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Chronic exposure to cigarette smoke transiently worsens the disease course in a mouse model of pulmonary paracoccidioidomycosis

ABSTRACT

Paracoccidioidomycosis (PCM) may present as an acute/subacute clinical form, characterized by a progressive disease arising from the airborne initial infection, or, most often, as an asymptomatic or subclinical infection that may manifest later during an individual’s life, the chronic form. Epidemiological studies show the existence of a strong association between smoking and the development of the chronic form. Current evidence demonstrates that cigarette smoke (CS) has immunosuppressive properties that could be implicated in the increasing susceptibility to the chronic form of PCM. To address this issue, we developed a murine model of a non-progressive pulmonary form of PCM that was exposed to CS at a magnitude that mimicked a moderate smoker. The chronic CS exposure started after 2 weeks and lasted up until 20 weeks post-infection, with the aim of mimicking human natural history, since it is estimated that individuals from endemic areas are infected early in life. The control group consisted of infected but not CS-exposed mice. We assessed the lung fungal burden (colony forming units [CFU]) and the area affected by the granulomatous inflammatory response, fungal dissemination to spleen and liver, and, by immunohistochemistry, the presence of CD4 and CD8 lymphocytes, CD68 and MAC-2 macrophages, and IFN-γ, IL-10 and TNF expressing cells within the granulomatous response. We detected a CS effect as early as 2 weeks after exposure (four weeks post-infection) when the lung CFU of exposed animals was significantly higher than in their non-exposed counterparts. At 12 weeks, the CS-exposed animals presented a more severe disease, as witnessed by the persistent higher lung fungal load (although it did not reach statistical significance [ p = 0.054]), greater dissemination to other organs, greater affected area of the lung, decreased IFN-γ/IL-10 ratio, and higher TNF expression within the granulomas, compared with CS-non-exposed mice. The number of CD4 and CD8 lymphocytes infiltrating the granulomas was similar between both mice groups, but there was a decrease in the number of MAC-2+ macrophages. No difference was noted in the CD68+ macrophage number. However, the follow-up in week 20 showed that the immunological effects of exposure to CS ceased, with both CS and NCS mice showing the same infectious features, i.e., a trend for resolution of the infection. In conclusion, we show that chronic CS-exposure alters the course of the disease in an experimental model of subclinical pulmonary PCM, confirming the epidemiological link between CS-exposure and the chronic form of PCM. However, we also show that this effect is transitory, being detected between 4- and 12-weeks post-infection but not thereafter. The possible immune mechanisms that mediate this effect and the reasons for its transitory effect are discussed.

Paracoccidioidomycosis; Cigarette smoke; Subclinical infection; Immune response; Risk factors

INTRODUCTION

Paracoccidioidomycosis (PCM) may present as an acute/subacute clinical form, characterized by a progressive disease arising from the airborne initial infection, or, most often, as an asymptomatic or subclinical infection that may manifest later on in an individual’s life, the chronic form. Epidemiological studies of PCM aiming to identify factors that lead the infected individual to manifest the disease have shown a strong association between smoking and the development of the chronic form of PCM11. Lemle A, Wanke B, Miranda JL, Kropf GL, Mandel MB, Mandel S. Pulmonary function in paracoccidioidomycosis (South American blastomycosis): an analysis of the obstructive defect. Chest. 1983;83:827-8.

2. Vieira GD, Alves TC, Lima SM, Camargo LM, Sousa CM.. Paracoccidioidomycosis in a western Brazilian Amazon State: clinical-epidemiologic profile and spatial distribution of the disease. Rev Soc Bras Med Trop. 2014;47:63-8.

3. Pina DR, Alvarez M, Giacomini G, Pavan AL, Guedes CI, Cavalcante RS, et al. Paracoccidioidomycosis: level of pulmonary sequelae in high resolution computed tomography images from patients of two endemic regions of Brazil. Quant Imaging Med Surg. 2017;7:318-25.
- 44. Paniago AM, Aguiar JI, Aguiar ES, Cunha RV, Pereira GR, Londero AT, et al. Paracoccidioidomicose: estudo clínico e epidemiológico de 422 casos observados no Estado de Mato Grosso do Sul. Rev Soc Bras Med Trop. 2003;36:455-9. . The epidemiological link between PCM and smoking is reinforced by a case-control study in which the authors demonstrated that smokers were 14 times more likely to develop the disease among patients from an endemic area in Brazil. Besides, the intensity of tobacco consumption also influences the disease, indicating that individuals who smoked twenty or more cigarettes/day became ill on average eight years earlier than the others55. Santos WA, Silva BM, Passos ED, Zandonade E, Falqueto A. Associação entre tabagismo e paracoccidioidomicose: um estudo de caso-controle no Estado do Espírito Santo, Brasil. Cad Saude Publica. 2003;19:245-53. .

It is well established that cigarette smoke has a strong impact on immunity, affecting several aspects of both the innate and adaptive immune responses66. Strzelak A, Ratajczak A, Adamiec A, Feleszko W. Tobacco smoke induces and alters immune responses in the lung triggering inflammation, allergy, asthma and other lung diseases: a mechanistic review. Int J Environ Res Public Health. 2018;15:1033. , 77. Qiu F, Liang CL, Liu H, Zeng YQ, Hou S, Huang S, et al. Impacts of cigarette smoking on immune responsiveness: up and down or upside down? Oncotarget. 2017;8:268-84. . Current evidence demonstrates that some cigarette components have immunosuppressive properties that could be implicated in increasing susceptibility to PCM66. Strzelak A, Ratajczak A, Adamiec A, Feleszko W. Tobacco smoke induces and alters immune responses in the lung triggering inflammation, allergy, asthma and other lung diseases: a mechanistic review. Int J Environ Res Public Health. 2018;15:1033. , 77. Qiu F, Liang CL, Liu H, Zeng YQ, Hou S, Huang S, et al. Impacts of cigarette smoking on immune responsiveness: up and down or upside down? Oncotarget. 2017;8:268-84. , as it has been demonstrated for tuberculosis (TB)88. Quan DH, Kwong AJ, Hansbro PM, Britton WJ. No smoke without fire: the impact of cigarette smoking on the immune control of tuberculosis. Eur Respir Rev. 2022;31:210252. , 99. López-Hernández Y, Rivas-Santiago CE, López JA, Mendoza-Almanza G, Hernandez-Pando R. Tuberculosis and cigarette smoke exposure: an update of in vitro and in vivo studies. Exp Lung Res. 2018;44:113-26. , a chronic granulomatous disease with some similarities to PCM. Strikingly, up to 10% of the CF PCM patients in Brazil concurrently present pulmonary TB1010. Quagliato Júnior R, Grangeia TA, Massucio RA, De Capitani EM, Rezende SM, Balthazar AB. Association between paracoccidioidomycosis and tuberculosis: reality and misdiagnosis. J Bras Pneumol. 2007;33:295-300. . Moreover, a previous study showed that monocytes isolated from CF PCM patients displayed an enhanced expression of components of the NLRP3-inflammasome, probably associated with the patients’ exposure to smoke and hypoxemia1111. Amorim BC, Pereira-Latini AC, Golim MA, Ruiz Júnior RL, Yoo HH, Arruda MS, et al. Enhanced expression of NLRP3 inflammasome components by monocytes of patients with pulmonary paracoccidioidomycosis is associated with smoking and intracellular hypoxemia. Microbes Infect. 2020;22:137-43. .

In order to mitigate PCM within the smoke-exposed population, it is important to understand the mechanisms through which cigarette smoke (CS) exposure increases the risk of developing the CF of the disease. In the current study, we addressed this significant knowledge gap and investigated the effects of cigarette smoke exposure (CS) experimentally in a murine model of a non-progressive form of PCM.

MATERIALS AND METHODS

Animal use and ethics statement

Specific-pathogen-free 6- to 8-week-old male C57BL/6 mice were obtained from the Animal Facility at the School of Medicine of the University of Sao Paulo (USP) in pathogen-free conditions with ad libitum access to chow and water. The National Council for the Control of Animal Experimentation (CONCEA) guidelines were strictly followed over the course of this project. All experimental protocols were approved by the Review Board for Human and Animal Studies at the School of Medicine of the University of Sao Paulo (USP), registry Nº 066/17, and the Institute of Tropical Medicine, registry Nº 000169A.

Fungus and inoculum preparation

The standard, virulent isolate P. brasiliensis 18 (Pb18) was used. It was obtained from reinoculated mice as a means to ensure its virulence as previously described1212. Brummer E, Restrepo A, Hanson LH, Stevens DA. Virulence of Paracoccidiodes brasiliensis: the influence of in vitro passage and storage. Mycopathologia. 1990;109:13-7. , and was maintained on semisolid Fava Netto’s culture medium for 7 days at 37 °C. To prepare the inoculum used in the experiments, Pb18 was harvested from the tubes and washed three times with phosphate buffered saline (PBS, pH 7.2). Thereafter, large and agglutinated yeast cells were separated by decanting and the small, isolated yeast cells were collected and counted by hemocytometer. The yeast cells used for intratracheal (IT) infection were >95% viable as determined by Trypan blue staining.

Inoculum standardization and intratracheal infection

Infection by the IT route was performed in C57BL/6 mice anesthetized intraperitoneally using a 200 μL solution of 80 mg/kg of Ketamine and 10 mg/kg of Xylazine. Under anesthesia, a small longitudinal incision was made in the neck, exposing the trachea, and 50 μL of PBS containing variable concentrations of the Pb18 were injected. The inoculum size (1022. Vieira GD, Alves TC, Lima SM, Camargo LM, Sousa CM.. Paracoccidioidomycosis in a western Brazilian Amazon State: clinical-epidemiologic profile and spatial distribution of the disease. Rev Soc Bras Med Trop. 2014;47:63-8. yeast cells/mouse) used throughout the study was determined in pilot experiments (see Results). After 2 weeks of infection, the mice were randomly divided into two groups and transferred to the conventional area in the animal facility at the Institute of Tropical Medicine, USP. The experiments were performed twice independently, with each experimental group (NC and NCS) comprising 36 animals, with similar results. The data shown represent the combined results of the two experiments.

Cigarette smoke exposure

The C57BL/6 mice that had been IT infected two weeks earlier were split into two groups, one of which was exposed to CS in a whole animal exposure system using a modified cigarette smoke exposure device from Biselli et al .1313. Biselli PJ, Lopes FD, Moriya HT, Rivero DH, Toledo AC, Saldiva PH, et al. Short-term exposure of mice to cigarette smoke and/or residual oil fly ash produces proximal airspace enlargements and airway epithelium remodeling. Braz J Med Biol Res. 2011;44:460-8. . The cigarette smoke exposure was performed in a 28-L plastic box with two inlets (for air and smoke), one outlet and a fan (for the enhancing air and smoke mixture inside the box). In the first inlet, we applied synthetic air (10 L/min). The second inlet received synthetic air (1.5 L/min) that passed through a Venturi valve connected to a lit cigarette that suctioned the cigarette smoke and pumped it inside the box. The mice were exposed twice daily for 120 min, 5 days a week to 12-16 cigarettes with filters removed for 20 weeks (100 days of actual CS exposure). The flow rate was set at 1.5 L/min, which generally resulted in blood levels of carboxyhemoglobin (COHb) of 10–15% after the first exposure, as determined in pilot experiments to mimic the levels found in moderate smokers1414. Stewart RD, Baretta ED, Platte LR, Stewart EB, Kalbfleisch JH, Van Yserloo B, et al. Carboxyhemoglobin levels in American blood donors. JAMA. 1974;229:1187-95. . Peripheral blood COHb was measured up to 20 min after blood collection using the Radiometer ABL800 Flex equipment (Radiometer, Bronshoi, Denmark). The other group, not exposed to CS, formed the infected-only control group (NCS). All mice were weighed before and at the end of the CS exposure period.

Fungal burden in the lungs of infected mice

Mice from each group were euthanized 4, 12 and 20 weeks after infection and one of the lungs (left and right side) was removed to analyze the fungal burden by colony forming units (CFU), and the other for histopathological analysis (below). A total of 20-24 animals from each group and time point was used. The lung was weighed, mashed and homogenized in PBS. Aliquots of 100 μL were plated onto Brain Heart Infusion (BHI) agar supplemented with 5% spent supernatant from P. brasiliensis Pb192 cultures, 10 IU/ml Streptomycin-Penicillin (Cultilab, Brazil), 4% fetal bovine serum (Gibco), and 500 mg/ml Cycloheximide (Sigma, St. Louis, MO, United States)1515. Singer-Vermes LM, Burger E, Russo M, Vaz CA, Calich VL. Advances in experimental paracoccidioidomycosis using an isogenic murine model. Arch Med Res. 1993;24:239-45. . CFUs were determined every 5 days for 15 days of incubation at 37 °C. The number (log10) of viable P. brasiliensis colonies per gram of tissue was expressed as mean ± standard error (SE). To assess fungal dissemination to liver and spleen, these organs were removed from 12 animals from each group and time point and processed as above.

Histological analysis

Histopathology studies were performed in weeks 4, 12 and 20 after infection in the other lung ( n = 20-24 for each group and time point). The lung was removed, fixed in 10% formalin and embedded in paraffin. Five-micrometer sections were stained with hematoxylin-eosin (H&E) for histological analysis. Pathological changes were analyzed based on the size, morphology and cell composition of granulomatous lesions, presence of fungi and intensity of the inflammatory infiltrates. Morphometrical analysis was performed using the AxioVision release 4.8.2 SP3 software (Carl Zeiss Microscopy, White Plains, NY, USA) (×10 magnification). The area of granulomatous lesions (µm22. Vieira GD, Alves TC, Lima SM, Camargo LM, Sousa CM.. Paracoccidioidomycosis in a western Brazilian Amazon State: clinical-epidemiologic profile and spatial distribution of the disease. Rev Soc Bras Med Trop. 2014;47:63-8. ) was measured in 20 microscopic high-power fields per slide. Results were expressed as the mean ± standard error (SEM) of the total area of lesions for each animal. The area corresponding to 25% of the total lung area was determined by the AxioVision software.

Immunohistochemical analysis (IHC)

Deparaffinized lung sections (4 µm thick) from the lungs excised from 12 animals from each group and time point were incubated for 18h with the primary antibody at 4 °C, to detect the antigens CD4 (Santa Cruz, clone sc-13573, dilution 1:50); CD8 (Invitrogen, clone MA170041, dilution 1:50); TNF (Santa Cruz, clonesc-52746, dilution 1:90000); IFN-γ (Bioss, clone bs-0480R, dilution 1:40); IL-10 (Santa Cruz, clone sc-73309, dilution 1:50); MAC-2 (Cedarlene, clone CL8942AP, dilution 1:25000) and CD68 (Biorbyt, clone Orb-10343, dilution 1:200). Antigen retrieval was performed with TRIS-EDTA at pH 9.0, except for MAC2 and CD68, for which antigen retrieval was performed with citrate at pH 6.0. The amplification of the reaction was achieved by the polymer molecules conjugated to peroxidase (Reveal, Biotin-Free Polyvalent horseradish peroxidase diaminobenzidine; Spring Bioscience, Pleasanton, CA, USA, code SPD-125) and chromogen (Dako Liquid DAB (DAB) + substrate chromogen system; Dako, Carpenteria, CA, USA, code K3460). Finally, the slides were counterstained with Harris hematoxylin (Merck, Darmstadt, Germany). The immunostained cells in the granulomas, with brownish cytoplasm, were counted as positive for each primary antibody, also considering the lymphocyte typical morphology for CD4/CD8 staining, and the histiocyte typical morphology for CD68/MAC-2 staining. Consecutive high power fields (400x) were used to quantify positive immune cells, considering the entire circumference of each granuloma. IHC counts were performed in a blinded fashion regarding the CS exposure by two pathologists (ANDN and FLL).

Statistical analysis

GraphPad Prism 7 software (GraphPad, San Diego, CA, United States) was used to build the graphics. The results were expressed as the mean values ± standard error of the mean (SEM). The variables analyzed were weight, CFU, affected granulomas area and IHC markers. To run the statistical analysis, Student’s t-test was used for continuous or quantitative variables with normal distribution as determined by the Shapiro–Wilk’s normality test. The Mann–Whitney’s test was used for variables that do not have a normal distribution. Fisher’s exact test was used for non-continuous variables. The level of statistical significance adopted was 0.05.

RESULTS

Inoculum standardization

The established mouse models of PCM aimed to mimic the human disease are based on the IT challenge with high fungal inoculum, usually ranging from 105to 106 yeast cells1515. Singer-Vermes LM, Burger E, Russo M, Vaz CA, Calich VL. Advances in experimental paracoccidioidomycosis using an isogenic murine model. Arch Med Res. 1993;24:239-45. . Although we know neither the initial steps of the infection in humans nor the fungal burden to which infected people from endemic areas have been exposed, it goes without saying that 105 to 106 yeast cells inoculum is greatly overestimated. For instance, the TB mouse models typically employ intratracheal inoculum of 102 M. tuberculosis bacilli1616. Gonzalez-Juarrero M, Turner OC, Turner J, Marietta P, Brooks JV, Orme IM. Temporal and spatial arrangement of lymphocytes within lung granulomas induced by aerosol infection with Mycobacterium tuberculosis. Infect Immun. 2001;69:1722-8. , 1717. Shang S, Ordway D, Henao-Tamayo M, Bai X, Oberley-Deegan R, Shanley C, et al. Cigarette smoke increases susceptibility to tuberculosis: evidence from in vivo and in vitro models. J Infect Dis. 2011;203:1240-8. . We thus sought to establish an inoculum size that could achieve a subclinical-like pulmonary infection. Inoculation experiments with 104 to 106 yeast cells all resulted in the involvement of ≥25% of the lung area and lung CFU ≥ 5x105/g tissue, by week 12 post-infection (Supplemental Figure S1). Surprisingly, the 103-inoculum also yielded a CFU around 5x105 cells/g of tissue, reaching 25% of the lung area affected. Only a 102-inoculum resulted in a low degree, “subclinical”-like, infection, with ≤ 104 CFU and <25% area involved at all three times of the study: 4, 12 and 20 weeks post infection.

Effects of CS on fungal burden, area of lung involvement, and fungal dissemination

We obtained an average level of COHb of 14.45 ± 1.50% in CS mice during the experiments, which is within the range expected for moderate smokers1414. Stewart RD, Baretta ED, Platte LR, Stewart EB, Kalbfleisch JH, Van Yserloo B, et al. Carboxyhemoglobin levels in American blood donors. JAMA. 1974;229:1187-95. . The group that was not exposed showed an average COHb level of 1.22 ± 0.24%. During the 20 weeks of experiments, we observed a marked weight increment in the NCS group while the CS group did not show significant weight gain, which resulted in a significantly lower weight at 12 and 20 weeks ( Figure 1A ). Nevertheless, we observed neither increased mortality nor altered behavior (e.g., water and food intake, nocturnal dynamic activity, etc.) during the study period in the CS mice compared to the NCS mice.

Figure 1
Body weight (A), lung colony forming units (B), total lung area affected by the granulomatous inflammatory response (C), and rate of fungal dissemination (D) over time in the cigarette smoke exposed (●) and non-exposed (●) groups. A, B and C: data shown as mean and standard error; Mann-Whitney or Student’s t-test was used. D) Fisher’s exact test was used. *p < 0.05; **p < 0.01; ***p < 0.001; + p = 0.054.

The NCS group showed small but detectable foci of granulomatous response as early as 4 weeks. These foci had markedly expanded by 12 weeks, slightly decreasing by week 20; at all three time-points, the involvement corresponded to less than 25% of the total lung area ( Figure 1B ). The CS group also presented small foci of granulomatous response at 4 weeks, which increased to involve >25% of the total lung area at 12 weeks, returning to lower levels by week 20 ( Figure 1B ). CFU counts showed as early as 4 weeks, a significantly higher fungal burden in the CS than the NCS group ( Figure 1C ). The fungal burden consistently increased in both groups from week 4 to week 12, with the CS group persisting with higher values, although this difference did not reach statistical significance (5.18±0.22 vs 4.49±0.27, p = 0.054). We observed a reduction in the CFU at 20 weeks, when both groups were comparable.

Dissemination to other organs (spleen and liver) was virtually absent in both groups at week 4 ( Figure 1D ). However, by week 12, dissemination to either one organ was observed in 66.7% of the mice in the CS group compared with only 16.7% in NCS mice ( p = 0.0078). By week 20 the dissemination rate in the CS group decreased to 24.0%, close to that of the NCS group (16.7%).

Histology and IHC analyses

Within four weeks, we observed diffuse foci of histiocytes, epithelioid cells, lymphocytes, and some neutrophils aggregated around the yeast cells, identifiable as the granuloma’s initial structures, usually close to an airway ( Figure 2 ). With disease progression, from week 12 onwards, well-organized granulomas composed of histiocytes, epithelioid cells, and multinuclear giant cells aggregated around the yeasts, and surrounded by a border of lymphocytes were observed. It should be noted that no alterations outside the granulomatous response could be found in the lung parenchyma, such as scarring fibrosis or other evidence of tissue repair, using H&E and Gomori’s stainings. The overall 4-week lung cell counts tended to be low, in line with the smaller affected areas due to smaller and less mature granulomas ( Figure 2 ). IHC analyses showed, in both groups and at the three-time points, a marked infiltration of cells of the myeloid lineage (either recently migrated monocytes, macrophages or dendritic cells), identified as MAC-2+ or CD68+ cells. Interestingly, after 4 weeks, MAC-2+ cells predominated over CD68+ cells, but from week 12 onwards, with granuloma maturation, CD68+ cells started to predominate ( Figure 3 ) while MAC-2 counts tended to decrease. Furthermore, the CS group exhibited significantly lower density of MAC+ cells than the NCS group at the three-time points, while no differences between the two groups were seen with CD68 staining ( Figure 3 ).

Figure 2
HE staining of the lung granulomatous response over time in the cigarette smoke exposed (top row) and non-exposed (bottom row) groups. At week 4, most granulomas in both groups were small and/or incipient; P. brasiliensis yeast cells were already found within them, more numerous in exposed (arrows) than non-exposed mice; at 12 and 20 weeks, the granulomas were in general well-formed, with epithelioid histiocytes, multinucleated giant cells and an external rime of lymphocytes surrounding numerous typical P. brasiliensis yeast cells. Granulomas were in general associated with an airway (indicated by the symbol ∗), as the animals were infected through intratracheal inoculation. Magnification: 200x. Scale bar: 100 mm.

Figure 3
Immunohistochemistry analyses of the lungs. Counts over time of CD68 and MAC-2 expressing cells with histiocyte morphology within the granulomas in the cigarette smoke exposed (●) and non-exposed (●) groups. Data shown as mean and standard error; Mann–Whitney’s test was used; *p < 0.05.

In contrast, there were small numbers of infiltrating CD4 and CD8 lymphocytes, ranging between 10-30 cells/mm3, without significant differences between CS and NCS groups at the three-time points for both subsets (data not shown). Some differences were noted regarding cytokine expression within the granulomas at week 12, but not with week 4 and week 20 lungs in the two groups ( Figure 4 ). Higher expression of IL-10 was seen in CS than NCS mice, while there was no difference regarding IFN-γ expression. Interestingly, the IFN-γ vs. IL-10 balance, critical for the regulation of the immune response in both human and experimental PCM1818. Benard G. Pathogenesis and classification of paracocidioidomycosis: new insights from old good stuff. Open Forum Infect Dis. 2021;8:ofaa624.

19. Benard G, Romano CC, Cacere CR, Juvenale M, Mendes-Giannini MJ, Duarte AJ. Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis. Cytokine. 2001;13:248-52.
- 2020. Cacere CR, Romano CC, Mendes Giannini MJ, Duarte AJ, Benard G. The role of apoptosis in the antigen-specific T cell hyporesponsiveness of paracoccidioidomycosis patients. Clin Immunol. 2002;105:215-22. , distinguished the two groups: by week 4 the IFN-γ/IL-10 ratio was lower in NCS compared with the CS (3.0 vs 8.4, p = 0.02) ( Figure 5 ). With disease progression (12 weeks) the ratio sharply increased to 9.4 in NCS mice but sharply decreased to 1.8 in CS mice ( p = 0.0047), correlating with the less controlled course of the disease in the former. By week 20, both groups had similar IFN-γ/IL-10 ratios (~3.5). In addition, significantly greater TNF expression was found in week 12 lungs of CS mice, possibly reflecting the enhanced inflammatory status at this time point in CS-exposed animals ( Figure 4 ).

Figure 4
Immunohistochemistry analyses of the lungs. Counts over time of IFN-γ, IL-10 and TNF expressing cells within the granulomas in the cigarette smoke exposed (●) and non-exposed (●) groups. Data shown as mean and standard error; Mann–Whitney’s test was used; *p < 0.05; **p < 0.01.

Figure 5
Ratio of IFN-g-expressing cells/IL-10-expressing cells within the granulomas over time in the cigarette smoke exposed (●) and non-exposed (●) groups; Data shown as mean and standard error; Mann–Whitney’s test was used; *p < 0.05.

Histological changes related to CS

In the fourth week of the experiment, the control group’s lung tissue, not exposed to cigarette smoke, had alveolar septa and bronchiolar epithelium within the normal morphological aspect. In contrast, in the lung tissue of mice exposed to cigarette smoke for two weeks, we could already observe high columnar epithelium and a slight dysplasia in it. At 12 and 20 weeks, we did not observe any lung tissue changes in the unexposed group’s respiratory epithelium. In contrast, among the group exposed to cigarette smoke, we observed moderate dysplasia of the columnar epithelium and increased mucus production ( Figure 6 ).

Figure 6
HE staining of the airway epithelial alterations along the cigarette smoke exposure (4, 12 and 20 weeks). The left column (A-C) shows non- P. brasiliensis infected/non-exposed control mice. The middle column (D-F) shows the dysplasia of the ciliary epithelium (multilayered cells, with larger and hyperchromatic nuclei and coarse chromatin) in the exposed group (arrows), and the right column (G-I) shows increased mucus production in this group (indicated by the symbol ∗). Magnification: 100x (A, B); 200x (G); 400x (C, D, E, F, H, I). Scale bar: 20 mm (C, D, E, F, H, I); 50 mm (G); 100 mm (A, B).

DISCUSSION

Although the epidemiological evidence strongly supports the hypothesis that smoking may lead to the development of the pulmonary CF of PCM11. Lemle A, Wanke B, Miranda JL, Kropf GL, Mandel MB, Mandel S. Pulmonary function in paracoccidioidomycosis (South American blastomycosis): an analysis of the obstructive defect. Chest. 1983;83:827-8.

2. Vieira GD, Alves TC, Lima SM, Camargo LM, Sousa CM.. Paracoccidioidomycosis in a western Brazilian Amazon State: clinical-epidemiologic profile and spatial distribution of the disease. Rev Soc Bras Med Trop. 2014;47:63-8.

3. Pina DR, Alvarez M, Giacomini G, Pavan AL, Guedes CI, Cavalcante RS, et al. Paracoccidioidomycosis: level of pulmonary sequelae in high resolution computed tomography images from patients of two endemic regions of Brazil. Quant Imaging Med Surg. 2017;7:318-25.

4. Paniago AM, Aguiar JI, Aguiar ES, Cunha RV, Pereira GR, Londero AT, et al. Paracoccidioidomicose: estudo clínico e epidemiológico de 422 casos observados no Estado de Mato Grosso do Sul. Rev Soc Bras Med Trop. 2003;36:455-9.
- 55. Santos WA, Silva BM, Passos ED, Zandonade E, Falqueto A. Associação entre tabagismo e paracoccidioidomicose: um estudo de caso-controle no Estado do Espírito Santo, Brasil. Cad Saude Publica. 2003;19:245-53. , no study to date has demonstrated the possible mechanism for this association. In our study, we evaluated for the first time the influence of smoking in a murine experimental model of CF pulmonary PCM. We showed that cigarette smoke exposure alters, albeit transitorily, the course of the mycosis in our mouse model.

A CS effect was discernible as early as 2 weeks after exposure, i.e., four weeks post-infection, when the lung CFU in exposed animals was significantly higher than in their non-exposed counterparts. However, the magnitude and characteristics of lung inflammation were still comparable. The granulomatous response was sparse (as expected for 4 weeks of infection2121. Cano LE, Kashino SS, Arruda C, André D, Xidieh CF, Singer-Vermes LM, et al. Protective role of gamma interferon in experimental pulmonary paracoccidioidomycosis. Infect Immun. 1998;66:800-6. ), affecting only ~3% of the total lung area. Furthermore, IHC analyses of this initial granulomatous response in CS and NCS mice showed that both were populated predominantly by myeloid lineage cells rather than by CD4 and CD8 lymphocytes. Monocytes predominated at week 4 (but were overcome onwards by macrophages, with granuloma maturation). 4-week CS and NCS lungs exhibited higher expression of IFN-γ than IL-10 and TNF; however, the IFN-γ/IL-10 ratio was much higher in the CS group. Strikingly, by 12 weeks, although the CD4 and CD8 lymphocytes infiltration did not change from 4 weeks, CS led to significantly wider affected lung areas, persistently higher CFUs, and a sharp reduction in the IFN-γ/IL10 ratio. As a correlate of the less controlled infectious process in the CS mice, TNF expression was also higher than in NCS mice.

However, the follow-up in week 20 showed that the CS exposure immunological effects ceased, with both CS and NCS mice showing the same infectious features. The affected lung area and CFU decreased to levels comparable with those of NCS (which also decreased slightly), as did the IFN-γ/IL10 ratio. Furthermore, TNF expression by the granulomas persisted highly, but was also of similar magnitude between CS and NCS mice. Our hypothesis for the transitory CS effect is that it was overcome by the genetic background of the mouse strain, which characteristically leads to the control of the infection in the long term2222. Calich VL, Singer-Vermes LM, Siqueira AM, Burger E. Susceptibility and resistance of inbred mice to Paracoccidioides brasiliensis. Br J Exp Pathol. 1985;66:585-94. .

Considering our experimental model, mice were exposed to cigarette smoke after two weeks of infection with P. brasiliensis , more truly mimicking what happens to patients: it is estimated that the patients are infected at young ages, before starting a smoking habit2323. Martinez R. Epidemiology of paracoccidioidomycosis. Rev Inst Med Trop Sao Paulo. 2015;57 Suppl 19:11-20. , 2424. Franco M, Montenegro MR, Mendes RP, Marques SA, Dillon NL, Mota NG. Paracoccidioidomycosis: a recently proposed classification of its clinical forms. Rev Soc Bras Med Trop. 1987;20:129-32. . We decided to wait 14 days, the time necessary for the infection to be established2121. Cano LE, Kashino SS, Arruda C, André D, Xidieh CF, Singer-Vermes LM, et al. Protective role of gamma interferon in experimental pulmonary paracoccidioidomycosis. Infect Immun. 1998;66:800-6. . Furthermore, exposure to cigarette smoke was carried out according to the modified protocol by Biselli et al .1313. Biselli PJ, Lopes FD, Moriya HT, Rivero DH, Toledo AC, Saldiva PH, et al. Short-term exposure of mice to cigarette smoke and/or residual oil fly ash produces proximal airspace enlargements and airway epithelium remodeling. Braz J Med Biol Res. 2011;44:460-8. and regardless of when the infection occurred, our mice maintained COHb levels within the desired range, which would correspond to an at least moderate smoker during the 20 weeks of experiments. This monthly control of COHb is crucial when discussing our results’ validity since these mice were not exposed to toxic doses related to smoking. As observed in other experimental models2525. Gentry-Nielsen MJ, Top EV, Snitily MU, Casey CA, Preheim LC. A rat model to determine the biomedical consequences of concurrent ethanol ingestion and cigarette smoke exposure. Alcohol Clin Exp Res. 2004;28:1120-8. , the exposed animals lost more weight than the unexposed group regardless of the inoculum administered, demonstrating that our experiment correctly simulated a whole-body exposure to cigarette smoke.

The possibility that the increase in the prevalence of diseases associated with cigarette smoke may, in part, be due to changes induced by tobacco smoke in the immune and inflammatory processes was recognized for the first time in the 1960s2626. Holt PG, Keast D. Environmentally induced changes in immunological function: acute and chronic effects of inhalation of tobacco smoke and other atmospheric contaminants in man and experimental animals. Bacteriol Rev. 1977;41:205-16. . However, recent reviews recognize that the influence of cigarette smoke on the immune system is complex due to its chemical heterogeneity and dual nature, pro-inflammatory and immunosuppressive66. Strzelak A, Ratajczak A, Adamiec A, Feleszko W. Tobacco smoke induces and alters immune responses in the lung triggering inflammation, allergy, asthma and other lung diseases: a mechanistic review. Int J Environ Res Public Health. 2018;15:1033. , 77. Qiu F, Liang CL, Liu H, Zeng YQ, Hou S, Huang S, et al. Impacts of cigarette smoking on immune responsiveness: up and down or upside down? Oncotarget. 2017;8:268-84. . Understanding CS-induced effects is further complicated by the sometimes-contradictory results of human vs. experimental studies, the individual genetic susceptibility, and the variability in experimental designs such as time, frequency and mode of exposure66. Strzelak A, Ratajczak A, Adamiec A, Feleszko W. Tobacco smoke induces and alters immune responses in the lung triggering inflammation, allergy, asthma and other lung diseases: a mechanistic review. Int J Environ Res Public Health. 2018;15:1033. . CS effects in our PCM model concern particularly adaptive immune response, since the mice were first infected and only 2 weeks later submitted to CS. Regarding this response, the weight of evidence points to CSE dampening the Th1 type and promoting the Th2 type immune responses66. Strzelak A, Ratajczak A, Adamiec A, Feleszko W. Tobacco smoke induces and alters immune responses in the lung triggering inflammation, allergy, asthma and other lung diseases: a mechanistic review. Int J Environ Res Public Health. 2018;15:1033. , which markedly impact the course and outcome of some infectious diseases. This was best studied in TB88. Quan DH, Kwong AJ, Hansbro PM, Britton WJ. No smoke without fire: the impact of cigarette smoking on the immune control of tuberculosis. Eur Respir Rev. 2022;31:210252. , 99. López-Hernández Y, Rivas-Santiago CE, López JA, Mendoza-Almanza G, Hernandez-Pando R. Tuberculosis and cigarette smoke exposure: an update of in vitro and in vivo studies. Exp Lung Res. 2018;44:113-26. , where it was shown that M. tuberculosis -mediated inhibition of DC function is exacerbated by smoking. CS-exposure led to decreased IL-12-production but enhanced IL-10-production by DCs, which was linked to reduced influx of IFN-γ-producing and TNF-producing CD4+ and CD8+ effector and memory T-cells1717. Shang S, Ordway D, Henao-Tamayo M, Bai X, Oberley-Deegan R, Shanley C, et al. Cigarette smoke increases susceptibility to tuberculosis: evidence from in vivo and in vitro models. J Infect Dis. 2011;203:1240-8. . In addition, CS has several direct detrimental impacts on T-cell function and proliferation and can induce T-cell apoptosis2727. Hernandez CP, Morrow K, Velasco C, Wyczechowska DD, Naura AS, Rodriguez PC. Effects of cigarette smoke extract on primary activated T cells. Cell Immunol. 2013;282:38-43. . The net result is enhanced permissiveness to M. tuberculosis , with increased mycobacterial loads88. Quan DH, Kwong AJ, Hansbro PM, Britton WJ. No smoke without fire: the impact of cigarette smoking on the immune control of tuberculosis. Eur Respir Rev. 2022;31:210252. , 99. López-Hernández Y, Rivas-Santiago CE, López JA, Mendoza-Almanza G, Hernandez-Pando R. Tuberculosis and cigarette smoke exposure: an update of in vitro and in vivo studies. Exp Lung Res. 2018;44:113-26. .

CD68+ is a specific marker of blood monocytes and tissue macrophages as it spares DC, while MAC-2 is expressed by all three cell types2828. Chistiakov DA, Killingsworth MC, Myasoedova VA, Orekhov AN, Bobryshev YV. CD68/macrosialin: not just a histochemical marker. Lab Invest. 2017;97:4-13. , 2929. Dumic J, Dabelic S, Flögel M. Galectin-3: an open-ended story. Biochim Biophys Acta. 2006;1760:616-35. . Interestingly, we observed in both the CS and NCS groups greater granuloma infiltration by MAC-2+ cells than by CD68+ cells early in the infection, and the reverse after its progression (20 weeks), when mature CD68+ macrophages predominate. A conceivable explanation for the higher MAC-2 counts is DC infiltration in the granulomas early in the infection. In addition, we also found sustained higher MAC-2+ cell counts in the NCS group than the CS group. This suggests an impaired infiltration of DC in the latter, which concurs with the described defective antigen presentation by DC caused by cigarette smoke exposure88. Quan DH, Kwong AJ, Hansbro PM, Britton WJ. No smoke without fire: the impact of cigarette smoking on the immune control of tuberculosis. Eur Respir Rev. 2022;31:210252. , 1717. Shang S, Ordway D, Henao-Tamayo M, Bai X, Oberley-Deegan R, Shanley C, et al. Cigarette smoke increases susceptibility to tuberculosis: evidence from in vivo and in vitro models. J Infect Dis. 2011;203:1240-8. . This putative DC deficiency caused by CS may be further linked to the findings observed from week 4 to week 12 of the infection in the CS group: (i) higher fungal load, (ii) increase in the lung area affected by the granulomatous response, (iii) increased dissemination to other organs, and (iv) sharp decrease in the IFN-γ/IL10 ratio as compared with non-exposed mice. Unfortunately, no staining with DC specific markers was included in the study to explore this mechanism. The net CS effects were, however, less persistent than those seen in TB models since by week 20 there were no marked differences between CS and NCS mice.

Some factors may help to explain the differences between PCM and TB. Firstly, M. tuberculosis is likely more pathogenic than Paracoccidioides spp: 10% of the M. tuberculosis -infected persons will develop the disease, while it is roughly estimated that the PCM-disease will develop in less than 1% of the Paracoccidioides -infected population in endemic areas1818. Benard G. Pathogenesis and classification of paracocidioidomycosis: new insights from old good stuff. Open Forum Infect Dis. 2021;8:ofaa624. , 1919. Benard G, Romano CC, Cacere CR, Juvenale M, Mendes-Giannini MJ, Duarte AJ. Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis. Cytokine. 2001;13:248-52. . Secondly, as opposed to our experimental protocol, the TB-CS association studies in mice were designed with CS taking place before infection, thus deeply affecting innate immune responses. Third, the mouse strain used, C57Bl/6 was shown to resist, at least partially, to a 1055. Santos WA, Silva BM, Passos ED, Zandonade E, Falqueto A. Associação entre tabagismo e paracoccidioidomicose: um estudo de caso-controle no Estado do Espírito Santo, Brasil. Cad Saude Publica. 2003;19:245-53. yeast cells intraperitoneal inoculum2121. Cano LE, Kashino SS, Arruda C, André D, Xidieh CF, Singer-Vermes LM, et al. Protective role of gamma interferon in experimental pulmonary paracoccidioidomycosis. Infect Immun. 1998;66:800-6.: C57Bl/6 mice inoculated showed no difference in survival rate as compared with control animals, and our unpublished data show that the fungal load decreases further after 20 weeks in both CS and NCS animals (unpublished data). We thus may infer that our CS protocol was not able to overcome the innate resistant behavior of this strain, as witnessed by the progressive increase in the density of mature CD68+ macrophage cells within the granulomas in both CS and NCS mice, potentially contributing to the progressive control of the infection (along with other ill-defined features of the protective anti- Paracoccidioides adaptive immune response of this strain). This is in contrast with the TB mice model, where a chronic infection persists with up to 300 days of infection characterized by a high M. tuberculosis load1717. Shang S, Ordway D, Henao-Tamayo M, Bai X, Oberley-Deegan R, Shanley C, et al. Cigarette smoke increases susceptibility to tuberculosis: evidence from in vivo and in vitro models. J Infect Dis. 2011;203:1240-8. . In the human PCM infection, it was clearly shown that the balance between the IL-12-IFN-γ axis and IL-10 drives the clinical presentation of the disease1919. Benard G, Romano CC, Cacere CR, Juvenale M, Mendes-Giannini MJ, Duarte AJ. Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis. Cytokine. 2001;13:248-52. , 2020. Cacere CR, Romano CC, Mendes Giannini MJ, Duarte AJ, Benard G. The role of apoptosis in the antigen-specific T cell hyporesponsiveness of paracoccidioidomycosis patients. Clin Immunol. 2002;105:215-22. , 3030. Castro LF, Ferreira MC, Silva RM, Blotta MH, Longhi LN, Mamoni RL. Characterization of the immune response in human paracoccidioidomycosis. J Infect. 2013;67:470-85. , while in mice IL-10 has also been shown to be a major player3131. Costa TA, Bazan SB, Feriotti C, Araújo EF, Bassi E, Loures FV, et al. In pulmonary paracoccidioidomycosis IL-10 deficiency leads to increased immunity and regressive infection without enhancing tissue pathology. PLoS Negl Trop Dis. 2013;7:e2512. .

When comparing with the TB study by Shang et al .1717. Shang S, Ordway D, Henao-Tamayo M, Bai X, Oberley-Deegan R, Shanley C, et al. Cigarette smoke increases susceptibility to tuberculosis: evidence from in vivo and in vitro models. J Infect Dis. 2011;203:1240-8. , we observed similarities in the initial inflammatory response characterized by a mild perivascular infiltrate and predominant peribronchiolar infiltrates of lymphocytes. The exposed mice showed thickening of the alveolar septa wall during the experiment, a more diffuse inflammatory infiltrate of macrophages, lymphocytes, and neutrophils. However, unlike the experimental model of tuberculosis, granulomas remained well-formed and compact in our study despite being more prominent in size in the exposed group. It is also interesting to note that the histological changes related to smoking were shown earlier, with 30 days of exposure, compared with the result of the area occupied by granulomas, in which the differences between the groups were more evident only after 10 weeks of exposure.

Our work has some limitations. Firstly, we observed individual variability in the results within the groups. This likely occurred due to the 1022. Vieira GD, Alves TC, Lima SM, Camargo LM, Sousa CM.. Paracoccidioidomycosis in a western Brazilian Amazon State: clinical-epidemiologic profile and spatial distribution of the disease. Rev Soc Bras Med Trop. 2014;47:63-8. -inoculum used: Paracoccidioides spp have a marked tendency to form clumps, eventually altering the final inoculum, despite our efforts and checks to avoid this issue. This variability may have precluded the observation of more significant differences between the two groups. In the same token, although we observed that the value of 100 yeasts apparently met the goal of generating a subclinical infection, perhaps the inoculum dose was too low to overcome the genetic background of the C57Bl/6 strain: the mice were able to control the disease at 20 weeks, despite the additional effect of smoking. Thirdly, it is worth noting that the mouse model of IT infection does not truly mimic the pathology of the human chronic pulmonary infection: the typical mouse’s large granulomas with nests of numerous yeast cells are not usually found in the lungs of patients, while many features of the patients’ lung histopathology are absent in mice, such as tissue necrosis, fibrosis, caseous necrosis and granulomatous inflammation, all intermixed and with a dispersed, lower number of fungal elements3232. Tuder RM, el Ibrahim R, Godoy CE, De Brito T. Pathology of the human pulmonary paracoccidioidomycosis. Mycopathologia. 1985;92:179-88. .

CONCLUSION

In conclusion, we showed that CS exposure can alter the course of the disease in a subclinical pulmonary PCM mouse model, providing a putative mechanism underlying the close link between a smoking habit and the development of the chronic form of PCM. However, the CS exposure probably did not overcome the background for resolution of the infection in the long-term, characteristic of the mouse strain. While further studies are warranted to further clarify this issue, we believe that public health policies aimed at reducing the cigarette smoke burden in highly endemic areas will likely also reduce the PCM burden.

ACKNOWLEDGMENTS

We would like to thank Tania A. Costa for the gift of the Pb18 isolate, Vera L. Calich, Fernanda D. T. Q. S. Lopes and Rafael Raposo for support with the experimental design, and Thalyta N. C. Pinto for expert editorial assistance.

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  • FUNDING: This work was supported by Fapesp, grant Nº 2016/08730-6. GB and CPT are Senior Researchers from CNPq. FLBS and GCH had a Scientific Initiation fellowship from Fapesp (grant Nº 2017/12374-3) and Capes, respectively.

Publication Dates

  • Publication in this collection
    14 Nov 2022
  • Date of issue
    2022

History

  • Received
    22 July 2022
  • Accepted
    26 Sept 2022
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