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 playing 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 subunit I and IV in a time dependent manner through early phosphorylation of the PI3kinase/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), nitric oxide production and cell death. The TLR4 mediated AKT activation and mitochondrial biogenesis required activation of adaptor protein MyD88 and Toll-IL-1R-containing adaptor inducing IFN-β (TRIF). Importantly, using a genetic approach, we show that the AKT1 isoform is pivotal in regulating mitochondrial biogenesis in response to TLR4 agonist.

Rationale: Sarcoidosis is a systemic inflammatory disorder characterized by distinct up-regulation of Th1 cytokines, such as tumor necrosis factor (TNF)-a 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-a 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-a. 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-a 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.

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.