Matrix metalloproteinase (MMP)-9 a realiable marker for inflammation in acute human trichinel-losis
Bruschi F.*, D’Amato C., Piaggi S., Bianchi C., Castagna B., Paolicchi A., Pinto B. Department of Translational Research, N.T.M.S., Università di Pisa, Pisa, Italy
*Corresponding author:
Department of Translational Research, N.T.M.S. Università di Pisa School of Medicine Via Roma, 55 56126 Pisa ITALY Tel. +39(050)2218547 Fax +39(050) 2210624 E-mail: fabrizio.bruschi@med.unipi.it
Abstract
Matrix Metalloproteinases (MMPs) are involved in many physiological and pathological processes. As regards parasitic infections, the role of these proteins has been particularly studied in malaria, neurocysticercosis and angiostrongyloidosis.
Recently, we evaluated serum levels of MMP-9 and -2 (gelatinases) in mice experimentally infected with Trichinella spiralis or Trichinella pseudospiralis, which cause different degrees of myositis and we found their significant increase in the former and, at a lesser extent, in the latter, thus sug-gesting the possibility that these gelatinases, particularly MMP-9, represent a marker of inflamma-tion.
Our aim was to evaluate the levels of MMP-9 and 2 in trichinellosis patients, to assess their possible clinical significance.
Serum samples from 31 Trichinella britovi-infected individuals (20 males and 11 females), living in Tuscany, Central Italy, were analysed for MMP-9 and MMP-2 serum levels. Patients acquired in-fection with Trichinella after consuming raw or undercooked meat of wild boar. Their median age was 49 ± 0.33 years (range from 7 to 91). Sera were collected before starting anti-inflammatory treatment, aliquoted and stored at -20°C until use. Sera from healthy subjects were considered as controls.
The gelatinolytic activity of MMPs was analyzed by gelatin zymography on 8% polyacrylamide-SDS gels containing 0.1% porcine gelatin, under non-reducing conditions. Clear bands correspond-ing to the digested areas were evaluated with an appropriate software. MMP-9 levels were addition-ally determined in 15 patients using a commercial ELISA kit for human MMP-9.
The zymographic analysis of the gels showed the presence in serum samples of gelatinase bands at approximately 125-kDa, 92-kDa and 72-kDa, corresponding to the MMP-9/ Neutrophil gelatinase-associated lipocalin (NGAL) complex and proenzyme forms of MMP-9 and MMP-2, respectively. A significant (p<0.01) increase in gelatinolytic activity in patients compared to the control group was observed for pro-MMP-9 in 25 out of 31. The mean increase in activity was 39.25% ± 16.67%.
No significant differences were observed for pro-MMP-2 activity. The MMP-9 levels detected by ELISA showed significant correlation with zymographic data (r2=0.62, p< 0.003) and were higher in more affected patients (suffering diarrhea, facial edemas and myalgia).
In conclusion, MMP-9 should be considered as a marker of inflammation in T. britovi patients. On the contrary, MMP-2 did not result significantly different in patients, compared to controls.
1.Introduction
Matrix metalloproteinases (MMPs) are endopeptidases belonging to a family of multi-domain Ca++ and Zn++ dependent enzymes, strictly related, able to degrade almost all components of the extracellular matrix (ECM), but also non-matrix proteins (Visse et al., 2003).
MMPs are involved in many physiological and pathological processes such as cancer, tissue regen-eration/repair and inflammation. As regards parasitic infections, the role of these proteins has been particularly studied in malaria, neurocysticercosis and angiostrongyloidosis where they are involved in the pathogenesis of the central nervous system involvement, playing a crucial role in the disrup-tion of the blood-brain barrier (Bruschi and Pinto, 2013; Chiu and Lai, 2014).
Among the different MMPs, the gelatinase group, consisting of MMP-2 (or gelatinase A) and MMP-9 (or gelatinase B), mainly digests gelatin, the denatured form of collagen (Singh et al., 2015).
MMPs are regulated in vivo by endogenous inhibitors, among them the tissue inhibitors of metallo-proteinases (TIMPs) are the major endogenous regulators of MMPs activities in tissues. They form tightly bound inhibitory complexes with MMPs and are produced by a variety of cell types and in-duced or constitutively expressed in most tissues and body fluids (Nagase et al., 2006).
In particular, TIMP-1 inhibits the activity of most MMPs but it works as a natural inhibitor of MMP-9 (Nagase et al., 2006).
Recently, we evaluated serum levels of MMP-9 and -2 in mice experimentally infected with Trichinella spiralis or Trichinella pseudospiralis. These two Trichinella species cause different de-grees of myositis, probably because of the presence of the capsule in the former and its absence in the latter case (Bruschi and Chiumiento, 2011). It was observed that the MMP serum levels in-creased significantly in mice experimentally infected with T. spiralis and at a lesser extent in those infected with T. pseudospiralis, suggesting the possibility that these MMPs (gelatinases) might rep-resent a marker of inflammation. Furthermore, the TIMP -1 mean serum levels in T. spiralis in-fected mice were significantly higher than in control mice (Bruschi et al., 2014).
The aim of the present work was to evaluate MMP-9 and 2 levels in trichinellosis patients, address-ing two questions: i) are these gelatinases useful as possible markers of inflammation, as already observed in experimental models; ii) is it possible to correlate the levels of these enzymes with the clinical manifestations to establish a possible clinical relevance of their increase?
2. MATERIALS AND METHODS 2.1 Patients
Patients acquired infection after the consumption of raw or undercooked wild boar meat, during an outbreak caused by Trichinella britovi in Italy in 2012, previously described (Fichi et al., 2015). This study included 31 patients (20 males and 11 females) with clinical and serological (see later) diagnosis of trichinellosis. After the informed consent was given, serum samples were collected at the moment of clinical diagnosis (aliquoted and stored at -20°C until use).
In November 2012, uncooked sausages made with meat from wild boar were consumed by 38 per-sons (23 males and 15 females; median age 47.8 years, range 7–91 years) living in a village of the Lucca province (Tuscany region, Italy). Of the serologically positive (34 in total after the second sampling), 32 met case definition of trichinellosis by having exposure to infected sausages, clinical signs and/or symptoms of the infection and positive serology, whereas two were asymptomatic. Among symptomatic patients, some reported a history of fever (71.0%), myalgia (52.6%), diarrhea (47.4%), facial oedemas (47.4%), abdominal pain (28.9%). Furthermore, eosinophilia was found in 73.7% and increased CPK/LDH serum levels in 66% of cases (Fichi et al., 2015).
Patients were analyzed on the basis of clinical (diarrhea, myalgia and facial oedemas) or laboratory signs (eosinophilia and increased CPK/LDH serum levels) and stratified in two categories, accord-ing to serum MMP-9 levels as detected by ELISA (normal <705 ng/ml) and increased (>705 ng/ml).
Controls: Sera from healthy subjects with erythrocyte sedimentation rate normal values were col-lected as a control group (median age xx.x years, range x–xx years).
2.2 Detection of anti-Trichinella antibodies
Serum samples were tested to detect anti-Trichinella IgG using an ELISA commercial kit (DRG In-ternational, USA) which employs excretory/secretory antigens. According to manufacturers’ in-structions, this kit is validated with a sensitivity of 93.8% and a specificity of 94.4%.
2.3 EnzChek Gelatinase/collagenase Assay (ECGCA)
Sera of patients were tested for gelatinase/collagenase activity using a quenched fluorogenic DQ-gelatin (Molecular Probes, Enz-Check® Gelatinase Assay Kit, E-12055). Fluorescence intensity was read on a microplate reader (Victor3, PerkinElmer, Turku, Finland) equipped with standard flu-orescein filters for excitation at 485 ± 10 nm and emission detection at 515 ± 15 nm. Background fluorescence was subtracted from each value.
2.4 Detection of MMP-2 and MMP-9 activities by gel zymography
We analysed serum samples from Trichinella britovi-infected patients and healthy individuals for MMP-9 and MMP-2 activities.
The gelatinolytic activity of MMPs was tested using a SDS-page gelatin zymography. Briefly, en-zymes were separated based on their molecular weight on 8% polyacrylamide-SDS gels (PA) con-taining 0.1% porcine gelatin (1mg/ml, Merck), under non-reducing conditions. For the zymography, a Mini-PROTEAN® III system (Bio-Rad Laboratories, Hercules, CA, USA) was used. Sera were diluted 1:150 in sample buffer. Molecular weight of the gelatinolytic areas was estimated by using unstained Precision Plus Protein KaleidoscopeTM SDS-PAGE molecular marker (Bio-Rad, Milan, Italy). Human 92-kDa-MMP-9, 72- and 66-kDa-MMP-2 were also used as standards (Calbiochem-Merck, Milan, Italy).
After migration, gels were washed twice with 2.5% Triton-X100 at room temperature for 30 min, and incubated with an activation buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl2, pH 7.6) overnight at 37 °C. Gels were then stained with Coomassie Brilliant Blue-R250 (250mg/100ml H2O). The gels were digitally scanned with a resolution of 600 dpi or higher and images were saved
in the TIFF format. The gelatinolytic activity was visualized as clear bands corresponding to the di-gested areas. A brighter or a larger band indicated a more concentrated proteinase in the sample. The optical density (relative integrated optical density) of the bands, indicating the gelatinolytic ac-tivity of MMPs, was measured as area of selected peaks (gel pixel values per gray levels of the lysis bands) by the Fiji-win32 ImageJ 1.41 software (NIH, USA). The activity was expressed as arbitrary units.
2.5 Detection of MMP-9 by ELISA
The levels of MMP-9 were evaluated in the serum of patients by a quantitative sandwich im-munoassay with a commercially available kit (R&D Systems, Milan, Italy). The range of values in healthy controls is 169-705 ng/ml. The intra- and inter-assay coefficients of variation were 2.0% and 7.8%, respectively, according to the manufacturer’s instructions.
2.6 Statistical analysis
Differences in the gelatinolytic activity between the two groups (patients and controls) were as-sessed using a two-tailed Student’s t-test assuming equal variances. Correlation analysis was used to quantify how well two variables relate to each other. Univariate analysis was performed by simple linear regression analysis using MMP-9 (ng/ml), as dependent variable.The correlation between the presence of symptoms and visible signs of disease was assessed by Fisher exact test. The signifi-cance level was set at p < 0.05. Statistical evaluations were performed using the statistical program GraphPad prism, version 4 (GraphPad Software, Inc. CA, USA).
3. RESULTS
Median time of onset of symptoms was 12 days (range 4-30 days). All patients were positive for Trichinella-specific IgG.
The zymographic analysis of the gels showed the presence in serum samples of gelatinase bands at approximately 125-kDa, 92-kDa and 72-kDA, corresponding to the MMP-9-neutrophil gelatinase-associated lipocalin (NGAL) complex, and the proenzyme forms of MMP-9 and MMP-2, respec-tively (Fig. 1), from now on named for simplicity as MMP-9/NGAL, MMP-9 and MMP-2. Western
blot carried out with a rabbit polyclonal antibody specific for human MMP-9 showed the specificity of gelatinolytic areas (data not shown).
Insert Fig. 1.
Areas of lysis of T. britovi- infected patients were compared to controls. A significant (p< 0.0001) increase in gelatinolytic activity in patients compared to the control group was observed for MMP-9 as well as for MMP-9/NGAL (p< 0.0005) (Fig. 2). The mean increase in activity was 39.25% ± 16.67%. No significant differences were observed for MMP-2 activity (p>0.05).
Insert Fig. 2
For this reason, we evaluated by ELISA only the MMP-9 serum levels which were higher than nor-mal values in 60% of the evaluated patients, with a statistically significant difference (p= 0.0037) compared to the healthy controls (Fig. 3A).
In Figure 3B the correlation between the serum MMP-9 levels as detected by ELISA or zymo-graphic analysis is shown. The MMP-9 levels detected by ELISA showed a significant concordance with data from zymographic analysis (r = 0.77; p< 0.001).
Insert Fig. 3.
Serum samples were also tested for proteolytic activity using a quenched fluorogenic DQ-gelatin (ECGCA) and measuring fluorescence intensity developed after incubation with the substrate. No significant difference in fluorogenic activity was observed between patient and the control group samples (p=0.42). No correlation was found between proteolytic activity as detected by ECGCA and MMP-9 levels detected by the ELISA (p=0.74) (data not shown).
In order to verify the existence of a possible relation of increased MMP-9 levels with the clinical manifestations, patients with normal and high MMP-9 values (as detected by the ELISA) were eval-uated separately for the presence of diarrhea, myalgia, facial oedemas, eosinophilia and increased CPK/LDH serum levels.
The patients with high MMP-9 values suffered of diarrhea, myalgia and facial oedemas, differently in a significant way from those with normal MMP-9 values. On the contrary, no difference was
ob-served between the two patient groups as regards eosinophilia and increased CPK/LDH serum lev-els. (Fig. 4).
Insert Fig. 4.
Similar results were obtained when patient groups were defined on the basis of high or low arbitrary units of gelatinolytic activity (data not shown).
4. DISCUSSION
In trichinellosis, inflammation affects the host during both enteral and parenteral phases, causing important modifications at intestinal and muscle levels, respectively (Bruschi and Chiumiento, 2012). In particular, myositis differs depending on the Trichinella species involved, being more se-vere when infection is caused by encapsulated species (Bruschi et al., 2009 and Bruschi and Chiu-miento, 2011). At present, no serological marker is available to evaluate the severity of myositis and CPK/LDH levels are evaluated as indirect markers, being them the result of the muscle damage (Br-uschi and Dupouy-Camet, 2014).
If the ECGCA did not prove to be a reliable method to evaluate possible differences in proteolytic activity in patient sera in comparison with healthy individuals, due to scarce correlation with ELISA results of MMP-9, gel zymography has been found to be a valid method, even for semiquantitative analyses, as confirmed by the significant relation with ELISA results.
As previously observed in our experimental model (Bruschi et al., 2014), MMP-9 is also found to be a good marker of inflammation in patients infected with T. britovi. On the contrary, MMP-2 did not show any significant difference in patients compared to controls. Our results differ from results observed in mice experimentally infected with T. spiralis in which gelatinase A significantly in-creased starting two weeks after infection, i.e. during the parenteral phase. It is noteworthy, that sera from our patients were collected (at the moment of the diagnosis) when the parenteral phase was not yet started and inflammation was mainly involving the gastrointestinal tract.
MMP-9/NGAL serum level, evaluated by zymography, significantly increased in patients, com-pared to healthy controls. This result is in disagreement with data arisen from previous studies car-ried out observed in experimental models of trichinellosis in mice infected with T. spiralis. In murine models, where nosignificant variation in serum NGALlevel, obtained (evaluated) using a with the Luminex® method, was found.
Currently, we are not able to explain the significance of the observed increase of MMP-9/NGAL complex in the sera of patients. NGAL, also known as oncogene 24p3, uterocalin, siderocalin or lipocalin 2, is a 24 kDa secreted glycoprotein originally purified from a culture of mouse kidney cells infected with simian virus 40. However, this protein is normally synthesized as a component of the late granules of neutrophils azurophilic granules where it is located in the (or (MPO) positive) where it co-localized with myloperoxidase peroxidase (MPO) (Chakraborty et al., 2012). As a mat-ter of fact, Indeed, a neutrophils activation might explain our results.
Furthermore, MMP-9 serum levels were increased only in patients with relevant symptoms (diar-rhea, myalgia and facial oedemas) suggesting a clinical significance of the increase of this gelati-nase. Our results are partially consistent with data obtained in other helminth infections, for exam-ple, in neurocysticercosis (NCC) where MPPs were deeply investigated for their possible clinical significance (Bruschi and Pinto, 2013). In patients with NCC, serum levels and enzymatic activities of MMP-2 and MMP-9 showed association with the clinical manifestations of the disease (Verma et al., 2011).
Mean serum MMP-2 levels were higher both in asymptomatic and symptomatic NCC cases com-pared to healthy controls; however, no difference was found in the levels of MMP-2 between symp-tomatic and asympsymp-tomatic NCC patients, resulting this gelatinase not clinically significant, as ob-served also in our study in trichinellosis patients.
On the contrary, MMP-9 serum levels were significantly higher in symptomatic NCC patients than in asymptomatic NCC cases or healthy controls. Levels of both MMPs positively correlated with symptomatic (presence of seizures) NCC (Verma et al., 2011).
Recent studies have shown that higher levels of MMP-9 correlated with epilepsy (Heuser et al., 2010; Yin et al., 2011), suggesting a possible causal effect. In consideration of that, symptomatic NCC patients might underlie the seizures, because of increased levels of MMP-9.
In patients with a solitary cysticercus granuloma, MMP-2 and MMP-9 in the cerebrospinal fluid and serum were elevated. Different patterns of immunological changes were observed in patients fol-lowing resolution or calcification of the lesion (Lalla et al., 2015).
In conclusion, the clinical significance (relevance) of serum MMP-9 levels seems to be relevant in the group of patients evaluated in our study, however these results should be confirmed in larger co-horts of patients involved in outbreaks caused by different Trichinella species, and in a late phase of infection (to understand the relation of MMP-9 level increase with myositis), before this laboratory parameter is routinely searched during the management of a trichinellosis outbreak.
According to the studies in NCC patients, this evaluation will result particularly interesting in those patients presenting with neurotrichinellosis. oppure
In conclusion, our results show a possible clinical relevance of serum MMP-9 level in the develop-ment of symptoms in this group of T. britovi infected patients. However, …
(In conclusion, our results show that abnormal serum MMP-9 level may represent a sensitive mea-sure of clinical manifestation However,…)
Figure legends
Fig. 1. Zymographic analysis of sera from T. britovi-infected patients and healthy individuals. Lane 1-3 and lane 15, Molecular standards; lane 4-8, healthy controls; lane 9-14 T. britovi-infected pa-tients. MMP-9/NGAL complex (125-kDa), Pro-MMP-9 (92-kDa), Pro-MMP-2 (72-kDa), Active-MMP-2 (66-kDa).
Fig. 2. Comparison of the MMP gelatinolytic activity (MMP-9/NGAL complex, MMP-9 and MMP-2) as determined by zymographic analysis in Trichinella britovi infected patients compared to healthy controls. The optical density of the bands indicating the gelatinase activity of MMPs was measured as areas of selected peaks and expressed as arbitrary units (A.U.). A significant difference was observed for both MMP-9/NGAL complex (**p< 0.0005) and MMP-9 (***p< 0.0001), but not for MMP-2 (p<0.05).
Fig. 3. (a): MMP-9 serum levels in trichinellosis patients. ** indicates the significant difference compared to healthy controls (p= 0.0037). (b): simple regression analysis of MMP-9 activity (A.U.) and serum MMP-9 levels (ng/ml). High correlation between both measures was observed (r = 0.77, p < 0.001) in patients. Solid line indicates the linear regression line. Dot lines indicate the 95% Confidence Intervals. MMP-9 levels are expressed as concentration (ng/ml) in ELISA test and as arbitrary units of gelatinolytic activity (A.U.) x 106 in the gelatin zymography.
Fig. 4. Occurrence of diarrhea, myalgia, facial oedemas, eosinophilia, and increased CPK/LDH serum levels in patients according to high and normal MMP-9 serum levels as quantified by the ELISA (ng/ml). A statistically significant difference was observed between the two patient groups regarding diarrhea (p<0.005), myalgia (p=0.007), and facial oedemas (p<0.05).
Acknowledgents
The Authors greatly appreciated the technical assistance of Stefano Mazzoni and Valentina Rocchi. They also acknowledge Drs. Michele de Gennaro and Sauro Luchi for providing clinical informa-tions on the patients.
References
Bruschi, F., Marucci, G., Pozio, E., Masetti, M., 2009. Evaluation of inflammatory responses against muscle larvae of different Trichinella species by an image analysis system. Vet. Parasitol. 159, 258-262.
Bruschi, F., Chiumiento, L., 2011. Trichinella inflammatory myopathy: host or parasite strategy? Parasit Vectors 4, 42-47.
Bruschi, F., Chiumiento, L., 2012. Immunomodulation in trichinellosis: does Trichinella really es-cape the host immune response? Endocr., Metabol. Immune Disord. Drug Targets 12, 4-15.
Bruschi, F., Pinto, B., 2013. The significance of Matrix Metalloproteinases in parasitic infections involving the Central Nervous System. Pathogens 2, 105-129.
Bruschi, F., Dupouy-Camet, J., 2014. Trichinellosis. In: Bruschi, F. (Ed.), Helminth infections and their impact on Global Public Health. Springer, Wien, pp. 229-273.
Bruschi, F., Bianchi, C., Fornaro, M., Naccarato G., Menicagli, M., Gomez-Morales, M.A., Pozio, E., Pinto, B., 2014. Matrix metalloproteinase (MMP)-2 and MMP-9 as inflammation markers of Trichinella spiralis and Trichinella pseudospiralis infections in mice. Parasite Immunol. 36, 540-549.
Chakraborty, S., Kaur, S., Guha, S., Batra, S.K., 2012. The multifaceted roles of neutrophils gelati-nase associated lipocalin (NGAL) in inflammation and cancer. Biochim. Biophys. Acta 1826, 129– 169.
Chiu, P.S., Lai, S.C., 2014. Matrix metalloproteinase-9 leads to blood-brain barrier leakage in mice with eosinophilic meningoencephaliteis caused by Angiostrongylus cantoniensis. Acta Trop. 140, 141-150.
Fichi, G., Stefanelli, S., Pagani, A., Luchi, S., De Gennaro, M., Gómez-Morales, M.A., Selmi, M., Rovai, D., Mari, M., Fischetti, R., Pozio, E., 2015. Trichinellosis outbreak caused by meat from a wild boar hunted in an Italian region considered to be at negligible risk for Trichinella. Zoonoses Public Health. 62, 285-291.
Nagase, H., Visse, R., Murphy, G., 2006. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc. Res. 69, 562–573.
Singh, D., Srivastava, S.K., Chaudhuri, T.K., Upadhyay, G., 2015. Multifaceted role of matrix met-alloproteinases (MMPs). Front Mol Biosci. 2, 1-5.
Verma, A., Prasad, K.N., Nyati, K.K., Singh, S.K., Singh, A.K., Paliwal, V.K., Gupta, R.K., 2011. As-sociation of MMP-2 and MMP-9 with clinical outcome of neurocysticercosis. Parasitology 138, 1423– 1428.
Visse, R., Nagase, H., 2003. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ. Res. 92, 827–839.
Heuser, K., Hoddevik, E.H., Taubøll, E., Gjerstad, L., Indahl, U., Kaczmarek, L., Berg, P.R., Lien, S., Nagelhus, E.A., Ottersen, O.P., 2010. Temporal lobe epilepsy and matrix metalloproteinase 9: a tempt-ing relation but negative genetic association. Seizure 19, 335–338.
Lalla, R.S., Garg, R.K., Malhotra, H.S., Jain, A., Verma, R., Pandey, C.M., Singh, G.P., Sharma, P.K., 2015. Cytokines, MMP-2, and MMP-9 levels in patients with a solitary cysticercus granuloma. Neurol India. 63,190-196.
Yin, P., Yang, L., Zhou, H.Y., Sun, R.P., 2011. Matrix metalloproteinase9 may be a potential therapeu -tic target in epilepsy. Med. Hypotheses 76, 184–186.
