Tulathromycin exerts proresolving effects in bovine neutrophils by inhibiting phospholipases and altering leukotriene B4, prostaglandin E2, and lipoxin A4 production.

The accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4 (LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4 [LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytes in vitro and in Mannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammation in vivo and characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4 and prostaglandin E2 (PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2 (PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4 in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.

Direct and indirect anti-inflammatory effects of tulathromycin in bovine macrophages: inhibition of CXCL-8 secretion, induction of apoptosis, and promotion of efferocytosis.

Recent evidence indicates that immunomodulation by antibiotics may enhance their clinical efficacy. Specifically, drug-induced leukocyte apoptosis and macrophage efferocytosis have been shown to promote the resolution of inflammation in a variety of disease settings. Tulathromycin is a new macrolide antibiotic for the treatment of bovine respiratory disease. The direct antimicrobial effects of the drug alone do not fully justify its superior clinical efficacy, and we hypothesize that tulathromycin may have immunomodulating properties. We recently reported that tulathromycin promotes apoptosis and inhibits proinflammatory NF-κB signaling in bovine neutrophils. In this study, we investigated the direct and indirect anti-inflammatory effects of tulathromycin in bovine macrophages. The findings indicate that bovine monocyte-derived macrophages and alveolar macrophages readily phagocytose tulathromycin-induced apoptotic neutrophils both in vitro and in the airways of Mannheimia haemolytica-infected calves. Moreover, tulathromycin promotes delayed, concentration-dependent apoptosis, but not necrosis, in bovine macrophages in vitro. Activation of caspase-3 and detection of mono- and oligonucleosomes in bovine monocyte-derived macrophages treated with tulathromycin was observed 12 h posttreatment; pretreatment with a pan-caspase inhibitor (ZVAD) blocked the proapoptotic effects of the drug. Lastly, tulathromycin inhibited the secretion of proinflammatory CXCL-8 in lipopolysaccharide (LPS)-stimulated bovine macrophages; this effect was independent of caspase activation or programmed cell death. Taken together, these immunomodulating effects observed in bovine macrophages help further elucidate the mechanisms through which tulathromycin confers anti-inflammatory and proresolution benefits. Furthermore, these findings offer novel insights on how antibiotics may offer anti-inflammatory benefits by modulating macrophage-mediated events that play a key role in inflammation.

The relationship between NMDA receptor function and the high ammonia tolerance of anoxia-tolerant goldfish.

Acute ammonia toxicity in vertebrates is thought to be characterized by a cascade of deleterious events resembling those associated with anoxic/ischemic injury in the central nervous system. A key event is the over-stimulation of neuronal N-methyl-D-aspartate (NMDA) receptors, which leads to excitotoxic cell death. The similarity between the responses to acute ammonia toxicity and anoxia suggests that anoxia-tolerant animals such as the goldfish (Carassius auratus Linnaeus) may also be ammonia tolerant. To test this hypothesis, the responses of goldfish were compared with those of the anoxia-sensitive rainbow trout (Oncorhynchus mykiss Walbaum) during exposure to high external ammonia (HEA). Acute toxicity tests revealed that goldfish are ammonia tolerant, with 96 h median lethal concentration (LC(50)) values of 199 µmol l(-1) and 4132 µmol l(-1) for NH(3) and total ammonia ([T(Amm)]=[NH(3)]+[NH(4)(+)]), respectively. These values were ~5-6 times greater than corresponding NH(3) and T(Amm) LC(50) values measured in rainbow trout. Further, the goldfish readily coped with chronic exposure to NH(4)Cl (3-5 mmol l(-1)) for 5 days, despite 6-fold increases in plasma [T] to ~1300 µmol l(-1) and 3-fold increases in brain [T(Amm)] to 6700 µmol l(-1). Muscle [T(Amm)] increased by almost 8-fold from ~900 µmol kg(-1) wet mass (WM) to greater than 7000 µmol kg(-1) WM by 48 h, and stabilized. Although urea excretion rates (J(Urea)) increased by 2-3-fold during HEA, the increases were insufficient to offset the inhibition of ammonia excretion that occurred, and increases in urea were not observed in the brain or muscle. There was a marked increase in brain glutamine concentration at HEA, from ~3000 µmol kg(-1) WM to 15,000 µmol kg(-1) WM after 48 h, which is consistent with the hypothesis that glutamine production is associated with ammonia detoxification. Injection of the NMDA receptor antagonists MK801 (0.5-8 mg kg(-1)) or ethanol (1-8 mg kg(-1)) increased trout survival time by 1.5-2.0-fold during exposure to 2 mmol l(-1) ammonia, suggesting that excitotoxic cell death contributes to ammonia toxicity in this species. In contrast, similar doses of MK801 or ethanol had no effect on ammonia-challenged (8-9.5 mmol l(-1) T(Amm)) goldfish survival times, suggesting that greater resistance to excitotoxic cell death contributes to the high ammonia-tolerance of the goldfish. Whole-cell recordings measured in isolated brain slices of goldfish telencephalon during in vitro exposure to 5 mmol l(-1) or 10 mmol l(-1) T(Amm) reversibly potentiated NMDA receptor currents. This observation suggested that goldfish neurons may not be completely resistant to ammonia-induced excitotoxicity. Subsequent western blot and densitometric analyses revealed that NMDA receptor NR1 subunit abundance was 40-60% lower in goldfish exposed to 3-5 mmol l(-1) T(Amm) for 5 days, which was followed by a restoration of NR1 subunit abundance after 3 days recovery in ammonia-free water. We conclude that the goldfish brain may be protected from excitotoxicity by downregulating the abundance of functional NMDA receptors during periods when it experiences increased internal ammonia.

Immunomodulatory effects of tulathromycin on apoptosis, efferocytosis, and proinflammatory leukotriene B4 production in leukocytes from Actinobacillus pleuropneumoniae-or zymosan-challenged pigs.

OBJECTIVE: To investigate the anti-inflammatory and immunomodulatory properties of tulathromycin in vitro and in experimental models of Actinobacillus pleuropneumoniae-induced pleuropneumonia and zymosan-induced pulmonary inflammation in pigs. ANIMALS: Blood samples from six 8- to 30-week-old healthy male pigs for the in vitro experiment and sixty-five 3-week-old specific pathogen-free pigs. PROCEDURES: Neutrophils and monocyte-derived macrophages were isolated from blood samples. Isolated cells were exposed to tulathromycin (0.02 to 2.0 mg/mL) for various durations and assessed for markers of apoptosis and efferocytosis. For in vivo experiments, pigs were inoculated intratracheally with A pleuropneumoniae, zymosan, or PBS solution (control group) with or without tulathromycin pretreatment (2.5 mg/kg, IM). Bronchoalveolar lavage fluid was collected 3 and 24 hours after inoculation and analyzed for proinflammatory mediators, leukocyte apoptosis, and efferocytosis. RESULTS: In vitro, tulathromycin induced time- and concentration-dependent apoptosis in neutrophils, which enhanced their subsequent clearance by macrophages. In the lungs of both A pleuropneumoniae- and zymosan-challenged pigs, tulathromycin promoted leukocyte apoptosis and efferocytosis and inhibited proinflammatory leukotriene B4 production, with a concurrent reduction in leukocyte necrosis relative to that of control pigs. Tulathromycin also attenuated the degree of lung damage and lesion progression in A pleuropneumoniae-inoculated pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Tulathromycin had immunomodulatory effects in leukocytes in vitro and anti-inflammatory effects in pigs in experimental models of A pleuropneumoniae infection and nonmicrobial-induced pulmonary inflammation. These data suggested that in addition to its antimicrobial properties, tulathromycin may dampen severe proinflammatory responses and drive resolution of inflammation in pigs with microbial pulmonary infections.

Anti-inflammatory effects of retinoids and carotenoid derivatives on caspase-3-dependent apoptosis and efferocytosis of bovine neutrophils.

OBJECTIVE: To evaluate immunomodulatory properties of all-trans retinoic acid and a fully oxidized ß-carotene dietary product in calves with Mannheimia haemolytica-induced pneumonia. ANIMALS: Twenty-five 6- to 10-week-old male Holstein calves for experimental inoculations and three 8- to 30-week-old Angus heifers for blood donations. PROCEDURES: In vitro, neutrophils and monocyte-derived macrophages isolated from blood of healthy Angus heifers were treated with all-trans retinoic acid (1 µM) or fully oxidized ß-carotene (8.3 µg/mL) for various times and assessed for markers of cellular death, antimicrobial function, and production of proinflammatory leukotriene B4. Following 28 days of dietary supplementation with fully oxidized ß-carotene, Holstein calves were experimentally inoculated with M haemolytica. Bronchoalveolar lavage fluid was collected at 3 and 24 hours after challenge inoculation and analyzed for markers of apoptosis. RESULTS: In vitro, all-trans retinoic acid and fully oxidized ß-carotene induced cell-selective, caspase-3-dependent apoptosis in neutrophils, which subsequently enhanced efferocytosis in macrophages. Conversely, neither treatment altered phorbol 12-myristate 13-acetate-induced oxidative burst, phagocytosis of nonopsonized zymosan (complement or antibody independent), or M haemolytica-induced leukotriene B4 production in bovine neutrophils. In vivo, fully oxidized ß-carotene enhanced leukocyte apoptosis in bronchoalveolar lavage fluid as well as subsequent efferocytosis by macrophages without altering numbers of circulating leukocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophil apoptosis and subsequent efferocytosis by macrophages are key mechanisms in the resolution of inflammation. Findings for the present study indicated that all-trans retinoic acid and fully oxidized ß-carotene could be novel nutraceutical strategies that may confer anti-inflammatory benefits for cattle with respiratory tract disease.