Role of the NF-kB transcription pathway in thehaemozoin- and 15-HETE-mediated activation of matrixmetalloproteinase-9 in human adherent monocytes

Mauro Prato, Valentina Gallo, Giuliana Giribaldi,Elisabetta Aldieri and Paolo Arese*Dipartimento di Genetica, Biologia e Biochimica,Università di Torino, Via Santena 5 bis, 10126 Torino,Italy.

Summary

Haemozoin (HZ, malarial pigment) is a crystallineferriprotoporphyrin IX polymer derived fromundigested host haemoglobin haem, present inlate stages of Plasmodium falciparum-parasitizedRBCs and in residual bodies shed afterschizogony. It was shown previously that phago-cytosed HZ or HZ-containing trophozoitesincreased monocyte matrix metalloproteinase-9(MMP-9) activity and enhanced production of MMP-9-related cytokines TNF and IL-1beta. Here weshow that in human monocytes the HZ/trophozoitephagocytosis effects and their recapitulationby 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid(15-HETE), a potent lipoperoxidation derivativegenerated by HZ from arachidonic acid via haemcatalysis, were mediated via activation of NF-kBtranscription pathway. After phagocytosis ofHZ/trophozoites or treatment with 15-HETE, theNF-kB complex migrated to the nuclear fractionwhile the inhibitory cytosolic IkBalpha protein wasphosphorylated and degraded. All HZ/trophozoite/15-HETE effects on MMP-9 activity and TNF/IL-1beta production were abrogated by quercetin,artemisinin and parthenolide, inhibitors of IkBal-pha phosphorylation and subsequent degradation,NF-kB nuclear translocation, and NF-kB-p65binding to DNA respectively. In conclusion,enhanced activation of MMP-9, and release of pro-inflammatory cytokines TNF and IL-1beta, a triad ofeffects involved in malaria pathogenesis, elicitedin human monocytes by trophozoite and HZphagocytosis and recapitulated by 15-HETE,

appear to be causally connected to persisting acti-vation of the NF-kB system.

Introduction

Phagocytosis of Plasmodium falciparum native haemozoin(HZ, malarial pigment) by human monocytes was shownto impair several cell activities such as oxidativeburst (Schwarzer et al., 1992), repeated phagocytosis(Schwarzer et al., 1992), bacterial killing (Fiori et al., 1993),antigen presentation (Schwarzer et al., 1998; Scorzaet al., 1999) and dendritic cell function (Skorokhod et al.,2004; Millington et al., 2006). On the other hand, HZ-fedhuman monocytes produced higher levels of pro-inflammatory cytokines and pyrogens such as IL-1beta,IL-10, IL-12, TNF, MIP-1alpha and MIP-1beta (Pichyangkulet al., 1994; Sherry et al., 1995; Mordmüller et al., 1998;Biswas et al., 2001) and displayed enhanced expression,release and enzyme activity of matrix metalloproteinase-9(MMP-9) that in turn increased production of IL-1beta andTNF (Prato et al., 2005; 2008; 2009). MMP-9 (gelatinaseB) is a proteolytic enzyme that degrades matrix proteinsand disrupts the basal lamina in endothelia (see Nagaseand Woessner, 1999; Sternlicht and Werb, 2001 forreviews). Besides its proteolytic activity, MMP-9 shedsIL-1beta and TNF from their cell-bound precursors increas-ing the production of the free cytokines which in turn arealso positive regulators of the MMP-9 gene (Gearing et al.,1995; Ito et al., 1996; Bond et al., 1998).cmi_1508 1780..1791

Data showing increased protein levels of cerebralMMP-1 and MMP-9 in post-mortem studies of cerebralmalaria (CM) cases (Brown et al., 2000; Deininger et al.,2002; 2003), increased MMP-2, MMP-7 and MMP-9 activ-ity and increased numbers of MMP-9-positive monocyticcells in the brain of mice with CM (Van den Steen et al.,2006; Szklarczyk et al., 2007) indicated a possible con-nection between MMPs and CM, a severe complication ofmalaria, characterized by sequestration of parasitizedRBCs in brain microvessels, breakdown of the blood–brain barrier and brain oedema, ring-haemorrhages andDürck granulomas infiltrated with partially HZ-ladenmacrophages (Turner, 1997; Coltel et al., 2004; Medanaand Turner, 2006), and frequently (Kwiatkowski et al.,

Received 25 March, 2010; revised 14 July, 2010; accepted 14 July,2010. *For correspondence. E-mail paolo.arese@unito.it; Tel. (+39)11 790 5846; Fax (+39) 11 790 5845.

Cellular Microbiology (2010) 12(12), 1780–1791 doi:10.1111/j.1462-5822.2010.01508.xFirst published online 27 August 2010

© 2010 Blackwell Publishing Ltd

cellular microbiology

1990; Akanmori et al., 2000), but not constantly (Shafferet al., 1991; Lovegrove et al., 2009), increased systemiclevels of TNF and IL-1.

Native HZ and HZ-fed human monocytes were found togenerate via haem catalysis (Schwarzer et al., 2003)large amounts of peroxidation and hydroxylation productsof polyunsaturated fatty acids (PUFAs) such as hydroxye-icosatetraenoic acids (HETEs), hydroxyoctadecadienoicacids and the terminal aldehyde 4-hydroxynonenal(Schwarzer et al., 2003). Lipid derivatives are possibleinducers of present HZ effects, as delipidized HZ (dHZ) orthe lipid-free synthetic HZ beta-haematin (BH) were notactive, while 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoicacid (15-HETE) mimicked the HZ effects on MMP-9,IL-1beta and TNF (Prato et al., 2008; 2010).

Here we show that the HZ/15-HETE enhancement ofMMP-9 and related cytokines was mediated by activationof the NF-kB system. NF-kB is a transcription factor familythat regulates the expression of numerous genes playingkey roles in growth and differentiation; in tumorigenesisand tumour metastasis; and in immune and inflammatoryresponses and apoptosis (see Ali and Mann, 2004;May et al., 2004; Hagemann et al., 2009; Shen andTergaonkar, 2009; Vallabhapurapu and Karin, 2009 forreviews). The NF-kB system responds to multiple stimuliincluding oxidizing agents and lipoperoxidation products(Flohé et al., 1997; McGowan et al., 1998; Bonizzi et al.,2000), and modulates activity of MMP-9 and production ofpro-inflammatory cytokines (Bond et al., 2001; Chaseet al., 2002; Rhee et al., 2007). Involvement of the NF-kBsystem was evidenced by the nuclear translocation of theNF-kB complex, and phosphorylation and subsequentdegradation of the inhibitory IkBalpha protein, observed inHZ/trophozoite-fed and 15-HETE-treated monocytes, andby the inhibition of the HZ/15-HETE-mediated effects byquercetin, artemisinin or parthenolide, known inhibitors ofthe NF-kB system (García-Piñeres et al., 2001; Aldieriet al., 2003; Nair et al., 2006).

Results

Adherent human monocytes (approximately 70% pureand NF-kB quiescent, see Experimental procedures)were allowed to phagocytose trophozoites, HZ andcontrol meals non-parasitized opsonized RBCs and latexparticles during 2 h. This time period maximized phago-cytosis and was not sufficient to induce haem oxygenase-mediated degradation of ingested haem (Schwarzeret al., 1999). As an average, each monocyte ingested~8–10 trophozoites, HZ equivalent to ~8–10 trophozoitesin term of ingested haem, or ~8–10 non-parasitized anti-DIgG-opsonized RBCs, as shown previously (Schwarzeret al., 1994). After phagocytosis, elimination of non-ingested phagocytic meals and further incubation up to

24 h, study parameters were measured in cell superna-tants and cell lysates as indicated.

Time-dependent nuclear translocation of the NF-kBcomplex in adherent monocytes fed with trophozoitesor HZ

Adherent monocytes were allowed to phagocytose tro-phozoites, HZ and control meals during 2 h. After termi-nation of phagocytosis, nuclear translocation of the NF-kBcomplex was assayed time-dependently in the nuclearprotein fraction by EMSA. Figure 1 shows that the NF-kBcomplex was absent in the nuclear protein fraction at theend of the phagocytic period but was detected 2 h and24 h post phagocytosis in HZ- and trophozoite-fed mono-cytes. No translocation occurred in monocytes fed withthe control meals anti-D IgG-opsonized RBCs and latexparticles. Nuclear translocation of either p65 or p50 NF-kBprotein 2 h and 24 h post phagocytosis was confirmed byWestern blotting (not shown), while artemisinin and par-thenolide (both 10 mM), inhibitors of NF-kB nuclear trans-location and p65 binding to DNA, respectively, added atthe end of the phagocytic period inhibited nuclear trans-location of the NF-kB complex, in agreement with litera-ture data (Aldieri et al., 2003) (not shown).

Fig. 1. Phagocytosis of trophozoites and HZ induces nucleartranslocation of the NF-kB complex in human monocytes. Adherenthuman monocytes were unfed (control) or fed for 2 h withtrophozoites, HZ and control meals anti-D IgG-opsonized RBCs(oRBC) and latex particles. After termination of phagocytosis andelimination of non-ingested phagocytic meals by repeatedwashings, cells were immediately lysed or incubated at 37°C for 2and 24 h before lysis. The NF-kB complex was detected by EMSAin the nuclear protein fraction. The bands in the assay correspondto the NF-kB complex. Data from one representative experiment offour with similar results. Additional densitometric analysis of bandswas performed (not shown) and densitometric values wereanalysed by Student’s t-test. The differences between trophozoite-and HZ-fed cells versus unfed controls, RBCs- and latex-fed cellswere statistically significant with P < 0.0001.

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Degradation of IkBalpha protein in adherent monocytesfed with trophozoites or HZ. Abrogation by quercetin

NF-kB activation is preceded by phosphorylation and sub-sequent degradation of inhibitory proteins called IkB pro-teins, allowing NF-kB dimers to enter the nucleus andinduce gene expression (Verma and Stevenson, 1997).Phagocytosis of trophozoites (Fig. 2A) or HZ (Fig. 2B)induced extensive degradation of IkBalpha protein, themain representative of the IkB inhibitory protein family.IkBalpha protein degradation was fully inhibited by quer-cetin (15 mM), an inhibitor of IkBalpha protein phosphory-lation and subsequent degradation (Nair et al., 2006).

Enhancement of MMP-9 enzyme activity in adherentmonocytes fed with trophozoites or HZ. Abrogation ofthe effect by the NF-kB inhibitors quercetin, artemisininand parthenolide

The next experiments were designed to show the involve-ment of NF-kB activation in the enhancement of MMP-9mediated by trophozoite and HZ phagocytosis. Adherentmonocytes were allowed to phagocytose trophozoites, HZand control meals. After termination of phagocytosis, cellswere incubated for 24 h with or without quercetin (15 mM),artemisinin (10 mM) or parthenolide (10 mM), known inhibi-tors of IkBalpha protein phosphorylation, NF-kB complexnuclear translocation and NF-kB complex/DNA bindingrespectively (García-Piñeres et al., 2001; Aldieri et al.,2003; Nair et al., 2006). As shown in Fig. 3A–C, HZ- andtrophozoite-mediated increase of MMP-9 activity wasabrogated by all three NF-kB inhibitors.

Enhancement of TNF and IL-1beta production byadherent monocytes fed with trophozoites or HZ.Abrogation of the effect by NF-kB inhibitors quercetin,artemisinin and parthenolide

As previous data have shown a relationship betweentrophozoite/HZ phagocytosis, MMP-9 activation andrelease, and production of TNF and IL-1beta in humanmonocytes (Prato et al., 2005; 2008; 2009), we checkedwhether stimulation of cytokine production was also underNF-kB control. Adherent monocytes were allowed tophagocytose trophozoites, HZ and control meals during2 h. After termination of phagocytosis, cells were treatedor not with quercetin (15 mM), artemisinin (10 mM) andparthenolide (10 mM). Cells were then further incubatedfor 24 h and the production of TNF (Fig. 4A–C) andIL-1beta (Fig. 5A–C) was assayed in the supernatantsand expressed as end-point levels at 24 h. Data shown inFigs 4A–C and 5A–C indicate that HZ- and trophozoite-mediated increase of TNF and IL-1beta production wasremarkably inhibited by all three NF-kB inhibitors.

Role of NF-kB in 15-HETE-mediated enhancement ofMMP-9 enzyme activity and TNF/IL-1beta production inadherent monocytes

PUFAs stably adherent to the crystalline poly-haem coreof native HZ generate a number of potent lipoperoxidationderivatives by non-enzymatic haem catalysis (pseudo-lipoxygenase catalysis) (Schwarzer et al., 2003). Amongthose, 15-HETE was previously shown to recapitulateHZ-mediated effects on MMP-9 and related cytokines

Fig. 2. Phagocytosis of trophozoites and HZ induces degradation of IkBalpha protein in human monocytes. Abrogation of the effect byquercetin, an inhibitor of IkBalpha phosphorylation. Adherent human monocytes were unfed (control) or fed for 2 h with trophozoites, HZ andcontrol meals opsonized RBCs (oRBC) or latex particles. After termination of phagocytosis and elimination of non-ingested phagocytic mealsby repeated washings, cells were supplemented or not with quercetin (15 mM) and further incubated at 37°C for 24 h. IkBalpha protein(37 kDa band in the blot) was assayed in the monocyte cytosolic lysates by Western blotting and densitometric quantification (arbitrarydensitometric units, solid columns corresponding to the bands in the gel). Column data are mean values of densitometric units � SD of fourindependent experiments. Blot data (upper panels) are from one representative experiment. Column data were analysed by Student’s t-test.The differences between trophozoite- and HZ-fed cells versus unfed controls were statistically significant with P < 0.02 and P < 0.01respectively. Differences between unfed versus control meal-fed cells were not significant. Differences between quercetin-treated versusuntreated trophozoite/HZ-fed monocytes were statistically significant with P < 0.01 and P < 0.005 respectively.

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(Prato et al., 2008; 2010). 15-HETE effects also appear tobe mediated by the NF-kB system. The role of NF-kB inthe HZ and 15-HETE effects is underscored by studies(Fig. 6) showing similar behaviour in phosphorylation anddisappearance of IkBalpha, nuclear translocation of theNF-kB complex and appearance of the p50 and p65(NF-kB) subunits in both monocytes fed with HZ and treatedwith 15-HETE, and abrogation of the effects by NF-kB

inhibitors. Figure 6 also shows that phagocytosis of dHZand the lipid-free HZ analogue BH had no effect on theNF-kB system. Figure 7 shows the parallelism betweenthe stimulatory effect of HZ phagocytosis and 15-HETEsupplementation on TNF and IL-1beta production (Fig. 7Aand B) and MMP-9 activity (Fig. 7C) and the remarkableabrogation of stimulatory effects by NF-kB inhibitors(Fig. 7A–C).

Fig. 3. Phagocytosis of trophozoites and HZ enhances MMP-9 activity in monocytes. Abrogation of the effect by the NF-kB inhibitorsquercetin, artemisinin and parthenolide. Adherent human monocytes were unfed (control) or fed for 2 h with trophozoites, HZ and controlmeals opsonized RBCs (oRBC) and latex beads. After termination of phagocytosis and elimination of non-ingested phagocytic meals byrepeated washings, cells were supplemented or not with (A) quercetin (15 mM), (B) artemisinin (10 mM) or (C) parthenolide (10 mM) and furtherincubated at 37°C for 24 h. Thereafter, MMP-9 enzyme activity was assayed in the monocyte supernatants by gelatin zymography (83 kDanegative bands in the gel) and densitometric quantification (arbitrary densitometric units, solid columns corresponding to the negative bands inthe gel). Column data are mean values of densitometric units � SD of four independent experiments. Gel data (upper panels) are from onerepresentative experiment. Column data were analysed for significance by Student’s t-test. The differences between trophozoite-/HZ-fed cellsversus unfed controls and versus all other conditions were statistically significant with worst P: P < 0.001. The difference between trophozoite-versus HZ-fed cells (A–C, lane/column 3 versus lane/column 5) and the differences between unfed controls versus quercetin-, artemisinin-,parthenolide-treated cells were not significant.

Fig. 4. Phagocytosis of trophozoites and HZ enhances TNF production in monocytes. Abrogation of the effect by the NF-kB inhibitorsquercetin, artemisinin and parthenolide. Adherent human monocytes were unfed (control) or fed for 2 h with trophozoites, HZ and controlmeals opsonized RBCs (oRBC) and latex particles. After termination of phagocytosis and elimination of non-ingested phagocytic meals byrepeated washings, cells were supplemented or not with (A) quercetin (15 mM), (B) artemisinin (10 mM) or (C) parthenolide (10 mM) and furtherincubated at 37°C for 24 h. The level of soluble TNF was assayed in the monocyte supernatants by specific ELISA and expressed as ng TNFper ml cell supernatant. Data are mean values � SD of four, five and five independent experiments for each panel respectively. Data wereanalysed for significance by Student’s t-test. The differences between trophozoite-/HZ-fed cells versus unfed controls and versus all otherconditions were statistically significant with worst P: P < 0.05. Differences between latex-fed cells versus controls were statistically significantwith P < 0.05. Differences between trophozoite- versus HZ-fed cells and between RBCs-fed versus controls were not significant.

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Discussion

The main role of zinc-dependent MMPs is to degradestructural proteins of the extracellular matrix and disruptsubendothelial basal membrane. In addition, they also

cleave cell surface molecules, such as membrane-boundcytokines and activate cytokines and metalloproteinasesfrom their inactive precursors. MMPs influence diversephysiological and pathological processes includingembryonic and fetal development, tissue morphogenesis,

Fig. 5. Phagocytosis of trophozoites and HZ enhances IL-1beta production in monocytes. Abrogation of the effect by the NF-kB inhibitorsquercetin, artemisinin and parthenolide. Adherent human monocytes were unfed (control) or fed for 2 h with trophozoites, HZ, and controlmeals opsonized RBCs (oRBC) and latex particles. After termination of phagocytosis and elimination of non-ingested phagocytic meals byrepeated washings, cells were supplemented or not with (A) quercetin (15 mM), (B) artemisinin (10 mM) or (C) parthenolide (10 mM) and furtherincubated at 37°C for 24 h. The level of soluble IL-1beta was assayed in the monocyte supernatants by specific ELISA and expressed as ngIL-1beta per ml cell supernatant. Data are mean values � SD of three, four and five independent experiments for each panel respectively.Data were analysed for significance by Student’s t-test. The differences between trophozoite-/HZ-fed cells versus unfed controls and versus allother conditions were statistically significant with worst P: P < 0.05. Differences between trophozoite- and HZ-fed cells versus controls, andbetween RBCs-/latex-fed cells versus controls were not significant.

Fig. 6. Phagocytosis of HZ and 15-HETE treatment enhances IkBalpha protein phosphorylation and degradation, and nuclear translocation ofthe NF-kB complex in monocytes. Lack of effect after phagocytosis of dHZ and BH. Abrogation of the HZ/15-HETE effects by the NF-kBinhibitors quercetin, artemisinin and parthenolide. Adherent human monocytes were unfed (control) or fed for 2 h with HZ, dHZ and BH. Aftertermination of phagocytosis and elimination of non-ingested phagocytic meals by repeated washings (fed cells), unfed cells were treated ornot with 15-HETE (1 mM), 15-HETE (1 mM) plus quercetin (15 mM), 15-HETE (1 mM) plus artemisinin (10 mM) and 15-HETE (1 mM) plusparthenolide (10 mM) added at time 0, and further incubated at 37°C for 2 h (A) or 24 h (B–E). In the 24 h studies 15-HETE was re-addedafter 12 h incubation. Thereafter, cells were lysed and cytosolic (A and B) and nuclear (C–E) lysates were isolated for following studies.A. Western blot with anti-phospho-IkBalpha Ab in cytosolic lysates. The 37 kDa band corresponds to phosphorylated IkBalpha.B. Western blot with anti-IkBalpha Ab in cytosolic lysates. The 37 kDa band corresponds to IkBalpha protein.C. EMSA in nuclear lysates. The band corresponds to NF-kB/DNA complex.D. Western blot with anti-NF-kB-p50 subunit Ab in monocyte nuclear lysates. The 50 kDa band corresponds to NF-kB-p50) subunit.E. Western blot with anti-NF-kB-p65 subunit Ab in monocyte nuclear lysates. The 65 kDa band corresponds to NF-kB-p65 subunit.Data in each panel are from one representative experiment of three with similar results.

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wound repair, neuroinflammatory diseases and cancer(see Nagase and Woessner, 1999; Sternlicht and Werb,2001; Rosenberg, 2002; Van den Steen et al., 2002;Cauwe et al., 2007 for reviews). Recently, MMPs weresuggested to play a role in the pathogenesis of humanand murine CM (see below).

Cerebral malaria, a severe complication of malaria,is characterized by cytoadhesion and sequestrationin endothelia of brain microvessels of parasitizedRBCs, phagocytes often containing HZ, and free HZ;breakdown of the blood–brain barrier, brain oedemaand focal endothelial damage; ring-haemorrhagesand Dürck granulomas infiltrated with partially HZ-ladenmacrophages (see Turner, 1997; Medana and Turner,2006 for reviews). According to several studies (seeGimenez et al., 2003 for review) CM is causally con-

nected to increased focal production of inflammatorycytokines, notably TNF and IL-1 (Udomsangpetch et al.,1997; Brown et al., 1999; Armah et al., 2005),while systemic levels of TNF and IL-1 are frequently(Kwiatkowski et al., 1990; Akanmori et al., 2000) but notconstantly increased (Shaffer et al., 1991; Lovegroveet al., 2009).

A possible role of MMP in CM was suggested byincreased protein levels of cerebral MMP-1 and MMP-9,both observed in post-mortem studies of CM cases(Brown et al., 2000; Deininger et al., 2002; 2003). Inmurine CM Van den Steen et al. (2006) have shown over-expression and increased gelatinolytic activity of MMP-9by brain cells of monocytic lineage. Lastly, induction ofMMP-9 gene by P. falciparum has been reported inmicroarray studies (Griffiths et al., 2005).

Fig. 7. Phagocytosis of HZ and 15-HETE treatment enhances TNF and IL-1beta production and MMP-9 enzyme activity in monocytes.Abrogation of the 15-HETE effects by the NF-kB inhibitors quercetin, artemisinin and parthenolide. Adherent human monocytes were fed ornot for 2 h with HZ and (data not shown) dHZ and BH. After termination of phagocytosis and elimination of non-ingested phagocytic meals byrepeated washings (fed cells), unfed cells were treated or not with 15-HETE (1 mM), 15-HETE (1 mM) plus quercetin (15 mM), 15-HETE (1 mM)plus artemisinin (10 mM) and 15-HETE (1 mM) plus parthenolide (10 mM) added at time 0, and further incubated at 37°C for 2, 4 (short-term)and 24 h (long-term studies). In long-term studies 15-HETE was re-added after 12 h incubation. The level of soluble TNF (A) and IL-1beta(B) was assayed in the monocyte supernatants by specific ELISA. TNF and IL-1beta data are given as pg ml-1 or as ng ml-1 supernatant[short-term experiments: mean values � SD of six (TNF) and three (IL-1beta) independent experiments; long-term experiments: meanvalues � SD of five (TNF) and three (IL-1beta) independent experiments]. MMP-9 enzyme activity (C) was assayed in the monocytesupernatants by gelatin zymography (83 kDa negative bands in the gel) and densitometric quantification (arbitrary densitometric units, solidcolumns corresponding to the negative bands in the gel). Column data are mean values of densitometric units � SD of four independentexperiments. Gel data are from one representative experiment. All column data were analysed for significance by Student’s t-test. Differencesbetween 15-HETE-treated versus HZ-fed cells were not significant; differences between HZ-fed/15-HETE-treated versus control/unfed cellswere significant (worst P for TNF: P < 0.02; worst P for IL-1beta: P < 0.05; P < 0.01 for MMP-9); differences between 15-HETE-treated versus15-HETE plus quercetin-, artemisinin- and parthenolide-treated cells were always significant (worst P for TNF: P < 0.05; worst P for IL-1beta:P < 0.05; P < 0.01 for MMP-9). Phagocytosis of dHZ and BH did not enhance MMP-9 activity and formation of TNF and IL-1beta (not shown).

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Phagocytosed HZ was recently suggested as the pos-sible mediator linking induction of MMP-9 expression,degranulation and activity with enhanced production ofTNF and IL-1beta (Prato et al., 2005; 2008; 2009), addingto the pleiotropic HZ activities described in humanand murine phagocytes. Biologically potent peroxidationand hydroxylation products of PUFAs such as 15-HETEare generated in large amounts by HZ (Schwarzeret al., 2003) by non-enzymatic haem catalysis (‘pseudo-lipoxygenase catalysis’). Those lipid derivatives may bethe final effector molecules of HZ. In the present contextand similarly to other HZ effects, HZ-bound lipids werenecessary for upregulation of TNF, IL-1beta and MMP-9,and 15-HETE recapitulated the HZ effects (Prato et al.,2008; 2010).

Involvement of the NF-kB system was shown here bydirect and indirect evidence. Direct evidence indicatedthat both HZ phagocytosis and 15-HETE treatmentinduced NF-kB binding to DNA already after 2 h. Twenty-four hours after HZ/trophozoite phagocytosis and15-HETE treatment nuclear binding persisted, NF-kB sub-units p50 and p65 were found in the nuclear fraction andIkBalpha protein was phosphorylated and destroyed. Indi-rect evidence was provided by using known inhibitors ofthe system: quercetin, a natural antioxidant flavonoid withantimalaria properties (Oliveira et al., 2004; Lehane andSaliba, 2008) that inhibits the phosphorylation of theinhibitory IkBalpha protein, preventing its dissociationfrom NF-kB complex and its subsequent degradation(Nair et al., 2006); artemisinin, also a potent antimalariaagent, recently shown to inhibit nuclear translocation ofNF-kB complex (Aldieri et al., 2003); and parthenolide, asesquiterpene lactone with anti-leishmania activity(Tiuman et al., 2005) that inhibits NF-kB/DNA binding byalkylating NF-kB-p65 subunit at Cys38 (García-Piñereset al., 2001). All reported effects of HZ/15-HETE onMMP-9 activity and TNF/IL-1beta production were abol-ished by each one of above inhibitors, confirming involve-ment of the NF-kB pathway.

Literature data are in accordance with present results.First, it has been shown that MMP-9 gene expression wascontrolled by NF-kB (Bond et al., 2001; Chase et al.,2002; Rhee et al., 2007); second, NF-kB was involvedin HZ-induced enhancement of inflammatory cytokines(Jaramillo et al., 2003); lastly, genome-wide expressionprofiles in peripheral mononuclear cells in malariapatients indicated upregulation of signalling throughNF-kB pathway and enhancement of inflammatory cytok-ines (Ockenhouse et al., 2006). Studies have shown thatthe MMP-9 gene can be induced through other transcrip-tion pathways (see Van den Steen et al. 2002 for review).As the three inhibitors used here are not specific for theNF-kB pathway, we cannot exclude the involvementof non-NF-kB-dependent transduction pathways, such

as ERK1/ERK2, JNK1/JNK2, p38, STAT1/STAT2 andSMAD4.

Present data suggest that 15-HETE may be one of theHZ products instrumental in the NF-kB-mediated upregu-lation of MMP-9 and related cytokines. Studies haveshown that HETEs and parent compounds hydroxyocta-decadienoic acids are peroxisome proliferator-activatedreceptor gamma ligands inhibiting the production ofinflammatory cytokines and suppressing the NF-kBsystem, in apparent conflict with present study. Literaturedata reported below indicate however that HETEs mayactivate the NF-kB system by alternative mechanisms. Afirst alternative activation of NF-kB is based on HETE-mediated activation of protein kinase C (Sharma et al.,2005; Wyke et al., 2005; Chen et al., 2008), which in turnactivates IKK (Smith et al., 2004), the kinase responsiblefor IkBalpha phosphorylation and degradation (Verma andStevenson, 1997) and subsequent activation of NF-kB.Transient activation of protein kinase C by HZ (Schwarzeret al., 1993) may thus explain the first NF-kB activationpeak revealed by EMSA analysis 2 h after HZ phagocy-tosis and the short-term enhancement of cytokine produc-tion observed here. A second alternative activation ofNF-kB (Di Mari et al., 2007) was found to involve MAPkinases, ERK and p38 cascade. The p38 cascade wasalso proposed for HETEs-dependent modulation of TNF(Wen et al., 2007) and MMP-9 (Nguyen et al., 2006).Interestingly, ERK-1/2 phosphorylation by HZ has beendocumented in a murine system (Jaramillo et al., 2005). Athird alternative activation of NF-kB may result from thedirect oxidation and inactivation of IkB by lipid hydroper-oxides (Flohé et al., 1997).

Based on present data, following sequence of eventscan be suggested. First, 15-HETE [possibly togetherwith co-generated and similarly active 12-HETE andother parent molecules (Schwarzer et al., 2003)] mayinduce production of IL-1beta and TNF via the NF-kBpathway, by promoting cytosolic IkBalpha protein phos-phorylation and degradation, NF-kB complex nucleartranslocation and final formation of the NF-kB/DNAcomplex. Subsequently, IL-1beta and TNF would inducepersistent upregulation of the NF-kB-dependent expres-sion and activity of MMP-9. Finally, TNF and MMP-9would start the positive loop described previously (Pratoet al., 2005) further enhancing MMP-9 activity and theshedding of soluble, active TNF from its membrane-bound precursors.

In conclusion, upregulation and activation of MMP-9,and enhanced formation of pro-inflammatory cytokinesTNF and IL-1beta, a triad of effects that play importantroles in malaria pathogenesis, elicited in human mono-cytes by trophozoite and HZ phagocytosis and recapitu-lated by 15-HETE, appear to be causally connected topersisting activation of the NF-kB system.

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Experimental procedures

Materials

All materials were from Sigma-Aldrich, St Louis, MO, unlessotherwise stated. Cell culture media RPMI, TRIzol, M-MLV,oligo-dT, sense and anti-sense primers, Platinum Taq DNAPolymerase were from Invitrogen, Carlsbad, CA; Panserin 601monocyte medium was from PAN Biotech, Aidenbach,Germany; electrophoresis reagents were from Bio-Rad Labora-tories, Hercules, CA; ELISA kit for TNF and IL-1beta assay and15-HETE were from Cayman, Ann Arbor, MI; anti-D IgG werefrom Immuno AG, Vienna, Austria; Percoll was from Pharmacia,Uppsala, Sweden; Diff-Quik parasite stain was from BaxterDade AG, Dudingen, Switzerland; sterile plastics were fromCostar, Cambridge, UK; bicinchoninic acid protein assay wasfrom Pierce, Rockford, IL; anti-IkBalpha and anti-NF-kB-p65polyclonal antibodies and anti-phospho-IkBalpha and anti-NF-kB-p50 monoclonal Ab were from Santa Cruz Biotechnology,Santa Cruz, CA; DNA-free kit was from Ambion, Austin, TX;Beacon Designer 7.0 software was from Premier Biosoft Inter-national, Palo Alto, CA; dNTPs were from Applied Biosystem,Foster City, CA.

Cultivation of P. falciparum, isolation oftrophozoite-parasitized RBCs and native or delipidizedHZ and preparation of BH

Plasmodium falciparum parasites (Palo Alto strain, Mycoplasma-free) were kept in culture as described (Prato et al., 2005). Aftercentrifugation at 5000 g on a discontinuous Percoll-mannitoldensity gradient, native HZ was collected from the 0–40% inter-phase and trophozoites/schizonts from the 40–80% interphase.After isolation, trophozoites enriched to 95–97% parasitaemiawere washed twice and reincubated in RPMI for 1 h at 37°Cbefore opsonization and phagocytosis. Native HZ was washedfive times with 10 mM Hepes (pH 8.0) containing 10 mM mannitolat 4°C and once with PBS. After quantification of haem content byluminescence (Schwarzer et al., 1992), native HZ was stored at20% (v/v) in PBS at -20°C or immediately used for opsonizationand phagocytosis. For dHZ, lipid extraction was performed aspreviously reported (Prato et al., 2008). BH (synthetic HZ) wasprepared according to the Slater et al. (1991) procedure, modi-fied as indicated (Prato et al., 2008).

Preparation and handling of monocytes

Human monocytes were separated by Ficoll centrifugation(Schwarzer et al., 1992) from freshly collected buffy coats dis-carded from blood donations by healthy adult donors of bothsexes provided by the local blood bank (AVIS, AssociazioneVolontari Italiani Sangue, Torino, Italy). Separated lympho-monocytes were resuspended in RPMI medium and plated onwells of six-well plates. Each well received 8 ¥ 106 cells. Theplates were incubated in a humidified CO2/air incubator at 37°Cfor 60 min. Thereafter, non-adherent cells were removed by threewashes with RPMI and adherent cells reincubated at 37°C over-night. Shortly before starting phagocytosis, wells were washedwith RPMI and Panserin 601 monocyte medium was added (2 mlper well).

Pre-selection of NF-kB-quiescent monocytes byFACScan analysis and real-time RT-PCR

Adherent monocytes prepared by Ficoll separation weredetached from the plates by scraping, stained with specificantibodies and analysed on a FACScan flow cytometer(Becton-Dickinson, San Jose, CA). In a random sample, mono-cytes (CD14+ cells) were 70 � 4%, lymphocytes 30 � 4%(mean values � SD, n = 4). To avoid the use of NF-kB pre-activated monocytes, in each cell preparation a cell aliquot wasstimulated or not with LPS (1 mg ml-1) for 4 h, and TNF RNAproduction measured in lysates by real-time RT-PCR beforestarting the experiments. RNA extraction and real-time RT-PCRassay were performed as previously reported (Prato et al.,2005). GAPDH was used as housekeeping gene. Only unstimu-lated monocyte preparations (defined as ‘NF-kB-quiescent’cells) showing at least a three-PCR-cycle gap of cDNAamplification between controls and LPS-stimulated cells wereutilized.

Phagocytosis of opsonized trophozoites, native HZ,dHZ, BH, non-parasitized opsonized RBCs andlatex particles

Fifty microlitres of trophozoites (10% haematocrit), native HZ ordHZ (120 nmoles of HZ haem, an amount comparable to 50 mlof trophozoites on haem content basis), 50 ml of BH(120 nmoles of haem), 50 ml of anti-D IgG-opsonized RBCs(10% haematocrit) and 50 ml of amine-modified, red-fluorescentlatex particles (2.5% solids, diameter 0.105 mm) were addedto each well of a six-well plate with approximately 1 ¥ 106

adherent monocytes. Trophozoites, native HZ, dHZ, BH andlatex particles were opsonized with fresh autologous serum,and non-parasitized RBCs were opsonized with anti-D IgG asindicated (Schwarzer et al., 1992). After opsonization, allphagocytic meals were suspended in Panserin 601 monocytemedium. The plates were centrifuged at low speed for 5 s tostart phagocytosis and incubated in a humidified CO2/air incu-bator at 37°C for 2 h, a time period maximizing phagocytosisbut not sufficient to induce haem oxygenase-mediated degra-dation of ingested haem (Schwarzer et al., 1999). Thereafter,non-ingested cells, HZ, latex and BH particles were removed byfour washes with RPMI. The plates were then incubated inPanserin 601 medium in a humidified CO2/air incubator at 37°Cfor the indicated times. In selected experiments, cells wereincubated with quercetin 15 mM, artemisinin 10 mM and par-thenolide 10 mM, dissolved in DMSO (final solvent concentra-tion less than 0.1% v/v).

Assay of MMP-9 activity by gelatin zymography

After termination of phagocytosis, monocytes were further incu-bated with Panserin 601 monocyte medium in a humidified CO2/air incubator at 37°C for 24 h. Thereafter, the activity of MMP-9was evaluated by gelatin zymography in the cell supernatants asindicated (Mitola et al., 2003). Briefly, supernatants were loadedon 8% polyacrylamide gels containing 0.1% gelatin undernon-denaturing and non-reducing conditions. Following electro-phoresis, gels were washed and incubated for 18 h at 37°Cin a collagenase buffer. Densitometric analysis of the bands

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considered to reflect total enzymatic activity of MMP-9 was per-formed using a computerized densitometer (Chemidoc, Biorad,Hercules, CA) with activity presented in relative units comparedwith background.

Assay of TNF and IL-1beta production

After termination of phagocytosis, monocytes were further incu-bated with Panserin 601 monocyte medium in a humidified CO2/air incubator at 37°C for 2, 4 and 24 h. The level of active solubleTNF or IL-1beta was assayed in monocyte supernatants byspecific ELISA. A standard calibration curve was generatedwith rhTNF and rhIL-1beta, according to the manufacturer’sinstructions.

Isolation of cytosolic and nuclear protein fractions incell lysates

Cells were mechanically scraped in PBS and washed, thenresuspended in lysis buffer (15 mM KCl, 10 mM Hepes, 2 mMMgCl2, 0.1 mM EDTA, 1 mM PMSF, 1 mM DTT, 10 mg ml-1 apro-tinin, 2 mg ml-1 leupeptin, 0.1% NP-40, pH 7.6). Cell suspensionswere then incubated for 15 min on ice with occasional vortexing,and centrifuged for 30 s to pellet nuclei. Supernatants with cyto-solic proteins were collected and stored at -20°C until usedfor Western blotting. Nuclei were rinsed with wash buffer (2 mMKCl, 25 mM Hepes, 0.1 mM EDTA, 1 mM PMSF, 1 mM DTT,10 mg ml-1 aprotinin, 2 mg ml-1 leupeptin, pH 7.6) and incubatedat 4°C for 20 min. Nuclear extracts were then prepared by cen-trifugation at 20 000 g for 15 min in lysis buffer (25 mM Hepes,0.1 mM EDTA, 20% glycerol, pH 7.6) and stored at -80°C untilused for EMSA or Western blotting. Protein concentration in thecell cytosolic or nuclear lysates was determined using a bicin-choninic acid assay.

Assay of NF-kB complex nuclear translocation by EMSA

Probes containing the NF-kB oligonucleotide consensussequence were labelled with g-32P (3000 Ci mmol-1, 250 mCi)using T4 polinucleotide kinase. Ten micrograms of nuclearextract was incubated for 20 min with 20 000 cpm of 32P-labelleddouble-stranded oligonucleotide at 4°C in a 25 ml reactionmixture containing 10 mg ml-1 BSA, 10¥ buffer (100 mM KCl,20 mM Hepes, 0.5 mM EDTA, 2 mM DTT, 0.1 PMSF, 20% glyc-erol, 0.25% NP-40, pH 7.6), 5¥ buffer (300 mM KCl, 100 mMHepes, 10 mM DTT, 100 mM PMSF, 20% Ficoll, pH 7.6) and1 mg ml-1 poly(dI-dC). The DNA–protein complex was separatedon a non-denaturing 4% polyacrylamide gel in TBE buffer (0.4 MTris, 0.45 M boric acid, 0.5 M EDTA, pH 8.0). After electrophore-sis, the gel was dried and autoradiographed by overnight expo-sure to X-ray film.

Assay of IkBalpha, phospho-IkBalpha, p50(NF-kB) andp65(NF-kB) subunit protein expression byWestern blotting

Ten micrograms of nuclear or cytosolic proteins were separatedon a 10% or 12% polyacrylamide gel, blotted on a polyvinylidenedifluoride membrane, and probed with rabbit anti-IkBalpha and

anti-NF-kB-p65 Ab or mouse anti-phospho-IkBalpha and anti-NF-kB-p50 Ab. Bands were visualized by ECL staining.

Acknowledgements

Thanks are due to Elena Valente and Denisa Baci for help withthe parasite cultures and handling of HZ; and to AssociazioneVolontari Italiani Sangue (AVIS, Torino, Italy) for providing freshlydiscarded buffy coats. The work was supported by theCompagnia di San Paolo, Torino, in the context of the ItalianMalaria Network and Regione Piemonte, Ricerca Sanitaria Final-izzata grants to M.P. and P.A.

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