the two main components of the host immune response to fungi , namely , resistance ( the ability to limit fungal burden ) and tolerance ( the ability to limit host damage caused by immune response or other mechanisms ) highlight the bipolar nature of the inflammatory process in fungal infections . both components must be considered when developing any therapeutic or prophylactic antifungal procedure . dendritic cells ( dcs ) are primarily responsible for antigen recognition , decoding this information , and then stimulating various t cell pathways using specific cytokine signals . the t cell subsets in turn secrete cytokines that mediate protective or detrimental / pathogenic effects on phagocytes and the inflammatory process . the primary protective response against fungal disease is the cell - mediated th1 response ( calich and kashino , 1998 ; netea et al . th1 lymphocytes produce ifn- , which stimulates the antifungal activity of pmn and macrophages . otherwise , some cytokines such as il-4 and il-13 provide signals that favor a th2-mediated immune response by lymphocytes . by diminishing the th1 cell response and promoting antibody production and t regulatory cells , it favors fungal infections , fungus - associated allergic responses , and disease relapse ( benard et 
 al . , 1997 ; therefore , th2 immunity is associated with severe and disseminated forms of fungal infections . this pattern is well established and reported in cryptococcosis ( mller et al . , 2007 ) , paracoccidioidomycosis ( pcm ; calich and kashino , 1998 ; ruas et al . , 2009 ) , and candidiasis ( netea et al . , 2004 ; haraguchi et al . , 2010 ) . in this review , we discuss the role of a plant lectin named artinm in a murine model of paracoccidioides brasiliensis infection , highlighting its immunomodulatory properties and the importance of the modulation of a cell - mediated immune response in the resistance to the fungus . we discuss the aspects that make this lectin an excellent candidate for further studies as a potential therapeutic for severe cases of pcm in human patients or for development as a prophylactic for individuals at risk for severe disease . the most common human systemic mycosis in latin america is pcm , which is caused by the dimorphic fungus p. brasiliensis . infection occurs by inhalation of fungal spores or particles , which transform into the pathogenic yeast form after reaching the pulmonary alveolar epithelium ( restrepo - moreno , 1993 ) . yeast can either be eliminated by immune - competent cells or disseminate to other tissues through lymphatic and hematogenous routes , resulting in a wide spectrum of clinical manifestations , which vary from asymptomatic , benign and localized to severe and disseminated forms ( borges - walmsley et al . , 2002 ) . clinical and experimental evidences indicate that , similar to other systemic mycosis , th1 immunity exerts a singular role in the asymptomatic form of pcm , while a th2 pattern is associated with progression to the severe disease form ( cano et al . , 1998 ; , 2000 ; benard et al . , 2001 ; oliveira et al . , 2002 ; peraoli et al . , 2003 ; ruas et al . , these immune patterns of resistance or susceptibility to fungal infections have been studied in murine models of infection that simulate human mycosis . resistant mice produce early and sustained levels of ifn- and il-2 , whereas susceptible mice produce low levels of ifn- , but significant levels of il-5 and il-10 ( calich and kashino , 1998 ; kashino et al . , 2000 ) . murine models have also showed that ifn- and tnf- activate macrophages to exert effects against p. brasiliensis ( brummer et al . . the essential role of these cytokines has been further demonstrated using mice that are genetically deficient in either the ifn- or the tnf- receptor ( cano et al . , 1998 ; souto et al . , 2000 ) . indeed , the presence of cytokines accounting for the activation of macrophages , which is necessary for fungal killing , has been consistently documented ( gonzales et al . , 2000 ; moreira et al . , 2008 , 2010 ) . the importance of innate immunity in the recognition of fungi has been extensively reviewed elsewhere ( roeder et al . , 2004 ; romani , 2004 ) , and it has been recently characterized for p.brasiliensis infection ( loures et al . , 2009 , 2010 , 2011 ) . the lack of the receptors toll - like receptor 2 ( tlr2 ) or tlr4 did not alter the survival rates of mice infected with p. brasiliensis . tlr2 knockout ( ko ) mice infected with p. brasiliensis presented with increased th17 immunity , associated with an impaired regulatory t cell expansion , which resulted in an uncontrolled inflammatory reaction . therefore , the authors concluded that the presence of tlr2 in p. brasiliensis infection is important to downregulate th17 immunity and lung pathological condition ( loures et al . , 2009 ) . tlr4-deficient mice presented lower fungal loads than the tlr4-normal mice , but these mice were unable to clear the infection completely owing to enhanced regulatory t cells and low inflammation ( loures et al . , clinically , the antifungal drugs most commonly used for pcm treatment include amphotericin b , sulfa derivatives , and azoles , but their toxicity can be a limiting factor in the treatment ( mendes et al . , 1994 ) . treatment regimens with these agents often require extended periods of maintenance therapy , which may range from months to years , and are usually associated with relapses ( shikanai - yasuda et al . , 2006 ) . moreover , even after prolonged administration of these drugs , there is no guarantee that the fungus will be completely eradicated . based on these data , there is a strong need for alternative clinical treatments to chemotherapy . researchers have focused their efforts in investigating fungal components able to promote cellular immune responses and host protection . immunization with heat - shock proteins ( hsps ) from p. brasiliensis has also been shown to provide some degree of protection against experimental disease ( soares et al . , 2008 ; ribeiro et al . , 2009 , 2010 ) . recently , it was shown that plasmid immunization with a peptide derived from the 43-kda glycoprotein antigen from the fungus , called p10 , was shown to be protective against pcm , inducing a reduction in fungal load in the lungs of experimentally infected mice ( rittner et al . , 2012 ) . although these studies focused on the use of fungal components to immunize mice against p. brasiliensis infection , it was shown that immunotherapy with a th1-inducing adjuvant that was independent of pb antigens has a beneficial effect against pcm ( oliveira et al . , 2008 ) . a single - dose administration of the adjuvant in infected mice was sufficient to restore their ability to mount an effective immune response to the fungus . these data support that stimulation of the host th1 immune response is a promising approach toward expanding available treatment options for systemic fungal diseases , including pcm . moreover , th1 stimulation may be achieved irrespective of whether p. brasiliensis antigens are used , providing new possibilities for the use of alternative drugs against the disease artinm ( also known as km or artocarpin ) ( pereira da silva et al . , 2008 ) is a lectin from artocarpus heterophyllus seeds that specifically recognizes the trisaccharide man13 [ man16 ] man core of n - glycans . artinm is a homotetramer formed by 13-kda subunits , each one corresponding to a -barrel , with a -prism folding , which includes a carbohydrate - recognition domain ( crd ) . artinm cdna has been cloned and heterologously expressed in saccharomyces cerevisiae and escherichia coli ( silva et al . , 2005 ) . native ( artinm ) and recombinant ( rartinm ) proteins share the same sugar recognition specificity and are equivalents in terms of the kinetics of binding affinity to a glycoligand ( pesquero et al . , 2010 ) . the artinm crd is preserved in rartinm , and the recombinant protein retains the same biological properties as the native form , with the advantage that it does not form oligomers . artinm possesses many relevant biological properties in cells of the immune system , which is reflected in the modulation of immunity during infection with intracellular pathogens . the lectin acts on mast cells and induces degranulation ( moreno et al . , 2003 ) . it also acts on neutrophils and induces haptotactic migration , as well as phenotypic and functional changes , which include intracellular tyrosine phosphorylation , shedding of l - selectin , release of inflammatory mediators , phagocytic and cell - killing activities , and increased expression of tlr2 ( ganiko et al . , 2005 ; toledo et al . , 2009 the pioneering observation on the artinm immunomodulatory activity was its ability to induce il-12 production in murine macrophages . this cytokine production then promoted a switch in the balb / c mouse immune response from th2- to th1-mediated immunity against leishmania major antigens . cytokine production was dependent on the crd of the lectin , since il-12 production was selectively inhibited by d - mannose , which is an artinm - specific ligand ( panunto - castelo et al . , 2001 ) . additional studies have shown that the benefits provided by the immunomodulation induced by artinm can be extended to several infections in which a th1-biased immunity is necessary for resistance , including the murine model of candida albicans infection . infected mice that were treated with artinm developed th1- and th17-mediated immune responses ; their macrophages and neutrophils exhibited increased phagocytical and candidacidal activities ( custodio et al . , 2011 ) . the augmented phagocytosis of yeast cells by macrophages from artinm - treated mice occurred via mannose and dectin-1 . this effect explains the faster clearance of c. albicans in the initial phase of infection in mice , which favors artinm - induced protection against disseminated candidiasis ( loyola et al . , 2012 ) . knowledge about the immunomodulatory effects of artinm on pcm is derived from studies that used an experimental model developed in balb / c mice that had been intravenously infected with p. brasiliensis . trials involving several protocols for the therapeutic and prophylactic administration of artinm showed that the most effective therapeutic protocol consisted of a single subcutaneous injection of artinm 10 days after infection , whereas the best prophylaxis was attained by the administration of two subcutaneous injections of artinm on day 10 and day 3 before infection . . , 2008 , 2010 ) of artinm on the severity of p. brasiliensis infection , which manifested on day 30 post - infection , included marked decrease in fungal burden and absence of granulomas in the lungs , which exhibited a well - preserved bronchoalveolar architecture . this pattern was in contrast to what was observed in the untreated mice , which had disseminated infection and multiple sites of focal and confluent epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and non - viable yeast cells ( figure 1 ) . the lesions were larger and still disseminated on day 60 after infection , while artinm - treated mice had no granulomas or yeast cells in the liver , spleen or lung tissue . lung histopathology of uninfected mice ( a ) , p.brasiliensis-infected mice ( b ) , and p. brasiliensis - infected mice treated with artinm ( c ) . brasiliensis - infected mice display extensive and confluent lesions in the lungs , with epithelioid granulomas surrounding a large number of yeast cells . infected mice treated with artinm present no granulomas , and lung architecture is similar to that of uninfected mice . the lung sections were stained with h&e ( modified from coltri et al . , 2010 ) lung homogenates from mice that were infected with p. brasiliensis and then subjected to prophylactic or therapeutic artinm regimen showed higher levels of the pro - inflammatory cytokines il-12 and tnf- , and no . artinm administration drove cytokine production from a th2 immune response pattern to a th1 immune response pattern . high concentrations of il-4 and low concentrations of ifn- were detected in untreated control mice , whereas in artinm - treated mice , lower il-4 and higher ifn- concentrations were stably produced during the course of the disease , as illustrated in figure 2 . it was clear that a drive toward th1-mediated immunity is stimulated in vivo by artinm . interestingly , stable il-10 production was also verified in the artinm - treated mice , indicating that the induced th1 response is balanced by the effects of this anti - inflammatory cytokine . studies involving il-12 ko mice have demonstrated the importance of il-12 for artinm - mediated beneficial effects on experimental pcm . when these mice were infected with p. brasiliensis , treatment with artinm exerted no protective effects against the infection . the parallel utilization of an artinm recombinant form to treat the p.brasiliensis-infected mice has provided evidence that the administration of artinm or its recombinant form ( rartinm ) exerts an equally protective effect against p. brasiliensis infection ( coltri et al . , 2008 , 2010 ) . mice were infected with p. brasiliensis yeast cells , and then treated or not with artinm . on day 30 after infection , the mouse lung tissue was analyzed for il-4 , ifn- and no concentrations . artinm treatment was associated with lower il-4 and higher ifn- and no pulmonary levels , which reflected in lower fungal load the role of the 70-kda heterodimeric cytokine il-12 in the activation of type 1 immune response is largely recognized . its bioactive il-12p70 form is composed of two disulfide - linked subunits : a 40-kda heavy chain of ( p40 ) and a 35-kda light chain ( p35 ) . macrophages and dcs are the major cell types producing this cytokine , which is released as the biologically inactive peptide il-12p40 as well as the biologically active il-12p70 . il-12 acts on t lymphocytes and natural killer ( nk ) cells , and induces ifn- production . this hallmark th1 cytokine is responsible for t cell proliferation and enhancement of macrophage cytotoxic activity ( kobayashi et al . , 1989 ; wolf et al . , 1991 ) il-12 production by phagocytes is generally initiated by the interaction of cell - surface tlrs with pathogen - associated molecular patterns ( pamps ) . tlrs constitute a protein family of cellular receptors that mediate recognition of microbial pathogens and subsequent inflammatory response in vertebrates . these receptors confer pamp recognition and their signaling triggers synthesis followed by release of pro - inflammatory cytokines , and induces expression of co - stimulatory molecules for promoting activation of adaptive immunity during antigen presentation ( janeway and medzhitov , 2002 ) . upon recognition of respective pamps , tlrs recruit a specific set of adaptor molecules that harbor tir domains , such as myd88 and trif , and initiate downstream signaling events that lead to the activation of the transcription factor and its translocation into the nucleus to induce the expression of pro - inflammatory genes , including the il-12-coding gene . inflammatory cytokines are released from the cell into the extracellular matrix , and they promote the recruitment of neutrophils to the site of infection , activation of macrophages , and induction of ifn--stimulated genes , resulting in direct killing of invading pathogens . moreover , activation of tlr signaling leads to the maturation of dcs , which contributes to the induction of adaptive immunity ( west et al . , 2006 ) . the involvement of myd88-mediated signaling in the enhanced secretion of artinm - induced il-12 was proved by the fact that macrophages from myd88ko mice did not respond to in vitro stimulation with the lectin . to investigate whether tlr2 was involved in artinm - induced il-12 production , an in vitro assay was performed to quantify the il-12 concentrations released by artinm - stimulated macrophages from the tlr2ko or tlr4-deficient mice . macrophages from tlr2ko mice , distinctly from those from tlr4-deficient or wt mice , were unable to produce il-12 in response to artinm stimulus . moreover , il-12 production by artinm - stimulated macrophages was inhibited by d - mannose , which indicates that its production is dependent on the lectin crd . these results demonstrate that tlr2 plays a critical role in artinm - mediated production of il-12 ( coltri et al . , 2008 ) . potential n - linked glycosylation sites have been revealed by amino acid sequencing analysis of all known tlrs . several lines of evidence indicate that oligosaccharides attached to tlrs play important roles in the recognition of pamps , and in the formation of a functional receptor complex on the cell surface ( ohnishi et al . , 2001 , 2003 ; da silva correia and ulevitch , 2002 ; weber et al . , 2004 ) . concerning human tlr2 , its ectodomain contains n - glycans linked to the residues asn114 , asn199 , asn414 , and asn442 ; among them , the glycan linked to asn442 was reported to contribute to efficient secretion of the tlr2 ectodomain ( weber et al . , 2004 ) and cellular recognition of pamps ( kataoka et al . , 2006 ) . direct interaction of artinm with tlr2 was further demonstrated by a gene reporter assay involving tlr2-transfected cells ( unpublished data ) . nicholas gay s laboratory , university of cambridge , uk ) are being used to identify the glycan(s ) targeted by artinm . artinm administration interferes with the outcome of p. brasiliensis infection by modulating host immunity according to the following events : ( a ) recognition of tlr2 glycans by the lectin , ( b ) induction of il-12 production , ( c ) generation of th1-balanced immunity , and ( d ) protection against p. brasiliensis , mainly manifested by the occurrence of milder lung lesions and low fungal burden . detection of il-10 production in artinm - treated animals reveals that the induced th1-prone immune response is regulated in a way that prevents systemic immune pathology , as indicated by the absence of exacerbated inflammatory lesions in artinm - treated animals ( coltri et al . , 2008 , 2010 ) . as part of a study on the pleiotropic activities of artinm , we are trying to identify the il-10-producing cells . observations concerning the immunomodulatory effects of artinm support the use of this protein , in its native or recombinant form , as an immunomodulatory agent that can stimulate balanced th1 immunity , which is required to protect the host against fungal infection . otherwise , complete characterization of the n - glycan(s ) recognized by artinm in tlr2 molecules may provide an adequate target for the development of novel antifungal therapies . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
the thermally dimorphic fungus paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis ( pcm ) , the most frequent systemic mycosis that affects the rural populations in latin america . despite significant developments in antifungal chemotherapy , its efficacy remains limited since drug therapy is prolonged and associated with toxic side effects and relapses . in response to these challenges , it is now recognized that several aspects of antifungal immunity can be modulated to better deal with fungal infections . a common idea for halting fungal infections 
 has been the need to activate a cell - based , pro - inflammatory th1 immune response to improve the fungal elimination . 
 artinm , a d - mannose binding lectin from artocarpus heterophyllus , has the property of modulating immunity against several intracellular pathogens . here 
 , we review the immunomodulatory activity of artinm during experimental pcm in mice . 
 both prophylactic and therapeutic protocols of artinm administration promotes a th1 immune response balanced by il-10 , which outstandingly reduces the fungal load in organs of the treated mice while maintaining a controlled inflammation at the site of infection . 
 a carbohydrate recognition - based interaction of artinm with toll - like receptor 2 ( tlr2 ) accounts for initiating the immunomodulatory effect of the lectin . 
 the precise identification of the tlr2 n - glycan(s ) targeted by artinm may support novel basis for the development of antifungal therapy .