Extracellular adenosine signaling induces CX3CL1 expression in the brain to promote experimental autoimmune encephalomyelitis

BACKGROUND: Multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) are debilitating neuroinflammatory diseases mediated by lymphocyte entry into the central nervous system (CNS). While it is not known what triggers lymphocyte entry into the CNS during neuroinflammation, blockade of lymphocyte migration has been shown to be effective in controlling neuroinflammatory diseases. Since we have previously shown that extracellular adenosine is a key mediator of lymphocyte migration into the CNS during EAE progression, we wanted to determine which factors are regulated by adenosine to modulate EAE development. METHODS: We performed a genetic analysis of wild type and CD73−/− (that are unable to produce extracellular adenosine and are protected from EAE development) to identify factors that are both important for EAE development and controlled by extracellular adenosine signaling. RESULTS: We show that extracellular adenosine triggered lymphocyte migration into the CNS by inducing the expression of the specialized chemokine/adhesion molecule CX3CL1 at the choroid plexus. In wild type mice, CX3CL1 is upregulated in the brain on Day 10 post EAE induction, which corresponds with initial CNS lymphocyte infiltration and the acute stage of EAE. Conversely, mice that cannot synthesize extracellular adenosine (CD73−/− mice) do not upregulate CX3CL1 in the brain following EAE induction and are protected from EAE development and its associated lymphocyte infiltration. Additionally, blockade of the A2A adenosine receptor following EAE induction prevents disease development and the induction of brain CX3CL1 expression. The CX3CL1 induced during EAE is found on the choroid plexus, which is the barrier between the blood and cerebral spinal fluid in the brain and is a prime entry point into the CNS for immune cells. Furthermore, CX3CL1 expression can be induced in the brains of mice and in choroid plexus cell line following A2A adenosine receptor agonist administration. Most importantly, we show that CX3CL1 blockade protects against EAE development and inhibits lymphocyte entry into the CNS. CONCLUSIONS: We conclude that extracellular adenosine is an endogenous modulator of neuroinflammation during EAE that induces CX3CL1 at the choroid plexus to trigger lymphocyte entry into the brain

CD73 Is Critical for the Resolution of Murine Colonic Inflammation

CD73 is a glycosyl-phosphatidylinositol-(GPI-) linked membrane protein that catalyzes the extracellular dephosphorylation of adenosine monophosphate (AMP) to adenosine. Adenosine is a negative regulator of inflammation and prevents excessive cellular damage. We investigated the role of extracellular adenosine in the intestinal mucosa during the development of Dextran-Sulfate-Sodium-(DSS-)salt-induced colitis in mice that lack CD73 (CD73−/−) and are unable to synthesize extracellular adenosine. We have found that, compared to wild-type (WT) mice, CD73−/− mice are highly susceptible to DSS-induced colitis. CD73−/− mice exhibit pronounced weight loss, slower weight recovery, an increase in gut permeability, a decrease in expression of tight junctional adhesion molecules, as well as unresolved inflammation following the removal of DSS. Moreover, colonic epithelia in CD73−/− mice exhibited increased TLR9 expression, high levels of IL-1β and TNF-α, and constitutive activation of NF-κB. We conclude that CD73 expression in the colon is critical for regulating the magnitude and the resolution of colonic immune responses.National Institutes of Health (U.S.) (grant A1072434-A2)National Institutes of Health (U.S.) (grant R01NS063011

The human male liver is predisposed to inflammation via enhanced myeloid responses to inflammatory triggers

BACKGROUND & AIM: Men have a higher prevalence of liver disease. Liver myeloid cells can regulate tissue inflammation, which drives progression of liver disease. We hypothesized that sex alters the responsiveness of liver myeloid cells, predisposing men to severe liver inflammation. METHODS: Luminex was done on plasma from Hepatitis B Virus infected patients undergoing nucleoside analogue cessation in 45 male and female patients. We collected immune cells from the sinusoids of uninfected livers of 53 male and female donors. Multiparametric flow cytometry was used to phenotype and characterize immune composition. Isolated monocytes were stimulated with TLR ligands to measure the inflammatory potential and the expression of regulators of TLR signaling. RESULTS: We confirmed that men experienced more frequent and severe liver damage upon Hepatitis B Virus reactivation, which was associated with inflammatory markers of myeloid activation. No differences were observed in the frequency or phenotype of sinusoidal myeloid cells between male and female livers. However, monocytes from male livers produced more inflammatory cytokines and chemokines in response to TLR stimulation than female monocytes. We investigated negative regulators of TLR signaling and found that TOLLIP was elevated in female liver-derived monocytes. CONCLUSIONS: Our data show that enhanced responsiveness of myeloid cells from the male liver predisposes men to inflammation, which was associated with altered expression of negative regulators of TLR signaling

HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell

Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses

Gamma Interferon Positively Modulates Actinobacillus actinomycetemcomitans-Specific RANKL(+) CD4(+) Th-Cell-Mediated Alveolar Bone Destruction In Vivo

Recent studies have shown the biological and clinical significance of signaling pathways of osteogenic cytokines RANKL-RANK/OPG in controlling osteoclastogenesis associated with bone pathologies, including rheumatoid arthritis, osteoporosis, and other osteolytic disorders. In contrast to the inhibitory effect of gamma interferon (IFN-γ) on RANKL-mediated osteoclastogenesis reported recently, alternative new evidence is demonstrated via studies of experimental periodontitis using humanized NOD/SCID and diabetic NOD mice and clinical human T-cell isolates from diseased periodontal tissues, where the presence of increasing IFN-γ is clearly associated with (i) enhanced Actinobacillus actinomycetemcomitans-specific RANKL-expressing CD4(+) Th cell-mediated alveolar bone loss during the progression of periodontal disease and (ii) a concomitant and significantly increased coexpression of IFN-γ in RANKL(+) CD4(+) Th cells. Therefore, there are more complex networks in regulating RANKL-RANK/OPG signaling pathways for osteoclastogenesis in vivo than have been suggested to date

Extracellular adenosine signaling induces CX3CL1 expression in the brain to promote experimental autoimmune encephalomyelitis

Abstract Background Multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) are debilitating neuroinflammatory diseases mediated by lymphocyte entry into the central nervous system (CNS). While it is not known what triggers lymphocyte entry into the CNS during neuroinflammation, blockade of lymphocyte migration has been shown to be effective in controlling neuroinflammatory diseases. Since we have previously shown that extracellular adenosine is a key mediator of lymphocyte migration into the CNS during EAE progression, we wanted to determine which factors are regulated by adenosine to modulate EAE development. Methods We performed a genetic analysis of wild type and CD73−/− (that are unable to produce extracellular adenosine and are protected from EAE development) to identify factors that are both important for EAE development and controlled by extracellular adenosine signaling. Results We show that extracellular adenosine triggered lymphocyte migration into the CNS by inducing the expression of the specialized chemokine/adhesion molecule CX3CL1 at the choroid plexus. In wild type mice, CX3CL1 is upregulated in the brain on Day 10 post EAE induction, which corresponds with initial CNS lymphocyte infiltration and the acute stage of EAE. Conversely, mice that cannot synthesize extracellular adenosine (CD73−/− mice) do not upregulate CX3CL1 in the brain following EAE induction and are protected from EAE development and its associated lymphocyte infiltration. Additionally, blockade of the A2A adenosine receptor following EAE induction prevents disease development and the induction of brain CX3CL1 expression. The CX3CL1 induced during EAE is found on the choroid plexus, which is the barrier between the blood and cerebral spinal fluid in the brain and is a prime entry point into the CNS for immune cells. Furthermore, CX3CL1 expression can be induced in the brains of mice and in choroid plexus cell line following A2A adenosine receptor agonist administration. Most importantly, we show that CX3CL1 blockade protects against EAE development and inhibits lymphocyte entry into the CNS. Conclusions We conclude that extracellular adenosine is an endogenous modulator of neuroinflammation during EAE that induces CX3CL1 at the choroid plexus to trigger lymphocyte entry into the brain.</p

Earlier onset of HIV gene expression in PBMCs by coculture infection.

<p>(<b>A</b>) Gating strategy for detectably infected target cell frequency. Donors were labelled with CTFR and infection was assayed by flow cytometry following p24 staining for HIV Gag. Top row is coculture infection, bottom row is cell-free infection. Percent of infected targets in the population (bottom right quadrant) shown in red, and values for other subpopulations in black. (<b>B</b>) Timing of coculture (red) versus cell-free (blue) infection. Shown are means and standard errors of duplicates from one of three experiments with different blood donors. Frequencies of infected target cells were normalized by infected target cell frequency at 50 hours post infection. Means±std of best fit Gamma distributions were 22.1±9.3 hours for coculture and 34.2±9.1 hours for cell-free infection. Best fit MOI for the coculture infection was 5.0 (dashed red line). Inset: Infection of purified CD4⁺ T cells by cell-free HIV or autologous infected CD4⁺ T cells. Shown are mean and standard errors of duplicates of the frequencies of infected target cells normalized to the frequency of infected target cells at 48 hours post-infection. One of three experiments, each performed on purified CD4⁺ T cells from a different individual. Timing differences between cell-free and coculture in CD4⁺ T cells were statistically significant at 12, 24 and 36 hours post-infection (p<0.01, two-tailed unpaired t-test corrected for multiple comparisons using the Sidak-Bonferroni method).</p

Modelling faster onset of HIV gene expression by multiple infections per cell.

<p>(<b>A</b>) Proposed probabilistic mechanism for the more rapid onset of HIV gene expression. (<b>B</b>) Simulation of cell-free infection times at different MOI by random draws from a Gamma distribution parametrizing the best fit cell-free infection times of single virion infections. Circles are experimental data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005964#ppat.1005964.g003" target="_blank">Fig 3A</a>, dashed lines represent the simulation results for MOI of 0.1 (blue), 0.5 (green), 2 (red), and 4 (purple). Fitted means±std of infection times were 26.4±5.2, 25.2±5.1, 22.7±4.8, and 21.9±4.9 hours. (<b>C</b>) Fit of coculture versus cell-free infection. Circles are experimental data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005964#ppat.1005964.g001" target="_blank">Fig 1C</a>, dashed lines represent the simulation results. Means±std for the fits were 28.0±5.0 hours for coculture and 34.5±6.2 hours for cell-free infections. Best fit MOI for the coculture infection was 4.6.</p

Decreased sensitivity of coculture HIV spread to RAL predicts multiple infections per cell.

<p>Sensitivity of coculture (red) and cell-free (blue) infection to the integrase inhibitor raltegravir (RAL) in PBMCs. Points are means and standard errors of duplicates from one of three experiments with different blood donors. Transmission index (Tx) was calculated as the number of infected target cells with RAL divided by the number of infected target cells without RAL. Blue dashed line represents parametrization of the cell-free infection response to RAL in terms of IC₅₀ (1.9 nM) and hill coefficient (0.7). Red dashed line represents the fit of coculture infection using the decreased sensitivity to RAL (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005964#sec008" target="_blank">Materials and methods</a>). An effective MOI of 4.8 is predicted for coculture infection. Inset: Sensitivity of primary CD4⁺ T cell infection to RAL. Points are Tx means and standard errors of duplicates from one of three experiments with different blood donors. The drug sensitivity differences in CD4⁺ T cells between cell-free and coculture was statistically significant (p < 0.001, two-tailed unpaired t-test).</p