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Toll-like receptor 4 mediates mitochondrial DNA damage and biogenic responses after heat-inactivated E. coli

Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA

¹Correspondence: Box 3315, Room 0590, CR2 Building, Duke University Medical Center Durham, NC 27710, USA. E-mail: piant001{at}mc.duke.edu

The body’s innate immune system closely monitors and contains bacterial products, but in pathological states the host response damages tissues by releasing inflammatory mediators that contribute to cell damage and cell death. In systemic infection by enteric bacteria, the liver’s immune role is to clear circulating microbes and their products and to produce the acute-phase response. Resident hepatic macrophages, Kuppfer cells (KC), are activated by lipopolysaccharide (LPS) and generate cytokines, especially tumor necrosis factor- (TNF-) and interleukins (IL), which induce reactive oxygen species (ROS) and nitric oxide (NO) production, as well as chemokines that recruit leukocytes. Elaborated factors activate receptor and nonreceptor pathways that involve mitochondria in hepatocyte necrosis and apoptosis.

Mammalian cells recognize conserved microbial motifs by means of at least 10 transmembrane Toll-like receptors (TLR). TLR activation leads to immediate antimicrobial peptide and cytokine production in myeloid and some nonmyeloid cells, including hepatocytes. In particular, TLR2 and TLR4 in juxtaposition with CD14 share a common pathway mediated through the Toll-IL-1R (TIR) signaling domain and the adaptor molecule, MyD88, that activates nuclear factor B (NF-B) for inflammatory cytokine synthesis. TLR4 mediates the critical LPS response against gram-negative bacteria, while TLR2, integral to gram-positive bacteria recognition, also influences specificity for gram-negatives.

We considered that TLR activation by bacterial challenge in vivo would intensify mitochondrial damage in the liver of the mouse. TNF- stimulation promotes mitochondrial ROS formation by the electron transport chain (ETC) that can be enhanced by NO binding to cytochrome c oxidase. In this state, NO-heme interactions may make mitochondria prone to lipid, protein, or nucleic acid oxidation. To prevent such damage, mitochondrial anti-oxidant defenses are up-regulated, e.g., by orchestrated co-regulation of TNF- and the major mitochondrial anti-oxidant enzyme, superoxide dismutase-2 (MnSOD). Nevertheless, oxidative damage to mitochondria, and specifically to mtDNA, can be sufficient to impair ETC function. Mitochondrial transcription and mtDNA genomic integrity depend on importation of nuclear-encoded proteins, notably mitochondrial transcription factor A (Tfam), a high mobility group box protein that binds the light and heavy strand promoter regions, and is critical to mitochondrial DNA transcription and biogenesis.

This background hypothetically links TLR activation to mitochondrial oxidative and nitrosative stress. TLR4 recognition of LPS and its connection to TLR2 suggest an influence of the common receptor pathway on mitochondrial ROS generation, mitochondrial function, and perhaps ultimately hepatic function. These concepts were tested in mice with targeted knockouts of these receptors, focusing on the liver to take advantage of its immune response to bacteria, high metabolic activity, and mitochondrial susceptibility to oxidative stress.

SPECIFIC AIMS

The hypothesis was tested that TLR4 activation by enteric bacteria causes excessive NO and ROS production that damages mtDNA by performing a quantitative comparison of mtDNA damage after a single dose of heat-killed E. coli bacteria (HkEC) in four strains of mice: wild-type (Wt), TLR2^–/–, TLR4^–/–, and TLR2/4^–/– deficient strains. Resolution of the mtDNA damage was characterized in the four strains of mice and the fidelity of the components of the common signaling pathway that leads to NF-B activation were compared. It was expected that differences would be detected among strains if these receptors play unique roles in activating innate defenses that cause hepatocellular injury.

PRINCIPAL FINDINGS

TLR4 activation was defined as a major cause of depletion of hepatic mtDNA by HkEC through a mechanism that involves NF-B up-regulation of iNOS in concert with TNF- expression. The absence of TLR4-dependent NO synthesis abrogated but did not fully prevent mtDNA damage; however with no TLR4, the residual damage was not cleared nor was copy number restored at a normal rate due in part to a lack of mitochondrial transcription factor A (Tfam) expression. The latter demonstrated a new role of TLR4’s downstream cascade in apposite restoration of organ mtDNA content by mtDNA replication and/or biogenesis because the receptor knockout disrupted the temporal linkage of mtDNA depletion to Tfam expression after the challenge. The key findings are illustrated in Fig. 1 .

View larger version (23K): [in this window] [in a new window]   Figure 1. TLR4-mediated mtDNA depletion in 4 strains of mice challenged with heat-inactivated E. coli (HkEC). A) Accumulation of 3,810 bp mtDNA deletion in livers of mice after HkEC. The percentage of deleted mtDNA by real-time PCR was greatest in Wt and TLR2–/– mice at 24 h after exposures. The percent mtDNA deletion in TLR4–/– and TLR2/4–/– mice was significantly less than in Wt and TLR2–/– mice. B) Quantification of mtDNA in liver by real-time PCR expressed as log copy number. Copy number decreased by almost 1 log in Wt and TLR2–/– mice 6 and 24 h after HkEC exposure but had recovered by 48 h. The depletion of copy number was attenuated and delayed in TLR4–/– and TLR2/4–/– mice, reaching statistical significance only at 48 h. C) Tfam mRNA measured by real-time RT-PCR and normalized to 18S rRNA CT values. Tfam expression increased significantly only in Wt and TLR2–/– strains, not in the TLR4 or double knockout strains. A–C) *Value > at P < 0.05 than baseline control mice and TLR4–/– and TLR2/4–/– groups at the same time, > at P < 0.05 than baseline control values, < than baseline control at P < 0.05; bars indicate SD.

It was found that HkEC stimulated the expression of TLR2 and TLR4; TLR2 was restricted primarily to KC whereas TLR4 was expressed in KC, endothelial cells, and hepatocytes on the plasma membrane and within cells. TLR4 was necessary and fully independent of TLR2 for the local TNF- expression, as the TNF mRNA was absent or its levels greatly attenuated only in TLR4^–/– mice. Other inducible inflammatory stressors that emerge in response to E. coli in the Wt and TLR2^–/– mice, most notably IL-6, iNOS and Nox2, were delayed and attenuated greatly in TLR4^–/– strains.

The sentinel responses to infection are associated with oxidative and nitrosative stress, and indeed hepatic iNOS transcript levels correlated with nitrite appearance in plasma. Selective iNOS inhibition significantly suppressed nitrite accumulation and mtDNA damage, thereby defining the role of NO in mtDNA depletion. But it was observed that repression of NO synthesis increased TNF- expression, previously reported as an effect of increased NADPH oxidase activity.

Mitochondrial protection from oxidants is normally provided by NF-B up-regulation of MnSOD, as seen in Wt and TLR2^–/– mice, but TLR4^–/– and TLR2/4^–/– strains lacked MnSOD induction, further implicating TLR4 activation in mitochondrial ROS generation and confirming that E. coli challenge per se does not block nuclear transcription of mitochondrial protein. Thus, selective mtDNA damage is caused by ETC-generated ROS (e.g., base pair conversions, large-scale deletions, and DNA rearrangements) that underlie the emergence of a 3810 bp mtDNA deletion marker and a decrease in mtDNA copy number (25%) that is sufficient in Wt mice after one HkEC exposure to interfere with in organello mitochondrial transcription. Although TLR4 null mice were more mtDNA damage-resistant than Wt, resolution of damage in the null strains was impaired relative to TLR4-competent strains, reflected by a failure of Tfam induction, a critical regulator of mtDNA transcription, replication, and biogenesis. Only in Wt and TLR2^–/– mice was mtDNA copy number fully restored, typically in 3 days (Fig. 1) .

As to the source of the NO stress, NF-B activation was a clear choice because it is required for iNOS and TNF- expression, and NF-B activation has mitochondrial ROS implicated as a regulatory factor. It was found that NF-B activation correlated with mtDNA damage, but we cannot tell whether lack of NF-B activation in TLR4-deficient strains or other factors prevented Tfam expression and mtDNA recovery. The mice were checked for MyD88 recruitment to the IL-1R-associated kinase (IRAK) and activation of TNF receptor-associated factor (TRAF) -6. These MyD88-dependent events at a minimum activate NF-B via the IB kinase (IKK) -ß- complex. In Wt and TLR2^–/– knockout mice, hepatic NF-B activation by HkEC far exceeded that of TLR4^–/– and TLR2/4^–/– mice, thereby implicating NF-B activation by TLR4 in cytokine-mediated mtDNA damage.

It was found that TLR4 through MyD88/IRAK-1/TRAF6 activated Akt, which enhances NF-B-dependent transcription via p65 phosphorylation. Although NF-B is a target of the kinase, Akt is regulated by NF-B and its transcription by p65. In TLR4^–/– and TLR2/4^–/– mice, Akt phosphorylation in response to HkEC was impaired, suggesting but not providing a definite cause of the lack of Tfam expression.

TLR4 activation in Wt and TLR2^–/– mice induced hepatic IRAK-1 phosphorylation and activity whereas TLR4-deficient mice lacked IRAK-1 phosphorylation and kinase activation. Because IRAK-1 is downstream of TLR4/MD-2/MyD88, the association of IRAK-1 with MyD88 was sought after HkEC, but virtually none was found in the TLR4^–/– and TLR2/4^–/– strains. To the best of our knowledge, this evidence that IRAK-1/MyD88 association is disrupted in TLR4 null mice is unique. Thus, loss of receptor coupling inhibits IRAK-1 phosphorylation and activation for TRAF-6 engagement and NF-B activation in TLR4^–/– and TLR2/4^–/– mice, which are needed to protect mitochondria from the inflammatory response.

CONCLUSIONS AND SIGNIFICANCE

This report integrates four new findings: 1) hepatocytes and KC in the murine liver respond in vivo to heat-inactivated E. coli by increasing TLR4 expression, which regulates critical early TNF- transcription almost exclusively, 2) this TLR4 activation governs the hepatic oxidative stress and iNOS expression that causes mtDNA damage, 3) TLR4-dependent iNOS expression requires MyD88, IRAK, and TRAF6 to develop NF-B signaling in the liver commensurate with myeloid cells, and 4) TLR4-mediated mtDNA damage recovers upon Tfam induction that is not fully competent in TLR-4 null strains.

Figure 2 is a diagram summarizing the current findings in the context of known TLR4 signaling pathways and our current idea of how TLR4 activation produces mitochondrial oxidative and nitrosative stress after the E. coli challenge in vivo. TLR4 activates NF-B signaling and generates TNF- and iNOS-mediated RNS production, which together stimulate mitochondrial ROS and mtDNA oxidation and deletion after a sublethal, subnecrotic dose of an important enteric strain of bacteria. The source of TNF- and possibly iNOS includes KC and hepatocytes, but the mtDNA damage occurs primarily in hepatocytes because almost a quarter of the mtDNA is depleted after challenge, while the KC only contributes 1% of hepatic cell mass and is not enriched in mitochondria. This mtDNA damage is greatly abrogated but not abolished in mice genetically deficient in TLR4. But despite less mtDNA depletion, TLR4-deficient mice show persistence of low copy number due in part to failure of Tfam up-regulation.

View larger version (36K): [in this window] [in a new window]   Figure 2. Simplified illustration of the TLR4 common signaling pathway that activates NF-B and triggers mitochondrial DNA (mtDNA) damage. NF-B is responsible for TNF- and iNOS gene transcription central to the early hepatic response, but it is also responsible for mtDNA damage. Hepatocytes and Kuppfer cells both produce TNF- and iNOS, but mtDNA damage occurs primarily in the hepatocytes (see text). NF-B regulates transcription of the major mitochondrial antioxidant enzyme, MnSOD, which scavenges superoxide but may allow increased export of mitochondrial H2O2 that could serve as a signal to promote transcription of genes such as Tfam involved in mtDNA replication and biogenesis. Specialized pathways that activate MAP kinases or MyD88-independent receptor selectivity (e.g., involving the interferons) have been omitted for clarity.

The significance of the main finding is that TLR4 activation not only triggers oxidative damage to liver mitochondria but also coordinates Tfam induction and the mitochondrial biogenic responses that stem from NF-B and perhaps Akt activation. The implication is that coupling of Tfam expression to mtDNA damage through the iNOS response anticipates the need to protect mitochondria during enteric and possibly other infections by initiating biogenesis before a critical degradation of mitochondrial function occurs.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3500fje;

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