced by bleomycin. RS-504393 was administered via intradermal injection 6 hours prior to bleomycin injection, in the same sites. Histopathological examination showed that RS-504393 treatment suppressed dermal fibrosis and decreased dermal thickness. The numbers of mast cells and myofibroblasts in the skin of RS-504393-treated mice were significantly lower compared with those in PBS-treated mice. Moreover, the amount of collagen in the skin of RS-504393-treated mice was significantly lower compared with that in the PBS-treated mice. Additionally, mRNA levels of TGF-ß1 and collagen I alpha 1 in sclerotic skin were significantly decreased by RS-504393, and semiquantitative histopathological scoring of the lungs showed inhibition of fibrosis in RS-504393-treated mice. The amount of collagen in the lung of the RS-504393-treated mice was lower compared with that in the PBS-treated mice. These data suggest that CCR2 antagonist RS-504393 may be a therapeutic agent for human scleroderma.

Crimean-Congo hemorrhagic fever virus nucleocapsid protein harbors distinct RNA-binding sites in the stalk and head domains.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus that causes severe hemorrhagic fever with a mortality rate of up to 30% in certain outbreaks worldwide. The virus has wide endemic distribution. There is no effective antiviral therapeutic or FDA approved vaccine for this zoonotic viral illness. The multifunctional CCHFV nucleocapsid protein (N protein) plays a crucial role in the establishment of viral infection and is an important structural component of the virion. Here we show that CCHFV N protein has a distant RNA-binding site in the stalk domain that specifically recognizes the vRNA panhandle, formed by the base pairing of complementary nucleotides at the 5' and 3' termini of the vRNA genome. Using multiple approaches, including filter-bonding analysis, GFP reporter assay, and biolayer interferometry we observed an N protein-panhandle interaction both in vitro and in vivo The purified WT CCHFV N protein and the stalk domain also recognize the vRNA panhandle of hazara virus, another Nairovirus in the family Bunyaviridae, demonstrating the genus-specific nature of N protein-panhandle interaction. Another RNA-binding site was identified at the head domain of CCHFV N protein that nonspecifically recognizes the single strand RNA (ssRNA) of viral or nonviral origin. Expression of CCHFV N protein stalk domain active in panhandle binding, dramatically inhibited the hazara virus replication in cell culture, illustrating the role of N protein-panhandle interaction in Nairovirus replication. Our findings reveal the stalk domain of N protein as a potential target in therapeutic interventions to manage CCHFV disease.

Role of Maraviroc in minimizing the risk of graft rejection in HIV-infected kidney transplant recipients.

BACKGROUND: Kidney transplantation in HIV-infected patients is characterized by a concerning high rate of allograft rejections. The etiological mechanisms leading to this increased immunoreactivity are still unknown. Maraviroc is a new antiretroviral agent that has been associated with immunomodulatory proprieties; therefore, its use may be a promising strategy to minimize the rate of rejections in HIV-infected kidney transplant (KT) recipients. METHODS: We conducted a retrospective study in our cohort of HIV-KT recipients with the aim to explore the effects of maraviroc in reducing the risk of graft rejection. RESULTS: Twenty-two HIV-infected KT recipients predominantly of Caucasian origin (86%) and with a median age of 49 (IQR, 51.9-42.2) years were evaluated. Ten HIV-infected patients were treated with maraviroc and 12 with a maraviroc-free antiretroviral regimen. After a median follow-up of 3.01 years, half of the maraviroc-treated patients (n = 5) developed seven episodes of graft rejection, most of them were T cell-mediated rejections (85.7%). Five episodes were recorded in the maraviroc-free group. The difference in the rate of graft rejections was not statistically significant (P = .23). CONCLUSIONS: The administration of maraviroc was ineffective in preventing graft rejections in our cohort of patients.

Potential roles of the CCL17-CCR4 axis in immunopathogenesis of oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is a T cell-mediated chronic inflammatory disease. C-C chemokine receptor type 4 (CCR4) and its cognate C-C motif chemokine ligand 17 (CCL17) play a key role in T-cell activation and trafficking, but their implication in OLP pathogenesis has not been explored. Our study was designed to analyze the expression and function of the CCL17-CCR4 axis in OLP. METHODS: The mRNA expression levels of CCL17 and CCR4 in the circulating T cells of OLP subjects were examined by quantitative real-time PCR. The protein levels of CCL17 and CCR4 in the peripheral blood of OLP subjects were detected by enzyme-linked immunosorbent assay (ELISA) and Simple Western assay, respectively. The functional relevance of increased expression of CCL17 and CCR4 in OLP was demonstrated in proliferation, apoptosis, and migration assays. RESULTS: The mRNA and protein expression levels of CCL17 and CCR4 in the peripheral blood of patients with OLP were significantly upregulated compared with those of controls. CCL17 induced the migration of OLP T cells. In addition, blocking CCR4 with a small molecule CCR4 antagonist not only inhibited the proliferation and migration of OLP T cells but also promoted the apoptosis of OLP T cells. CONCLUSION: Our findings indicate that the CCL17-CCR4 axis might be responsible for the inflammatory infiltration of T cells in OLP.

CCR5 and CCR5Δ32 in bacterial and parasitic infections: Thinking chemokine receptors outside the HIV box.

The CCR5 molecule was reported in 1996 as the main HIV-1 co-receptor. In that same year, the CCR5Δ32 genetic variant was described as a strong protective factor against HIV-1 infection. These findings led to extensive research regarding the CCR5, culminating in critical scientific advances, such as the development of CCR5 inhibitors for the treatment of HIV infection. Recently, the research landscape surrounding CCR5 has begun to change. Different research groups have realized that, since CCR5 has such important effects in the chemokine system, it could also affect other different physiological systems. Therefore, the effect of reduced CCR5 expression due to the presence of the CCR5Δ32 variant began to be further studied. Several studies have investigated the role of CCR5 and the impacts of CCR5Δ32 on autoimmune and inflammatory diseases, various types of cancer, and viral diseases. However, the role of CCR5 in diseases caused by bacteria and parasites is still poorly understood. Therefore, the aim of this article is to review the role of CCR5 and the effects of CCR5Δ32 on bacterial (brucellosis, osteomyelitis, pneumonia, tuberculosis and infection by Chlamydia trachomatis) and parasitic infections (toxoplasmosis, leishmaniasis, Chagas disease and schistosomiasis). Basic information about each of these infections was also addressed. The neglected role of CCR5 in fungal disease and emerging studies regarding the action of CCR5 on regulatory T cells are briefly covered in this review. Considering the "renaissance of CCR5 research," this article is useful for updating researchers who develop studies involving CCR5 and CCR5Δ32 in different infectious diseases.

Rapid and low-cost multiplex synthesis of chemokine analogs.

Peptides represent a promising source of new medicines, but improved technologies are needed to facilitate discovery and optimization campaigns. In particular, longer peptides with multiple disulfide bridges are challenging to produce, and producing large numbers of structurally related variants is dissuasively costly and time-consuming. The principal cost and time drivers are the multiple column chromatography purification steps that are used during the multistep chemical synthesis procedure, which involves both ligation and oxidative refolding steps. In this study, we developed a method for multiplex parallel synthesis of complex peptide analogs in which the structurally variant region of the molecule is produced as a small peptide on a 384-well synthesizer with subsequent ligation to the longer, structurally invariant region and oxidative refolding carried out in-well without any column purification steps. To test the method, we used a panel of 96 analogs of the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 (69 residues, two disulfide bridges), which had been synthesized using standard approaches and characterized pharmacologically in an earlier study. Although, as expected, the multiplex method generated chemokine analogs of lower purity than those produced in the original study, it was nonetheless possible to closely match the pharmacological attributes (anti-HIV potency, capacity to elicit G protein signaling, and capacity to elicit intracellular receptor sequestration) of each chemokine analog to reference data from the earlier study. This rapid, low-cost approach has the potential to support discovery and optimization campaigns based on analogs of other chemokines as well as those of other complex peptide and small protein targets of a similar size.

Regulation of C-C chemokine receptor 5 (CCR5) stability by Lys197 and by transmembrane protein aptamers that target it for lysosomal degradation.

C-C motif chemokine receptor 5 (CCR5) is a cell surface-associated, immune-regulatory G protein-coupled receptor (GCPR) with seven transmembrane helices. We previously reported the isolation and initial characterization of short artificial transmembrane protein aptamers, named "traptamers," that specifically down-regulate CCR5 expression and inhibit infection of human T cells by HIV strains that use CCR5 as a co-receptor. Here, we investigated the mechanism of traptamer-mediated CCR5 down-regulation and show that most of the traptamers (designated class 1 traptamers) form a stable complex with CCR5 and target it for lysosome-mediated degradation. The ability of these traptamers to down-regulate CCR5 depended on Lys197 in the fifth transmembrane helix of CCR5. In the absence of traptamers, substitution of Lys197 to an uncharged amino acid increased CCR5 stability, and introduction of a lysine at the homologous position in CCR2b, a related chemokine receptor, decreased CCR2b levels. The prototypic class 2 traptamer BY6M4 also formed a complex with CCR5, but CCR5 down-regulation caused by class 2 traptamers did not depend on the lysosome or on Lys197 These results demonstrate that traptamers use diverse mechanisms to down-regulate CCR5 and identify a specific amino acid that plays a central role in controlling chemokine receptor stability. Further studies of these traptamers are likely to provide new insights into CCR5 metabolism and biology and may suggest new therapeutic approaches to modulate the levels of CCR5 and other GPCRs.