fundTPL-2 – ERK1/2 Signaling Promotes Host Resistance against Intracellular Bacterial Infection by Negative Regulation of Type I Interferon Production

Abstract

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality and morbidity worldwide, causing approximately 1.4 million deaths per year. Key immune components for host protection during tuberculosis include the cytokines IL-12, IL-1 and TNF-α, as well as IFN-γ and CD4+ Th1 cells. However, immune factors determining whether individuals control infection or progress to active tuberculosis are incompletely understood. Excess amounts of type I interferon have been linked to exacerbated disease during tuberculosis in mouse models and to active disease in patients, suggesting tight regulation of this family of cytokines is critical to host resistance. In addition, the immunosuppressive cytokine IL-10 is known to inhibit the immune response to Mtb in murine models through the negative regulation of key pro-inflammatory cytokines and the subsequent Th1 response. We show here, using a combination of transcriptomic analysis, genetics and pharmacological inhibitors that the TPL-2-ERK1/2 signaling pathway is important in mediating host resistance to tuberculosis through negative regulation of type I interferon production. The TPL-2-ERK1/2 signalling pathway regulated production by macrophages of several cytokines important in the immune response to Mtb as well as regulating induction of a large number of additional genes, many in a type I IFN dependent manner. In the absence of TPL-2 in vivo, excess type I interferon promoted IL-10 production and exacerbated disease. These findings describe an important regulatory mechanism for controlling tuberculosis and reveal mechanisms by which type I interferon may promote susceptibility to this important disease.

Introduction

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb) infection, is a leading cause of mortality and morbidity (1). Listeriosis, resulting from infection with Listeria monocytogenes (Listeria) causes severe infection in immune compromised individuals, with high levels of mortality (2, 3). Both Mtb (4-6) and Listeria (2, 3, 7) are intracellular pathogens, with phagocytes, including macrophages, thought to be the major infected cell types. Although macrophages alone are incapable of clearing these infections, common effector functions employed by these cells are crucial to host resistance against both pathogens (2, 3, 7) (4-6).

Key innate cytokines for host protection against Mtb (4-6) and Listeria (2, 3, 7) infection include IL-12, TNFα and IL-1. Both human and mouse studies indicate that IL-12 is critical for activation of CD4+ T cells and induction of IFNγ production from a variety of cellular sources during tuberculosis (8-10) and Listeria (11, 12) infection. IFNγ in turn is essential to activate macrophage bactericidal functions and promote further innate cytokine production, including IL-12 (8, 12-17). Mice lacking TNF or IL-1 or their receptors are highly susceptible to Mtb (18-22) and Listeria (23-26) infection. Furthermore, anti-TNF antibody therapy in arthritis and Crohn’s disease patients is associated with increased reactivation of latent tuberculosis (27).

Not all aspects of the immune response are host protective, with some cytokines contributing to chronic disease during Mtb (6) and Listeria (3) infection. Members of the type I interferon (IFN) family of cytokines, signaling through a common heterodimeric receptor (IFNαβR or IFNAR), seem to play a detrimental rather than protective role during tuberculosis and Listeria infection (28, 29). Several studies have shown that Ifnar1−/− mice have greatly reduced bacterial loads following Listeria infection (30-33). A number of mechanisms have been suggested for the exacerbated disease induced by type I IFN during Listeria infection, including induction of IL-10 following T cell apoptosis and down-regulation of the IFNγ receptor (30-34).

Results from Mtb infection of Ifnar1−/− mice have varied between studies (35-40), but overall suggest a detrimental role for type I IFN during tuberculosis (37-39). Increased levels of type I IFN were induced by infection of mice with several hyper-virulent strains of Mtb and resulted in exacerbated disease (36, 37). Furthermore, in mouse models where large amounts of type I IFN were induced following administration of the polyinosinic-polycytidylic acid derivative Poly-ICLC during Mtb infection, tuberculosis was more severe (40). Direct instillation of IFNα/β into the lung of Mtb infected mice also resulted in exacerbated disease (36). We have recently reported a potential role of type I IFN in human tuberculosis, since active tuberculosis patients showed a prominent type I IFN-inducible gene signature in their blood, which correlated with extent of radiographic disease and was diminished upon successful treatment (41).

The mechanisms by which type I IFN exacerbates tuberculosis are less clear than for Listeria infection, however a recent report has shown that type I IFN suppresses the host protective cytokine IL-1, an effect that was partially due to IL-10 (20). IL-10 is a broadly immune-suppressive cytokine produced by cells of both the innate and adaptive immune systems (42) which can suppress the host protective immune response during Mtb (4, 43-45) and Listeria (46-48) infection.

The regulation of cytokine production by macrophages and other innate cells during Mtb infection is likely to shape the downstream immune response and determine disease outcome. Pattern recognition receptors (PRR), such as the Toll-like receptors (TLR), expressed by innate immune cells are critical in the activation of these cells and induction of cytokines in response to infection (49). Signaling pathways triggered by these PRR variously lead to activation of NF-κB, mitogen-activated protein kinases (MAPK) such as extracellular signal-related kinases 1/2 (ERK1/2), Interferon regulatory factors (IRF), and the inflammasome (49). Together these control induction of pro-inflammatory cytokines (such as IL-12, TNF and IL-1), IL-10 and type I IFN (49).

We show here that infection of macrophages with Mtb results in early induction of an ERK-specific cluster of genes. This prompted us to investigate the role of this pathway in disease outcome upon Mtb infection. The MAPK ERK1/2 is known to regulate production of multiple cytokines by macrophages, including those important in the response to Mtb and Listeria, downstream of TLR and other PRR (50-60). In macrophages, activation of ERK1/2 following ligation of TLRs is known to depend on the MAP 3-kinase Tumour progression locus 2 (TPL-2) (61). We here report, by analysis of Tpl2−/− macrophages, that TPL-2 – ERK1/2 signaling differentially regulates the production of multiple cytokines following Mtb infection. Furthermore, we show that in vivo, dysregulation of cytokine production in Tpl2−/− mice led to increased susceptibility to both Mtb and Listeria infection. This increased susceptibility was dependent on type I IFN signaling. Our findings define an important role for the TPL-2 – ERK1/2 signaling pathway in control of intracellular bacterial infections through negative regulation of type I IFN production.

Materials and Methods

Mice & Ethics statement

C57BL/6 WT mice and Tpl2−/−, Ifnar1−/−, Tpl2−/−Ifnar1−/−, Rag1−/− and Rag1−/−Tpl2−/− mice (all backcrossed ≥N10 generations to the C57BL/6 background) were bred and housed under specific pathogen-free conditions at the MRC NIMR. All protocols for breeding and experiments were performed in accordance with Home Office (UK) requirements and the Animal Scientific Procedures Act, 1986. Mice were sex and age matched for use in experiments.

In vivo infection protocols

Mtb stocks were grown and Mtb experiments were performed under containment level-3 conditions as previously described (44). Listeria experiments were performed under containment level-2 conditions. Listeria monocytogenes stocks were grown in BHI broth and stocks frozen in PBS/glycerol. Mice were injected intravenously with ~5 × 10³ Listeria.

Removal of Organs for determination of bacterial load

Bacterial loads from Mtb infected mice were determined as previously described (44). For Listeria infections cell suspensions were serially diluted onto BHI plates and after 1 day at 37°C, visible CFU counted and bacterial loads per organ calculated.

Reagents

Culture medium was RPMI 1640 with 5% heat-inactivated FCS, 0.05mM 2-ME, 10mM HEPES buffer, 2mM L-glutamine, and 1mM sodium pyruvate. IFNβ was obtained from PBL and IFNγ from R & D systems. DMSO was from Sigma Aldrich.

Generation and infection of bone marrow-derived macrophages & sorting and infection of bone marrow monocytes

Bone marrow cells were flushed from the femurs and tibia of mice and plated at 0.5×10⁶ cells/ml on bacterial plates (Sterilin), in culture medium containing 10% FCS and 20% L929 cell-conditioned medium. At day 6, macrophages were harvested and seeded into 24-well tissue culture plates (Corning) at 1×10⁶ cells/ml in culture medium. Cells were rested overnight, washed once with PBS, and infected at multiplicity of infection (MOI) 2:1 with Mtb H37Rv. Where indicated, IFNβ (2ng/ml) was added concomitantly with Mtb H37Rv. For inhibitor experiments, cells were pre-treated for 30 minutes with 0.1μM of either the MEK1/2 inhibitor PD0325901 or DMSO vehicle control. 0.1μM PD0325901 was chosen based on data presented in Bain et al (65) and on a detailed titration of the inhibitor on BMDM, done for this study (data not shown), to determine a dose that gave robust inhibition but was low enough to avoid toxicity or potential off target effects. For Listeria infection, macrophages were generated as described above and infected with Listeria at an MOI of 1:1. At 1hr post-infection cells were washed once with PBS and media containing 10ug/ml gentamycin added to inhibit growth of extra-cellular bacteria. Where indicated, IFNα or IFNβ (both 2ng/ml) was added concomitantly with Listeria.

Bone Marrow monocytes were isolated by fluorescence activated cell sorting on a Beckman-Coulter MoFlo. Bone Marrow cell suspensions were stained with antibodies against CD3, CD19, CD11c, F4/80, Ly6G and Ly6C, purchased from eBioscience. Monocytes were sorted as CD3-CD19-CD11c^loF4/80-Ly6G⁻Ly6C⁺. Cells were then plated at 1×10⁶ cells/ml and infected with H37Rv at MOI 2:1.

Measurement of supernatant & serum cytokines

Cytokine concentrations in the supernatants of infected cells were determined using commercial kits for IFNβ and IFNα (PBL), TNFα and IL-12p70 (eBioscience) and IL-1β (R & D systems). IL-12p40 was detected using Ab clone C15.6.7 for capture and biotinylated Ab clone C17.8 for detection. IL-10 was detected using Ab clone JES5-2A5 for capture and biotinylated anti-IL-10 for detection (BD Biosciences). Custom magnetic bead arrays to measure IL-10, IL-12p40, IL-12p35, and IFNγ in serum were purchased from Millipore and used according to manufacturer’s instructions. Samples were run on a Bio-Rad Luminex200 machine.

Processing of macrophage RNA for qPCR and microarray analysis

At indicated times post-infection, supernatants were removed and cells were washed once with PBS. RNA was harvested in 350μl RLT buffer (Qiagen) and stored at −80°C before processing. RNA was processed using RNeasy mini-kits (Qiagen). RNA integrity was assessed using an Agilent 2100 Bioanalyser (Agilent technologies). Samples had an RNA integrity number (RIN) of 9.3 to 10.

For microarray analysis, biotinylated, amplified antisense complementary RNA (cRNA) was prepared from 300ng of total RNA using the Illumina TotalPrep RNA amplification kit (Applied Biosystems/Ambion). 750ng of labelled cRNA was hybridized overnight to Illumina Mouse WG-6 V2.0 Beadchip arrays (Illumina). The arrays were then washed, blocked, stained and scanned on an Illumina BeadStation 500 following the manufacturer’s protocols. Illumina BeadStudio software (Illumina) was used to generate signal intensity values from the scans. For qPCR, RNA was reverse transcribed with a high-capacity reverse transcription kit (Applied Biosystems) to cDNA. The expression of indicated genes was quantified by real-time PCR (ABI PRISM 7900 (Applied Biosystems)) and normalised against Hprt1 mRNA levels. Murine primers were all purchased from Applied Biosystems.

Microarray data analysis

Illumina Beadstudio software was used to subtract background from signal intensity values. GeneSpring GX version 11 (Agilent Technologies) was used to perform further analysis. All signal intensity values less than 10 were set to equal 10. This threshold step is a standard analysis step done for Illumina microarray data. Due to the lower sensitivity of the lower intensity values obtained from Illumina microarray data it is necessary in order to reduce Type 1 error (false positives) occurring when applying fold change analysis. Data was then normalised by shifting to the 75^th percentile. Per-gene normalization was then applied by dividing the signal intensity of each probe in each sample by the median intensity for that specific probe which is obtained across all samples. This is also a standard step for Illumina microarray data. Since this calculation is done in the log scale it acts as a subtraction. This step is performed to reduce non-biological differences and although it may affect the absolute values, it does not affect the fold change between values: the measurement used here.

All transcripts were filtered first to select detected transcripts: All transcripts were filtered first to select detected transcripts: detected transcripts were defined as those ‘present’ (p<0.01) in 100% of triplicate samples within any one group. Present calls were selected if the signal precision (a probe’s intensity value compared to the background intensity value) was statistically significantly different (p<0.01). Statistical and fold-change analyses were then applied as described in the figure legends, to generate lists of differentially expressed genes. These were subjected to hierarchical clustering, using Pearson Centered distance metric and complete linkage, and visualised using a heat map. Microarray data has been deposited at the GEO database (http://www.ncbi.nlm.nih.gov/geo/), accession number GSE47674.

Statistics

Statistical tests, as described in the figure legends, were used to determine significance. * p<0.05, **p<0.01, ***p<0.001.

Results

The TPL-2 - ERK1/2 pathway is rapidly activated in macrophages infected with Mtb

To investigate the transcriptional regulation of cytokine production in response to Mtb infection, we infected bone marrow derived macrophages with Mtb and carried out microarray analysis of purified RNA from cells harvested at various times post-infection. Removal of undetectable genes and those which did not show a greater than two-fold-change upon infection, together with statistical filtering, resulted in 6479 differentially regulated transcripts, which were then subjected to supervised hierarchical clustering (Figure 1A). We used K-means clustering to separate these 6479 transcripts into discrete clusters of genes that had similar expression kinetics (Supplementary Figure 1). The clusters showed a range of expression patterns, with some induced rapidly (between 30mins and 1hr post-infection) and others expressed relatively late (between 6hr and 24hr post-infection) (Supplementary Figure 1). Two clusters in particular were upregulated between 30mins and 1hr post-infection. The first remained elevated throughout the course of infection, and contained several pro-inflammatory cytokines and chemokines (Cluster 8, Supplementary Figure 1). In contrast, the genes in the second early cluster (Cluster 15, Supplementary Figure 1) were only transiently upregulated, with most genes returning to baseline by 3-6hr post-infection (Figure 1B, 1C). Interestingly, many of the genes in this cluster have previously been described as targets of the ERK1/2 MAPK pathway, including Fos (54, 62, 63), Egr1 (62, 63) and Dusp1 (64) (Figure 1C, 1D).

Figure 1. The TPL2-ERK pathway is rapidly activated in macrophages infected with Mtb.

WT macrophages were infected with Mtb H37Rv, RNA was harvested at the indicated time-points post-infection, processed and analysed by microarray. Following normalisation as described in the materials and methods, differentially regulated transcripts were obtained by taking those that were at least 2-fold up- or down-regulated in infected samples vs. controls at any time-point, and those that were significantly different by 2-way ANOVA p<0.05 with Benjamini-Hochberg multi-test correction.

(A) This left 6479 differentially regulated genes. These were separated into 24 clusters based on expression profile, using K-means clustering with Pearson Centered distance metric (See Supplementary figure 1).

(B) One of the 24 clusters with early but transient expression.

(C) Heat map of early cluster.

(D) qRT-PCR validation of several ERK inducible genes in this cluster found by microarray analysis. RNA was reverse transcribed to cDNA and expression of the indicated genes analysed by qRT-PCR. Values were normalised relative to Hprt expression levels.

TPL-2 negatively regulates IL-12 and IFNβ, but positively regulates IL-10, IL-1β and TNFα, in Mtb infected macrophages and monocytes

The TPL-2-ERK MAPK pathway has previously been shown to be a key regulator of cytokine production in innate immune cells, in response to a variety of TLR ligands including LPS, Pam-3-Cys and CpG (51, 52, 54, 55). We next investigated if this pathway also regulated cytokines similarly in response to Mtb infection, which activates multiple PRRs, including both TLR and non-TLR (4, 5). Addition of PD0325901, which specifically inhibits the catalytic activity of the MAP 2-kinases MEK1/2 and abrogates subsequent ERK activation (65), resulted in increased production of IL-12p40, IL-12p70 and IFNβ and decreased production of IL-10, TNFα and IL-1β, in Mtb infected macrophages (Figure 2A). This effect was phenocopied in Tpl2−/− macrophages which secreted markedly increased levels of IL-12p40, IL-12p70 and IFNβ, but less IL-10, TNFα and IL-1β, in response to Mtb infection, as compared to WT controls (Figure 2B). The effects of TPL-2 deficiency on cytokine production following Mtb infection, therefore, appeared to result from impaired ERK activation, similar to previous studies using purified TLR ligands (51, 52, 54, 55). Finally, to confirm that similar responses were seen in ex vivo-derived cells, monocytes were sorted from the bone marrow of WT and Tpl2−/− mice, and infected with Mtb. The phenotype of these cells (Figure 2C) mirrored that seen using Tpl2−/− macrophages, or following MEK1/2 inhibition.

Figure 2. TPL-2 negatively regulates IL-12 and IFNβ, but positively regulates IL-10, IL-1β and TNFα, in Mtb infected macrophages and monocytes.

(A) WT macrophages were treated with 0.1μM of the MEK inhibitor PD0325901, or vehicle control, beginning 30 minutes prior to infection with Mtb H37Rv. Cytokine levels in culture supernatant were determined by ELISA at 24hr post-infection. Significance was determined using an unpaired t test.

(B) Macrophages derived from WT or Tpl2−/− mice were infected with Mtb H37Rv. Cytokine levels in culture supernatant were determined by ELISA at 24hr post-infection. Significance was determined using an unpaired t test.

(C) Monocytes (Ly6C+Ly6G−) were sorted from the bone-marrow of WT or Tpl2−/− mice, and infected with Mtb H37Rv. Cytokine levels in culture supernatant were determined by ELISA at 24hr post-infection. Significance was determined using an unpaired t test.

Graphs show one representative experiment of at least 4 for parts (A) & (B) and one of three for part (C) with triplicate wells per group.

The TPL-2 – ERK1/2 pathway is an important regulator of transcription in Mtb infected macrophages

Having shown that the TPL-2 – ERK1/2 pathway regulates cytokine protein production in Mtb-infected macrophages, we next carried out microarray analysis of RNA from infected WT and Tpl2−/− macrophages, to determine in an unbiased fashion how the TPL-2-ERK pathway regulates gene expression at a global level. We chose 6 hr post-infection for this analysis, as this was the peak of transcription of the majority of genes observed in WT macrophages infected with Mtb (Figure 1A).

To determine which Mtb induced genes were significantly differentially regulated in WT versus Tpl2−/− macrophages, we carried out fold-change analysis of genes that were over or under expressed upon Mtb infection of macrophages to firstly determine those genes regulated by Mtb infection. This set of Mtb regulated genes was then further analysed by statistical filtering and fold change analyses, now comparing the WT and Tpl2−/− macrophages, to determine which of the Mtb regulated genes were significantly regulated by TPL-2 upon Mtb infection. This identified 104 differentially expressed genes (Figure 3A), containing a prominent cluster of genes that were upregulated to a greater extent in Tpl2−/− macrophages (Figure 3A). Genes present in this cluster included the class-2 transactivator (C2ta), plasmin (Plat) and chloride intracellular ion channel 5 (Clic5). Importantly, Il12a (encoding the p35 subunit of IL-12) and several members of the type I IFN family including Ifna2, Ifna5 and Ifna6 were also found in this cluster of elevated genes (Figure 3A). A second cluster contained genes that were downregulated to a greater extent in Tpl2−/− macrophages, including the genes encoding Arginase (Arg1) and dual specificity phosphatase 6 (Dusp6) (Figure 3A). Interestingly, this down-regulated cluster of genes also included that encoding a subunit of the interferon-gamma receptor (Ifngr1), which is crucial in activation of myeloid cells for resistance to Mtb infection (Figure 3A). We confirmed the elevated expression levels of Il12a, Ifna2, Ifna5, Ifna6 mRNAs and reduced expression of Ifngr1 mRNA by qRT-PCR of RNA taken from the same Mtb-infected Tpl2−/− and WT control macrophages used for microarray (Figure 3B). Although not detected in the microarray, qRT-PCR for Il12b mRNA (encoding the IL-12p40 subunit) revealed elevated expression levels in Tpl2−/− macrophages (Supplementary Figure 2), similarly to the results seen for Il12a. A third cluster of differentially regulated transcripts was also found, which contained the immediate early genes, Ier3 and Ier5 (Figure 3A).

Figure 3. The TPL-2-ERK pathway regulates many genes in Mtb infected macrophages, including IL-12 and several subtypes of IFNα.

Macrophages derived from WT or Tpl2−/− mice were infected with Mtb H37Rv. At indicated time-points RNA was harvested, processed and analysed by microarray.

(A) Following normalisation as described in the materials and methods, differentially regulated transcripts were obtained by taking those that were at least 2-fold up- or down-regulated in infected samples compared to controls at 6hr in either strain, and those that were significantly different by 2-way ANOVA p<0.05 with Benjamini-Hochberg multi-test correction. A further filter was applied to remove genes less than 2-fold different between WT and Tpl2−/− infected samples. This left 104 genes. A cluster of 52 genes was more strongly upregulated in Tpl2−/− macrophages (upper box) and a cluster of 25 was more strongly downregulated (lower box). A third cluster of differentially regulated genes was also present.

(B) RNA from the same experiment was reverse transcribed to cDNA and expression of the indicated genes analysed by qRT-PCR. Values are normalised relative to Hprt expression levels.

The list of 104 differentially expressed genes shown by microarray did not contain IFNβ itself, IL-1β, TNFα or IL-10, despite differences being observed at the protein level (see Figure 2) and in contrast to our and others’ previous reports showing these genes to be regulated by TPL-2 in macrophages in response to TLR ligands (50-60). However, qRT-PCR revealed that the mRNA encoding IFNβ was actually significantly upregulated in Tpl2−/− macrophages compared to WT (Figure 3B & Supplementary Figure 2). TPL-2 deficiency also significantly decreased the levels of Il10 and Il1b mRNAs, although the differences compared to WT were relatively small, in contrast to our previous results with LPS stimulation (54). Given the significant decreases in protein concentrations of IL-10 and IL-1β in infected Tpl2−/− macrophages (Figure 2), this may suggest a role for post-transcriptional regulation of these cytokines. No difference in the abundance of the mRNA encoding TNFα was detected by qRT-PCR (Figure 3B), in line with earlier reports with LPS-stimulated macrophages showing that TNFα is largely regulated at the post-transcriptional level by TPL-2 (52, 56).

TPL-2 deficient mice are more susceptible to intracellular bacterial infection

Given the complex regulation by TPL-2 – ERK1/2 signaling in Mtb-infected macrophages of cytokines known to be either host protective or favouring chronic infection, we investigated the net effect of abrogation of TPL-2 – ERK1/2 signaling during intracellular bacterial infection in vivo. WT and Tpl2−/− mice were aerosol infected with Mtb H37Rv, or alternatively intravenously infected with Listeria. Organs from Mtb infected mice were harvested at days 28, 56 and 100 post-infection for enumeration of bacterial loads (Figure 4 and data not shown). Although no difference in lung bacterial loads were found early (day 28) following Mtb infection (data not shown), at day 56 Tpl2−/− mice had significantly higher bacterial loads in the lung (Figure 4A) and spleen (data not shown) compared with wild-type mice, with 5 to 10 fold more bacteria in the lung (Figure 4A). This increase in bacterial load in lungs of Tpl2−/− compared with WT mice was also seen at day 100 post-infection (data not shown). Tpl2−/− mice were also highly susceptible to Listeria infection; having bacterial loads in the spleen more than ten fold higher than WT controls at day 3 post-infection (Figure 4B). Bacterial loads were also significantly higher in Tpl2−/− mice than WT controls at days 1 and 2 post-infection, although differences were smaller than at day 3 (data not shown).

Figure 4. TPL-2 deficient mice are more susceptible to intracellular bacterial infection.

(A) WT and Tpl2−/− (top row) or Rag−/− and Rag−/−Tpl2−/− (bottom row) mice were infected with Mtb H37Rv via the aerosol route. At the indicated times post-infection lungs were harvested and bacterial loads enumerated via serial dilution and plating. Graphs show one representative experiment of at least two, with at least 5 mice per group. Significance was determined using unpaired t test.

(B) WT and Tpl2−/− (top row) or Rag−/− and Rag−/−Tpl2−/− (bottom row) mice were infected with Listeria via the intravenous route. At the indicated times post-infection spleens were harvested and bacterial loads enumerated via serial dilution and plating. Graphs show one representative experiment of at least three, with 4 mice per group for Listeria infections. Significance was determined using unpaired t test.

(C) Serum from Listeria infected WT and Tpl2−/− mice was harvested at day 2 post-infection and the indicated cytokines measured by ELISA or bead array. Graphs show one representative experiment of at least two, with at least 4 mice per group. Significance was determined using unpaired t test. ND - Not detectable.

(D) WT or Tpl2−/− macrophages infected with Listeria were treated concomitantly with 2 ng/ml of recombinant IFNα or IFNβ. Cytokine levels in culture supernatant were determined by ELISA at 24hr post-infection. Graphs show one representative experiment of at least three, with triplicate wells per group. Significance was determined using one-way ANOVA with tukey post-hoc test.

To determine whether the increased susceptibility of TPL-2 deficient mice involved impaired innate immune responses, WT and Tpl2−/− mice on a Rag1 deficient background were infected with Mtb H37Rv or with Listeria (Figure 4A & 4B, bottom rows). Similar to Tpl2−/− versus WT mice, infected Rag−/−Tpl2−/− mice had significantly higher bacterial loads compared to their counterpart Rag−/− controls (Figure 4A & 4B, bottom rows). These data demonstrate that the increased susceptibility of Tpl2−/− mice could be in part accounted for by a defect in the innate immune response.

TPL-2 negatively regulates expression of type I Interferon during bacterial infection

To investigate how TPL-2 deficiency might increase susceptibility of mice to bacterial infection, we measured levels of IL-12, TNFα and IL-1β, which are important for protection, and levels of type I IFN and IL-10, known to promote bacterial infection. Cytokine levels in the serum of WT and Tpl2−/− mice infected with Listeria were measured (Figure 4C). Levels of IL-12p40 and IL-12p35, IL-1β and TNFα in sera of Listeria infected mice were not significantly different between groups (Figure 4C). Although IFNβ was not detectable, strikingly, IFNα levels were significantly higher in sera from Listeria infected Tpl2−/− mice compared to WT (Figure 4C). Unexpectedly, IL-10 levels were also higher in serum of Tpl2−/− mice, in contrast to the reported requirement for TPL-2 – ERK1/2 in endogenous IL-10 production by TLR stimulated (51, 54, 60) or Mtb infected (Figure 2) innate immune cells in vitro. This suggested that other factors elevated in Tpl2−/− mice in vivo positively regulate IL-10 production in a TPL-2 – ERK1/2 independent manner. Indeed, type I IFN are known inducers of IL-10 (20, 66-68). To test whether Tpl2−/− cells were responsive to type I IFN, which might help to explain the elevated IL-10 levels seen in the serum of Tpl2−/− mice infected in vivo, Listeria infected WT or Tpl2−/− macrophages were treated with exogenous IFNα or IFNβ at the time of infection and IL-10 levels measured (Figure 4D). While levels of IL-10 overall were, as expected, lower in Tpl2−/− compared with WT macrophages, addition of type I IFNs to Tpl2−/− cells led to a fold increase in IL-10 production equivalent to that seen in WT macrophages treated with type I IFN (Figure 4D). Similar results were seen in WT and Tpl2−/− macrophages infected with Mtb (Supplementary Figure 2). This showed that Tpl2−/− cells were not completely deficient in IL-10 production and that type I IFN could at least partially overcome the requirement for TPL-2 – ERK1/2 signaling in vitro and possibly fully overcome the requirement for TPL-2 – ERK1/2 in vivo. In addition, we found that type I IFN treatment of WT and Tpl2−/−, Listeria infected, macrophages significantly suppressed production of both IL-12p40 and IL-12p70 by these cells (Figure 4D). None of the cytokines measured were detectable in the serum of Mtb infected mice (data not shown). Collectively, these results suggested that elevated levels of type I IFN and IL-10 may contribute to the increased susceptibility of Tpl2−/− mice to intracellular bacterial infection.

Type I IFN signaling regulates transcription in Tpl2−/− macrophages

Given that protein and mRNA levels of type I IFN were greatly increased in in vitro Mtb infected TPL-2 deficient macrophages, and that levels of type I IFN were increased in vivo in mice lacking TPL-2, we hypothesised that type I IFN may be contributing to transcriptional changes in innate cells following infection in the absence of TPL-2, leading to increased susceptibility. We analysed by microarray the expression of the genes previously found to be up- or down-regulated by TPL-2 deficiency in Mtb-infected macrophages (see Figure 3) in Tpl2−/− or WT macrophages additionally crossed to Ifnar1−/−, to determine whether any TPL-2 regulated genes were strongly regulated by type I IFN signalling during Mtb infection (Figure 5). A large number of the genes up-regulated in Tpl2−/− macrophages were dramatically lower in Tpl2−/−Ifnar1−/− macrophages, in many cases down to levels observed in Ifnar1−/− control Mtb-infected macrophages (Figure 5A, genes in bold). These genes included Il12a, which intriguingly has a protective role in Mtb infection, but also several subtypes of Ifna: Ifna2, 5 and 6, associated with disease exacerbation, as well as Tapblp, Clic5, Snn and Hmgn3 (Figure 5A). This IFNAR dependent regulation also applied to a cluster of genes which were down-regulated by TPL-2 deficiency following infection with Mtb; with many returning to WT levels in Tpl2−/−Ifnar1−/− macrophages (Figure 5B). These included Ifngr1, Dusp6 and to a lesser extent Arg1 (Figure 5B). We validated the microarray results for Il12a, Ifna2, Ifna5, Ifna6, and Ifngr1 by qRT-PCR, and IL-12p70 by ELISA (Supplementary Figure 3B, C), confirming that the perturbation of expression of these genes/ proteins in Tpl2−/− macrophages infected with Mtb resulted from enhanced IFNAR signaling. We also carried out qRT-PCR and ELISA to examine levels of IFNβ, IL-1β, TNFα and IL-10 (Supplementary Figure 3B, C). IL-1β transcriptional levels were not rescued in the Tpl2−/−Ifnar1−/− cells but protein levels were much higher, suggesting post-transcriptional regulation of IL-1β by type I IFN (Supplementary Figure 3). IL-10 levels, both at the transcriptional and protein level, were shown to be reduced in Mtb-infected Tpl2−/− macrophages and Ifnar1−/− macrophages as compared to WT controls, and lowest levels of IL-10 mRNA and protein were detected in Tpl2−/−Ifnar1−/− Mtb-infected macrophages (Supplementary Figure 3). Elevated IFNβ mRNA expression and protein levels in Mtb-infected Tpl2−/− macrophages were dependent on IFNAR expression (Supplementary Figure 3B, C). This experiment demonstrates that a large component of the transcriptional changes observed in Tpl2−/− macrophages result from the increased levels of type I IFN signaling, affecting immunologically important genes such as Il12a, Ifna and Ifngr1.

Figure 5. Type I IFN signaling regulates transcription in Tpl2−/− macrophages.

(A) & (B) Macrophages derived from WT, Ifnar1−/−, Tpl2−/− and Tpl2−/−Ifnar1−/− mice were infected with Mtb H37Rv. At indicated time-points RNA was harvested, processed and analysed by microarray. Expression levels for the gene lists originally generated by comparison of Mtb infected WT and Tpl2−/− macrophages and detailed in Figure 3 were analysed across the four strains and are shown here. See also Supplementary figure 2.

Type I Interferon signaling contributes significantly to the increased susceptibility of Tpl2−/− mice to intracellular bacterial infection

Given the raised levels of type I IFN in Listeria infected Tpl2−/− mice, and the IFNAR dependent transcriptional perturbation of genes observed in Tpl2−/− macrophages, we hypothesised that type I IFN over-production in TPL-2 deficient animals may be contributing to their increased susceptibility to Mtb and Listeria infection in vivo. To test this hypothesis we infected Tpl2−/−Ifnar1−/− mice with Mtb or Listeria and compared their bacterial load with similarly infected single knockout Tpl2−/− and Ifnar1−/− mice and WT control mice (Figure 6). We chose the Mtb strains HN878 and BTB02-171 for infection as these strains are hyper-IFN inducing and would enhance any IFN dependent difference between groups ((36, 37) & J. Carmona and M. Saraiva, unpublished data).

Figure 6. Type I IFN signaling contributes significantly to the increased susceptibility of Tpl2−/− mice to intracellular bacterial infection.

(A) WT, Tpl2−/−, Ifnar1−/− and Tpl2−/−Ifnar1−/− mice were infected with Mtb HN878 or Mtb BTB02-171 via the aerosol route. At the indicated times post-infection lungs were harvested and bacterial loads enumerated via serial dilution and plating. Graphs show one representative experiment of at least two for each Mtb strain, with at least 4 mice per group. Significance was determined using one-way ANOVA with tukey post-hoc test.

(B) WT, Tpl2−/−, Ifnar1−/− and Tpl2−/−Ifnar1−/− mice were infected with Listeria via the intravenous route. At the indicated time post-infection spleens were harvested and bacterial loads enumerated via serial dilution and plating. Graph shows one representative experiment of three, with at least 3 mice per group. Significance was determined using one-way ANOVA with tukey post-hoc test.

(C) Serum from Listeria infected WT, Tpl2−/−, Ifnar1−/− and Tpl2−/−Ifnar1−/− mice was harvested at day 2 post-infection and cytokines measured by ELISA or bead array. Graphs show one representative experiment of at least two, with at least 4 mice per group. Significance was determined using one-way ANOVA with tukey post-hoc test.

As previously seen, Tpl2−/− mice had higher bacterial loads compared to WT mice following infection with the different strains of Mtb or Listeria (Figure 6A & 6B). Ifnar1−/− mice had similar lung bacterial levels to WT mice following infection with Mtb. Following Listeria infection, Ifnar1−/− mice had lower bacterial burdens in the spleen compared to WT mice (Figure 6B), in line with previous reports (30-32). In all three infections the bacterial loads in Tpl2−/−Ifnar1−/− mice were significantly reduced compared with Tpl2−/− mice (Figure 6A & 6B). Following Mtb infection the increased bacterial burden in Tpl2−/− mice was completely abrogated in the absence of type I IFN signaling, with bacterial loads in the lungs of Tpl2−/−Ifnar1−/− mice equivalent to those in WT and Ifnar1−/− control mice (Figure 6A). Listeria infected Tpl2−/−Ifnar1−/− also had reduced bacterial loads compared with Tpl2−/− controls, to a level between the WT and Ifnar1−/− control groups (Figure 6B). Measurement of IL-10 in the serum of the Listeria infected mice revealed elevated concentrations of IL-10 in Tpl2−/− mice compared with WT controls (Figure 6C), as shown earlier (Figure 4C). This increase in IL-10 protein was abrogated in sera of Tpl2−/− mice crossed to Ifnar1−/− mice infected with Listeria (Figure 6C). These levels reverted to that of WT and Ifnar1−/− mice, demonstrating that this elevation of IL-10 in Tpl2−/− mice sera was totally attributable to IFNα/β. By contrast, IL-12p40 and IL-12p70 levels were significantly elevated in the serum of Listeria infected Tpl2−/−Ifnar1−/− mice, compared to WT, Tpl2−/− and Ifnar1−/− controls (Figure 6C), indicating an important role for type I IFN signaling in inhibiting IL-12 production, even in the absence of TPL-2 – ERK1/2 signaling. Similar results were also seen in in vitro Listeria infected macrophages (Supplementary Figure 4).

These data demonstrate a significant contribution of type I IFN signaling to the increased susceptibility of Tpl2−/− mice to intracellular bacterial infections.

Discussion

The macrophage plays a central role in the host response to intracellular bacterial infection. How macrophages perceive infection varies depending on the specific array of PRR engaged by a particular pathogen and on the signaling pathways activated downstream of these (49). We show here that following infection of macrophages with Mtb a set of gene transcripts are rapidly expressed, many of which have previously been described as targets of the TPL-2 – ERK1/2 MAPK pathway (62-64). We now show that the TPL-2 – ERK1/2 signaling pathway is important in mediating host resistance to tuberculosis and Listeria through negative regulation of type I IFN production.

Importantly, many of the ERK inducible genes expressed, such as Fos, Egr1 and Jmjd3, are transcription factors or transcriptional regulators implicated in regulation of cytokines in immune cells (50, 51, 54, 69, 70). Indeed, following TLR stimulation of macrophages, expression of the transcription factor FOS, downstream of TPL-2 – ERK1/2 signaling, has been shown to regulate production of several cytokines, including IL-12, IL-10 and type I IFN (50, 51, 54). Dusp1 and Ier3, known regulators of signaling pathways including MAPK pathways, were also activated (71, 72). This profile suggested that the TPL-2 – ERK1/2 pathway downstream of PRR may play an important role in the response to Mtb. We further investigated this by looking at the later transcriptional response in Mtb infected macrophages. Several type I IFN transcripts were upregulated in Tpl2−/− cells, as was Il-12a. Despite this increase in type I IFN transcripts in Tpl2−/− cells many classically type I IFN inducible transcripts (eg. Mx1) were not upregulated in Tpl2−/− cells compared with WT sufficiently to pass the microarray filtering. This probably reflects the early timepoint (6hr) at which the analysis was done as relatively little type I IFN feedback will have occurred by 6hr. Expression of Il-10, IFNβ and IL-1β was transcriptionally different in Tpl2−/− macrophages, as measured by qRT-PCR, despite not being sufficiently different to pass the microarray filtering. Tnf mRNA levels were not affected by TPL-2 deficiency. However, it is likely that TNFα and IL-1β and possibly IL-10 are also regulated at the post-transcriptional level. Interestingly, TPL-2 also appeared to regulate mRNA levels of Ifngr1 (which were reduced in Tpl2−/− macrophages), encoding a subunit of the IFNγ receptor. Together, these findings further suggested that TPL-2 – ERK1/2 signaling had important regulatory functions in the macrophage response to Mtb.

At the protein level TPL-2 – ERK1/2 was a positive regulator of TNFα, IL-10 and IL-1β following Mtb infection of macrophages. In contrast, this pathway negatively regulated IL-12 and IFNβ. Importantly, this finding was not limited to bone marrow-derived macrophages as ex vivo monocytes displayed the same phenotype. These findings are similar to results from previous reports that used purified TLR ligands to stimulate Tpl2−/− macrophages (52, 54-57, 59), with the exception of one report, where TPL-2 was a positive regulator of IFNβ (58). However, type I IFN protein levels were only investigated at very early time points in that study [58], which might account for some of the difference with our results. Furthermore, that report examined mice lacking TPL-2 through an ENU induced point-mutation (58), a process which may induce only partial reductions in signaling pathways.

The net effects of dysregulated cytokine production in mice lacking the TPL-2 – ERK1/2 pathway resulted in increased susceptibility to intracellular bacterial infection, as seen by increased bacterial loads. We observed this finding following Listeria infection of mice, in agreement with a recent study (55). We now report the novel findings that loss of the TPL-2 – ERK1/2 pathway resulted in exacerbated infection with Mtb, where multiple strains with varying levels of virulence all showed similar phenotypes. The observation that Tpl2−/− mice on a Rag1−/− background are also more susceptible to Mtb and Listeria infection suggests that a defect in innate immunity is important for increased susceptibility. Interestingly, this provides an additional mechanism for increased pathogen load in infection of Tpl2−/− mice to that of a previously reported model of Toxoplasma gondii infection, where T cell intrinsic defects in the response to IL-12, IL-18 and TCR stimuli, leading to reduced IFNγ production, in Tpl2−/− mice were suggested to be solely responsible for impaired host immunity (73). In our system we did not see differences in IFNγ protein levels in the serum of infected mice or by mRNA in lung (data not shown). It remains possible that any defect in CD4+ T cell IFNγ production is obscured by IFNγ produced by other sources that are not TPL-2 – ERK1/2 regulated. Alternatively, a recent study found that CD4+ T cells that were unable to produce IFNγ were still protective during tuberculosis (74). Our findings, while not wholly excluding a role for T cell defects in exacerbated Mtb and Listeria infection, support the idea that innate defects in immunity are important for lack of bacterial control in Tpl2−/− animals.

Because of the complex regulation of cytokine production in innate cells by the TPL-2 – ERK1/2 pathway, the perturbation to the immune response in Tpl2−/− mice that leads to increased bacterial load in vivo could be attributed to a number of sources. Likely candidates to explain the heightened disease seen in Tpl2−/− mice infected with Mtb or Listeria were reduced levels of the host protective cytokines TNF and IL-1β, or increased levels of type I IFN, which likely play a detrimental role. We found evidence of increased levels of type I IFN in Tpl2−/− mice and macrophages following infection. Further dissection of the response of Mtb infected macrophages, using Tpl2−/−Ifnar1−/− cells, revealed that many of the changes seen in Tpl2−/− macrophages, both at the transcriptional and protein levels, were controlled by type I IFN signaling. Of note, this group included transcription of Ifngr1, suggesting that type I IFN may be at least partially responsible for down-regulation of the IFNγR during Mtb infection. This may in turn reduce macrophage activation in response to IFNγ. In support of this, type I IFN has been shown to suppress macrophage activation through down-regulating expression of the IFNγR during Listeria infection (34, 75, 76). Altogether these results suggested that type I IFN signaling played a significant role in the dysfunctional response seen in the absence of TPL-2.

This led us to test the involvement of type I IFN signaling in the increased susceptibility of Tpl2−/− mice to bacterial infection. Upon infection with either Mtb or Listeria we found that abrogation of type I IFN signaling restored protection of Tpl2−/− mice to WT or near WT levels. These results strongly point to the loss of negative regulation of type I IFN production as primarily responsible for the increased susceptibility of Tpl2−/− mice. Increased levels of type I IFN were not solely responsible for exacerbated bacterial loads in Tpl2−/− mice in all cases, however, as bacterial loads in Tpl2−/−Ifnar1−/− mice were elevated slightly above Ifnar1−/− in some infections.

A previous report has suggested that loss of IL-1β production but not TNFα is responsible for increased bacterial loads in Tpl2−/− mice following Listeria infection (55). IL-1 is known to be important in host protection from Listeria (23, 24) and Mtb (19-22). However, it was not directly shown whether levels of IL-1β were reduced following in vivo Listeria infection of Tpl2−/− mice, nor whether the loss of IL-1β seen in vitro was a direct effect of TPL-2 deficiency or was mediated through another factor. We saw little difference in the transcriptional levels of TNFα in the lung (data not shown) or protein levels in serum. However, we also saw little difference in IL-1β. Nonetheless, this does not exclude a role for reduced levels of IL-1β contributing to exacerbated disease. Our results suggest, however, that reduced IL-1β in Tpl2−/− mice is likely to be an indirect effect of TPL-2 deficiency mediated by increased amounts of type I IFN. Indeed, we find, and others have also reported, that type I IFN is a potent suppressor of IL-1 production, in a partially IL-10 dependent manner (Supplementary Figure 2, data not shown and (20, 66)).

Unexpectedly, levels of IL-10 were increased in serum of infected Tpl2−/− animals, in contrast to results seen with macrophages in vitro. This increase in IL-10 was dependent on type I IFN signaling, as infected Tpl2−/−Ifnar1−/− mice did not have increased serum levels of IL-10. This suggests that increased levels of type I IFN are capable of overcoming the requirement for TPL-2 – ERK1/2 signaling for IL-10 production in vivo. Further supporting this idea, Listeria or Mtb infected Tpl2−/− macrophages in vitro produced increased levels of IL-10 in response to exogenous IFNα or IFNβ indicating that these cells are still responsive to type I IFN, although IL-10 production was still impaired compared with WT macrophages. In addition, it is possible that increased type I IFN produced by myeloid cells in vivo induces IL-10 production from other cell types that do not require TPL-2 for IL-10 induction but that are responsive to type I IFN or that very high levels of type I IFN can overcome the TPL-2 – ERK1/2 dependency of IL-10 production. Altogether, these results suggest that in vivo, promotion of IL-10 production is at least one mechanism by which the excess levels of type I IFN seen in TPL-2 deficient mice could lead to immunosuppression and hence increased susceptibility to Mtb and Listeria infection (20, 66-68).

In summary, we show that the TPL-2 – ERK1/2 signaling pathway in innate immune cells plays an important role in controlling intracellular bacterial infections caused by Mtb and Listeria. Despite the role of this pathway in regulating production of multiple cytokines, it is its ability to negatively regulate the production of type I IFN that is crucial in promoting host resistance to these bacterial pathogens. Because of the role of the TPL-2 – ERK1/2 pathway in regulating TNFα production it has attracted attention as a possible therapeutic target for treating inflammatory conditions such as rheumatoid arthritis (77). Our results suggest that great care will be necessary in targeting this pathway as it may lead to unwanted exacerbation of, or increased susceptibility to, certain bacterial infections.