TLR4-mediated AKT activation is MyD88/TRIF dependent and critical for induction of oxidative phosphorylation and mitochondrial transcription factor A in murine macrophages.

Mitochondria play a critical role in cell survival and death. Mitochondrial recovery during inflammatory processes such as sepsis is associated with cell survival. Recovery of cellular respiration, mitochondrial biogenesis, and function requires coordinated expression of transcription factors encoded by nuclear and mitochondrial genes, including mitochondrial transcription factor A (T-fam) and cytochrome c oxidase (COX, complex IV). LPS elicits strong host defenses in mammals with pronounced inflammatory responses, but also triggers activation of survival pathways such as AKT pathway. AKT/PKB is a serine/threonine protein kinase that plays an important role in cell survival, protein synthesis, and controlled inflammation in response to TLRs. Hence we investigated the role of LPS-mediated AKT activation in mitochondrial bioenergetics and function in cultured murine macrophages (B6-MCL) and bone marrow-derived macrophages. We show that LPS challenge led to increased expression of T-fam and COX subunits I and IV in a time-dependent manner through early phosphorylation of the PI3K/AKT pathway. PI3K/AKT pathway inhibitors abrogated LPS-mediated T-fam and COX induction. Lack of induction was associated with decreased ATP production, increased proinflammatory cytokines (TNF-α), NO production, and cell death. The TLR4-mediated AKT activation and mitochondrial biogenesis required activation of adaptor protein MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-ß. Importantly, using a genetic approach, we show that the AKT1 isoform is pivotal in regulating mitochondrial biogenesis in response to TLR4 agonist.

Dysregulation of p38 and MKP-1 in response to NOD1/TLR4 stimulation in sarcoid bronchoalveolar cells.

RATIONALE: Sarcoidosis is a systemic inflammatory disorder characterized by distinct up-regulation of Th1 cytokines, such as tumor necrosis factor (TNF)-α and IL-12. The mechanism underlying this up-regulation remains unclear. Recognition of microbial moieties through Toll-like or Nod-like receptors evokes sequential activation of mitogen-activated protein kinases (MAPKs), which plays a role in Th1-immune response. OBJECTIVES: To test the hypothesis that dysregulation in MAPK signaling in response to microbial stimulation is important in mediating Th1 response in sarcoidosis. METHODS: Ex vivo cultured bronchoalveolar lavage (BAL) cells isolated from patients with sarcoidosis and control subjects were stimulated with low-dose Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 1 (NOD1) ligands as a model of microbial stimulation, and MAPK signaling and inflammatory response were analyzed. MEASUREMENTS AND MAIN RESULTS: BAL cells from patients with sarcoidosis exhibited higher basal p38 activity, greater p38 phosphorylation, and more robust production of TNF-α and IL-12/IL-23p40 on stimulation with NOD1 and TLR4 agonists than cells isolated from control subjects. In contrast, control BAL cells had greater basal extracellular signal-regulated kinase (ERK) activity and NOD1 and TLR4 agonists preferentially activated the ERK pathway. Inhibition of p38, but not ERK, attenuated production of both IL12/IL23p40 and TNF-α. Interestingly, stimulation of cells from patients with sarcoidosis with either NOD1 or TLR4 ligand failed to induce MAPK phosphatase 1 (MKP-1). Adenovirus-mediated overexpression of MKP-1 attenuated p38 activation and decreased the production of IL12/IL23p40 and TNF-α in sarcoid BAL cells. CONCLUSIONS: Our results suggest that enhanced p38 signaling in response to microbial products is caused by abnormal regulation of MKP-1 and contributes to heightened inflammation in sarcoidosis.

Nasal-associated lymphoid tissue is not an absolute requirement for the induction of rat tear IgA antibody responses.

UNLABELLED: PURPOSE/AIM OF STUDY: The purpose of this work was to determine whether rat nasal-associated lymphoid tissue is required for the induction of tear IgA responses. MATERIALS AND METHODS: Particulate antigen in the form of DNP-BSA encapsulated in cationic microparticles was applied topically to the eyes (ocular topically) of rats that had the nasolacrimal ducts temporarily plugged with chromic gut suture material. Eye washes and serum were monitored for development of antigen specific IgA and IgG, respectively. To track the particulate uptake, fluorescent latex beads were applied topically to the eyes of plugged and unplugged animals. The nasal-associated lymphoid tissue and the draining lymph nodes were then examined for the presence of the fluorescent beads. RESULTS: It was found that the chromic gut suture was effective in blocking the passage of antigen into the nasopharyngeal cavity for at least 24 hr. Tear antigen-specific IgA levels found in the eyes of plugged animals were not significantly lower from those of unplugged animals. Serum IgG antibody levels were also similar between the two groups. In animals with plugged nasolacrimal ducts, fluorescent beads were found predominately in the superficial cervical lymph nodes, which have been shown to drain the surface of the eye. CONCLUSIONS: These results indicate that particulate antigen can be taken up by the conjunctiva and transported to the draining lymph nodes, showing that antigen does not need to access nasal-associated lymphoid tissue to induce tear IgA antibody responses.