Thermal stability of the human immunodeficiency virus type 1 (HIV-1) receptors, CD4 and CXCR4, reconstituted in proteoliposomes

Background

The entry of human immunodeficiency virus (HIV-1) into host cells involves the interaction of the viral exterior envelope glycoprotein, gp120, and receptors on the target cell. The HIV-1 receptors are CD4 and one of two chemokine receptors, CCR5 or CXCR4.

Methodology/Principal Findings

We created proteoliposomes that contain CD4, the primary HIV-1 receptor, and one of the coreceptors, CXCR4. Antibodies against CD4 and CXCR4 specifically bound the proteoliposomes. CXCL12, the natural ligand for CXCR4, and the small-molecule CXCR4 antagonist, AMD3100, bound the proteoliposomes with affinities close to those associated with the binding of these molecules to cells expressing CXCR4 and CD4. The HIV-1 gp120 exterior envelope glycoprotein bound tightly to proteoliposomes expressing only CD4 and, in the presence of soluble CD4, bound weakly to proteoliposomes expressing only CXCR4. The thermal stability of CD4 and CXCR4 inserted into liposomes was examined. Thermal denaturation of CXCR4 followed second-order kinetics, with an activation energy (E_a) of 269 kJ/mol (64.3 kcal/mol) and an inactivation temperature (T_i) of 56°C. Thermal inactivation of CD4 exhibited a reaction order of 1.3, an E_a of 278 kJ/mol (66.5 kcal/mol), and a T_i of 52.2°C. The second-order denaturation kinetics of CXCR4 is unusual among G protein-coupled receptors, and may result from dimeric interactions between CXCR4 molecules.

Conclusions/Significance

Our studies with proteoliposomes containing the native HIV-1 receptors allowed an examination of the binding of biologically important ligands and revealed the higher-order denaturation kinetics of these receptors. CD4/CXCR4-proteoliposomes may be useful for the study of virus-target cell interactions and for the identification of inhibitors.     

Myocardial Chemokine Expression and Intensity of Myocarditis in Chagas Cardiomyopathy Are Controlled by Polymorphisms in CXCL9 and CXCL10

Background

Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium.

Methods and Results

Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2–6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes.

Conclusions

Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.     

CXCR7 Functions as a Scavenger for CXCL12 and CXCL11

Background

CXCR7 (RDC1), the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues.

Methodology/Principal Findings

We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium.

Conclusions/Significance

The finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.     

The role of fibrocytes in sickle cell lung disease

Background

Interstitial lung disease is a frequent complication in sickle cell disease and is characterized by vascular remodeling and interstitial fibrosis. Bone marrow-derived fibrocytes have been shown to contribute to the pathogenesis of other interstitial lung diseases. The goal of this study was to define the contribution of fibrocytes to the pathogenesis of sickle cell lung disease.

Methodology/Principal Findings

Fibrocytes were quantified and characterized in subjects with sickle cell disease or healthy controls, and in a model of sickle cell disease, the NY1DD mouse. The role of the chemokine ligand CXCL12 in trafficking of fibrocytes and phenotype of lung disease was examined in the animal model. We found elevated concentration of activated fibrocytes in the peripheral blood of subjects with sickle cell disease, which increased further during vaso-occlusive crises. There was a similar elevations in the numbers and activation phenotype of fibrocytes in the bone marrow, blood, and lungs of the NY1DD mouse, both at baseline and under conditions of hypoxia/re-oxygenation. In both subjects with sickle cell disease and the mouse model, fibrocytes expressed a hierarchy of chemokine receptors, with CXCR4 expressed on most fibrocytes, and CCR2 and CCR7 expressed on a smaller subset of cells. Depletion of the CXCR4 ligand, CXCL12, in the mouse model resulted in a marked reduction of fibrocyte trafficking into the lungs, reduced lung collagen content and improved lung compliance and histology.

Conclusions

These data support the notion that activated fibrocytes play a significant role in the pathogenesis of sickle cell lung disease.     

A Novel Mechanism of Soluble HLA-G Mediated Immune Modulation: Downregulation of T Cell Chemokine Receptor Expression and Impairment of Chemotaxis

Background

In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations.

Methodology/Principal Findings

T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4⁺ T cells, ii) CXCR3 in CD8⁺ T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vδ2γ9 T cells, and upregulated CXCR4 expression in TCR Vδ2γ9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4⁺ T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8⁺ T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vδ2γ9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T_FH) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T_FH and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T_FH cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, β-arrestin and SHP2 was modulated by sHLA-G treatment.

Conclusions/Significance

Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions.     

Cyclophosphamide-Induced Cystitis Increases Bladder CXCR4 Expression and CXCR4-Macrophage Migration Inhibitory Factor Association

Background

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in cystitis and a non-cognate ligand of the chemokine receptor CXCR4 in vitro. We studied whether CXCR4-MIF associations occur in rat bladder and the effect of experimental cystitis.

Methods and Findings

Twenty male rats received saline or cyclophosphamide (40 mg/kg; i.p.; every 3^rd day) to induce persistent cystitis. After eight days, urine was collected and bladders excised under anesthesia. Bladder CXCR4 and CXCR4-MIF co-localization were examined with immunhistochemistry. ELISA determined MIF and stromal derived factor-1 (SDF-1; cognate ligand for CXCR4) levels. Bladder CXCR4 expression (real-time RTC-PCR) and protein levels (Western blotting) were examined. Co-immunoprecipitations studied MIF-CXCR4 associations.Urothelial basal and intermediate (but not superficial) cells in saline-treated rats contained CXCR4, co-localized with MIF. Cyclophosphamide treatment caused: 1) significant redistribution of CXCR4 immunostaining to all urothelial layers (especially apical surface of superficial cells) and increased bladder CXCR4 expression; 2) increased urine MIF with decreased bladder MIF; 3) increased bladder SDF-1; 4) increased CXCR4-MIF associations.

Conclusions

These data demonstrate CXCR4-MIF associations occur in vivo in rat bladder and increase in experimental cystitis. Thus, CXCR4 represents an alternative pathway for MIF-mediated signal transduction during bladder inflammation. In the bladder, MIF may compete with SDF-1 (cognate ligand) to activate signal transduction mediated by CXCR4.     

CX3CL1/CX3CR1 and CCL2/CCR2 Chemokine/Chemokine Receptor Complex in Patients with AMD

Purpose

The chemokine receptors CX3CR1 and CCR2 have been implicated in the development of age-related macular degeneration (AMD). The evidence is mainly derived from experimental cell studies and murine models of AMD. The purpose of this study was to investigate the association between expression of CX3CR1 and CCR2 on different leukocyte subsets and AMD. Furthermore we measured the plasma levels of ligands CX3CL1 and CCL2.

Methods

Patients attending our department were asked to participate in the study. The diagnosis of AMD was based on clinical examination and multimodal imaging techniques. Chemokine plasma level and chemokine receptor expression were measured by flow-cytometry.

Results

A total of 150 participants were included. We found a significantly lower expression of CX3CR1 on CD8⁺ T cells in the neovascular AMD group compared to the control group (p = 0.04). We found a significant positive correlation between CCR2 and CX3CR1 expression on CD8⁺ cells (r = 0.727, p = 0.0001). We found no difference in plasma levels of CX3CL1 and CCL2 among the groups.

Conclusions

Our results show a down regulation of CX3CR1 on CD8⁺ cells; this correlated to a low expression of CCR2 on CD8⁺ cells. Further studies are needed to elucidate the possible role of this cell type in AMD development.     

Bioluminescence resonance energy transfer reveals ligand-induced conformational changes in CXCR4 homo- and heterodimers

Homo- and heterodimerization have emerged as prominent features of G-protein-coupled receptors with possible impact on the regulation of their activity. Using a sensitive bioluminescence resonance energy transfer system, we investigated the formation of CXCR4 and CCR2 chemokine receptor dimers. We found that both receptors exist as constitutive homo- and heterodimers and that ligands induce conformational changes within the pre-formed dimers without promoting receptor dimer formation or disassembly. Ligands with different intrinsic efficacies yielded distinct bioluminescence resonance energy transfer modulations, indicating the stabilization of distinct receptor conformations. We also found that peptides derived from the transmembrane domains of CXCR4 inhibited activation of this receptor by blocking the ligand-induced conformational transitions of the dimer. Taken together, our data support a model in which chemokine receptor homo- and heterodimers form spontaneously and respond to ligand binding as units that undergo conformational changes involving both protomers even when only one of the two ligand binding sites is occupied.     

Lack of chemokine signaling through CXCR5 causes increased mortality, ventricular dilatation and deranged matrix during cardiac pressure overload

Rationale

Inflammatory mechanisms have been suggested to play a role in the development of heart failure (HF), but a role for chemokines is largely unknown. Based on their role in inflammation and matrix remodeling in other tissues, we hypothesized that CXCL13 and CXCR5 could be involved in cardiac remodeling during HF.

Objective

We sought to analyze the role of the chemokine CXCL13 and its receptor CXCR5 in cardiac pathophysiology leading to HF.

Methods and Results

Mice harboring a systemic knockout of the CXCR5 (CXCR5^−/−) displayed increased mortality during a follow-up of 80 days after aortic banding (AB). Following three weeks of AB, CXCR5^−/− developed significant left ventricular (LV) dilatation compared to wild type (WT) mice. Microarray analysis revealed altered expression of several small leucine-rich proteoglycans (SLRPs) that bind to collagen and modulate fibril assembly. Protein levels of fibromodulin, decorin and lumican (all SLRPs) were significantly reduced in AB CXCR5^−/− compared to AB WT mice. Electron microscopy revealed loosely packed extracellular matrix with individual collagen fibers and small networks of proteoglycans in AB CXCR5^−/− mice. Addition of CXCL13 to cultured cardiac fibroblasts enhanced the expression of SLRPs. In patients with HF, we observed increased myocardial levels of CXCR5 and SLRPs, which was reversed following LV assist device treatment.

Conclusions

Lack of CXCR5 leads to LV dilatation and increased mortality during pressure overload, possibly via lack of an increase in SLRPs. This study demonstrates a critical role of the chemokine CXCL13 and CXCR5 in survival and maintaining of cardiac structure upon pressure overload, by regulating proteoglycans essential for correct collagen assembly.     

Epigenetics underpinning the regulation of the CXC (ELR+) chemokines in non-small cell lung cancer

Background

Angiogenesis may play a role in the pathogenesis of Non-Small Cell Lung cancer (NSCLC). The CXC (ELR⁺) chemokine family are powerful promoters of the angiogenic response.

Methods

The expression of the CXC (ELR⁺) family members (CXCL1-3/GROα-γ, CXCL8/IL-8, CXCR1/2) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of these chemokines was examined in normal bronchial epithelial and NSCLC cell lines.

Results

Overall, expression of the chemokine ligands (CXCL1, 2, 8) and their receptors (CXCR1/2) were down regulated in tumour samples compared with normal, with the exception of CXCL3. CXCL8 and CXCR1/2 were found to be epigenetically regulated by histone post-translational modifications. Recombinant CXCL8 did not stimulate cell growth in either a normal bronchial epithelial or a squamous carcinoma cell line (SKMES-1). However, an increase was observed at 72 hours post treatment in an adenocarcinoma cell line.

Conclusions

CXC (ELR⁺) chemokines are dysregulated in NSCLC. The balance of these chemokines may be critical in the tumour microenvironment and requires further elucidation. It remains to be seen if epigenetic targeting of these pathways is a viable therapeutic option in lung cancer treatment.     

IFN-γ-inducible protein of 10 kDa upregulates the effector functions of eosinophils through β2 integrin and CXCR3

Background

Eosinophils play an important role in the pathogenesis of bronchial asthma and its exacerbation. Recent reports suggest the involvement of IFN-γ-inducible protein of 10 kDa (IP-10) in virus-induced asthma exacerbation. The objective of this study was to examine whether CXCR3 ligands including IP-10 modify the effector functions of eosinophils.

Methods

Eosinophils isolated from the blood of healthy donors were stimulated with CXCR3 ligands and their adhesion to rh-ICAM-1 was then measured using eosinophil peroxidase assays. The generation of eosinophil superoxide anion (O₂^-) was examined based on the superoxide dismutase-inhibitable reduction of cytochrome C. Eosinophil-derived neurotoxin (EDN) release was evaluated to determine whether CXCR3 ligands induced eosinophil degranulation. Cytokine and chemokine production by eosinophils was examined using a Bio-plex assay.

Results

Eosinophil adhesion to ICAM-1 was significantly enhanced by IP-10, which also significantly induced eosinophil O₂^- generation in the presence of ICAM-1. Both the enhanced adhesion and O₂^- generation were inhibited by an anti-β₂ integrin mAb or an anti-CXCR3 mAb. Other CXCR3 ligands, such as monokine induced by IFN-γ (Mig) and IFN-inducible T cell α chemoattractant (I-TAC), also induced eosinophil adhesion and O₂^- generation in the presence of ICAM-1. IP-10, but not Mig or I-TAC, increased the release of EDN. IP-10 increased the production of a number of cytokines and chemokines by eosinophils.

Conclusions

These findings suggest that CXCR3 ligands such as IP-10 can directly upregulate the effector functions of eosinophils. These effects might be involved in the activation and infiltration of eosinophils in the airway of asthma, especially in virus-induced asthma exacerbation.     

Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice

Background

Monocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents.

Methodology/Principal Findings

We created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6C^hi/CCR2^hi monocytes. Surprisingly, neutrophils, not Ly6C^lo monocytes, largely replaced Ly6C^hi cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia.

Conclusion/Significance

These results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations.     

CCR5 Haplotypes Influence HCV Serostatus in Caucasian Intravenous Drug Users

Background

Up to 90% HIV-1 positive intravenous drug users (IDUs) are co-infected with HCV. Although best recognized for its function as a major co-receptor for cell entry of HIV, CC chemokine receptor 5 (CCR5) has also been implicated in the pathogenesis of HCV infection. Here, we investigated whether CCR5 haplotypes influence HIV-1 and HCV seropositivity among 373 Caucasian IDUs from Estonia.

Methods

Of these IDUs, 56% and 44% were HIV and HCV seropositive, respectively, and 47% were coinfected. 500 blood donors seronegative for HIV and HCV were also evaluated. CCR5 haplotypes (HHA to HHG*2) were derived after genotyping nine CCR2–CCR5 polymorphisms. The association between CCR5 haplotypes with HIV and/or HCV seropositivity was determined using logistic regression analysis. Co-variates included in the models were length of intravenous drug use, HBV serostatus and copy number of CCL3L1, the gene encoding the most potent HIV-suppressive chemokine and ligand for CCR5.

Results

Compared to IDUs seronegative for both HCV and HIV (HCV−/HIV-), IDUs who were HCV+/HIV- and HCV+/HIV+were 92% and 82%, respectively, less likely to possess the CCR5-HHG*1 haplotype, after controlling for co-variates (P_adjusted = 1.89×10⁻⁴ and 0.003, respectively). This association was mostly due to subjects bearing the CCR5 HHE and HHG*1 haplotype pairs. Approximately 25% and<10% of HCV−/HIV- IDUs and HCV−/HIV- blood donors, respectively, possessed the HHE/HHG*1 genotype.

Conclusions

Our findings suggest that HHG*1-bearing CCR5 genotypes influence HCV seropositivity in a group of Caucasian IDUs.     

Abrogation of CC chemokine receptor 9 ameliorates collagen-induced arthritis of mice

Introduction

Biological drugs are effective in patients with rheumatoid arthritis (RA), but increase severe infections. The CC chemokine receptor (CCR) 9 antagonist was effective for Crohn’s disease without critical adverse effects including infections in clinical trials. The present study was carried out to explore the pathogenic roles of chemokine (C-C motif) ligand (CCL) 25 and its receptor, CCR9, in autoimmune arthritis and to study if the CCR9 antagonist could be a new treatment for RA.

Methods

CCL25 and CCR9 expression was examined with immunohistochemistry and Western blotting. Concentration of interleukin (IL)-6, matrix metalloproteinase (MMP)-3 and tumor necrosis factor (TNF)-α was measured with enzyme-linked immunosorbent assays. Effects of abrogating CCR9 on collagen-induced arthritis (CIA) was evaluated using CCR9-deficient mice or the CCR9 antagonist, CCX8037. Fluorescence labeled-CD11b⁺ splenocytes from CIA mice were transferred to recipient CIA mice and those infiltrating into the synovial tissues of the recipient mice were counted.

Results

CCL25 and CCR9 proteins were found in the RA synovial tissues. CCR9 was expressed on macrophages, fibroblast-like synoviocytes (FLS) and dendritic cells in the synovial tissues. Stimulation with CCL25 increased IL-6 and MMP-3 production from RA FLS, and IL-6 and TNF-α production from peripheral blood monocytes. CIA was suppressed in CCR9-deficient mice. CCX8037 also inhibited CIA and the migration of transferred CD11b⁺ splenocytes into the synovial tissues.

Conclusions

The interaction between CCL25 and CCR9 may play important roles in cell infiltration into the RA synovial tissues and inflammatory mediator production. Blocking CCL25 or CCR9 may represent a novel safe therapy for RA.     

Toward the Discovery of Vaccine Adjuvants: Coupling In Silico Screening and In Vitro Analysis of Antagonist Binding to Human and Mouse CCR4 Receptors

Background

Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses.

Methodology

Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4⁺ Tregs.

Significance

Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.     

The CXCR3(+)CD56Bright phenotype characterizes a distinct NK cell subset with anti-fibrotic potential that shows dys-regulated activity in hepatitis C

Background

In mouse models, natural killer (NK) cells have been shown to exert anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Chemokines and chemokine receptors critically modulate hepatic recruitment of NK cells. In hepatitis C, the chemokine receptor CXCR3 and its ligands have been shown to be associated with stage of fibrosis suggesting a role of these chemokines in HCV associated liver damage by yet incompletely understood mechanisms. Here, we analyzed phenotype and function of CXCR3 expressing NK cells in chronic hepatitis C.

Methods

Circulating NK cells from HCV-infected patients (n = 57) and healthy controls (n = 27) were analyzed with respect to CXCR3 and co-expression of different maturation markers. Degranulation and interferon-γ secretion of CXCR3(+) and CXCR3(−) NK cell subsets were studied after co-incubation with primary human hepatic stellate cells (HSC). In addition, intra-hepatic frequency of CXCR3(+) NK cells was correlated with stage of liver fibrosis (n = 15).

Results

We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. In healthy controls CXCR3(+)CD56Bright NK cells displayed strongest activity against HSC. Chronic hepatitis C was associated with a significantly increased frequency of CXCR3(+)CD56Bright NK cells which showed impaired degranulation and impaired IFN-γ secretion in response to HSC. Of note, we observed intra-hepatic accumulation of this NK cell subset in advanced stages of liver fibrosis.

Conclusion

We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. Intra-hepatic accumulation of the functionally impaired CXCR3(+)CD56Bright NK cell subset might be involved in HCV-induced liver fibrosis.     

The Oral Commensal Streptococcus mitis Shows a Mixed Memory Th Cell Signature That Is Similar to and Cross-Reactive with Streptococcus pneumoniae

Background

Carriage of and infection with Streptococcus pneumoniae is known to predominantly induce T helper 17 (Th17) responses in humans, but the types of Th cells showing reactivity towards commensal streptococci with low pathogenic potential, such as the oral commensals S. mitis and S. salivarius, remain uncharacterized.

Methods

Memory CD4⁺ T helper (Th) cell subsets were isolated from healthy human blood donors according to differential expression of chemokine receptors, expanded in vitro using polyclonal stimuli and characterized for reactivity against different streptococcal strains.

Results

Th cells responding to S. mitis, S. salivarius and S. pneumoniae were predominantly in a CCR6⁺CXCR3⁺ subset and produced IFN-γ, and in a CCR6⁺CCR4⁺ subset and produced IL-17 and IL-22. Frequencies of S. pneumoniae-reactive Th cells were higher than frequencies of S. mitis- and S. salivarius-specific Th cells. S. mitis and S. pneumoniae isogenic capsule knock-out mutants and a S. mitis mutant expressing the serotype 4 capsule of S. pneumoniae showed no different Th cell responses as compared to wild type strains. S. mitis-specific Th17 cells showed cross-reactivity with S. pneumoniae.

Conclusions

As Th17 cells partly control clearance of S. pneumoniae, cross-reactive Th17 cells that may be induced by commensal bacterial species may influence the immune response, independent of capsule expression.