Persistent Toll-like receptor 7 stimulation induces behavioral and molecular innate immune tolerance.

Toll-like receptors 7 and 8 (TLR7 and TLR8) are endosomal pattern recognition receptors that detect a variety of single-stranded RNA species. While TLR7/8 agonists have robust therapeutic potential, clinical utility of these agents is limited by sickness responses associated with treatment induction. To understand the kinetics and mechanism of these responses, we characterized the acute and chronic effects of TLR7 stimulation. Single-cell RNA-sequencing studies, RNAscope, and radiolabeled in situ hybridization demonstrate that central nervous system gene expression of TLR7 is exclusive to microglia. In vitro studies demonstrate that microglia are highly sensitive to TLR7 stimulation, and respond in a dose-dependent manner to the imidazoquinoline R848. In vivo, both intraperitoneal (IP) and intracerebroventricular (ICV) R848 induce acute sickness responses including hypophagia, weight loss, and decreased voluntary locomotor activity, associated with increased CNS pro-inflammatory gene expression and changes to glial morphology. However, chronic daily IP R848 resulted in rapid tachyphylaxis of behavioral and molecular manifestations of illness. In microglial in vitro assays, pro-inflammatory transcriptional responses rapidly diminished in the context of repeated R848. In addition to TLR7 desensitization, we found that microglia become partially refractory to lipopolysaccharide (LPS) following R848 pretreatment, associated with induction of negative regulators A20 and Irak3. Similarly, mice pre-treated with R848 demonstrate reduced sickness responses, hypothalamic inflammation, and hepatic inflammation in response to LPS. These data combined demonstrate that TLR7 stimulation induces acute behavioral and molecular evidence of sickness responses. Following prolonged dosing, R848 induces a refractory state to both TLR7 and TLR4 activation, consistent with induced immune tolerance.

TLR2 activation causes tachyphylaxis to ß2 -agonists in vitro and ex vivo: modelling bacterial exacerbation.

BACKGROUND: Asthma is a widespread chronic health problem exacerbated by common viral and bacterial infections. Further research is required to understand how infection worsens asthma control in order to advance therapeutic options in the future. Recent research has revealed that ß2 -adrenergic receptor (ß2 -AR) agonists lose bronchodilatory efficacy because the receptor-mediated molecular pathways responsible for their beneficial actions are desensitized by infection. To date, most studies have focussed on viral infection, leaving the impact of bacterial infection on ß2 -AR desensitization relatively under-investigated. We address this in this study. METHODS AND RESULTS: Utilizing an in vitro model of bacterial exacerbation in airway smooth muscle (ASM) cells, we show that activation of toll-like receptor 2 (TLR2; mimicking bacterial infection) in the presence of an inflammatory stimulus leads to ß2 -AR desensitization. This occurs via TLR2-dependent upregulation of cyclooxygenase 2 (COX-2) mRNA expression and increased secretion of PGE2 . Importantly, PGE2 causes heterologous ß2 -AR desensitization and reduces cAMP production in response to short-acting (salbutamol) and long-acting (formoterol) ß2 -agonists. Thus, bacterial infectious stimuli act in a PGE2 -dependent manner to severely curtail the beneficial actions of ß2 -agonists. The impact of ß2 -AR desensitization is demonstrated by reduced gene expression of the critical anti-inflammatory molecule MKP-1 in response to ß2 -agonists, as well as impaired bronchodilation in a mouse lung slices. CONCLUSIONS: Taken together, our results show that, like viruses, bacteria induce prostanoid-dependent ß2 -AR desensitization on ASM cells. Notably, COX-2 inhibition with the specific inhibitor celecoxib represses PGE2 secretion, presenting a feasible pharmacological option for treatment of infectious exacerbation in asthma in the future.

Prevention of infusion reactions to PEGylated liposomal doxorubicin via tachyphylaxis induction by placebo vesicles: a porcine model.

PEGylated liposomal doxorubicin (Doxil) has been used in cancer chemotherapy for 16 years. Clinical experience shows that it can cause mild-to-severe hypersensitivity (infusion) reactions, which are manifestations of complement (C) activation-related pseudoallergy (CARPA). Although in most cases CARPA is inconsequential, a main symptom, cardiopulmonary distress, may be life threatening in hypersensitive individuals. To date, the prevention of Doxil-induced CARPA is based on premedication and a slow infusion protocol. The present study suggests desensitization by Doxil-like empty liposomes, called placebo Doxil (Doxebo), as an alternative strategy, which is based on the tachyphylactic nature of Doxil reactions. Doxebo-induced tolerance to Doxil was shown to develop within minutes and to be specific to Doxil-like PEGylated liposomes. The procedure of desensitization involves slow, low-dose pre-infusion of Doxebo before Doxil treatment which minimizes the ensuing physiological changes or keeps them subclinical. Although the mechanism of tolerance induction is not yet clear, the effector arm of C response is unlikely to be affected, as the vascular reactivity of desensitized pigs to zymosan remains intact. Desensitization with empty vesicles represents a novel approach for reducing the risk of anaphylactic reactions to drug carrier liposomes. The underlying immediate, most likely passive silencing of an innate immune response may represent a novel mechanism of tolerance induction which may work for other reactogenic nanosystems as well.

Interferon-beta responders and non-responders. A biological approach.

The therapeutic benefits of interferon-beta are limited, as some patients with multiple sclerosis (MS) do not respond to therapy. Based on their biological characteristics, non-responsive patients can be divided into three subgroups: genetic, pharmacological and pathogenetic non-responders. In order to tailor the best treatment in both newly diagnosed MS patients and those already receiving treatment, the neurologist must carefully consider the risk of treating non-responders.

Botulinum toxin, immunologic considerations with long-term repeated use, with emphasis on cosmetic applications.

Botulinum toxin is a unique pharmaceutical agent in wide-scale use for cosmetic and multiple therapeutic applications. Physicians using these agents for esthetic purpose must realize the potential for future therapeutic needs. Because most indications require repeated injections, the recipient is at risk for immunologic reactions with possible formation of neutralizing antibodies. An individual who is injected with botulinum toxin for cosmetic purposes could someday require an effective form of type A toxin for dystonia, pain, or spasticity.

IFN-alpha 2B enhances Th1 cytokine responses in bladder cancer patients receiving Mycobacterium bovis bacillus Calmette-Guérin immunotherapy.

Combination therapy with intravesical bacillus Calmette-Guérin (BCG) plus IFN-alpha for superficial bladder cancer has been demonstrated to be more effective than either single agent alone in animal studies and of suggested greater efficacy in clinical studies. However, the mechanism by which IFN-alpha enhances BCG-mediated antitumor activity is poorly understood. Using PBMCs from bladder cancer patients, IFN-alpha was found to substantially enhance the efficacy of BCG to induce IFN-gamma production. Among 34 patients tested, 80% showed >4-fold increase. This effect of IFN-alpha was observed in both initial and memory responses to BCG. In addition, IFN-alpha up-regulated BCG-induced IL-12 and TNF-alpha and down-regulated BCG-induced IL-10. Neutralizing endogenous IL-10 or adding exogenous IL-12 provided further synergy for IFN-gamma production. In clinical practice, intravesical IFN-alpha 2B (50 million units (MU)/dose) was observed to accelerate urinary IFN-gamma production to low-dose BCG (one-tenth or one-third of a full dose) in patients treated with combination therapy compared with BCG alone. These results suggest that IFN-alpha is a potent BCG enhancer that polarizes the BCG-induced immune response toward the cellular immune pathway by promoting Th1 cytokine expression and reducing Th2 cytokine expression. This study provides an immunological basis for future rational use of IFN-alpha in conjunction with intravesical BCG for bladder cancer immunotherapy.

Nicotine induces leukocyte rolling and adhesion in the cerebral microcirculation of the mouse.

Nicotine and several related metabolites were examined for their ability to induce leukocyte rolling and adhesion in the cerebral microcirculation of the mouse. A cranial window was surgically prepared for the visualization of the pial microcirculation using an intra-vital microscopy imaging system. Using this technique rhodamine-labeled leukocytes could be visualized and video-recorded as they traveled within the microvessels, and the quantitation of their rolling and adhesion along the pial venule walls was assessed during an off-line video playback analysis. Nicotine was found to produce significant dose-related increases in leukocyte rolling and adhesion. Cotinine, a major nicotine metabolite, did not induce the same degree of leukocyte rolling and adhesion. Mecamylamine, a nicotine antagonist, was found to inhibit the nicotine-induced leukocyte rolling and adhesion. Anti-P-selectin antibody blocked nicotine-induced leukocyte rolling, while anti-CD18 antibody effectively inhibited leukocyte adhesion, but not rolling in similar experiments. Nicotine-induced leukocyte rolling and adhesion were also inhibited by superoxide dismutase and catalase. These data suggest that nicotine, the principle pharmacological agent in cigarette smoke and related tobacco products, acts via a ganglionic-type nicotinic receptor to enhance leukocyte rolling via P-selectin and reactive oxygen radical-dependent mechanisms in cerebral microcirculation of the mouse.

Extracellular guanosine 3',5'-cyclic monophosphate and disodium cromoglycate share a similar spectrum of activity in the inhibition of histamine release from isolated mast cells and basophils.

In comparison to adenosine 3',5'-cyclic monophosphate (cAMP), surprisingly little is known regarding the role of guanosine 3',5'-cyclic monophosphate (cGMP) in the functional modulation of mast cells and basophils. In the present study, the ability of cGMP, cAMP, and a range of related compounds, to inhibit immunologically induced histamine release from these cells was investigated and compared to the anti-asthmatic drug disodium cromoglycate (DSCG). Exogenously applied cGMP produced a potent inhibition of histamine release from rat peritoneal mast cells, but cAMP had a negligible effect. The attenuation noted with cGMP was markedly reduced if cells were pretreated with the nucleotide before stimulation. Similar results were obtained with DSCG. The inhibitory and tachyphylactic effects noted with cGMP were mimicked by direct derivatives of the compound but not by a range of other cyclic or guanosine nucleotides. The time courses of the induced tachyphylaxis seen with cGMP and DSCG were similar, and cross-tachyphylaxis between the two compounds was observed. In addition, both cGMP and DSCG showed a comparable spectrum of activity against mast cells isolated from the mouse, guinea pig, and human. The parallel effects of the two agents suggests that they may inhibit mediator release from mast cells through similar mechanisms.

Inhibition profiles of sodium cromoglycate and nedocromil sodium on mediator release from mast cells of human skin, lung, tonsil, adenoid and intestine.

We have studied an aspect of the functional heterogeneity of human mast cells, namely responsiveness to the inhibitory effects of sodium cromoglycate and nedocromil sodium. The effects of these drugs were examined on the release of histamine and PGD2 from mast cells of human skin, lung, tonsils, adenoids and intestine. A high concentration, 1000 microM, of sodium cromoglycate was required to significantly inhibit histamine release from lung and tonsillar mast cells. Nedocromil sodium, 1000 microM, was more effective than sodium cromoglycate against histamine release from lung, tonsillar and adenoidal cells. Both compounds showed tachyphylaxis in lung and tonsillar mast cells but not in adenoidal and intestinal mast cells. In contrast, in intestinal mast cells, the effect of nedocromil sodium was weaker and more variable than sodium cromoglycate. Skin mast cells differed from mast cells of the other anatomical sites in being unresponsive to sodium cromoglycate and nedocromil sodium. Our results confirm that high concentrations of sodium cromoglycate and nedocromil sodium are required to achieve even modest inhibition of mediator release from human mast cells under in vitro conditions. Notwithstanding this, the results also indicate that differences exist among skin, lung, tonsillar, adenoidal and intestinal mast cells with respect to their sensitivity to sodium cromoglycate and nedocromil sodium, thus extending our knowledge of functional heterogeneity within the human mast cell populations.

Tachyphylaxis to repeated challenges with inhaled adenosine in asthmatic subjects.

Adenosine, when administered as an aerosol to asthmatic subjects, provokes a prompt dose-dependent bronchoconstriction. The aim of this study was to establish whether tachyphylaxis in response to repeated inhalations with adenosine occurs in asthmatic subjects. Eight asthmatic patients were studied on three separate days (days A, B, and C) at the same time of day for each subject. Three inhalation tests were performed, separated by one hour, on each study day. The study was conducted in a double-blind fashion and performed in random order. On day A, three adenosine inhalation tests were performed to assess adenosine tachyphylaxis. On day B, a saline inhalation test was carried out between two adenosine challenges to assess the time course of the adenosine tachyphylaxis. On day C, three methacholine inhalation tests were performed to confirm that tachyphylaxis does not occur to methacholine. Repeated inhalation with adenosine, but not methacholine, produced a progressive loss of responsiveness to the nucleoside, which was particularly manifest with the third challenge.