A clindamycin acetylated derivative with reduced antibacterial activity inhibits articular hyperalgesia and edema by attenuating neutrophil recruitment, NF-κB activation and tumor necrosis factor-α production.

We recently demonstrated that clindamycin exhibits activities in acute and chronic models of pain and inflammation. In the present study, we investigated the effects of clindamycin and a clindamycin acetylated derivative (CAD) in models of acute joint inflammation and in a microbiological assay. Joint inflammation was induced in mice by intraarticular (i.a.) injection of zymosan or lipopolysaccharide (LPS). Clindamycin or CAD were administered via the intraperitoneal route 1 h before zymosan or LPS. Paw withdrawal threshold, joint diameter, histological changes, neutrophil recruitment, tumor necrosis factor-α (TNF-α) production and phosphorylation of the IκBα and NF-κB/p65 were evaluated. In vitro assays were used to measure the antibacterial activity of clindamycin and CAD and also their effects on zymosan-induced TNF-α production by RAW264.7 macrophages. Clindamycin exhibited activity against Staphylococcus aureus and Salmonella Typhimurium ATCC® strains at much lower concentrations than CAD. Intraarticular injection of zymosan or LPS induced articular hyperalgesia, edema and neutrophil infiltration in the joints. Zymosan also induced histological changes, NF-κB activation and TNF-α production. Responses induced by zymosan and LPS were inhibited by clindamycin (200 and 400 mg/kg) or CAD (436 mg/kg). Both clindamycin and CAD inhibited in vitro TNF-α production by macrophages. In summary, we provided additional insights of the clindamycin immunomodulatory effects, whose mechanism was associated with NF-κB inhibition and reduced TNF-α production. Such effects were extended to a clindamycin derivative with reduced antibacterial activity, indicating that clindamycin derivatives should be investigated as candidates to drugs that could be useful in the management of inflammatory and painful conditions.

S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity.

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.

Phosphoinositide-3 kinase gamma regulates caspase-1 activation and leukocyte recruitment in acute murine gout.

This study investigates the participation of PI3Kγ in the development of joint inflammation and dysfunction in an experimental model of acute gout in mice. Acute gout was induced by injection of monosodium urate (MSU) crystals into the tibiofemoral joint of mice. The involvement of PI3Kγ was evaluated using a selective inhibitor and mice deficient for PI3Kγ (PI3Kγ-/- ) or with loss of kinase activity. Neutrophils recovered from the inflamed joint were quantified and stained for phosphorylated Akt (pAkt) and production of reactive oxygen species (ROS). The adherence of leukocytes to the joint microvasculature was assessed by intravital microscopy and cleaved caspase-1 by Western blot. Injection of MSU crystals induced massive accumulation of neutrophils expressing phosphorylated Akt. In the absence of PI3Kγ, there was reduction of pAkt expression, chemokine production, and neutrophil recruitment. Genetic or pharmacological inhibition of PI3Kγ reduced the adherence of leukocytes to the joint microvasculature, even in joints with established inflammation. Neutrophils from PI3Kγ-/- mice produced less ROS than wild-type neutrophils. There was decreased joint damage and dysfunction in the absence of PI3Kγ. In addition, in the absence of PI3Kγ activity, there was reduction of cleaved caspase-1 and IL-1ß production in synovial tissue after injection of MSU crystals and leukotriene B4 . Our studies suggest that PI3Kγ is crucial for MSU crystal-induced acute joint inflammation. It is necessary for regulating caspase-1 activation and for mediating neutrophil migration and activation. Drugs that impair PI3Kγ function may be useful to control acute gout inflammation.