Complex regulation of neutrophil-derived MMP-9 secretion in central nervous system tuberculosis

Abstract

Background: Central nervous system tuberculosis (CNS-TB) may be fatal even with treatment. Neutrophils are the key mediators of TB immunopathology, and raised CSF matrix metalloproteinase-9 (MMP-9) which correlates to neutrophil count in CNS-TB is associated with neurological deficit and death. The mechanisms by which neutrophils drive TB-associated CNS matrix destruction are not clearly defined.

Methods: Human brain biopsies with histologically proven CNS-TB were stained for neutrophils, neutrophil elastase, and MMP-9. Neutrophil MMP-9 secretion and gene expression were analyzed using Luminex and real-time PCR. Type IV collagen degradation was evaluated using confocal microscopy and quantitative fluorescent assays. Intracellular signaling pathways were investigated by immunoblotting and chemical inhibitors.

Results: MMP-9-expressing neutrophils were present in tuberculous granulomas in CNS-TB and neutrophil-derived MMP-9 secretion was upregulated by Mycobacterium tuberculosis (M.tb). Concurrent direct stimulation by M.tb and activation via monocyte-dependent networks had an additive effect on neutrophil MMP-9 secretion. Destruction of type IV collagen, a key component of the blood-brain barrier, was inhibited by neutralizing neutrophil MMP-9. Monocyte-neutrophil networks driving MMP-9 secretion in TB were regulated by MAP-kinase and Akt-PI₃ kinase pathways and the transcription factor NF-kB. TNFα neutralization suppressed MMP-9 secretion to baseline while dexamethasone did not.

Conclusions: Multiple signaling paths regulate neutrophil-derived MMP-9 secretion, which is increased in CNS-TB. These paths may be better targets for host-directed therapies than steroids currently used in CNS-TB.

Keywords: Immunopathology; Matrix metalloproteinase; Neutrophils; Tuberculosis.

Fig. 1

Neutrophils secrete MMP-9 and are present in CNS-TB. a M.tb infection with MOI of 1 upregulates neutrophil MMP-9 secretion. b Neutrophils from healthy volunteers secrete MMP-9 in response to CoMTB. c Kinetics of Neutrophil MMP-9 gene expression after CoMTB stimulation. d M.tb and CoMTB stimulation have an additive effect on neutrophil MMP-9 secretion. e NGAL and MMP-9 concentrations closely correlate despite being located in different neutrophil granules. f–h Neutrophils expressing MMP-9 are present in CNS TB granulomas and present in vessel walls and perivascular interstitial space. f H&E, scale bar 250 μm. g Elastase immunoperoxidase, scale bar 250 μm and h MMP-9 immunoperoxidase, ×20 magnification, scale bar 50 μm. Inset shows neutrophils within lesions (×40). Positive control on acute appendicitis with i elastase, scale bar 50 μm and j MMP-9 on blood vessel wall and interstitium, scale bar 200 μm. k Negative control with omission of primary antibodies, scale bar represents 100 μm. Bars represent mean ± s.d and represent at least two independent experiments performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 2

Collagen is degraded by neutrophils stimulated with M.tb in an MMP-9 dependent manner. a Confocal microscopy of Type IV DQ collagen coated slides demonstrating collagen degradation in the presence of M.tb stimulation. DQ collagen fluorescence increases after degradation. b Type IV collagenase activity is inhibited by MMP-9 neutralizing antibody. Bars represent mean ± s.d and represent two independent experiment performed in triplicate. **P < 0.01

Fig. 3

MAP-kinase pathways are activated in neutrophils in TB and regulate MMP-9 secretion. a Representative immunoblots of p-38, ERK, and JNK MAP kinases in neutrophils stimulated with CoMTB. CoMTB increases phosphorylation of all pathways from 15 min. Densitometric analysis of response to CoMCont and CoMTB stimulation of b phospho-p38 to total p38, c phospho-ERK to total ERK, and d phospho-JNK to total JNK. e CoMTB-stimulated MMP-9 secretion is suppressed by p-38 inhibitor SB203580. f MMP-9 secretion with CoMTB is inhibited by ERK inhibitor PD98059. g Representative immunoblots to detect p-38, ERK MAP kinases in neutrophils stimulated with M.tb. Densitometric analysis of response to control (PBS) and M.tb infection of h phospho-p-38 to total p-38 and i phospho-JNK to total JNK. j Pre-incubation with p-38 inhibitor SB does not suppress MMP-9 secretion in M.tb-infected neutrophils. k Pre-incubation with ERK inhibitor PD does not affect MMP-9 secretion by M.tb-stimulated neutrophils. All chemical inhibitor experiments were performed by pre-incubating neutrophils for 30 min before stimulating for 4 h. Bars represent mean ± s.d and are representative of at least two independent experiments performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

The Akt pathway regulates neutrophil MMP-9 secretion in TB. a Pre-incubation with PI3-kinase inhibitor LY294002 suppresses MMP-9 secretion with CoMTB. b Representative immunoblots to detect p-Akt in neutrophils infected with M.tb show increased phosphorylation with corresponding densitometric analysis. c Pre-incubation with PI3-kinase inhibitor LY does not affect MMP-9 secretion after M.tb infection. Bars represent mean ± s.d and are representative of at least two independent experiments performed in triplicate. *P < 0.05; **P < 0.01

Fig. 5

Neutrophil MMP-9 secretion is regulated by NF-kB. a Pre-incubation with helenalin inhibits MMP-9 secretion after CoMTB stimulation. b Pre-incubation with IKK2 inhibitor SC-514 inhibits MMP-9 secretion with CoMTB stimulation. c Pre-incubation with NF-kB inhibitor helenalin inhibits MMP-9 secretion after M.tb infection. Bars represent mean ± s.d and are representative of at least two independent experiments performed in triplicate. **P < 0.01; ***P < 0.001

Fig. 6

Anti-TNFα suppresses neutrophil MMP-9 secretion, but dexamethasone does not. a MMP-9 secretion is inhibited by anti-TNFα pre-treatment of neutrophils. CoMTB was incubated with anti-TNFα antibodies for 1 h before neutrophil stimulation. b Anti-TNFα inhibits MMP-9 gene expression. c TIMP-1 secretion is not affected by anti-TNFα. d TIMP-2 secretion is reduced by anti-TNFα. e MMP-9 secretion is not affected by dexamethasone. f Dexamethasone inhibits neutrophil MMP-8 secretion. Bars represent mean ± s.d and are representative of at least two independent experiments performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001