Toso controls encephalitogenic immune responses by dendritic cells and regulatory T cells.

The ability to mount a strong immune response against pathogens is crucial for mammalian survival. However, excessive and uncontrolled immune reactions can lead to autoimmunity. Unraveling how the reactive versus tolerogenic state is controlled might point toward novel therapeutic strategies to treat autoimmune diseases. The surface receptor Toso/Faim3 has been linked to apoptosis, IgM binding, and innate immune responses. In this study, we used Toso-deficient mice to investigate the importance of Toso in tolerance and autoimmunity. We found that Toso(-/-) mice do not develop severe experimental autoimmune encephalomyelitis (EAE), a mouse model for the human disease multiple sclerosis. Toso(-/-) dendritic cells were less sensitive to Toll-like receptor stimulation and induced significantly lower levels of disease-associated inflammatory T-cell responses. Consistent with this observation, the transfer of Toso(-/-) dendritic cells did not induce autoimmune diabetes, indicating their tolerogenic potential. In Toso(-/-) mice subjected to EAE induction, we found increased numbers of regulatory T cells and decreased encephalitogenic cellular infiltrates in the brain. Finally, inhibition of Toso activity in vivo at either an early or late stage of EAE induction prevented further disease progression. Taken together, our data identify Toso as a unique regulator of inflammatory autoimmune responses and an attractive target for therapeutic intervention.

Lymphocyte-derived ACh regulates local innate but not adaptive immunity.

Appropriate control of immune responses is a critical determinant of health. Here, we show that choline acetyltransferase (ChAT) is expressed and ACh is produced by B cells and other immune cells that have an impact on innate immunity. ChAT expression occurs in mucosal-associated lymph tissue, subsequent to microbial colonization, and is reduced by antibiotic treatment. MyD88-dependent Toll-like receptor up-regulates ChAT in a transient manner. Unlike the previously described CD4(+) T-cell population that is stimulated by norepinephrine to release ACh, ChAT(+) B cells release ACh after stimulation with sulfated cholecystokinin but not norepinephrine. ACh-producing B-cells reduce peritoneal neutrophil recruitment during sterile endotoxemia independent of the vagus nerve, without affecting innate immune cell activation. Endothelial cells treated with ACh in vitro reduced endothelial cell adhesion molecule expression in a muscarinic receptor-dependent manner. Despite this ability, ChAT(+) B cells were unable to suppress effector T-cell function in vivo. Therefore, ACh produced by lymphocytes has specific functions, with ChAT(+) B cells controlling the local recruitment of neutrophils.

Involvement of Toso in activation of monocytes, macrophages, and granulocytes.

Rapid activation of immune responses is necessary for antibacterial defense, but excessive immune activation can result in life-threatening septic shock. Understanding how these processes are balanced may provide novel therapeutic potential in treating inflammatory disease. Fc receptors are crucial for innate immune activation. However, the role of the putative Fc receptor for IgM, known as Toso/Faim3, has to this point been unclear. In this study, we generated Toso-deficient mice and used them to uncover a critical regulatory function of Toso in innate immune activation. Development of innate immune cells was intact in the absence of Toso, but Toso-deficient neutrophils exhibited more reactive oxygen species production and reduced phagocytosis of pathogens compared with controls. Cytokine production was also decreased in Toso(-/-) mice compared with WT animals, rendering them resistant to septic shock induced by lipopolysaccharide. However, Toso(-/-) mice also displayed limited cytokine production after infection with the bacterium Listeria monocytogenes that was correlated with elevated presence of Listeria throughout the body. Accordingly, Toso(-/-) mice succumbed to infections of L. monocytogenes, whereas WT mice successfully eliminated the infection. Taken together, our data reveal Toso to be a unique regulator of innate immune responses during bacterial infection and septic shock.

2-Methoxyestradiol inhibits experimental autoimmune encephalomyelitis through suppression of immune cell activation.

The endogenous metabolite of estradiol, 2-Methoxyestradiol (2ME2), is an antimitotic and antiangiogenic cancer drug candidate that also exhibits disease-modifying activity in animal models of rheumatoid arthritis (RA). We found that 2ME2 dramatically suppresses development of mouse experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). 2ME2 inhibits in vitro lymphocyte activation, cytokine production, and proliferation in a dose-dependent fashion. 2ME2 treatment of lymphocytes specifically reduced the nuclear translocation and transcriptional activity of nuclear factor of activated T-cells (NFAT) c1, whereas NF-κB and activator protein 1 (AP-1) activation were not adversely affected. We therefore propose that 2ME2 attenuates EAE through disruption of the NFAT pathway and subsequent lymphocyte activation. By extension, our findings provide a molecular rationale for the use of 2ME2 as a tolerable oral immunomodulatory agent for the treatment of autoimmune disorders such as MS in humans.

Adenylyl Cyclase 6 Mediates Inhibition of TNF in the Inflammatory Reflex.

Macrophage cytokine production is regulated by neural signals, for example in the inflammatory reflex. Signals in the vagus and splenic nerves are relayed by choline acetyltransferase+ T cells that release acetylcholine, the cognate ligand for alpha7 nicotinic acetylcholine subunit-containing receptors (α7nAChR), and suppress TNF release in macrophages. Here, we observed that electrical vagus nerve stimulation with a duration of 0.1-60 s significantly reduced systemic TNF release in experimental endotoxemia. This suppression of TNF was sustained for more than 24 h, but abolished in mice deficient in the α7nAChR subunit. Exposure of primary human macrophages and murine RAW 264.7 macrophage-like cells to selective ligands for α7nAChR for 1 h in vitro attenuated TNF production for up to 24 h in response to endotoxin. Pharmacological inhibition of adenylyl cyclase (AC) and knockdown of adenylyl cyclase 6 (AC6) or c-FOS abolished cholinergic suppression of endotoxin-induced TNF release. These findings indicate that action potentials in the inflammatory reflex trigger a change in macrophage behavior that requires AC and phosphorylation of the cAMP response element binding protein (CREB). These observations further our mechanistic understanding of neural regulation of inflammation and may have implications for development of bioelectronic medicine treatment of inflammatory diseases.

Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit.

Neural circuits regulate cytokine production to prevent potentially damaging inflammation. A prototypical vagus nerve circuit, the inflammatory reflex, inhibits tumor necrosis factor-α production in spleen by a mechanism requiring acetylcholine signaling through the α7 nicotinic acetylcholine receptor expressed on cytokine-producing macrophages. Nerve fibers in spleen lack the enzymatic machinery necessary for acetylcholine production; therefore, how does this neural circuit terminate in cholinergic signaling? We identified an acetylcholine-producing, memory phenotype T cell population in mice that is integral to the inflammatory reflex. These acetylcholine-producing T cells are required for inhibition of cytokine production by vagus nerve stimulation. Thus, action potentials originating in the vagus nerve regulate T cells, which in turn produce the neurotransmitter, acetylcholine, required to control innate immune responses.

Phase I combination of sorafenib and erlotinib therapy in solid tumors: safety, pharmacokinetic, and pharmacodynamic evaluation from an expansion cohort.

The aims of this study were to further define the safety of sorafenib and erlotinib, given at their full approved monotherapy doses, and to correlate pharmacokinetic and pharmacodynamic markers with clinical outcome. In addition, a novel pharmacodynamic marker based on the real-time measurement of RAF signal transduction capacity (STC) is described. Sorafenib was administered alone for a 1-week run-in period, and then both drugs were given together continuously. RAF STC was assessed in peripheral blood monocytes prior to erlotinib initiation. Epidermal growth factor receptor (EGFR) expression and K-RAS mutations were measured in archival tumor samples. Changes in pERK and CD31 were determined in fresh tumor biopsies obtained pretreatment, prior to erlotinib dosing, and during the administration of both drugs. In addition, positron emission tomography-computed tomography scans and pharmacokinetic assessments were done. Eleven patients received a total of 57 cycles (median, 5; range, 1-10). Only four patients received full doses of both drugs for the entire study course, with elevation of liver enzymes being the main reason for dose reductions and delays. Among 10 patients evaluable for response, 8 experienced tumor stabilization of >or=4 cycles. Pharmacokinetic analysis revealed no significant interaction of erlotinib with sorafenib. Sorafenib-induced decrease in RAF-STC showed statistically significant correlation with time-to-progression in seven patients. Other pharmacodynamic markers did not correlate with clinical outcome. This drug combination resulted in promising clinical activity in solid tumor patients although significant toxicity warrants close monitoring. RAF-STC deserves further study as a predictive marker for sorafenib.

Differential requirement of MALT1 for BAFF-induced outcomes in B cell subsets.

B cell activation factor of the TNF family (BAFF) activates noncanonical nuclear factor kappaB (NF-kappaB) heterodimers that promote B cell survival. We show that although MALT1 is largely dispensable for canonical NF-kappaB signaling downstream of the B cell receptor, the absence of MALT1 results in impaired BAFF-induced phosphorylation of NF-kappaB2 (p100), p100 degradation, and RelB nuclear translocation in B220(+) B cells. This corresponds with impaired survival of MALT1(-/-) marginal zone (MZ) but not follicular B cells in response to BAFF stimulation in vitro. MALT1(-/-) MZ B cells also express higher amounts of TRAF3, a known negative regulator of BAFF receptor-mediated signaling, and TRAF3 was found to interact with MALT1. Furthermore, phenotypes associated with overexpression of BAFF, including increased MZ B cell numbers, elevated serum immunoglobulin titers, and spontaneous germinal center formation, were found to be dependent on B cell-intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction.

Histamine h(4) and h(2) receptors control histamine-induced interleukin-16 release from human CD8(+) T cells.

Histamine is known to trigger the release of interleukin (IL)-16 from human CD8(+) cells. However, the individual roles of the presently known histamine receptor subtypes (H(1)-H(4)) in this inflammatory response have not been fully characterized. Histamine stimulation of human CD8(+) T lymphocytes purified from peripheral blood led to a 5- to 8-fold increase in the basal release of IL-16 within 24 h, and this increase was significantly blocked by the H(2)-selective antagonist, cimetidine, or by thioperamide, an antagonist of H(3) and H(4) receptors, respectively. The H(1) antagonist pyrilamine showed limited effects. Agonists selective for H(2) (dimaprit), H(3/4) (R-(-)-alpha-methylhistamine), and H(4) (clobenpropit) were capable of inducing the release of bioactive IL-16 because CD8(+) cell supernatants induced CD4(+) cell migration, which was abrogated by an anti-IL-16 antibody. Furthermore, preincubation of lymphocytes with pertussis toxin abolished IL-16 release triggered by activation of the G(i/o)-coupled H(4) receptor but not by the H(2) receptor. Messenger RNA expression studies confirmed H(4), H(2), and H(1) expression in human CD8(+) lymphocytes, whereas H(3) mRNA was completely absent. All leukocyte populations investigated expressed mRNA for H(4), with highest levels found in eosinophils, dendritic cells, and tonsil B cells. H(4) expression was also detected in human lung, trachea, and various cells of human lung origin, such as fibroblasts, bronchial smooth muscle cells, epithelial, and endothelial cells. Since many of those are known sources of IL-16, immune cell- and lung cell-expressed H(4) receptors may have a general role in the control of this mediator of inflammatory disorders such as asthma.