Luisa Saraiva

MMP-1 drives immunopathology in human tuberculosis and transgenic mice

Mycobacterium tuberculosis can cause lung tissue damage to spread, but the mechanisms driving this immunopathology are poorly understood. The breakdown of lung matrix involves MMPs, which have a unique ability to degrade fibrillar collagens at neutral pH. To determine whether MMPs play a role in the immunopathology of tuberculosis (TB), we profiled MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), in sputum and bronchoalveolar lavage fluid from patients with TB and symptomatic controls. MMP-1 concentrations were significantly increased in both HIV-negative and HIV-positive patients with TB, while TIMP concentrations were lower in HIV-negative TB patients. In primary human monocytes, M. tuberculosis infection selectively upregulated MMP1 gene expression and secretion, and Ro32-3555, a specific MMP inhibitor, suppressed M. tuberculosis-driven MMP-1 activity. Since the mouse MMP-1 ortholog is not expressed in the lung and mice infected with M. tuberculosis do not develop tissue destruction equivalent to humans, we infected transgenic mice expressing human MMP-1 with M. tuberculosis to investigate whether MMP-1 caused lung immunopathology. In the MMP-1 transgenic mice, M. tuberculosis infection increased MMP-1 expression, resulting in alveolar destruction in lung granulomas and significantly greater collagen breakdown. In summary, MMP-1 may drive tissue destruction in TB and represents a therapeutic target to limit immunopathology.

Human γδ T Lymphocytes Are Licensed for Professional Antigen Presentation by Interaction with Opsonized Target Cells

Activated human blood γδ T cells have also been previously demonstrated to behave as professional APCs, although the processes that control APC function have not been characterized. n this study, we show that the acquisition of potent APC function by human blood γδ T cells is achieved after physical interaction with an Ab-coated target cell, a process that we refer to as licensing. In cancer models, licensing of γδ T cells by tumor-reactive mAbs promotes the uptake of tumor Ags and professional presentation to tumor-reactive αβ T cells. We propose that licensing by Ab is a mechanism whereby the adaptive properties of γδ T cells are induced by their innate functions in a spatially and temporally controlled manner.

Doxycycline and HIV infection suppress tuberculosis-induced matrix metalloproteinases.

RATIONALE Tuberculosis kills more than 1.5 million people per year, and standard treatment has remained unchanged for more than 30 years. Tuberculosis (TB) drives matrix metalloproteinase (MMP) activity to cause immunopathology. In advanced HIV infection, tissue destruction is reduced, but underlying mechanisms are poorly defined and no current antituberculous therapy reduces host tissue damage. OBJECTIVES To investigate MMP activity in patients with TB with and without HIV coinfection and to determine the potential of doxycycline to inhibit MMPs and decrease pathology. METHODS Concentrations of MMPs and cytokines were analyzed by Luminex array in a prospectively recruited cohort of patients. Modulation of MMP secretion and Mycobacterium tuberculosis growth by doxycycline was studied in primary human cells and TB-infected guinea pigs. MEASUREMENTS AND MAIN RESULTS HIV coinfection decreased MMP concentrations in induced sputum of patients with TB. MMPs correlated with clinical markers of tissue damage, further implicating dysregulated protease activity in TB-driven pathology. In contrast, cytokine concentrations were no different. Doxycycline, a licensed MMP inhibitor, suppressed TB-dependent MMP-1 and -9 secretion from primary human macrophages and epithelial cells by inhibiting promoter activation. In the guinea pig model, doxycycline reduced lung TB colony forming units after 8 weeks in a dose-dependent manner compared with untreated animals, and in vitro doxycycline inhibited mycobacterial proliferation. CONCLUSIONS HIV coinfection in patients with TB reduces concentrations of immunopathogenic MMPs. Doxycycline decreases MMP activity in a cellular model and suppresses mycobacterial growth in vitro and in guinea pigs. Adjunctive doxycycline therapy may reduce morbidity and mortality in TB.

Receptor-targeted liposome-peptide nanocomplexes for siRNA delivery

RNA interference induced by double-stranded, small interfering RNA (siRNA) molecules has attracted great attention as a genetic therapeutic approach. Despite major advances in this field, new nanoparticle formulations are required for in vivo delivery of siRNA, particularly for tissue-specific delivery of siRNA reagents. We have developed and optimized LYR nanocomplex formulations for siRNA delivery that consist of a liposome (DOTMA/DOPE; L) and a targeting peptide (K₁₆GACYGLPHKFCG; Y) which self-assemble on mixing at optimal ratios with siRNA (R). Biophysical measurements indicated that LYR nanocomplexes were strongly cationic, mainly spherical particles of less than 100 nm. These formulations packaged and protected siRNA on incubation with RNAseA with >90% intact siRNA recovery. In addition, intact siRNA was recovered from LYRs upon heparin treatment. A critical synergy was observed between the lipid and peptide components for LYR particle stability and transfection efficiency. To evaluate targeting, transfections were compared with non-targeted formulations containing K₁₆ with no targeting ligand. Gene knockdown efficiencies with targeted formulations were more than two-fold better in all cell lines tested (p < 0.01). LYR formulations with liposomes containing DOTMA, which has an 18-carbon (C18) alkyl tail, were significantly better in silencing than formulations containing cationic lipids with shorter alkyl tails. LYRs with siRNA against endogenous luciferase and GAPDH were successful in silencing these genes in 3 cell lines (1HAEo- human airway epithelial, B104 rat neuroblastoma, Neuro2A-Luc mouse neuroblastoma) in vitro with 80% efficiency, similar in efficiency to Lipofectamine 2000. Confocal microscopy analysis with LYRs containing fluorescently labelled siRNA (Cy3) showed that the siRNA was located in the perinuclear region of the cytoplasm, where the RNA-induced silencing complex (RISC) is likely to be found. The LYR formulations may have applications for the further development of siRNA-based therapeutics.

Cell Electrospinning: An In Vitro and In Vivo Study

Cell electrospinning and aerodynamically assisted bio-threading are novel bioplatforms for directly forming large quantities of cell-laden scaffolds for creating living sheets and vessels in three-dimensions. The functional biological architectures generated will be useful in both the laboratory and the clinic.

Matrix Metalloproteinase-1 Is Regulated in Tuberculosis by a p38 MAPK-Dependent, p-Aminosalicylic Acid-Sensitive Signaling Cascade

Mycobacterium tuberculosis (M. tb) must cause lung disease to spread. Matrix metalloproteinases (MMPs) degrade the extracellular matrix and are implicated in tuberculosis-driven tissue destruction. We investigated signaling pathways regulating macrophage MMP-1 and -7 in human pulmonary tuberculosis and examine the hypothesis that the antimycobacterial drug p-aminosalicylic acid acts by inhibiting such pathways. In primary human macrophages, M. tb up-regulates gene expression and secretion of MMP-1 (interstitial collagenase) and MMP-7 (matrilysin). In tuberculosis patients, immunohistochemical analysis of lung biopsies demonstrates that p38 MAPK is phosphorylated in macrophages surrounding granulomas. In vitro, M. tb drives p38 phosphorylation. p38 inhibition suppresses M. tb-dependent MMP-1 secretion by 57.8% and concurrently increases secretion of its specific inhibitor TIMP-1 by 243.7%, demonstrating that p38 activity regulates matrix degradation by macrophages. p38 signals downstream to the cyclooxygenase 2/PGE2 pathway. p-Aminosalicyclic acid, an agent used to treat drug-resistant tuberculosis, inhibits M. tb-driven MMP-1 but not MMP-7 gene expression and secretion. PAS acts by blocking PGE2 production without affecting M. tb growth. In summary, p-aminosalicyclic acid decreases MMP-1 activity by inhibiting a p38 MAPK-PG signaling cascade, suggesting that this pathway is a therapeutic target to reduce inflammatory tissue destruction in tuberculosis.

Comparison of nanocomplexes with branched and linear peptides for siRNA delivery.

Efficient delivery of small interfering RNA (siRNA) remains the greatest technological barrier to the clinical implementation of RNA interference strategies. We are investigating the relationship between the biophysical properties of siRNA nanocomplexes and their transfection efficiency as an approach to the generation of improved formulations. Peptide-based formulations are of great interest, and so in this study we have compared nanocomplex formulations for siRNA delivery containing linear and branched oligolysine or oligoarginine peptides. Peptides were combined with cationic liposomes in siRNA formulations and compared for transfection efficiency, siRNA packaging efficiency, biophysical properties, and particle stability. Nanocomplexes containing linear peptides were more condensed and stable than branched peptide formulations; however, their silencing activity was lower, suggesting that their greater stability might limit siRNA release within the cell. Thus, differences in transfection appeared to be associated with differences in packaging and stability, indicating the importance of optimizing this feature in siRNA nanocomplexes.

Regulation of matrix metalloproteinase-1, -3, and -9 in Mycobacterium tuberculosis-dependent respiratory networks by the rapamycin-sensitive PI3K/p70S6K cascade

This study was designed to investigate the role of the phosphatidyl inositol 3‐kinase (PI3K)/AKT/p70S6K signaling path on regulation of primary normal human bronchial epithelial cell‐derived matrix metalloproteinase (MMP)‐1, ‐3, and ‐9 expression in tuberculosis (TB). These MMPs are key in pathological extracellular matrix degradation in TB. Normal human bronchial epithelials were stimulated with conditioned medium from monocytes infected with virulent TB (CoMTb) and components of the PI3K/AKT signaling pathway blocked using specific chemical inhibitors and siRNA. MMP gene expression was measured by RT‐PCR and secretion by ELISA, luminex, or zymography. Phospho‐p70 S6Kwas detected by Western blot analysis and activity blocked by rapamycin. Chemical blockade of the proximal catalytic PI3Kp110 subunit augmented MMP‐1 and MMP‐9 in a dose‐dependent manner (all P<0.001) but suppressed MMP‐3 (P<0.01). Targeted siRNA studies identified the p110α isoform as key causing 5‐fold increase in TB network‐dependent MMP‐1 secretion to 4900 ± 1100 pg/ml. Specific inhibition of the AKT node suppressed all 3 MMPs. Phospho‐p70S6K was identified in the cellular model, and rapamycin, a p70S6K inhibitor, inhibited MMP‐1 (P<0.001) and MMP‐3 (P<0.01) but not MMP‐9. Controls were epithelial cells that were unstimulated or exposed to conditioned medium from monocytes not exposed to TB. In summary, blockade of the proximal PI3K catalytic subunit increases MMP‐1 and MMP‐9, whereas rapamycin decreased both MMP‐1 and MMP‐3. The regulation of the PI3K path in TB is complex, MMP specific, and a potential immunotherapeutic target in diseases characterized by tissue destruction.—Singh, S., Saraiva, L., Elkington, P. T. G., Friedland, J. S. Regulation of matrix metalloproteinase‐1, ‐3, and ‐9 in Mycobacterium tuberculosis dependent respiratory networks by the rapamycin‐sensitive PI 3‐kinase/p70S6K cascade. FASEB J. 28, 85–93 (2014). www.fasebj.org

Membrane Type 1 Matrix Metalloproteinase Regulates Monocyte Migration and Collagen Destruction in Tuberculosis

Tuberculosis (TB) remains a global pandemic and drug resistance is rising. Multicellular granuloma formation is the pathological hallmark of Mycobacterium tuberculosis infection. The membrane type 1 matrix metalloproteinase (MT1-MMP or MMP-14) is a collagenase that is key in leukocyte migration and collagen destruction. In patients with TB, induced sputum MT1-MMP mRNA levels were increased 5.1-fold compared with matched controls and correlated positively with extent of lung infiltration on chest radiographs (r = 0.483; p < 0.05). M. tuberculosis infection of primary human monocytes increased MT1-MMP surface expression 31.7-fold and gene expression 24.5-fold. M. tuberculosis–infected monocytes degraded collagen matrix in an MT1-MMP–dependent manner, and MT1-MMP neutralization decreased collagen degradation by 73%. In human TB granulomas, MT1-MMP immunoreactivity was observed in macrophages throughout the granuloma. Monocyte–monocyte networks caused a 17.5-fold increase in MT1-MMP surface expression dependent on p38 MAPK and G protein–coupled receptor-dependent signaling. Monocytes migrating toward agarose beads impregnated with conditioned media from M. tuberculosis–infected monocytes expressed MT1-MMP. Neutralization of MT1-MMP activity decreased this M. tuberculosis network–dependent monocyte migration by 44%. Taken together, we demonstrate that MT1-MMP is central to two key elements of TB pathogenesis, causing collagen degradation and regulating monocyte migration.

In Vitro and In Vivo Interrogation of Bio-sprayed Cells

Bio-sprays can directly form pre-organized cell-bearing structures for applications ranging from engineering functional tissues to the forming of cultures, most useful for modeling disease, to the discovery and development of drugs. Bio-electrosprays and aerodynamically assisted bio-jets, are leading approaches that have been demonstrated as having far-reaching ramifications for regenerative biology and medicine.