Importância da molécula CD28 (molécula co-estimulatória de linfócitos T) na Paracoccidioidomicose...

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9048

DOI: 10.1007/s11046-007-9048-1

Re: Innate immunity to Paracoccidioides brasiliensis infection

By: Vera Lúcia Garcia Calich · Tânia da Costa · Maíra Felonato · Celina Arruda · Simone Bernardino · Flávio Loures · Laura Ribeiro · Rita de Cássia Valente-Ferreira · Adriana Pina

To be published in: Mycopathologia

Vera Lúcia Garcia Calich,

50

Vera Lúcia Garcia Calich Departamento de Imunologia

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Mycopathologia DOI: 10.1007/s11046-007-9048-1

Innate immunity to Paracoccidioides brasiliensis infection

Vera Lúcia Garcia Calich · Tânia da Costa · Maíra Felonato · Celina Arruda · Simone Bernardino · Flávio Loures · Laura Ribeiro · Rita de Cássia Valente-Ferreira · Adriana Pina

Springer Science+Business Media B.V.

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Vera Lúcia Garcia Calich,

Mycopathologia

9048

Innate immunity to Paracoccidioides brasiliensis infection

Vera Lúcia Garcia Calich

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UNCORRECTED

PROOF

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PROOF

1 2

3

Innate immunity to

Paracoccidioides brasiliensis

infection

4 Vera Lu´cia Garcia Calich Taˆnia Alves da Costa

5 Maı´ra Felonato Celina Arruda Simone Bernardino

6 Fla´vio Vieira Loures Laura Raquel Rios Ribeiro

7 Rita de Ca´ssia Valente-Ferreira Adriana Pina

8 Received: 8 May 2007 / Accepted: 7 August 2007

9 ÓSpringer Science+Business Media B.V. 2007

10 Abstract Innate immunity is based in pre-existing 11 elements of the immune system that directly interact 12 with all types of microbes leading to their destruction 13 or growth inhibition. Several elements of this early 14 defense mechanism act in concert to control initial 15 pathogen growth and have profound effect on the 16 adaptative immune response that further develops. 17 Although most studies in paracoccidioidomycosis 18 have been dedicated to understand cellular and 19 humoral immune responses, innate immunity remains 20 poorly deﬁned. Hence, the main purpose of this 21 review is to present and discuss some mechanisms of 22 innate immunity developed by resistant and suscep-23 tible mice toParacoccidioides brasiliensisinfection, 24 trying to understand how this initial host-pathogen 25 interface interferes with the protective or deleterious 26 adaptative immune response that will dictate disease 27 outcome. An analysis of some mechanisms and 28 mediators of innate immunity such as the activation 29 of complement proteins, the microbicidal activity of 30 natural killer cells and phagocytes, the production of 31 inﬂammatory eicosanoids, cytokines, and chemokines

32 among others, is presented trying to show the

33 important role played by innate immunity in the host

34 response toP. brasiliensisinfection.

35

Keywords CytokinesDendritic cells

36 Innate immunityMacrophages

37 NK and PMN Cells

38 Paracoccidioides brasiliensis infection

39

Introduction

40 Innate immunity has been deﬁned as the ﬁrst phase of

41 immune response and is based in pre-existing

42 elements of the immune system that directly interact

43 with all types of microbes leading to their destruction

44 or growth inhibition. Innate immunity, which is not

45 clonally speciﬁc for a particular pathogen and does

46 not generate speciﬁc memory, is mediated by

phys-47 ical barriers, chemical elements, and cell components

48 of the immune system. The adaptative immunity,

49 involving more slowly developing, long-lived, and

50 highly antigen-speciﬁc responses are mediated by

51 cell-mediated immunity and antibody production.

52 Several elements of innate immunity act in concert to

53 control initial pathogen growth and have profound

54 effect on the adaptative immune response that further

55 develops. Furthermore, most effector mechanisms of

56 innate immunity are identical to those of adaptative

57 immunity that are activated at later phases of immune

58 response.

A1 V. L. G. Calich (&₎

T. A. da Costa

A2 M. FelonatoC. ArrudaS. Bernardino

A3 F. V. LouresL. R. R. Ribeiro

A4 R. de Ca´ssia Valente-FerreiraA. Pina

A5 Departamento de Imunologia, Instituto de Cieˆncias A6 Biome´dicas da Universidade de Sa˜o Paulo (USP), A7 Av. Prof. Lineu Prestes 1730, CEP 05508-900, Sao Paulo, A8 SP, Brazil

A9 e-mail: vlcalich@icb.usp.br

123

Journal :Medium 11046 Dispatch : 11-8-2007 Pages : 14

Article No. : 9048 h LE h TYPESET

MS Code : MYCO629 h4CP h4DISK

Mycopathologia

DOI 10.1007/s11046-007-9048-1

(15)

UNCORRECTED

PROOF

UNCORRECTED

PROOF

59 Several mechanisms of innate immunity such as 60 the activation of complement proteins, the microbi-61 cidal activity of natural killer (NK) cells and 62 phagocytes, the production of inflammatory cytokines 63 and chemokines among others, have been shown to 64 play an important role in the early host response to 65 pathogens [1]. Besides their intrinsic complexities, 66 innate immunity mechanisms present important 67 peculiarities which depend on the site they take 68 place [2]. The innate immune response against 69 Paracoccidioides brasiliensis, Coccidioides immitis, 70 Blastomyces dermatitides, and Histoplasma capsula-71 tum, primary fungal pathogens which infect hosts 72 through the respiratory tract, occurs in the lungs. The 73 lung response to infection is initiated by the secretion 74 of several antimicrobial proteins by the pulmonary 75 epithelium and the phagocytic activity of resident 76 alveolar macrophages. The cell-wall-degrading 77 enzyme lyzozyme, the iron-chelating protein lacto-78 ferrin and the membrane-permeabilizing members of 79 the defensin, cathelicidin, and pentraxin families are 80 the initial antimicrobial proteins secreted in the 81 alveolar lining layer of the pulmonary epithelium. 82 Innate immunity recognition of microorganism is 83 mediated by germ-line encoded receptors (‘‘pattern 84 recognition receptors, PRR’’) which interact with 85 conserved pathogen structures, the so-called ‘‘patho-86 gen associated molecular patterns’’ or ‘‘PAMP’’ 87 [3–5]. The initial macrophage-pathogen interaction 88 results in internalization by the activated cell which 89 can kill the organism through the action of reactive 90 oxygen species and lytic enzymes or extracellular 91 microbial containment. In addition, the secretion of 92 chemokines and cytokines orchestrates the expression 93 of cell adhesion and chemotactic molecules which 94 further control the influx and activation of inflamma-95 tory cells to the site of infection [1–5].

96 Although most studies in human PCM have been 97 dedicated to understand cellular and humoral immune 98 responses, innate immunity remains poorly defined. 99 This is easily understood when one reminds that PCM 100 infection and disease in human beings are recognized 101 at a later and undefined period after initial infection, 102 making difficult to evoke the early events which 103 resulted in controlled infection or overt disease. In 104 this aspect, experimental models are powerful tools to 105 study the initial events that govern hosts-P.

brasili-108 108 108 ensis interactions. Thus, the main purpose of this

109 review is to present and discuss some mechanisms of

110 innate immunity toP. brasiliensisinfection, trying to

111 understand how this initial host-pathogen interface

112 interferes with the protective or deleterious

adapta-113 tive immune response that will dictate disease

114 outcome. This review does not intend to be a

115 comprehensive revision of the PCM literature that

116 has been reported elsewhere [6–10], but to present a

117 personal view, mainly based in the murine model of

118 genetic resistance and susceptibility toP. brasiliensis,

119 of how innate immunity can inﬂuence PCM severity

120 and the adaptative immune response to this pathogen.

121

The isogenic murine model of resistance/

122

susceptibility of paracoccidioidomycosis mimics

123

the human disease

124 Our laboratory established a genetically controlled

125 murine model of paracoccidioidomycosis (PCM),

126 which allowed us to investigate several parameters of

127 host-parasite interactions. Most of these studies were

128 recently reviewed [10–13] and clearly showed the

129 diverging immune responses mounted by genetically

130 susceptible (B10.A) and resistant (A/Sn or A/J) mice to

131 P. brasiliensisinfection. One important characteristic of

132 our model is the similarity with the human disease,

133 B10.A mice mimicking the progressive, severe forms of

134 the disease and A/Sn mice showing similar features of

135 the regressive or localized forms of the infection

136 (Fig.1). As in the human disease, our experimental

137 model demonstrated that resistance is associated with

138 immune responses that favor cellular immunity and

139 activation of phagocytes, whereas susceptibility is

140 associated with impairment of cellular immune

141 responses and preferential activation of B cells [10–12].

142 After an intra-tracheal (i.t.) infection, the

suscep-143 tibility and resistance patterns observed following i.p.

144 infection were maintained, as reﬂected by the high

145 mortality rates of B10.A mice and the regressive

146 disease developed by the A/J strain. The susceptible

147 mice were not able to restrain the infection to the

148 lungs and, 2 months after infection, dissemination to

149 liver and spleen was seen, characterizing a chronic,

150 progressive and disseminated form of the disease; in

151 the resistant mice, on the other hand, no organ

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152 dissemination occurred and a pulmonary-restricted 153 chronic disease was observed. Unexpectedly, early in 154 the i.t. infection (2nd and 4th weeks), A/J mice 155 presented higher pulmonary CFU counts than B10.A 156 mice suggesting that susceptible mice developed a 157 more efﬁcient innate immunity than resistant animals. 158 The adaptative immunity of resistant mice appears to 159 compensate their ineffective innate immunity 160 (Fig.2). Accordingly, from week 8 of infection 161 onward, positive DTH responses, marked control of 162 fungal burdens, secretion of pulmonary type 1 and 163 type 2 cytokines and preferential production of IgG2a 164 antibodies were seen, leading to a regressive pattern 165 of disease. On the contrary, the anergy of DTH 166 reactions, the preferential synthesis of IgG1 and 167 IgG2b antibodies and the progressively increased 168 fungal burdens of susceptible mice resulted in severe 169 disseminated disease leading to decreased survival 170 times [14,15].

171

Innate immunity

172 Genetic control of susceptibility

173 Clinical studies suggested that susceptibility to P.

174 brasiliensisis dependent on several factors, including

175 genetic background, and host’s hormonal function

176 [4–7]. A fungal receptor for estrogen was identiﬁed

177 and appears to block the conversion of conidia or

178 mycelium to the infecting yeast form [16]. This

179 ﬁnding was further explored in an animal model of

180 infection demonstrating the enhanced resistance of

181 female animals [17,18] and may explain the unusual

182 susceptibility of male individuals of endemic areas

183 [6,8,19].

184 Genetic studies performed by our group have

185 shown the existence of an autosomal dominant gene

186 (Pbr gene), which control P. brasiliensis resistance

187 [20] and appears to be similar to theNrampgene, that

188 control resistance to Mycobacterium sp, Leishmania

189 sp, andSalmonella spinfection [21]. Further studies

190 with Nramp1 congenic macrophages (B10R and

191 B10S expressing or not the Nramp1 protein,

respec-192 tively) showed that B10R macrophages, in

193 comparison with B10S cells, expressed higher levels

194 of mannose receptors, presented higher phagocytic

195 ability and increased inhibitory effect on the conidia

196 to yeast conversion [22].

197 Complement system and chemokines

198 P. brasiliensiscells are able to activate the alternative

199 pathway of complement and yeast cells-adherent C3b Fig. 1 Main features of the isogenic murine model of

resistance and susceptibility to Paracoccidioides brasiliensis infection

Fig. 2 At the onset of infection, susceptible (B10.A) mice show a better control of pulmonary fungal loads than resistant (A/J) mice. A/J and B10.A mice were i.t. infected with one millionP. brasiliensisyeast cells. The graph on the left shows

the recovery of viable fungal cell from lungs (colony forming unit counts, CFU), and the graph on the right the delayed hypersensitivity (DTH) responses measured during 16 weeks of infection

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200 molecules can contribute to fungi phagocytosis by 201 macrophages [23–25]. On the other hand, chemokin-202 es which play a major role in regulating the migration 203 of speciﬁc leukocytes subsets in both the acute and 204 chronic inﬂammatory processes [26], were shown to 205 control mononuclear cell recruitment to the lungs of 206 P. brasiliensis-infected C57BL/6 mice [27]. Unpub-207 lished results from our laboratory suggest that 208 increased and sustained expression of IP-10, RAN-209 TES and the chemokine receptor CXCR3 is 210 associated with the resistant behavior of A/Sn mice 211 (C. Arruda and V. L. G. Calich, unpublished obser-212 vations). This is in accordance with the sustained T 213 cell response mounted by resistant mice at the 214 acquired phase of the immune response [14,15].

215 Lipid mediators (Eicosanoids) andP. brasiliensis 216 lipids

217 During an inﬂammatory reaction, the enzymatic 218 oxidation of aracdonic acid (AA) by cyclooxigenase 219 produces prostaglandins, thromboxanes, and prosta-220 cyclins, whereas the 5-LO is an enzyme that catalyzes 221 the oxidation of AA for the synthesis of leukotrienes 222 (LT). The importance of LT as cellular activators and 223 chemotactic factors for neutrophils and eosinophils is 224 very well established, however, little is known about 225 the function of these lipid mediators in the host 226 defense against infectious agents [28,29].

227 As the role of LT in pulmonary PCM was never 228 investigated, we asked whether they would have a 229 regulatory function in the severity of PCM of 230 resistant (A/J) and susceptible (B10.A) mice and in 231 the fungicidal and secretory ability of their macro-232 phages. Our results showed that in vivo and in vitro 233 P. brasiliensisinfection induces LT synthesis. Com-234 pared with A/J mice, levels of pulmonary LT was 235 higher in B10.A animals and increases in the course 236 of infection. To evaluate the importance of LT in 237 PCM, an inhibitor of LT synthesis (MK-0591) and an 238 antagonist of LT receptor (montelukast) were studied 239 afterP. brasiliensisinfection. In vitro, LT inhibitors 240 signiﬁcantly reduced the recovery of P. brasiliensis 241 yeasts from normal and IFN-cprimed macrophages. 242 At 48 h of in vivo infection, montelukast treatment of 243 B10.A mice induced diminished fungal loads, 244 impaired inﬂux of PMN leukocytes, and increased 245 number of monocytes in the lungs ofP. brasiliensis

-246 infected mice. Furthermore, in susceptible mice

247 montelukast treatment led to increased levels of

248 pulmonary IL-10 concomitant with diminished

249 amounts of IL-12, TNF-a, and GM-CSF. In contrast,

250 at the chronic phase of the disease, LT inhibition did

251 not alter the fungal loads of B10.A and A/J mice. In

252 conclusion, our results showed for the ﬁrst time that

253 LT are important mediators of the acute inﬂammatory

254 reaction induced byP. brasiliensisinfection affecting

255 fungal recovery, cellular inﬂux, and cytokines

syn-256 thesis by susceptible mice [30, L. R. R. Ribeiro and

257 V. L. G. Calich, unpublished observations].

Impor-258 tantly, our ﬁndings with LT inhibition appear to

259 demonstrate that the activation of innate immunity

260 can result in increased ingestion and survival of

261 P. brasiliensis yeasts which can evolve to a more

262 severe disease.

263 Several lines of evidence suggest that

prostag-264 landins production has a deleterious role for

265 P. brasiliensis-infected hosts. In murine PCM, at

266 early steps of infection, secretion of PGE2was shown

267 to have an immunossupressive activity by inhibiting

268 IL-12 production and up-regulating IL-4 and IL-10

269 synthesis [31]. In addition, studies with normal and

270 IFN-c activated human macrophages demonstrated

271 that prostaglandins secretion inhibited their

fungi-272 cidal ability which depends on the levels of hydrogen

273 peroxide produced [32, 33]. Interestingly, recent

274 studies showed that virulent and low virulence strains

275 of P. brasiliensis are able to synthesize

prostaglan-276 dins by a cyclooxigenase-dependent pathway and that

277 these lipid mediators are required for P. brasiliensis

278 survival [33].

279 Besides the importance of hosts lipid mediators

280 such as the eicosanoids in innate immunity, other

281 lipid components of pathogen membranes or walls

282 have also been shown to play a role in the

host-283 parasite interaction [34,35]. Studies on the inﬂuence

284 ofP. brasiliensislipid fractions in the fungicidal and

285 secretory activities of B10.A macrophages were

286 developed in our laboratories. Although allP.

brasil-287 iensis lipid fractions are potent inducers of NO

288 synthesis, they can inhibit or enhance the fungicidal

289 ability of macrophages. The previous in vitro

treat-290 ment of macrophages by F1 (phospholipids + neutral

291 lipids) and F2 (short chain glycolipids) fractions

292 resulted in increased phagocytic activity of cells, and

293 recovery of higher numbers of viable yeasts from

294 infected macrophages, despite the presence of high

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295 NO levels. On the other hand, fractions 3a (glyco-296 sylphosphatidylinositol-anchored glycoproteins) and 297 F3b (long chain glycolipids) caused an opposite 298 behavior; they inhibited the phagocytic ability of 299 macrophages leading to decreased recovery of viable 300 yeasts. As a whole, secretion of IL-10, IL-12, MCP-1, 301 and GM-CSF induced byP. brasiliensisinfection was 302 inhibited by the previous pre-incubation with all lipid 303 fractions [36, F. V. Loures, I. Almeida and V. L. G. 304 Calich, unpublished data]. The different behavior 305 of the studied lipid fractions could be attributed to 306 the different physicochemical structures of these 307 components which would interact with macropha-308 ges membranes through different PRR, and the 309 subsequent balance of pro- and anti-inﬂammatory 310 cytokines and chemokines secreted. Indeed, a further 311 characterization of these lipid fractions will permit 312 us to better understand the innate host response to 313 P. brasiliensisinfection.

314 Toll Like and other macrophage receptors

315 In mammalian cells, the Toll-Like Receptors (TLR) 316 are transmembrane proteins, which interact with 317 invariant molecular structures from pathogens 318 (PAMP) and are involved in the activation of the 319 innate immune system. Several typical pathogen 320 components such as lipopolysaccharides, flagelin, 321 peptidoglycans, DNA motifs, among others, are 322 recognized by different TLR [37–42]. Early TLR 323 activation results in the production of several inflam-324 matory mediators and the final balance among pro-325 and anti-inflammatory components will regulate the 326 type of adaptative immune response [37–42]. The 327 TLR 4 is the key receptor that recognizes bacterial 328 lipopolysaccharides, whereas TLR 2 is involved in 329 the interaction with bacterial peptidoglycans and 330 lipoproteins [38, 39]. TLR have been implicated in 331 the resistance of mammalian hosts to several micro-332 organisms [42–44] including fungal pathogens such 333 as Candida albicans, Aspergillus fumigatus, and 334 Cryptococcus neoformans[5]. Interestingly, our pre-335 vious studies with P. brasiliensis infection showed 336 that the LPS-resistant, TLR 4 deficient, C3H/HeJ 337 strain is more resistant to i.p. infection than the 338 congenic LPS-susceptible, TLR 4 normal, C3HeB/ 339 FeJ strain [45]. Our recent in vitro studies with TLR 340 4 normal (C3HeB/FeJ) and deficient (C3H/HeJ)

341 macrophages have demonstrated that this receptor

342 interacts with P. brasiliensis cells resulting in

mac-343 rophage activation as shown by increased synthesis of

344 nitric oxide, IL-12, MCP-1, and enhanced phagocytic

345 activity; this activation, however, was associated with

346 augmented recovery of viable yeast cells from

347 infected macrophages. In the acute phase of

pulmo-348 nary infection, the presence of TLR 4 induces a more

349 severe disease, with increased numbers of viable

350 yeasts in the lungs associated with elevated synthesis

351 of NO and IL-12. Moreover, even in the chronic

352 phase, higher fungal burdens were seen in the lungs

353 of TLR-4-normal mice, associated with increased

354 levels of pulmonary IL-12 and serum antibodies (IgM

355 and IgG). Thus, the early macrophage activation

356 induced by TLR 4 usage is not able to control

357 P. brasiliensis infection [36, F. V. Loures and V. L.

358 G. Calich, unpublished observations]. As LPS

unre-359 sponsiveness of C3H/HeJ mice was linked to a point

360 mutation in theTLR 4gene, it is tempting to suggest

361 that recognition of P. brasiliensis components (LPS

362 like?) by TLR 4 has a not yet described contribution

363 to the control of PCM. We have also preliminary

364 in vitro and in vivo studies with TLR 2 knockout

365 mice in a C57Bl/6 background demonstrating a more

366 severe infection in TLR-normal hosts or cells.

367 Altogether, our ﬁndings with TLR-deﬁcient animals

368 are unusual since PRR are most commonly used by

369 phagocytes to recognize molecular patterns of

patho-370 gens, and their interaction usually results in cell

371 activation, enhanced secretion of pro-inﬂammatory

372 cytokines and chemokines, and increased

microbi-373 cidal activity. In our model, the increase production

374 of nitric oxide and IL-12 by TLR-normal

macro-375 phages was not sufﬁcient to control fungal growth

376 and subsequent disease severity (F. V. Loures and V.

377 L. G. Calich, unpublished results). These receptors

378 appear to be used byP. brasiliensisyeast cells to gain

379 access into macrophages and to escape from other

380 fungicidal or fungistatic mechanisms of innate

381 immunity.

382 Some reports have described the importance of

383 mannose receptors in P. brasiliensis ingestion by

384 phagocytic cells. Phagocytosis of yeasts by adherent

385 peritoneal macrophages of susceptible and resistant

386 mice was inhibited by gp-43, a P. brasiliensis

387 glycoprotein most recognized by patients antibodies,

388 as well as by Saccharomyces cerevisiae derived

389 a-mannan. Gp 43 was also shown to inhibit NO

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390 production and killing ability of cytokine-stimulated 391 macrophages [46]. Immature dendritic cells of resis-392 tant mice appear to use mannose receptors to 393 internalize P. brasiliensis yeasts [47]. In addition, 394 comparative studies with Nramp1 gene congenic 395 macrophages (B10R and B10S) have demonstrated 396 that B10R cells were better inhibitors of conidia to 397 yeast conversion and expressed more mannose 398 receptors than B10S macrophages, whereas both cell 399 lines expressed similar levels of complement receptor 400 3 (C3R) [20]. As described below, CR3 was also 401 shown to play an important function inP. brasiliensis 402 adherence and ingestion by phagocytic cells [23,24].

403 Polymorphonuclear leukocytes and NK cells

404 Differently from macrophages, murine PMN leuko-405 cytes are able to killP. brasiliensisyeasts through the 406 oxidative metabolism [48,49]. In an air-pouch model 407 of infection and compared with PMN leukocytes 408 from susceptible mice, cells from A/J mice presented 409 superior fungicidal ability associated with their 410 enhanced oxidative burst [50]. The antifungal activity 411 of murine and human PMN leukocytes was shown to 412 be enhanced by IFN-c, GM-CSF, or IL-1b, but not by 413 TNF-aor IL-8 [51]. In contrast, TNF-awas shown to 414 better enhance P. brasiliensis killing by human 415 macrophages than IFN-c[52].

416 Comparing the early influx of inflammatory cells 417 to the lungs of susceptible and resistant mice, Cano 418 [53] demonstrated an equivalent mononuclear cell 419 influx, but a more prominent migration of neutrophil 420 and eosinophil PMN cells into the lung of suscep-421 tible mice. This early PMN influx was also seen 422 early in the infection of BALB/c mice [54]. 423 Furthermore, only in susceptible mice this early 424 (24 h after infection) PMN influx affects disease 425 outcome and acquired immunity further established. 426 Interestingly, the more severe disease of PMN-427 depleted susceptible mice was associated with the 428 increased presence of pulmonary IL-12 and IFN-c 429 suggesting that the production of pro-inflammatory 430 mediators not always leads to immunoprotection. 431 Differently from primary infection, neutrophil deple-432 tion did not alter immunoprotection in secondary 433 paracoccidioidomycosis. As a whole, our data 434 showed that the genetic pattern of hosts exerts an 435 important influence on the immunoprotective and

436 immunoregulatory functions of neutrophils which

437 appear to be essential in situations devoid of

cell-438 mediated immunity [55].

439 The role of NK cell has not been well studied in

440 P. brasiliensis infection, but the few available

441 investigations in this area suggest that this

lympho-442 cyte subpopulation has a complex function in PCM

443 that varies according to the type of host or site where

444 these cells were obtained. In the peripheral blood of

445 PCM patients, NK cells were found in elevated

446 number but they displayed low cytotoxic activity

447 [56]. In vitro studies showed a direct inhibitory effect

448 of murine NK cells onP. brasiliensisgrowth [57] and

449 in a hamster model of infection, NK cells were shown

450 to be activated at the ﬁrst weeks of infection followed

451 by an impairment of its activity associated with

452 depressed cell-mediated immunity [58].

453 Our ﬁndings of illness exacerbation after in vivo

454 depletion of IL-12 or IFN-c(in euthymic and

athy-455 mic BALB/c mice [59, 60] suggested that NK cells

456 would have a protective role in pulmonary PCM.

457 In vivo depletion of NK cells by anti-Asialo GM1

458 polyclonal antibody resulted in a more severe

459 disease of both mouse strains, but the depletion

460 effect was more pronounced in the NK-depleted

461 athymic than euthymic mice. Anti-NK cell treatment

462 led to increased antibody production by the former

463 strain but did not modify the humoral immunity of

464 euthymic animals, indicating that the isotype class

465 switch in T cell deﬁcient mice is inﬂuenced by NK

466 cells cytokines. In addition, NK cells were shown to

467 control PMN leukocytes inﬂux to the lungs of

468 infected mice. Hence, NK cells seem to have a

469 protective effect in pulmonary PCM and their

470 function appears to be more prominent in T-cell

471 deﬁcient than in T-cell sufﬁcient mice [61, R. C.

472 Valente-Ferreira and VLG Calich, unpublished

473 data].

474 Macrophages and nitric oxide

475 The crucial role of the mononuclear phagocytic

476 system in the resistance to P. brasiliensis infection

477 was demonstrated by the fact that reticuloendothelial

478 system blockade, induced by colloidal carbon

inoc-479 ulation previous to P. brasiliensis infection (i.p.

480 route), increased the severity of the disease in both

481 resistant and susceptible animals [62].

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482 The infection by P. brasiliensisoccurs by inhala-483 tion of airborne propagules of the mycelial phase of 484 the fungus, which reach the lungs, eventually evade 485 the host defenses and disseminate via the bloodstream 486 and/or lymphatics to virtually all parts of the body [4, 487 8,10,19]. Alveolar macrophages (AM) are believed 488 to be important in the initial containment of the 489 microorganisms through nonspeciﬁc or innate 490 immune mechanisms. AM or dendritic cells (DC) 491 also phagocytose particles and microbial organisms 492 and carry them via lymphatics to regional hilar lymph 493 nodes, where speciﬁc immune responses are believed 494 to be generated.

495 P. brasiliensis proliferates ex vivo in a variety of 496 mouse macrophages, including resident peritoneal 497 alveolar and peripheral blood derived monocytes 498 until the cells are lysed and killed by a yet unknown 499 mechanism. However, the immunological activation 500 of these cells efﬁciently inhibits fungal growth [48]. 501 When alveolar macrophages were analyzed after 502 pulmonary infection, absence of hydrogen peroxide 503 production was observed with cells obtained from 504 susceptible mice, whereas macrophages from resis-505 tant mice produced increasing levels of this 506 metabolite in the course of disease [14]. These 507 different activities parallel the DTH anergy and the 508 evident DTH reactivity developed by susceptible and 509 resistant mice, respectively.

510 Brummer et al. [48, 63] have demonstrated that 511 activation of mouse peritoneal macrophages by IFN-c 512 enhances the fungicidal activity of these cells but 513 fungal killing is independent of the respiratory burst. 514 Further investigations showed the fundamental role 515 of nitric oxide in the fungicidal ability of activated 516 macrophages, which appear to use an iron-restriction 517 mechanism to inhibit the transformation of ingested 518 conidia to yeast cells [64,65]. We have conﬁrmed the 519 fundamental role of NO in the murine PCM [64]. In 520 the course of infection, peritoneal macrophages from 521 resistant mice secrete low levels of NO associated 522 with high amounts of TNF-a; the opposite was seen 523 with glass adherent cells from susceptible mice. 524 Interestingly, in vitro inhibition of NO production by 525 aminoguanidine treatment of B10.A macrophages led 526 to increased production of TNF-a indicating the 527 inhibitory role of NO on cytokine secretion. More 528 importantly, the disease of i.p. infected C57BL/6 529 mice genetically deﬁcient for inducible nitric oxide-530 synthase (iNOS KO) and in resistant and susceptible

531 mice in vivo treated with aminoguanidine and

inca-532 pable of secreting NO, is more severe [66].

533 The dual role of NO in murine PCM was further

534 conﬁrmed in the pulmonary model of infection.

535 Compared with wild type mice, a lower fungal load

536 was observed at week 2, although at week 10,

537 increased number of fungi was detected in the lungs

538 of mice genetically deﬁcient of inducible

NO-539 synthase (iNOS KO). The better control of fungal

540 loads by iNOS KO mice at week 2 of infection

541 appeared to be TNF-a mediated, since its in vivo

542 neutralization abolished this difference [67, S.

Ber-543 nardino and V. L. G. Calich, unpublished results). In

544 agreement, Gonzales et al. [68] showed that

TNF-a-545 activated peritoneal macrophages, although not

pro-546 ducing NO, were able to inhibit the transition of P.

547 brasiliensis conidia to yeast cells. Interestingly, our

548 studies also demonstrated that iNOS KO mice,

549 despite the more intense fungal infection by week

550 10 of infection, developed better organized

granulo-551 mas. Thus, the increased secretion of TNF-a, the

552 increased inﬂux of activated T cells to the lungs, and

553 the better organized lesions appear to compensate the

554 genetic deﬁciency of NO. This was further conﬁrmed

555 by the equivalent survival times showed by iNOS KO

556 and WT mice, despite the higher fungal loads in the

557 former strain [67, S. Bernardino and V. L. G. Calich,

558 unpublished data).

559 Recent studies were also performed aimed to

560 understand the interaction between alveolar

macro-561 phages from resistant and susceptible mice and

562 P. brasiliensis. Normal alveolar macrophages of

563 B10.A mice, in vitro infected with P. brasiliensis

564 yeasts, can be activated by small doses of

exoge-565 nously added IFN-c, secrete high levels of IL-12,

566 nitric oxide and display a very efﬁcient fungal killing

567 activity. In contrast, macrophages from A/J mice

568 were poorly activated by low doses of IFN-c, secrete

569 low amounts of IL-12, NO and present a poor

570 fungicidal ability concomitant with the production of

571 high levels of active TGF-b. The fungicidal ability of

572 B10.A macrophages was modulated by

aminoguani-573 dine, whereas TGF-bwas the main negative regulator

574 of A/J macrophages. Thus, alveolar macrophages of

575 susceptible mice seem to be more efﬁcient than those

576 of resistant mice and interaction of P. brasiliensis

577 with these cells probably occurs through different

578 macrophage receptors [69 A. Pina and V. L. G.

579 Calich, unpublished observations]. These ﬁndings

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580 appear to explain the apparently discrepant result we 581 had observed when the pulmonary model of infection 582 was first described: at the beginning of infection 583 higher number of viable yeast cells were recovered 584 from lungs of resistant mice as compared with 585 susceptible ones [14]. Furthermore, IFN-c, the most 586 efficient macrophage activator was found in higher 587 levels in the lung homogenates of susceptible mice 588 [59]. Thus, the innate immunity appears to be much 589 more efficient in the susceptible strain than in the 590 resistant one. This hyperactivity is concomitant with 591 high levels of NO production that is able to restrain 592 fungal growth but also interferes with acquired 593 immune responses leading to a subsequent immuno-594 suppression of T-cell mediated immunity [66, A. Pina 595 and V. L. G. Calich, unpublished data].

596 Dendritic cells and other APCs

597 T cells have clonal receptors (TCR) educated to see 598 antigen epitopes presented by major histocompatibil-599 ity complex (MHC) molecules of antigen presenting 600 cells (APC). Several cell types can exert the APC 601 function such as macrophages, B cells and endothe-602 lial cells, but the dendritic cells (DC) are considered 603 the ‘‘professional APC’’ due to their special ability to 604 activate T cells. DC are derived from hematopoietic 605 stem cells in the bone marrow and form a network of 606 a heterogeneous cell populations. Many DC reside 607 and traffic through nonlymphoid peripheral tissues, 608 continuously surveying the environment for invading 609 microorganisms [70]. During infection, DCs in the 610 periphery are activated by interaction with microor-611 ganisms or inflammatory mediators to increase their 612 expression of MHC and co-stimulatory molecules 613 such as CD80, CD86, and CD40. They also modify 614 their expression of chemokines receptors and adhe-615 sion molecules, causing migration from the periphery 616 to the T cell zone of draining lymph nodes. Activated 617 DC then display pathogen encoded antigens to naı¨ve 618 antigen-specific T cells which initiate primary T cell 619 responses [71,72]. In the course of maturation, DC 620 are subject to profound changes. The endocytic 621 capacity is downmodulated, while there is a marked 622 up-regulation of MHC class II expression, from an 623 already high constitutive level [72].

624 As with other infectious pathologies, some studies 625 on the importance of different antigen presenting

626 cells (DC, macrophages, and B cells) in the resistance

627 to P. brasiliensis infection were reported. It was

628 shown that gp43, the immunodominant antigen for

629 humoral immunity in PCM [73], was mainly

pre-630 sented by macrophages and stimulated a preferential

631 Th1 cytokine production in resistant mice. In

con-632 trast, in susceptible mice gp43 was predominantly

633 presented by B lymphocytes and led to preferential

634 secretion of Th2 cytokines. In addition, no

differ-635 ences in T cell reactivity of resistant and susceptible

636 mice were detected. [74]. Another report from the

637 same group showed that the s.c. injection of mature

638 DC, macrophages and B cells primed naı¨ve

suscep-639 tible and resistant mice and induced T cell

640 proliferation. In this study, however, macrophages

641 and B cells from both mouse strains displayed

642 equivalent stimulatory activity inducing a preferential

643 secretion of IL-10 and IL-4; DC from resistant

644 animals, however, when compared with B10.A DC,

645 stimulated a higher production of IFN-c,(equivalent

646 levels of IL-12 and higher expression of MHC class II

647 and CD80 molecules. B10.A macrophages were also

648 shown to secrete high levels of IL-6 while IL-12 was

649 secreted in similar levels by DC of both strains.

650 Hence, it was suggested that DC of resistant mice

651 preferentially drive Th1 development while B cells

652 and macrophages from both mouse strains appeared

653 to induce the differentiation of a Th0 or Th2

654 phenotype [75]. Further studies with resistant [76]

655 and susceptible mice derived DC [77] demonstrated

656 an equivalent behavior of gp-43 stimulated DC. Thus,

657 gp43 treatment as well as P. brasiliensis infection

658 down-regulated MHC class II, CD80, CD86, CD54,

659 and CD40 expression as well as IL-12 and

TNF-660 a(secretion by LPS-treated DC. So, no major

differ-661 ences were reported in the activities of DC obtained

662 from resistant and susceptible mice, unless they were

663 previously activated by LPS. The i.v. infusion of DC

664 previously treated with gp-43 plus LPS, but not with

665 each of these components individually, increased

666 pulmonary CFU counts and altered granulomas

667 morphology ofP. brasiliensis-infected mice [77].

668 We have also preliminary results comparing the

669 behavior of DC from resistant and susceptible mice.

670 Bone marrow derived DC were obtained and

acti-671 vated with LPS, P. brasiliensis yeast cells or a

672 soluble whole yeast cells antigen. DC from both

673 mouse strains exhibited MHC class II and

co-674 stimulatory molecules (CD80, CD86, CD11c,

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675 CD40) when activated by LPS or fungal yeast cells 676 although A/J DC presented higher CD11c and 677 CD86 expression. Compared with A/J cells, B10.A 678 DC secreted higher levels of IL-12, IL-10, and NO, 679 whereas the former are more able to produce active 680 TGF-b. DC of susceptible mice induced a high 681 proliferative activity of A/J T cells but not of 682 B10.A lymphocytes while A/J DC stimulated T cell 683 proliferation of both mouse strains. Thus, T cell 684 anergy was only detected when B10.A DC were 685 co-cultivated with homologous lymphocytes indi-686 cating that B10.A DC does not lack the ability to 687 properly present P. brasiliensis antigens and that 688 B10.A lymphocytes are appropriately activated 689 when P. brasiliensis antigens were presented by 690 A/J DCs (A. Pina and V. L. G. Calich, unpublished 691 results).

692 Cytokines

693 As a more detailed review on the role of cytokines 694 in PCM was reported elsewhere [10, 13], only a 695 brief analysis of those studies will be presented 696 here. IFN-cis the most important protective cytokine 697 to susceptible, intermediate, and resistant mice to 698 P. brasiliensis infection [59, 78]. TNF-a and IL-12 699 are also very important protective cytokines [60,78, 700 79]. IL-4 has a dual role (protective or disease 701 promoting) in pulmonary PCM depending on the 702 genetic pattern of the host [80,81]. Despite the less 703 severe disease induced by administration of rIL-12 704 [79], the strong inﬂammatory reaction in the lungs 705 demonstrated the harmful effect of this cytokine. IL-706 10 appears to be one important macrophage-deacti-707 vating cytokine in pulmonary PCM, and its genetic 708 absence appears to result in the aseptic cure of 709 infected mice (Fig.3) (T. A. Costa and V. L. G. 710 Calich, unpublished results). Altogether, studies with 711 cytokine-deﬁcient mice showed that the Th1/Th2 712 paradigm can be applied to explain fungal growth (or 713 dissemination) in liver and spleen: IL-4 and IL-10 are 714 disease-promoting cytokines while IL-12 and IFN-c 715 are protective ones. However, the control of fungal 716 growth in the lungs is more complex and both, Th1 717 (e.g., IL-12) and Th2 cytokines (e.g., IL-4) can have 718 antagonistic effects. IL-10 is a disease-promoting 719 cytokine and appears to have a more prominent role 720 in the control of pulmonary PCM than IL-4.

721 Summarizing, our studies on innate immunity to

722 P. brasiliensisinfection suggest that a highly efﬁcient

723 innate immunity can lead to severe

paracoccidioid-724 omycosis. The following ﬁndings appear to support

725 such inference: at the onset of infection, susceptible

726 mice display a better control of lung fungal loads;

IL-727 4 protects susceptible mice from severe infection;

728 exogenous IL-12 leads to increased lung pathology;

729 TLR usage leads to increased macrophage activation

730 associated with increased fungal loads; susceptible

731 mice secrete higher levels of LT and its inhibition

732 results in milder pathology; PMN depletion causes

733 more severe PCM associated with increased secretion

734 of pro-inﬂammatory cytokines; early NO secretion

735 can induce more severe infection. As a whole, it

736 appears that ‘‘the more reactive the host innate

737 immunity the more severe is the initialP. brasiliensis

738 infection.’’

739

The influence of innate immunity in the resistance

740

to P. brasiliensis_infection

741 Protective immunity in paracoccidioidomycosis

742 (PCM) is believed to be mainly mediated by cellular

743 immunity [82]. In the human disease the Th1/Th2

744 dichotomy of CD4+ T cells appears to partially

745 explain the behavior of PCM patients and healthy

746 infected individuals. So, the most evident Th1

747 immunity is observed when lymphocytes from

748 healthy infected subjects or cured patients are in vitro

749 activated by gp 43 and a clear production of IL-2 and

750 IFN-cis concomitant with a vigorous

lymphoprolif-751 erative response [83, 84]. The acute form of the

752 disease appears to be the Th2 pole of reactivity,

753 where IL-4, IL-5, and IL-10 are produced and

754 associated with low T cell proliferation which,

755 however, can be reverted by in vitro treatment with

756 rIL-12 and anti-IL-10 antibodies [85]. The severe

757 form of the chronic disease also appears to present a

758 Th2 pattern of reactivity. Most individuals of the

759 chronic form of PCM, however, do not display

760 polarized Th1/Th2 immune responses and their

761 hyporesponsiveness appears to be not linked to

762 imbalanced cytokine synthesis and may be due to

763 other immunoregulatory mechanisms such as T cell

764 anergy, T cell deletion by apoptosis or suppressive

765 activity of natural regulatory T cells [10, 86–88].

766 Indeed, a recent paper showed a direct correlation

Mycopathologia