Jonathan Worthington

Introduction: The fluoroquinolone levofloxacin is often selected for use prophylactically as well as during respiratory infections. However, studies on how these antibiotics may alter innate immunity, as opposed to their bactericidal activity, are limited. Materials & Methods: We employed a murine model of therapeutically relevant antibiotic dosing to investigate the effect of prophylactic levofloxacin treatment on innate immunity. Results: We observed mild pathology at the barrier sites of both the lung and colon in terms of alveolar space and goblet cell numbers, respectively. Although we saw no alteration in lung immune populations of neutrophils, eosinophils, or dendritic cells, we did see heightened expression of macrophage inducible nitric oxide synthase (iNOS). Interestingly this was only present in the shorter-lived CD206− interstitial macrophage subset and not observed in the long-lived resident alveolar population. Within the large intestine levofloxacin also targeted iNOS expression in the shorter-lived TIM4-CD4+ population but conversely inhibiting expression in the microbially rich colon. We therefore utilized the bone marrow-derived macrophage system, devoid of microbial interactions and demonstrated that levofloxacin had a direct effect on driving iNOS expression and increasing phagocytosis but only when present in developing macrophages and not mature macrophage populations. Our macrophage observations were replicated in ciprofloxacin, but not doxycycline-treated animals, indicating a fluoroquinolone specific action. Mechanistically, fluoroquinolone treatment was associated with mitochondrial hyperpolarization, indicating a direct alteration of macrophage immunity via off target effects. Conclusion: Collectively, this study demonstrates a direct action of fluoroquinolones on macrophage immunity, which should be considered when selecting antibiotics for tissue specific and prophylactic use.

Neuromedin U (NMU) belongs to a family of multifunctional neuropeptides that modulate the activity of several neural networks of the brain. Acting via metabotropic receptor NMUR2, NMU plays a role in the regulation of multiple systems, including energy homeostasis, stress responses, circadian rhythms, and endocrine signaling. The involvement of NMU signaling in the central regulation of important neurophysiological processes and its disturbances is a potential target for pharmacological modulation. Number of preclinical studies have proven that both modified NMU analogues such as PASR8‐NMU or F4R8‐NMU and designed NMUR2 agonists, for example, CPN‐116, CPN‐124 exhibit a distinct pharmacological activity especially when delivered transnasally. Their application can potentially be useful in the more convenient and safe treatment of obesity, eating disorders, Alzheimer's disease‐related memory impairment, alcohol addiction, and sleep disturbances. Accumulating findings suggest that pharmacomodulation of the central NMU signaling may be a promising strategy in the treatment of several neuropsychiatric disorders.

Background Newly identified multifunctional peptidergic modulators of stress responses: neuromedin U (NMU) and neuropeptide S (NPS) are involved in the wide spectrum of brain functions. However, there are no reports dealing with potential molecular relationships between the action of diverse anxiolytic or antidepressant drugs and NMU and NPS signaling in the brain. The present work was therefore focused on local expression of the aforementioned stress-related neuropeptides in the rat brain after long-term treatment with escitalopram and clonazepam. Methods Studies were carried out on adult, male Sprague-Dawley rats that were divided into 3 groups: animals injected with saline (control) and experimental individuals treated with escitalopram (at single dose 5 mg/kg daily), and clonazepam (at single dose 0.5 mg/kg). All individuals were sacrificed under anaesthesia and the whole brain excised. Total mRNA was isolated from homogenized samples of amygdala, hippocampus, hypothalamus, thalamus, cerebellum and brainstem. Real time-PCR method was used for estimation of related NPS, NPS receptor (NPSR), NMU, NMU and receptor 2 (NMUR2) mRNA expression. The whole brains were also sliced for general immunohistochemical assessment of the neuropeptides expression. Results Chronic administration of clonazepam resulted in an increase of NMU mRNA expression and formation of NMU-expressing fibers in the amygdala, while escitalopram produced a significant decrease in NPSR mRNA level in hypothalamus. Long-term escitalopram administration affects the local expression of examined neuropeptides mRNA in a varied manner depending on the brain structure. Conclusions Pharmacological effects of escitalopram may be connected with local at least partially NPSR-related alterations in the NPS/NMU/NMUR2 gene expression at the level selected rat brain regions. A novel alternative mode of SSRI action can be therefore cautiously proposed.

Background Neuropeptide S (NPS) is a multifunctional regulatory factor that exhibits a potent anxiolytic activity in animal models. However, there are no reports dealing with the potential molecular relationships between the anxiolytic activity of selective serotonin reuptake inhibitors (SSRIs) and NPS signaling, especially in the context of novel stress-related neuropeptides action. The present work therefore focused on gene expression of novel stress neuropeptides in the rat brain after acute treatment with escitalopram and in combination with neuropeptide S receptor (NPSR) blockade. Methods Studies were carried out on adult, male Sprague–Dawley rats that were divided into five groups: animals injected with saline (control) and experimental rats treated with escitalopram (at single dose 10 mg/kg daily), escitalopram and SHA-68, a selective NPSR antagonist (at a single dose of 40 mg/kg), SHA-68 alone and corresponding vehicle (solvent SHA-68) control. To measure anxiety-like behavior and locomotor activity the open field test was performed. All individuals were killed under anaesthesia and the whole brain was excised. Total mRNA was isolated from homogenized samples of the amygdala, hippocampus, hypothalamus, thalamus, cerebellum, and brainstem. Real-time PCR was used for estimation of related NPS, NPSR, neuromedin U (NMU), NMU receptor 2 (NMUR2) and nesfatin-1 precursor nucleobindin-2 (NUCB2) gene expression. Results Acute escitalopram administration affects the local expression of the examined neuropeptides mRNA in a varied manner depending on brain location. An increase in NPSR and NUCB2 mRNA expression in the hypothalamus and brainstem was abolished by SHA-68 coadministration, while NMU mRNA expression was upregulated after NPSR blockade in the hippocampus and cerebellum. Conclusions The pharmacological effects of escitalopram may be connected with local NPSR-related alterations in NPS/NMU/NMUR2 and nesfatin-1 gene expression at the level of selected rat brain regions. A novel alternative mode of SSRI action can be therefore cautiously proposed.

Background: Neuropeptide S (NPS) is a multifunctional regulatory factor that exhibits a potent anxiolytic activity in animal models. However, there are no reports dealing with the potential molecular interactions between the activity of selective serotonin reuptake inhibitors (SSRIs) and NPS signaling, especially in the context of adult neurogenesis and the expression of noncanonical stress-related neuropeptides such as neuromedin U (NMU). The present work therefore focused on immunoexpression of neuromedin U receptor 2 (NMUR2) and doublecortin (DCX) in the rat hippocampus after acute treatment with escitalopram and in combination with selective neuropeptide S receptor (NPSR) blockade. Methods: Studies were carried out on adult, male Sprague-Dawley rats that were divided into five groups: animals injected with saline (control) and experimental individuals treated with escitalopram (at single dose 10 mg/kg daily), escitalopram + SHA-68, a selective NPSR antagonist (at single dose 40 mg/kg), SHA-68 alone, and corresponding vehicle control. All animals were sacrificed under halothane anaesthesia. The whole hippocampi were quickly excised, fixed, and finally sliced for general qualitative immunohistochemical assessment of the NPSR and NMUR2 expression. The number of immature neurons was enumerated using immunofluorescent detection of doublecortin (DCX) expression within the subgranular zone (SGZ). Results: Acute escitalopram administration affects the number of DCX and NMUR2-expressing cells in the adult rat hippocampus. A decreased number of DCX-expressing neuroblasts after treatment with escitalopram was augmented by SHA-68 coadministration. Conclusions: Early pharmacological effects of escitalopram may be at least partly connected with local NPSR-related alterations of neuroblast maturation in the rat hippocampus. Escitalopram may affect neuropeptide and DCX-expression starting even from the first dose. Adult neurogenesis may be regulated via paracrine neuropeptide S and NMU-related signaling.

Background: Neurosteroids are involved in several important brain functions and have recently been considered novel players in the mechanic actions of neuropsychiatric drugs. There are no reports of murine studies focusing on the effect of chronic neurosteroid treatment in parallel with antipsychotics on key steroidogenic enzyme expression and we therefore focused on steroidogenic enzyme gene expression in the brainstem of rats chronically treated with olanzapine and haloperidol. Methods and results: Studies were carried out on adult, male Sprague–Dawley rats which were divided into 3 groups: control and experimental animals treated with olanzapine or haloperidol. Total mRNA was isolated from homogenized brainstem samples for RealTime-PCR to estimate gene expression of related aromatase, 3β-HSD and P450scc. Long-term treatment with the selected antipsychotics was reflected in the modulation of steroidogenic enzyme gene expression in the examined brainstem region; with both olanzapine and haloperidol increasing aromatase, 3β-HSD and P450scc gene expression. Conclusions: The present findings shed new light on the pharmacology of antipsychotics and suggest the existence of possible regulatory interplay between neuroleptic action and steroidogenesis at the level of brainstem neuronal centres.

Background: Phoenixin, spexin and nesfatin-1 belong to a family of newly discovered multifunctional neuropeptides that play regulatory roles in several brain structures and modulate the activity of important neural networks. However, little is known about their expression and action at the level of brainstem. The present work was, therefore, focused on gene expression of the aforementioned peptides in the brainstem of rats chronically treated with olanzapine, a second generation antipsychotic drug. Methods: Studies were carried out on adult, male Sprague–Dawley rats that were divided into 2 groups: control and experimental animals treated with olanzapine (28-day-long intraperitoneal injection, at dose 5 mg/kg daily). All individuals were killed under anesthesia and the brainstem excised. Total mRNA was isolated from homogenized samples of both structures and the RT-PCR method was used for estimation of related SMIM20/phoenixin, NPQ/spexin and NUCB2/nesfatin-1 gene expression. Results: Long-term treatment with olanzapine is reflected in qualitatively different changes in expression of examined neuropeptides mRNA in the rat brainstem. Olanzapine significantly decreased NPQ/spexin mRNA expression, but increased SMIM20/phoenixin mRNA level in the rat brainstem; while NUCB2/nesfatin-1 mRNA expression remained unchanged. Conclusions: Olanzapine can affect novel peptidergic signaling in the rat brainstem. This may cautiously suggest the presence of an alternative mode of its action.