Liver X receptors contribute to the protective immune response against Mycobacterium tuberculosis in mice (original) (raw)
Infection kinetics of the experimental pulmonary tuberculosis model. We used an airway infection model in which mice were instilled with 5 × 103 mRLU of luminescent M. tuberculosis H37Rv intratracheally (i.t.) and monitored for bacillary growth at different time intervals. This inoculum corresponded to 104 CFU by standard plating techniques (data not shown) and established an initial load of 3 × 103 mRLU in the lungs. The infection slowly progressed into an exponential growth phase (days 6–14), followed by a chronic phase, as the bacterial numbers started to stabilize at close to 5 × 104 mRLU/lung (days 20–35; Figure 1A). The infection remained local during the 5 weeks of the experiment, as evidenced by minimal dissemination to the spleen (Figure 1A). Another characteristic feature of M. tuberculosis infection is a strong proinflammatory response triggered in the airways, keeping the bacterial growth in check. Increased mRNA levels of typical inflammatory mediators IFN-γ, TNF-α, IL-12p40, and iNOS were observed from day 14 after infection, corresponding to the bacterial growth arrest initiating the chronic phase (Figure 1B). After day 20, expression levels were either sustained or decreased slightly. Expression of IFN-γ in particular was triggered strongly by M. tuberculosis infection, as reflected by cytokine concentrations in the BAL fluid of up to 800 pg/ml on day 14 after infection (data not shown). Similar results were obtained with transcriptional analysis of these parameters in lung tissue (data not shown). Interestingly, increased mRNA levels of IL-17 and IL-23p19 were also observed after airway challenge with the pathogen (Figure 1B), thus underscoring the mixed Th1/Th17 nature of the cytokine function in the airways of this experimental model. In line with these findings, the cellular composition of the airway response to M. tuberculosis infection featured early infiltration of neutrophils and mononuclear cells. These 2 cell populations steadily increased during the course of the infection, mononuclear cells always representing the majority of the cells present in the airway lumen. Lymphocytes became apparent only at day 14 after the initial pathogen encounter and increased until day 20 (Figure 1C).
Characteristic features of the murine pulmonary tuberculosis model. (A) Bacterial counts in the lungs and spleen at different time points after i.t. instillation of luminescent M. tuberculosis (1 × 104 CFU). Values are expressed as log10 mRLU per organ (n = 4). Dotted lines indicate the detection limit of the bioluminescence assay. (B) RT-qPCR analysis of the indicated inflammatory mediators within BAL cells. Data are expressed as relative mRNA levels, normalized against reference the housekeeping gene. Mice were tested individually (n = 4). Dotted lines denote mRNA levels obtained from background-matched naive BAL cells. (C) Cellular infiltration in the airways after exposure to the pathogen. Shown are absolute numbers of total cells, mononuclear cells, neutrophils, and lymphocytes. Dotted lines denote cell numbers in naive animals. Data in C are mean ± SEM (n = 5).
LXRs and LXR target genes are induced in response to airway infection with M. tuberculosis. To investigate a possible role for LXR-dependent pathways during M. tuberculosis infection, we assessed the expression levels of LXRs and LXR target genes in the total cell population isolated from the bronchoalveolar lumen. LXRα mRNA was induced as early as 3 days after infection, and remained high (despite fluctuating) at all time points tested (Figure 2A). In contrast, expression levels of LXRβ were in general much lower than those observed for LXRα and remained basal for the first 10 days after infection, after which a moderate increase was observed (Figure 2A). Because increased expression cannot always be correlated to functional activity of LXR (33), we evaluated relative mRNA levels of well-documented LXR target genes — cholesterol transporter, ABCA1, and ApoE — as readout for LXR activation. Markedly increased expression levels in BAL cells for both LXR target genes were observed throughout the course of infection (Figure 2A). Analysis of the CD11c+ cell fraction from the BAL, as well as analysis of lung tissue, at day 21 after infection confirmed this assessment, showing strong induction of LXRα in contrast to moderate induction of LXRβ (Figure 2B). The CD11c+ population was selected because it contains the majority of macrophages together with some DCs (37) and represents the cellular target of M. tuberculosis. Additionally, mRNA levels for ApoE and ABCA1 markedly increased as a result of M. tuberculosis infection (Figure 2B). The dependence on LXR transcriptional activity of the infection-induced increment was confirmed by the partial to complete abolishment of induction with single-knockout Lxra–/– and double-knockout Lxra–/–Lxrb–/– mice, respectively (Figure 2B).
Expression of LXRs and LXR-dependent genes during M. tuberculosis infection. (A and C) RT-qPCR analysis of expression levels of LXRα and LXRβ isoforms, LXR-dependent target genes, and PPARγ and SREBP-1c within BAL cells isolated at the indicated times after infection. Dotted lines denote mRNA levels obtained from background-matched naive BAL cells. Mice were tested individually (n = 4–5). (B and D) After 3 weeks of infection, CD11c+ cells were isolated from the BAL, and lung tissue digests and relative mRNA levels of the indicated parameters were determined by RT-qPCR. (B, top) Infected (black bars) CD11c+ BAL and lung cells or naive controls (white bars) from WT mice. (B, bottom, and D) Transcriptional analysis of CD11c+ BAL cells isolated from infected WT, Lxra–/–, Lxrb–/–, and Lxra–/–Lxrb–/– mice (n = 5). Dotted lines denote mRNA levels obtained from background-matched naive CD11c+ BAL cells.
Besides LXRs, airway challenge with M. tuberculosis also triggered the expression of PPARγ and SREBP-1c, additional transcription factors prominent in the regulation of lipid metabolism pathways. Increased mRNA levels of these transcription factors were observed in cells from the BAL at different time points after infection and, similar to the results for LXRα, were highly induced in CD11c+ cells from the airway lumen (Figure 2, C and D). However, LXR deficiency did not alter the expression levels of PPARγ, a nuclear receptor upstream of LXRα. On the other hand, loss of both LXR isoforms did result in a pronounced reduction in the relative mRNA levels of SREBP-1c, which is by itself a known LXR target (Figure 2D). These results demonstrate prominent activation of LXRs and their downstream pathways during infection with M. tuberculosis, implicating a possible role for them in the host response.
Mice lacking LXRs are more susceptible to M. tuberculosis infection. We verified the involvement of LXRs during experimental pulmonary tuberculosis by challenging Lxra–/–Lxrb–/– mice with M. tuberculosis using the same model described above. Whereas WT mice effectively stabilized bacillary growth in the lungs at around 5 × 104 mRLU, the bacterial burden in the lungs of background-matched Lxra–/–Lxrb–/– mice was approximately 10-fold higher after 3 weeks of infection (Figure 3A). Susceptibility to M. tuberculosis infection was associated primarily with the loss of LXRα and not LXRβ, since the bacterial load in the lungs of Lxra–/– mice was almost identical to that of Lxra–/–Lxrb–/– mice, while bacterial replication in Lxrb–/– mice was not different from that in WT animals. In line with these results, only Lxra–/– mice and Lxra–/–Lxrb–/– mice showed accelerated cachexia 14–20 weeks after infection (Figure 3A). After 21 weeks, Lxra–/–Lxrb–/– mice reached a point at which they had to be euthanized, having lost more than 20% of their initial body weights. Previous experiments demonstrated that background-matched animals survive up to a year without any physical sign of disease with this i.t. infection dose (our unpublished observations). Histological analysis at 5 weeks of infection revealed a marked increase in the size and number of granulomatous lesions that was most pronounced in Lxra–/–Lxrb–/– mice (Figure 3, B and C). Moreover, a high percentage of lesions in Lxra–/–Lxrb–/– as well as Lxra–/– mice contained numerous acid fast bacteria within foamy macrophages and showed multiple lipid-loaded cells, as demonstrated by Oil Red O staining of the lung tissue sections (Figure 3, B and C).
Mice lacking LXRs are more susceptible to M. tuberculosis infection. (A) Changes in bacterial load in the lungs and total body mass of WT, Lxra–/–, Lxrb–/–, and Lxra–/–Lxrb–/– mice infected i.t. with M. tuberculosis (1 × 104 CFU). Values are expressed as log10 mRLU per organ and as percent of original weight (n = 5–7). *P < 0.05, **P < 0.01, Lxra–/–Lxrb–/– versus WT. (B) Histological analysis of H&E-stained lungs sections at 5 weeks after infection revealed more extensive granulomatous inflammation (red arrows) as well as total inflammation in the in Lxra–/–Lxrb–/– mice. Acid fast staining of the sections showed the presence of mycobacteria as pink rods. Frequent events of multiple bacteria per cell were observed in the lung sections from Lxra–/– and Lxra–/–Lxrb–/– mice (black arrows). Foamy macrophages containing multiple red lipid droplets after staining with Oil Red O was most prominent in Lxra–/– and Lxra–/–Lxrb–/– mice (white arrows). Original magnification (left to right), ×20, ×100, ×1,000, ×600. (C) Quantification of the lung inflammation index, number of bacilli, and Oil Red–positive cells, as described in Methods (n = 5). *P < 0.05, **P < 0.01 versus WT.
Airway infection models mimic human pulmonary tuberculosis in the sense that the bacilli remain locally in the lungs with minimal dissemination to other organs, as we observed in Figure 1A. In order to verify whether Lxra–/–Lxrb–/– mice are able to restrict infection to the pulmonary cavity, we challenged the airways with a 10-fold higher inoculum (105 CFU) of luminescent M. tuberculosis and analyzed the bacterial burden in lungs, spleen, and liver after 5 weeks of infection. The data obtained confirmed the previous result, showing increased bacterial load in the lungs of Lxra–/–Lxrb–/– mice compared with WT animals. In addition, Lxra–/–Lxrb–/– mice failed to contain the infection: significant levels of bacteria were detected in the spleen (Figure 4A). Interestingly, histological analysis of spleen sections by standard H&E staining revealed numerous macrophages from Lxra–/–Lxrb–/– mice containing lipofuscin-like particles (Figure 4B). This could be indicative of an overload of lipid oxidation products that interact with cell proteins and lead to the formation of yellow-brown oxidized protein aggregates. Alternatively, these particles could be indicative of increased phagocytosis of red blood cells, resulting in the accumulation of hemosiderin as the brown iron-containing catabolic product. In the liver, in which bacterial replication after i.t. infection with M. tuberculosis was tightly controlled in WT mice, bacterial numbers from Lxra–/–Lxrb–/– mice were clearly above the basal level (Figure 4A). These data support a crucial role for LXR-dependent pathways, and especially for LXRα, in the control and containment of infection with M. tuberculosis.
Infection of Lxra–/–Lxrb–/– mice results in disseminated systemic infection. (A) Bacterial load in the lungs, spleens, and livers of WT, Lxra–/–, Lxrb–/–, and Lxra–/–Lxrb–/– mice infected i.t. with luminescent M. tuberculosis (1 × 105 CFU) for 5 weeks. Values are expressed as log10 mRLU per organ (n = 3). Dotted lines indicate the detection limit of the bioluminescence assay. *P < 0.05, **P < 0.01 versus WT. (B) Histological analysis of spleen sections prepared after 5 weeks of infection revealed the presence in Lxra–/–Lxrb–/– mice of multiple macrophages with a brown reaction product after standard H&E staining (arrows). Original magnification, ×100.
LXRs regulate the innate response to M. tuberculosis infection. Mycobacterial infection typically results in the induction of a local inflammatory response that culminates in granuloma formation. As documented above, this response, initiated shortly after the bacterium-macrophage interaction, was characterized by the recruitment of innate effector cells, such as macrophages, polymorphonuclear neutrophils, and NK cells, to the infectious foci. To verify whether the increased susceptibility of the Lxra–/–Lxrb–/– mice could be linked to a defective innate response, we analyzed the acute (day 7) airway inflammatory response to pulmonary M. tuberculosis infection. Lxra–/– and Lxra–/–Lxrb–/– mice failed to mount an effective neutrophilic inflammatory response, as demonstrated by the minimal neutrophil cell numbers in the BAL compared with WT mice (Figure 5A). The number of total as well as mononuclear cells from the airway lumen remained stable in all the groups tested, while lymphocyte counts were marginal (data not shown). Real-time quantitative PCR (RT-qPCR) analysis of myeloperoxidase (MPO), a marker for activated neutrophils (38), confirmed the defective neutrophilic inflammation in Lxra–/–Lxrb–/– and Lxra–/– mice 7 days after infection, showing only basal mRNA levels in the lung tissue (Figure 5B). Furthermore, CD11c+ lung cells from Lxra–/–Lxrb–/– mice expressed lower mRNA levels of the neutrophil-attracting chemokines keratinocyte-derived chemokine (KC) and LPS-induced chemokine (LIX) as well as of IL-23, a cytokine implicated in the regulation of granulopoiesis and the prevalence of IL-17–producing cells (39), compared with cells from WT mice (Figure 5C). In agreement with our bacillary growth findings, CD11c+ lung cells from Lxra–/– mice, but not Lxrb–/– mice, mimicked the blunted neutrophil-attracting chemokine and cytokine response observed in the Lxra–/–Lxrb–/– mice.
LXR-dependent regulation of the innate immune response after i.t. challenge with M. tuberculosis. WT, Lxra–/–, Lxrb–/–, and Lxra–/–Lxrb–/– mice were infected i.t. with luminescent M. tuberculosis (1 × 104 CFU) and sacrificed 7 days after infection. (A) Differential cell infiltration in the BAL. Shown are the absolute numbers of total cells and neutrophils. Data in A are mean ± SEM (n = 5). (B) Relative mRNA levels of MPO from total lung tissue samples, as determined by RT-qPCR (n = 5). (C) CD11c+ cells were isolated 7 days after infection from lung tissue digests, and relative mRNA levels were determined by RT-qPCR. Dotted lines denote values obtained from CD11c+ cells of naive mice. Data are expressed as relative mRNA levels, normalized against reference housekeeping genes (n = 5), and are representative of 2 separate experiments. Arg, arginase. *P < 0.05, **P < 0.01.
Finally, mRNA levels of the characteristic inflammatory mediators iNOS and TNF-α were still at basal levels in isolated CD11c+ lung cells of both WT and LXR-deficient animals after 7 days of infection (data not shown). However, IL-12p40 was induced at this early time point in CD11c+ cells from WT animals, but showed no induction or was induced only marginally in CD11c+ cells from Lxra–/– and Lxra–/–Lxrb–/– mice (Figure 5C). In contrast to this apparent reduction in the IL-12/IL-23 inflammatory axis, mRNA levels of arginase and found in inflammatory zone–1 (Fizz1), both prominent IL-4– and IL-13–dependent antiinflammatory macrophage markers (40–43), were highly increased in CD11c+ lung cells from Lxra–/–Lxrb–/– mice (Figure 5C). Interestingly, expression of these markers has previously been shown to be detrimental in the context of M. tuberculosis infection (44). These results indicate that not only may failure to raise neutrophilic effector functions contribute to the increased susceptibility of Lxra–/–Lxrb–/– mice observed in this study, but modulation of macrophage activation may also be a contributing factor.
Decreased Th1 and Th17 function in the lungs of Lxra–/–Lxrb–/– mice. We also analyzed the CD4+ T cell response in the lungs at days 21 and 35 after infection — at which time bacterial numbers start to stabilize and the chronic phase develops. The isolated CD4+ T cells were not further stimulated ex vivo, and therefore represent the in vivo status of the lung CD4+ T cell subset. Importantly, we did not observe any difference in the number of CD4+ lung T cells recruited to the lungs after infection among the experimental groups (data not shown). RT-qPCR analysis of normalized numbers of T cells showed a strong inhibition of the type 1 cytokine IFN-γ and of the Th1-specific transcription factor T-bet in Lxra–/–Lxrb–/– mice (Figure 6). Levels of the type 2 cytokine IL-4 and the Th2-specific transcription factor Gata3 remained basal in WT mice, and only Gata3 mRNA levels increased in Lxra–/–Lxrb–/– mice, especially at day 35 (Figure 6). Expression of immunoregulatory cytokines IL-10 and TGF-β and of regulatory T cell markers FoxP3 and GITR was unchanged compared with infected WT animals (data not shown). However, similar to the results for IFN-γ, CD4+ lung T cells from Lxra–/–Lxrb–/– mice featured low mRNA levels of IL-17 and the Th17-specific transcription factor retinoic acid receptor–related orphan receptor–γt (RORγt) at both time points tested (Figure 6). Interestingly, Lxra–/– mice closely mimicked the reduced Th1 and Th17 immune function in the lungs of Lxra–/–Lxrb–/– mice at day 21, when the host immune defense started to stabilize bacterial expansion. However, at day 35, when the infection had become chronic, IFN-γ and T-bet expression levels in Lxra–/– mice were restored to normal, leaving a defective Th17 response as the most prominent feature shared between the more susceptible Lxra–/– and Lxra–/–Lxrb–/– mouse strains (Figure 6).
The role of LXRs in the modulation of T cell function during M. tuberculosis infection. WT, Lxra–/–, Lxrb–/–, and Lxra–/–Lxrb–/– mice were infected i.t. with luminescent M. tuberculosis (1 × 104 CFU). After 21 and 35 days of infection, CD4+ lung T cells were isolated, and the relative mRNA levels of Th1 (IFN-γ and T-bet), Th2 (IL-4 and Gata3), and Th17 (IL-17 and RORγt) markers were analyzed by RT-qPCR. Data are expressed as relative mRNA levels, normalized against reference housekeeping genes (n = 5). Results are representative of 2 separate experiments.
Treatment with LXR agonist increases resistance to M. tuberculosis infection. To address the consequences of further LXR signaling pathway activation on susceptibility to M. tuberculosis infection, we administered the LXR agonist TO91317 to WT mice. Treatment consisted of i.p. injection of C57BL/6 mice with 50 μg TO91317 3 times a week for 4 weeks. In a prophylactic protocol, treatment started 1 week before i.t. instillation of the bacteria. Bacillary growth in the lungs was monitored after 3 weeks of infection (Figure 7A). In a control experiment, the efficacy of the agonist in triggering the expression of the LXR target genes ABCA1 and ApoE was confirmed in mock-infected mice following the same treatment schedule (Figure 7B). Upon challenge of TO91317-treated mice with live M. tuberculosis, a pronounced 10-fold reduction in bacterial numbers in the lungs was observed compared with the control group (Figure 7C). Treatment with the highly LXR-specific agonist GW3965 similarly increased resistance of the mice to infection with M. tuberculosis (Figure 7C). In line with previous results, this further activation of LXR activity resulted in increased Th1 and Th17 responses, as evidenced by the increased mRNA levels of IFN-γ and T-bet — and even more so of IL-17 and RORγt — in CD4+ lung T cells from agonist-treated mice (Figure 7D). The mRNA expression levels of IL-4 and Gata3 remained largely unchanged.
Prophylactic LXR agonist treatment protects mice against airway challenge with M. tuberculosis. (A) Schedule of prophylactic treatment with LXR agonist TO91317 (TO) or GW3965 (GW) in conjunction with the i.t. M. tuberculosis challenge model. WT C57BL/6 mice were injected i.p. 3 times per week with 50 μg agonist. (B) RT-qPCR analysis of LXR-dependent target gene mRNA in total BAL cells from mock-infected animals using the same treatment schedule. (C) Bacterial load in the lungs of treated and control groups at day 21 after infection. No difference was observed in the vehicle-treated control group and the placebo-treated group (not shown). Values are expressed as log10 mRLU per organ (n = 5). (D) CD4+ lung T cells were isolated after 21 days of infection, and the relative mRNA levels of Th1, Th2, and Th17 markers (described in Figure 6) were analyzed by RT-qPCR. Data are expressed as relative mRNA levels, normalized against reference housekeeping genes (n = 5). Results are representative of 2 separate experiments. *P < 0.05, **P < 0.01 versus control.
Therapeutic postinfection treatment with the LXR agonist, in which we allowed the infection to establish for a period of 14 days before initiating a 3-week treatment (Figure 8A), produced a significant drop in bacterial load in the lungs (Figure 8, A and B). In order to verify that treatment with the LXR agonist was also capable of repressing a long-term, persistent pulmonary infection, mice were instilled i.t. with low-dose M. tuberculosis (1 × 102 CFU), and the infection was allowed to establish itself for 8 months before starting agonist treatment (Figure 8C). In this model of persisting tuberculosis, the 4-week treatment schedule reduced the bacterial burden in the lungs approximately 10-fold compared with control-treated animals (Figure 8D). These results highlight the importance of LXR-dependent pathways in the protective immune response both at the onset of and during further propagation of M. tuberculosis infection.
Therapeutic treatment with LXR agonist protects mice against airway challenge with M. tuberculosis. (A and C) Schedule of therapeutic treatment with TO91317 in conjunction with the i.t. M. tuberculosis challenge model. WT C57BL/6 mice were injected i.p. 3 times per week with 50 μg TO91317. (B) Bacterial load in the lungs at 35 days after infection. Values are expressed as log10 mRLU per organ (n = 5). (D) Bacterial load in the lungs at 28 days after infection. Values are expressed as log10 CFU per organ (n = 5). *P < 0.05, **P < 0.01 versus control.