Diabetic patients experience a higher risk for severe periodontitis; however, the underlying mechanism remains unclear. We investigated the contribution of antibacterial T-cell-mediated immunity to enhanced alveolar bone loss during periodontal infection in nonobese diabetic (NOD) mice by oral inoculation with Actinobacillus actinomycetemcomitans, a G(؊) anaerobe responsible for juvenile and severe periodontitis. The results show that 1) inoculation with A. actinomycetemcomitans in pre-diabetic NOD mice does not alter the onset, incidence, and severity of diabetes; 2) after A. actinomycetemcomitans inoculation, diabetic NOD mice (blood glucose >200 mg/dl and with severe insulitis) exhibit significantly higher alveolar bone loss compared with pre-diabetic and nondiabetic NOD mice; and 3) A. actinomycetemcomitans-reactive CD4 ؉ T-cells in diabetic mice exhibit significantly higher proliferation and receptor activator of nuclear factor B ligand (RANKL) expression. When diabetic mice are treated with the RANKL antagonist osteoprotegerin (OPG), there is a significant reversal of alveolar bone loss, as well as reduced RANKL expression in A. actinomycetemcomitans-reactive CD4 ؉ T-cells. This study clearly describes the impact of autoimmunity to anaerobic infection in an experimental periodontitis model of type 1 diabetes. Thus, microorganism-reactive CD4 ؉ T-cells and the RANKL-OPG axis provide the molecular basis of the advanced periodontal breakdown in diabetes and, therefore, OPG may hold therapeutic potential for treating bone loss in diabetic subjects at high risk. Diabetes

A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states.DOI: http://dx.doi.org/10.7554/eLife.22028.001

Within the past decade, the critical roles of T cells and T cell-mediated immunity in inflammation-induced osteoclastogenesis and subsequent bone loss have been extensively studied, thereby establishing the new paradigm of osteoimmunology. Therefore, dendritic cells (DCs), the most potent antigenpresenting cells, responsible for activation of naïve T cells and orchestration of the immune response, became critically situated at the osteo-immune interface. Today, emerging new evidence suggests that DC may be directly involved in inflammation-induced osteoclastogenesis and bone loss, by acting as osteoclast (OC) precursors that can further develop into DC-derived OCs (DDOC) under inflammatory conditions. These findings have tremendous implications, because in addition to DC's important roles in regulating innate and adaptive immunity, a direct contribution by these cells to inflammation-induced bone loss may provide a promising therapeutic target not only for controlling inflammation but also for modulating bone destruction.

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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.

BackgroundMultiple 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.MethodsWe 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.ResultsWe 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.ConclusionsWe 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.

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.

Toxoplasma gondii is an obligate intracellular protozoan pathogen that traffics to the central nervous system (CNS) following invasion of its host. In the CNS, T. gondii undergoes transformation from a rapidly dividing tachyzoite to a long-lived, slow-dividing bradyzoite contained within cysts. The role of extracellular adenosine in T. gondii pathogenesis has not been previously investigated. T. gondii uses host purines such as adenosine for its energy needs, as it is unable to make its own. Here, we show that CD73 −/− mice, which lack the ability to generate extracellular adenosine, are protected from T. gondii chronic infection, with significantly fewer cysts and reduced susceptibility to reactivation of infection in the CNS independent of host effector function. Parasite dissemination to the brain was unimpaired in CD73 −/− hosts, suggesting that the reduced cyst number is due to impaired parasite differentiation in the CNS. Confirming this, T. gondii tachyzoites formed fewer cysts following alkaline pH stress in astrocytes isolated from CD73 −/− mice compared with wild type, and in fibroblasts treated with a CD73 inhibitor. Cyst formation was rescued in CD73 −/− astrocytes supplemented with adenosine, but not with adenosine receptor agonist 5′- N -ethylcarboxamidoadenosine. Furthermore, mice lacking adenosine receptors had no defect in cyst formation. Based on these findings, we conclude that CD73 expression promotes Toxoplasma bradyzoite differentiation and cyst formation by a mechanism dependent on the generation of adenosine, but independent of adenosine receptor signaling. Overall, these findings suggest that modulators of extracellular adenosine may be used to develop therapies aimed at defending against human toxoplasmosis.