Constitutive Differences in Gene Expression Profiles Parallel Genetic Patterns of Susceptibility to Tuberculosis in Mice

The primary host cells for Mycobacterium tuberculosis, which is the causative agent of tuberculosis (TB), are mature tissue macrophages. The specific host response pathways allowing M. tuberculosis to take up residence in macrophages and the host cell factors that underlie the M. tuberculosis-macrophage interplay are largely unknown. We have previously demonstrated that in A/Sn and I/St mice, which are genetically resistant and susceptible to tuberculosis, respectively (7, 16), only freshly isolated interstitial lung macrophages, and not peritoneal or spleen- or bone marrow-derived macrophages, strictly followed the genetic pattern of tuberculosis susceptibility/resistance (11). In addition, the resistance phenotype can be readily transferred with bone marrow cells from resistant F₁ donors into irradiated susceptible I/St recipients (12). To further identify host response genes involved in early M. tuberculosis-macrophage interactions, we conducted a series of microarray gene expression experiments employing lung macrophages from A/Sn and I/St mice.

Interstitial lung macrophages, isolated as described earlier (11), were either infected with M. tuberculosis H37Rv at a multiplicity of infection of 5:1 for 24 h or cultured under the same conditions without infection (control). The efficiency of infection was 50% to 60%, as demonstrated by auramine staining of fixed macrophages, with no observed interstrain difference in mycobacterial uptake (data not shown). RNA extracted from infected and control macrophages of I/St and A/Sn mice (RNeasy minikit; QIAGEN, California) was hybridized to murine genome U74Av2 microarrays (www.affymetrix.com). The data were analyzed with the Significance Analysis of Microarrays software (SAM; http://www-stat.stanford.edu/∼tibs/SAM//index.html). For the analysis, the gene expression levels in macrophages of I/St and A/Sn mice were compared either before or after infection. We considered genes that had d scores (absolute values) of ≥2.0 to be significant. The d score is similar to a t statistic, but a small constant is added to the standard error to reduce the variability in its estimate. A better measure of statistical significance can be obtained by examining the false detection rate (FDR) associated with the magnitude of the differences between strains, with adjustment for the number of genes tested (24). Accurate empirical estimates of the FDR were obtained from the permutation analysis built into the SAM software, employing a d score (absolute value) of 2.0 corresponding to an estimated FDR of 1% (24). Microarray data analysis led to the identification of 152 genes with significant differentials in expression either before or after infection of lung macrophages of the two strains (Table 1; see also supplemental material S1).

Generally, lung macrophages from susceptible I/St mice demonstrated significantly higher expression levels of cytokine/chemokine genes, including the genes for interleukin 11 (Il-11), Il-6, Cxcl-13, and Cxcl-14 (Table 1), than did their A/Sn counterparts. In contrast, only three cytokine/chemokine genes (Cxcl-10, Cxcl-9, and Il-17) were expressed at significantly higher levels in macrophages from resistant A/Sn mice. In the group of immune response genes, I/St macrophages expressed only three genes (Ifi205, Ifi202, and Saa3) at a higher level than did A/Sn macrophages. Conversely, a large number of genes belonging to this class were expressed at significantly higher levels in A/Sn macrophages (Table 1), suggesting their critical role in the development of the immune response at an early stage of infection. The majority of genes encoding receptor/cell surface molecules that are potentially important for the on-time activation of protective mechanisms after infection were highly expressed in lung macrophages of A/Sn mice. Likewise, genes encoding signal transduction molecules were generally expressed at higher levels in A/Sn macrophages (see supplemental material S1). Interestingly, matrix metalloproteinase 8, one of the extracellular matrix proteins involved in the processing of extracellular matrices and wound healing (20), was shown to be expressed at significantly higher levels in A/Sn macrophages. Differences in constitutive expression levels for selected genes (Il-11, Il-6, Mmp8, Cxcl-14, Cxcl-13, and Saa3) were confirmed by real-time reverse transcription-PCR (RT-PCR) (Table 2; see also supplemental material S2) using mRNAs obtained in three additional independent experiments.

Constitutive higher expression of Il-6 by macrophages of susceptible I/St mice is consistent with the data of Keller and colleagues, who demonstrated an approximately 10-fold increase in Il-6 expression in infected macrophages from TB-susceptible but not from TB-resistant mice (10). IL-6 is a pleiotropic cytokine which is produced by a variety of cells, including macrophages (14, 26), with numerous types of cell targets. M. tuberculosis-infected macrophages produce IL-6, which inhibits gamma interferon-responsive genes in macrophages and inhibits eradication of infection (14).

Remarkably, the high expression level of Il-6 by macrophages of I/St mice is accompanied by elevated levels of Cxcl-13 (Scyb13) expression (Tables 1 and 2). Cxcl-13, the B-cell-homing chemokine, is produced by macrophages (2, 9) and dendritic cells (3). Goya and colleagues (8) have shown that prolonged production of IL-6 in the lungs leads to formation of pulmonary lesions that have lymphoid tissue-like structure, where the chemokine gene Cxcl-13 is highly expressed. Significantly higher expression levels of Il-6 and Cxcl-13 by lung macrophages of susceptible I/St mice (Tables 1 and 2), in conjunction with extremely high levels of specific immunoglobulin G2a antibody responses in these mice (18), strongly suggest that severe TB inflammation in the lungs of these mice involves a nonprotective B-cell component. This suggestion is further supported by a recent finding of Ulrichs et al. (25), who demonstrated the formation of well-organized B-cell foci in the vicinity of tuberculous lesions in lung tissue surgically removed from TB patients with a rapidly progressing severe form of the disease.

An exciting new finding obtained in this study is the high level of Il-11 expression by lung macrophages. IL-11 is a pleiotropic cytokine with anti-inflammatory activity when expressed at moderate levels (23, 27), but its overexpression may have a significant proinflammatory effect (22, 28). The production of IL-11 had previously been described for lung fibroblasts, airway epithelial cells (5, 6), and antigen-presenting cells after infection with respiratory syncytial virus (1). To demonstrate that Il-11 is indeed expressed by lung macrophages and not by contaminating lung fibroblasts, we developed fibroblast cultures from lung stroma of I/St and A/Sn mice and compared the levels of expression of Il-11 and Cxcl-14 in these cells and in interstitial lung macrophages. Cxcl-14 is the mouse ortholog of the human breast- and kidney-expressed chemokine gene (BRAK) and is constitutively expressed by fibroblasts in a number of mouse organs, including lungs. The results of this comparison are presented in Fig. 1. I/St and A/Sn lung macrophages expressed, respectively, 60- and 30-fold-higher levels of Il-11 than their corresponding lung fibroblasts. Conversely, I/St and A/Sn lung fibroblasts expressed 8- and 50-fold-higher levels of Cxcl-14 than their corresponding lung macrophages. These results show that lung macrophages are major producers of Il-11 and that the high expression levels of Il-11 in macrophages of tuberculosis-susceptible I/St mice compared to expression levels of Il-11 in tuberculosis-resistant A/Sn mice offer a possible explanation for the development of severe pathology in the lungs of M. tuberculosis-infected I/St mice (7, 16, 18).

While several studies have analyzed the response of host macrophages to mycobacterial infection (4, 10, 13, 15, 17, 19, 21,), none of these studies employed ex vivo-isolated lung macrophages, the predominant cell type naturally infected with M. tuberculosis, and only one study used a combination of resistant and susceptible strains of mice (10). However, all of these studies reported significant M. tuberculosis-triggered host gene expression changes. Surprisingly, we did not observe major changes in gene expression by lung macrophages of either I/St or A/Sn mice following 24-h infection with M. tuberculosis H37Rv. Hence, we tested whether overly conservative criteria for significant gene expression changes underlie this finding. It appeared that even a reduction in stringency of the analysis with an FDR up to 75% did not allow the reproduction of previously reported results (4, 10, 13, 17, 21). In a final set of experiments, we selected eight genes (Il-6, Saa3, Slpi, Ccl5, Cxcl-5, Cxcl-10, Mrc1, and Mmp9) that had been reported to undergo significant expression changes following M. tuberculosis infection of murine bone marrow-derived macrophages (4, 21, 10, 17). We found that infection of interstitial lung macrophages with M. tuberculosis does not lead to changes in the expression level of these genes (change of ≤1.5-fold [absolute value]) (data not shown). These results support the hypothesis that different types of macrophages respond differently to M. tuberculosis infection and argue against the suggestion that too-stringent criteria had been used in the microarray analysis.

In summary, by employing global analysis of gene expression, we observed a statistically well-defined signature of gene expression differences among interstitial macrophages from A/Sn and I/St mice. These interstrain gene expression differences provide a rational basis for a mechanistic framework of the genetically controlled tuberculosis resistance and susceptibility displayed by A/Sn and I/St mice. By contrast, we were unable to reveal significant M. tuberculosis-triggered gene expression changes in interstitial lung macrophages. It is possible that the in vitro infection experiments are not a correlate of the response of the whole animal. This possibility appears unlikely since lung macrophages faithfully repeat the pattern of resistance and susceptibility observed at the whole-animal level. It is more likely that intrinsic gene expression levels are an important determinant of TB pathogenesis in the mouse and that constitutive genetically controlled gene expression in lung macrophages is an area that requires more careful consideration in the study of TB pathogenesis.

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FIG. 1.

Expression of Il-11 and Cxcl-14 by lung macrophages and fibroblasts isolated from TB-susceptible I/St mice and TB-resistant A/Sn mice. Normalized Il-11 and Cxcl-14 gene expression levels are shown as severalfold differences relative to Hprt gene expression. (A) Lung macrophages (gray bars) express higher levels of Il-11 than lung fibroblasts (hatched bars) from both I/St and A/Sn mice. (B) Lung fibroblasts (hatched bars) express higher levels of Cxcl-14 than macrophages (gray bars) from mice of both strains. In each experiment, syngeneic lung macrophages or lung fibroblasts extracted from several mice were pooled. Expression levels of Il-11 and Cxcl-14 were measured by quantitative RT-PCR. Results are expressed as means (±standard errors) of triplicate assays from one of three experiments with similar results.

• American Society for Microbiology