Development of a Guinea Pig Immune Response-Related Microarray and Its Use To Define the Host Response following Mycobacterium bovis BCG Vac
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感染与免疫杂志 2006年第2期
Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom
ABSTRACT
Immune responses in the guinea pig model are understudied because of a lack of commercial reagents. We have developed a custom-made guinea pig oligonucleotide microarray (81 spots) and have examined the gene expression profile of splenocytes restimulated in vitro from Mycobacterium bovis BCG-vaccinated and naive animals. Eleven genes were significantly (P < 0.05) up-regulated following vaccination, indicating a Th1-type response. These results show that microarrays can be used to more fully define immune profiles of guinea pigs.
TEXT
The guinea pig (Cavia porcellus) animal model is used widely in medical research for the study of many human diseases caused by bacteria, including tuberculosis (8), anthrax (24), and Q fever (44). Guinea pigs are also used to study viruses (e.g., genital herpes [13], Ebola [35], and cytomegalovirus [31]) and human immune disorders, such as asthma (25), allergy (39), and osteoarthritis (3). In this work, guinea pigs are frequently utilized to study disease pathology, vaccine efficacy, and treatment effectiveness. However, despite their widespread use, few commercial reagents are available for studying their immune response (27).
The guinea pig model of tuberculosis is widely recognized as an important preclinical indicator of the protective capacity of new vaccines (28). New vaccine candidates are tested against the existing "gold standard" vaccine, Mycobacterium bovis BCG (bacille Calmette-Guerin, an attenuated strain of M. bovis). The immune response evoked by these novel tuberculosis vaccines is rarely assessed because of the shortage of guinea pig immune reagents. By using the few immunological tools available, such as bioassays (6, 43), guinea pig-specific antibodies (21, 23, 41), and molecular techniques, e.g., real-time PCR (1, 18, 42), Northern blot analysis (14, 15, 26), and semiquantitative PCR (19, 26), our understanding of tuberculosis immunology in this model has improved. Many of the molecular studies, however, have analyzed the mRNA expression of only a small number of genes (one to nine genes) at any one time. Consequently, we have developed a custom-made oligonucleotide microarray to analyze the mRNA expression of multiple cytokines and immune-related genes in the guinea pig.
Studies of mice and humans have shown that protective immunity against tuberculosis depends on an acquired cellular immune response (2, 29), involving various T-cell subsets (16) where a Th1-type response is considered favorable (29). This develops in a complex environment of chemokines and proinflammatory cytokines secreted by macrophages, dendritic cells, and inflammatory cells that reside in, or are recruited to, the site of infection (7). This complexity makes it difficult to define a precise immunological correlate of protective immunity as induced by vaccination. It is possible, therefore, that a pattern of cytokines and chemokines may be more representative of a diseased or immune status.
In this study, we used BCG vaccination as a model to investigate the utility of our guinea pig 50-mer oligonucleotide microarray (81 spots). The immune profile of splenocytes from BCG-vaccinated guinea pigs restimulated in vitro with purified protein derivative of Mycobacterium tuberculosis (PPD) was analyzed and compared with the immune pattern from splenocytes of naive animals. The microarray data were supplemented with information about total bioactive interferon (IFN), bioactive tumor necrosis factor alpha (TNF-), and interleukin-8 (IL-8) proteins in order to understand the whole biological system. The microarray approach enabled the definition of an immune profile of BCG vaccination in the guinea pig model.
Animals and vaccination. Outbred Hartley strain guinea pigs from David Hall, Burton-on-Trent, United Kingdom, were either vaccinated subcutaneously (n = 5) with 5 x 104 CFU BCG Pasteur (36) or left untreated (n = 5) (naive controls). Guinea pigs were rested for 20 weeks before being used in these studies.
Preparation of splenocytes and culture. Splenocytes were prepared and treated with ACK lysis buffer to remove red blood cells as described previously (14). Cells (4 x 107 in 6 ml medium) were either stimulated with 1,670 units/ml PPD (Evans Vaccines Ltd., Liverpool, United Kingdom) or left untreated (unstimulated). The cultures were incubated in a 5% CO2 atmosphere at 37°C for 24 h. Smaller cultures of 1 x 107 cells in 1.5 ml medium were stimulated with either concanavalin A (10 μg/ml; Sigma) (internal positive control) for 18 and 24 h or PPD (1,670 units/ml) for 18 h or left untreated for 18 h. After incubation, all cells were centrifuged for 7 min at 145 x g. The supernatant was removed and stored at –70°C in aliquots for protein testing at a later time. The cell pellet from the 24-h cultures was resuspended in RLT buffer (QIAGEN, Crawley, United Kingdom) supplemented with 1% -2 mercaptoethanol (Sigma). RNeasy columns (QIAGEN) were used to purify total RNA from the cells, and this RNA was further concentrated using RNA MinElute columns (QIAGEN).
Microarray procedures. The LabelStar kit (QIAGEN) was used for the labeling and purification of target cDNA according to the manufacturer's instructions; sample total RNA (5 μg) was used as a template for reverse transcriptase in the presence of an oligonucleotide deoxyribosylthymine primer and Cy3- and Cy5-labeled dCTPs (Pfizer-Pharmacia, Kent, United Kingdom). Dye swap slide procedures, for which the Cy3 and Cy5 labels were reversed (two slides), were performed using the RNA from each animal.
The purified cDNA was mixed with 120 μl of preheated (42°C) salt-based hybridization solution (MWG Biotech, Milton Keynes, United Kingdom), and the mixture was heated in a water bath at 95°C for 3 min. The reaction mixture was cooled slightly and centrifuged before being added to the small Gene Frame (21 by 22 mm) (ABgene, Epsom, United Kingdom) on the microarray slide. The slide was incubated in a sealed hybridization cassette (TeleChem International) and submerged in a water bath at 42°C for 17 h. After hybridization, the slides were washed three times and dried by centrifugation at 520 x g for 5 min. Scanning of the slides was performed using a dual-laser scanner (Affymetrix 428; MWG Biotech) at various amplification (6-7) settings (gains) above and below saturation of the most intensely fluorescent spots on each array.
ImaGene 5.5.4 (BioDiscovery, CA) software was used to quantify the scanned images. The median pixel intensity of the spot was taken after background correction. These data were processed using the MAVI Pro 2.6.0 software package (MWG Biotech) and then imported into R/BioConductor (9) and analyzed using functions in the linear models for microarray data (limma) library (33, 34). The data were normalized using print-tip loess normalization, with significant up-weighting given to control spots. Then, a representative value for each gene for each slide was obtained by taking the median of the normalized log ratios of the six replicate spots. Moderated t statistics were generated, and resulting P values were adjusted using the Benjamini and Hochberg step-up method for controlling the false discovery rate (4). Genes were considered to be statistically significant if they had at least 1.4-fold up- or down-regulation and had a P value of <0.05 after correction for multiple testing. The normalized data were also analyzed using significance analysis of microarrays (SAM) (37). One-class SAM t tests were performed on each group, and a two-sample SAM t test was used to compare the BCG and naive groups.
Microarray design and production. Published gene sequences of guinea pig cytokines were selected from the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/). The accession numbers were submitted to MWG Biotech for 50-mer oligonucleotide design. Sixty-five genes from C. porcellus were selected (see the supplemental material), including those for 11 cytokines, 2 cytokine receptors, 6 chemokines, and 3 chemokine receptors as well as antigen presentation genes and antimicrobial genes. The positive controls were printed once in each array; these included rat (Rattus norvegicus) cytoplasmic -actin, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and polyubiquitin. The other positive controls were C. porcellus GAPDH, -actin, -actin, 5S rRNA, and cytoplasmic actin, and serial dilutions of the -actin oligonucleotide (50 μM, 30 μM, 20, μM, and 10 μM) were placed at the end of the array. Several negative controls were used; one was derived from the plant Arabidopsis thaliana (Brassicaceae family) and was printed three times throughout each array, and two other negative control genes (for M. bovis 16S ribosome and M. bovis GAPDH) were printed once.
The guinea pig genome has not been fully sequenced; therefore, it was not possible to design oligonucleotide probes to those genes not already sequenced, e.g., the IL-4 gene. In this instance, oligonucleotides were designed to cat (Felius catus), dog (Canis familiaris), rat, and human (Homo sapiens) sequences for IL-4 in the event that some sequence homology occurs between species. However, the absence of any signal from these probes does not necessarily mean that guinea pig IL-4 was not present.
During design, the probes were targeted to the 3' end of a gene sequence. It was not always possible to design oligonucleotides that could differentiate between two genes with high homology, as reported elsewhere (20). For example, it was difficult to design individual oligonucleotides to CD1 subtypes (e.g., CD1-b4, CD1-b1, and CD1-c2) due to their high degree of sequence similarity, raising the potential for cross-hybridization between each of these oligonucleotides.
All the probes were designed to avoid cross-hybridization with known sequences from M. tuberculosis and M. bovis as well as the guinea pig, rat, and human genomes (as a proxy for the guinea pig genome). The array contained 81 spots and was printed six times on epoxy resin-coated glass slides by MWG Biotech.
Bioassays and enzyme-linked immunosorbent assays for cytokines and chemokines. Aliquots of frozen supernatant were thawed and spun at 9,300 x g for 3 min to remove any residual cell debris. The concentration of bioactive total interferon in the culture supernatants was assayed using a viral inhibition bioassay, as described previously (43), where the guinea pig fetal cell line 104C1 (ATCC CRL-1405) and the challenge virus encephalomyocarditis (ATCC VR-129B) was used. Guinea pig IL-8 protein was measured by using the human IL-8 enzyme-linked immunosorbent assay (R&D Systems) as described previously (23). The concentration of total bioactive TNF was measured by the amount of cytotoxicity on WEHI-13 Var cells (ATCC CRL-2148) using the method described by Espevik and Nissen-Meyer (6) with some minor modifications. To verify the specificity of TNF in the culture supernatants, the samples taken at 24 h were incubated with a 1-in-116 dilution of rabbit anti-guinea pig TNF- polyclonal antiserum (kind gift from D. McMurray, Texas A&M University) for 30 min (21) prior to the addition to WEHI cells and then assayed in the normal way.
The mRNA expression profile of PPD-stimulated splenocytes from BCG-vaccinated guinea pigs. Microarrays were used to compare the mRNA transcript expression of splenocytes stimulated with PPD to that of nonstimulated cells from 24-h in vitro cultures. Eleven genes that were expressed at statistically significantly higher levels in the PPD-stimulated cells than in unstimulated cells from the same animal were identified (one-class analysis) (Table 1), using limma analysis. The most-highly differentially expressed transcript encoded IFN- (30-fold change). Other genes, such as those for secretory type II phospholipase A2, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, GRO, MCP-3, RANTES, IL-1, and IL-2 (also IL-2 precursor), were significantly up-regulated; the same gene list was generated using a one-class SAM t test.
Eight gene transcripts that were expressed at significantly lower levels in PPD-stimulated cells than in unstimulated cells at the same time point were identified using limma analysis (Table 1). A similar list was obtained using SAM except that the last four genes listed had a down-regulation of less than 1.4-fold.
The mRNA expression profile of PPD-stimulated splenocytes from naive guinea pigs. The up- or down-regulated mRNA transcripts seen in BCG-vaccinated animals (Table 1) may be due to nonspecific effects of the PPD preparation rather than as a result of a memory response following BCG vaccination. In order to investigate this, microarrays were used to compare the RNA transcript expression level of splenocytes from naive animals stimulated with PPD to that of splenocytes from nonstimulated cells at the same time point (24 h) from the same animal. In the limma analysis, the mRNA transcripts of two genes (for IL-8 and IL-1) were expressed at higher levels in the PPD-stimulated cells than in unstimulated cells. (one-class analysis) (Table 1). The down-regulated transcripts were the IL-12p40 and MCP-1 genes. The same gene list (both up- and down-regulated genes) was obtained using SAM except that the down-regulation of MCP-1 was considered not to be statistically significant (P > 0.05).
Comparison of mRNA expression from BCG-vaccinated and naive animals following PPD stimulation. A comparison was made between the up- and down-regulated transcripts from the naive and BCG-vaccinated animals, and similar results were obtained using both limma and SAM. Many of the genes that were up-regulated in the BCG-vaccinated animals (e.g., those for IFN- and IL-2) upon PPD stimulation were found to be significantly different from those seen in the naive animals (two-class analysis) (Table 1). Similarly, many of the down-regulated genes, such as those for neutrophil cationic peptide 2 and ferritin heavy chain, were significantly down-regulated in BCG-vaccinated animals compared to those in nave animals.
IL-8 mRNA was up-regulated in both BCG-vaccinated and naive animals upon PPD stimulation; hence, there was no significant (P > 0.05) difference between the groups (Table 1). This suggests that some of the M. tuberculosis proteins or peptides in the PPD preparation caused a nonspecific up-regulation of IL-8. Similarly, IL-12p40 was significantly down-regulated in both BCG and naive animals, and therefore, overall there was no significant difference (P > 0.05) between the naive and vaccinated groups in the two-class analysis.
Secretion of cytokine protein from PPD-stimulated and unstimulated cells. In order to understand the whole biological system, the level of secreted protein in the culture supernatant was assessed at 18 h and 24 h. Assessment at the earlier time point (18 h) was performed to determine the amount of preexisting protein in the culture supernatant before the microarray analysis was performed at 24 h. The amounts of total TNF and total interferon protein detected at 18 h (data not shown) were similar (P > 0.05) to those detected at 24 h.
IL-8 protein was detected in supernatants from both BCG-vaccinated and naive animals (Fig. 1a) when cells were stimulated with PPD. This is consistent with the microarray data, where an increase in the mRNA was seen in stimulated cells from both naive and BCG-vaccinated guinea pigs. Significantly more IL-8 protein was detected from the BCG-vaccinated animals (P < 0.01) than from the nave animals, and this increased (P < 0.05) with time, from 570 pg/ml at 18 h to 754 pg/ml at 24 h.
In response to PPD stimulation, significant amounts of bioactive TNF- were secreted from cells of BCG-vaccinated animals (Fig. 1b). A corresponding significant up-regulation of TNF- mRNA was not seen in the microarray analysis.
Secreted total bioactive IFN protein was detected in the culture supernatant of the cells stimulated with PPD from BCG-vaccinated animals (Fig. 1c). The viral inhibition seen here could have been caused by the presence of type I interferons (IFN- and IFN-) as well as type II interferon (IFN-). However, since there was a 30-fold up-regulation of the mRNA for IFN-, this strongly suggests that some viral inhibition was caused by the presence of IFN-.
Discussion and conclusions. We have developed a custom-made oligonucleotide array focused on those guinea pig genes with published sequences and applied this to examine the regulation of multiple cytokines and immune response-related genes induced by PPD stimulation following BCG vaccination. This microarray allowed a detailed definition of the Th1-type cytokine immune profile, which was strongly characterized by the up-regulation of IFN-, IL-2, and IL-2 precursor. The finding of significantly up-regulated IFN- mRNA was supported by the increased quantities of bioactive IFN protein in the culture supernatant. In contrast, although TNF-, which synergizes with IFN- in activating macrophages (17), was detected in the culture supernatant of BCG-vaccinated animals following PPD stimulation, there was no detectable up-regulation in mRNA after 24 h. It is possible that the mRNA was up-regulated at a time point earlier than the one used for our microarray experiment. The release of significant amounts of TNF- from PPD-stimulated cells was seen in BCG-vaccinated animals and not in the naive animals, confirming similar observations where live M. tuberculosis was used as the stimulant (22). Interleukin-12 is important in the host defense against M. tuberculosis as it strongly influences the development of a Th1-type profile (2). In this study, IL-12p40 was down-regulated in BCG-vaccinated and naive animals at 24 h upon exposure to PPD. Recently, studies of guinea pigs have shown that the expression of IL-12p40 mRNA is influenced by the concentration of TNF-; higher concentrations of TNF- suppressed IL-12p40 mRNA expression (5). It was likely, however, that additional mechanisms influenced the down-regulation of IL-12p40 in this work since little TNF was detected in the cell culture supernatant from naive animals.
There was an up-regulation of the mRNA transcripts of other immune response-related genes, such as those for GM-CSF, IL-8, MCP-3, RANTES, and GRO, to PPD stimulation in BCG-vaccinated guinea pigs. IL-8 and RANTES have been shown to be involved in the immune response to M. tuberculosis in previous guinea pig studies (14, 23). GM-CSF has also been shown to be important in the immune response to M. tuberculosis in mice (12).
An increase of sixfold in the mRNA of type II phospholipase A2 was seen in the BCG-vaccinated animals when cells were stimulated with PPD. Phospholipase A2 catalyzes the hydrolysis of ester bonds at the sn-2 position of membrane phospholipids and plays a key role in the production of proinflammatory, lipid-derived mediators (38). Type II phospholipase A2 exhibits bactericidal activity especially towards gram-positive bacteria (40), including both spores and encapsulated bacilli of Bacillus anthracis (10). The bactericidal activity of type II phospholipase A2 against M. tuberculosis has yet to be determined.
A down-regulation of ferritin heavy and light chains was seen when PPD-stimulated cells from BCG-vaccinated animals were compared with those from naive animals. Ferritin is an intracellular storage molecule for iron (30). Iron is essential for the intracellular growth of M. tuberculosis (32), and carboxymycobactin (from M. tuberculosis) can remove iron from transferrin and ferritin to promote cell growth (11). Our findings suggest that part of the success of BCG vaccination might be a signal that causes the down-regulation of the host's available iron in the form of ferritin.
In this study, microarray technology has enabled the examination of multiple cytokines, chemokines, and immune response-related genes in the guinea pig model at one time point. The mRNA immune profile derived by PPD stimulation of cells from BCG-vaccinated guinea pigs clearly indicated a Th1-type immune response. This type of profiling may be helpful in determining protective immunity, following vaccination, against infection with M. tuberculosis rather than studying individual cytokines. This focused microarray can also be used for studying the host response to other pathogenic microorganisms or immune disorders that are assessed in the guinea pig animal model.
ACKNOWLEDGMENTS
We are very grateful to Simon Clark and the Biological Investigations Group at the HPA for conducting the animal procedures. We thank Ruth Thom for setting up the bioassays. We also thank Lorenz Wernisch, Birkbeck College, University of London, for his advice on the statistical analysis of microarray data.
This work was funded by the Department of Health, United Kingdom, and the views in this paper are of the authors and not the funding body.
Supplemental material for this article may be found at http://iai.asm.org/.
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ABSTRACT
Immune responses in the guinea pig model are understudied because of a lack of commercial reagents. We have developed a custom-made guinea pig oligonucleotide microarray (81 spots) and have examined the gene expression profile of splenocytes restimulated in vitro from Mycobacterium bovis BCG-vaccinated and naive animals. Eleven genes were significantly (P < 0.05) up-regulated following vaccination, indicating a Th1-type response. These results show that microarrays can be used to more fully define immune profiles of guinea pigs.
TEXT
The guinea pig (Cavia porcellus) animal model is used widely in medical research for the study of many human diseases caused by bacteria, including tuberculosis (8), anthrax (24), and Q fever (44). Guinea pigs are also used to study viruses (e.g., genital herpes [13], Ebola [35], and cytomegalovirus [31]) and human immune disorders, such as asthma (25), allergy (39), and osteoarthritis (3). In this work, guinea pigs are frequently utilized to study disease pathology, vaccine efficacy, and treatment effectiveness. However, despite their widespread use, few commercial reagents are available for studying their immune response (27).
The guinea pig model of tuberculosis is widely recognized as an important preclinical indicator of the protective capacity of new vaccines (28). New vaccine candidates are tested against the existing "gold standard" vaccine, Mycobacterium bovis BCG (bacille Calmette-Guerin, an attenuated strain of M. bovis). The immune response evoked by these novel tuberculosis vaccines is rarely assessed because of the shortage of guinea pig immune reagents. By using the few immunological tools available, such as bioassays (6, 43), guinea pig-specific antibodies (21, 23, 41), and molecular techniques, e.g., real-time PCR (1, 18, 42), Northern blot analysis (14, 15, 26), and semiquantitative PCR (19, 26), our understanding of tuberculosis immunology in this model has improved. Many of the molecular studies, however, have analyzed the mRNA expression of only a small number of genes (one to nine genes) at any one time. Consequently, we have developed a custom-made oligonucleotide microarray to analyze the mRNA expression of multiple cytokines and immune-related genes in the guinea pig.
Studies of mice and humans have shown that protective immunity against tuberculosis depends on an acquired cellular immune response (2, 29), involving various T-cell subsets (16) where a Th1-type response is considered favorable (29). This develops in a complex environment of chemokines and proinflammatory cytokines secreted by macrophages, dendritic cells, and inflammatory cells that reside in, or are recruited to, the site of infection (7). This complexity makes it difficult to define a precise immunological correlate of protective immunity as induced by vaccination. It is possible, therefore, that a pattern of cytokines and chemokines may be more representative of a diseased or immune status.
In this study, we used BCG vaccination as a model to investigate the utility of our guinea pig 50-mer oligonucleotide microarray (81 spots). The immune profile of splenocytes from BCG-vaccinated guinea pigs restimulated in vitro with purified protein derivative of Mycobacterium tuberculosis (PPD) was analyzed and compared with the immune pattern from splenocytes of naive animals. The microarray data were supplemented with information about total bioactive interferon (IFN), bioactive tumor necrosis factor alpha (TNF-), and interleukin-8 (IL-8) proteins in order to understand the whole biological system. The microarray approach enabled the definition of an immune profile of BCG vaccination in the guinea pig model.
Animals and vaccination. Outbred Hartley strain guinea pigs from David Hall, Burton-on-Trent, United Kingdom, were either vaccinated subcutaneously (n = 5) with 5 x 104 CFU BCG Pasteur (36) or left untreated (n = 5) (naive controls). Guinea pigs were rested for 20 weeks before being used in these studies.
Preparation of splenocytes and culture. Splenocytes were prepared and treated with ACK lysis buffer to remove red blood cells as described previously (14). Cells (4 x 107 in 6 ml medium) were either stimulated with 1,670 units/ml PPD (Evans Vaccines Ltd., Liverpool, United Kingdom) or left untreated (unstimulated). The cultures were incubated in a 5% CO2 atmosphere at 37°C for 24 h. Smaller cultures of 1 x 107 cells in 1.5 ml medium were stimulated with either concanavalin A (10 μg/ml; Sigma) (internal positive control) for 18 and 24 h or PPD (1,670 units/ml) for 18 h or left untreated for 18 h. After incubation, all cells were centrifuged for 7 min at 145 x g. The supernatant was removed and stored at –70°C in aliquots for protein testing at a later time. The cell pellet from the 24-h cultures was resuspended in RLT buffer (QIAGEN, Crawley, United Kingdom) supplemented with 1% -2 mercaptoethanol (Sigma). RNeasy columns (QIAGEN) were used to purify total RNA from the cells, and this RNA was further concentrated using RNA MinElute columns (QIAGEN).
Microarray procedures. The LabelStar kit (QIAGEN) was used for the labeling and purification of target cDNA according to the manufacturer's instructions; sample total RNA (5 μg) was used as a template for reverse transcriptase in the presence of an oligonucleotide deoxyribosylthymine primer and Cy3- and Cy5-labeled dCTPs (Pfizer-Pharmacia, Kent, United Kingdom). Dye swap slide procedures, for which the Cy3 and Cy5 labels were reversed (two slides), were performed using the RNA from each animal.
The purified cDNA was mixed with 120 μl of preheated (42°C) salt-based hybridization solution (MWG Biotech, Milton Keynes, United Kingdom), and the mixture was heated in a water bath at 95°C for 3 min. The reaction mixture was cooled slightly and centrifuged before being added to the small Gene Frame (21 by 22 mm) (ABgene, Epsom, United Kingdom) on the microarray slide. The slide was incubated in a sealed hybridization cassette (TeleChem International) and submerged in a water bath at 42°C for 17 h. After hybridization, the slides were washed three times and dried by centrifugation at 520 x g for 5 min. Scanning of the slides was performed using a dual-laser scanner (Affymetrix 428; MWG Biotech) at various amplification (6-7) settings (gains) above and below saturation of the most intensely fluorescent spots on each array.
ImaGene 5.5.4 (BioDiscovery, CA) software was used to quantify the scanned images. The median pixel intensity of the spot was taken after background correction. These data were processed using the MAVI Pro 2.6.0 software package (MWG Biotech) and then imported into R/BioConductor (9) and analyzed using functions in the linear models for microarray data (limma) library (33, 34). The data were normalized using print-tip loess normalization, with significant up-weighting given to control spots. Then, a representative value for each gene for each slide was obtained by taking the median of the normalized log ratios of the six replicate spots. Moderated t statistics were generated, and resulting P values were adjusted using the Benjamini and Hochberg step-up method for controlling the false discovery rate (4). Genes were considered to be statistically significant if they had at least 1.4-fold up- or down-regulation and had a P value of <0.05 after correction for multiple testing. The normalized data were also analyzed using significance analysis of microarrays (SAM) (37). One-class SAM t tests were performed on each group, and a two-sample SAM t test was used to compare the BCG and naive groups.
Microarray design and production. Published gene sequences of guinea pig cytokines were selected from the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/). The accession numbers were submitted to MWG Biotech for 50-mer oligonucleotide design. Sixty-five genes from C. porcellus were selected (see the supplemental material), including those for 11 cytokines, 2 cytokine receptors, 6 chemokines, and 3 chemokine receptors as well as antigen presentation genes and antimicrobial genes. The positive controls were printed once in each array; these included rat (Rattus norvegicus) cytoplasmic -actin, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and polyubiquitin. The other positive controls were C. porcellus GAPDH, -actin, -actin, 5S rRNA, and cytoplasmic actin, and serial dilutions of the -actin oligonucleotide (50 μM, 30 μM, 20, μM, and 10 μM) were placed at the end of the array. Several negative controls were used; one was derived from the plant Arabidopsis thaliana (Brassicaceae family) and was printed three times throughout each array, and two other negative control genes (for M. bovis 16S ribosome and M. bovis GAPDH) were printed once.
The guinea pig genome has not been fully sequenced; therefore, it was not possible to design oligonucleotide probes to those genes not already sequenced, e.g., the IL-4 gene. In this instance, oligonucleotides were designed to cat (Felius catus), dog (Canis familiaris), rat, and human (Homo sapiens) sequences for IL-4 in the event that some sequence homology occurs between species. However, the absence of any signal from these probes does not necessarily mean that guinea pig IL-4 was not present.
During design, the probes were targeted to the 3' end of a gene sequence. It was not always possible to design oligonucleotides that could differentiate between two genes with high homology, as reported elsewhere (20). For example, it was difficult to design individual oligonucleotides to CD1 subtypes (e.g., CD1-b4, CD1-b1, and CD1-c2) due to their high degree of sequence similarity, raising the potential for cross-hybridization between each of these oligonucleotides.
All the probes were designed to avoid cross-hybridization with known sequences from M. tuberculosis and M. bovis as well as the guinea pig, rat, and human genomes (as a proxy for the guinea pig genome). The array contained 81 spots and was printed six times on epoxy resin-coated glass slides by MWG Biotech.
Bioassays and enzyme-linked immunosorbent assays for cytokines and chemokines. Aliquots of frozen supernatant were thawed and spun at 9,300 x g for 3 min to remove any residual cell debris. The concentration of bioactive total interferon in the culture supernatants was assayed using a viral inhibition bioassay, as described previously (43), where the guinea pig fetal cell line 104C1 (ATCC CRL-1405) and the challenge virus encephalomyocarditis (ATCC VR-129B) was used. Guinea pig IL-8 protein was measured by using the human IL-8 enzyme-linked immunosorbent assay (R&D Systems) as described previously (23). The concentration of total bioactive TNF was measured by the amount of cytotoxicity on WEHI-13 Var cells (ATCC CRL-2148) using the method described by Espevik and Nissen-Meyer (6) with some minor modifications. To verify the specificity of TNF in the culture supernatants, the samples taken at 24 h were incubated with a 1-in-116 dilution of rabbit anti-guinea pig TNF- polyclonal antiserum (kind gift from D. McMurray, Texas A&M University) for 30 min (21) prior to the addition to WEHI cells and then assayed in the normal way.
The mRNA expression profile of PPD-stimulated splenocytes from BCG-vaccinated guinea pigs. Microarrays were used to compare the mRNA transcript expression of splenocytes stimulated with PPD to that of nonstimulated cells from 24-h in vitro cultures. Eleven genes that were expressed at statistically significantly higher levels in the PPD-stimulated cells than in unstimulated cells from the same animal were identified (one-class analysis) (Table 1), using limma analysis. The most-highly differentially expressed transcript encoded IFN- (30-fold change). Other genes, such as those for secretory type II phospholipase A2, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, GRO, MCP-3, RANTES, IL-1, and IL-2 (also IL-2 precursor), were significantly up-regulated; the same gene list was generated using a one-class SAM t test.
Eight gene transcripts that were expressed at significantly lower levels in PPD-stimulated cells than in unstimulated cells at the same time point were identified using limma analysis (Table 1). A similar list was obtained using SAM except that the last four genes listed had a down-regulation of less than 1.4-fold.
The mRNA expression profile of PPD-stimulated splenocytes from naive guinea pigs. The up- or down-regulated mRNA transcripts seen in BCG-vaccinated animals (Table 1) may be due to nonspecific effects of the PPD preparation rather than as a result of a memory response following BCG vaccination. In order to investigate this, microarrays were used to compare the RNA transcript expression level of splenocytes from naive animals stimulated with PPD to that of splenocytes from nonstimulated cells at the same time point (24 h) from the same animal. In the limma analysis, the mRNA transcripts of two genes (for IL-8 and IL-1) were expressed at higher levels in the PPD-stimulated cells than in unstimulated cells. (one-class analysis) (Table 1). The down-regulated transcripts were the IL-12p40 and MCP-1 genes. The same gene list (both up- and down-regulated genes) was obtained using SAM except that the down-regulation of MCP-1 was considered not to be statistically significant (P > 0.05).
Comparison of mRNA expression from BCG-vaccinated and naive animals following PPD stimulation. A comparison was made between the up- and down-regulated transcripts from the naive and BCG-vaccinated animals, and similar results were obtained using both limma and SAM. Many of the genes that were up-regulated in the BCG-vaccinated animals (e.g., those for IFN- and IL-2) upon PPD stimulation were found to be significantly different from those seen in the naive animals (two-class analysis) (Table 1). Similarly, many of the down-regulated genes, such as those for neutrophil cationic peptide 2 and ferritin heavy chain, were significantly down-regulated in BCG-vaccinated animals compared to those in nave animals.
IL-8 mRNA was up-regulated in both BCG-vaccinated and naive animals upon PPD stimulation; hence, there was no significant (P > 0.05) difference between the groups (Table 1). This suggests that some of the M. tuberculosis proteins or peptides in the PPD preparation caused a nonspecific up-regulation of IL-8. Similarly, IL-12p40 was significantly down-regulated in both BCG and naive animals, and therefore, overall there was no significant difference (P > 0.05) between the naive and vaccinated groups in the two-class analysis.
Secretion of cytokine protein from PPD-stimulated and unstimulated cells. In order to understand the whole biological system, the level of secreted protein in the culture supernatant was assessed at 18 h and 24 h. Assessment at the earlier time point (18 h) was performed to determine the amount of preexisting protein in the culture supernatant before the microarray analysis was performed at 24 h. The amounts of total TNF and total interferon protein detected at 18 h (data not shown) were similar (P > 0.05) to those detected at 24 h.
IL-8 protein was detected in supernatants from both BCG-vaccinated and naive animals (Fig. 1a) when cells were stimulated with PPD. This is consistent with the microarray data, where an increase in the mRNA was seen in stimulated cells from both naive and BCG-vaccinated guinea pigs. Significantly more IL-8 protein was detected from the BCG-vaccinated animals (P < 0.01) than from the nave animals, and this increased (P < 0.05) with time, from 570 pg/ml at 18 h to 754 pg/ml at 24 h.
In response to PPD stimulation, significant amounts of bioactive TNF- were secreted from cells of BCG-vaccinated animals (Fig. 1b). A corresponding significant up-regulation of TNF- mRNA was not seen in the microarray analysis.
Secreted total bioactive IFN protein was detected in the culture supernatant of the cells stimulated with PPD from BCG-vaccinated animals (Fig. 1c). The viral inhibition seen here could have been caused by the presence of type I interferons (IFN- and IFN-) as well as type II interferon (IFN-). However, since there was a 30-fold up-regulation of the mRNA for IFN-, this strongly suggests that some viral inhibition was caused by the presence of IFN-.
Discussion and conclusions. We have developed a custom-made oligonucleotide array focused on those guinea pig genes with published sequences and applied this to examine the regulation of multiple cytokines and immune response-related genes induced by PPD stimulation following BCG vaccination. This microarray allowed a detailed definition of the Th1-type cytokine immune profile, which was strongly characterized by the up-regulation of IFN-, IL-2, and IL-2 precursor. The finding of significantly up-regulated IFN- mRNA was supported by the increased quantities of bioactive IFN protein in the culture supernatant. In contrast, although TNF-, which synergizes with IFN- in activating macrophages (17), was detected in the culture supernatant of BCG-vaccinated animals following PPD stimulation, there was no detectable up-regulation in mRNA after 24 h. It is possible that the mRNA was up-regulated at a time point earlier than the one used for our microarray experiment. The release of significant amounts of TNF- from PPD-stimulated cells was seen in BCG-vaccinated animals and not in the naive animals, confirming similar observations where live M. tuberculosis was used as the stimulant (22). Interleukin-12 is important in the host defense against M. tuberculosis as it strongly influences the development of a Th1-type profile (2). In this study, IL-12p40 was down-regulated in BCG-vaccinated and naive animals at 24 h upon exposure to PPD. Recently, studies of guinea pigs have shown that the expression of IL-12p40 mRNA is influenced by the concentration of TNF-; higher concentrations of TNF- suppressed IL-12p40 mRNA expression (5). It was likely, however, that additional mechanisms influenced the down-regulation of IL-12p40 in this work since little TNF was detected in the cell culture supernatant from naive animals.
There was an up-regulation of the mRNA transcripts of other immune response-related genes, such as those for GM-CSF, IL-8, MCP-3, RANTES, and GRO, to PPD stimulation in BCG-vaccinated guinea pigs. IL-8 and RANTES have been shown to be involved in the immune response to M. tuberculosis in previous guinea pig studies (14, 23). GM-CSF has also been shown to be important in the immune response to M. tuberculosis in mice (12).
An increase of sixfold in the mRNA of type II phospholipase A2 was seen in the BCG-vaccinated animals when cells were stimulated with PPD. Phospholipase A2 catalyzes the hydrolysis of ester bonds at the sn-2 position of membrane phospholipids and plays a key role in the production of proinflammatory, lipid-derived mediators (38). Type II phospholipase A2 exhibits bactericidal activity especially towards gram-positive bacteria (40), including both spores and encapsulated bacilli of Bacillus anthracis (10). The bactericidal activity of type II phospholipase A2 against M. tuberculosis has yet to be determined.
A down-regulation of ferritin heavy and light chains was seen when PPD-stimulated cells from BCG-vaccinated animals were compared with those from naive animals. Ferritin is an intracellular storage molecule for iron (30). Iron is essential for the intracellular growth of M. tuberculosis (32), and carboxymycobactin (from M. tuberculosis) can remove iron from transferrin and ferritin to promote cell growth (11). Our findings suggest that part of the success of BCG vaccination might be a signal that causes the down-regulation of the host's available iron in the form of ferritin.
In this study, microarray technology has enabled the examination of multiple cytokines, chemokines, and immune response-related genes in the guinea pig model at one time point. The mRNA immune profile derived by PPD stimulation of cells from BCG-vaccinated guinea pigs clearly indicated a Th1-type immune response. This type of profiling may be helpful in determining protective immunity, following vaccination, against infection with M. tuberculosis rather than studying individual cytokines. This focused microarray can also be used for studying the host response to other pathogenic microorganisms or immune disorders that are assessed in the guinea pig animal model.
ACKNOWLEDGMENTS
We are very grateful to Simon Clark and the Biological Investigations Group at the HPA for conducting the animal procedures. We thank Ruth Thom for setting up the bioassays. We also thank Lorenz Wernisch, Birkbeck College, University of London, for his advice on the statistical analysis of microarray data.
This work was funded by the Department of Health, United Kingdom, and the views in this paper are of the authors and not the funding body.
Supplemental material for this article may be found at http://iai.asm.org/.
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