Megakaryocytes possess a STING pathway that is transferred to platelets to potentiate activation.

Platelets display unexpected roles in immune and coagulation responses. Emerging evidence suggests that STING is implicated in hypercoagulation. STING is an adaptor protein downstream of the DNA sensor cyclic GMP-AMP synthase (cGAS) that is activated by cytosolic microbial and self-DNA during infections, and in the context of loss of cellular integrity, to instigate the production of type-I IFN and pro-inflammatory cytokines. To date, whether the cGAS-STING pathway is present in platelets and contributes to platelet functions is not defined. Using a combination of pharmacological and genetic approaches, we demonstrate here that megakaryocytes and platelets possess a functional cGAS-STING pathway. Our results suggest that in megakaryocytes, STING stimulation activates a type-I IFN response, and during thrombopoiesis, cGAS and STING are transferred to proplatelets. Finally, we show that both murine and human platelets contain cGAS and STING proteins, and the cGAS-STING pathway contributes to potentiation of platelet activation and aggregation. Taken together, these observations establish for the first time a novel role of the cGAS-STING DNA sensing axis in the megakaryocyte and platelet lineage.

Alpha-2-macroglobulin prevents platelet aggregation induced by brain-derived neurotrophic factor.

The brain-derived neurotrophic factor (BDNF) has been recently shown to have activating effects in isolated platelets. However, BDNF circulates in plasma and a mechanism to preclude constant activation of platelets appears necessary. Hence, we investigated the mechanism regulating BDNF bioavailability in blood. Protein-protein interactions were predicted by molecular docking and validated through immunoprecipitation. Platelet aggregation was assessed using light transmission aggregometry with washed platelets in response to classical agonists or BDNF, in the absence or presence of alpha-2-macroglobulin (α2M), and in platelet-rich plasma. BDNF signaling was assessed with phospho-blots. As little as 25% autologous plasma was sufficient to completely abolish platelet aggregation in response to BDNF. Docking predicted two forms of BDNF binding to native or activated α2M, in parallel and perpendicular arrangements, and the model suggested that the BDNF-α2M complex cannot bind to the high-affinity BDNF receptor, tropomyosin receptor kinase B (TrkB). Experimentally, native and activated α2M formed stable complexes with BDNF preventing BDNF-induced TrkB activation and signal transduction. Both native and activated α2M inhibited BDNF induced-platelet aggregation in a concentration-dependent manner with comparable half-maximal inhibitory concentrations (IC50≈ 125-150 nM). Our study implicates α2M as a physiological regulator of BDNF bioavailability, and as an inhibitor of BDNF-induced platelet activation in blood.

Clinical Correlates Identify ProBDNF and Thrombo-Inflammatory Markers as Key Predictors of Circulating p75NTR Extracellular Domain Levels in Older Adults.

The p75NTR receptor binds all neurotrophins and is mostly known for its role in neuronal survival and apoptosis. Recently, the extracellular domain (ECD) of p75NTR has been reported in plasma, its levels being dysregulated in numerous neurological diseases. However, the factors associated with p75NTR ECD levels remain unknown. We investigated clinical correlates of plasma p75NTR ECD levels in older adults without clinically manifested neurological disorders. Circulating p75NTR levels were measured by enzyme-linked immunosorbent assay in plasma obtained from participants in the BEL-AGE cohort (n = 1,280). Determinants of plasma p75NTR ECD levels were explored using linear and non-linear statistical models. Plasma p75NTR ECD levels were higher in male participants; were positively correlated with circulating concentrations of pro-brain-derived neurotrophic factor, and inflammatory markers interleukin-6 and CD40 Ligand; and were negatively correlated with the platelet activation marker P-selectin. While most individuals had p75NTR levels ranging from 43 to 358 pg/ml, high p75NTR levels reaching up to 9,000 pg/ml were detectable in a subgroup representing 15% of the individuals studied. In this cohort of older adults without clinically manifested neurological disorders, there was no association between plasma p75NTR ECD levels and cognitive performance, as assessed by the Montreal Cognitive Assessment score. The physiological relevance of high p75NTR ECD levels in plasma warrants further investigation. Further research assessing the source of circulating p75NTR is needed for a deeper understanding of the direction of effect, and to investigate whether high p75NTR ECD levels are predictive biomarkers or consequences of neuropathology.

The brain-derived neurotrophic factor prompts platelet aggregation and secretion.

Brain-derived neurotrophic factor (BDNF) has both autocrine and paracrine roles in neurons, and its release and signaling mechanisms have been extensively studied in the central nervous system. Large quantities of BDNF have been reported in circulation, essentially stored in platelets with concentrations reaching 100- to 1000-fold those of neurons. Despite this abundance, the function of BDNF in platelet biology has not been explored. At low concentrations, BDNF primed platelets, acting synergistically with classical agonists. At high concentrations, BDNF induced complete biphasic platelet aggregation that in part relied on amplification from secondary mediators. Neurotrophin-4, but not nerve growth factor, and an activating antibody against the canonical BDNF receptor tropomyosin-related kinase B (TrkB) induced similar platelet responses to BDNF, suggesting TrkB could be the mediator. Platelets expressed, both at their surface and in their intracellular compartment, a truncated form of TrkB lacking its tyrosine kinase domain. BDNF-induced platelet aggregation was prevented by inhibitors of Ras-related C3 botulinum toxin substrate 1 (Rac1), protein kinase C, and phosphoinositide 3-kinase. BDNF-stimulated platelets secreted a panel of angiogenic and inflammatory cytokines, which may play a role in maintaining vascular homeostasis. Two families with autism spectrum disorder were found to carry rare missense variants in the BDNF gene. Platelet studies revealed defects in platelet aggregation to low concentrations of collagen, as well as reduced adenosine triphosphate secretion in response to adenosine diphosphate. In summary, circulating BDNF levels appear to regulate platelet activation, aggregation, and secretion through activation of a truncated TrkB receptor and downstream kinase-dependent signaling.

Brain-Derived Neurotrophic Factor Mitigates the Association Between Platelet Dysfunction and Cognitive Impairment.

Background: Platelet hyperactivity is deleterious in coronary artery disease (CAD), requiring lifelong antiplatelet therapy, and is associated with worse cognitive outcomes. Upon activation, platelets release Brain-Derived Neurotrophic Factor (BDNF), a neurotrophin protective against cognitive decline. Given these apparently opposing effects of platelet activation on cognitive health, we investigated whether BDNF levels intercede in the relationship between platelet activation and cognitive function; and whether this relationship is moderated by the presence of CAD. Methods: In this cross-sectional study, 1,280 participants with (n = 673) and without CAD (n = 607) completed the Montreal Cognitive Assessment (MoCA). Plasma BDNF and soluble P-selectin (a marker of platelet activity) levels were assessed using multiplex flow cytometry. Results: In a mediation model, platelet activity was correlated with higher plasma BDNF concentrations (b = 0.53, p < 0.0001). The relationship between sP-selectin and BDNF concentrations was stronger for individuals without CAD (b = 0.71, p < 0.0001) than for CAD participants (b = 0.43, p < 0.0001; p interaction <0.0001). Higher BDNF concentrations were associated with higher MoCA scores (b = 0.26, p = 0.03). The overall effect of platelet activity on cognitive performance was non-significant (total effect: b = -0.12, p = 0.13), and became significant when accounting for BDNF as a mediating factor (direct effect: b = -0.26, p = 0.01). This resulted in a positive indirect effect of platelet activity (via BDNF) on MoCA scores (b = 0.14, CI 95% 0.02-0.30), that was smaller in CAD participants than in non-CAD participants [Δ -0.07 (95% CI -0.14 to -0.01)]. Conclusions: BDNF released from activated platelets could be a mitigating factor in a negative association between platelet activity and cognitive function.

Tissue-Specificity of Antibodies Raised Against TrkB and p75NTR Receptors; Implications for Platelets as Models of Neurodegenerative Diseases.

Platelets and neurons share many similarities including comparable secretory granule types with homologous calcium-dependent secretory mechanisms as well as internalization, sequestration and secretion of many neurotransmitters. Thus, platelets present a high potential to be used as peripheral biomarkers to reflect neuronal pathologies. The brain-derived neurotrophic factor (BDNF) acts as a neuronal growth factor involved in learning and memory through the binding of two receptors, the tropomyosin receptor kinase B (TrkB) and the 75 kDa pan-neurotrophic receptor (p75NTR). In addition to its expression in the central nervous system, BDNF is found in much greater quantities in blood circulation, where it is largely stored within platelets. Levels 100- to 1,000-fold those of neurons make platelets the most important peripheral reservoir of BDNF. This led us to hypothesize that platelets would express canonical BDNF receptors, i.e., TrkB and p75NTR, and that the receptors on platelets would bear significant resemblance to the ones found in the brain. However, herein we report discrepancies regarding detection of these receptors using antibody-based assays, with antibodies displaying important tissue-specificity. The currently available antibodies raised against TrkB and p75NTR should therefore be used with caution to study platelets as models for neurological disorders. Rigorous characterization of antibodies and bioassays appears critical to understand the interplay between platelet and neuronal biology of BDNF.

Severity of Megakaryocyte-Driven Osteosclerosis in Mpig6b-Deficient Mice Is Sex-Linked.

Patients with chronic myelofibrosis often suffer from osteosclerosis, which is associated with bone pain and may lead to bone marrow failure. The pathogenesis of myelofibrosis is linked to aberrant megakaryocyte development and function. Null and loss-of-function mutations in MPIG6B, which codes for the inhibitory heparan sulfate receptor G6b-B, result in severe macrothrombocytopenia, large megakaryocyte clusters, and focal primary myelofibrosis in mice and humans. We investigated the development of osteosclerosis in Mpig6b null (Mpig6b-/- ) mice. Although male and female Mpig6b-/- mice presented with elevated bone marrow megakaryocyte number and macrothrombocytopenia, female Mpig6b-/- mice developed progressive splenomegaly starting at 8 weeks of age. Micro-computed tomography (µCT) of femurs showed that female Mpig6b-/- mice had increased cortical thickness and reduced bone marrow area starting at 8 weeks of age and developed occlusion of the medullary cavity by trabeculae by 16 weeks of age. In contrast, male Mpig6b-/- mice developed only a small number of trabeculae in the medullary cavity at the proximal diaphysis and demonstrated a temporary decrease in bone volume fraction and trabecular thickness at 16 weeks. Ovariectomy of 10-week-old female Mpig6b-/- mice prevented the development of medullary cavity osteosclerosis, whereas orchiectomy of male Mpig6b-/- mice did not exacerbate their disease. Importantly, ovariectomized female Mpig6b-/- mice also demonstrated improvement in spleen weight compared to sham-operated Mpig6b-/- mice, establishing estrogen as a contributing factor to the severity of the megakaryocyte-driven osteosclerosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Differential correlation of serum BDNF and microRNA content in rats with rapid or late onset of heavy alcohol use.

Heavy alcohol use reduces the levels of the brain-derived neurotrophic factor (BDNF) in the prefrontal cortex of rodents through the upregulation of microRNAs (miRs) targeting BDNF mRNA. In humans, an inverse correlation exists between circulating blood levels of BDNF and the severity of psychiatric disorders including alcohol abuse. Here, we set out to determine whether a history of heavy alcohol use produces comparable alterations in the blood of rats. We used an intermittent access to 20% alcohol using the two-bottle choice paradigm (IA20%2BC) and measured circulating levels of BDNF protein and miRs targeting BDNF in the serum of Long-Evans rats before and after 8 weeks of excessive alcohol intake. We observed that the drinking profile of heavy alcohol users is not unified, whereas 70% of the rats gradually escalate their alcohol intake (late onset), and 30% of alcohol users exhibit a very rapid onset of drinking (rapid onset). We found that serum BDNF levels are negatively correlated with alcohol intake in both rapid onset and late onset rats. In contrast, increased expression of the miRs targeting BDNF, miR30a-5p, miR-195-5p, miR191-5p and miR206-3p, was detected only in the rapid onset rats. Finally, we report that the alcohol-dependent molecular changes are not due to alterations in platelet number. Together, these data suggest that rats exhibit both late and rapid onset of alcohol intake. We further show that heavy alcohol use produces comparable changes in BDNF protein levels in both groups. However, circulating microRNAs are responsive to alcohol only in the rapid onset rats.

Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF.

Background: Brain-derived neurotrophic factor (BDNF) plays a role in synaptic plasticity and neuroprotection. BDNF has well-established pro-survival effects, whereas its precursor protein, proBDNF, induces apoptosis. Thus, it has been suggested that the proBDNF/BDNF ratio could be an indicator of neuronal health. Access to neurons is, understandably, limited. Because of their similarities, platelets have been put forward as a non-invasive biomarker of neuronal health; indeed, they store large quantities of BDNF and can release it into circulation upon activation, similarly to neurons. However, whether platelets also express the precursor proBDNF protein remains unknown. We therefore sought to characterize proBDNF levels in human platelets and plasma. Methods: The presence of proBDNF was assessed by immunoblotting, cell fractionation, flow cytometry, and confocal microscopy in washed platelets from 10 healthy volunteers. Platelets from 20 independent healthy volunteers were activated with several classical agonists and the release of BDNF and proBDNF into plasma was quantified by ELISA. Results: Platelets expressed detectable levels of proBDNF (21 ± 13 fmol/250 x 106 platelets). ProBDNF expression was mainly localized in the intracellular compartment. The proBDNF to BDNF molar ratio was ~1:5 in platelets and 10:1 in plasma. In stark contrast to the release of BDNF during platelet activation, intraplatelet and plasma concentrations of proBDNF remained stable following stimulation with classical platelet agonists, consistent with non-granular expression. Conclusions: Platelets express both the mature and the precursor form of BDNF. Whether the intraplatelet proBDNF to BDNF ratio could be used as a non-invasive biomarker of cognitive health warrants further investigation.

Head-to-Head Comparison of Consensus-Recommended Platelet Function Tests to Assess P2Y12 Inhibition-Insights for Multi-Center Trials.

The vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation level is a highly specific method to assess P2Y12 receptor inhibition. Traditionally, VASP phosphorylation is analyzed by flow cytometry, which is laborious and restricted to specialized laboratories. Recently, a simple ELISA kit has been commercialized. The primary objective of this study was to compare the performance of VASP assessment by ELISA and flow cytometry in relation to functional platelet aggregation testing by Multiplate® whole-blood aggregometry. Blood from 24 healthy volunteers was incubated with increasing concentration of a P2Y12 receptor inhibitor (AR-C 66096). Platelet function testing was carried out simultaneously by Multiplate® aggregometry and by VASP assessment through ELISA and flow cytometry. As expected, increasing concentrations of the P2Y12 receptor inhibitor induced a proportional inhibition of platelet aggregation and P2Y12 receptor activation across the modalities. Platelet reactivity index values of both ELISA- and flow cytometry-based VASP assessment methods correlated strongly (r = 0.87, p < 0.0001) and showed minimal bias (1.05%). Correlation with Multiplate® was slightly higher for the flow cytometry-based VASP assay (r = 0.79, p < 0.0001) than for the ELISA-based assay (r = 0.69, p < 0.0001). Intraclass correlation (ICC) was moderate for all the assays tested (ICC between 0.62 and 0.84). However, categorization into low, optimal, or high platelet reactivity based on these assays was strongly concordant (κ between 0.86 and 0.92). In conclusion, the consensus-recommended assays with their standardized cut-offs should not be used interchangeably in multi-center clinical studies but, rather, they should be standardized throughout sites.

Reassessing GWAS findings for the shared genetic basis of insomnia and restless legs syndrome.

Two genome-wide association studies (GWAS) suggest that insomnia and restless legs syndrome (RLS) share a common genetic basis. While the identified genetic variation in the MEIS1 gene was previously associated with RLS, the two GWAS suggest a novel and independent association with insomnia symptoms. To test the potential pleiotropic effect of MEIS1, we genotyped three MEIS1 variants in 646 chronic insomnia disorder (CID) patients with and without RLS. To confirm our results, we compared the allelic and genotypic distributions of the CID cohort with ethnically matched controls and RLS cases in the French Canadian cohort. The CID cohort was diagnosed by sleep medicine specialists and 26% of the sample received the combined diagnosis of CID+RLS. We find significant differences in allele and genotype distributions between CID-only and CID+RLS groups, suggesting that MEIS1 is only associated with RLS. Genotype distributions and minor allele frequencies of the three MEIS1 SNPs of the CID-only and control groups were similar (rs113851554: 5.3% vs. 5.6%; rs2300478: 25.3% vs. 26.5%; rs12469063: 23.6% vs. 24.4%; all p > 0.05). Likewise, there were no differences between CID+RLS and RLS-only groups (all p > 0.05). In conclusion, our data confirms that MEIS1 is a genetic risk factor for the development of RLS, but it does not support the pleiotropic effect of MEIS1 in CID. While a lack of power precluded us from refuting small pleiotropic effects, our findings emphasize the critical importance of isolating CID from other disorders that can cause sleep difficulties, particularly RLS, for future genetic studies.

Protective role against hydrogen peroxide and fibroblast stimulation via Ce-doped TiO2 nanostructured materials.

BACKGROUND: Cerium oxide (CeO2) and Ce-doped nanostructured materials (NMs) are being seen as innovative therapeutic tools due to their exceptional antioxidant effects; nevertheless their bio-applications are still in their infancy. METHODS: TiO2, Ce-TiO2 and CeO2-TiO2 NMs were synthesized by a bottom-up microemulsion-mediated strategy and calcined during 7h at 650°C under air flux. The samples were compared to elucidate the physicochemical characteristics that determine cellular uptake, toxicity and the influence of redox balance between the Ce(3+)/Ce(4+) on the cytoprotective role against an exogenous ROS source: H2O2. Fibroblasts were selected as a cell model because of their participation in wound healing and fibrotic diseases. RESULTS: Ce-TiO2 NM obtained via sol-gel reaction chemistry of metallic organic precursors exerts a real cytoprotective effect against H2O2 over fibroblast proliferation, while CeO2 pre-formed nanoparticles incorporated to TiO2 crystalline matrix lead to a harmful CeO2-TiO2 material. TiO2 was processed by the same pathways as Ce-TiO2 and CeO2-TiO2 NM but did not elicit any adverse or protective influence compared to controls. CONCLUSIONS: It was found that the Ce atoms source and its concentration have a clear effect on material's physicochemical properties and its subsequent influence in the cellular response. It can induce a range of biological reactions that vary from cytotoxic to cytoprotective. GENERAL SIGNIFICANCE: Even though there are still some unresolved issues and challenges, the unique physical and chemical properties of Ce-based NMs are fascinating and versatile resources for different biomedical applications.

Specific inhalation challenge with flour induced release of brain-derived neurotrophic factor in nasal fluid.

BACKGROUND: Neurotrophins may play a role in the pathophysiology of allergic occupational rhinitis (OR). We sought to investigate whether an immediate allergic reaction that induces nasal inflammation is also able to induce changes in levels of brain-derived neurotrophic factor (BDNF) in nasal lavage (NAL) fluid from patients with allergic OR. METHODS: Ten patients sensitized to flour underwent control and active specific inhalation challenge (SIC) on consecutive days. Nasal response to SIC was monitored with acoustic rhinometry and symptoms recording. NAL was performed before and 30 minutes, 6 hours, and 24 hours after control and active challenge for the assessment of levels of BDNF and inflammatory cells in NAL fluid. RESULTS: In contrast to control day, flour challenge induced immediate clinical reactions in all subjects. After flour challenge, a significant increase in levels of BDNF in NAL fluid was observed at 6 hours after challenge (p < 0.05). Also, a significant increase in the number of eosinophils in NAL fluid at 30 minutes (p < 0.01), 6 hours (p < 0.01), and 24 hours (p = 0.05) postchallenge was observed. Also, levels of BDNF in NAL fluid were significantly higher at 30 minutes after flour challenge (p = 0.02) in comparison to levels on the control day at the same postchallenge time. A marginally significant positive correlation between BDNF levels and eosinophil counts at 30 minutes (r = 0.60, p = 0.06) and at 6 hours (r = 0.50, p = 0.08) after flour challenge was noted. CONCLUSION: We showed that BDNF is released in nasal fluid after SIC with flour. Results support the suggestion that neurotrophins may play a role in the pathogenesis of allergic OR.

Perturbation of adhesion molecule-mediated chondrocyte-matrix interactions by 4-hydroxynonenal binding: implication in osteoarthritis pathogenesis.

INTRODUCTION: Objectives were to investigate whether interactions between human osteoarthritic chondrocytes and 4-hydroxynonenal (HNE)-modified type II collagen (Col II) affect cell phenotype and functions and to determine the protective role of carnosine (CAR) treatment in preventing these effects. METHODS: Human Col II was treated with HNE at different molar ratios (MR) (1:20 to 1:200; Col II:HNE). Articular chondrocytes were seeded in HNE/Col II adduct-coated plates and incubated for 48 hours. Cell morphology was studied by phase-contrast and confocal microscopy. Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and α1ß1 integrin at protein and mRNA levels were quantified by Western blotting, flow cytometry and real-time reverse transcription-polymerase chain reaction. Cell death, caspases activity, prostaglandin E2 (PGE2), metalloproteinase-13 (MMP-13), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were assessed by commercial kits. Col II, cyclooxygenase-2 (COX-2), MAPK, NF-κB-p65 levels were analyzed by Western blotting. The formation of α1ß1 integrin-focal adhesion kinase (FAK) complex was revealed by immunoprecipitation. RESULTS: Col II modification by HNE at MR approximately 1:20, strongly induced ICAM-1, α1ß1 integrin and MMP-13 expression as well as extracellular signal-regulated kinases 1 and 2 (ERK1/2) and NF-κB-p65 phosphorylation without impacting cell adhesion and viability or Col II expression. However, Col II modification with HNE at MR approximately 1:200, altered chondrocyte adhesion by evoking cell death and caspase-3 activity. It inhibited α1ß1 integrin and Col II expression as well as ERK1/2 and NF-κB-p65 phosphorylation, but, in contrast, markedly elicited PGE2 release, COX-2 expression and p38 MAPK phosphorylation. Immunoprecipitation assay revealed the involvement of FAK in cell-matrix interactions through the formation of α1ß1 integrin-FAK complex. Moreover, the modification of Col II by HNE at a 1:20 or approximately 1:200 MR affects parameters of the cell shape. All these effects were prevented by CAR, an HNE-trapping drug. CONCLUSIONS: Our novel findings indicate that HNE-binding to Col II results in multiple abnormalities of chondrocyte phenotype and function, suggesting its contribution in osteoarthritis development. CAR was shown to be an efficient HNE-snaring agent capable of counteracting these outcomes.

Outcome of occupational asthma after removal from exposure: A follow-up study.

BACKGROUND: Despite being removed from their workplace, the majority of workers with occupational asthma (OA) remain afflicted with asthma. OBJECTIVES: To assess the time course of clinical, functional and inflammatory parameters in subjects with OA over a four-year period, and whether the airway inflammation observed at the time of the diagnosis predicts the outcome of OA. METHODS: The present study was a four-year, prospective, longitudinal investigation of workers with OA. Spirometry, methacholine challenge and sputum induction were performed at two weeks, and followed up at six months, and one, two, three and four years after the performance of specific inhalation challenges. RESULTS: A total of 24 subjects were enrolled. Overall, clinical and functional characteristics remained stable during the four-year follow-up period. Sputum eosinophil (Eos) counts  decreased within two weeks after exposure. Two groups of subjects were identified according to low (less than 2%, Eos-) or high (2% or greater, Eos+) Eos counts after exposure to the offending agent. The Eos+ group decreased their dose of inhaled corticosteroids, had a trend toward an improvement of airway responsiveness as well as a stable forced expiratory volume in 1 s (FEV1), whereas the Eos- group showed a decrease in FEV1, without any improvement in their functional parameters. The Eos- group also had an increase in sputum neutrophils after exposure to the occupational agents as well as during the follow-up period. CONCLUSION: There was a rapid decrease in eosinophilic inflammation after removal from exposure. Subjects with a noneosinophilic asthmatic reaction during specific inhalation challenge seemed to have a poorer prognosis than subjects with eosinophilic airway  inflammation.

Expression and regulation of CCR1 by airway smooth muscle cells in asthma.

C-C chemokines such as CCL11, CCL5, and CCL3 are central mediators in the pathogenesis of asthma. They are mainly associated with the recruitment and the activation of specific inflammatory cells, such as eosinophils, lymphocytes, and neutrophils. It has recently been shown that they can also activate structural cells, such as airway smooth muscle and epithelial cells. The aims of this study were to examine the expression of the CCL3 receptor, CCR1, on human airway smooth muscle cells (ASMC) and to document the regulation of this receptor by cytokines involved in asthma pathogenesis. We first demonstrated that CCR1 mRNA is increased in the airways of asthmatic vs control subjects and showed for the first time that ASMC express CCR1 mRNA and protein, both in vitro and in vivo. Calcium mobilization by CCR1 ligands confirmed its functionality on ASMC. Stimulation of ASMC with TNF-alpha and, to a lesser extent, IFN-gamma resulted in an up-regulation of CCR1 expression, which was totally suppressed by both dexamethasone or mithramycin. Taken together, our data suggest that CCR1 might be involved in the pathogenesis of asthma, through the activation of ASMC by its ligands.

Role of envelope processing and gp41 membrane spanning domain in the formation of human immunodeficiency virus type 1 (HIV-1) fusion-competent envelope glycoprotein complex.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is directed by the envelope (Env) glycoproteins, which are present on the surface of HIV-1 virion or infected cells in the form of trimers consisting of gp120/gp41 complexes. The surface subunit, gp120, initiates the entry process by interacting sequentially with the CD4 receptor and a co-receptor, thereby inducing a conformational change that allows the transmembrane (TM) gp41 subunit to mediate fusion between viral and target cell membranes. Cleavage of Env into its gp120 and gp41 components is necessary for activation of its fusogenic activity. Here, the gp41 TM glycoprotein was altered by either deleting an isoleucine residue (DeltaI642) in a critical region of its ectodomain or by substituting its membrane spanning domain (MSD) by that of the influenza hemagglutinin (HA) glycoprotein (TM-HA) to examine the contribution of these regions to Env functions. Characterization of these mutant forms of gp41 revealed that they both affected the infectivity of pseudotyped virions, however, through distinct defects in Env functions. While deletion of Ile 642 drastically altered processing of Env, replacement of gp41 MSD by that of HA led to a marked fusion defect even though the TM-HA Env was efficiently processed and incorporated into viral particles. Interestingly, both DeltaI642 and TM-HA Env were found to act as trans dominant-negative mutant of viral infectivity, presumably via their ability to form hetero-oligomers with wild type Env. Together, these results support a previously proposed model whereby all three gp120-gp41 monomers must be cleaved for the Env homo-trimer to function and suggest that the gp41 MSD plays a critical role in the formation of fusion-competent Env trimers.

CD4/CXCR4 co-expression allows productive HIV-1 infection in canine kidney MDCK cells.

The Madin-Darby canine kidney (MDCK) cell line has become the prototypic cell type for studying the mechanisms involved in viral glycoproteins transport and viral assembly in polarized cells. This cell line has been used in our laboratories for studying human immunodeficiency virus (HIV-1), despite the fact that MDCK cells cannot be infected by HIV. In transfected MDCK cells, HIV-1 glycoproteins are specifically transported to the basolateral cell surface where viral budding also mostly occurs. However, this model is of limited use when viral propagation, infection of most cells, or larger production of virions, is needed. The initial objective of this work was thus to establish an MDCK-derived cell line that could be productively infected by HIV-1, in order to pursue our studies on the polarization of viral budding. Expression of both receptor and co-receptor for T-tropic strains of the virus showed that canine cells are rendered permissive once virus binding and entry is allowed. In addition, a reduced infectivity of the viral particles released from the basolateral surface was observed. This observation most likely reflects the interference mediated by CD4 molecules that accumulate at the basolateral domain. Accordingly, this effect was largely prevented when using viruses that down-regulate cell surface CD4 by expression of both viral accessory proteins Vpu and Nef. This is a further evidence that the function of different viral proteins depends of the site of viral budding, which is itself determined by the presence of targeting signal(s) harbored by viral envelope glycoproteins.