Clinical and Experimental Immunology

OR I G INA L A RTI CLE

doi:10.1111/cei.12725

Matrix metalloproteinases contribute to the regulation of chemokine expression and pulmonary inflammation in Cryptococcus infection

O. Supasorn,*‡ N. Sringkarin,† P. Srimanote‡ and P. Angkasekwinai*† *Department of Medical Technology, Faculty of Allied Health Sciences, †Graduate Program in Medical Technology, Faculty of Allied Health Sciences, and ‡Graduate Program in Biomedical Science, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand

Accepted for publication 6 October 2015 Correspondence: P. Angkasekwinai, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand. E-mail: [email protected]; [email protected]

Summary Matrix metalloproteinases (MMPs) are a family of extracellular proteases that play roles in regulating the immune response in inflammatory processes. Previous studies indicated that different MMPs were involved in the host defence and tissue damage in response to different pathogens. However, the contributions of MMPs during Cryptococcus infection have not been addressed clearly. Here, we examined the expression and activity of MMPs during Cryptococcus infection. Among MMP family members, we found significant increases of MMP-3 and MMP-12 mRNA levels and MMP12 zymographic activities in response to C. neoformans but not C. gattii infection. The expression of MMP12 was induced in RAW cells after C. neoformans treatment and in alveolar macrophages purified from C. neoformans-infected mice. Interestingly, administration of MMP inhibitor GM6001 into C. neoformans-infected mice resulted in a significantly increased pulmonary fungal burden with attenuated inflammatory cell infiltration. Corresponding to this finding, the expression of the macrophage- and neutrophil-attracting chemokines CCL2 and CXCL1 was inhibited in the GM6001-treated group and MMP12 levels were found to be correlated strongly with CCL2 mRNA expression. Thus, our data suggest that the induction of MMPs by C. neoformans infection potentiates inflammatory cell infiltration by modulating pulmonary chemokines, thereby promoting effective host immunity to pulmonary Cryptococcus infection. Keywords: Cryptococcus infection, MMP-12, pulmonary inflammation

Introduction Cryptococcal disease is a fungal infection caused by Cryptococcus spp. The disease patterns vary greatly, ranging from asymptomatic airway colonization and severe pneumonia with respiratory failure to life-threatening meningoencephalitis [1–3]. C. neoformans is the most common cause of mortality in HIV-infected individuals, whereas C. gattii is distinct from its sibling species in that it more commonly infects immunocompetent hosts. In a mouse model of cryptococcosis, it has been indicated that mice infected with C. gattii displayed less pulmonary inflammation with reduced neutrophil infiltration and proinflammatory cytokine production [4]. Moreover, the highly virulent C. gattii infection was able to dampen T helper type 1 (Th1) induction and recruitment [5]. Although these studies suggest that the host inflammatory response altered by fungus may contribute to the disease pathogenesis, the immunological mediators involved in this regulation remain unclear.

Matrix metalloproteinases (MMPs) are a group of enzymes that play important roles in multiple physiological and pathological processes, including regulating inflammatory responses and modulating immune function [6,7]. Several lines of evidence indicate that MMPs, including MMP-3, MMP-8, MMP-9 and MMP-12, have multiple effects on inflammation through their action in cleaving, activating or inactivating chemokines, cytokines and their receptors [8,9]. In normal tissues MMP expression is constitutively low, but their synthesis is induced rapidly during inflammation and tissue remodelling [10]. Indeed, several MMPs have been shown to be up-regulated during infection and contribute to host response to pathogens and tissue damage [11]. Moreover, some MMPs such as the C-terminal cathelicidin-like domain of MMP-12 could also be involved in the direct killing of pathogens by macrophages [12]. It has been reported that MMP-2 was expressed in the lungs of C. neoformans-infected mice and

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its expression was correlated with the type of immune response and lung granuloma formation [13]; however, the roles of MMPs family members in Cryptococcus spp. infection have not yet been elucidated fully. In this study, we aim to characterize the expression of MMPs and determine the roles of MMPs during Cryptococcus infection by using an MMP inhibitor. We showed that mRNA expression and activity of MMP-12 were highly upregulated during C. neoformans infection and were found to be associated with the expression of inflammatory cellattracting chemokines. More importantly, inhibition of MMP family members attenuated inflammatory cell infiltration, resulting in an increased lung fungal burden.

Materials and methods Animals C57BL/6 and BALB/C mice were obtained from The National Laboratory Animal Center, Mahidol University. Female 6–8-week-old mice were used for experiments because females were used previously in immunology studies in our laboratory. Mice were housed in enclosed filtertop cages in the animal facility at the Faculty of Allied Health Science, Thammasat University. All animal studies were approved by the Thammasat University Animal Care and Use Committee.

Cryptococcal strains The highly virulent Cryptococcus neoformans strain H99 (serotype A, VNI) and Cryptococcus gattii strain R265 (serotype B, VGII) was used in this study. The reference strains were stored in 25% glycerol at 2808C until use and were maintained on Sabouraud dextrose agar (SDA) at 258C during study.

Murine model of fungal infections For murine infection, yeast cells were grown for 24 h in Sabouraud dextrose broth in a shaking incubator. The yeast cells were then washed in phosphate-buffered saline (PBS), enumerated using a haemocytometer and resuspended in phosphate-buffered saline (PBS) at a concentration of 1 3 106 yeast cells/ml. After mice were anaesthetized with isofluorene, a final volume of 50 ml of PBS or yeast cell suspension (5 3 104 yeast cells/mouse) was then administered intranasally to the mice, as described previously [4]. Infected mice were euthanized using CO2 at day 7 postinfection. For studies of MMP expression kinetics, mice were infected and euthanized at days 1, 7 and 14 postinfection. For fungal burden analysis, lungs were homogenized at day 7 post-infection in sterile PBS, diluted and plated on yeast peptone dextrose (YPD) agar for colony counts. Colony-forming units (CFU) were enumerated following incubation at 308C for 48 h. 432

MMP inhibitor treatment A broad-spectrum MMP inhibitor GM6001 (10 mg; Calbiochem, San Diego, CA, USA) was reconstituted in dimethylsulphate (DMSO) and diluted with low salt buffer (containing 50 mM Tris pH 7
5, 150 mM NaCl and 20 mM CaCl2). Mice were administered 100 ll of diluted GM6001 intraperitoneally (i.p.) to yield a final concentration of 100–125 mg/kg body weight [14]. Control mice received an identical volume of diluted DMSO vehicle. Inhibitors or vehicle were given daily for 7 days after intranasal inoculation of Cryptococcus neoformans strain H99.

Lung leucocyte analysis Fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (M1/70), phycoerythrin (PE)-conjugated anti-CD11c (HL3) and allophycocyanin (APC)-conjugated-anti-Gr.1 (RB6-8C5) antibodies (BD Pharmingen, San Jose, CA, USA) were used for leucocyte analysis. Single cell suspensions of lungs were prepared, stained and analysed using a BD FACSVerseTM cytometer (BD Biosciences, San Jose, CA, USA), as described previously [5]. Neutrophils were analysed based on the expression of Gr-1 and CD11b [4]; dendritic cells express high levels of CD11b and CD11c and macrophages express high levels of CD11b and low levels of CD11c [4].

Alveolar macrophage cell isolation Alveolar macrophage cells were isolated as described previously [15]. C57BL/6 mice were treated with PBS or infected with C. neoformans or C. gattii for 7 days. Lungs were lavaged five times with an infusion of cold 0
5-ml PBS (pH 7
2). Bronchoalveolar lavage (BAL) cells were subjected to CD11b1 cell isolation using magnetic bead positive selection according to the manufacturer’s directions (Miltenyi Biotec, Auburn, CA, USA). Enriched CD11b1 cells were determined for viability by trypan blue exclusion. For MMP activity analysis of alveolar macrophages, purified CD11b1 cells were isolated from BAL fluid of BALB/C mice treated with PBS or infected with C. neoformans strain H99 or C. gattii strain R265 for 7 days. Purified alveolar macrophage cells were then cultured in serum-free medium for 24 h and culture supernatant was collected for further analysis of MMP zymography.

In-vitro stimulation of macrophages RAW 264.7 with C. neoformans RAW 264
7 macrophage-like cells (American Type Culture collection, Manassas, CA, USA) were cultured as described previously in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum, penicillin and streptomycin (Life Technologies, Carlsbad, CA, USA). Cells were removed from plates by gentle scraping in fresh medium and counted by haemocytometer. For in-vitro stimulation,

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C. neoformans were cultured, rinsed and enumerated as described above. Macrophages (5 3 105 cells) were plated into each well of a 24-well tissue culture plate. After 24 h, macrophages were then infected with 5 3 106 C. neoformans cells (for 10: one yeast per macrophage) for 6, 18 and 24 h, and then extracellular yeasts were washed away. Cultures were then harvested in Trizol for RNA extraction.

family gene and chemokine expressions in which each value of mRNA expression was estimated as log-transformed before statistical analysis. Fungal burden analysis was evaluated using an unpaired two-tailed t-test. All statistical analysis was performed with GraphPad Prism version 5 software. A value of P < 0
05 was considered significant.

Zymography

Results

For zymography analysis, BAL fluid or pooled culture supernatants of alveolar macrophages were concentrated with Amicon Ultra Centrifugal Filter Units (Millipore Ltd., Tullagreen, Carrigtwohill, Ireland). Gelatin and casein zymography were performed using concentrated BAL fluid, as described previously [16]. Equal amounts of concentrated BAL fluid proteins underwent electrophoresis in sodium dodecyl sulphate (SDS) under non-reducing conditions (10% polyacrylamide gels containing 2 mg/ml gelatin (Biorad, Hercules, CA, USA) or 12% polyacrylamide gels containing 1 mg/ml casein (Sigma, St Louis, MO, USA). After electrophoresis, gels were washed twice each for 30 min in 2
5% Triton X-100 and washed twice each for 10 min in 10 mM Tris-HCl (pH 7
5) and then incubated in 50 mM Tris-HCl (pH 7
5), 10 mM CaCl2, 150 mM NaCl, 0
05% Brij35 and 1 lM ZnCl2 at 378C overnight. Following incubation, the gels were stained with 0
5% Coomassie brilliant blue R-250 for 3–4 h and destained in a solution of 40% methanol and 10% acetic acid. Enzyme activity was detected as clear bands against a blue background. The intensity of the bands in the inverted image of the zymogram was estimated using densitometry and analysed by Image Lab software (Biorad).

The induction of matrix metalloproteinases (MMPs) in lungs of C. neoformans-infected mice

Real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis Lungs were removed from naive or infected mice and homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA extracted using TRIzol reagent was used to generate cDNA using oligo-dT, random hexamers and Moloney murine leukaemia virus (MMLV) reverse transcriptase (Invitrogen) [17]. To detect MMP and chemokine expression, cDNA samples were amplified in IQTM SYRBV Green Supermix (Biorad). The data were normalized to actin expression (Actb). The primer pairs for the analysis of MMPs, tissue inhibitors of metalloproteinases (TIMPs) and chemokine were used as described previously [5,17–20]. R

Statistical analysis Each experiment was conducted two or three times. Data are presented as mean value 6 standard deviation (s.d.). Data were analysed using one-way analysis of variance (ANOVA) with Tukey’s post-hoc analysis. A Pearson’s correlation test was used to assess the correlation between MMP

To determine the roles of MMPs during Cryptococcus infection, we first analysed the expression of several MMPs known previously to be involved in lung infection in a mouse model of cryptococcosis. C57BL/6 mice were infected with C. neoformans and the kinetics of MMPs expression in the lungs were analysed following infection at days 1, 7 and 14 and were then compared with control mice treated with PBS by real-time PCR analysis. We detected the significant up-regulation of MMP-3 transcript at day 7 post-infection (Fig. 1a). Interestingly, among analysed MMP members, MMP-12 was found to be highly induced and remained up-regulated from days 7 to 14 post-infection (Fig. 1a). Because the proteolytic activities of MMPs could be regulated by TIMPs, we therefore further analysed the expression of TIMP-1 and TIMP-2 after infection. Indeed, we found that mRNA expression of TIMP-1, but not TIMP-2, was increased significantly at days 1 and 7 post-infection (Fig. 1a). We confirmed our data by assessing MMP enzyme activity in bronchoalveolar lavage fluid of C. neoformansinfected mice using gelatine (for MMP-2, MMP-9) or casein zymography (for MMP-3 and MMP-12). Consistent with the dramatically increased mRNA levels, we detected greater MMP-12 activity levels by casein zymography in BAL fluid of C. neoformans-infected mice at day 7 postinfection compared with PBS-treated mice (Fig. 1b). Although we detected enhanced MMP-3 transcripts, we could not detect a significant increase in MMP-3 activity in C. neoformans-infected BAL fluid (Fig. 1b). Altogether, these data suggest that C. neoformans induces the expression and activity of MMP-12 in the lungs.

Differential expression of pulmonary MMPs in mice infected with C. neoformans and C. gattii Besides C. neoformans, C. gattii is recognized as an important pathogenic Cryptococcus spp. that commonly affects healthy individuals. Mice infected with the highly virulent C. gattii strain R265, compared with those infected with C. neoformans, displayed attenuated pulmonary inflammation, suggesting that the highly virulent C. gattii strain may cause disease in immunocompetent hosts by suppressing the pulmonary immune response [3–5]. To understand the

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Fig. 1. The induction of pulmonary matrix metalloproteinases (MMPs) by Cryptococcus neoformans infection. C57BL/6 mice were treated with phosphate-buffered saline (PBS) (control) or infected with highly virulent strains of Cryptococcus neoformans (H99) and killed at 1, 7 and 14 days post-infection. Lungs were harvested and analysed for the mRNA expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) (a) and activity of MMPs (b). (a) Total RNA was isolated from lungs and subjected to cDNA synthesis and subsequent real-time polymerase chain reaction (PCR) analysis. Data are expressed as fold induction over actin (Actb) expression, with mRNA levels in the PBS-treated group set as 1. (b) Bronchoalveolar lavage (BAL) fluid from PBS-treated mice and mice infected with H99 for 7 days were harvested and subjected to gelatin (MMP-2 and MMP-9) and casein (MMP-3 and MMP-12) zymography. The results shown are representative data of three independent experiments. Graphs depict mean 6 standard deviation (s.d.) and are representative of three experiments with three mice per group. *P < 0
05; **P < 0
01.

roles of MMPs in the pathogenesis of Cryptococcus infection, we compared the expression of MMPs in the lungs of mice infected with the highly virulent C. neoformans strain H99 and those infected with the highly virulent C. gattii strain R265. Mice infected with C. neoformans, but not those with C. gattii, had a marked induction of MMP-3 mRNA levels at day 7 post-infection (Fig. 2a). Although MMP-12 mRNA levels in C. gattii-infected mice were up-regulated when compared to PBS-treated mice, the expression levels were significantly lower than those in C. neoformans-infected mice (Fig. 2a,b). We also detected the expression of TIMP-1 after C. gattii infection, although to a lesser extent, than after C. neoformans infection at day 7 post-infection (Fig. 2a). As MMP-12 was highly induced following C. neoformans infection, we confirmed these data by examining the MMP-12 activity in the BAL fluid of C. neoformans- and C. gatii-infected mice. As shown in Fig. 2c, levels of active MMP12 detected by zymography at 22 kDa were increased in BAL fluid of C. neoformans strain 434

H99-infected mice but not in those of C. gattii-infected mice (Fig. 2c). These data suggest that the induction of MMP-12 may be involved in the pathogenesis of Cryptococcus infection.

C. neoformans induces MMP-12 expression by macrophages To investigate the roles of MMP-12 in Cryptococcus infection, we further tested the direct effect of C. neoformans infection on the expression of MMP-12 by macrophages. First, we stimulated the RAW264
7 murine macrophage cell line with C. neoformans for 6, 18 and 24 h and determined the expression of MMP-12 mRNA by using real-time PCR analysis. As expected, we could detect the significant up-regulation of MMP-12 after 24 h of stimulation with C. neoformans (Fig. 3a). To confirm whether or not MMP-12 was indeed produced by macrophages during Cryptococcus infection, we assessed the expression and

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Fig. 2. Cryptococcus gattii-infected mice demonstrate reduced induction of matrix metalloproteinases (MMPs) in the lungs. C57BL/6 mice were treated with phosphate-buffered saline (PBS) for control or infected with highly virulent strains of C. neoformans (H99) or C. gattii (R265). (a,b) Lungs were harvested and analysed for mRNA expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) at (a) day 7 and (b) day 14 post-infection. Total RNA was isolated from lungs and subjected to cDNA synthesis and subsequent real-time polymerase chain reaction (PCR) analysis. Data are expressed as fold induction over actin (Actb) expression, with mRNA levels in the PBS-treated group set as 1. (c) Bronchoalveolar lavage (BAL) fluid from PBS-treated mice and mice infected with H99 or R265 for 7 days were harvested and subjected to casein zymography analysis. The results shown are representative data of three independent experiments. Graphs depict mean 6 standard deviation (s.d.) and are representative of three experiments with three mice per group. *P < 0
05; **P < 0
01.

activity of MMP-12 in purified CD11b1 macrophages from BAL fluid of mice infected with Cryptococcus spp. for 7 days. The expression and activity of MMP-12 were elevated significantly in CD11b1 macrophage cells purified from C. neoformans H99-infected mice (Fig. 3b,c). Besides MMP12, which is known to be produced by macrophages, we also detected the induction of MMP-9 in the enriched macrophages of C. neoformans-infected mice (Fig. 3b,c). Consistent with total lung gene expression analysis, CD11b1 macrophages from BAL fluid of C. gattii-infected mice expressed much lower levels of MMP-12 transcript and activity than those from lungs of C. neoformans-infected mice (Fig. 3b,c). Interestingly, purified macrophages of C. gattii-infected mice exhibited lower levels of MMP-9 mRNA and activity than those of C. neoforman-infected mice (Fig. 3b,c), although this difference was not observed in total lung cell analysis. Our data indicate that C. neoformans infection may stimulate the expression of MMP-12,

and possibly MMP-9 by macrophages, which then play a role in cryptococcal disease pathogenesis.

Effect of matrix metalloproteinase inhibition in a mouse model of C. neoformans infection To test the function of MMPs during C. neoformans infection, we utilized the broad-spectrum matrix metalloproteinase inhibitor GM6001 in our study. GM6001 is known to inhibit both MMP activity (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-14) and disease development in murine models of asthma [21], emphysema [22] and asbestos-induced lung injury [23]. The MMP inhibitor GM6001 or DMSO (as control) was given daily by i.p. injection into C. neoformans-infected mice. Mice receiving GM6001 or vehicle were then subjected to fungal burden analysis in lungs after 7 days of infection. Interestingly, administration of the MMP inhibitor during

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O. Supasorn et al. Fig. 3. The induction of matrix metalloproteinase (MMP)-12 by macrophages infected with Cryptococcus neoformans but not C. gattii. (a) RAW 264.7 cells were treated with highly virulent C. neoformans (H99) or left untreated for 6, 18 and 24 h and analysed for the mRNA expression of MMP-12 by real-time polymerase chain reaction (PCR) analysis. (b,c) Alveolar macrophages purified from bronchoalveolar lavage cells in PBStreated mice, C. neoformans-infeced mice and C. gattii-infected mice were subjected to RNA extraction, cDNA synthesis and analysis of MMP-12 transcript level by real-time PCR analysis (b) and to MMP zymography (c). Data are expressed as fold induction over actin (Actb) expression, with the mRNA levels in the PBS-treated group set as 1. Graphs depict mean 6 standard deviation (s.d.) and are representative of at least two to three independent experiments with three to five mice per group. *P < 0
05; **P < 0
01.

Fig. 4. The effect of a broad spectrum matrix metalloproteinase (MMP) inhibitor GM6001 treatment in Cryptococcus neoformans-infected mice on lung fungal burdens and inflammatory cell infiltration. C57BL/6 mice were infected with C. neoformans (H99) and daily treated with GM6001 or vehicle (DMSO). At day 7 post-infection, whole lungs were harvested and analysed for (a) pulmonary fungal burdens and (b) inflammatory cell infiltration. (b) Total cell numbers, neutrophils (Gr.11CD11b1), dendritic cells (CD11bhiCD11chi) and macrophages (CD11b1CD11c2) were assessed by flow cytometry analysis. The results are presented as dot-plots and absolute numbers of cells. Flow cytometric data shown are a representative dot-plot of one of three independent experiments. Graphs depict mean 6 standard deviation (s.d.) and are representative of three experiments with three to four mice per group. *P < 0
05; **P < 0
01.

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Fig. 5. Matrix metalloproteinase (MMP) expression is associated with the pulmonary chemokine expression level in Cryptococcus infection. (a) C57BL/6 mice were infected with C. neoformans (H99) and treated daily with GM6001 or vehicle [dimethylsulphoxide (DMSO)]. At day 7 post-infection, total RNA of lungs was isolated and subjected to cDNA synthesis and subsequent real-time polymerase chain reaction (PCR) analysis of chemokine gene expression. Data are expressed as fold induction over actin (Actb) expression, with mRNA levels in the phosphate-buffered saline (PBS)-treated group set as 1. (b) The correlation between MMP-12 expression and the expression of CXCL1, CCL2 and CCL11 in mice infected with C. neoformans (H99) and C. gattii (R265) was evaluated. A Pearson’s correlation test was used to assess the correlation between chemokine and MMP family gene expressions in which each value of mRNA expression was estimated as log-transformed before statistical analysis. Graphs depict mean 6 standard deviation (s.d.) and are representative of three experiments with three to four mice per group. *P < 0
05.

C. neoformans infection resulted in a significantly enhanced lung fungal burden (Fig. 4a). The numbers of inflammatory cells in the lungs of C. neoformans-infected mice treated with GM6001 and DMSO were analysed further by flow cytometry. As shown in Fig. 4b, total lung-infiltrating cells, macrophages, neutrophils and myeloid dendritic cells in the GM6001-treated group were reduced compared to those in the vehicle-treated group. Thus, our data suggest that MMP activity is involved in promoting inflammatory cell infiltration to control pulmonary fungal load during Cryptococcus infection. As giving GM6001 in C. neoformans-infected mice inhibited pulmonary inflammation, we therefore assessed whether MMPs could regulate the immune response by modulating chemokine expression during Cryptocccus infection. Indeed, C. neoformans-infected mice treated with GM6001 had attenuated expression of CXCL-1 and CCL-2 but not CCL-3, CCL-11 and CCL-20 (Fig. 5a). To further determine the involvement of MMP-12 function in chemokine expression, we examined the correlation between MMP-12 transcripts and mRNA levels of chemokines CXCL-1 and CCL-2 in lungs of mice infected with C. neoformans and C. gattii. As indicated in Fig. 5b, there was a

significant correlation between the level of MMP-12 and chemokine expression. The strongest correlation was observed between MMP-12 and CCL-2, which is known to be a macrophage-attracting chemokine. Altogether, these data indicated that MMPs play a role in controlling Cryptococcus fungal burden by regulating chemokine expression and inflammatory cell recruitment into infected sites.

Discussion The MMP family members are known to contribute to the host immune response and pathogenesis of acute and chronic diseases, including allergic asthma, emphysema, lung cancer and infection [6,7]. Although previous studies implicated the involvement of MMP members in several infectious diseases [11,24–26], the function of MMPs in response to Cryptococcus fungus infection has not been addressed clearly. In the present study, we examined the expression of different MMPs during Cryptococcus infection and the effect of MMP inhibition in regulating host immune responses and disease pathogenesis. We found that MMPs played an important role in promoting inflammatory cell infiltration into Cryptococcus-infected lungs,

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thereby enhancing effective host immunity to pulmonary Cryptococcus infection. Numerous reports described an increased expression of a variety of MMPs in response to lung infection. For example, mice deficient of MMP-2 and MMP-9 failed to clear S. pneumoniae bacterial infection effectively [27]. MMP-9 was required for controlling influenza viral replication in the respiratory tract [28]. MMP-3 was induced by an infectious bronchitis virus and was suggested to regulate cytokine and chemokine expression [29]. Recent evidence indicated that MMP-12 was involved in respiratory syncytial virus (RSV)induced respiratory symptoms and plays a role in regulating interferon (IFN)-a production in the anti-viral response [30,31]. In our study, we found a dramatic increase in mRNA expression of MMP-3 and MMP-12 and activity of MMP-12 after pulmonary Cryptococcus neoformans infection. Similar to a previous study, the transcript levels of MMP-2 was not induced in the lungs of C. neoformans-infected C57BL/6 mice [13]. However, it was suggested that MMP-2 was involved in the lung pathology of Cryptococcus infection, as the expression of MMP-2 was detected in CB17 mice which caused well-defined granulomas after infection [13]. Studies in patients with cryptococcosis showed high levels of MMP-9 in the cerebrospinal fluid (CSF) of patients infected with C. neoformans [32]. In our study, there was a trend towards an increase of MMP9 mRNA in the lungs following infection, although was not statistically significant. Our data indicated that in addition to MMP-2 and MMP-9, MMP-3 and MMP-12 might be involved in the host immune response to Cryptococcus infection. Besides C. neoformans, C. gattii has been shown recently to be an important fungal pathogen, as it is able to infect hosts with normal immune responses. Some evidence suggests that the difference in their target organs after C. gattii infection often caused pulmonary rather than central nervous system (CNS) infection [3]. Moreover, it has been suggested that the mechanisms of that this highly virulent strain of fungus could use to infect immunocompetent hosts may be through inhibition of both pulmonary inflammation and the T helper cell response [4,5]. By comparing the expression and activity of MMPs in mouse lung infected with C. neoformans and C. gattii, we detected the profound difference in both expression and activity of MMP-12 after infection for 7 and 14 days. Besides MMP12, MMP-3 transcript levels of C. gattii-infected mice were lower than those of C. neoformans-infected mice. In the lungs, MMP-12 is produced primarily by alveolar macrophages and to a lesser extent by dendritic cells, lung epithelial cells and smooth muscle cells. Indeed, we found that MMP-12 and MMP-9 were expressed in purified macrophage cells in response to C. neoformans but not to C. gattii stimulation. These data implied that the induced expression of MMPs may contribute to cryptococcal disease pathogenesis. MMPs may function by augmenting host 438

immune responses to Cryptococcus infection. Lower expression of MMPs after C. gattii infection may thereby result in an ineffective control of fungal growth in lungs. The imbalance between MMPs and tissue endogenous inhibitors of MMPs (TIMPs) activities is known to be involved in several lung disease pathologies [33]. After Cryptococcus infection, only TIMP-1 but not the TIMP-2 transcript was induced. The expression of TIMP-1 in mice infected with C. gattii, similar to that of MMP-12, is increased to a lesser extent than in those infected with C. neoformans. TIMP-1 is known to inhibit the activity of most MMPs, including MMP-9 and MMP-12 [33]. As shown by previous studies, the increased expression of MMP-9 and MMP-12 was associated with enhanced TIMP1 expression [34,35]. Induced TIMP-1 expression after Cryptococcus infection might play a role in balancing the activity of MMPs during inflammation. Moreover, we observed that the up-regulation of MMP mRNA did not always reflect the level of enzyme activity in the zymography after Cryptococcus infection. It is likely that the activity of MMPs might be modulated by the regulation of TIMPs or other post-transcriptional mechanisms [36,37]. Although MMPs were suggested to be involved in the lung pathology of Cryptococcal diseases, in-vivo experiments confirmed that MMP functions in this disease have not been addressed. Interestingly, by using the broadspectrum MMP inhibitor GM6001 in mice infected with C. neoformans for 7 days, we detected the enhanced fungal burden in GM6001-treated mice compared to those receiving vehicle, suggesting that the MMP inhibitor GM6001 could limit the growth of fungus but does not eradicate fungal burden. In the GM6001-treated mice, we observed attenuated inflammatory cell infiltration. This reduction in the infiltration of inflammatory cells was associated with a dampened chemokine response after MMP inhibitor was given. Previously, it has been demonstrated that rhMMP12 instillation in mouse airways resulted in the increased levels of IL-6, tumour necrosis factor (TNF)-a, CCL-2 [monocyte chemotactic protein 1 (MCP-1)], CCL-3 [macrophage inflammatory protein 1 (MIP-1a)] and CXCL-1 (KC) [38]. In our study, we observed a strong association between the expression of MMP-12 and chemokines CCL2 and CXCL-1, which are known to be important factors for monocyte, dendritic cells, macrophage and neutrophil recruitment, respectively. Mechanistically, we proposed that MMPs may function by modulating chemokine expression, allowing the activation of chemokines to promote inflammatory cell recruitment into the lungs after Cryptococcus infection. In conclusion, we provide important evidence that MMPs are important mediators in regulating the host immune response to pulmonary cryptococcosis. Despite the fact that several MMPs were known to be involved in tissue damage and MMP-2 was reported to be associated with lung granuloma formation in Cryptococcus infection,

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it is possible that MMPs may play a dual function in response to Cryptococcus infection. Early expression of MMPs may function by promoting inflammatory cell recruitment, but they may also be involved in tissue damage during chronic infection. Further studies on MMP inhibitor treatment during chronic infection may provide better understanding in the roles of MMPs in Cryptococcus infection. Furthermore, targeting specific MMP inhibitors, particularly MMP-12, could be an interesting subject for future studies and may help in the better design of therapeutic strategies.

Acknowledgements We thank the Faculty of Allied Health Sciences, Thammasat University for their support, the Institute of Drug Discovery and Development, Thammasat University for the Flow Cytometry Facility and Hoainam T. Nguyen-Jackson for her critical reading of the manuscript. This work was supported by grants from Thammasat University, Thailand.

Disclosure The authors declare no financial disclosures.

Author contributions O. S., N. S. and P. A. performed the experiments. P. A. and P. S. designed the study. P. A. and O. S. prepared the manuscript. All authors read and approved the final manuscript.

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