Systemic Lupus Erythematosus (SLE) is a common and devastating autoimmune disease, characterized by a dysregulated adaptive immune response against intracellular antigens, which involves both autoreactive T and B cells. In SLE, mainly intracellular autoantigens generate autoantibodies and these assemble into immune complexes and activate the classical pathway of the complement system enhancing inflammation. Matrix metalloproteinase-9 (MMP-9) levels have been investigated in the serum of SLE patients and in control subjects. On the basis of specific studies, it has been suggested to treat SLE patients with MMP inhibitors. However, some of these inhibitors induce SLE. Analysis of LPR^−/−MMP-9^−/− double knockout mice suggested that MMP-9 plays a protective role in autoantigen clearance in SLE, but the effects of MMP-9 on immune complexes remained elusive. Therefore, we studied the role of MMP-9 in the clearance of autoantigens, autoantibodies and immune complexes and demonstrated that the lack of MMP-9 increased the levels of immune complexes in plasma and local complement activation in spleen and kidney in the LPR^−/− mouse model of SLE. In addition, we showed that MMP-9 dissolved immune complexes from plasma of lupus-prone LPR^−/−/MMP-9^−/− mice and from blood samples of SLE patients. Surprisingly, autoantigens incorporated into immune complexes, but not immunoglobulin heavy or light chains, were cleaved by MMP-9. We discovered Apolipoprotein-B 100 as a new substrate of MMP-9 by analyzing the degradation of immune complexes from human plasma samples. These data are relevant to understand lupus immunopathology and side-effects observed with the use of known drugs. Moreover, we caution against the use of MMP inhibitors for the treatment of SLE.

Inclusion bodies (IBs) are protein nanoclusters obtained during recombinant protein production processes, and several studies have demonstrated their potential as biomaterials for therapeutic protein delivery. Nevertheless, IBs have been, so far, exclusively sifted by their biological activity in vitro to be considered in further protein-based treatments in vivo. Matrix metalloproteinase-9 (MMP-9) protein, which has an important role facilitating the migration of immune cells, was used as model protein. The MMP-9 IBs were compared with their soluble counterpart and with MMP-9 encapsulated in polymeric-based micelles (PM) through ionic and covalent binding. The soluble MMP-9 and the MMP-9-ionic PM showed the highest activity values in vitro. IBs showed the lowest activity values in vitro, but the specific activity evolution in 50% bovine serum at room temperature proved that they were the most stable format. The data obtained with the use of an air-pouch mouse model showed that MMP-9 IBs presented the highest in vivo activity compared to the soluble MMP-9, which was associated only to a low and a transitory peak of activity. These results demonstrated that the in vivo performance is the addition of many parameters that did not always correlate with the in vitro behavior of the protein of interest, becoming especially relevant at evaluating the potential of IBs as a protein-based nanomaterial for therapeutic purposes.

A fundamental part of the immune response to infection or injury is leukocyte migration. Matrix metalloproteinases (MMPs) are a class of secreted or cell-bound endopeptidases, implicated in every step of the process of inflammatory cell migration. Hence, specific inhibition of MMPs is an interesting approach to control inflammation. We evaluated the potential of a bivalent carboxylate inhibitor to selectively inhibit the trimeric proteoform of MMP-9 and compared this with a corresponding monovalent inhibitor. The bivalent inhibitor efficiently inhibited trimeric MMP-9 (IC₅₀ = 0.1 nM), with at least 500-fold selectivity for MMP-9 trimers over monomers. Surprisingly, in a mouse model for chemotaxis, the bivalent inhibitor amplified leukocyte influxes towards lipopolysaccharide-induced inflammation. We verified by microscopic and flow cytometry analysis increased amounts of neutrophils. In a mouse model for endotoxin shock, mice treated with the bivalent inhibitor had significantly increased levels of MMP-9 in plasma and lungs, indicative for increased inflammation. In conclusion, we propose a new role for MMP-9 trimers in tempering excessive neutrophil migration. In addition, we have identified a small molecule inhibitor with a high selectivity for the trimeric proteoform of MMP-9, which will allow further research on the functions of MMP-9 proteoforms.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) triggers multiple sclerosis (MS) and the animal model of experimental autoimmune encephalomyelitis (EAE) by the breakdown of the blood-brain barrier. Interestingly, MMP-9 is beneficial in systemic autoimmunity caused by Fas-deficiency. Fas-deficient (fas^lpr) and Fas-ligand-deficient mice are protected against EAE. We here investigated the interaction between Fas and MMP-9 in the setting of induction of EAE and compared short- and long-term effects. We provoked EAE with myelin oligodendrocyte glycoprotein (MOG) peptide and compared EAE development in four genotypes (wild-type (WT), single knockout mmp-9^-/-, fas^lpr, and mmp-9^-/-/fas^lpr) and monitored leukocytes, cytokines and chemokines as immunological parameters. As expected, fas^lpr mice were resistant against EAE induction, whereas MMP-9 single knockout mice were not. In the double mmp-9^-/-/ fas^lpr mice the effects on disease scores pointed to independent rather than interrelated disease mechanisms. On a short term, after EAE induction leukocytes infiltrated into the brain and cytokine and chemokine levels were significantly higher in all the four genotypes studied, even in the fas^lpr and mmp-9^-/-/fas^lpr, which did not develop clinical disease. The levels of MMP-9 but not of MMP-2 were increased in the brain and in the peripheral organs after EAE induction. After 40 days all the animals recovered and did not show signs of EAE. However, the absence of MMP-9 in the remission phase suggested a protective role of MMP-9 in the late phase of the disease, because single mmp-9^-/- mice presented a delayed remission in comparison with WT animals suggesting a phase-dependent role of MMP-9 in the disease. Nevertheless, the levels of some cytokines and chemokines remained higher than in control animals even 100 days after EAE induction, attesting to a prolonged state of immune activation. We thus yielded new insights and useful markers to monitor this activated immune status. Furthermore, MMP-9 but not MMP-2 levels remained increased in the brains and, to a higher extend, in the spleens of the WT mice even during the remission phase, which is in line with the role of MMP-9 as a useful marker and a protective factor for EAE in the remission phase.