Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages
Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved June 29, 2015 (received for review December 11, 2014)
Significance
Inflammatory signaling is a central mechanism controlling host defenses to pathogens. Members of Mycobacterium avium complex cause disease in immunocompromised patients and in individuals with predisposing lung abnormalities. We provide evidence of a mechanism in human primary macrophages whereby the oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) negatively regulates inflammatory responses and thus facilitates intracellular growth of M. avium. Our findings are of high biological and clinical significance, as opportunistic infections with nontuberculous mycobacteria are receiving renewed attention because of increased incidence and difficulties in treatment. Altered Keap1 gene expression may also have vital clinical implications for other inflammation-associated conditions, opening novel research venues for translational research, for instance in the expanding field of host-targeted therapy for infectious diseases.
Abstract
Several mechanisms are involved in controlling intracellular survival of pathogenic mycobacteria in host macrophages, but how these mechanisms are regulated remains poorly understood. We report a role for Kelch-like ECH-associated protein 1 (Keap1), an oxidative stress sensor, in regulating inflammation induced by infection with Mycobacterium avium in human primary macrophages. By using confocal microscopy, we found that Keap1 associated with mycobacterial phagosomes in a time-dependent manner, whereas siRNA-mediated knockdown of Keap1 increased M. avium-induced expression of inflammatory cytokines and type I interferons (IFNs). We show evidence of a mechanism whereby Keap1, as part of an E3 ubiquitin ligase complex with Cul3 and Rbx1, facilitates ubiquitination and degradation of IκB kinase (IKK)-β thus terminating IKK activity. Keap1 knockdown led to increased nuclear translocation of transcription factors NF-κB, IFN regulatory factor (IRF) 1, and IRF5 driving the expression of inflammatory cytokines and IFN-β. Furthermore, knockdown of other members of the Cul3 ubiquitin ligase complex also led to increased cytokine expression, further implicating this ligase complex in the regulation of the IKK family. Finally, increased inflammatory responses in Keap1-silenced cells contributed to decreased intracellular growth of M. avium in primary human macrophages that was reconstituted with inhibitors of IKKβ or TANK-binding kinase 1 (TBK1). Taken together, we propose that Keap1 acts as a negative regulator for the control of inflammatory signaling in M. avium-infected human primary macrophages. Although this might be important to avoid sustained or overwhelming inflammation, our data suggest that a negative consequence could be facilitated growth of pathogens like M. avium inside macrophages.
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The Mycobacterium avium complex consists of widely distributed opportunistic mycobacteria that cause nontuberculous pulmonary disease in immunocompromised individuals (1, 2). Like the more virulent Mycobacterium tuberculosis (Mtb), M. avium invades host macrophages and may escape killing by preventing phagosomal maturation while retaining access to essential nutrients (2–4). Initial infection is sensed by various pattern recognition receptors (PRRs) from surface Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), to endosomal TLRs, cytosolic NOD-like receptors (NLRs), and nucleic acid sensors (5–8). Engagement of these PRRs initiates production of inflammatory mediators and type I IFNs through activation of the canonical IκB kinase (IKK)-α/β kinases and the transcription factor NF-κB or the IKK-related kinase TBK1 and IFN regulatory factors (IRFs) (9, 10). Individual PRRs initiate overlapping and distinct signaling pathways in various cell types. Endosomal TLRs act as sensors of foreign nucleic acids and trigger the production of type I IFNs and inflammatory cytokines. In plasmacytoid dendritic cells (DCs), this results from RNA and DNA activation of NF-κB, IRF5, and IRF7, whereas IRF1, rather than IRF7, drives type I IFNs downstream of TLR7 and TLR9 in myeloid DCs (11–14). IFN-β production induced by Candida albicans in DCs is largely dependent on the CLR Dectin-1 and signaling through the Syk kinase and IRF5 (15). Listeria monocytogenes and Mtb may disrupt the phagosomal membrane of macrophages and trigger type I IFN responses through cytosolic Nod1/Nod2/RIP2 and IRF3 (L. monocytogenes) or IRF5 (Mtb) responding to bacterial cell-wall peptidoglycan, or STING/TBK1/IRF3 responding to bacterial DNA (6, 8). There is more evidence that TBK1 and IKKε are also activated by TLR ligands that signal via MyD88, leading to IFN-β production without the activation of IRF3 (16), and we recently showed that IRF5 activation in human monocytes by Staphylococcus aureus ssRNA was dependent on IKKβ and not TBK1 (17).
Inflammatory mediators shape the immune response and the outcome of infection (reviewed in refs. 2, 3, 5, 7, 18). Mice deficient in the adaptor MyD88 that conveys signals from TLRs and IL-1 and IL-18 receptors are extremely susceptible to infections with Mtb and M. avium. Mice deficient in TNF or IL-1 receptor die rapidly after Mtb infection, and people receiving anti-TNF therapy have increased risk for reactivation of latent tuberculosis (3). On the contrary, mice deficient in type I IFNs actually do better than WT mice, arguing for a role of IFN-α/β in supporting Mtb survival (19). PRR activation induces the production of reactive oxygen species (ROS) via phagosomal NADPH oxidase (NOX) (20). However, excessive oxidative signaling may be damaging to cells; hence, an efficient mechanism for modulation is imperative. Kelch-like ECH-associated protein 1 (Keap1) plays a well-established role as a sensor for ROS for the protection of cells against oxidative damage (21). Keap1 is characterized by several distinct binding domains, allowing its self-interaction and interaction with other proteins such as cullin-ring 3 E3 ubiquitin ligase (Cul3), the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and p62 (22–25). Modification of Keap1 by electrophilic or oxidative stress leads to stabilization of Nrf2. Nrf2 translocates to the nucleus and activates the transcription of cytoprotective genes, including p62, heme oxygenase, and NADPH dehydrogenase, quinone 1. The Keap1–p62 interaction may lead to Keap1 degradation by autophagy, resulting in a noncanonical mechanism of regulation of Nrf2 (24, 26, 27). There is some evidence suggesting a role for the Keap1 E3-ligase complex in the regulation of NF-κB signaling in the context of tumorigenesis (26–28). Although the role of Keap1 in ROS signaling has been well established, its role in regulation of inflammatory signaling has not been clearly elucidated. We show here that Keap1 down-regulates the inflammatory response necessary for the control of M. avium infection through the canonical IKK complex and its homolog, TBK1.
Results
M. avium Induces ROS and the Recruitment of Keap1 to Its Phagosomes in Human Primary Macrophages.
ROS can be triggered by invading bacteria, and, despite the fact that pathogenic mycobacteria have developed strategies to counter it, ROS modulates antimycobacterial host defenses, including inflammation (29–31). We investigated whether M. avium induced ROS production in human primary monocyte-derived macrophages (MDMs). Cells were infected with M. avium, and, 1 h after infection, ROS was significantly increased as shown by confocal fluorescence microscopy (Fig. 1 A and B). Small amounts of ROS were detected in untreated macrophages. When ROS was inhibited by using N-acetyl cysteine (NAC), which enhances the production of the antioxidant glutathione, ROS levels were significantly decreased in M. avium-infected macrophages (Fig. 1A, Lower, and B), with a similar trend for the positive control, tert-butyl hydroperoxide (TBHP). As Keap1 is well described as a sensor for ROS and M. avium triggered ROS generation, we wondered if Keap1 was recruited to mycobacterial phagosomes. We found that Keap1 was recruited to CFP-M. avium phagosomes 4 h after infection (Fig. 1C). This recruitment significantly decreased by approximately 50% after 24 h (Fig. 1 C and D). In addition, when ROS was inhibited by NAC or diphenyleneiodonium (DPI), which inhibits ROS generated by NOX2, there was a significant reduction of Keap1 association with M. avium phagosomes (Fig. 1E). However, this recruitment did not seem to depend on p62, a well-known Keap1 interaction partner and a receptor for autophagy (23, 24), as there was no difference in Keap1 association with M. avium phagosomes when p62 was knocked down by using siRNA (Fig. 1F). These observations show a recruitment of Keap1 to M. avium phagosomes that is partly ROS-dependent, and suggest a role for Keap1 in M. avium infections.
Fig. 1.
Keap1 Knockdown Increased Inflammatory Cytokine Production During M. avium Infection of Human Primary Macrophages.
We next investigated if Keap1 may affect mechanisms that contribute in controlling M. avium infections. We knocked down Keap1 in MDMs by using targeting siRNA (siKeap1) and screened for the expression levels of 578 human genes known to be differentially expressed in immunology (nCounter GX Human Immunology Kit; NanoString Technologies) 4 h after infection with M. avium. The expression of some of these genes is shown in Table S1. Keap1 knockdown efficiency of 50–80% was achieved at mRNA and protein levels in the MDMs (Fig. 2 A and B). Most of the genes induced by M. avium infection seemed to be regulated by Keap1, such as IL-1β, IL-18, and IRF1 (Table S1), and apparently NF-κB–driven (www.bu.edu/nf-kb/gene-resources/target-genes/). However, several genes were expressed but not further induced by M. avium infection and also not regulated by Keap1, like MyD88 and the scavenger receptor CD36 (Table S1). We selected a few genes important in mycobacterial immune response for further evaluation (2, 18). Infection induced significantly higher fold expression of TNF, IL-6, IL-1β, CXCL10, and IFN-β mRNA levels measured by quantitative PCR in Keap1 knockdown cells than in nontargeting control siRNA (siNTC)-treated cells (Fig. 2C). Protein levels of cytokines were measured by ELISA in cell supernatants and showed similarly significant results in fold changes (Fig. 2D and Fig. S1A). However, CXCL10 and IFN-β induction by infection were often low and highly variable from donor to donor when absolute protein levels were measured (Fig. S1A). As expected, Keap1 inhibition also led to increased transcription of some Nrf2 target genes (Fig. S1B) (22). We thus show that Keap1 regulates NF-κB signaling and specifically represses inflammatory cytokine and type I IFN expression induced by M. avium infection in human primary macrophages.
Fig. 2.
Table S1.
| Gene | Fold induction by M. avium infection | |
|---|---|---|
| siNTC | siKeap1 | |
| CCL4 | 140.4 | 168.7 |
| IL-1β | 98.0 | 343.8 |
| PTGS2 (COX2) | 42.1 | 45.9 |
| TRAF1 | 14.3 | 11.5 |
| CXCL2 | 11.5 | 19.4 |
| CCR7 | 7.9 | 9.8 |
| IL-18 | 6.9 | 10.9 |
| TNFAIP3 (A20) | 5.1 | 7.9 |
| CD274 | 5.1 | 6.0 |
| CD40 | 3.4 | 4.1 |
| IRF1 | 2.7 | 5.0 |
| CLEC4E (Mincle) | 2.6 | 2.8 |
| NFKB2 | 2.6 | 3.5 |
| GBP1 | 2.3 | 4.0 |
| CD83 | 1.6 | 2.8 |
| SLAMF7 | 1.6 | 1.9 |
| GPR183 | 1.5 | 1.5 |
| CD36 | 1.0 | 1.0 |
| MYD88 | 0.9 | 1.0 |
| HLA-B | 0.9 | 1.1 |
| HLA-C | 0.8 | 1.0 |
| HLA-A | 0.8 | 1.1 |
| CD81 | 0.8 | 1.0 |
| IRAK1 | 0.7 | 0.9 |
Human primary macrophages were treated with siKeap1 or siNTC control and infected with M. avium for 4 h. Induction of immunology-related genes using NanoString Technology. Excerpt of genes from a 578-gene array is shown as fold induction by infection.
Fig. S1.
Keap1 Knockdown Increased Nuclear Translocation of NF-κB-p65 and IRF1, -3, and -5 in Human Macrophages.
Having determined that Keap1 inhibits inflammatory cytokines and type I IFNs during M. avium infection, we further investigated the point in the signaling cascade at which Keap1 may interfere. We first examined Keap1 regulation of transcription factor activation by using a recently developed nuclear translocation assay whereby transcription factors are stained with specific antibodies and nuclear accumulation is assayed (17). We infected cells for 1 h and 4 h and analyzed the nuclear translocation of NF-κB-p65 and IRF1, -3, and -5 (Fig. 3). We saw a strong early and sustained nuclear accumulation of NF-κB (p65, 45% and 30% at 1 and 4 h post infection, respectively) and IRF5 (from 26% to 48% and 62% 1 h and 4 h post infection, respectively) and a later IRF1 translocation (10% and 38% at 1 and 4 h post infection, respectively), whereas IRF3 was hardly detected (1–4% nuclear translocation) upon M. avium infection (Fig. 3A). In Keap1 knockdown cells, however, and in line with our cytokine observations (Fig. 2), there was approximately a two- to threefold increase in nuclear accumulation compared with control siRNA-treated cells for NF-κB and IRF1 and a twofold increase in IRF5 1 h after infection. IRF3 translocation was still apparently quite low and seemed unaffected by Keap1 knockdown 1 h after M. avium infection (Fig. 3B). Antibodies to IRF7 and -8 showed high nonspecific staining and were thus unsuitable for use in the nuclear translocation assay. Furthermore, knocking down NF-κB (RelA) reduced M. avium-induced TNF and IFN-β, whereas only modest effects were seen with siIRF1 or IRF5 (Fig. S2), possibly as a result of knockdown efficiencies of only 40–70% of messenger levels. These data are indicative of the fact that the cytokines were driven by NF-κB and possibly IRF1 and -5.
Fig. 3.
Fig. S2.
Keap1 Inhibits Inflammatory Cytokine Responses during M. avium Infection Through the Canonical IKK Complex and TBK1.
Upstream of NF-κB and IRFs are central kinases, canonical IKKs and TBK1, respectively, that control signaling and inflammatory cytokine and type I IFN production. Translocation of NF-κB and IRF1 were significantly inhibited at 1 h and 4 h after infection when cells were treated with kinase inhibitors for IKKβ (IKKVIII) or TBK1 (MRT67307 or BX795; Fig. 3C). Similar trends were also observed for IRF5, but results were more variable and did not reach significance (Fig. 3C). These data suggest that IKKβ and TBK1 are involved in signaling pathways engaged by M. avium.
We next measured the effect of Keap1 knockdown in MDMs on the phosphorylated and total protein levels of these kinases and nine other different proteins in the NF-κB and IRF signaling pathways (IKKα, IKKβ, IKKγ, IκB, NF-κB-p65, TBK1, IRF3, IRF5, IRF7, IRF8, and p38) subsequent to M. avium infection (Fig. 4 and Fig. S3). There was an overall significant increase in total and phosphorylated levels of members of the IKK complex and NF-κB in Keap1 knockdown cells compared with controls and relative to uninfected samples. These regulatory effects were quite rapid, as significant differences could be detected as early as 30–60 min after infection (Fig. 4). Notably, expression of the inhibitor of NF-κB, IκB, was not affected by Keap1 knockdown after infection (Fig. S3A). In addition to this, and interestingly so, we also observed a significant increase in phosphorylated (as early as 30 min post infection) and total TBK1 levels in Keap1 knockdown cells after infection (Fig. 4). However, total IRF3, IRF5, IRF7, and IRF8 levels were not affected with Keap1 knockdown (Fig. S3B). Expression of the p38 MAPK in response to infection remained unchanged by Keap1 knockdown (Fig. S3B). p38 regulates the transcription factors CREB and c/EBPβ and does not use IKK or TBK1 (32). Of important note is the observation that, in Keap1 knockdown cells, changes in the mRNA levels of IKKβ and TBK1 were insignificant (Fig. S3C).
Fig. 4.
Fig. S3.
Taken together our results show evidence that Keap1 may negatively regulate activation of the IKK complex and TBK1, leading to reduced nuclear translocation of NF-κB, IRF1, and IRF5 driving the expression of inflammatory cytokines and type I IFNs induced by M. avium infection.
Disruption of Cul3 and Rbx1 Genes Up-Regulates Inflammatory Cytokine Responses During M. avium Infection.
Under normal conditions, the Keap1/Cul3-Rbx1 complex constantly facilitates Nrf2 degradation. However, when a cell encounters oxidative or electrophilic stress, Nrf2 dissociates from the complex and translocates into the nucleus, where it activates myriad antioxidant and cytoprotective genes. Along with Keap1, Cul3 and Rbx1 make up the ubiquitin ligase complex that is responsible for the ubiquitination and degradation of Nrf2 (22). We thus investigated if the regulatory effect of Keap1 on inflammation induced by M. avium was also observed when the other members of the ubiquitin ligase complex or Nrf2 were disrupted. Indeed, TNF and IFN-β mRNA levels significantly increased at 4 h aftr infection with the knockdown of both Cul3 and Rbx1 (Fig. 5A), thus phenocopying Keap1. Specificity of the siRNAs was verified, as they silenced only the target gene and did not affect expression of the other partners (Fig. S4A). However, silencing of Nrf2 did not change TNF levels induced by M. avium infection (Fig. 5B). p62 has also been shown to regulate Nrf2 via the Keap1/Cul3/Rbx1 complex (22, 23). When we knocked down p62, there was rather a trend of decreased TNF mRNA levels (Fig. 5B) in response to infection, opposite from what we observed with Keap1 knockdown. These data suggest that Keap1 may regulate the inflammatory response induced by M. avium via members of the ubiquitin ligase complex Cul3/Rbx1, and that this effect is independent of Nrf2 and p62, which are other important interaction partners.
Fig. 5.
Fig. S4.
Keap1 Plays a Role in the Ubiquitination and Degradation of IKKβ During M. avium Infection.
Given that Keap1 knockdown led to IKKβ and TBK1 accumulation and Keap1 is an adaptor in the Cul3-ubiquitin ligase complex, we further investigated whether Keap1 might contribute in the ubiquitination and degradation of activated IKKβ and TBK1 to terminate inflammatory signaling. Previous studies in cancer cell lines indicate that IKKβ directly interacts with Keap1 through a conserved motif and that Keap1/Cul3 contributes in regulating IKKβ (27). We infected control siRNA- and siKeap1-treated macrophages from four different donors with M. avium in the presence and absence of the proteasome inhibitor MG132, followed by immunoprecipitation of IKKβ or TBK1 and Western blot analyses of ubiquitinated IKKβ and TBK1. M. avium infection induced phosphorylation of IKKβ, and ubiquitinated IKKβ accumulated when MG132 was added (Fig. 5C). In Keap1-knockdown cells, ubiquitination of IKKβ was significantly lower, even in the presence of MG132, supporting a mechanism whereby Keap1 regulates IKKβ activity through ubiquitination and degradation, and that accumulation of active IKKβ (i.e., p-IKKβ) results in increased translocation of key transcription factors and production of cytokines in Keap1-knockdown macrophages. We also observed an accumulation of TBK1 during Keap1 knockdown suggestive of Keap1 regulation of TBK1. However, unlike IKKβ, TBK1 did not seem to be ubiquitinated during M. avium infection in macrophages (Fig. S4B). These results suggest that the Keap1-Cul3-Rbx1 ubiquitin ligase complex is specifically responsible for ubiquitin-mediated degradation of IKKβ but not TBK1. Hence, the mechanism of Keap1 regulation of TBK1 may be indirect, possibly mediated through IKKβ (33).
Increased Inflammatory Responses Resulting from Keap1 Knockdown Restrict Intracellular Growth of M. avium in Human Macrophages.
Our results show that Keap1 negatively regulates inflammation induced by M. avium; hence, we wanted to investigate if this affected mycobacterial growth. We knocked down Keap1 and infected MDMs with luciferase-expressing M. avium. Bacteria were then enumerated by measuring luciferase enzyme activity over a 72-h period. Keap1 knockdown was done with four individual and pooled siRNA duplexes (Fig. 6A). Four hours after infection, all infected cells showed approximately equal level of luciferase activity, indicating an equal uptake of bacteria. Forty-eight and 72 h after infection, bacterial counts from all Keap1-knockdown samples were significantly lower than in controls (P < 0.01 and P < 0.001, respectively). The effect of Keap1 knockdown on bacterial growth was also confirmed by cfu counts (Fig. S5A). To investigate the contribution of the inflammatory response in controlling infection during Keap1 knockdown, we used inhibitors for IKKβ (IKKVIII) and TBK1 (MRT67307 and BX795), and an inactive MRT67307 analog, MRT166. As shown in Fig. 6B, both IKKβ and TBK1 inhibitors significantly increased bacterial growth (P < 0.05) in Keap1-knockdown samples.
Fig. 6.
Fig. S5.
We conclude that Keap1 is a negative regulator of IKK- and TBK1-mediated inflammatory signaling, which is important to avoid chronic or overwhelming inflammation. A negative consequence seems to be facilitated growth of M. avium in human primary macrophages.
Discussion
Inflammatory signaling is central in controlling host defenses to intracellular pathogens. Here, we have pioneered a role of the oxidative stress sensor Keap1 in regulating inflammatory signaling and intracellular survival of M. avium in human primary macrophages. Silencing of Keap1 enhanced M. avium-induced inflammatory cytokine and type I IFN responses, resulting in better control of mycobacterial growth. We show evidence of a mechanism whereby the Keap1/Cul3-Rbx E3 ubiquitin ligase complex regulates IKKβ activity through ubiquitination and degradation, and that accumulation of p-IKKβ results in increased translocation of transcription factors NF-κB, IRF1, and IRF5 and production of target gene expression in Keap1-knockdown macrophages. We also observed Keap1 regulation of TBK1, shown by increased TBK1 and p-TBK1 upon Keap1 knockdown in M. avium-infected macrophages. However, unlike IKKβ, TBK1 did not seem to be ubiquitinated, suggesting the mechanism may be indirect, possibly mediated through IKKβ.
We found that M. avium can induce intracellular ROS generation and observed, to our knowledge for the first time, that Keap1 was recruited to mycobacterial phagosomes in an ROS-dependent manner. Komatsu and coworkers have recently shown the recruitment of Keap1 to Salmonella phagosomes in a p62-dependent fashion (34). However, a physiological significance of this association remains to be determined. We did not find in the present study that Keap1 recruitment to mycobacterial phagosomes depended on p62, and thus the identity of the recruiting partner protein remains elusive. Nonetheless, we addressed the possible outcomes of Keap1 interactions and recruitment to mycobacterial phagosomes, in particular because the formation of signaling complexes on endomembranes seems to be a common theme for inflammatory signaling.
Cytokines play important roles as effectors and regulators of mycobacterial immunity, although their mechanism of regulation is complex and continues to be poorly understood. We observed that Keap1 down-regulated NF-κB–driven cytokines and show evidence that Keap1 also inhibits type I IFN, IFN-β, and the IFN-inducible gene CXCL10 during M. avium infection. Similar results were observed with Cul3 and Rbx1, members of the Keap1/Cul3-Rbx1 E3 ubiquitin ligase complex. Notably, there were usually quite low amounts of IFN-β and CXCL10 induced by M. avium that varied widely among blood donors. Nonetheless, in addition to our findings, several studies are also highlighting a role of the type I IFN response in bacterial infections, including Escherichia coli (35) and Mtb (6, 36–38). There is mounting evidence of association of excessive production of type I IFNs and exacerbated Mtb infections in mouse models and humans, possibly via an eicosanoid imbalance (19, 39), although the mechanisms behind this are not well understood. In Mtb-infected macrophages, production of cytokines such as TNF, IL-12, and IL-1β was inhibited by recombinant IFN-β (40), and, indeed, when we added recombinant IFN-β to M. avium-infected macrophages, we observed a trend of increased bacterial growth (Fig. S5B). It might thus be important to maintain low IFN-β levels in macrophages particularly early in infection, as observed in our experiments, to curtail the inhibitory effects on the other rather host-protective cytokines.
The present results further clearly implicate Keap1 in the control of the IKK complex and TBK1, suggesting a role for Keap1 in inhibiting the activation of infection-induced inflammatory signaling and type I IFNs. Our results are in line with reports citing a role of Keap1 in the regulation of NF-κB signaling and showing a consequence thereof in tumorigenesis (26, 27). These studies showed that Keap1 regulated TNF-induced NF-κB activity via IKKβ. The E(T/S)GE motif, which is found only in the IKKβ subunit of the IKK complex, was essential for direct interaction with the C-terminal Kelch domain of Keap1 (27, 41). However, we show evidence that the entire IKK complex, along with its homolog TBK1, is significantly affected by Keap1, even though only IKKβ seemed to be ubiquitinated and possibly degraded through Keap1. The suggested mechanism is further supported by the fact that similar effects as for Keap1 on inflammation were observed with Cul3 and Rbx1. The regulation of TBK1 and the other IKK homologs by Keap1 has not been shown before to our knowledge, and could be indirect. IKKs and their homologs have been described to regulate each other through the phosphorylation of their catalytic and regulatory subunits during an innate immune response (16, 42). Sustained kinase activity of IKKβ in Keap1-silenced macrophages could thus impact the activity and phosphoprotein levels of TBK1 and the other IKKs, but that remains to be further elucidated in our settings. Although we did not find ubiquitination of TBK1, other studies have described regulation of IFN signaling through the degradation of TBK1 by the PRR NLRP4 by using another E3 ligase, deltex homolog 4, in response to viral ligands (43). Alternatively, because of the inherent difficulties in immunoprecipitation (IP) and ubiquitin blotting for endogenous proteins in primary macrophages (low levels, high background), we cannot conclude that TBK1 is not directly regulated by Keap1 similar to IKKβ. However, TBK1 does not have the proposed Keap1 interaction domain (D/N)XE(T/S)GE as found in IKKβ, and could be indirectly regulated through IKKβ. Our study thus clearly places the IKKβ/NF-κB signaling axis as the central pathway that is regulated by Keap1.
The main pathways leading to IFN-α/β induction during mycobacterial infections remain unclear and quite varied. Pandey et al. (6) demonstrated that phagosomal Mtb peptidoglycan triggers the expression of type I IFNs in a mainly TBK1/IRF5-dependent manner. Others have shown a requirement of the TBK1/IRF3 or -5 signaling axis in response to cytosolic Mtb DNA (8, 13). In our hands, M. avium induced a generally low IFN-β response and no IFN-α. In addition to NF-κB, nuclear translocation assays suggested that IRF1 and -5 were activated and also further increased by Keap1 knockdown. IRF1 is shown to be involved in IFN-β production by TNF (44). Chemical inhibition of IKKβ or TBK1 both reduced M. avium induced nuclear translocation of NF-κB, IRF1, and possibly IRF5. We could not detect significant activation of IRF3 in our experiments, and, because of unspecific staining by available antibodies, we could not address the role of IRF7 and -8. To address the role of transcription factors in driving cytokine responses, we individually silenced NF-κB (RelA), IRF1, and IRF5 before infection with M. avium and measured cytokine mRNA transcription. These experiments proved challenging, with highly variable knockdown of 40–70% and low induction of IFN-β. siRelA inhibited M. avium-induced TNF and IFN-β mRNA, and, although there was a tendency of reduced IFN-β by siIRF1 and siIRF5, results were not significant. Together, we show evidence that IKKβ and TBK1 are involved in M. avium-induced activation of NF-κB, IRF1, and possibly IRF5. NF-κB is driving TNF and IFN-β expression, but the role of IRF1 and IRF5 are less clear and need further elucidation. Some studies also suggest a coregulation of IFN-β production by NF-κB and IRF5 (14), and we recently showed an IKKβ/IRF5-dependent pathway in response to S. aureus ssRNA (17). Our data indicate that IKK and TBK1 regulation by Keap1 might have even broader consequences in the innate immune response to mycobacterial infections, as these kinases play important roles in different arms of the immune response to infection.
To determine if the role of Keap1 in inflammation was linked to its recruitment to M. avium phagosomes, we investigated the role of p62 through siRNA knockdown. p62 is well known for its role in protein aggregation and trafficking of ubiquitinated cargo to autophagy, and intricately linked to Keap1 (23, 24, 45). p62 is also reported to regulate various signaling events, including those activated by TNF, IL-1β and nerve growth factor receptors through scaffolding TRAF6 and atypical protein kinase C with these receptors in different cell types (46–48). Keap1 interacts with p62 (23–25), and p62 and LC3 are recruited to bacterial phagosomes to mediate autophagic clearance of the bacteria (49–51). However, Keap1 recruitment to mycobacterial phagosomes seemed independent of p62 in the present study. We also found that knockdown of p62 showed a trend of reduced expression of TNF in line with other studies (47, 48, 52), and opposite from what we observed with Keap1 knockdown. We thus found no evidence that suggests that the effect seen on NF-κB signaling by Keap1/Cul3-Rbx1 might be dependent on p62. Finally, we observed that silencing of Keap1 reduced bacterial growth in MDMs. The mechanism behind it may be more complex but can be explained at least in part by the role of Keap1 in modulating the inflammatory responses, as inhibition of IKKβ and TBK1 complemented the growth impairment observed with Keap1 silencing. Inhibiting inflammatory responses improved bacterial growth even in mock-treated cells. However, pretreatment of infected cells with recombinant TNF and IL-1β showed no effect on mycobacterial growth, and IFN-β showed increased bacterial growth (Fig. S5B). Results could thus not be ascribed to auto- and paracrine activity of individual cytokines despite the well-characterized importance of these cytokines in mycobacterial defenses (2, 3, 7, 18, 39, 40). We therefore propose that Keap1 acts as a negative regulator for controlling inflammatory signaling in M. avium-infected human primary macrophages. Although this might be important to avoid sustained or overwhelming inflammation, our data suggest that a negative consequence could be facilitated growth of pathogens like M. avium inside macrophages.
Recent studies have reported frequent loss-of-function mutations in the Keap1-Cul3-Rbx1 complex in several human cancers that could be associated with pathological NF-κB activation in addition to increased transcription of Nrf2 cytoprotective target genes (21, 28). Our results indicate that altered Keap1 gene expression may have vital clinical implications also for other inflammation-associated conditions, including mycobacterial infections, which opens novel research venues for translational research, for instance in the expanding field of host-targeted therapy for infectious diseases.
Materials and Methods
Materials.
Rabbit polyclonal antibody against Keap1 (cat. no. 10503–2-AP) was obtained from ProteinTech, and Keap1 antibody (sc-15246) was obtained from Santa Cruz Biotechnology. Anti-rabbit Alexa 647 and 546 was from Molecular Probes, and nitrocellulose membrane was from GE Healthcare Life Science. Cell culture media including RPMI and OptiMEM were obtained from Sigma and Invitrogen, respectively. Protease inhibitor mixture was from Roche. GAPDH, IRF5, and IRF7 antibodies were from Abcam; IRF8 antibody was from Atlas Antibodies; and all other antibodies, including anti-IRF1, anti-IRF3, anti–NF-κB-p65, anti–phospho-NF-κB-p65, anti-IKKβ, anti–phospho-IKKα/β, anti-TBK1, and anti–phospho-TBK1 were purchased from Cell Signaling Technology. Ubiquitin antibody α-Ub FK2 was from Enzo (BML-PW8810). COX IV antibody (ab33985) for blotting and controlling for loading control in immunoprecipitation assays was obtained from Abcam. Proteasome inhibitor MG132 was from Sigma (C2211). Nuclear stains DRAQ5 and Hoechst 33342 were from Alexis Biochemicals and Life Technologies, respectively. NAC, DPI, MRT67307, and MRT166 were purchased from Sigma. BX795 was from Axon MedChem and IKK inhibitor VIII was from Merck Millipore. RNA was extracted from cells using RNeasy Mini Kits (Qiagen) and reverse-transcribed to cDNA by using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Transfection reagents used were Lipofectamine RNAiMax (Invitrogen) and siLentFect Lipid Reagent for RNAi (Bio-Rad). All primer/probes were from Applied Biosystems, and qPCR was done by using the StepOne Plus system from Applied Biosystems.
Methods.
Cells.
Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from the blood bank of St Olavs Hospital (Trondheim, Norway). MDMs were generated from monocytes enriched from total PBMCs by plastic adherence and maintenance in RPMI 1640 medium (Gibco) supplemented with 30% (vol/vol) (before stimulation) or 10% (vol/vol) (after stimulation) human serum (blood bank of St Olavs Hospital, Trondheim, Norway).
ROS detection assay.
The Image-iT LIVE Green ROS detection kit (Molecular Probes) was used to detect ROS in human macrophages infected with luciferase-expressing M. avium. The manufacturer’s protocol was followed, and control and test samples were incubated for 1 h before detection of ROS. ROS inhibition was achieved by pretreating cells with NAC or DPI for 30 min before infection. Fluorescence intensity per cell was measured by using the Bitplane Imaris Cell module.
Mycobacteria and macrophage infection.
Transformants of the virulent M. avium clone 104 expressing firefly luciferase or CFP are described elsewhere (4). Mycobacteria were cultured in Middlebrook 7H9 medium (Difco/Becton Dickinson) supplemented with glycerol, Tween 80, and albumin dextrose catalase (ADC). Single-cell suspensions of mycobacteria from log-phase broth cultures were added to macrophages differentiated from human PBMCs at a multiplicity of infection of 10 for appropriate times. In some experiments, macrophages were pretreated with proteasome inhibitor, MG132, inhibitors MRT67307 (MRT67307 analog; MRT166) or BX795 (TBK1, Sigma, and Axon MedChem, respectively), or IKK inhibitor VIII (Merck Millipore) for 30 min before infection and throughout the entire time course of infection. Mycobacterial survival was assessed by colony counting after plating on 7H10/oleic acid ADC agar or by luciferase activity (Promega).
Immunofluorescence Assays.
Macrophages grown on glass-bottomed dishes or plates (MatTek) were fixed with 2% paraformaldehyde, and then saponin-based permeabilization buffer or ice-cold methanol was used for cell permeabilization. Unspecific binding was blocked with 20% human serum. Cells were stained with Keap1 and transcription factor-specific primary and Alexa-fluorochrome secondary antibodies. The cells were imaged by using a Zeiss LSM 510 META microscope (Carl Zeiss Microimaging) equipped with a 63×/1.45 oil-immersion objective. Quantitative protein recruitment analysis from confocal images was done with Bitplane Imaris software. In the nuclear translocation assay, human primary macrophages were prepared and stained as described earlier, also including nuclear stain Hoechst 33342. Cells were then imaged by using the Olympus Scan^R microscope, and nuclear translocation was quantified as overlapping fluorescence of transcription factor and nuclear stain by using the accompanying software (Olympus) (17). The specificity of the transcription factor staining was verified by using specific siRNAs.
siRNA Transfection Assay.
Transfection with siRNA was performed by using Lipofectamine RNAiMAX transfection reagent or siLentFect Lipid Reagent for RNAi according to the manufacturer’s protocol at 0 and 48 h. Gene knockdown was evaluated by quantitative real-time PCR or Western blotting. Keap1 pooled HP Validated siRNA (a pool of four duplexes; Qiagen) and ON-TARGETplus human siRNAs (Dharmacon/Thermo Scientific) were used to target Keap1, p62, IKKβ, TBK1, and Nrf2. In case of siKeap1, individual siRNAs were tested and found equally efficient in silencing Keap1, with resulting increased inflammation and reduced mycobacterial growth (Fig. 5). Nontargeting control siRNAs from both manufacturers (Qiagen and Dharmacon/Thermo Scientific) were also included. Cells were treated for a total of 72 h with 20 nM siRNA.
RNA Extraction and qPCR Assessment of mRNA Levels.
Total RNA was extracted from cells by using RNeasy Mini kits including DNase digestion with the QIAcube robot (all from Qiagen). cDNA synthesis was performed with the High-Capacity RNA-to-cDNA Kit according to the recommended protocol.
A total of 578 immunology-related human genes and 14 internal reference genes were included in the digital transcript counting (nCounter GX Human Immunology kit assay; NanoString Technologies). Total RNA (100 ng) was assayed on nCounter Digital Analyzer (NanoString) according to the manufacturer’s instructions. Data were normalized by scaling with the geometric mean of the built-in control gene probes for each sample. Gene expression was analyzed by using the accompanying nSolver software. qPCR was performed with the StepOnePlus System, TaqMan Gene Expression Assays, and TaqMan Universal Master Mix (ABI). The level of GAPDH mRNA was used for normalization. All primer/probe sets used for qPCR were obtained from and validated by Qiagen and used according to the manufacturer’s recommendations.
Assessment of Protein Levels.
Inflammatory mediators in cell supernatants were analyzed by ELISA according to the manufacturer’s protocols (R&D Systems). IFN-β ELISAs were from PBL Assay Science. Western blots were performed following standard procedures. Protein lysates were quantified using the bicinchoninic acid assay (Fisher Scientific) and run in 10% NuPAGE mini gels (Invitrogen), then transferred to nitrocellulose membrane by using Invitrogen iBlot system and incubated with primary antibody at 4 °C for 24–48 h. The blots were developed with SuperSignal West Femto (Thermo Scientific) and visualized with Image Estimation 2000R (Kodak). The Kodak 1D Image Analysis software was used for band intensity quantification.
For IP and detection of ubiquitination, after Keap1 knockdown, macrophages were pretreated with 10 µM MG132 for 30 min before infection for 2 h. Macrophages were lysed in RIPA buffer [50 mM Tris⋅HCl, 200 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.05% SDS, 10 mM EDTA, 10 mM EGTA, phosphatase inhibitor mixture 2 and 3 (Sigma), and complete protease inhibitor mixture (Roche)]. Dynabeads Protein A (Life Technologies) were incubated with anti-IKKβ or normal rabbit IgG (R&D Systems) for 1 h at room temperature. Dynabead/antibody complexes were crosslinked with BS3 reagent (Life Technologies) for 15 min according to the manufacturer’s protocol. Lysates were adjusted for similar concentration and volume and immunoprecipitated with the prepared beads at 4 °C overnight. After IP, beads were washed three times with RIPA buffer and once with 1 M urea, transferred to new tubes, and eluted by denaturation with LDS sample buffer. Eluates were run in 10% NuPAGE mini gels, transferred to nitrocellulose membranes using Invitrogen’s iBlot system, and then incubated with primary antibodies at 4 °C for 24 h. Membranes were then incubated with IRDye secondary antibodies and visualized by the Odyssey Imaging System (Licor). Image Studio 3.1 software was used for quantification.
Statistical Analysis.
When cells were counted by microscopy, at least 50 cells were counted for each condition in each experiment. Unless otherwise stated, at least three independent experiments were performed for all figures. The means ± SD or SEM is shown in the figures, and P values were calculated by using a paired two-tailed Student t test of log-transformed data with GraphPad Prism software. Statistical significance was set at P < 0.05.
Ethics Statement.
The Regional Committees for Medical and Health Research Ethics at Norwegian University of Science and Technology (NTNU) approved use of PBMCs from healthy adult blood donors after informed consent (identification number 2009/2245–2). All donors provided written informed consent.
Acknowledgments
We thank professors T. Johansen (University of Tromsø) and D. M. Underhill (Cedars–Sinai, Los Angeles) for carefully reading through the manuscript. All imaging was performed at the Cellular & Molecular Imaging Core Facility at NTNU. This work was supported by the Research Council of Norway through Centres of Excellence Funding Scheme Project 223255/F50, Outstanding Young Investigator Project 180578/V50 (to T.H.F.), and the Liaison Committee between NTNU and the Central Norway Regional Health Authority (T.H.F., J.A.A., M.H., and M.S.).
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Published online: July 20, 2015
Published in issue: August 4, 2015
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Acknowledgments
We thank professors T. Johansen (University of Tromsø) and D. M. Underhill (Cedars–Sinai, Los Angeles) for carefully reading through the manuscript. All imaging was performed at the Cellular & Molecular Imaging Core Facility at NTNU. This work was supported by the Research Council of Norway through Centres of Excellence Funding Scheme Project 223255/F50, Outstanding Young Investigator Project 180578/V50 (to T.H.F.), and the Liaison Committee between NTNU and the Central Norway Regional Health Authority (T.H.F., J.A.A., M.H., and M.S.).
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This article is a PNAS Direct Submission.
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The authors declare no conflict of interest.
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Keap1 regulates inflammatory signaling in Mycobacterium avium-infected human macrophages, Proc. Natl. Acad. Sci. U.S.A.
112 (31) E4272-E4280,
https://doi.org/10.1073/pnas.1423449112
(2015).
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