nocardia brasiliensis induces an immunosuppressive
TRANSCRIPT
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Nocardia brasiliensis induces an immunosuppressive 1
microenvironment in BALB/c mice favoring chronic infection 2
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Adrian G. Rosas-Taraco, Amira R. Perez-Liñan, Paola Bocanegra-Ibarias, Luz I. Perez-4
Rivera and Mario C. Salinas-Carmona. 5
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Universidad Autonoma de Nuevo Leon, UANL, Department of Immunology, Faculty of 7
Medicine and University Hospital. Monterrey, Nuevo Leon. Mexico. 8
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Running Title: Immunosuppression in Nocardia brasiliensis infection 10
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Keywords: Nocardia brasiliensis, Cytokines, Lymphocyte subpopulations. 12
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Address correspondence to Mario Cesar Salinas-Carmona, Universidad Autonoma de 15
Nuevo Leon, UANL, Department of Immunology, Faculty of Medicine and University 16
Hospital. Gonzalitos 235 Norte. Mitras Centro. Monterrey, Nuevo Leon. Mexico. Phone 17
+52(81)83294211, Fax +52(81)833310-58. E-mail address: [email protected] 18
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.06307-11 IAI Accepts, published online ahead of print on 30 April 2012
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ABSTRACT 26
Nocardia brasiliensis (Nbr) is an intracellular microorganism and the most common 27
etiologic agent of actinomycetoma in the Americas. Several intracellular pathogens 28
induce an immunosuppressive microenvironment through increases in CD4+Foxp3+ 29
regulatory T cells (Treg), thus downregulating other T cell subpopulations and assuring 30
survival in the host. In this study, we determined if Nbr modulates T lymphocyte 31
responses and their related cytokine profiles in a murine experimental model. We also 32
examined the relationship between Nbr immunomodulation and pathogenesis and 33
bacteria survival. In early infection, Th17/Tc17 cells were increased at day 3 (P<0.05) 34
in footpad tissue and spleen. Treg subpopulations peaked at days 7 and 15 (P<0.01) in 35
footpad and spleen, respectively. TGF-β1 and IL-10 are cytokines known for their 36
immunosuppressive effects. During early and chronic infection, these cytokines were 37
elevated with increased TGF-β1 levels from days 3 to 30 (P<0.01) and sustained IL-10 38
expression throughout infection as compared with uninfected mice. IL-6 production was 39
increased at day 3 (P<0.01), whereas IFN-γ, IL-17A and IL-23 were highest at day 15 40
post-infection (P<0.01) when a decrease in the bacterial load (>1 log) was also observed 41
(P<0.05). Following these changes, at 30-60 days post-infection, IFN-γ production was 42
decreased while the expression of anti-inflammatory cytokines and the bacterial load 43
again increased (P<0.05). The increment in Treg cells and the related cytokine profile 44
correlated with reduced inflammation at day 15 (P<0.05) in the footpad. We concluded 45
that Nbr modulates the immune system to induce an immunosuppressive 46
microenvironment that benefits its survival during the chronic stage of infection. 47
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Introduction 51
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Nocardia brasiliensis (Nbr) is a gram-positive, partially acid-fast bacterium belonging 53
to the order of actinomycetales. In Mexico, Nbr has been isolated in 86.6% of 54
actinomycetoma cases (22). The infection occurs by accidental inoculation of bacteria in 55
the skin or cutaneous tissue (32). Actinomycetoma caused by Nbr is a chronic disease 56
that occurs in immune-competent individuals, however, some studies suggest the 57
possibility that subtle and continuous decay in immune protection may be responsible 58
for the chronic infection (8). 59
60
Others studies have shown that the protective host immune response to Nbr involves 61
cell-mediated immunity (24). IgM, but not IgG, monoclonal antibodies also confer 62
protection against experimental actinomycetoma (10). In the early stages of infection, 63
there is a local infiltration of polymorphonuclear cells followed by phagocytic and 64
mononuclear cells to the site of infection. Nbr is an intracellular bacterium that survives 65
and multiplies inside macrophages (34). Little is known concerning how Nbr escapes 66
from microbicidal mechanisms developed by the host. 67
68
Using a murine model of Nbr, Salinas-Carmona et al. (1999) demonstrated that the Th1 69
and Th2 type immunity are involved in the host response against Nbr. The same study 70
reported the presence of Nbr antibodies in the sera of mice after infection with Nbr (34). 71
Studies have also reported that IgM is involved in the partial protection against 72
experimental actinomycetoma in BALB/c mice (10, 33). 73
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Th17 and Tc17 lymphocytes are characterized by their production of IL-17, which is a 75
proinflammatory cytokine with an important role in chronic inflammatory diseases, 76
such as arthritis, myocarditis, asthma and allergies (47). IL-17 induces the secretion of 77
antimicrobial peptides, including defensins and S100 proteins, which are important 78
factors in the host response against bacteria (14, 18, 20). Th17 cell differentiation in 79
murine models of chronic infection with Mycobacterium tuberculosis (Mtb) and 80
Mycobacterium bovis (16, 17, 47) is dependent on transforming growth factor β (TGF- 81
β), IL-6 and IL-23. Early protection in the lungs against influenza A (12) is also 82
dependent on the presence of Tc17 and Th17 cells. 83
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Treg cells maintain the balance between anti-inflammatory and pro-inflammatory 85
responses to protect the host from exacerbated inflammation and subsequent pathology. 86
Treg produce IL-10 and TGF-β cytokines, which are associated with anti-inflammatory 87
processes (5). These cells can suppress Th1, Th2 and Th17 functions (9) and may also 88
inhibit anti-microbial activity in phagocytic cells, thus promoting microorganism 89
survival. Treg are also found during the chronic stages of Mtb infection (35), suggesting 90
that they may contribute to the persistent stages of Mtb infection by suppressing the 91
immune response. Little is known about the microenvironment induced by Nbr in an 92
experimental actinomycetoma model, especially with regard to T cell 93
immunomodulation. 94
95
In this study, we evaluated changes in the Treg, Th17 and Tc17 populations, along with 96
their related cytokine profiles, during the course of an experimental intracellular Nbr 97
infection in BALB/c mice and whether these changes play a role in the 98
immunopathogenesis of the disease. Here, we demonstrated for the first time that Nbr 99
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induces high levels of Th17 and Tc17 lymphocytes and inflammatory cytokines (IL-6, 100
IFN-γ, IL-17A and IL-23) in the early stages of infection in situ. However, evidence of 101
an immunosuppressive microenvironment (IL-10 and TGF-β1) was found in both the 102
early and chronic stages of infection. Therefore, we demonstrated that Nbr modulates 103
the immune system to favor its survival and persistence in the host. 104
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Materials and Methods 106
107
Mice 108
Eight- to twelve-week-old female BALB/c mice were used for this study. Animals were 109
derived from the colony kindly donated by Carl Hansen (Small Animal Section, 110
Veterinary Resources Branch, National Institutes of Health, Bethesda, MD) and 111
maintained under BSL2 conditions at the Department of Immunology. Mice received 112
sterile water and Purina rodent food ad libitum. Protocols for animal care and infections 113
were developed according to the International Review Board regulations and Mexican 114
regulations (NOM-062-ZOO-1999) and were approved by The Bioethics Committee of 115
the Facultad de Medicina at the Universidad Autonoma de Nuevo Leon . 116
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Experimental Infection 118
Actinomycetoma infection was induced in mice by injecting 1x106 CFU of Nbr into a 119
rear footpad as previously described (5). Footpad (n=5) and spleen tissues from each 120
animal were surgically removed at 0, 3, 7, 15, 30, and 60 days post-infection. Footpads 121
and spleens were processed to obtain cell suspensions for flow cytometry analysis. 122
Footpads were homogenized for bacterial load (in 5 ml of saline solution), cytokine (in 123
1 ml of 10 mM of PBS pH 7.2-7.4) or gene expression analysis (in 1 ml of Trizol). 124
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Inflammation observed in the footpads and spleen of each mouse was registered at each 125
time point indicated during the course of the study. The results shown in this study are 126
representative of three independent experiments with similar experimental designs. 127
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Footpad digestion 129
Footpads were surgically removed and placed in complete RPMI medium (cRPMI: 130
RPMI 1640 supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 10 mM 131
HEPES, 1% nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin and 132
10% fetal bovine serum) containing collagenase XI (0.7 mg/ml; Sigma-Aldrich) and 133
type IV bovine pancreatic DNase (30 μg/ml; Sigma-Aldrich) prior to incubation for 30 134
minutes at 37°C. The digested tissue was disrupted by gently pushing the tissue sample 135
through a cell strainer (BD Biosciences, Lincoln Park, NJ). Red blood cells were lysed 136
with Gey’s solution, washed and resuspended in cRPMI. Total cell numbers per foot 137
pad were determined using a hemocytometer. 138
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Flow cytometric analysis 140
Cell suspensions from the footpad and spleen containing 1x106 cells/ml from each 141
mouse were incubated with mAbs conjugated to Fluorescein isothiocyanate [FITC], 142
Phycoerythrin [PE], or Allophycocyanin [APC] purchased from eBioscience (San 143
Diego, CA). These cells were stained for cell surface markers by incubating with mAbs 144
recognizing CD4 (clone RM4-5) and CD8 (clone eBioH35-17.2) molecules at 4°C for 145
30 minutes in the dark. After washing with PBS, intracellular staining was performed 146
using mAbs against IL-17A (clone eBio17B7) or Foxp3 (clone FJK-16s). Samples were 147
incubated again at 4°C for 30 minutes in the dark then washed with PBS and 148
resuspended in 500µl of FACS flow solution (BD Biosciences). Data acquisition and 149
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analysis for this study were completed using a FACS Calibur cytometer (BD 150
Biosciences, Mountain View, CA) and Cell Quest Pro software (BD Biosciences, San 151
Jose, CA), respectively. Analyses were performed with an acquisition of at least 10,000 152
total events in the cell gate. Results are reported following background subtraction. 153
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ELISA assays 155
Footpad samples were homogenized and cytokine quantification was determined by 156
ELISA following the manufacturer’s protocol for TGF-β1 (R&D Systems, MN), IL-6, 157
IL-17A, and IL-23 (eBioscience, CA). Each sample was analyzed in triplicate. 158
159
Gene expression assays 160
The footpad was homogenized in Trizol and immediately frozen at -80°C. RNA was 161
extracted following the manufacturer’s protocol (Invitrogen, Carlsbad, CA). DNA was 162
digested with RQ1 RNase-free DNase (Promega, Madison,WI) and RNA was re-163
isolated with Trizol. Finally, the concentration of RNA in each sample was determined 164
by spectrophotometry, and 2 µg of RNA was reverse transcribed with M-MuLV 165
(Invitrogen, Carlsbad, CA) and random hexamers (Invitrogen, Carlsbad, CA). Real-time 166
PCR was performed using 1 µl of cDNA, iQ supermix (Biorad, Hercules, CA) and 167
specific primers and Taqman probes (table 1 supplied by Applied Biosystems, Foster 168
City, CA) in a CFX96 thermocycler (Biorad, Hercules, CA) to evaluate the relative 169
mRNA expression of IL-10, TGF-β1, and IFN-γ. GAPDH was used to normalize the 170
expression levels. Amplification conditions were as follows: 50°C for 2 min, 95°C for 2 171
min and 40 cycles of denaturation at 95°C for 15 s, annealing and extension at 58°C for 172
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30 s. Cytokine mRNA level were normalized to GAPDH levels using the ΔΔCt method 173
to calculate relative changes. 174
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Bacterial load 176
Bacterial loads in the footpads of mice were determined by plating serial dilutions of the 177
tissue homogenized on brain heart infusion agar and counting colony-forming units 178
(CFU) after one-week incubation at 37°C. 179
180
Statistical Analysis 181
The results presented are representative of three experiments. The data are expressed as 182
the mean± SEM values (n=5) from triplicate assays. One-way analysis of variance and 183
Tukey’s post-hoc test were used. Calculations were performed using GraphPad Prism 184
version 4.00 for Windows (San Diego, CA). P-values <0.05 were considered 185
significant. 186
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RESULTS 198
Treg and Th17/Tc17 lymphocytes are present during the early stages of N. 199
brasiliensis infection 200
Treg and Th17/Tc17 lymphocytes in the footpads of experimentally infected BALB/c 201
mice were studied by flow cytometry. Treg and Th17/Tc17 lymphocytes were identified 202
by their characteristic phenotypes of CD4+Foxp3+, CD4+IL-17A+ and CD8+IL-17A+, 203
respectively. The percentage of Treg in the footpads (figure 1B) of Nbr-infected mice 204
were higher in samples obtained after day 7 post-infection when compared with cell 205
suspensions obtained from the control group (0.4340% ± 0.0777 vs. 0.002% ± 0.002, 206
P<0.001). However, the percentage of Treg lymphocytes (figure 2B) in spleens 207
appeared to be highest in the samples obtained at 15 days post-infection when compared 208
with the same population from the control group mice (0.1220 ± 0.02780 vs. 0± 0, 209
P<0.001). Cell suspensions obtained from the footpads at 3 days post-infection 210
contained the highest percentages of Th17 and Tc17 cells (0.2840 ± 0.03250 vs. 0.0040 211
± 0.0024, P<0.001 and 0.2120 ± 0.0140 vs. 0.0040 ± 0.0024, P<0.001) when compared 212
with the control group mice (P<0.05) (figure 1C and D). Meanwhile, cells analyzed 213
from spleens demonstrated that the percentages of Th17 and Tc17 lymphocytes (figure 214
2C and D) obtained at 3 days post-infection were higher than those observed in control 215
mice (0.0960 ± 0.0175 vs. 0 ± 0, P<0.05 and 0.43 ± 0.04 vs. 0 ± 0, P<0.05, 216
respectively). 217
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Immunosuppressive microenvironment during Nbr infection 221
We studied whether the changes in the percentages of Treg and Th17/Tc17 cells 222
observed in the footpad during the early stages of Nbr infection in BALB/c mice were 223
also accompanied by changes in the cytokine profile in the same tissue. Using ELISA 224
and qPCR, we investigated the levels of IL-6, IL-17A, IL-10, IL-23, and TGF-β1 225
cytokines in samples obtained from homogenized footpad tissue. The concentration of 226
IL-6 in samples from day 3 post-infection footpads was higher than in the control group 227
(358.1 ± 38.75 vs. 1.097 ± 0.0975, P<0.001). After day 3 post-infection, the 228
concentration of IL-6 decreased (figure 3A). The levels of IL-17A in control mice were 229
higher than those observed in the footpads of mice in the early stages of infections 230
(P<0.05); however, once the infection was established, IL-17A increased at 15 days 231
post-infection (P<0.05) (figure 3B). The concentration of IL-23 cytokine in the same 232
samples was found to be higher than in the control group at day 15 post-infection (155.6 233
± 35.07 vs. 53.15 ± 13.29, P<0.05) (figure 3C). Interestingly, the expression of IL-10 in 234
the Nbr-infected animals was higher than that of the controls at every time point 235
(P<0.05) (figure 3D). The concentrations of TGF-β1 protein and mRNA were elevated 236
in all samples obtained between 3-30 days post-infection and decreased in samples 237
obtained at day 60 post-infection (P<0.05) (figure 3E and 3F). 238
239
Reduction in inflammation paralleled with Treg increment 240 241 Progressive inflammation, abscess formation and tissue destruction were observed 242
during Nbr infection in mouse footpads as previously reported (34). The inflammation 243
in the footpad was measured throughout the infection using a vernier, and the 244
inflammation score was calculated using the ellipsoid equation. At 7 days post-245
infection, the lesions reached 0.23 ± 0.03 cm. The lesions decreased 70% in size at day 246
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15 post-infection when Treg cells were present in high numbers as compared with 247
infected mice at day 7 (figure 4A). However, the lesions increased in size again at day 248
60 post-infection (1.10 ± 0.37 cm; P<0.05). Infection with Nbr also resulted in 249
splenomegaly, thus, we measured the size of the spleen from each mouse. Interestingly, 250
the changes in spleen size followed a similar trend as that described for the footpads. 251
Thus, the size of the spleen was lower after 15 days post-infection than at 60 days when 252
the size of the spleen increased by 75% (figure 4B). 253
254
IFN-γ is not capable of controlling bacterial load 255
IFN-γ is essential in the control of bacteria growth due to activation of bactericidal 256
mechanisms in macrophages (7). Thus, we questioned if the levels of expression of 257
IFN-γ correlated with bacterial load. For this purpose, samples obtained from the 258
footpads of each mouse were processed for qPCR or CFU analysis and used to 259
determine the levels of IFN-γ expression and bacterial load, respectively. These 260
analyses demonstrated that in samples obtained from the footpads at 3-15 days post-261
infection, IFN-γ expression was higher than in similar samples from the control group 262
(P<0.05). Increased IFN-γ expression in these samples correlated with a >1 log 263
reduction in bacterial load (P<0.05). Most importantly, after 15 days of infection the 264
bacterial load in the footpads samples increased again, while IFN-γ expression 265
decreased in the following days (P<0.05) (figure 5A). Meanwhile, higher levels of IL-266
10 were correlated with bacterial load during chronic infection (figure 5B). 267
268
Discussion 269
Our studies have demonstrated that BALB/c mice experimentally infected with Nbr 270
develop actinomycetoma. This chronic infection is characterized by progressive 271
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inflammation, abscess formation and tissue destruction. We found alterations in the 272
Treg, Th17, and Tc17 cell populations and cytokine profiles in the footpads of mice at 273
different stages of Nbr infection. Furthermore, these changes correlated with the course 274
of the immunopathogenesis of actinomycetoma induced by Nbr. 275
276
We found that the percentages of Th17 and Tc17 cells increased at 3 days post-infection 277
but then were down-regulated when the Treg population emerged. This close 278
relationship of Th17, Tc17 and Treg cells during the early stages of infection suggests 279
that Treg lymphocytes may participate in the regulation of Th17 and Tc17 lymphocytes. 280
Furthermore, the cytokine profile of immunosuppressive (TGF-β1 and IL-10) and 281
protective cytokines (IFN-γ) along with sustained bacterial load observed during the 282
infection also support a regulatory role for Treg lymphocytes during this infection. 283
Thus, TGF-β1 and IL-10 cytokines are known to be expressed by Treg cells and can 284
suppress IFN-γ function with subsequent increases in bacterial load. Our data 285
demonstrated the same trend in the upregulation of Treg numbers, TGF-β1, and IL-10 286
with concomitant downregulation of IFN-γ and augmentation of bacterial load in the 287
lesions developed in the footpads of Nbr-infected mice. 288
289
It is well known that TGF-β1 and IL-6 are important cytokines involved in Th17 and 290
Tc17 lymphocyte differentiation (11, 13). As shown here, elevated levels of TGF-β1 291
and IL-6 at day 3 post-infection in footpad and spleen tissues correlated with our 292
findings of increased numbers of Th17 and Tc17 lymphocytes. 293
294
In the spleen, Tc17 lymphocytes were higher than Th17 and Treg cells, at least in the 295
first 7 days post-infection. Differences among these three subpopulations were 296
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measured until day 30, however, Nbr has not been observed in the spleen (data not 297
shown), which could explain the apparent T cell activation. A possible explanation for 298
these findings is that the soluble antigens produced by Nbr may reach the blood flow 299
and activate T cells in this organ. 300
301
In our study, IL-6 peaked at 3 days post-infection and decreased significantly thereafter, 302
which is in agreement with the emergence of Treg cells (mainly IL-10-producing cells) 303
and upregulated IL-10 expression. This finding may be explained by the suppressive 304
actions of IL-10 on IL-6 as IL-10 is known to downregulate IL-6 production in 305
monocytes activated by lipopolysaccharide/IFN-γ in vitro (6). However, decreased IL-6 306
levels do not explain the presence of the Th17 and Tc17 subpopulations after 3 days 307
post-infection. In this study, IL-23 was elevated at days 7 and 15, which may be related 308
to Th17 lymphocyte maintenance during Nbr infection. It has been demonstrated that 309
IL-23 produced by Mtb-infected dendritic cells stimulates the production of IL-17 by γδ 310
T cells and other populations (21). 311
312
We found elevated levels of IL-17A expression in uninfected mice when compared with 313
Nbr-infected mice. In agreement with our results, other authors have detected high 314
levels of IL-17A in uninfected mice with γδ T cells as the source of this cytokine (39, 315
48). Those findings could partially explain the high concentration of IL-17A at day 0 in 316
our experiment. In flow cytometry analysis, we observed other cell populations positive 317
for IL-17A. IL-17A-producing alveolar macrophages in asthmatic patients have been 318
reported and it has been suggested that those cells are also involved in the inflammatory 319
process (40). In the experimental actinomycetoma model, macrophages are important 320
cells recruited into the lesion site to phagocytose the bacteria. Our data suggests that 321
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recruited macrophages may produce IL-17A and contribute to the inflammatory process 322
in this model. Recent publications demonstrated that IL-17 deficient mice are unable to 323
control Mtb infection. In the present work, we report high levels of IL-17 in early stage 324
of the Nbr infection, suggesting that this cytokine may be playing a role in early control 325
of infection. IL-17 is a potent inducer of chemokines, some of them participate in the 326
recruitment of neutrophil and macrophages, that in the mycobacterial animal model 327
contribute to control infection and granuloma formation (26, 37, 44). Nbr induces 328
granuloma formation characterized by recruitment of neutrophils, macrophages and 329
lymphocytes similar to tuberculosis. In the Nbr early infection, IL-17 is increased 330
suggesting that they are responsible for recruiting neutrophils. 331
332
High levels of CD4+Foxp3+ Treg cells were found at days 7 and 15, when IL-10 333
expression and TGF-β1 levels were also high. During day 15, footpad inflammation was 334
reduced, which may be associated with the high levels of Treg cells and related 335
cytokines. Therefore, the environment produced by the Treg lymphocytes could favor 336
Nbr replication after inoculation as has been demonstrated in several tuberculosis 337
models. Marin et al. found high levels of Treg lymphocytes in patients with active 338
pulmonary tuberculosis when compared with latent TB patients. In the same study, it 339
was reported that Treg lymphocytes inhibited IFN-γ production, but not Th17 340
production (23). Those findings are in agreement with our results, which show the 341
downregulation of IFN-γ expression by the immunosuppressive environment produced 342
by Treg lymphocytes and a reduced expression of IL-17A during infection. Another 343
study reported an association between the IL-10 produced by Treg cells with anergy 344
status to tuberculin skin test (TST) found in patients with active tuberculosis (3). 345
Shafiani et al. reported that Treg cells depress T cell-mediated immune responses to the 346
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protective mycobacterial antigen HBHA during active TB in humans (38). These data 347
have demonstrated the role of Treg lymphocytes in infection and chronic disease. Other 348
studies using parasites have demonstrated that Plasmodium vivax and Strongyloides 349
ratti induce regulatory T cells, which have been associated with high parasite load and 350
impaired parasite clearance, respectively (2, 4, 15). Furthermore, it was demonstrated 351
that regulatory T cells favor persistent Salmonella infection (15). However, inactivation 352
of Treg cells during Mtb infection increases cytokine production but not the bacterial 353
load (29). 354
355
Our results suggest that there is a synergistic effect between TGF-β1 and IL-10 during 356
Nbr infection. However, TGF-β1 is also an important cytokine involved in the 357
differentiation of the studied lymphocyte subpopulations. In tuberculosis studies, it has 358
been described that TGF-β1 downregulates phagocytic receptors and suppresses 359
antimicrobial activity (43, 45, 46). Our results are in agreement with those found in 360
tuberculosis models. Although Treg cells are important source of IL-10, many other 361
cells produce this cytokine, including macrophages (25) and dendritic cells (42). In the 362
early stage of infection, a high number of Treg cells and elevated IL-10 were detected; 363
however, these cells were diminished after 15 days post-infection. These data suggest 364
that IL-10 detected during the following days could have been produced by 365
macrophages and dendritic cells in the lesion site. IL-10 has been associated with the 366
susceptibility with several intracellular infections, such as Mtb (41), Listeria 367
monocytogenes (27) and Salmonella (28). It is known that IL-10 suppresses the 368
mycobactericide mechanisms, and IL-10 deficient mice showed an increase protection 369
to Mtb infection (30, 36). Moreover, IL-10 has been implicated in the promotion of the 370
tuberculosis progression (1). Here we demonstrated IL-10 levels increased during Nbr 371
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infection, and a correlation between IL-10 and bacterial load was observed in chronic 372
infection. Redford et al. proposed that IL-10 is associated with Mtb ability to evade the 373
immune response and its persistence in the lung (31). The findings presented here are in 374
agreement with these authors using an intracellular bacterium such as Nbr that also 375
induces high levels of IL-10 creating an immunosuppressive environment to evade the 376
immune response favoring its growth and persistence in the footpad. 377
378
Although Th1 lymphocytes were not assessed in our study, IFN-γ expression was 379
analyzed (main Th1 cytokine). IFN-γ levels were highest at 15 days post-infection when 380
the bacterial load decreased by >1 log thereafter suggesting an effective host Th1 381
response. Elevated levels of IL-17A and IL-23 were found at the same time point. 382
Interestingly, a significant increment in bacterial load in the subsequent days was 383
associated with a reduction in the levels of IFN-γ and Th17-related cytokines and a 384
predominantly immunosuppressive environment in the lesion site. These findings 385
suggest that those Th1- and Th17-related cytokines could be playing an important role 386
in bacterial load control. Regulatory T cells inhibit γδ T cells to produce IFN-γ in 387
response to Mtb antigen (19). 388
389
In summary, we report here that Nbr modulates the Th17/Tc17 and Treg cells in the 390
early stages of infection and induces an immunosuppressive microenvironment in 391
BALB/c mice (in the early and chronic stages of the infection) favoring bacterial growth 392
and chronic disease. 393
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ACKNOWLEDGMENTS 397
This work was supported by a grant to AGRT from Programa para el mejoramiento del 398
Profesorado (PROMEP). Paola Bocanegra-Ibarias received a scholarship from 399
PROMEP. We thank Dr. Mercedes Gonzalez-Juarrero for reading the manuscript and 400
providing insightful comments. We are grateful to Alejandra Gallegos and Reynaldo 401
Rodriguez for technical support. 402
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macrophages by suppressing STAT1 activation and accelerating iNOS protein 571 degradation. Genes to cells : devoted to molecular & cellular mechanisms 572 11:871-882. 573
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588 589 590
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Table 1. Primers and Taqman probes were purchased from Applied Biosystem. 606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
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629
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Figures 631
632
Figure 1. T cells dynamic during Nocardia brasiliensis infection in mice footpad. 633
Percentage of T cells subpopulations were obtained using specific monoclonal 634
antibodies and flow cytometry analysis. A) Representative dot plots show selected 635
region and specific staining to studied T cell subpopulation. B) CD4+Foxp3+ (Treg) 636
cells were increased at day 7 of infection. C) and D) CD4+IL-17A+ (Th17) and 637
CD8+IL-17A+ (Tc17) cells were highest at day 3. E) Three subpopulations dynamic 638
during Nbr infection. Data represent the mean of five mice per group ± SEM. Statistics 639
analysis were done using one-way ANOVA and the post-hoc test was Tukey’s, 640
*P<0.05, **P<0.01. The data are representative of three experiments of similar 641
experimental design. 642
643
644
Figure 2. Spleen T cells dynamic during Nocardia brasiliensis infection. Percentage 645
of T cell subpopulations were obtained using specific monoclonal antibodies and flow 646
cytometry analysis. A) Representative dot plots show selected region and specific 647
staining to studied T cell subpopulation. B) CD4+Foxp3+ (Treg) cells were increased at 648
day 15 of infection. C and D) CD4+IL-17A+ (Th17) and CD8+IL-17A+ (Tc17) cells 649
were highest at day 3. E) Three subpopulations dynamic during Nbr infection. Data 650
represent the mean of five mice per group ± SEM. Statistics analysis were done using 651
one-way ANOVA and the post-hoc test was Tukey’s, *P<0.05, **P<0.01. The data 652
presented in this figure are representative of three experiments of similar experimental 653
design. 654
655
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Figure 3. Immunosuppressive environment is predominant during Nbr. Footpad 656
samples were collected and processed for ELISA or qPCR. A) Graph shows 657
concentration of IL-6 in footpad homogenates from each group of mice. B) and C) IL-658
17A and IL-23 show higher levels at day 15 of infection. D) IL-10 expression was 659
elevated during N. brasiliensis infection. E and F) TGF-β1 levels by ELISA and qPCR 660
increase during the first 30 days of infection. Data represent the mean of five mice per 661
group ± SEM. Statistics analysis were done using one-way ANOVA and the post-hoc 662
test was Tukey’s, *P<0.05, **P<0.01. The data are representative of three experiments 663
of similar experimental design. 664
665
Figure 4. CD4+Foxp3+ Treg cells reduce inflammation of mice footpad and spleen. 666
A) The continuous line represents the foot inflammation calculated though ellipse 667
formula and the discontinuous line represent the percentage of CD4+Foxp3+ (Treg) 668
cells. B) The continuous line represents the spleen inflammation calculated though 669
ellipse formula and the discontinuous line represent the percentage of CD4+Foxp3+ 670
(Treg) cells. Data represent the mean of five mice per group ± SEM. Statistics analysis 671
were done using one-way ANOVA and the post-hoc test was Tukey’s, *P<0.05. The 672
data are representative of three experiments of similar experimental design. 673
674
675
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677
678
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Figure 5. Increase of IFN-γ was not capable to control bacterial load in N. 680
brasiliensis infection. A) The continuous line represents the bacterial load obtained 681
from mice footpad using plating serial dilutions and the discontinuous line represents 682
the IFN-γ expression during Nbr infection. B) The continuous line represents the 683
bacterial load obtained from mice footpad using plating serial dilutions and the 684
discontinuous line represents the IL-10 expression during Nbr infection. Data represent 685
the mean of five mice per group ± SEM. Statistics analysis were done using one-way 686
ANOVA and the post-hoc test was Tukey’s, *P<0.05. The data are representative of 687
three experiments of similar experimental design. 688
689
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Table 1.
Gene
Product
(Applied Biosystem)
GAPDH Mm03302249_g1
IL-10 Mm00439614_m1
TGF-β1 Mm00441729_g1
IFN-γ Mm01168134_m1
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