Tumour necrosis factor (TNF)-mediated NF-κB activation facilitates cellular invasion of...
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Tumour necrosis factor (TNF)-mediated NF-kB activation facilitates cellular invasion of non-professional phagocytic epithelial cell lines by Trypanosoma cruzi Andrea M. T. Pinto, Paula C. M. Sales, Elizabeth R. S. Camargos † and Aristóbolo M. Silva* † Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627 – ICB, UFMG, 31270-901, Belo Horizonte, MG, Brazil. Summary At the site of infection, pro-inflammatory cytokines locally produced by macrophages infected with Trypanosoma cruzi can activate surrounding non- professional phagocytes such as fibroblasts, epi- thelial and endothelial cells, which can be further invaded by the parasite. The effect of secreted soluble factors on the invasion of these cells remains, however, to be established. We show here that two epithelial cell lines become significantly susceptible to the infection by the Y strain of T. cruzi after tumour necrosis factor (TNF) treat- ment. The increase in the invasion was correlated with the increasing concentration of recombinant TNF added to cultures of HEK293T or LLC-MK2 cells. Supernatants taken from PMA-differentiated human monocytes infected with T. cruzi also increased the permissiveness of epithelial cells to subsequent infection with the parasite, which was inhibited by a TNF monoclonal antibody. Further- more, the permissiveness induced by TNF was inhibited by TPCK, and led to significant decrease in the number of intracellular parasites, providing evidence that activation of NF-kB induced by TNF favours the invasion of the epithelial cell lines by T. cruzi through yet an unidentified mechanism. Our data indicate that soluble factors released from macrophages early in the infection favours T. cruzi invasion of non-professional phagocytic cells. Introduction Chagas’ disease or American trypanosomiasis is a life-long infection caused by the haemoflagellate protozoan Trypano- soma cruzi and affects millions of people in Latin America (WHO, 2002). The acute phase is characterized by circula- tion of infective trypomastigotes in the blood and proliferation of amastigotes inside cells. T. cruzi can invade and prolifer- ate in different cell types, mainly muscle cells (Tanowitz et al., 1992). Afterwards, circulating trypomastigotes are rarely seen but the persistence of the parasite has been proved to occur in many tissues. Chagasic cardiomyopa- thy (CCC) is the most important clinical manifestation of the chronic form and affects 10–30% of the infected patients (Zhang and Tarleton, 1999). The majority of infected indi- viduals pass through an asymptomatic chronic phase (indeterminate clinical form). The current view considers that factors related to both parasite and host genetic char- acteristics have a role in the pathogenesis of this infection (Macedo et al., 2004; Gutierrez et al., 2009).The parasite entry into non-professional phagocytic cells depends on Ca 2+ signalling triggered by a G protein pathway (Tardieux et al., 1994; Leite et al., 1998). Adhe- sion of the parasite activates PLC (phospholipase C) and releases Ca 2+ from IP3 (Inositol Triphosphate)-sensitive intracellular stores (Rodriguez et al., 1995). The increased cytosolic Ca 2+ levels induce the recruitment of lysosomes to the site of T. cruzi adhesion and its subse- quent internalization (reviewed by Andrade and Andrews, 2005). An alternative pathway has been proposed by Woolsey and co-workers in 2003, which leads to the enrichment of cell membrane with lipids produced by PI3K (phosphoinositide 3-kinase) as one step for cell invasion, where fusion with lysosomes would occur after parasite invasion (Woolsey et al., 2003). During the initial period of infection, cellular immune response plays an essential role in the host resistance to infection (Gazzinelli et al., 1992; Camargo et al., 1997). Afterwards, development of a polarized Th1-acquired immune response becomes fundamental for controlling parasite replication (Tarleton et al., 1992; Aliberti et al., 1996; Michailowsky et al., 2001). tGPI (T. cruzi-glycosylphosphatydilinositol) anchors are the main surface macromolecule from trypomastigotes Received 26 October, 2010; revised 12 May, 2011; accepted 6 June, 2011. *For correspondence. E-mail [email protected]; Tel. (+55) 31 34092802; Fax (+55) 31 34092771. † These authors have contributed equally to this work. Cellular Microbiology (2011) 13(10), 1518–1529 doi:10.1111/j.1462-5822.2011.01636.x First published online 12 July 2011 © 2011 Blackwell Publishing Ltd cellular microbiology
Transcript of Tumour necrosis factor (TNF)-mediated NF-κB activation facilitates cellular invasion of...
Tumour necrosis factor (TNF)-mediated NF-kBactivation facilitates cellular invasion ofnon-professional phagocytic epithelial cell lines byTrypanosoma cruzi
Andrea M. T. Pinto, Paula C. M. Sales,Elizabeth R. S. Camargos† and Aristóbolo M. Silva*†
Departamento de Morfologia, Instituto de CiênciasBiológicas, Universidade Federal de Minas Gerais,Av. Antonio Carlos, 6627 – ICB, UFMG, 31270-901,Belo Horizonte, MG, Brazil.
Summary
At the site of infection, pro-inflammatory cytokineslocally produced by macrophages infected withTrypanosoma cruzi can activate surrounding non-professional phagocytes such as fibroblasts, epi-thelial and endothelial cells, which can be furtherinvaded by the parasite. The effect of secretedsoluble factors on the invasion of these cellsremains, however, to be established. We show herethat two epithelial cell lines become significantlysusceptible to the infection by the Y strain ofT. cruzi after tumour necrosis factor (TNF) treat-ment. The increase in the invasion was correlatedwith the increasing concentration of recombinantTNF added to cultures of HEK293T or LLC-MK2cells. Supernatants taken from PMA-differentiatedhuman monocytes infected with T. cruzi alsoincreased the permissiveness of epithelial cells tosubsequent infection with the parasite, which wasinhibited by a TNF monoclonal antibody. Further-more, the permissiveness induced by TNF wasinhibited by TPCK, and led to significant decreasein the number of intracellular parasites, providingevidence that activation of NF-kB induced by TNFfavours the invasion of the epithelial cell lines byT. cruzi through yet an unidentified mechanism.Our data indicate that soluble factors releasedfrom macrophages early in the infection favoursT. cruzi invasion of non-professional phagocyticcells.
Introduction
Chagas’ disease or American trypanosomiasis is a life-longinfection caused by the haemoflagellate protozoan Trypano-soma cruzi and affects millions of people in Latin America(WHO, 2002). The acute phase is characterized by circula-tion of infective trypomastigotes in the blood and proliferationof amastigotes inside cells. T. cruzi can invade and prolifer-ate in different cell types, mainly muscle cells (Tanowitzet al., 1992). Afterwards, circulating trypomastigotes arerarely seen but the persistence of the parasite has beenproved to occur in many tissues. Chagasic cardiomyopa-thy (CCC) is the most important clinical manifestation of thechronic form and affects 10–30% of the infected patients(Zhang and Tarleton, 1999). The majority of infected indi-viduals pass through an asymptomatic chronic phase(indeterminate clinical form). The current view considersthat factors related to both parasite and host genetic char-acteristics have a role in the pathogenesis of this infection(Macedo et al., 2004; Gutierrez et al., 2009).cmi_1636 1518..1529
The parasite entry into non-professional phagocyticcells depends on Ca2+ signalling triggered by a G proteinpathway (Tardieux et al., 1994; Leite et al., 1998). Adhe-sion of the parasite activates PLC (phospholipase C) andreleases Ca2+ from IP3 (Inositol Triphosphate)-sensitiveintracellular stores (Rodriguez et al., 1995). Theincreased cytosolic Ca2+ levels induce the recruitment oflysosomes to the site of T. cruzi adhesion and its subse-quent internalization (reviewed by Andrade and Andrews,2005). An alternative pathway has been proposed byWoolsey and co-workers in 2003, which leads to theenrichment of cell membrane with lipids produced by PI3K(phosphoinositide 3-kinase) as one step for cell invasion,where fusion with lysosomes would occur after parasiteinvasion (Woolsey et al., 2003).
During the initial period of infection, cellular immuneresponse plays an essential role in the host resistance toinfection (Gazzinelli et al., 1992; Camargo et al., 1997).Afterwards, development of a polarized Th1-acquiredimmune response becomes fundamental for controllingparasite replication (Tarleton et al., 1992; Aliberti et al.,1996; Michailowsky et al., 2001).
tGPI (T. cruzi-glycosylphosphatydilinositol) anchors arethe main surface macromolecule from trypomastigotes
Received 26 October, 2010; revised 12 May, 2011; accepted 6 June,2011. *For correspondence. E-mail [email protected]; Tel.(+55) 31 34092802; Fax (+55) 31 34092771.†These authors have contributed equally to this work.
Cellular Microbiology (2011) 13(10), 1518–1529 doi:10.1111/j.1462-5822.2011.01636.xFirst published online 12 July 2011
© 2011 Blackwell Publishing Ltd
cellular microbiology
able to induce the synthesis of pro-inflammatory cytokineby macrophages. tGPI recognition by Toll-like receptor 2(TLR2) stimulates TLR2/MyD88 pathways, includingmitogen-activated protein kinases (MAPK) phosphoryla-tion and NF-kB transcription factor activation (Camposet al., 2001; Takeda and Akira, 2005; Ishii et al., 2008).Ropert et al. have shown that NF-kB activation is impor-tant for tumour necrosis factor (TNF) secretion by T. cruzi-infected professional phagocytic cells (Ropert et al.,2001). Thus, in an autocrine fashion, released TNF mayinduce the trypanocidal function of macrophages bystimulating NO production in IFN-gamma-activated mac-rophages (Silva et al., 1995). The action of recombinantTNF alone was also shown to inhibit intracellular multipli-cation of blood trypomastigotes of T. cruzi in murine peri-toneal macrophages (De Titto et al., 1986). Therefore, thiscytokine exerts an essential role in host defence toT. cruzi infection by triggering macrophage activation andthe inflammatory processes.
Non-professional phagocytic cells are also able tosecrete a broad range of inflammatory molecules inresponse to T. cruzi infection. Mouse cardiomyocytesexpress high levels of TNF, IL-1b (interleukin-1beta), IL-6(interleukin-6) and iNOS (inducible nitric oxide synthase)mRNA, as well as ICAM-1 (intercellular molecule adhe-sion type 1) and VCAM-1 (vascular adhesion moleculetype 1) (Machado et al., 2000). Vascular endothelial cellsalso exhibits increased expression of pro-inflammatorycytokines (Tanowitz et al., 1992), chemokines (Teixeiraet al., 2002), vascular adhesion molecules (Huang et al.,1999), nitric oxide synthase (Chandrasekar et al., 2000)and endothelin-1 (Wittner et al., 1995; Petkova et al.,2000). Activation of MAPKs, transcription factors AP-1and NF-kB and cell cycle regulatory molecules are alsodescribed in non-professional phagocytes during T. cruziinfection in vitro (Huang et al., 2003).
Studies about the impact of inflammatory molecules onT. cruzi invasion and persistence in non-professionalphagocytic cells are missing. Early in the infection, epi-thelial cells interact with trypomastigotes in a pro-inflammatory environment mainly in mucosal layers andwounded skin. As the mechanisms related to the infectionof non-professional phagocytic cells are still unknown, wehave initiated studies to investigate whether TNF-mediated activation of NF-kB transcription factor inter-feres with T. cruzi infection in mammalian epithelial cells.
Results
TNF treatment increases the invasion of humanepithelial cells by T. cruzi
To investigate whether TNF treatment of epithelial cellscan affect the number of intracellular parasites, we carried
out in vitro infections of HEK293T and LLC-MK2 cells withthe Y strain of T. cruzi. The results of the infection experi-ments (Fig. 1A–F) indicate that the number of parasiteswithin 100 cells was significantly increased in the TNF-treated HEK293T and LLC-MK2 cells. Compared with theHEK293T cells, before and after TNF treatment, thenumber of intracellular parasites in LLC-MK2 cells wastwofold increased. The increased number of intracellularparasites in the TNF-treated cells was clearly dependenton the concentration of the cytokine (Fig. 2A–D). Thenumber of intracellular parasites in HEK293T cells startedto increase with the concentration of 5 ng ml-1 of TNF andreached a peak at the 40 ng ml-1 concentration. In LLC-MK2 cells, the progressive increase of the amount ofparasites started with TNF at 10 ng ml-1. Next, we soughtto determine at which time interval upon infection theincrease in the number of intracellular parasites wasoccurring in the TNF-treated cells. As shown in Fig. 3, asimilar increase in that number occurred in all time pointsinvestigated. Importantly, this result demonstrates thatsuch increase occurs at the beginning of the infection inboth cell types, with no significant fluctuation in subse-quent periods. These results indicate that TNF facilitatesthe cell invasion by the parasite without affecting signifi-cantly the intracellular multiplication. This observation iscorroborated by the adhesion studies where T. cruzi try-pomastigotes seem to adhere more to TNF-treated thanuntreated cells (Fig. S2). Mechanistic studies of host cellinvasion have shown that activation of G protein-coupledreceptors induces an endocytic uptake of T. cruzi causedby IP3-mediated intracellular Ca2+ release (Tardieux et al.,1994; Leite et al., 1998). To determine whether this isinvolved in the parasite uptake by TNF-alpha primed cells,we preincubated HEK293T and LLC-MK2 cells with 1 or3 mM thapsigargin to deplete Ca2+ stores, and then treatedwith TNF, and infected with T. cruzi. As shown in Fig. S1,addition of thapsigargin in both concentrations dramati-cally reduced invasion in the TNF-treated cells. This resultsuggests that the parasite can invade TNF-primed cells byintracellular Ca2+-dependent pathways.
TNF-mediated NF-kB activation is required for theinvasion of epithelial cell by T. cruzi
To gain a better understanding of the mechanismsinvolved in the increased cell invasion, we examinedwhether this was a phenomenon that required signallingpathways activated by TNF. First, TNF-mediated cytotox-icity was determined in the HEK293T and LLC-MK2 cells.As depicted in the Fig. 4, MTT assays indicate that TNFtreatment did not induce cytotoxicity at the concentrationstested, providing evidence that the increased number ofintracellular parasites seen in the results shown above isnot due to low cell viability. We next evaluated whether by
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inhibiting NF-kB activation resulted in decreased numberof amastigotes within TNF-treated cells. To this, HEK293Tor LLC-MK2 cells were first treated with increasing con-centrations of the NF-kB inhibitor TPCK for 30 min andfurther treated with TNF for 6 h, and then infected withT. cruzi. After 24 h, the number of amastigotes per 100cells was determined. As shown in Fig. 5A–D, in the pres-ence of TPCK the number of intracellular parasites wasclearly reduced in the TNF-treated cells. Such decreasewas more prominent in LLC-MK2 cells at low concentra-tions of TPCK. TPCK treatment alone did not significantlyaffect the number of intracellular parasites. In addition, nosignificant changes were seen in the number of amastig-otes in the presence of different concentrations of theTPCK solvent, dimethylsulfoxide (DMSO) (Fig. S3), con-firming the specificity of the effect seen with TPCK. Theresults demonstrate that the increase in the number ofintracellular parasites in both types of epithelial cellsinvolves the activation of NF-kB induced by TNF. We haveconfirmed these observations by transfecting HEK293T
cells with mutant IkBa dominant negative, treating themwith TNF and further infecting with T. cruzi (Fig. S4A–C).
Supernatants taken from monocytes infected withT. cruzi increase the number of intracellular parasites inepithelial cells
We next determined if the supernatants taken fromhuman differentiated monocytes infected with T. cruziaffect the invasion of the epithelial cells by the parasite.For this purpose, THP-1 cells were first differentiatedwith PMA, infected with T. cruzi, and the supernatantscollected. Furthermore, the supernatants were used totreat HEK293T and LLC-MK2 cells followed by infectionwith T. cruzi and determination of the number of para-sites. As demonstrated in Fig. 6A, PMA-differentiatedTHP-1 cells can be infected with T. cruzi; moreover, theinfected cells can secrete TNF in the supernatants (unin-fected: 15.16 pg ml-1; 2 h post infection: 3280.65 pg ml-1.Data not shown). Yet, T. cruzi infection of these cells
Fig. 1. Increased number of intracellularparasites in epithelial cells treated with TNF.A. Number of parasites per 100 HEK293Tcells.B. Number of parasites per 100 LLC-MK2cells. Cells were treated for 6 h with TNF(20 ng ml-1) and further infected withTrypanosoma cruzi. After 24 h, cells werefixed and the parasites counted. The valuesrepresent mean � SD of three independentexperiments. *P < 0.05.C and D. Immunofluorescence of LLC-MK2cells untreated (C) or treated (D) with TNFand infected with Y strain of T. cruzi. Scalebar: 20 mM.E and F. Frequency of infected cells ofexperiments shown in (A) and (B).
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resulted in the activation of p38 MAPK (Fig. 6B). Asshown in Fig. 6C and D, and Fig. S5, an increase in thenumber of parasites was seen in both epithelial celltypes treated with the supernatants, suggesting thatsoluble factors secreted by the phagocytic cells infectedwith T. cruzi facilitate subsequent infection by the para-
site. Furthermore, the supernatants taken from the THP-1-infected cells retained ability to induce NF-kBactivation in HEK293T cells at comparable levels withTNF (Fig. S6), indicating the presence of inflammatorysoluble factors. To provide evidence for such observa-tion, the effects of Infliximab, a mouse-human chimeric
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Fig. 5. Inhibition of TNF-mediated NF-kB activation decreases infection in epithelial cells.A and B. Number of intracellular parasites in HEK293T (A) and LLC-MK2 (B) cells treated with increasing concentrations of TPCK for 30 minand treated or not for 6 h with of TNF (20 ng ml-1) followed by infection for 2 h with T. cruzi. The values represent mean � SD of threeindependent experiments. Different letters represent groups with statistical significance, P < 0.05.C and D. Frequency of infected cells taken from an independent experiment.
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neutralizing anti-TNF-alpha antibody, at 1 and 10 mg ml-1
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Fig. 6. Soluble factors secreted from human monocytes infected with Trypanosoma cruzi increase the invasion of epithelial cellsby the parasite.A. Number of intracellular parasites in PMA-differentiated THP-1 cells and further infected for 2 h with T. cruzi. The values representmean � SD of three independent experiments. Different letters represent groups with statistical significance, P < 0.05.B. Western Blot analysis of p38 MAPK phosphorylation in THP-1 cells infected with T. cruzi. After infections, the culture medium wasdiscarded, cells were washed with PBS 1¥ and the cell extracts obtained. The protein concentration was determined by Bradford assay. Thirtymicrograms of each cell extract was fractionated onto SDS-PAGE 12% and then transferred to a PVDF membrane. The membrane wasincubated with anti-phospho-p38 MAPK (1:1000) and secondary antibody anti-rabbit anti-IgG-HRP. Proteins were detected with the use of thechemiluminescent reagent and further exposure to autoradiogram film. The membrane was further washed several times and incubated withprimary anti-tubulin and secondary mouse anti-IgG-HRP antibodies similarly as above.C and D. Number of intracellular parasites in HEK293T (C) and LLC-MK2 (D) cells treated for 6 h with the supernatants taken from THP-1cells infected with T. cruzi.E. Effects of neutralizing anti-TNF antibody (Infliximab) at 1 and 10 mg ml-1 in the number of intracellular parasites in HEK293T (left panel) andLLC-MK2 (right panel) cells treated with the supernatants taken from THP-1 cells infected with T. cruzi. The values represent mean � SD ofthree independent countings. *P < 0.05.
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Discussion
Our study demonstrates that TNF action on non-professional phagocytic epithelial cell lines render themmore susceptible to the infection by T. cruzi. TNF is apleiotropic cytokine that displays a broad range of biologi-cal activities. During infections, the gene encoding TNF israpidly transcribed and the protein is finally secreted. Inthe extracellular environment, TNF will exert its action ontarget cells upon binding to one of its receptor TNFR1 thateventually results in the activation of the transcriptionfactor NF-kB (Hatada et al., 2000; Thommesen and Lae-greid, 2005) and regulation of a number of genes impor-tant for diverse biological processes mediated by TNF.
As T. cruzi binding and invasion of host cells is a complexprocess that involves multiple host and parasite molecules,we chose to use HEK293T cells because they almostcompletely lack expression of any of the TLRs (Hornunget al., 2002), as NF-kB activation induced by expression ofthe TLR adapter Mal/TIRAP molecule also increased per-missiveness to cell invasion (A.M. Pinto and A.M. Silva,unpubl. data). In addition, because we found that thesecells are permissive to T. cruzi infection, they can be usedin further transfection studies to examine potential surfacecandidate proteins (e.g. G protein-coupled receptors)involved in the permissiveness induced by TNF, such asbradykinin receptors, especially B1R and B2R that havebeen implicated in signalling mechanisms of T. cruzi inva-sion of non-professional phagocytes (Scharfstein et al.,2000; Todorov et al., 2003). The use of LLC-MK2 cells bymany investigators has shown the greatest success ininvasion studies of T. cruzi from tissue culture-derivedtrypomastigotes. Moreover, several invasion studies havederived T. cruzi trypomastigotes from these cells with nosignificant loss of infectivity, even after several passages(Andrews and Colli, 1982). Finally, as a particularly inter-esting cell type model to determine host cell receptors toT. cruzi surface molecules, a number of candidate ligandproteins have been suggested in studies using LLC-MK2(Magdesian et al., 2001; Ulrich et al., 2002). It will beinteresting to examine the regulation of their expression inresponse to TNF. Furthermore, these molecules could beeasily tested in further transfection studies or RNA interfer-ence approaches to determine host–parasite interaction inTNF-treated non-immune cells.
It has been shown in human macrophages that p38MAPK regulates TNF production mediated through a post-transcriptional mechanism requiring the 3′ untranslatedregion of the gene (Sun and Ding, 2006). Ropert et al.(2001) demonstrated that GPI-mucins of T. cruzi arecapable of triggering phosphorylation of ERK-1/ERK-2,MKK4 and p38 MAPKs as well as IkBa phosphorylation inmouse peritoneal macrophages. Moreover, they sug-gested that p38 MAPK pathway is involved in the synthesis
of TNF by stimulated macrophages. Therefore, we lookedat the ability of THP-1 to become infected with T. cruzi. Ourresult in Fig. 6B shows that after T. cruzi infection, p38MAPK is readily phosphorylated in THP-1 cells.
We have demonstrated that the increase in the numberof intracellular parasites in the TNF-treated cells occurs atthe first steps of the infection corroborating the idea thatTNF facilitated cell invasion by T. cruzi with no apparenteffects on the intracellular multiplication of the parasite.This observation is supported by the results of Fig. S2where parasites seem to adhere more to cells that werepreviously treated with TNF, suggesting the involvementof a TNF-regulated expression of an adhesion or receptormolecule, yet unidentified. Signalling pathways emanat-ing from TNF receptor include the NF-kB-mediated sur-vival and apoptotic response depending on the cellularcontext. In our experiments, at the time points tested, TNFdid not induce apoptosis in HEK293T and LLC-MK2 cells.Therefore, we evaluated the role of NF-kB in theincreased cell susceptibility to infection. NF-kB (p50/RelA)is a transcription factor that regulates the expression ofmany genes involved in immune and inflammatoryresponses, cell survival and proliferation. These biologicaleffects mediated by NF-kB can be inhibited by a numberof pharmacological compounds. N-Tosyl-L-phenylalaninechloromethyl ketone (TPCK), a serine and cysteine pro-teinase inhibitor, was shown to inhibit NF-kB activation byblocking signal-induced phosphorylation and subsequentdegradation of IkBa in various cell types (Henkel et al.,1993; Finco et al., 1994; Ha et al., 2009). We have shownthat TNF-mediated cell invasion by T. cruzi was markedlyinhibited by TPCK. At lower concentrations of TPCK, thedecrease in the number of intracellular parasites wasmore prominent in LLC-MK2 cells. These observationsprovide evidence that the increase in the number of intra-cellular parasites in both types of epithelial cells involvesthe activation of NF-kB induced by TNF.
Tumour necrosis factor has been reported to induceapoptosis in a number of cell lines. Robaye et al. (1991)demonstrated that this cytokine causes massive deathof bovine endothelial cells in primary culture in aconcentration- and time-dependent manner. Administra-tion of recombinant TNF exacerbated mortality (Blacket al., 1989) and the increased levels of endogenous TNFwere associated with increased susceptibility (Russoet al., 1989) and shown to mediate cachexia and inflam-matory damage during infection with T. cruzi (Truyenset al., 1995). However, our results demonstrate that TNFtreatment did not induce cytotoxicity at the concentrationsinvestigated, providing evidence that the increasednumber of intracellular parasites was not related to adecrease in cellular viability as demonstrated by the MTTassay. Interestingly, it has been demonstrated that T. cruziprotects cardiac cells in vitro from apoptosis triggered by
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TNF, and this effect correlates with the presence of theparasite intracellularly and the activation of NF-kB(Petersen et al., 2006). Although we have seen that TNFtreatment leads to NF-kB activation in the cell lines usedour study, it remained to be determined whether suchactivation is also regulated in the presence of the parasite.Nonetheless, similarly to what has been observed byPetersen and colleagues, it is possible that NF-kB signal-ling pathway triggered firstly by TNF and then by T. cruzicontributes to protect the parasite from cell death in epi-thelial cells.
NF-kB activation has been demonstrated in non-immune cells such as epithelial and endothelial cells, andfibroblasts following T. cruzi infection where the ability ofT. cruzi to activate NF-kB correlates inversely with sus-ceptibility to infection (Hall et al., 2000). Those authorsshowed a higher infection level in IkBaM/GFPtransfected-epithelial cells, i.e. cells unable to activateNF-kB. In contrast, our present work carried out in epithe-lial cells indicates that NF-kB activation induced by TNFincreases the susceptibility to infection. Hall and col-leagues demonstrated that NF-kB activation in non-phagocytic cell lines is related to resistance to theinfection by Silvio, Tulahuen and MV13 strains of T. cruzi.The major discrepancy with our findings is that in theirwork they have tested a number of non-phagocytic celllines whose resistance to T. cruzi infection was only cor-related with the ability of those cells to have an increasedNF-kB activation elicited by T. cruzi infection. Their workdid not demonstrate whether such resistance was asso-ciated to secreted factors from the infected cells, ratherthan soluble material released by trypomastigotes.
It is valuable to mention that several studies haveclearly demonstrated a close association between theparasite persistence and the intensity of inflammatory pro-cesses and the severity of the histopathological lesions inthe heart and digestive organs during the chronic phase ofthis infection, both in human (Palomino et al., 2000) andexperimental models (Zhang and Tarleton, 1999). Inter-estingly, CD8+ lymphocytes and TNF-expressing mac-rophages are described as the predominant cell types inthe inflammatory foci present in the heart of chronicpatients (Reis et al., 1993). It will now be relevant toinvestigate the effect of several cytokines on non-immunecells and define the molecular determinants of such phe-nomenon. It is also tempting to look for possible candidateadhesion molecules and membrane microdomains pro-teins that could be involved in the higher parasite adhe-sion observed in the TNF-treated epithelial cells.
Our results show that TNF/NF-kB axis participates inT. cruzi invasion of non-professional phagocytic epithelialcell lines, resulting in increased number of intracellularparasites, leading us to conclude that NF-kB activation innon-immune cells elicited by paracrine factors released
by immune cells represents a mechanism by whichT. cruzi persists in the host. Understanding of the mecha-nism of the invasion facilitated by inflammatory solublefactors will further aid in the development of immuneprotective therapy for Chagas’ disease.
Experimental procedures
Cells and treatments
LLC-MK2 (monkey kidney epithelial cell line) and HEK293T(human embryonic kidney cell line) were maintained in DMEM(Dulbecco’s modified Eagle’s medium – Invitrogen) with 10%fetal bovine serum (FBS, Invitrogen), 1% penicillin-streptomycin(Invitrogen) and 1% glutamine (Sigma-Aldrich). For infections,HEK293T and LLC-MK2 cells were grown in DMEM 2% FBSin 24-well plates with glass coverslips. In some experiments,before addition of TNF (20 ng ml-1 for 6 h), cells were exposedfor 30 min to different concentrations of TPCK (N-tosyl-L-phenylalanine chloromethyl ketone). Promonocytic THP-1 cells,which can be differentiated into macrophage-like cells (Tsuchiyaet al., 1980), were collected from culture media by centrifugationat 200 g, washed once in phosphate-buffered saline (PBS)(3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl,pH 7.4) and resuspended at 1 ¥ 106 cells ml-1 in RPMI-1640supplemented with 10% FBS (Gibco-Invitrogen), 1% penicillin-streptomycin (Gibco-Invitrogen) and 2 mM glutamine (Sigma-Aldrich), and 100 nM phorbol myristate acetate (PMA). Twomillilitres of cell suspension was added to each well of a 6-wellplate and allowed to differentiate and adhere to the culture platesfor 48 h at 37°C under 5% CO2. After this time, cells were washedtwice with PBS and fresh media (without PMA) containing para-sites were added for further infection studies. Following infection,culture media and cell lysates were collected and analysed forTNF secretion by ELISA and Western blot studies respectively. Forneutralizing TNF from the supernatants taken from T. cruzi-infected PMA-differentiated THP-1 cells, Infliximab (a kind gift ofProfessor Mauro M. Teixeira, UFMG, Brazil) was added at 1 or10 mg ml-1 to the supernatants and incubated on ice for 30 min.Then, the supernatants (500 ml per well) were added to HEK293Tor LLC-MK2 cell cultures and incubated for 6 h before infection.
Parasites
Trypanosoma cruzi trypomastigotes from the Y strain wereobtained from infected LLC-MK2 cells maintained in DMEMsupplemented with 2% FBS, 1% glutamine and penicillin-streptomycin. Infected cells were maintained in humidified incu-bator in 5% CO2 at 37°C. The supernatants containing thetrypomastigotes were collected in a polystyrene tube, centrifugedat 2200 r.p.m. for 10 min and then reincubated in the humidifiedincubator for at least 3 h. This procedure is crucial to separatetrypomastigotes forms from the amastigotes forms and cellulardebris. After 3 h, the supernatant was collected and the numberof viable trypomastigotes was determined by counting in neu-bauer chamber.
In vitro infection studies
Before infections, HEK293T and LLC-MK2 cells were leftuntreated or treated with 20 ng ml-1 TNF (Sigma) for 6 h. Medium
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was discarded and the monolayers washed three times withPBS. Cells were infected at the 10:1 ratio (parasites : cell) for 2 h,after which the parasites were removed, and the cells werewashed three times with PBS before adding fresh medium.Twenty-two hours after removal of the parasites (i.e. 24 h postinfection), the cells growing in coverslips were fixed with 4%paraformaldehyde (PFA) for 16 h and stained with 4,6-diamidino-2-phenylindole (DAPI) (1:1000). To determine the number ofintracellular parasites the amount of amastigotes in 250 cellsdistributed in 15 different fields was counted in a fluorescencemicroscope. The experiments were performed in triplicate. Theproportion of the number of intracellular parasites in 100 cells ispresented in the graphs. In some experiments, the percentage ofinfected cells was also evaluated in the same 15 fields. Thephotomicrographs were acquired in an Axioplan 2 Zeiss micro-scope through the Axion Vison software 4.7.2.
MTT viability assay
Cellular viability analysis of cells treated with TNF was deter-mined by MTT assay (3-(4,5-dimetiltiazol-2yl)-2,5-difenil tetrazo-lium bromide). HEK293T and LLC-MK2 cells were seeded in24-well plates and in the next day the cells were treated withdifferent concentration of TNF for 6 h. The medium was removedand freshly culture medium was added. After 18 h, the cell culturemedia were removed and 210 ml of fresh DMEM plus 170 ml ofMTT solution (5 mg ml-1) were added for each well. Plates wereincubated in humidified incubator for 2 h in 5% CO2 at 37°C. After2 h, 210 ml of SDS in 10% HCl (hydrochloric acid) solution wasadded for each well and the plate incubated for 18 h protectedfrom the light. The media containing MTT were collected and theabsorbance at 595 nm determined by colorimetric assay (Versa-max, Molecular Devices). The values obtained from the readingsare directly proportional to the number of viable cells.
TPCK treatment for NF-kB inhibition
N-Tosyl-L-phenylalanine chloromehylketone (TPCK, Sigma) wasdissolved in DMSO at 10 mM final concentration and stored at-20°C. For use in the cell cultures, a work solution of TPCK wasprepared at 1 mM concentration. Before TNF treatment, cellswere preincubated for 30 min with different concentration ofTPCK. Cells were then treated with TNF 20 ng ml-1 for 6 h,washed with PBS and then infected with T. cruzi. Twenty-fourhours post infection, cells were fixed, stained and the number ofintracellular parasites determined as described above.
Infection of THP-1 cells with T. cruzi
THP-1 cells seeded in 24-well plates with glass coverslips anddifferentiated with PMA for 48 h were infected with T. cruzi (10parasites : cell) for different time intervals. At the end of infectionscells were fixed. The number of intracellular parasites wasdetermined as described above. For harvesting supernatantsfrom THP-1 cells infected with T. cruzi, cells were seeded in10 cm plates and differentiated with PMA 100 nM. After 48 h,medium was removed and the cells infected with T. cruzi(10 parasites : cell) for 2 h. The supernatant was collected andcentrifuged at 300 g for 10 min at 4°C to pellet parasites and celldebris. Supernatants from non-infected PMA-differentiated cells
were also collected. The supernatants were stored at -20°C andfurther used to treat HEK293T and LLC-MK2 cells for 6 h, afterwhich cells were infected with T. cruzi as described above.
Cell lysis, SDS-PAGE and immunoblotting
Following infection, THP-1 cells were washed twice in ice-coldPBS. Cell extracts were obtained with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 50 mM sodium fluoride,10 mM b-glycerolphosphate, 0.1 mM EDTA, 10% glycerol, 1%Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 mM sodiumorthovanadate and 2 mg ml-1 of pepstatin, leupeptin, and aproti-nin). Following incubation on ice for 10 min, cell extracts wereclarified by centrifugation at 12 000 g for 10 min. Total proteinwas quantified using standard Bio-Rad protein assay (Bio-Rad/USA). Cells extracts were fractionated onto SDS-PAGE andtransferred to PVDF membranes (Immobilon-P, Millipore). Themembranes were blocked at room temperature for 1 h in TBS-Tween + 5% fat-free milk, washed three times in TBS-Tween andprobed with primary anti-phospho-p38 MAPK (Cell SignalingTechnology, Danvers, MA, USA) or alpha-tubulin (Santa CruzBiotechnology, Santa Cruz, CA, USA) antibodies diluted in TBS-Tween with either 5% BSA or 1% milk respectively. After over-night incubation at 4°C, membranes were washed three times inTBS-Tween, followed by the addition of the secondary antibodiesHRP-conjugated diluted in TBS-Tween + 1% milk, and furtherincubation at room temperature for 1 h. After washing the mem-branes, the proteins were detected with the use of the ECLPlus-Western Blotting Detection System (GE Healthcare, USA)and further exposure to autoradiogram film.
Statistical analyses
Results are expressed as mean � SD and for comparisonbetween groups; variance analysis one-way ANOVA was usedwith post-test application. Differences between groups were con-sidered significant if P < 0.05. All analyses were carried out withGraphPad Prism software (GraphPad Software).
Acknowledgements
We thank Dr Luciana Andrade for suggestions and criticalreading of the manuscript. This study was supported byFAPEMIG, CNPq and the National Institute of Science and Tech-nology for Vaccine Development (INCTV). AMTP and PCMS arerecipient of pre-doctoral fellowship from CAPES and CNPqrespectively. ERS and AMS are research fellows of CNPq.
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Supporting information
Additional Supporting Information may be found in the onlineversion of this article:
Fig. S1. Effects of thapsigargin on the invasion of Trypanosomacruzi in TNF-treated epithelial cells. HEK293T or LLC-MK2 cells(upper and lower graphs respectively) were firstly treated withthapsigargin (1 or 3 mM) for 30 min and then with TNF for 6 h.Cells were further infected with T. cruzi. After 24 h, cells werefixed and the parasites counted. All incubations were performedat 37°C in a 5% CO2 incubator.Fig. S2. Increased number of trypomastigotes adhered to epi-thelial cells treated with TNF.A. Number of trypomastigotes per 100 HEK293T cells.B. Number of trypomastigotes per 100 LLC-MK2 cells. Cells weretreated for 6 h with TNF (20 ng ml-1), prefixed with 2% glutaral-dehyde, incubated with 0.16 M ethanolamine and then incubatedwith Trypanosoma cruzi. After 2 h, cells were fixed and Giemsa-stained for counting the number of adhered trypomastigotes.*P < 0.05.C. Giemsa staining of LLC-MK2 cells untreated (left) or treated(right) with TNF and infected with Y strain of T. cruzi.Fig. S3. DMSO does not affect invasion of Trypanosoma cruzi inthe TNF-treated epithelial cells. Number of intracellular parasitesin HEK293T (A) and LLC-MK2 (B) cells treated with increasingequivalent concentrations of DMSO for 30 min and treated or notfor 6 h with TNF (20 ng ml-1) followed by infection for 2 h withT. cruzi. The values represent mean � SD of three independentexperiments. Different letters represent groups with statisticalsignificance, P < 0.05.Fig. S4. Effect of IkBa dominant negative on Trypanosoma cruziinfection in epithelial cell line.A. Semi-confluent HEK293T cells growing in 24-well plates wereleft untransfected (No Tfx) or transfected with the vector plasmid(pcDNA3.1) or pRcCMV-IkBa-mutant (IkBa-DN) as indicated.Twenty-four hours post transfection, cells were left untreated ortreated with TNF (20 ng ml-1) for 6 h, and further infected asfollowing: in one experiment (left panel) cells were infected withT. cruzi for 2 h, fixed and immunostained firstly with rabbit anti-T. cruzi antibody (1:500) followed by immunostaining with goatanti-rabbit IgG conjugated to Alexa546 in order to estimate thenumber of non-internalized parasites. Cells were stained with
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DAPI and the number of Alexa-stained parasites was subtractedfrom the number of DAPI-stained parasites. In the experiment inthe right panel, infections were performed as previously described.The lower graphs represent the percentage of infected cells.B. T. cruzi invasion facilitated by TNF in HEK293T cells is inhib-ited in the presence of increasing amounts of IkBa-DN. Similarprocedures for infections were carried out as described for thegraphs at the left in (A).C. Expression of IkBa-DN blocks NF-kB activation induced byTNF. HEK293T cells were co-transfected with NF-kB reporter(pGL3-E-Sel), pRL-TK (for normalization) and the plasmids asindicated in the presence of polyethylenimine. After 24 h posttransfection, cells were treated with TNF for 6 h, cell extractsobtained and the luciferase activity detected by the luminometer(Lumicount Packard BioSciences).Fig. S5. Effects of diffused soluble on Trypanosoma cruzi infec-tion. THP-1 cells were infected for 2 h, and the parasites-containing medium was harvested, centrifuged to remove theparasites, and the supernatants added back to THP-1 cells. After
6 h, the inserts were discarded, and the HEK293T (A) or LLC-MK2 (B) cells in lower chambers infected with T. cruzi for 2 h,fixed and counted. Medium of cells in the lower compartmentswas changed to 10% FBS RPMI 30 min before infection ofTHP-1. The volumes in the upper and lower chambers were200 ml and 500 ml respectively.Fig. S6. NF-kB activation induced by the supernatants fromPMA-differentiated THP-1 cells infected with Trypanosoma cruzi.HEK293T cells were transfected with NF-kB luciferase reporterand pRL-TK plasmids. Twenty-four hours post transfection, cellswere left untreated (medium only) or treated for 6 h with TNF(20 ng ml-1) or with the supernatants taken from uninfected andinfected PMA-treated THP-1 cells. Cell extracts were obtained todetermine luciferase activity in a luminometer.
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