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Nitric oxide synthase activity has limited influence on the control of Coccidioides infection in mice
Microbial Pathogenesis 51 (2011) 161e168
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Nitric oxide synthase activity has limited influence on the control of Coccidioides infection in miceq Angel Gonzalez a, b, c, d, *, Chiung-Yu Hung a, Garry T. Cole a a
Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas at San Antonio, San Antonio, TX 78249, USA Medical and Experimental Mycology Group, Corporación para Investigaciones Biologicas (CIB), Carrera 72 A, No. 78, B 141, Medellín, Colombia c Escuela de Microbiología, Universidad de Antioquia, Medellín, Colombia d Escuela de la Salud, Universidad Pontificia Bolivariana, Medellín, Colombia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2011 Received in revised form 28 February 2011 Accepted 2 March 2011 Available online 14 April 2011
The functions of inducible nitric oxide synthase (iNOS) activity in protection against microbial insults are still controversial. In this study, we explored the role of iNOS in protection against Coccidioides infection in mice. We observed that wild type (WT) and iNOS/ mice showed similar percent survival and fungal burden in their lungs at days 7 and 11 after intranasal challenge with Coccidioides. Vaccinated WT and iNOS/ mice revealed comparable fungal burden in their lungs and spleen at 7 and 11 days postchallenge. However, at 11 days the non-vaccinated, iNOS-deficient mice had significantly higher fungal burden in their spleen compared to WT mice. Additionally, higher numbers of lung-infiltrated neutrophils, macrophages and dendritic cells were observed in WT mice at day 11 postchallenge compared to iNOS/ mice. Moreover, no difference in numbers of T, B, NK or regulatory T cells, or concentrations of selected cytokines and chemokines were detected in lungs of both mouse strains at 7 and 11 days postchallenge. Although iNOS-derived NO production appears to influence the inflammatory response and dissemination of the fungal pathogen, our results suggest that iNOS activity does not play a significant role in the control of coccidioidal infection in mice and that other, still undefined mechanisms of host protection are involved. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Nitric oxide Coccidioides Inflammation Fungal burden
1. Introduction Nitric oxide (NO) has been demonstrated to play a diversity of roles during infection of the mammalian host. NO production catalyzed by the action of inducible nitric oxide synthase (iNOS) represents one of the major defense mechanisms of host phagocytic cells against certain pathogens [1]. Numerous reports have demonstrated that iNOS-derived NO helps to counteract excessive immune reactions to infection and functions both as an intra- and intercellular signaling molecule that contributes significantly to modulation of the immune response [2e4]. However, in some cases iNOS activity has been shown to exacerbate the course of disease due to such detrimental effects as NO-mediated cytotoxicity and
q This work was conducted at the University of Texas at San Antonio in the Department of Biology while the senior author (AG) was supported as a postdoctoral fellow by a National Institutes of Health grant (AI071118) awarded to GTC. * Corresponding author: Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas (CIB), Carrera 72 A, No. 78, B 141, Medellín, Colombia. Tel.: þ57 4 441 08 55; fax: þ57 4 441 55 14. E-mail address: [email protected] (A. Gonzalez). 0882-4010/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2011.03.013
host tissue damage, inhibition of T cell proliferation, and other immune regulatory properties which are detrimental to the host protective response to infection [5,6]. NO synthesis which is dependent on iNOS activity occurs in macrophages, neutrophils, dendritic and NK cells upon induction by exposure to interferongamma (IFN-g), tumor necrosis factor-alpha (TNF-a), certain bacterial products such as lipopolysaccharide (LPS), and other endogenous and exogenous stimuli [2,7]. NO has been implicated in the control of numerous infectious diseases caused by a variety of prokaryotic and eukaryotic pathogens, including Mycobacterium tuberculosis [8], Histoplasma capsulatum [9], Cryptococcus neoformans [10], Leishmania major [11], and Schistosoma mansoni [12]. On the other hand, NO has also been shown to be either detrimental or non-essential to host protection, or appears to play a dual role in infections caused by Paracoccidioides brasiliensis [13,14], Sporotrix schenckii [15], Candida albicans [16] and Trypanosoma brucei [6]. Coccidioidomycosis is an endemic human respiratory disease which occurs in southwestern regions of the United States, and is caused by the dimorphic fungus Coccidioides spp. This soilborne, fungal pathogen is also found in parts of Mexico, Central and South America [17]. Inhalation of its infectious, air-dispersed spores
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(arthroconidia) by the host is followed by the initiation of an elaborate parasitic cell cycle that is unique amongst the medically important fungi [18]. The spores differentiate into large, multinucleate spherules that give rise to a multitude of endospores which are responsible for dissemination of the pathogen from original sites of infection, typically in the lungs [19]. It is estimated 100,000 new infections of coccidioidomycosis occur in the United States each year, but only about 40% of individuals exposed to the fungus develop clinical symptoms. The latter range from an influenza-like illness to severe pneumonia or, rarely, to extrapulmonary disseminated disease and meningitis [20]. Risk factors known to contribute to symptomatic coccidioidal infection include pregnancy (third trimester), immunosuppression, advanced age (>65 years), and ethnicity [21]. Several studies have demonstrated that cellular immunity, mainly CD4þ and CD8þ T cell responses, is essential for protection against coccidioidomycosis [20,22e24]. Moreover, it has been shown that T helper (Th) 17 cells are essential for stimulation of protective immunity against pulmonary infections of Coccidioides [25]. In addition, interleukin (IL)-10 has been reported to play a key role in murine susceptibility to coccidioidomycosis; thus, transgenic mice producing high levels of human IL-10 are more susceptible to coccidioidal infection and expressed lower levels of interferon-gamma (IFN-g), IL-12p40 and iNOS mRNA in their lungs compared to control mice, implicating NO synthesis as a mechanism of resistance against coccidioidomycosis [26]. However, the role of iNOS-derived NO produced by Coccidioides is still not clear. We previously demonstrated that Coccidioides spp. has the ability to suppress both NO production and iNOS expression by murine primary macrophages in vitro that were previously activated by exposure to IFN-g þ LPS [27]. On this basis, we initially considered that the suppressive product (s) secreted by the fungal pathogen represented an important virulence factor. However, macrophages obtained from iNOS/ mice were as competent in their uptake and killing of parasitic cells of Coccidioides as macrophages isolated from wild type (WT) mice, suggesting that the fungicidal mechanism of the host phagocytes in vitro is not dependent on NO production. In the present study, we have compared the survival and immune response of WT and iNOS/ mice following intranasal challenge with Coccidioides in an attempt to further explore whether iNOS-derived NO is essential for protection against Coccidioides infection, and to determine if NO plays a role in modulation of cellular immunity during the course of pulmonary coccidioidomycosis. 2. Results 2.1. iNOS/ mice showed similar mortality but increased dissemination of Coccidioides compared to WT mice C57BL/6 WT and iNOS gene-deficient mice (iNOS/) were challenged with a potentially lethal inoculum of C. posadasii spores by the intranasal (i.n.) route. To assess the influence of the absence of iNOS-derived NO on disease outcome, we first examined the mortality of each group of infected mice recorded daily for a 30-day period (Fig. 1). The mean survival time for both strains was 12 days, and no statistically significant difference was observed between the mortality data for the two groups of infected mice (P ¼ 0.88). To further evaluate these results, comparative histopathology of the infected lungs of the two strains were conducted, and determination of the fungal burden in both the lungs and spleen of the two mouse strains was determined at 7 and 11 days postchallenge. A similar pattern of inflammatory response was observed in the infected, H&E-stained lungs of the WT and iNOS/ mice at 7 days (c.f. Fig. 2A and B, respectively). The inflammatory lesions appeared
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60 40 20 0 0
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Days postchallenge Fig. 1. Survival curves for C57BL/6 wild type mice (WT; n ¼ 10) and iNOS-deficient mice (iNOS/; n ¼ 10) following i.n. challenge with 80 viable spores of Coccidioides posadasii. These data are representative of two independent experiments.
to have replaced almost all the normal lung parenchyma tissue and were composed of poorly differentiated granulomas of various sizes surrounded by large numbers of neutrophils and mononuclear cells. The abscesses contained comparable numbers of parasitic cells of Coccidioides (spherules and endospores). At 11 days postchallenge, the infected lungs of the WT mice presented with incipient granulomas, characterized by a thick layer of mononuclear cells which contained the fungal cells (Fig. 2C). On the other hand, the iNOSdeficient mice at day 11 postchallenge showed a more diffuse distribution of inflammatory cells and absence of well-defined granulomatous structures (Fig 2D). Near equal numbers of colonyforming units (CFU) of Coccidioides were observed in lung homogenates of the iNOS/ and WT mice at days 7 and 11 after challenge (open bars in Fig. 3A and B,, respectively). However, significantly higher fungal burden was detected in the spleen of the iNOSdeficient mice at 11 days postchallenge when compared with the infected WT mice (P ¼ 0.006) (Fig. 3C). To determine whether the absence of iNOS-derived NO would influence acquired immunity to Coccidioides infection, both mouse strains were immunized with a genetically engineered, live attenuated vaccine strain of Coccidioides which had previously been reported to provide protection (100% survival) to C57BL/6 mice against coccidioidomycosis [28]. Vaccinated mice, irrespective of the strain, revealed comparable fungal burden in their lungs at 7 and 11 days postchallenge (Fig. 3A and B, respectively), and showed no statistically significant difference in dissemination of the pathogen to the spleen of the two groups of mice (Fig. 3C). 2.2. Elevated iNOS expression correlated with reduced dissemination of Coccidioides in WT mice We evaluated iNOS expression in the WT C57BL/6 mice at 0, 7 and 11 days postchallenge using QRT-PCR (Fig. 4). We detected a significantly higher number of iNOS transcripts on day 11 after challenge than at day 7 (P < 0.001). Elevated expression of iNOS in these mice correlates with the significantly lower CFU in their spleen compared with the spleen homogenates of iNOS/ mice at 11 days postchallenge (c.f., Fig. 3C). 2.3. WT and iNOS-deficient mice showed comparable numbers of infiltrated pulmonary leukocytes at 7 and 11 days postchallenge Leukocyte recruitment into the lungs of Coccidioides-infected WT and iNOS/ mice was examined by cytofluorometry. Not surprising, a significant increase in the total number of lung-
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Fig. 2. Histopathology of infected lungs of WT and iNOS/ mice at 7 days (A, B) and 11 days postchallenge (C, D), respectively. Arrow in (A) indicates dense population of neutrophils which surrounds the coccidioidal lesion. Arrow in (B) indicates the concentrated array of parasitic cells (spherules and endospores) of C. posadasii present in the pulmonary abscess. Bar in panel (B) represents 0.5 mm, and also applies to panels (A, C and D).
infiltrated leukocytes (LIL) was observed in both mouse strains between 7 and 11 days postchallenge (Fig. 5). However, no statistically significant difference existed between the total LIL detected in the infected lungs of the two strains at either 7 or 11 days after intranasal challenge. 2.4. Loss of iNOS expression correlated with changes in numbers of certain innate cell types which had infiltrated infected lung tissue We also employed cytofluorometry to detect numbers of selected subpopulations of leukocytes that had infiltrated the infected lungs of WT and iNOS/ mice at 7 and 11 days postchallenge (Fig. 6AeD). Significant changes were observed in the numbers of neutrophils (PMN) and tissue macrophages (T. Mac) between the two strains of mice during this period of infection. However, the higher numbers of these two innate cell types in the iNOS-deficient mice compared to WT mice at 7 days was reversed at 11 days after challenge. At day 7 the numbers of PMNs and tissue macrophages in iNOS/ mice compared to WT mice were 0.519 0.14 versus 0.253 0.0 (P < 0.01) and 1.0 0.26 versus 0.672 0.14 (P < 0.05), respectively (Fig. 6A and B; all values 106). At 11 days postchallenge, the numbers of these same two cell types were significantly higher (P < 0.001) in the WT mice (8.0 1.08 and 4.84 1.25, respectively) compared to the iNOS-deficient mice (5.44 0.475 and 2.97 0.37, respectively). The number of lunginfiltrated dendritic cells (DC) in the WT mice at 11 days also showed a significant increase compared to the iNOS/ mice (0.58 0.30 versus 0.23 0.14, respectively; P < 0.01), but no statistically significant difference was observed at 7 days postchallenge. The numbers of selected subpopulations of infiltrated lymphocytes (CD4þ, CD8þ, total B cells, regulatory T cells) and NK cells in WT and iNOS/ mice showed no significant difference at 7 or 11 days postchallenge (Fig. 6C and D).
2.5. Loss of iNOS expression had no effect on the concentration of selected cytokines/chemokines secreted in Coccidioides-infected lung tissue It is well appreciated that exogenous and endogenous stimulation of the host cytokine network (including secretion of proinflammatory cytokines/chemokines and Th1-, Th2-, and Th-17 type cytokines) impacts the efficiency and effectiveness of the inflammatory response to infection and determines the composition of activated immune cells which are directed to the sites of the microbial insult [25]. Inducible nitric oxide synthase activity has been reported to have a versatile role in the induction of cytokine secretion and immune cell differentiation and proliferation [2,3]. Although our results reported in Fig. 6 suggested that iNOS activity has limited influence on the infiltration of leukocytes into Coccidioides-infected lungs of mice, it is possible that this enzyme is involved in the induction of chemical signals that influence other pathways of immune response. On this basis, we set out to compare the concentrations of selected cytokines and chemokines present in lung homogenates of infected WT and iNOS-deficient mice which have been reported to be associated with the immune response of C57BL/6 mice to Coccidioides infection (unpublished data). The results presented in Table 1 reveal that no statistically significant differences were detected between concentrations of the selected array of cytokines and chemokines tested in lung homgenates of the Coccidioides-infected WT and iNOS/ strains of mice at 7 and 11 days postchallenge. 3. Discussion The mammalian host is equipped with a complexity of mechanisms that induce protective immune responses to microbial infections, while counter-balanced by feedback signals that can restrict the magnitude and duration of these responses in order to
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Fig. 3. Comparison of fungal burden in the lungs (A, B) and spleen (C) of wild type (WT) and iNOS/ mice at 7 and 11 days postchallenge. These studies were conducted using nonvaccinated and vaccinated mice. The median CFUs (log10) are indicated by horizontal lines inside each box. A statistically significant difference was revealed between the fungal burden in the spleen of non-vaccinated WT and iNOS/ mice at 11 days postchallenge (C) (*P ¼ 0.006). The results are representative of two independent experiments.
minimize tissue damage caused by persistent inflammation and/or autoimmune reactions [3]. Nitric oxide (NO) generated by activity of the inducible isoform of nitric oxide synthase (iNOS) is such a signaling molecule involved in immunomodulatory events during the course of infectious diseases, while also serving as a potent growth inhibitor of certain microbial pathogens [2,3,29]. Studies using NO donors, NO inhibitors or iNOS-deficient mice have provided evidence for the participation of iNOS-derived NO in
a broad spectrum of immune regulatory processes including apoptosis, production of cytokines and other soluble mediators, expression of co-stimulatory and adhesion molecules, and synthesis and deposition of extracellular matrix components [2,30,31]. Results of these investigations have also revealed that production of iNOS-derived NO can be detrimental in some disease processes [15], of little consequence in controlling pathogen proliferation [27], or beneficial in others [14]. For example, NO produced by macrophages has been reported to have both host
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Fig. 4. Relative levels of transcription of the iNOS gene were measured by QRT-PCR. Lungs were excised from separate groups of WT mice (n ¼ 4) prior to infection (0 days) or at 7 and 11 days after i.n. challenge and used as sources of total RNA. Relative amounts of detected iNOS gene transcript were normalized with that of the constitutive GADPH gene. Untreated (naïve) mice were included for determination of baseline amounts of transcripts. The expression level of the iNOS gene was significantly elevated at 11 days postchallenge compared to the other two selected time points (P < 0.001). The bars represent means SEM. The results are representative of two independent experiments.
0 7 Days postchallenge
11 Days postchallenge
Fig. 5. Total number of lung-infiltrated leukocytes (LIL) in non-vaccinated, Coccidioides-infected wild type (WT) and iNOS/ mice (n ¼ 5). Cytometric analyses of fluorescent anti-CD45 mAb-labeled cells was conducted to identify leukocytes within the pulmonary cell suspension. The total leukocyte numbers for both mouse strains were significantly higher at 11 days compared to 7 days postchallenge (P < 0.001), but were not significantly different from each other at either time point. The bars represent means SEM.
A WT iNOS -/-
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Fig. 6. Loss of iNOS activity results in both increased and decreased numbers of selected phagocytic cells, but no significant changes in lymphocytic cell populations. Numbers of lung-infiltrated leukocyte subpopulations (identified in the Section 4) were identified by cytometric analyses of total pulmonary cells isolated from the lungs of groups of Coccidioides-infected WT and iNOS/ mice (n ¼ 5) sacrificed at 7 and 11 days postchallenge. Asterisks indicate statistically significant differences between numbers of neutrophils (PMN), tissue macrophages (T. Mac) and dendritic cells (DC) of the two mouse strains, as described in Section 2. The bars represent the means SEM.
tissue damaging and anti-inflammatory effects as a result of its modulation of host secretion and function of certain cytokines, chemokines and growth factors in response to a microbial insult [2]. Previous studies of Candida infections have concluded that macrophages obtained from WT and iNOS/ mice showed no difference in their ability to kill the pathogen in vitro [16]. Regulation of iNOS activity occurs both at the transcriptional and post-
transcriptional levels. Arginase can also have a regulatory effect on iNOS activity by competing for the same substrate (L-arginine) and converting it to ornithine and urea. We have previously shown that expression of the arginase 1 gene of Coccidioides-infected C57BL/6 mice is highly up-regulated at 6 days postchallenge compared to non-infected control mice (190.2-fold increase) [32]. This observation, combined with our results reported here that no
Table 1 Concentrations of pulmonary proinflammatory cytokines and chemokines in Coccidioides-infected lungs of wild type (WT) and INOS/ mice at 7 and 11 days postchallenge (PC). Cytokines and chemokines (pg/ml)
7 Days PC
11 Days PC
WT
iNOS/
WT
iNOS/
1105.6 171.1 5622.9 1141.1 1067.2 422.4 1326.9 240.8 1755.9 187.6 13766.1 3035.2 1342.4 101.9
1269.2 55.3 7084.9 515.1 1188.4 529.6 1836.7 593.0 1528.8 294.6 14123.3 1443.4 1315.3 143.3
4343.5 976.6 11830.9 2154.2 2366.6 482.9 3116.4 1329.6 3867.3 1225.4 45326.5 9346.0 1283.1 235.6
4266.5 1562.6 12536.7 2976.9 3213.1 440.1 2402.6 929.7 4037.7 1742.8 51172.6 10807.8 1319.1 479.9
Th1 cytokines IFN-g IL-2 IL-3 IL-12p70
1212.6 80.1 774.7 108.3 332.7 68.5 562.2 68.9
1134.5 189.1 865.3 76.9 329.9 72.3 601.7 106.0
1426.8 133.9 551.4 40.4 250.4 17.8 1611.6 305.2
1634.2 431.1 572.6 95.5 259.4 55.4 1761.4 342.5
Th2 cytokines IL-4 IL-5 IL-10 IL-13
1477.5 465.1 1436.6 565.5 303.1 51.5 3977.3 260.5
1818.2 904.5 1318.0 412.0 313.7 78.2 4001.3 282.9
852.9 368.8 1807.3 615.5 785.2 344.5 4058.9 876.9
937.0 134.2 1833.8 273.6 936.4 462.1 3794.1 169.1
Chemokines CXCL1/KC CCL2/MCP-1 CCL3/MIP-1a CCL4/MIP-1b CCL5/RANTES CCL11/Eotaxin
5950.9 284.7 4678.7 810.9 6270.9 889.7 1198.6 258.3 1226.5 102.6 5271.7 417.9
5399.3 519.6 5168.3 866.9 7215.8 793.2 1369.1 197.8 1236.2 387.5 5128.4 748.5
8542.4 1856.7 5420.5 813.6 16042.4 3444.8 2299.7 463.8 527.1 93.0 5704.8 543.8
7771.7 2070.5 6137.9 458.8 17986.2 2972.3 2783.1 478.2 493.2 165.9 5643.7 990.2
Proinflammatory cytokines IL-1a IL-1b IL-6 IL-17 TNF-a G-CSF GM-CSF
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significant difference was evident in survival, lung histopathology or fungal burden between Coccidioides-infected WT and iNOS/ mice at 7 days postchallenge, argue that iNOS-derived NO has little to no effect on immune regulation or microbicidal activity during this early stage of pulmonary coccidioidomycosis. We had previously demonstrated that Coccidioides is able to suppress NO production by IFN-g þ LPS-activated mouse primary macrophages in vitro as a result of secretion of an undefined, soluble inhibitory factor(s). Intuitively, this suggests that inhibition of NO production is important for persistence of the pathogen within the hostile environment of the host. On the other hand, results of our comparative in vitro studies of Coccidioides killing efficiency by macrophages derived from WT and iNOS/ mouse strains indicated that NO is not an essential contributor to the fungicidal activity of these phagocytic cells [27]. We speculate, therefore, that production of iNOS-derived NO by alveolar and tissue macrophages, neutrophils, and dendritic cells in vivo does not have a direct effect on limiting growth of Coccidiodes in infected lungs of C57BL/6 mice, but instead contributes to modulation of the immune response to infection. The fact that we observed an approximate 6-fold increase in iNOS expression in the infected lungs of WT mice between 7 and 11 days postchallenge indicates that iNOS activity may still play a role in the course of progressive coccidiodal disease. This prompted us to compare the nature of the immune response between Coccidioides-infected WT and iNOS/ mice. Examinations of the lung histopathology at 7 days after challenge revealed that WT mice showed signs of containment of the pathogen, albeit within rudimentary granulomas, while the iNOS/ mice revealed diffuse pulmonary lesions with high concentrations of parasitic cells. In addition, the iNOS/ mice showed a slightly higher fungal burden in their spleen at 11 days postchallenge, a fact that would suggests the involvement of iNOS in the dissemination process observed with this fungal pathogen in mice lacking the corresponding enzyme. It is known that the genetic background of the host may influence the immune response against microbial pathogens, as has been previously described for DBA/2 mice that are resistant to Coccidioides infection and C57BL/6, C57BL/10 and BALB/c mice which are very susceptible [33]. Taking this into account, we used an animal model of pulmonary coccidioidomycosis based on susceptible wild type C57BL/6 mice and gender-matched mice with the same genetic background which lacked the iNOS gene. We designed an identical vaccination protocol which was applied to both mouse strains in an attempt to explore the role of inducible nitric oxide activity in experimental coccidioidomycosis. Vaccinated mice of both strains revealed comparable low CFU in their spleen and were apparently equally protected against dissemination of the fungal pathogen. In addition, total numbers of recruited leukocytes which had infiltrated the infected lungs of the nonvaccinated mice showed an approximate 4 to 5-fold increase between 7 and 11 days postchallenge. However, no difference was observed between the infected WT and iNOS/ mouse strains at these two stages in the course of the disease. These results suggest that iNOS is not needed for acquired immunity in C57BL/6 mice induced by vaccination against Coccidioides. On the other hand, when we assessed the numbers of selected lung-infiltrated leukocytes subpopulations by flow cytometry, a higher but transient number of neutrophils and tissue macrophages were detected in the Coccidioides-infected iNOS/ mice at day 7 postchallenge compared to the WT mice. The total numbers of these innate cells increased sharply at 11 days after challenge, but the relationship between the two strains of mice was reversed. The WT mice showed significantly higher numbers of infiltrated neutrophils, tissue macrophages and dendritic cells when compared with the iNOS-deficient mice, an observation that
correlates with the histopathologic findings, increased expression of iNOS in infected lung tissue and lower fungal burden in the spleen at 11 days in the same WT mouse strain. Although these preliminary results suggest that iNOS-derived NO may enhance recruitment of mononuclear defense cells to infection sites in the lungs during advanced stages of the disease, the data do not entirely support this conclusion. No difference between the two strains was recorded in the numbers of lymphocytes that had infiltrated the infected lung tissue. In addition, we did not observe any significant differences in the levels of selected proinflammatory cytokines or chemokines between the WT and iNOS/ mice at 7 or 11 days postchallenge. Similar results have been reported by Farah and coworkers [16] who observed that WT and iNOS/ mice infected with C. albicans showed equivalent levels of expression of inflammatory cytokines measured by QRT-PCR. In contrast, Livonesi et al., [14] found that iNOS/ mice infected with P. brasiliensis yeast cells showed higher levels of Th1 and Th2 cytokines than the WT strain, and proposed that elevated iNOS activity contributes significantly to both protection and regulation of the inflammatory response to this fungal pathogen. Jimenez and coworkers [26] proposed that a correlation exists between elevated IL-10 production, reduced expression of iNOS and increased susceptibility to coccidioidomycosis, and suggested a protective role for NO. However, in our previous study we observed that IFN-g þ LPSactivated murine primary macrophages from both WT and iNOSdeficient mice were able to produce comparable amounts of IL-10 in vitro [27], and in this study no differences in the pulmonary levels of IL-10 were observed. These reports illustrate the contradictory interpretations of the in vivo role of iNOS-derived NO. The contribution of iNOS activity in the control of coccidioidomycosis has not been previously investigated. The results of our comparative study of disease outcome and immune response of vaccinated and non-vaccinated WT and iNOS/ mice to pulmonary challenge with Coccidioides indicate that NO has limited influence on processes of defense against this fungal pathogen. We cannot rule out the possibility that Coccidioides utilizes energy to generate and secrete products that suppress NO production by macrophages [27] in order to modulate pathways of host immune response which aids in its survival in vivo. Additional studies are required to further explore this concept. 4. Materials and methods 4.1. Fungus C. posadasii (isolate C735) was used for all experimental procedures reported in this study. The saprobic phase of this fungus was grown on glucose-yeast-extract agar (GYE; 1% glucose, 0.5% yeastextract, 2% agar) at 30 C for 3e4 weeks to induce sporulation. Spores were harvested into a plastic centrifuge tube containing 10 ml sterile, endotoxin-free saline and glass beads (5 mm diam.). The tubes were shook by hand to disrupt the spore chains. The cell suspension was filtered through a layer of nylon fiber to remove hyphal fragments, washed twice and resuspended in endotoxinfree PBS. 4.2. Animals Breeding pairs of homozygous C57BL/6 iNOS-deficient mice (iNOS/; strain B6.129P2-Nos2tm1Lau/J) and wild type (WT) C57BL/ 6 mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and the National Cancer Institute/Charles River Laboratory (Wilmington, MA, USA), respectively. Mice were maintained for breeding in the Small Animal Laboratory at the University of Texas at San Antonio and handled according to guidelines approved by the
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University Institutional Animal Care and Use Committee. Infected animals were housed in a pathogen-free, Thoren caging system (Thoren, Hazelton, PA), which was accommodated in a Biological Safety Level (BSL3) laboratory. Coccidioides is designated as a “select agent” by the Centers for Disease Control and Prevention (Atlanta, GA, USA) and must be contained within a BSL3 facility. 4.3. Intranasal infection and mortality, histopathology and fungal burden analyses Mice of both strains (WT and iNOS/) were challenged by the natural, intranasal (i.n.) route with a potentially lethal inoculum of 60e80 viable spores of C. posadasii as previously reported [28]. Separate groups of ten mice were used to compare the percent survival over a 30-day period postchallenge. For histopathological examination, groups of 3 infected mice of each strain were sacrificed at 7 or 11 days postchallenge and their intact lungs were removed, fixed in 10% formalin, and embedded in paraffin. Tissue fixation and embedding procedures were performed as described [34]. Five-micrometer sections were cut and stained with hematoxylin and eosin (H&E) by standard procedure for examination of the lesions. Histopathological features were compared based on size, morphology and cell composition of the pulmonary lesions, concentration of the inflammatory infiltrates and density of the fungal elements. The two strains of infected mice were also evaluated for fungal burden at 7 and 11 days postchallenge as previously described [28]. Groups of five mice per time point were examined for colony-forming units (CFU) in homogenates of their lungs and spleen. The CFU determinations per organ were expressed on a log scale. 4.4. Vaccination protocol Separate groups of mice were immunized with a genetically engineered, live, attenuated vaccine strain of C. posadasii as previously reported [28]. Control mice were immunized with saline alone using the same protocol as employed for vaccination with the live, attenuated strain. Mice were challenged as above by the i.n. route with viable spores of the virulent, parental strain (isolate C735) at 4 weeks after completion of the vaccination schedule as previously described [28]. The mice were sacrificed at days 7 or 11 postchallenge to determine the CFU in their lungs and spleen. 4.5. Determination of levels of iNOS expression by QRT-PCR Total RNA was extracted from the lungs of non-vaccinated WT mice prior to challenge (day 0), and at 7 or 11 days after challenge with Coccidioides spores as above using a RNeasy Mini Kit (Qiagen, Valencia, CA). Reverse transcription and quantitative real time-PCR (QRT-PCR) analysis of iNOS expression (GenBank accession number NM_010927) were conducted as previously described [27]. Measurement of expression of the mouse constitutive gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GenBank accession number XM_001473623), was conducted for normalization of the data as previously reported [27]. 4.6. Assessment of total numbers of infiltrated pulmonary leukocytes and leukocyte subpopulations Five Coccidioides-infected WT and iNOS/ mice per group were used to evaluate the total numbers of lung-infiltrated leukocytes (LIL) or leukocyte subpopulations at each time point (7 and 11 days) postchallenge. The excised lungs of each mouse were placed in separate sterile cell strainers (70 mm diam. pore
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size; Fisher Scientific, Pittsburgh, PA, Cat. # 22363548) containing 5 ml of RPMI 1640 medium (HyClone, Logan, UT) plus 1% heatinactivated fetal bovine serum (FBS, HyClone). The lung tissue was then mashed using the thumb end of a 3-ml syringe plunger and the filtrate was collected in a 60 15 mm Petri dish. An additional 3 ml of RPMI 1640 þ 1% FBS was used to wash the filtrate, and the separate cell suspensions were then centrifuged at 500g for 10 min without application of the centrifuge brake. The supernatants were carefully aspirated and discarded. The pellets were resuspended in 3 ml of ACK lysing buffer (Lonza, Inc., Walkersville, MD) and incubated at room temperature for 3 min to lyse the erythrocytes. The cell suspensions were washed with two volumes of RPMI 1640 þ 1% FBS as above, and then filtered through a second sterile cell strainer (40 mm diam. pore size; Fisher, Cat. # 22363547) to remove epithelial cell contaminants. The pulmonary cells were centrifuged and resuspended in 1.0 ml of RPMI 1640 medium containing 10% heat-inactivated FBS for subsequent assays. An aliquot of the pulmonary cell suspension of each sample was subjected to the trypan blue exclusion test for cell viability, and the total number of live pulmonary cells per lung homogenate was determined by hemocytometer counts. Absolute numbers of pulmonary leukocytes in the total lung cell preparations were resolved by flow cytofluorometry using a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data were acquired with CellQuest Pro software (BD Biosciences) and analyzed using a FlowJo software package (Tree Star, Inc., Ashland, OR). Isolated pulmonary cells were suspended in FACS buffer (PBS containing 0.5% FBS). Unlabeled anti-CD16/CD32 (clone 2.4G2) monoclonal antibodies (mAb; BD Biosciences) were first incubated with the cell suspension for 15 min at 4 C to block nonspecific binding of cell surface antigen-specific mAb to Fc receptors. Cocktails containing appropriate fluorochrome-conjugated mAb (0.25e0.5 mg/106 cells in 50 ml FACS buffer) were added to the Fc-blocked pulmonary cells and incubated for 30 min at 4 C. All labeled mAb were obtained from BD Biosciences. Anti-CD45 (clone 30-F11) was employed to determine the total number of leukocytes in each preparation. Cocktails of labeled mAb were then used to determine absolute numbers of selected subpopulations of pulmonary leukocytes. Following incubation with the selected, labeled mAb the cells were washed twice with FACS buffer, and then fixed with 200 ml of 1% paraformaldehyde (Polysciences Inc., Warrington, PA) and stored in the dark at 4 C until analyzed in the flow cytometer. The mAb employed in the cocktail preparations included anti-Ly6G (clone IA8), anti-CD11b (clone M1/70), anti-CD11c (clone HL3), anti-Mac3 (clone M3/84), and anti-SiglecF (clone E50-2440), and were used to determine the absolute numbers of neutrophils (PMN; CD45þ/Ly6Gþ), alveolar mcrophages (AM; CD45þ/CD11bMedium/CD11cþ) tissue macrophages (T. Mac; CD45þ/Mac3þ/CD11cLow), dendritic cells (DC; CD45þ/CD11bHigh/CD11cþ), and eosinophils (Eos; CD45þ/CD11c/ SiglecFþ). Anti-CD3 (clone 17A2), anti-CD4 (clone RM4-5), antiCD8a (clone 53e67) and anti-NK1.1 (clone PK136) mAb were used to determine the absolute numbers of CD4þ T cells (CD3þ/CD4þ), CD8þ T cells (CD3þ/CD8þ) and natural killer cells (NK1.1þ). A cocktail which contained anti-CD19 (clone ID3), anti-CD23 (clone B3B4), and anti-sIgM (clone II/41) mAb was used to determine the absolute number of B cells present in the total leukocyte population. Regulatory T cells (Tregs) were resolved by intracellular staining of Foxp3 using a Treg staining kit from BD Biosciences. The detected levels of labeled CD25 at the cell surface and intracellular FoxP3 expression were back-gated on the CD4þ T cell population. The absolute number of each subpopulation of leukocytes was quantified by multiplying the total number of CD45þ cells by the percentage of the individual mAb cocktaillabeled cell types determined by flow cytofluorometry.
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4.7. Determination of pulmonary cytokine and chemokine concentrations The concentrations of Th1- and Th2-type cytokines and selected proinflammatory cytokines and chemokines were analyzed in lung homogenates of non-vaccinated, Coccidioides-infected WT and iNOS/ mice using a Bio-Plex suspension array system (Bio-Plex; Bio-Rad Laboratories, Hercules, CA). Briefly, excised lungs of individual mice sacrificed at 7 or 11 days postchallenge (5 mice per group) were homogenized in 0.5 ml of ice-cold sterile PBS and mixed with a cytokine extraction buffer (0.5 ml) containing a protease inhibitor cocktail lacking EDTA (Roche, Indianapolis, IN) to which 0.05% Triton X-100 was added. Each sample of the 4 groups of mice was then centrifuged at 4 C and 8000g for 10 min. The supernatants were recovered and stored at 80 C until assayed using the Bio-Plex system for concentration of the selected cytokines and chemokines following the manufacturer’s protocol. 4.8. Statistical analyses All data were expressed as mean values standard error of the mean (SEM). Analysis of variance (ANOVA) followed by the Tukey’s test or Student’s t test was applied to all in vitro experiments for determination of statistically significant difference between sets of data. Comparison of data from in vivo experiments was conducted by the ManneWhitney U test or ANOVA followed by the Student’s t test. Survival data were analyzed by the KaplaneMaier method (percentage of surviving animals) using log rank analysis to compare the survival curves. Statistical calculations were performed using GraphPad Prism version 4.0 software for Macintosh (GraphPad, San Diego, CA). Values of P < 0.05 were considered significant. Acknowledgements Support for this study was provided by Public Health Service grants AI071118 and AI070891 from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, awarded to GTC. Additional support was been provided by the Margaret Batts Tobin Foundation, San Antonio, TX. The authors are grateful for the technical assistance given by Ms. Natalia Castro-Lopez. References [1] Nathan C, Shiloh MU. Reactive oxygen and nitrogen intermediates in the relationship between mammalian host and microbial pathogens. Proc Natl Acad Sci USA 2000;97:8841e8. [2] Bogdan C. Nitric oxide and the immune response. Nat Immunol 2001;2: 907e16. [3] Bogdan C. Regulation of lymphocytes by nitric oxide. Methods Mol Biol 2011; 677:375e93. [4] Bogdan C, Rollinghoff M, Diefenbach A. The role of nitric oxide in innate immunity. Immunol Rev 2000;173:17e26. [5] Niedbala W, Wei X, Piedrafita D, Xu D, Liew FY. Effects of nitric oxide on the induction and differentiation of Th1 cells. Eur J Immunol 1999;29:2498e505. [6] Millar AE, Sternberg J, McSharry C, Wei XQ, Liew FY, Turner CM. T cell responses during Trypanosoma brucei infections in mice deficient in inducible nitric oxide synthase. Infect Immun 1999;67:3334e8. [7] Liew Y. Regulation of lymphocyte functions by nitric oxide. Curr Opin Immunol 1995;7:396e9. [8] MacMicking JD, North RJ, LaCourse R, Mudget JS, Shah SK, Nathan CF. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci USA 1997;94:5243e8.
[9] Lane TE, Otero GC, Wa-Hsieh B, Howard D. Expression of inducible nitric oxide synthase by stimulated macrophages correlates with their antihistoplasma activity. Infect Immune 1994;62:1940e5. [10] Rossi GR, Cervi LA, Garcia MM, Chiapello LS, Sastre DA, Mashi DT. Involvement of nitric oxide in protecting mechanism during experimental cryptococcosis. Clin Immunol 1990;90:256e65. [11] Liew FY, Millot S, Parkinson C, Palmer RMJ, Moncada S. Macrophage killing of Leishmania parasitic in vivo is mediated by nitric oxide from L-arginine. J Immunol 1990;144:4794e7. [12] James SL, Glaves J. Macrophage citotoxicity against schistosomula of Schistosoma mansoni involved arginine-dependent production of reactive nitrogen intermediates. J Immunol 1989;143:4208e12. [13] Nascimento FR, Calich VLG, Rodriguez D, Russo M. Dual role of nitric oxide in paracoccidioidomycosis: essential for resistance, but overproduction associated with susceptibility. J Immunol 2002;168:4593e600. [14] Livonesi MC, Rossi MA, De Souto JT, Campanelli AP, De Souza RLM, Maffei CML, et al. Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis. Microb Infect 2009;11:123e32. [15] Fernandes KSS, Neto EH, Brito MMS, Silva JS, Cunha FQ, Barja-Fidalgo C. Detrimental role of endogenous nitric oxide in host defence against Sporotrix schenckii. Immunology 2008;123:469e79. [16] Farah CS, Saunus JM, Hu Y, Kazoullis A, Ashman RB. Gene targeting demonstrates that inducible nitric oxide synthase is not essential for resistance to oral candidiasis in mice, or for killing of Candida albicans by macrophages in vitro. Oral Microbiol Immunol 2009;24:83e8. [17] Galgiani JN. Coccidioidomycosis: a regional disease of national importance: rethinking approaches for control. Ann Inter Med 1999;130:293e300. [18] Cole GT, Hung CY, Delgado N. Parasitic-phase gene expression in Coccidioides. ASM News 2002;68:603e11. [19] Delgado N, Xue J, Yu JJ, Hung CY, Cole GT. A recombinant b-1,3-glucanosyltransferase homolog of Coccidioides posadasii protects mice against coccidioidomycosis. Infect Immun 2003;71:3010e9. [20] Cole GT, Xue J, Okeke C, Tarcha EJ, Basrur V, Schaller RA, et al. A vaccine against coccidioidomycosis is justified and attainable. Med Mycol 2004;42: 189e216. [21] Tarcha EJ, Basrur V, Hung CY, Gardner MJ, Cole GT. Multivalent recombinant protein vaccine against coccidioidomycosis. Infect Immun 2006;74:5802e13. [22] Fierer J, Waters C, Walls L. Both CD4þ and CD8þ T cells can mediate vaccineinduced protection against Coccidioides immitis infection in mice. J Infect Dis 2006;193:1323e31. [23] Cox RA, Magee DM. Coccidioidomycosis: host response and vaccine development. Clin Microbiol Rev 2004;17:804e39. [24] Shubitz LF, Dial SM, Perrill R, Casement R, Galgiani JN. Vaccine-induced cellular immune responses differ from innate responses in susceptible and resistant strains of mice infected with Coccidioides posadasii. Infect Immun 2008;76:5553e64. [25] Wüthrich M, Gern B, Hung CY, Ersland K, Rocco N, Pick-Jacobs J, et al. Vaccineinduced protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice. J Clin Inv 2011;121:554e68. [26] Jimenez Mdel P, Wall L, Fierer J. High levels of interleukin-10 impair resistance to pulmonary coccidioidomycosis in mice in part through control of nitric oxide synthase 2 expression. Infect Immun 2006;74:3387e95. [27] Gonzalez A, Hung CY, Cole GT. Coccidioides releases a soluble factor that suppresses nitric oxide production by murine primary macrophages. Microb Pathogen 2011;50:100e8. [28] Xue J, Chen X, Selby D, Hung CY, Yu JJ, Cole GT. A genetically engineered live attenuated vaccine of Coccidioides posadasii protects BALB/c mice against coccidioidomycosis. Infect Immun 2009;77:3196e208. [29] Qidwai T, Jamal F. Inducible nitric oxide synthase (iNOS) gene polymorphism and disease prevalence. Scand J Immunol 2010;72:375e87. [30] Huang FP, Niedbala W, Wei XQ, Xu D, Feng GJ, Robinson JH, et al. Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages. Eur J Immunol 1998;28:4062e70. [31] Sternlicht MD, Werb Z. How matrix metalloproteinase’s regulate cell behavior. Annu Rev Cell Dev Biol 2001;17:463e516. [32] Cole GT, Xue J, Seshan K, Borra P, Borra R, Tarcha E, et al. Virulence mechanisms of Coccidioides. In: Heitman J, Filler S, Edwards J, Mitchell A, editors. Molecular principles of fungal pathogenesis. Washington, DC: American Society of Microbiology Press; 2006. p. 363e91. [33] Kirkland TN, Fierer J. Inbred mouse strains differ in resistance to lethal Coccidioides immitis infection. Infect Immun 1983;40:912e6. [34] Mirbod-Donovan F, Schaller R, Hung CY, Xue J, Reichard U, Cole GT. Urease produced by Coccidioides posadasii contributes to the virulence of this respiratory pathogen. Infect Immun 2006;74:504e15.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Nitric oxide synthase activity has limited influence on the control of Coccidioides infection in miceq Angel Gonzalez a, b, c, d, *, Chiung-Yu Hung a, Garry T. Cole a a
Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas at San Antonio, San Antonio, TX 78249, USA Medical and Experimental Mycology Group, Corporación para Investigaciones Biologicas (CIB), Carrera 72 A, No. 78, B 141, Medellín, Colombia c Escuela de Microbiología, Universidad de Antioquia, Medellín, Colombia d Escuela de la Salud, Universidad Pontificia Bolivariana, Medellín, Colombia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 January 2011 Received in revised form 28 February 2011 Accepted 2 March 2011 Available online 14 April 2011
The functions of inducible nitric oxide synthase (iNOS) activity in protection against microbial insults are still controversial. In this study, we explored the role of iNOS in protection against Coccidioides infection in mice. We observed that wild type (WT) and iNOS/ mice showed similar percent survival and fungal burden in their lungs at days 7 and 11 after intranasal challenge with Coccidioides. Vaccinated WT and iNOS/ mice revealed comparable fungal burden in their lungs and spleen at 7 and 11 days postchallenge. However, at 11 days the non-vaccinated, iNOS-deficient mice had significantly higher fungal burden in their spleen compared to WT mice. Additionally, higher numbers of lung-infiltrated neutrophils, macrophages and dendritic cells were observed in WT mice at day 11 postchallenge compared to iNOS/ mice. Moreover, no difference in numbers of T, B, NK or regulatory T cells, or concentrations of selected cytokines and chemokines were detected in lungs of both mouse strains at 7 and 11 days postchallenge. Although iNOS-derived NO production appears to influence the inflammatory response and dissemination of the fungal pathogen, our results suggest that iNOS activity does not play a significant role in the control of coccidioidal infection in mice and that other, still undefined mechanisms of host protection are involved. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Nitric oxide Coccidioides Inflammation Fungal burden
1. Introduction Nitric oxide (NO) has been demonstrated to play a diversity of roles during infection of the mammalian host. NO production catalyzed by the action of inducible nitric oxide synthase (iNOS) represents one of the major defense mechanisms of host phagocytic cells against certain pathogens [1]. Numerous reports have demonstrated that iNOS-derived NO helps to counteract excessive immune reactions to infection and functions both as an intra- and intercellular signaling molecule that contributes significantly to modulation of the immune response [2e4]. However, in some cases iNOS activity has been shown to exacerbate the course of disease due to such detrimental effects as NO-mediated cytotoxicity and
q This work was conducted at the University of Texas at San Antonio in the Department of Biology while the senior author (AG) was supported as a postdoctoral fellow by a National Institutes of Health grant (AI071118) awarded to GTC. * Corresponding author: Medical and Experimental Mycology Group, Corporación para Investigaciones Biológicas (CIB), Carrera 72 A, No. 78, B 141, Medellín, Colombia. Tel.: þ57 4 441 08 55; fax: þ57 4 441 55 14. E-mail address: [email protected] (A. Gonzalez). 0882-4010/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2011.03.013
host tissue damage, inhibition of T cell proliferation, and other immune regulatory properties which are detrimental to the host protective response to infection [5,6]. NO synthesis which is dependent on iNOS activity occurs in macrophages, neutrophils, dendritic and NK cells upon induction by exposure to interferongamma (IFN-g), tumor necrosis factor-alpha (TNF-a), certain bacterial products such as lipopolysaccharide (LPS), and other endogenous and exogenous stimuli [2,7]. NO has been implicated in the control of numerous infectious diseases caused by a variety of prokaryotic and eukaryotic pathogens, including Mycobacterium tuberculosis [8], Histoplasma capsulatum [9], Cryptococcus neoformans [10], Leishmania major [11], and Schistosoma mansoni [12]. On the other hand, NO has also been shown to be either detrimental or non-essential to host protection, or appears to play a dual role in infections caused by Paracoccidioides brasiliensis [13,14], Sporotrix schenckii [15], Candida albicans [16] and Trypanosoma brucei [6]. Coccidioidomycosis is an endemic human respiratory disease which occurs in southwestern regions of the United States, and is caused by the dimorphic fungus Coccidioides spp. This soilborne, fungal pathogen is also found in parts of Mexico, Central and South America [17]. Inhalation of its infectious, air-dispersed spores
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(arthroconidia) by the host is followed by the initiation of an elaborate parasitic cell cycle that is unique amongst the medically important fungi [18]. The spores differentiate into large, multinucleate spherules that give rise to a multitude of endospores which are responsible for dissemination of the pathogen from original sites of infection, typically in the lungs [19]. It is estimated 100,000 new infections of coccidioidomycosis occur in the United States each year, but only about 40% of individuals exposed to the fungus develop clinical symptoms. The latter range from an influenza-like illness to severe pneumonia or, rarely, to extrapulmonary disseminated disease and meningitis [20]. Risk factors known to contribute to symptomatic coccidioidal infection include pregnancy (third trimester), immunosuppression, advanced age (>65 years), and ethnicity [21]. Several studies have demonstrated that cellular immunity, mainly CD4þ and CD8þ T cell responses, is essential for protection against coccidioidomycosis [20,22e24]. Moreover, it has been shown that T helper (Th) 17 cells are essential for stimulation of protective immunity against pulmonary infections of Coccidioides [25]. In addition, interleukin (IL)-10 has been reported to play a key role in murine susceptibility to coccidioidomycosis; thus, transgenic mice producing high levels of human IL-10 are more susceptible to coccidioidal infection and expressed lower levels of interferon-gamma (IFN-g), IL-12p40 and iNOS mRNA in their lungs compared to control mice, implicating NO synthesis as a mechanism of resistance against coccidioidomycosis [26]. However, the role of iNOS-derived NO produced by Coccidioides is still not clear. We previously demonstrated that Coccidioides spp. has the ability to suppress both NO production and iNOS expression by murine primary macrophages in vitro that were previously activated by exposure to IFN-g þ LPS [27]. On this basis, we initially considered that the suppressive product (s) secreted by the fungal pathogen represented an important virulence factor. However, macrophages obtained from iNOS/ mice were as competent in their uptake and killing of parasitic cells of Coccidioides as macrophages isolated from wild type (WT) mice, suggesting that the fungicidal mechanism of the host phagocytes in vitro is not dependent on NO production. In the present study, we have compared the survival and immune response of WT and iNOS/ mice following intranasal challenge with Coccidioides in an attempt to further explore whether iNOS-derived NO is essential for protection against Coccidioides infection, and to determine if NO plays a role in modulation of cellular immunity during the course of pulmonary coccidioidomycosis. 2. Results 2.1. iNOS/ mice showed similar mortality but increased dissemination of Coccidioides compared to WT mice C57BL/6 WT and iNOS gene-deficient mice (iNOS/) were challenged with a potentially lethal inoculum of C. posadasii spores by the intranasal (i.n.) route. To assess the influence of the absence of iNOS-derived NO on disease outcome, we first examined the mortality of each group of infected mice recorded daily for a 30-day period (Fig. 1). The mean survival time for both strains was 12 days, and no statistically significant difference was observed between the mortality data for the two groups of infected mice (P ¼ 0.88). To further evaluate these results, comparative histopathology of the infected lungs of the two strains were conducted, and determination of the fungal burden in both the lungs and spleen of the two mouse strains was determined at 7 and 11 days postchallenge. A similar pattern of inflammatory response was observed in the infected, H&E-stained lungs of the WT and iNOS/ mice at 7 days (c.f. Fig. 2A and B, respectively). The inflammatory lesions appeared
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to have replaced almost all the normal lung parenchyma tissue and were composed of poorly differentiated granulomas of various sizes surrounded by large numbers of neutrophils and mononuclear cells. The abscesses contained comparable numbers of parasitic cells of Coccidioides (spherules and endospores). At 11 days postchallenge, the infected lungs of the WT mice presented with incipient granulomas, characterized by a thick layer of mononuclear cells which contained the fungal cells (Fig. 2C). On the other hand, the iNOSdeficient mice at day 11 postchallenge showed a more diffuse distribution of inflammatory cells and absence of well-defined granulomatous structures (Fig 2D). Near equal numbers of colonyforming units (CFU) of Coccidioides were observed in lung homogenates of the iNOS/ and WT mice at days 7 and 11 after challenge (open bars in Fig. 3A and B,, respectively). However, significantly higher fungal burden was detected in the spleen of the iNOSdeficient mice at 11 days postchallenge when compared with the infected WT mice (P ¼ 0.006) (Fig. 3C). To determine whether the absence of iNOS-derived NO would influence acquired immunity to Coccidioides infection, both mouse strains were immunized with a genetically engineered, live attenuated vaccine strain of Coccidioides which had previously been reported to provide protection (100% survival) to C57BL/6 mice against coccidioidomycosis [28]. Vaccinated mice, irrespective of the strain, revealed comparable fungal burden in their lungs at 7 and 11 days postchallenge (Fig. 3A and B, respectively), and showed no statistically significant difference in dissemination of the pathogen to the spleen of the two groups of mice (Fig. 3C). 2.2. Elevated iNOS expression correlated with reduced dissemination of Coccidioides in WT mice We evaluated iNOS expression in the WT C57BL/6 mice at 0, 7 and 11 days postchallenge using QRT-PCR (Fig. 4). We detected a significantly higher number of iNOS transcripts on day 11 after challenge than at day 7 (P < 0.001). Elevated expression of iNOS in these mice correlates with the significantly lower CFU in their spleen compared with the spleen homogenates of iNOS/ mice at 11 days postchallenge (c.f., Fig. 3C). 2.3. WT and iNOS-deficient mice showed comparable numbers of infiltrated pulmonary leukocytes at 7 and 11 days postchallenge Leukocyte recruitment into the lungs of Coccidioides-infected WT and iNOS/ mice was examined by cytofluorometry. Not surprising, a significant increase in the total number of lung-
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Fig. 2. Histopathology of infected lungs of WT and iNOS/ mice at 7 days (A, B) and 11 days postchallenge (C, D), respectively. Arrow in (A) indicates dense population of neutrophils which surrounds the coccidioidal lesion. Arrow in (B) indicates the concentrated array of parasitic cells (spherules and endospores) of C. posadasii present in the pulmonary abscess. Bar in panel (B) represents 0.5 mm, and also applies to panels (A, C and D).
infiltrated leukocytes (LIL) was observed in both mouse strains between 7 and 11 days postchallenge (Fig. 5). However, no statistically significant difference existed between the total LIL detected in the infected lungs of the two strains at either 7 or 11 days after intranasal challenge. 2.4. Loss of iNOS expression correlated with changes in numbers of certain innate cell types which had infiltrated infected lung tissue We also employed cytofluorometry to detect numbers of selected subpopulations of leukocytes that had infiltrated the infected lungs of WT and iNOS/ mice at 7 and 11 days postchallenge (Fig. 6AeD). Significant changes were observed in the numbers of neutrophils (PMN) and tissue macrophages (T. Mac) between the two strains of mice during this period of infection. However, the higher numbers of these two innate cell types in the iNOS-deficient mice compared to WT mice at 7 days was reversed at 11 days after challenge. At day 7 the numbers of PMNs and tissue macrophages in iNOS/ mice compared to WT mice were 0.519 0.14 versus 0.253 0.0 (P < 0.01) and 1.0 0.26 versus 0.672 0.14 (P < 0.05), respectively (Fig. 6A and B; all values 106). At 11 days postchallenge, the numbers of these same two cell types were significantly higher (P < 0.001) in the WT mice (8.0 1.08 and 4.84 1.25, respectively) compared to the iNOS-deficient mice (5.44 0.475 and 2.97 0.37, respectively). The number of lunginfiltrated dendritic cells (DC) in the WT mice at 11 days also showed a significant increase compared to the iNOS/ mice (0.58 0.30 versus 0.23 0.14, respectively; P < 0.01), but no statistically significant difference was observed at 7 days postchallenge. The numbers of selected subpopulations of infiltrated lymphocytes (CD4þ, CD8þ, total B cells, regulatory T cells) and NK cells in WT and iNOS/ mice showed no significant difference at 7 or 11 days postchallenge (Fig. 6C and D).
2.5. Loss of iNOS expression had no effect on the concentration of selected cytokines/chemokines secreted in Coccidioides-infected lung tissue It is well appreciated that exogenous and endogenous stimulation of the host cytokine network (including secretion of proinflammatory cytokines/chemokines and Th1-, Th2-, and Th-17 type cytokines) impacts the efficiency and effectiveness of the inflammatory response to infection and determines the composition of activated immune cells which are directed to the sites of the microbial insult [25]. Inducible nitric oxide synthase activity has been reported to have a versatile role in the induction of cytokine secretion and immune cell differentiation and proliferation [2,3]. Although our results reported in Fig. 6 suggested that iNOS activity has limited influence on the infiltration of leukocytes into Coccidioides-infected lungs of mice, it is possible that this enzyme is involved in the induction of chemical signals that influence other pathways of immune response. On this basis, we set out to compare the concentrations of selected cytokines and chemokines present in lung homogenates of infected WT and iNOS-deficient mice which have been reported to be associated with the immune response of C57BL/6 mice to Coccidioides infection (unpublished data). The results presented in Table 1 reveal that no statistically significant differences were detected between concentrations of the selected array of cytokines and chemokines tested in lung homgenates of the Coccidioides-infected WT and iNOS/ strains of mice at 7 and 11 days postchallenge. 3. Discussion The mammalian host is equipped with a complexity of mechanisms that induce protective immune responses to microbial infections, while counter-balanced by feedback signals that can restrict the magnitude and duration of these responses in order to
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Fig. 3. Comparison of fungal burden in the lungs (A, B) and spleen (C) of wild type (WT) and iNOS/ mice at 7 and 11 days postchallenge. These studies were conducted using nonvaccinated and vaccinated mice. The median CFUs (log10) are indicated by horizontal lines inside each box. A statistically significant difference was revealed between the fungal burden in the spleen of non-vaccinated WT and iNOS/ mice at 11 days postchallenge (C) (*P ¼ 0.006). The results are representative of two independent experiments.
minimize tissue damage caused by persistent inflammation and/or autoimmune reactions [3]. Nitric oxide (NO) generated by activity of the inducible isoform of nitric oxide synthase (iNOS) is such a signaling molecule involved in immunomodulatory events during the course of infectious diseases, while also serving as a potent growth inhibitor of certain microbial pathogens [2,3,29]. Studies using NO donors, NO inhibitors or iNOS-deficient mice have provided evidence for the participation of iNOS-derived NO in
a broad spectrum of immune regulatory processes including apoptosis, production of cytokines and other soluble mediators, expression of co-stimulatory and adhesion molecules, and synthesis and deposition of extracellular matrix components [2,30,31]. Results of these investigations have also revealed that production of iNOS-derived NO can be detrimental in some disease processes [15], of little consequence in controlling pathogen proliferation [27], or beneficial in others [14]. For example, NO produced by macrophages has been reported to have both host
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7
11
Fig. 4. Relative levels of transcription of the iNOS gene were measured by QRT-PCR. Lungs were excised from separate groups of WT mice (n ¼ 4) prior to infection (0 days) or at 7 and 11 days after i.n. challenge and used as sources of total RNA. Relative amounts of detected iNOS gene transcript were normalized with that of the constitutive GADPH gene. Untreated (naïve) mice were included for determination of baseline amounts of transcripts. The expression level of the iNOS gene was significantly elevated at 11 days postchallenge compared to the other two selected time points (P < 0.001). The bars represent means SEM. The results are representative of two independent experiments.
0 7 Days postchallenge
11 Days postchallenge
Fig. 5. Total number of lung-infiltrated leukocytes (LIL) in non-vaccinated, Coccidioides-infected wild type (WT) and iNOS/ mice (n ¼ 5). Cytometric analyses of fluorescent anti-CD45 mAb-labeled cells was conducted to identify leukocytes within the pulmonary cell suspension. The total leukocyte numbers for both mouse strains were significantly higher at 11 days compared to 7 days postchallenge (P < 0.001), but were not significantly different from each other at either time point. The bars represent means SEM.
A WT iNOS -/-
1.00 0.75
* 7 Days postchallenge
*
0.50 0.25 0.00
0.60
PMN
AM
T. Mac
DC
Eos
C
0.50
7 Days postchallenge
0.40 0.30 0.20 0.10 0.00
CD4+ CD8+
NK cells
LIL Subpop. (x 106)
1.25
B T reg cells
LIL Subpop. (x 106)
LIL Subpop. (x 106)
LIL Subpop. (x 106)
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10.0
165
B
*
*
6.00
11 Days postchallenge
2.00 1.00
*
0.50 0.00
0.60 0.50
PMN
AM
T. Mac
DC
Eos
D 11 Days postchallenge
0.40 0.30 0.20 0.10 0.00
CD4+ CD8+
NK cells
B T reg cells
Fig. 6. Loss of iNOS activity results in both increased and decreased numbers of selected phagocytic cells, but no significant changes in lymphocytic cell populations. Numbers of lung-infiltrated leukocyte subpopulations (identified in the Section 4) were identified by cytometric analyses of total pulmonary cells isolated from the lungs of groups of Coccidioides-infected WT and iNOS/ mice (n ¼ 5) sacrificed at 7 and 11 days postchallenge. Asterisks indicate statistically significant differences between numbers of neutrophils (PMN), tissue macrophages (T. Mac) and dendritic cells (DC) of the two mouse strains, as described in Section 2. The bars represent the means SEM.
tissue damaging and anti-inflammatory effects as a result of its modulation of host secretion and function of certain cytokines, chemokines and growth factors in response to a microbial insult [2]. Previous studies of Candida infections have concluded that macrophages obtained from WT and iNOS/ mice showed no difference in their ability to kill the pathogen in vitro [16]. Regulation of iNOS activity occurs both at the transcriptional and post-
transcriptional levels. Arginase can also have a regulatory effect on iNOS activity by competing for the same substrate (L-arginine) and converting it to ornithine and urea. We have previously shown that expression of the arginase 1 gene of Coccidioides-infected C57BL/6 mice is highly up-regulated at 6 days postchallenge compared to non-infected control mice (190.2-fold increase) [32]. This observation, combined with our results reported here that no
Table 1 Concentrations of pulmonary proinflammatory cytokines and chemokines in Coccidioides-infected lungs of wild type (WT) and INOS/ mice at 7 and 11 days postchallenge (PC). Cytokines and chemokines (pg/ml)
7 Days PC
11 Days PC
WT
iNOS/
WT
iNOS/
1105.6 171.1 5622.9 1141.1 1067.2 422.4 1326.9 240.8 1755.9 187.6 13766.1 3035.2 1342.4 101.9
1269.2 55.3 7084.9 515.1 1188.4 529.6 1836.7 593.0 1528.8 294.6 14123.3 1443.4 1315.3 143.3
4343.5 976.6 11830.9 2154.2 2366.6 482.9 3116.4 1329.6 3867.3 1225.4 45326.5 9346.0 1283.1 235.6
4266.5 1562.6 12536.7 2976.9 3213.1 440.1 2402.6 929.7 4037.7 1742.8 51172.6 10807.8 1319.1 479.9
Th1 cytokines IFN-g IL-2 IL-3 IL-12p70
1212.6 80.1 774.7 108.3 332.7 68.5 562.2 68.9
1134.5 189.1 865.3 76.9 329.9 72.3 601.7 106.0
1426.8 133.9 551.4 40.4 250.4 17.8 1611.6 305.2
1634.2 431.1 572.6 95.5 259.4 55.4 1761.4 342.5
Th2 cytokines IL-4 IL-5 IL-10 IL-13
1477.5 465.1 1436.6 565.5 303.1 51.5 3977.3 260.5
1818.2 904.5 1318.0 412.0 313.7 78.2 4001.3 282.9
852.9 368.8 1807.3 615.5 785.2 344.5 4058.9 876.9
937.0 134.2 1833.8 273.6 936.4 462.1 3794.1 169.1
Chemokines CXCL1/KC CCL2/MCP-1 CCL3/MIP-1a CCL4/MIP-1b CCL5/RANTES CCL11/Eotaxin
5950.9 284.7 4678.7 810.9 6270.9 889.7 1198.6 258.3 1226.5 102.6 5271.7 417.9
5399.3 519.6 5168.3 866.9 7215.8 793.2 1369.1 197.8 1236.2 387.5 5128.4 748.5
8542.4 1856.7 5420.5 813.6 16042.4 3444.8 2299.7 463.8 527.1 93.0 5704.8 543.8
7771.7 2070.5 6137.9 458.8 17986.2 2972.3 2783.1 478.2 493.2 165.9 5643.7 990.2
Proinflammatory cytokines IL-1a IL-1b IL-6 IL-17 TNF-a G-CSF GM-CSF
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significant difference was evident in survival, lung histopathology or fungal burden between Coccidioides-infected WT and iNOS/ mice at 7 days postchallenge, argue that iNOS-derived NO has little to no effect on immune regulation or microbicidal activity during this early stage of pulmonary coccidioidomycosis. We had previously demonstrated that Coccidioides is able to suppress NO production by IFN-g þ LPS-activated mouse primary macrophages in vitro as a result of secretion of an undefined, soluble inhibitory factor(s). Intuitively, this suggests that inhibition of NO production is important for persistence of the pathogen within the hostile environment of the host. On the other hand, results of our comparative in vitro studies of Coccidioides killing efficiency by macrophages derived from WT and iNOS/ mouse strains indicated that NO is not an essential contributor to the fungicidal activity of these phagocytic cells [27]. We speculate, therefore, that production of iNOS-derived NO by alveolar and tissue macrophages, neutrophils, and dendritic cells in vivo does not have a direct effect on limiting growth of Coccidiodes in infected lungs of C57BL/6 mice, but instead contributes to modulation of the immune response to infection. The fact that we observed an approximate 6-fold increase in iNOS expression in the infected lungs of WT mice between 7 and 11 days postchallenge indicates that iNOS activity may still play a role in the course of progressive coccidiodal disease. This prompted us to compare the nature of the immune response between Coccidioides-infected WT and iNOS/ mice. Examinations of the lung histopathology at 7 days after challenge revealed that WT mice showed signs of containment of the pathogen, albeit within rudimentary granulomas, while the iNOS/ mice revealed diffuse pulmonary lesions with high concentrations of parasitic cells. In addition, the iNOS/ mice showed a slightly higher fungal burden in their spleen at 11 days postchallenge, a fact that would suggests the involvement of iNOS in the dissemination process observed with this fungal pathogen in mice lacking the corresponding enzyme. It is known that the genetic background of the host may influence the immune response against microbial pathogens, as has been previously described for DBA/2 mice that are resistant to Coccidioides infection and C57BL/6, C57BL/10 and BALB/c mice which are very susceptible [33]. Taking this into account, we used an animal model of pulmonary coccidioidomycosis based on susceptible wild type C57BL/6 mice and gender-matched mice with the same genetic background which lacked the iNOS gene. We designed an identical vaccination protocol which was applied to both mouse strains in an attempt to explore the role of inducible nitric oxide activity in experimental coccidioidomycosis. Vaccinated mice of both strains revealed comparable low CFU in their spleen and were apparently equally protected against dissemination of the fungal pathogen. In addition, total numbers of recruited leukocytes which had infiltrated the infected lungs of the nonvaccinated mice showed an approximate 4 to 5-fold increase between 7 and 11 days postchallenge. However, no difference was observed between the infected WT and iNOS/ mouse strains at these two stages in the course of the disease. These results suggest that iNOS is not needed for acquired immunity in C57BL/6 mice induced by vaccination against Coccidioides. On the other hand, when we assessed the numbers of selected lung-infiltrated leukocytes subpopulations by flow cytometry, a higher but transient number of neutrophils and tissue macrophages were detected in the Coccidioides-infected iNOS/ mice at day 7 postchallenge compared to the WT mice. The total numbers of these innate cells increased sharply at 11 days after challenge, but the relationship between the two strains of mice was reversed. The WT mice showed significantly higher numbers of infiltrated neutrophils, tissue macrophages and dendritic cells when compared with the iNOS-deficient mice, an observation that
correlates with the histopathologic findings, increased expression of iNOS in infected lung tissue and lower fungal burden in the spleen at 11 days in the same WT mouse strain. Although these preliminary results suggest that iNOS-derived NO may enhance recruitment of mononuclear defense cells to infection sites in the lungs during advanced stages of the disease, the data do not entirely support this conclusion. No difference between the two strains was recorded in the numbers of lymphocytes that had infiltrated the infected lung tissue. In addition, we did not observe any significant differences in the levels of selected proinflammatory cytokines or chemokines between the WT and iNOS/ mice at 7 or 11 days postchallenge. Similar results have been reported by Farah and coworkers [16] who observed that WT and iNOS/ mice infected with C. albicans showed equivalent levels of expression of inflammatory cytokines measured by QRT-PCR. In contrast, Livonesi et al., [14] found that iNOS/ mice infected with P. brasiliensis yeast cells showed higher levels of Th1 and Th2 cytokines than the WT strain, and proposed that elevated iNOS activity contributes significantly to both protection and regulation of the inflammatory response to this fungal pathogen. Jimenez and coworkers [26] proposed that a correlation exists between elevated IL-10 production, reduced expression of iNOS and increased susceptibility to coccidioidomycosis, and suggested a protective role for NO. However, in our previous study we observed that IFN-g þ LPSactivated murine primary macrophages from both WT and iNOSdeficient mice were able to produce comparable amounts of IL-10 in vitro [27], and in this study no differences in the pulmonary levels of IL-10 were observed. These reports illustrate the contradictory interpretations of the in vivo role of iNOS-derived NO. The contribution of iNOS activity in the control of coccidioidomycosis has not been previously investigated. The results of our comparative study of disease outcome and immune response of vaccinated and non-vaccinated WT and iNOS/ mice to pulmonary challenge with Coccidioides indicate that NO has limited influence on processes of defense against this fungal pathogen. We cannot rule out the possibility that Coccidioides utilizes energy to generate and secrete products that suppress NO production by macrophages [27] in order to modulate pathways of host immune response which aids in its survival in vivo. Additional studies are required to further explore this concept. 4. Materials and methods 4.1. Fungus C. posadasii (isolate C735) was used for all experimental procedures reported in this study. The saprobic phase of this fungus was grown on glucose-yeast-extract agar (GYE; 1% glucose, 0.5% yeastextract, 2% agar) at 30 C for 3e4 weeks to induce sporulation. Spores were harvested into a plastic centrifuge tube containing 10 ml sterile, endotoxin-free saline and glass beads (5 mm diam.). The tubes were shook by hand to disrupt the spore chains. The cell suspension was filtered through a layer of nylon fiber to remove hyphal fragments, washed twice and resuspended in endotoxinfree PBS. 4.2. Animals Breeding pairs of homozygous C57BL/6 iNOS-deficient mice (iNOS/; strain B6.129P2-Nos2tm1Lau/J) and wild type (WT) C57BL/ 6 mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and the National Cancer Institute/Charles River Laboratory (Wilmington, MA, USA), respectively. Mice were maintained for breeding in the Small Animal Laboratory at the University of Texas at San Antonio and handled according to guidelines approved by the
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University Institutional Animal Care and Use Committee. Infected animals were housed in a pathogen-free, Thoren caging system (Thoren, Hazelton, PA), which was accommodated in a Biological Safety Level (BSL3) laboratory. Coccidioides is designated as a “select agent” by the Centers for Disease Control and Prevention (Atlanta, GA, USA) and must be contained within a BSL3 facility. 4.3. Intranasal infection and mortality, histopathology and fungal burden analyses Mice of both strains (WT and iNOS/) were challenged by the natural, intranasal (i.n.) route with a potentially lethal inoculum of 60e80 viable spores of C. posadasii as previously reported [28]. Separate groups of ten mice were used to compare the percent survival over a 30-day period postchallenge. For histopathological examination, groups of 3 infected mice of each strain were sacrificed at 7 or 11 days postchallenge and their intact lungs were removed, fixed in 10% formalin, and embedded in paraffin. Tissue fixation and embedding procedures were performed as described [34]. Five-micrometer sections were cut and stained with hematoxylin and eosin (H&E) by standard procedure for examination of the lesions. Histopathological features were compared based on size, morphology and cell composition of the pulmonary lesions, concentration of the inflammatory infiltrates and density of the fungal elements. The two strains of infected mice were also evaluated for fungal burden at 7 and 11 days postchallenge as previously described [28]. Groups of five mice per time point were examined for colony-forming units (CFU) in homogenates of their lungs and spleen. The CFU determinations per organ were expressed on a log scale. 4.4. Vaccination protocol Separate groups of mice were immunized with a genetically engineered, live, attenuated vaccine strain of C. posadasii as previously reported [28]. Control mice were immunized with saline alone using the same protocol as employed for vaccination with the live, attenuated strain. Mice were challenged as above by the i.n. route with viable spores of the virulent, parental strain (isolate C735) at 4 weeks after completion of the vaccination schedule as previously described [28]. The mice were sacrificed at days 7 or 11 postchallenge to determine the CFU in their lungs and spleen. 4.5. Determination of levels of iNOS expression by QRT-PCR Total RNA was extracted from the lungs of non-vaccinated WT mice prior to challenge (day 0), and at 7 or 11 days after challenge with Coccidioides spores as above using a RNeasy Mini Kit (Qiagen, Valencia, CA). Reverse transcription and quantitative real time-PCR (QRT-PCR) analysis of iNOS expression (GenBank accession number NM_010927) were conducted as previously described [27]. Measurement of expression of the mouse constitutive gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GenBank accession number XM_001473623), was conducted for normalization of the data as previously reported [27]. 4.6. Assessment of total numbers of infiltrated pulmonary leukocytes and leukocyte subpopulations Five Coccidioides-infected WT and iNOS/ mice per group were used to evaluate the total numbers of lung-infiltrated leukocytes (LIL) or leukocyte subpopulations at each time point (7 and 11 days) postchallenge. The excised lungs of each mouse were placed in separate sterile cell strainers (70 mm diam. pore
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size; Fisher Scientific, Pittsburgh, PA, Cat. # 22363548) containing 5 ml of RPMI 1640 medium (HyClone, Logan, UT) plus 1% heatinactivated fetal bovine serum (FBS, HyClone). The lung tissue was then mashed using the thumb end of a 3-ml syringe plunger and the filtrate was collected in a 60 15 mm Petri dish. An additional 3 ml of RPMI 1640 þ 1% FBS was used to wash the filtrate, and the separate cell suspensions were then centrifuged at 500g for 10 min without application of the centrifuge brake. The supernatants were carefully aspirated and discarded. The pellets were resuspended in 3 ml of ACK lysing buffer (Lonza, Inc., Walkersville, MD) and incubated at room temperature for 3 min to lyse the erythrocytes. The cell suspensions were washed with two volumes of RPMI 1640 þ 1% FBS as above, and then filtered through a second sterile cell strainer (40 mm diam. pore size; Fisher, Cat. # 22363547) to remove epithelial cell contaminants. The pulmonary cells were centrifuged and resuspended in 1.0 ml of RPMI 1640 medium containing 10% heat-inactivated FBS for subsequent assays. An aliquot of the pulmonary cell suspension of each sample was subjected to the trypan blue exclusion test for cell viability, and the total number of live pulmonary cells per lung homogenate was determined by hemocytometer counts. Absolute numbers of pulmonary leukocytes in the total lung cell preparations were resolved by flow cytofluorometry using a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data were acquired with CellQuest Pro software (BD Biosciences) and analyzed using a FlowJo software package (Tree Star, Inc., Ashland, OR). Isolated pulmonary cells were suspended in FACS buffer (PBS containing 0.5% FBS). Unlabeled anti-CD16/CD32 (clone 2.4G2) monoclonal antibodies (mAb; BD Biosciences) were first incubated with the cell suspension for 15 min at 4 C to block nonspecific binding of cell surface antigen-specific mAb to Fc receptors. Cocktails containing appropriate fluorochrome-conjugated mAb (0.25e0.5 mg/106 cells in 50 ml FACS buffer) were added to the Fc-blocked pulmonary cells and incubated for 30 min at 4 C. All labeled mAb were obtained from BD Biosciences. Anti-CD45 (clone 30-F11) was employed to determine the total number of leukocytes in each preparation. Cocktails of labeled mAb were then used to determine absolute numbers of selected subpopulations of pulmonary leukocytes. Following incubation with the selected, labeled mAb the cells were washed twice with FACS buffer, and then fixed with 200 ml of 1% paraformaldehyde (Polysciences Inc., Warrington, PA) and stored in the dark at 4 C until analyzed in the flow cytometer. The mAb employed in the cocktail preparations included anti-Ly6G (clone IA8), anti-CD11b (clone M1/70), anti-CD11c (clone HL3), anti-Mac3 (clone M3/84), and anti-SiglecF (clone E50-2440), and were used to determine the absolute numbers of neutrophils (PMN; CD45þ/Ly6Gþ), alveolar mcrophages (AM; CD45þ/CD11bMedium/CD11cþ) tissue macrophages (T. Mac; CD45þ/Mac3þ/CD11cLow), dendritic cells (DC; CD45þ/CD11bHigh/CD11cþ), and eosinophils (Eos; CD45þ/CD11c/ SiglecFþ). Anti-CD3 (clone 17A2), anti-CD4 (clone RM4-5), antiCD8a (clone 53e67) and anti-NK1.1 (clone PK136) mAb were used to determine the absolute numbers of CD4þ T cells (CD3þ/CD4þ), CD8þ T cells (CD3þ/CD8þ) and natural killer cells (NK1.1þ). A cocktail which contained anti-CD19 (clone ID3), anti-CD23 (clone B3B4), and anti-sIgM (clone II/41) mAb was used to determine the absolute number of B cells present in the total leukocyte population. Regulatory T cells (Tregs) were resolved by intracellular staining of Foxp3 using a Treg staining kit from BD Biosciences. The detected levels of labeled CD25 at the cell surface and intracellular FoxP3 expression were back-gated on the CD4þ T cell population. The absolute number of each subpopulation of leukocytes was quantified by multiplying the total number of CD45þ cells by the percentage of the individual mAb cocktaillabeled cell types determined by flow cytofluorometry.
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4.7. Determination of pulmonary cytokine and chemokine concentrations The concentrations of Th1- and Th2-type cytokines and selected proinflammatory cytokines and chemokines were analyzed in lung homogenates of non-vaccinated, Coccidioides-infected WT and iNOS/ mice using a Bio-Plex suspension array system (Bio-Plex; Bio-Rad Laboratories, Hercules, CA). Briefly, excised lungs of individual mice sacrificed at 7 or 11 days postchallenge (5 mice per group) were homogenized in 0.5 ml of ice-cold sterile PBS and mixed with a cytokine extraction buffer (0.5 ml) containing a protease inhibitor cocktail lacking EDTA (Roche, Indianapolis, IN) to which 0.05% Triton X-100 was added. Each sample of the 4 groups of mice was then centrifuged at 4 C and 8000g for 10 min. The supernatants were recovered and stored at 80 C until assayed using the Bio-Plex system for concentration of the selected cytokines and chemokines following the manufacturer’s protocol. 4.8. Statistical analyses All data were expressed as mean values standard error of the mean (SEM). Analysis of variance (ANOVA) followed by the Tukey’s test or Student’s t test was applied to all in vitro experiments for determination of statistically significant difference between sets of data. Comparison of data from in vivo experiments was conducted by the ManneWhitney U test or ANOVA followed by the Student’s t test. Survival data were analyzed by the KaplaneMaier method (percentage of surviving animals) using log rank analysis to compare the survival curves. Statistical calculations were performed using GraphPad Prism version 4.0 software for Macintosh (GraphPad, San Diego, CA). Values of P < 0.05 were considered significant. Acknowledgements Support for this study was provided by Public Health Service grants AI071118 and AI070891 from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, awarded to GTC. Additional support was been provided by the Margaret Batts Tobin Foundation, San Antonio, TX. The authors are grateful for the technical assistance given by Ms. Natalia Castro-Lopez. References [1] Nathan C, Shiloh MU. Reactive oxygen and nitrogen intermediates in the relationship between mammalian host and microbial pathogens. Proc Natl Acad Sci USA 2000;97:8841e8. [2] Bogdan C. Nitric oxide and the immune response. Nat Immunol 2001;2: 907e16. [3] Bogdan C. Regulation of lymphocytes by nitric oxide. Methods Mol Biol 2011; 677:375e93. [4] Bogdan C, Rollinghoff M, Diefenbach A. The role of nitric oxide in innate immunity. Immunol Rev 2000;173:17e26. [5] Niedbala W, Wei X, Piedrafita D, Xu D, Liew FY. Effects of nitric oxide on the induction and differentiation of Th1 cells. Eur J Immunol 1999;29:2498e505. [6] Millar AE, Sternberg J, McSharry C, Wei XQ, Liew FY, Turner CM. T cell responses during Trypanosoma brucei infections in mice deficient in inducible nitric oxide synthase. Infect Immun 1999;67:3334e8. [7] Liew Y. Regulation of lymphocyte functions by nitric oxide. Curr Opin Immunol 1995;7:396e9. [8] MacMicking JD, North RJ, LaCourse R, Mudget JS, Shah SK, Nathan CF. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci USA 1997;94:5243e8.
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