However, substrate overlap exists as dystro glycan is also cleaved by MMP 9 with Mmp2Mmp9 null mice protected from neuroinflammation selleck Alisertib in an EAE model. Cellular influx into areas of inflammation is regulated by MMP 2 activity. For example, HIV infected microglia macrophages secreted proMMP 2 which is activated by neuronal MMP 14 and cleaves astrocyte derived CXCL12 that is itself Inhibitors,Modulators,Libraries neurotoxic and Inhibitors,Modulators,Libraries HIV infected leucocytes up regulate MMP 2 secretion in order to cross the blood brain barrier. MMP 2 is thus an effector of tissue destruction in the CNS and mechanisms that lead to its suppression may be neuroprotective. There are few published data on MMP 2 in TB but we and others have shown that cerebrospinal fluid con centrations are raised in TBM with no concomitant rise in TIMP 1 or 2.
In addition, MMP 2 has been detected by immunohistochemistry in meninges of CNS TB patients. In CNS TB excess MMP 2 is likely to be secreted by microglial cells, the resident innate cells of the CNS, as TB infected monocytes macrophages do not secrete MMP 2 and astrocytes do Inhibitors,Modulators,Libraries not alter MMP 2 secretion in response to TB infection. Therefore we investigated mechanisms regulat ing MMP 2 gene expression and secretion in a simpli fied cellular model of CNS TB. Methods Chemicals for microglial inhibition, including the cas pase 8 inhibitor were from Sigma Aldrich, tissue culture materials from Invitro gen and tissue culture plastic from TPP unless otherwise stated. Anti TNF a neutralizing antibodies, recombinant TNF a, IL 1Ra, recombinant IL 1b were from Peprotech.
SB203580, PD98059, SP600125 and SC 514 were purchased from Calbiochem, helenalin from Biomol. M. tb culture and colony counting The virulent M. tb strain H37Rv Pasteur was cultured from frozen stocks in Middlebrook 7H9 broth with 10% OADC enrichment medium, 0. 2% glycerol, and Inhibitors,Modulators,Libraries 0. 02% Tween 80 in a shaking incubator. An optical densitometer was used to assess growth of M. tb. Experiments were performed with cul tures having an OD of 0. 6 after 30 seconds sonication. The endotoxin level was 0. 03 ng ml LPS as measured by amoebocyte lysate assay. Multiplicity of infection used in each experiment was checked by colony counts on Middlebrook 7H11 agar containing ADC enrichment medium and amphotericin 2. 5 ug ml. Tb medium was generated by centrifuging M. tb at OD 0. 6 at 11,700 RCF for 5 minutes and then sterile filtering the supernatant through an Anopore 0.
2 mM membrane. Monocyte isolation and M. tb infection PBMCs were isolated from single healthy blood donor residual buffy coats using Ficoll Paque density gradient centrifugation and a standard adherence protocol. Monocytes were stimulated by M. tb at an MOI of 1 or 7H9 medium alone. At 24 hours, cell cul Inhibitors,Modulators,Libraries ture medium selleck chem inhibitor was collected and filtered via a 0. 2 um pore size sterile filter to remove infectious particles. This conditioned medium from M.