Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7.

Chemokine receptors play important roles in the immune system and are linked to several human diseases. Targeting chemokine receptors have so far shown very little success owing to, to some extent, the promiscuity of the immune system and the high degree of biased signaling within it. CCR7 and its two endogenous ligands display biased signaling and here we investigate the differences between the two ligands, CCL21 and CCL19, with respect to their biased activation of CCR7. We use bystander bioluminescence resonance energy transfer (BRET) based signaling assays and Transwell migration assays to determine (A) how swapping of domains between the two ligands affect their signaling patterns and (B) how receptor mutagenesis impacts signaling. Using chimeric ligands we find that the chemokine core domains are central for determining signaling outcome as the lack of ß-arrestin-2 recruitment displayed by CCL21 is linked to its core domain and not N-terminus. Through a mutagenesis screen, we identify the extracellular domains of CCR7 to be important for both ligands and show that the two chemokines interact differentially with extracellular loop 2 (ECL-2). By using in silico modeling, we propose a link between ECL-2 interaction and CCR7 signal transduction. Our mutagenesis study also suggests a lysine in the top of TM3, K1303.26, to be important for G protein signaling, but not ß-arrestin-2 recruitment. Taken together, the bias in CCR7 between CCL19 and CCL21 relies on the chemokine core domains, where interactions with ECL-2 seem particularly important. Moreover, TM3 selectively regulates G protein signaling as found for other chemokine receptors.

Insulin Secretion Depends on Intra-islet Glucagon Signaling.

The intra-islet theory states that glucagon secretion is suppressed when insulin secretion is stimulated, but glucagon's role in intra-islet paracrine regulation is controversial. This study investigated intra-islet functions of glucagon in mice. We examined glucagon-induced insulin secretion using isolated perfused pancreata from wild-type, GLP-1 receptor (GLP-1R) knockout, diphtheria toxin-induced proglucagon knockdown, ß cell-specific glucagon receptor (Gcgr) knockout, and global Gcgr knockout (Gcgr-/-) mice. We found that glucagon stimulates insulin secretion through both Gcgr and GLP-1R. Moreover, loss of either Gcgr or GLP-1R does not change insulin responses, whereas combined blockage of both receptors significantly reduces insulin secretion. Active GLP-1 is identified in pancreatic perfusate from Gcgr-/- but not wild-type mice, suggesting that ß cell GLP-1R activation results predominantly from glucagon action. Our results suggest that combined activity of glucagon and GLP-1 receptors is essential for ß cell secretory responses, emphasizing a role for paracrine intra-islet glucagon actions to maintain appropriate insulin secretion.

Bile acids are important direct and indirect regulators of the secretion of appetite- and metabolism-regulating hormones from the gut and pancreas.

OBJECTIVE: Bile acids (BAs) facilitate fat absorption and may play a role in glucose and metabolism regulation, stimulating the secretion of gut hormones. The relative importance and mechanisms involved in BA-stimulated secretion of appetite and metabolism regulating hormones from the gut and pancreas is not well described and was the purpose of this study. METHODS: The effects of bile acids on the secretion of gut and pancreatic hormones was studied in rats and compared to the most well described nutritional secretagogue: glucose. The molecular mechanisms that underlie the secretion was studied by isolated perfused rat and mouse small intestine and pancreas preparations and supported by immunohistochemistry, expression analysis, and pharmacological studies. RESULTS: Bile acids robustly stimulate secretion of not only the incretin hormones, glucose-dependent insulinotropic peptide (GIP), and glucagon-like peptide-1 (GLP-1), but also glucagon and insulin in vivo, to levels comparable to those resulting from glucose stimulation. The mechanisms of GLP-1, neurotensin, and peptide YY (PYY) secretion was secondary to intestinal absorption and depended on activation of basolateral membrane Takeda G-protein receptor 5 (TGR5) receptors on the L-cells in the following order of potency: Lithocholic acid (LCA) >Deoxycholicacid (DCA)>Chenodeoxycholicacid (CDCA)> Cholic acid (CA). Thus BAs did not stimulate secretion of GLP-1 and PYY from perfused small intestine in TGR5 KO mice but stimulated robust responses in wild type littermates. TGR5 is not expressed on α-cells or ß-cells, and BAs had no direct effects on glucagon or insulin secretion from the perfused pancreas. CONCLUSION: BAs should be considered not only as fat emulsifiers but also as important regulators of appetite- and metabolism-regulating hormones by activation of basolateral intestinal TGR5.

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration.

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.

Truncation of CXCL12 by CD26 reduces its CXC chemokine receptor 4- and atypical chemokine receptor 3-dependent activity on endothelial cells and lymphocytes.

The chemokine CXCL12 or stromal cell-derived factor 1/SDF-1 attracts hematopoietic progenitor cells and mature leukocytes through the G protein-coupled CXC chemokine receptor 4 (CXCR4). In addition, it interacts with atypical chemokine receptor 3 (ACKR3 or CXCR7) and glycosaminoglycans. CXCL12 activity is regulated through posttranslational cleavage by CD26/dipeptidyl peptidase 4 that removes two NH2-terminal amino acids. CD26-truncated CXCL12 does not induce calcium signaling or chemotaxis of mononuclear cells. CXCL12(3-68) was chemically synthesized de novo for detailed biological characterization. Compared to unmodified CXCL12, CXCL12(3-68) was no longer able to signal through CXCR4 via inositol trisphosphate (IP3), Akt or extracellular signal-regulated kinases 1 and 2 (ERK1/2). Interestingly, the recruitment of ß-arrestin 2 to the cell membrane via CXCR4 by CXCL12(3-68) was abolished, whereas a weakened but significant ß-arrestin recruitment remained via ACKR3. CXCL12-induced endothelial cell migration and signal transduction was completely abrogated by CD26. Intact CXCL12 hardly induced lymphocyte migration upon intra-articular injection in mice. In contrast, oral treatment of mice with the CD26 inhibitor sitagliptin reduced CD26 activity and CXCL12 cleavage in blood plasma. The potential of CXCL12 to induce intra-articular lymphocyte infiltration was significantly increased in sitagliptin-treated mice and CXCL12(3-68) failed to induce migration under both CD26-inhibiting and non-inhibiting conditions. In conclusion, CD26-cleavage skews CXCL12 towards ß-arrestin dependent recruitment through ACKR3 and destroys the CXCR4-mediated lymphocyte chemoattractant properties of CXCL12 in vivo. Hence, pharmacological CD26-blockade in tissues may enhance CXCL12-induced inflammation.

Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28.

BACKGROUND: Some herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency. RESULTS: We investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection. CONCLUSIONS: We show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.

Natural nitration of CXCL12 reduces its signaling capacity and chemotactic activity in vitro and abrogates intra-articular lymphocyte recruitment in vivo.

The chemokine CXCL12/stromal cell-derived factor-1 is important for leukocyte migration to lymphoid organs and inflamed tissues and stimulates tumor development. In vitro, CXCL12 activity through CXCR4 is abolished by proteolytic processing. However, limited information is available on in vivo effects of posttranslationally modified CXCL12. Natural CXCL12 was purified from the coculture supernatant of stromal cells stimulated with leukocytes and inflammatory agents. In this conditioned medium, CXCL12 with a nitration on Tyr7, designated [3-NT7]CXCL12, was discovered via Edman degradation. CXCL12 and [3-NT7]CXCL12 were chemically synthesized to evaluate the biological effects of this modification. [3-NT7]CXCL12 recruited ß-arrestin 2 and phosphorylated the Akt kinase similar to CXCL12 in receptor-transfected cells. Also the affinity of CXCL12 and [3-NT7]CXCL12 for glycosaminoglycans, the G protein-coupled chemokine receptor CXCR4 and the atypical chemokine receptor ACKR3 were comparable. However, [3-NT7]CXCL12 showed a reduced ability to enhance intracellular calcium concentrations, to generate inositol triphosphate, to phosphorylate ERK1/2 and to induce monocyte and lymphocyte chemotaxis in vitro. Moreover, nitrated CXCL12 failed to induce in vivo extravasation of lymphocytes to the joint. In summary, nitration on Tyr7 under inflammatory conditions is a novel natural posttranslational regulatory mechanism of CXCL12 which may downregulate the CXCR4-mediated inflammatory and tumor-promoting activities of CXCL12.

Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo.

The use of receptor-ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX3CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX3CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX3CL1, CX3CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo.

Increased prevalence of HTLV-1 in patients with pulmonary tuberculosis coinfected with HIV, but not in HIV-negative patients with tuberculosis.

BACKGROUND: Few and inconclusive results have been presented regarding the influence of human T-lymphotropic virus 1 (HTLV-1) infection on the risk of acquiring tuberculosis (TB). METHODS: In 1994-1997, we performed a prospective study on hospitalized adult patients with pulmonary TB in Guinea-Bissau and compared the clinical outcome in HIV-2 and HIV-negative patients. We determined the prevalence of HTLV-1 in all patients screened and diagnosed with TB in that study and compared the infection rate with a serosurvey of HTLV-1 in a population sample from a community-based study conducted at the same time and in the same city. RESULTS: In the TB group, a total of 32 (11.4%) of 280 patients were positive for HTLV-1. This was significantly higher compared with the population-based group in which 74 (3.5%) of 2117 were HTLV-1 positive [crude odds ratio (OR) = 3.6; 95% confidence interval (CI) 2.2 to 5.6, P < 0.001]. However, in a logistic regression analysis controlling for age, gender, and HIV result, the difference was no longer significant (OR = 1.61; 95% CI 0.95 to 2.70, P = 0.074). In HIV-negative patients, no association was found between HTLV-1 and TB (OR = 1.18; 95% CI 0.48 to 2.89, P = 0.71), whereas a significant association was found in HIV-positive patients (OR = 2.41; 95% CI 1.26 to 4.61, P = 0.008). CONCLUSIONS: The immunosuppressive effect of HTLV-1 alone was not enough to increase the risk of TB in a highly endemic country, but HTLV-1 increased the risk of TB among HIV-infected individuals.

Mortality associated with HIV-1, HIV-2, and HTLV-I single and dual infections in a middle-aged and older population in Guinea-Bissau.

BACKGROUND: In Guinea-Bissau HIV-1, HIV-2, and HTLV-I are prevalent in the general population. The natural history of HIV/HTLV-I single and dual infections has not been fully elucidated in this population. Previous studies have shown that combinations of these infections are more common in older women than in men. The present study compares mortality associated with HIV-1, HIV-2, and HTLV-I single and dual infections in individuals over 35 years of age within an urban community-based cohort in Guinea-Bissau. RESULTS: A total of 2,839 and 1,075 individuals were included in the HIV and HTLV-I mortality analyses respectively. Compared with HIV-negative individuals, adjusted mortality rate ratios (MRRs) were 4.9 (95% confidence interval (CI): 2.3, 10.4) for HIV-1, 1.8 (95%CI: 1.5, 2.3) for HIV-2, and 5.9 (2.4, 14.3) for HIV-1/HIV-2 dual infections. MRR for HTLV-I-positive compared with HTLV-I-negative individuals was 1.7 (1.1, 2.7). Excluding all HIV-positive individuals from the analysis, the HTLV-I MRR was 2.3 (1.3, 3.8). The MRR of HTLV-I/HIV-2 dually infected individuals was 1.7 (0.7, 4.3), compared with HIV/HTLV-I-negative individuals. No statistically significant differences were found in retrovirus-associated mortality between men and women. CONCLUSION: HIV-1-associated excess mortality was low compared with community studies from other parts of Africa, presumably because this population was older and the introduction of HIV-1 into the community recent. HIV-2 and HTLV-I-associated mortality was 2-fold higher than the mortality in uninfected individuals. We found no significant differences between the mortality risk for HIV-2 and HTLV-I single infection, respectively, and HIV-2/HTLV-I dual infection. The higher prevalence of retroviral dual infections in older women is not explained by differential retrovirus-associated mortality for men and women.

Potent neutralizing serum immunoglobulin A (IgA) in human immunodeficiency virus type 2-exposed IgG-seronegative individuals.

The mechanisms behind the resistance to human immunodeficiency virus type 2 (HIV-2) infection are still not fully understood. In the present study, we explored the HIV-2-specific humoral serum immunoglobulin A (IgA) immune response in HIV-2-exposed IgG-seronegative (EGSN) individuals. Serum samples from heterosexual EGSN individuals and their known HIV-2-infected partners, as well as controls originating from Guinea-Bissau in Africa, were studied. Antibody reactivity to native and recombinant envelope glycoproteins was investigated, and the capacity of purified serum IgA to neutralize HIV-2(SBL6669) was tested. Our results showed that 16 of 25 EGSN samples exhibited reactivity against whole HIV-2 antigen, 6 of 25 samples reacted with recombinant gp36 (rgp36), and 3 of 25 samples were positive against HIV-2 rgp105; no reactivity to native HIV-2 gp125 was detected. Purified serum IgA antibodies from both EGSN and HIV-2-positive individuals, but not that from the negative controls, exhibited neutralization of HIV-2(SBL6669). The most potent neutralization activity was exhibited by IgA purified from EGSN compared to infected individuals' IgA. The antigenic pattern of the HIV-2-positive partners showed that all serum IgA samples were reactive to whole HIV-2 antigen, and 14 of 15 reacted with rgp36. For rgp105 and gp125, 5 of 15 and 4 of 15 samples exhibited binding, respectively. The serum of the EGSN group had a higher mean IgA concentration than that of the negative controls (P < 0.05). Thus, we describe HIV-2-specific serum IgA antigen reactivity and show a more potent serum IgA-mediated HIV-2-neutralizing activity in EGSN individuals than in HIV-2-infected patients.

Quantification of Human T-lymphotropic virus type I (HTLV-I) provirus load in a rural West African population: no enhancement of human immunodeficiency virus type 2 pathogenesis, but HTLV-I provirus load relates to mortality.

Human T-lymphotropic virus type I (HTLV-I) provirus load was examined in a cohort of a population in Guinea-Bissau among whom human immunodeficiency virus (HIV) type 2 is endemic. Geometric mean of HIV-2 RNA load among HTLV-I-coinfected subjects was significantly lower than that in subjects infected with HIV-2 alone (212 vs. 724 copies/mL; P=.02). Adjusted for age, sex, and HIV status, the risk of death increased with HTLV-I provirus load; mortality hazard ratio was 1.59 for each log10 increase in HTLV-I provirus copies (P=.038). There is no enhancing effect of HTLV-I coinfection on HIV-2 disease, but high HTLV-I provirus loads may contribute to mortality.