Quantification of the virus-host interaction in human T lymphotropic virus I infection

BACKGROUND: HTLV-I causes the disabling inflammatory disease HAM/TSP: there is no vaccine, no satisfactory treatment and no means of assessing the risk of disease or prognosis in infected people. Like many immunopathological diseases with a viral etiology the outcome of infection is thought to depend on the virus-host immunology interaction. However the dynamic virus-host interaction is complex and current models of HAM/TSP pathogenesis are conflicting. The CD8+ cell response is thought to be a determinant of both HTLV-I proviral load and disease status but its effects can obscure other factors. RESULTS: We show here that in the absence of CD8+ cells, CD4+ lymphocytes from HAM/TSP patients expressed HTLV-I protein significantly more readily than lymphocytes from asymptomatic carriers of similar proviral load (P = 0.017). A high rate of viral protein expression was significantly associated with a large increase in the prevalence of HAM/TSP (P = 0.031, 89% of cases correctly classified). Additionally, a high rate of Tax expression and a low CD8+ cell efficiency were independently significantly associated with a high proviral load (P = 0.005, P = 0.003 respectively). CONCLUSION: These results disentangle the complex relationship between immune surveillance, proviral load, inflammatory disease and viral protein expression and indicate that increased protein expression may play an important role in HAM/TSP pathogenesis. This has important implications for therapy since it suggests that interventions should aim to reduce Tax expression rather than proviral load per se

Quantification of the virus-host interaction in human T lymphotropic virus I infection

Abstract Background HTLV-I causes the disabling inflammatory disease HAM/TSP: there is no vaccine, no satisfactory treatment and no means of assessing the risk of disease or prognosis in infected people. Like many immunopathological diseases with a viral etiology the outcome of infection is thought to depend on the virus-host immunology interaction. However the dynamic virus-host interaction is complex and current models of HAM/TSP pathogenesis are conflicting. The CD8+ cell response is thought to be a determinant of both HTLV-I proviral load and disease status but its effects can obscure other factors. Results We show here that in the absence of CD8+ cells, CD4+ lymphocytes from HAM/TSP patients expressed HTLV-I protein significantly more readily than lymphocytes from asymptomatic carriers of similar proviral load (P = 0.017). A high rate of viral protein expression was significantly associated with a large increase in the prevalence of HAM/TSP (P = 0.031, 89% of cases correctly classified). Additionally, a high rate of Tax expression and a low CD8+ cell efficiency were independently significantly associated with a high proviral load (P = 0.005, P = 0.003 respectively). Conclusion These results disentangle the complex relationship between immune surveillance, proviral load, inflammatory disease and viral protein expression and indicate that increased protein expression may play an important role in HAM/TSP pathogenesis. This has important implications for therapy since it suggests that interventions should aim to reduce Tax expression rather than proviral load per se.</p

Impulse Conduction Increases Mitochondrial Transport in Adult Mammalian Peripheral Nerves <i>In Vivo</i>

<div><p>Matching energy supply and demand is critical in the bioenergetic homeostasis of all cells. This is a special problem in neurons where high levels of energy expenditure may occur at sites remote from the cell body, given the remarkable length of axons and enormous variability of impulse activity over time. Positioning mitochondria at areas with high energy requirements is an essential solution to this problem, but it is not known how this is related to impulse conduction <i>in vivo</i>. Therefore, to study mitochondrial trafficking along resting and electrically active adult axons <i>in vivo</i>, confocal imaging of saphenous nerves in anaesthetised mice was combined with electrical and pharmacological stimulation of myelinated and unmyelinated axons, respectively. We show that low frequency activity induced by electrical stimulation significantly increases anterograde and retrograde mitochondrial traffic in comparison with silent axons. Higher frequency conduction within a physiological range (50 Hz) dramatically further increased anterograde, but not retrograde, mitochondrial traffic, by rapidly increasing the number of mobile mitochondria and gradually increasing their velocity. Similarly, topical application of capsaicin to skin innervated by the saphenous nerve increased mitochondrial traffic in both myelinated and unmyelinated axons. In addition, stationary mitochondria in axons conducting at higher frequency become shorter, thus supplying additional mitochondria to the trafficking population, presumably through enhanced fission. Mitochondria recruited to the mobile population do not accumulate near Nodes of Ranvier, but continue to travel anterogradely. This pattern of mitochondrial redistribution suggests that the peripheral terminals of sensory axons represent sites of particularly high metabolic demand during physiological high frequency conduction. As the majority of mitochondrial biogenesis occurs at the cell body, increased anterograde mitochondrial traffic may represent a mechanism that ensures a uniform increase in mitochondrial density along the length of axons during high impulse load, supporting the increased metabolic demand imposed by sustained conduction.</p></div

Higher frequency (50 Hz) conduction is associated with shortening of stationary, and an increase in distance between stationary mitochondria.

<p>(A) The total number of stationary mitochondrial profiles did not change significantly in response to impulse conduction (<i>p</i>>0.05, Kruskal-Wallis test with Dunn's multiple comparison test). (B) Average length of stationary mitochondria was significantly lower in axons conducting impulses at 50 Hz (<i>n</i> = 366, <i>p</i><0.01), or at 1 Hz following 50 Hz (<i>n</i> = 231, <i>p</i><0.001) than in naive (<i>n</i> = 231) or sham-stimulated (<i>n</i> = 366) axons. (C). Frequency distribution of length of stationary mitochondria between groups showed a significantly lower number of long mitochondria (i.e., 4 µm) and higher number of short (i.e., 2 µm) in axons conducting at high frequency, than in sham-stimulated axons. (D) The number of mitochondria separated by 1.5 µm (measured between their mid-points) decreased in axons conducting at 50 Hz (<i>n</i> = 15 axons, <i>n</i> = 288 mitochondria), and was significantly lower for mitochondria separated by 3 µm (<i>p</i><0.001), than in sham-stimulated group (<i>n</i> = 13 axons, <i>n</i> = 309 mitochondria; three animals per group), i.e., mitochondria were less clustered in stimulated axons. In all groups axons were pulled from three independent experiments (animals).</p

In saphenous nerve axons stimulated at high frequency (50 Hz) mitochondria accumulate at the peripheral sensory terminals.

<p>Skin innervated by saphenous nerve was immunohistochemically labelled with VDAC1 (red) and neuron-specific β-III-tubulin (green). In comparison with sham-stimulated animals (A), axons were strongly labelled for VDAC1 in nerves stimulated at 1 Hz (B) and 50 Hz (C). (C′–C′″) Saphenous nerve from a YFP⁺ mouse (shown in low power in C), which expresses YFP (green) in a proportion of fibres, was stimulated with 50 Hz and labelled with VDAC1 (red). (D′–D′″) Saphenous nerve from a YFP⁻ mouse was stimulated with 50 Hz and double labelled with VDAC1 (red) and β-III-tubulin. The two markers were often found to co-localise. (E) Intensity of VDAC1 labelling was significantly higher within cutaneous fibres of saphenous nerve stimulated at 50 Hz (<i>n</i> = 5, <i>p</i><0.05) than in sham-stimulated (<i>n</i> = 3) or fibres stimulated at 1 Hz (<i>n</i> = 3). (F) There was no difference in VDAC1 labelling intensity in DRGs of saphenous nerves between the groups. Scale bars in (A–C) = 100 µm, in (C′–C′″) = 20 µm, in (D′–D′″) = 10 µm.</p

Mitochondria do not accumulate in the vicinity of Nodes of Ranvier.

<p>(A and B) Nodes of Ranvier in sham-stimulated (<i>n</i> = 35) and axons stimulated at high frequency (50 Hz, <i>n</i> = 28) appeared similar upon ultrastructural examination (bar = 1 µm), and (C) possessed similar numbers of mitochondria (<i>p</i> = 0.22, Mann-Whitney test).</p

Mitochondrial traffic during impulse conduction.

<p>(A) Confocal image of saphenous nerve exposed in an anaesthetised mouse, labelled with IB4-isolectin (green) and TMRM (red). (B) Typical image of saphenous fibres labelled with TMRM. Asterisks denote Schwann cell nuclei. (C) Kymograph analysis of mitochondrial movement in representative axons. (D) CAPs averaged every 30 s during 50 Hz stimulation for 75 min during time-lapse confocal imaging remained similar in form. (E) Impulse conduction significantly increased the number of trafficking mitochondria. (F) Anterograde and retrograde mitochondrial trafficking increased in axons conducting at 1 Hz (<i>n</i> = 30 axons) and 50 Hz (<i>n</i> = 31 axons) versus naive (<i>n</i> = 60 axons) (<i>p</i><0.001) and sham-stimulated animals (<i>n</i> = 32 axons) (<i>p</i><0.01): anterograde trafficking is selectively and markedly raised with 50 Hz stimulation (<i>p</i><0.001). (G) Trafficking velocity is significantly higher at 50 Hz than in naive, sham, and 1 Hz-stimulated axons (<i>p</i><0.001), and it decreases upon reducing stimulation to 1 Hz (<i>n</i> = 23 axons) (<i>p</i><0.05): anterograde velocity is particularly increased (H). In all groups axons were pulled from three independent experiments (animals). Data that followed normal (Gaussian) distribution are represented as mean ± SEM, whereas data that did not follow normal distribution are represented as median ± IQR. Kruskal-Wallis test with Dunn's multiple comparison test; *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001.</p

In vivo T lymphocyte dynamics in humans and the impact of human T-lymphotropic virus 1 infection

Human T-lymphotropic virus type 1 (HTLV-1) is a persistent CD4(+) T-lymphotropic retrovirus. Most HTLV-1-infected individuals remain asymptomatic, but a proportion develop adult T cell leukemia or inflammatory disease. It is not fully understood how HTLV-1 persists despite a strong immune response or what determines the risk of HTLV-1-associated diseases. Until recently, it has been difficult to quantify lymphocyte kinetics in humans in vivo. Here, we used deuterated glucose labeling to quantify in vivo lymphocyte dynamics in HTLV-1-infected individuals. We then used these results to address four questions. (i) What is the impact of HTLV-1 infection on lymphocyte dynamics? (ii) How does HTLV-1 persist? (iii) What is the extent of HTLV-1 expression in vivo? (iv) What features of lymphocyte kinetics are associated with HTLV-1-associated myelopathy/tropical spastic paraparesis? We found that CD4(+)CD45RO(+) and CD8(+)CD45RO(+) T lymphocyte proliferation was elevated in HTLV-1-infected subjects compared with controls, with an extra 10(12) lymphocytes produced per year in an HTLV-1-infected subject. The in vivo proliferation rate of CD4(+)CD45RO(+) cells also correlated with ex vivo viral expression. Finally, the inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis was associated with significantly increased CD4(+)CD45RO(+) cell proliferation. We suggest that there is persistent viral gene expression in vivo, which is necessary for the maintenance of the proviral load and determines HTLV-1-associated myelopathy/tropical spastic paraparesis risk