Curcumin increases gelatinase activity in human neutrophils by a p38 mitogen-activated protein kinase (MAPK)-independent mechanism

Abstract

Curcumin has been found to possess anti-inflammatory activities and neutrophils, key players in inflammation, were previously found to be important targets to curcumin in a few studies. For example, curcumin was found to induce apoptosis in neutrophils by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. However, the role of curcumin on the biology of neutrophils is still poorly defined. To study the role of curcumin on neutrophil degranulation and to determine the role of p38 MAPK, human neutrophils were freshly isolated from healthy individuals and incubated in vitro with curcumin. Degranulation was studied at three levels: surface expression of granule markers by flow cytometry; release of matrix metallopeptidase-9 (MMP-9 or gelatinase B) enzyme into supernatants by Western blot; and gelatinase B activity by zymography. Activation of p38 MAPK was studied by monitoring its tyrosine phosphorylation levels by western blot and its role by the utilization of a pharmacological inhibitor. The results indicate that curcumin increased the cell surface expression of CD35 (secretory vesicle), CD63 (azurophilic granules), and CD66b (gelatinase granules) in neutrophils. Also, curcumin increased the release and enzymatic activity of gelatinase B in the extracellular milieu and activated p38 MAP kinase in these cells. However, in contrast to fMLP, curcumin-induced enzymatic activity and secretion of gelatinase B were not reversed by use of a p38 inhibitor. Finally, it was found that curcumin was able to enhance phagocytosis. Taken together, the results here demonstrate that curcumin induced degranulation in human neutrophils and that the increased gelatinase activity is not dependent on p38 MAPK activation. Therefore, degranulation is another human neutrophil function that could be modulated by curcumin, as well as phagocytosis.

• Degranulation
• flow cytometry
• inflammation
• granulocytes
• zymography

Introduction

In humans, polymorphonuclear neutrophil cells (PMN) are the most abundant circulating leukocytes and are indispensable for innate immunity against invading micro-organisms (Antoine et al., 2013; Tak et al., 2013). PMN are key player cells in inflammation and are known to be among the first cells to arrive at an inflammatory site. They are known to exert several beneficial functions, including phagocytosis and production of a plethora of potent immunomodulatory molecules including chemokines, cytokines, eicosanoids, and reactive oxygen species (ROS) (Amulic et al., 2012; Duffin et al., 2010; Mocsai, 2013).

One important mechanism exerted by PMN is degranulation, leading to release of diverse immunomodulatory agents, many of which are pro-inflammatory (Amulic et al., 2012; Hoffstein et al., 1982; Witko-Sarsat et al., 2000). Neutrophils possess four types of granules containing different, but some overlapping, sets of proteins. Specific proteins in the lumen and on the membrane characterize the different types of granules. Azurophilic granules contain high amounts of lysosomal enzymes (Faurschou & Borregaard, 2003) and are characterized by expression of granulophysin (CD63) in their membranes (Cham et al., 1994). Gelatinase granules are the principal reservoir of degrading enzymes, including metalloproteinases (Borregaard, 2010; Kjeldsen et al., 1992). Specific granules are rich in anti-microbial peptides and participate in the anti-microbial activity of PMN (Borregaard, 2010; Mollinedo et al., 1997). These two latter granules subsets are characterized by the presence of specific markers, including CD66b, CD15, or CD67 (Borregaard, 2010; Borregaard & Cowland, 1997; Borregaard et al., 2001). Lastly, secretory vesicles, presumed formed by endocytosis (Borregaard, 2010; Borregaard et al., 1987), constitute the main source of plasma proteins and membrane-associated receptors essential for PMN inflammatory responses and are released easily in response to inflammatory agents. This latter type of granule is characterized by a presence of CD35 in the membrane (Perretti et al., 2000).

Curcumin is obtained from the dried rhizome powder of Curcuma longa and has been commonly used for centuries as a spice in curries, and more recently in food additives as well as a dietary pigment. Today, there is increased interest in the potential use of this plant as a nutraceutical due to knowledge emerging from laboratories worldwide that has confirmed its beneficial effects for both animal and human health. Curcumin has been found to possess potent immunomodulatory properties and is especially recognized for its anti-inflammatory activity (Manjunatha & Srinivasan, 2006; Moon et al., 2010; Shehzad et al., 2013). In PMN, curcumin has been found to induce apoptosis in a concentration-dependent manner (Hu et al., 2005), agreeing with its known anti-inflammatory activity. In addition, our laboratories recently demonstrated that, in vitro, curcumin inhibited agent-induced PMN functions including generation of ROS and cytokine production; in vivo, curcumin inhibited lipopolysaccharide (LPS)-induced neutrophilic inflammation (Antoine et al., 2013). Despite the fact that curcumin possesses strong immunomodulatory effects on PMN, how curcumin alters degranulation in human PMN has never been investigated. In this study, it was seen that curcumin increased the cell surface expression of the three markers of the principal subsets of granules in PMN. It was also observed that curcumin increased the enzymatic activity of gelatinase release in the supernatants of the PMN via a p38-independent mechanism.

Materials and methods

Chemicals

Otherwise specified, all chemicals were purchased from Sigma-Aldrich (St-Louis, MO). Based on prior experiments (Antoine et al., 2013; Hu et al., 2005), curcumin was used at 10 or 50 μM throughout these studies.

Isolation of human PMN

Neutrophils were isolated from venous blood from healthy volunteers by dextran sedimentation followed by centrifugation over Ficoll-Hypaque (Pharmacia Biotech Inc., Quebec, Canada), as described previously (Antoine et al., 2013; Babin et al., 2013). Blood donations were obtained from informed consenting individuals according to institutionally approved procedures. Cell viability was assessed by trypan blue exclusion (>99%); cell purity (>97%) was confirmed from cytocentrifuged preparations stained with HemaStain 3 (Fisher Scientific, Ottawa, ON).

Cell surface expression of granules markers

Cell surface expression of CD35, CD63, and CD66b was monitored by flow cytometry to assess the degranulation status, respectively, of secretory, azurophilic, and gelatinase granules as previously published (Babin et al., 2013; Simard et al., 2010). Non-specific binding of antibodies was prevented by incubating the cells (10⁶cells/ml) in a solution of phosphate-buffered saline (PBS, pH 7.4) +20% autologous serum for 30 min on ice. After several washes, primary antibodies directed against CD35 (clone 555451; BD Biosciences, Mississauga, ON), CD63 (clone 556019; BD Biosciences), CD66b (clone 80H3; AbD Serotec/BioRaD, Raleigh, NC), or an IgG₁ isotypic control antibody (R&D Systems Inc., Minneapolis, MN) were added to separate aliquots of the cells, each at a concentration of 0.5 μg/ml, and the cells were incubated on ice for 30 min. The cells were then washed twice and incubated with 10 μg/ml FITC-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA) for 30 min at 4 °C. After two washes with PBS, analysis was performed with a FACScan (BD Biosciences). All data were analyzed using Flowing Software v2.5.1 (Turku Center for Biotechnology, Turku, Finland). A minimum of 10 000 events was acquired for each sample.

Gelatin zymography

Isolated PMN (10 × 10⁶cells/ml in complete RPMI 1640 [containing 5 mM HEPES {N-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid)}, 100 U penicillin/ml, and 100 μg streptomycin/ml]) were incubated for 30 min under indicated conditions. Cells were then centrifuged at 13 000 rpm for 10 min and the pellets discarded. The supernatants (10 μl corresponded to materials derived from 0.1 × 10⁶ cells) were then mixed with non-reducing buffer (40% glycerol, 1 M Tris-HCl [pH 6.8], 8% sodium dodecyl sulfate [SDS]) and resolved over a 7.5% acrylamide gel containing 0.2% gelatin (Babin et al., 2013). The gel was then washed for 30 min (twice with 2.5% Triton X-100 in water) and incubated overnight in enzymatic digestion buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM CaCl₂). The gel was then stained with 0.1% Coomassie blue solution and then unstained as described previously (Babin et al., 2013).

Phosphorylation of p38 MAP kinases

Isolated PMN (10 × 10⁶cells/ml in complete RPMI-1640 were stimulated with indicated agonist for 30 min at 37 °C. The cells were then lysed in 4× Laemmli sample buffer, and aliquots corresponding to 10⁶ cells resolved over 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels and then electrotransferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature with TBS-Tween (Tris-buffered saline [TBS]-Tween 0.1%) containing 3% bovine serum albumin (BSA), and then incubated with a solution of TBS-Tween containing anti-phospho-specific p38 antibody (Bio-Source, Camarillo, CA; 1:1000 dilution) on a rocking platform overnight at 4 °C. Thereafter, the membranes were washed with TBS-Tween and incubated for 1 h at room temperature in a solution of TBS-Tween containing rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch; 1:25 000 dilution). After several washes with TBS-Tween, protein expression was revealed using an enhanced chemiluminescence Western blot analysis detection system (Amersham Biosciences, Piscataway, NJ). For protein loading determination, membranes were stripped and stained with antibody directed against the corresponding un-phosphorylated form of p38 (sc-535; Santa Cruz Biotechnology, Dallas, TX).

Expression of matrix metallopeptidase-9 (MMP-9) in the external milieu

Isolated PMN (10 × 10⁶cells/ml in complete RPMI 1640 were stimulated with curcumin (10 or 50 μM), N-formyl-Met-Leu-Phe (fMLP) (10⁻⁹M), or buffer/diluent (Hanks Balance Salt Solution [HBSS]) containing 1% dimethyl sulfoxide [DMSO]) for 30 min at 37 °C. In some conditions, cells were pre-incubated with 2 μM of p38 mitogen-activated protein kinase (MAPK) inhibitor (p38i) or diluent as control for 30 min prior to agonist stimulation. Cells were then centrifuged and pellets discarded. Supernatants were used to assess expression of MMP-9 by Western blot. In brief, a volume of 15 μl supernatant was mixed with 5 μl 4× Laemmli buffer, and aliquots corresponding to 0.15 × 10⁶ cells resolved over 7.5% SDS-PAGE gels and electrotransferred to nitrocellulose membranes. The membranes were blocked for 1 h as above and then the membranes were incubated with a solution of TBS-Tween containing anti-MMP-9 antibody (Abcam, Cambridge, UK; 1:1000) overnight on a rocking platform at 4 °C. The membranes were then washed with TBS-Tween and incubated for 1 h at room temperature in a solution of TBS-Tween containing goat anti-mouse HRP-conjugated secondary antibody (1:25 000 dilution). After several washes with TBS-Tween, protein expression was revealed using the enhanced chemiluminescence blot analysis detection system.

Phagocytosis of sheep red blood cells (SRBC)

SRBC (Quelab, Montreal, Canada) were opsonized by incubation with a 1:200 dilution of a rabbit IgG anti-SRBC antibody (Sigma-Aldrich) in PBS for 45 min at 37 °C. Isolated PMN (10⁶cells/ml in complete RPMI-1640) were treated for 30 min with the indicated agonists and then 10⁶ PMN/treatment were each incubated with 5 × 10⁶ opsonized SRBC for 30 min at 37 °C. Thereafter, the samples were centrifuged at 200 × g for 10 min at 4 °C. Resulting supernatants were discarded and osmotic shock was performed on the pellets by suspending the cells in 400 μl distilled water for 15 s, followed by addition of 4.5 ml PBS. The samples were then washed twice with PBS and the final pellets were suspended in 100 μl PBS. Aliquots of the materials were then placed on a slide and examined using a light microscope. The incidence of phagocytosis was expressed as the percentage of PMN having ingested at least one opsonized SRBC. A minimum of five different fields (corresponding to ∼200 PMN/slide) was randomly assessed; each slide was analyzed in duplicate.

Statistical analysis

Data are reported as mean ± SEM and analyzed by one-way analysis of variance (ANOVA). Differences between test groups and control were assessed using a Dunnett’s Multiple Comparison Test. All analyses were performed using GraphPad Prism (v5.00 for Windows; GraphPad Software, San Diego, CA). Statistical significance was established at a p value < 0.05.

Results

Curcumin increased cell surface expression of granule markers CD35, CD63, and CD66b

As illustrated in Figure 1, curcumin significantly induced cell surface expression of the three markers of the different subsets of granules, even at the lowest concentration used (10 μM; CUR10). As expected, positive control fMLP (0.1 μM) was able to induce cell surface expression of CD35 (secretory) and CD66b (gelatinases), but not CD63 (azurophilic) (Simard et al., 2010). When used in combination with fMLP, curcumin was able to potentiate cell surface expression of CD35 but not CD66b. Although fMLP itself did not increase CD63, the combination curcumin + fMLP further increased the surface expression of this marker when compared to that by curcumin alone. Of note, the effect of the combination fMLP + curcumin50 upon (50 µM; CUR50) CD63 was statistically significant, but not upon CD35 or CD66b (although not indicated in figure, for clarity).

Figure 1. Curcumin induces cell surface expression of granule markers CD35, CD63, and CD66b in human PMN. Freshly isolated human PMN (10 × 10⁶cells/ml in complete RPMI 1640) were incubated with buffer (Ctrl), 10 μM curcumin (CUR10), 50 μM curcumin (CUR50), 10⁻⁹M fMLP, or a mixture of CUR50 + fMLP for 30 min. The cell surface expression of (A) CD35, (B) CD63, and (C) CD66b was then assessed by flow cytometry. Results are in terms of the general mean (Gmean) of fluorescence expressed; shown are the mean ± SEM (n = 5) of these values. *p < 0.05 versus Ctrl; **p < 0.05 versus fMLP. Iso, isotypic control for the assay; ns = not significant.

Curcumin enhanced gelatinase (MMP-9) release/enzymatic activity in extracellular milieu

Knowing that curcumin increased the cell surface expression of granule markers, whether or not curcumin could induce release of MMP-9 was next assessed. Figure 2A illustrates that curcumin increased MMP-9 protein release into the supernatants of human PMN (assessed by Western blot). Of note, the combination of curcumin + fMLP did not result in an over-expression of MMP-9 protein compared to that induced by fMLP alone. Interestingly, zymography experiments revealed that curcumin increased gelatinase enzymatic activity (Figure 2B). As with the protein expression, enzymatic activity was not further increased by a curcumin + fMLP mixture.

Figure 2. Curcumin increases MMP-9 expression and gelatinase activity in extracellular milieu. Freshly isolated human PMN (10 × 10⁶cells/ml in complete RPMI 1640) were stimulated for 30 min with buffer (Ctrl), 10 μM curcumin (CUR10), 50 μM curcumin (CUR50), 10⁻⁹M fMLP, or a mixture of CUR50 + fMLP; supernatants were then harvested and used to perform (A) Western blots for analysis of MMP-9 protein expression or (B) zymography assays for determining enzymatic activity. (A, B) One representative experiments is shown (of six). the three gelatinase enzymatic activities (A, B, C) were similarly affected in all test conditions.

Role of p38 MAPK in release/enzymatic activity in curcumin-induced human PMN

Curcumin is known to activate p38 MAPK in human PMN; this kinase is also involved in the pro-apoptotic effect of curcumin (Hu et al., 2005). However, the activation of p38 MAPK under priming conditions is unknown. Further, the role of p38 MAPK in degranulation has never been investigated. As illustrated in Figure 3A, curcumin effectively activated p38 MAPK, but the activation state was not further increased when the PMN were treated with a mixture of curcumin + fMLP. When the role of p38 MAPK was evaluated by a pharmacological approach, it was seen that expression of MMP-9 protein was significantly decreased by treatment with curcumin + fMLP versus that by fMLP alone. Although MMP-9 expression was weaker in this cohort of blood donors, as opposed to in Figure 2A, the results are clear-cut and demonstrate that p38 MAPK inhibitor (p38i) did not significantly decrease the levels of MMP-9 in response to curcumin alone. As with MMP-9 protein expression, treatment with the p38 MAPK inhibitor significantly reversed the gelatinase activity induced by fMLP, but not that induced by curcumin (Figure 4).

Figure 3. Curcumin does not further increase fMLP-induced p38 MAPK in human PMN and does not increase MMP-9 protein expression via p38 MAPK-dependent mechanism. (A) Cells (10 × 10⁶cells/ml complete RPMI 1640) were stimulated for 30 min with buffer (Ctrl), 10 μM curcumin (CUR10), 50 μM curcumin (CUR50), 10⁻⁹M fMLP, or a mixture of CUR50 + fMLP; p38 MAPK activation was assessed by Western blot analysis. (B) Cells were treated as above but pre-incubated with diluent or 2 μM of p38 MAPK inhibitor (p38i) for 30 min; MMP-9 protein expression was studied by Western blot analysis. (A) Results from one representative experiment (of three). Equivalent loading was evaluated by expression of un-phosphorylated p38. (B) Densitometric analysis is plotted in a bar graph (integrated density value) to quantify MMP-9 protein expression (mean ± SEM, n = 3). Bottom: results from one representative experiment.

Figure 4. Ability of curcumin to increase gelatinase activity is unaffected by p38 MAPK inhibitor. PMN (10 × 10⁶cells/ml complete RPMI 1640) were pre-incubated with diluent or 2 μM of p38 MAPK inhibitor (p38i) for 30 min, and then treated for 30 min with buffer (Ctrl), 10 μM curcumin (CUR10), 50 μM curcumin (CUR50), or 10⁻⁹M fMLP; supernatants were then harvested and underwent zymography assays. (A) Results from one representative experiment showing the three gelatinase activities (A, B, C). (D) Densitometric analysis of the three detected gelatinase activities were added together as each was similarly affected. Results are mean ± SEM (n ≥ 3). *p < 0.05 versus Ctrl; **p < 0.05 fLMP without p38i versus fMLP with p38i.

Curcumin enhanced phagocytosis but did not prime the same fMLP-induced response

The effect of curcumin on F_cR-mediated phagocytosis of opsonized SRBC was examined to ascertain whether curcumin would enhance another fMLP-induced PMN response apart from degranulation. As illustrated in Figure 5, 50 μM curcumin (60.9 ± 3.6%), like fMLP (61.5 ± 6.3%), significantly enhanced phagocytosis compared to that seen with control cells (40.4 ± 3.1%). Use of 10 μM curcumin only resulted in an activity of 49.4 ± 5.8%. Of note, the curcumin + fMLP mixture did not cause any further increase in phagocytosis (61.8 ± 2.5%).

Figure 5. Curcumin enhanced PMN phagocytosis. Freshly isolated human PMN (10 × 10⁶cells/ml in complete RPMI 1640) were stimulated for 30 min with buffer (Ctrl), 10 μM curcumin (CUR10), 50 μM curcumin (CUR50), 10⁻⁹M fMLP, or a mixture of CUR50 + fMLP. After incubating 10⁶ PMN/treatment each with 5 × 10⁶ opsonized SRBC for 30 min at 37 °C, the extent of phagocytic activity among the cells was assessed by counting the number of PMN placed on a slide that had ingested at least one opsonized SRBC. A minimum of five different fields (corresponding to ∼200 PMN/slide) was randomly assessed; each slide was analyzed in duplicate. Results are expressed as mean ± SEM (n ≥ 3). *p < 0.05 versus control. Inset: a typical PMN that did (thin black arrows) or did not (large black arrows) ingest an SRBC. Magnification = 400×.

Discussion

In this study, curcumin was shown to be able to induce degranulation in human PMN. This was verified by the fact that curcumin increased cell surface expression of markers CD35, CD63, and CD66b of the different subsets of granules, release of MMP-9 (known constituent of gelatinase granules (Soehnlein et al., 2009)), and gelatinase activity in PMN culture supernatants. In addition to the identification of this novel property for curcumin in human PMN, this study provided evidence that degranulation occurred by a p38 MAPK-independent mechanism. Since this kinase was previously found to be involved in the pro-apoptotic activity of curcumin (Hu et al., 2005), this suggested to us that curcumin could probably induce other signaling pathways in human PMN. This remains to be investigated.

Over the years, several studies have reported on potent anti-inflammatory effects of curcumin (Aggarwal & Sung, 2009; Larmonier et al., 2011; Shehzad et al., 2013). However, the effect of curcumin on PMN degranulation has never been reported, and little is known about the precise mechanisms involved in the immunomodulatory effects of curcumin. Curcumin is a pleiotropic molecule having a plethora of biological targets (Aggarwal et al., 2007; Aggarwal & Sung, 2009). Although the degranulation process is often viewed as a part of the inflammatory response, one has to remember this is always context-dependent. Also, it is plausible that pro-apoptotic effects of curcumin could counteract pro-inflammatory ones. The capacity of curcumin to induce degranulation of azurophilic granules may be a considerable element in the mechanism responsible for its anti-inflammatory effects.

Given the ability of curcumin to trigger a rapid and strong degranulation of the azurophilic granules (and other subsets), one can imagine that this process could help the immune cells to resolve chronic inflammatory disorders set by recurrent infections or deficiencies of the immune system. Azurophilic granules contain high amounts of microbicidal proteins, including defensin, presenilin, stomatin, and myeloperoxidase (Bainton et al., 1971; Cham et al., 1994). Also, granulophysin (CD63) is a platelet lysosomal membrane protein commonly used as a marker of azurophilic granules (Cham et al., 1994) secreted only following intense stimulation of neutrophils (Faurschou & Borregaard, 2003). In fact, exocytosis of azurophilic granules after a stimulation with phorbol myristate acetate (PMA) or fMLP, two potent leukocyte activators, is limited in time and only occurs to a minimal extent (Estensen et al., 1974). Therefore, the capacity of curcumin to induce the degranulation of azurophilic granules can be viewed as a major mechanism differentiating curcumin than other molecules and might be the result of a specific mode of action, probably due to its hydrophobic molecular structure. In that perspective, the high lipid content of granules may explain the effects of curcumin on the exocytosis of granules, since curcumin was showed to be involved in intracellular lipid traffic (Canfran-Duque et al., 2013).

Canfran-Duque et al. (2013) also reported that curcumin stimulated release of cholesterol and lysosomal β-hexosaminidase enzyme, as well as exosome markers flotillin-2 and CD63, in HepG2 hepatocarcinoma cells and human THP-1 differentiated macrophages. This is in agreement with our present data that curcumin increased cell surface expression of CD63 on human PMN. Those authors attributed their findings to exosome/microvesicle secretion induced by curcumin. In PMN, CD63 marker refers to azurophilic granules known to be the major subset displaying microbicidal activity. Recently, we demonstrated that curcumin inhibited fMLP- or lipopolysaccharide (LPS)-induced suppression of human PMN apoptosis and reversed the ability of PMA to induce reactive oxygen species (Antoine et al., 2013). In this latter study, using an antibody array approach, curcumin was also found to inhibit LPS-induced cytokine production, including several important chemokines such as macrophage inflammatory proteins (MIP)-1α and -1β, interleukin (IL)-8 (CXCL-8), and GROα.

Interestingly, the inhibitory effect of curcumin on LPS-induced NF-κB activation in human PMN was also observed, fitting well with the potent inhibitory activity of curcumin against NF-κB previously reported (Zhou et al., 2011). Thus, the present data showing that curcumin induced degranulation by itself and that it could enhance the response to fMLP indicated that curcumin altered various PMN functions in vitro. In vivo, using a murine air pouch model of acute inflammation, we previously showed that intraperitoneal administration of curcumin inhibited LPS-induced neutrophilic infiltration and, interestingly, also decreased local production of several cytokines/chemokines induced by LPS, including, but not limit to, MIP-1α and MIP-1β (Antoine et al., 2013).

Acute inflammation occurs a few hours following trauma or infection, and is characterized by PMN adhesion to endothelial barriers and migration toward affected tissues (Kumar & Sharma, 2010). PMN are the first immune cells to arrive at the inflammatory site and are crucial for containment of pathogens within the infected site. In some cases, the acute response fails to eliminate pathogens and the response is exacerbated, leading to a chronic inflammatory state. This attests to the importance of a fast efficient clearance of pathogens. Degranulation and phagocytosis are certainly the two most important functions of PMN in fighting pathogens. Therefore, based on the ability of curcumin to induce these two responses, curcumin possesses important biological activities beneficial for human health. On the one hand, curcumin induced both degranulation and phagocytosis (this report), and, on the other hand, by its ability to induce apoptosis, curcumin simultaneously promoted a resolution of inflammation, largely known to occur via elimination of apoptotic PMN (Akgul et al., 2001; El Kebir & Filep, 2010).

Conclusions

The results of this present study help us to better understand previously reported beneficial effects of curcumin in a variety of inflammatory disorders (see Aggarwal & Sung, 2009; Larmonier et al., 2011; Shehzad et al., 2013; Taylor & Leonard, 2011). Curcumin increases important neutrophil functions involved in host defense, including degranulation and phagocytosis, and possesses anti-inflammatory activities by virtue of its pro-apoptotic effects. Therefore, although its mode of action is still not completely understood, it can be concluded that curcumin possesses important beneficial effects for human health related to its biological activity with regard to PMN.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

The study was partially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).