No abstract

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently discovered molecule that is expressed on the cell surface of monocytes and neutrophils. Engagement of TREM-1 triggers synthesis of proinflammatory cytokines in response to microbes, but the extent and mechanism by which TREM-1 modulates the inflammatory response is poorly defined. In the present study, we investigated the functional effects of blocking TREM-1 on the Toll-like receptor (TLR)4-mediated signaling pathway in macrophages. By transfecting cells with small hairpin interfering RNA molecules to TREM-1 (shRNA), we confirmed that TREM-1 mRNA and protein expression was greatly attenuated in RAW cells in response to treatment with LPS. PCR array for genes related to or activated by the TLR pathway revealed that although the expression of TLR4 itself was not significantly altered by silencing of TREM-1, expression of several genes, including MyD88, CD14, IkappaBalpha, IL-1beta, MCP-1, and IL-10 was significantly attenuated in the TREM-1 knockdown cells in response to treatment with LPS. These data indicate that expression of TREM-1 modulates the TLR signaling in macrophages by altering the expression of both adaptor and effector proteins that are critical to the endotoxin response.

Triggering receptor expressed on myeloid cells (TREM)‐1 is a recently identified immunoglobulin receptor that is expressed on neutrophils and monocytes where it amplifies the acute inflammatory response to bacteria. We examined the transcriptional regulation of TREM‐1 in macrophages. Treatment of RAW cells with Escherichia coli LPS or Pseudomonas aeruginosa led to the induction of TREM‐1 within 1 h with an expression lasting up to at least 24 h in vitro as detected by RT‐PCR. Since the promoter of TREM‐1 has multiple binding sites for NF‐κB and PU.1 (one of the members of the ets family of transcription factors), we investigated the role of these transcription factors in the induction of TREM‐1. Treatment of cells with NF‐κB inhibitors abolished the expression of message of TREM‐1 induced by LPS and P. aeruginosa. In contrast, the expression of TREM‐1 was increased after stimulation with LPS or P. aeruginosa in cells that had gene of PU.1 silenced. Additionally, over‐expression of PU.1 led to inhibition of TREM‐1 induction in response to LPS and P. aeruginosa. These data suggest that both these transcription factors are involved in the expression of TREM‐1. NF‐κB functions as a positive regulator whereas PU.1 is a negative regulator of the TREM‐1 gene.

Cyclooxygenase (COX)-2 expression and release of prostaglandins (PGs) by macrophages are consistent features of lipopolysaccharide (LPS)-induced macrophage inflammation. The two major PGs, PGE2 and PGD2, are synthesized by the prostanoid isomerases, PGE synthases (PGES) and PGD synthases (PGDS), respectively. Since the expression profile and the individual role of these prostanoid isomerases-mediated inflammation in macrophages has not been defined, we examined the LPS-stimulated PGs production pattern and the expression profile of their synthases in the primary cultured mouse bone marrow derived macrophages (BMDM). Our data show that LPS induced both PGE2 and PGD2 production, which was evident by ∼8 hrs and remained at a similar ratio (∼1∶1) in the early phase (≤12 hrs) of LPS treatment. However, PGE2 production continued increase further in the late phase (16–24 hrs); whereas the production of PGD2 remained at a stable level from 12 to 24 hrs post-treatment. In response to LPS-treatment, the expression of both COX-2 and inducible nitric oxide synthase (iNOS) was detected within 2 to 4 hrs; whereas the increased expression of microsomal PGES (mPGES)-1 and a myeloid cell transcription factor PU.1 did not appear until later phase (≥12 hrs). In contrast, the expression of COX-1, hematopoietic-PGDS (H-PGDS), cytosolic-PGES (c-PGES), or mPGES-2 in BMDM was not affected by LPS treatment. Selective inhibition of mPGES-1 with either siRNA or isoform-selective inhibitor CAY10526, but not mPGES-2, c-PGES or PU.1, attenuated LPS-induced burst of PGE2 production indicating that mPGES-1 mediates LPS-induced PGE2 production in BMDM. Interestingly, selective inhibition of mPGES-1 was also associated with a decrease in LPS-induced iNOS expression. In summary, our data show that mPGES-1, but not mPGES-2 or c-PGES isomerase, mediates LPS-induced late-phase burst of PGE2 generation, and regulates LPS-induced iNOS expression in BMDM.

The role of NADPH oxidase (NOX) was recently suggested in cardiac diseases including hypertrophy. Overexpression of a DN mutant of p67 phox , a cytosolic subunit of NOX, DN-p67 with a V204A point mutation completely inhibited NOX enzymatic function of superoxide generation. We found that overexpression of DN-p67 in cultured adult rat cardiac myocytes attenuated α 1 -adrenoceptor-induced hypertrophy , we thus hypothesized that NOX/p67 phox plays a critical role in cardiac hypertrophy and remodeling in vivo . Accordingly, we recently generated a Tg mouse model in C57BL/6 strain with cardiac-specific overexpression of this DN-p67 mutant along with an IRES-initiated EGFP tag protein using the cardiac-specific α-MHC promoter. Hearts from 1.5-, 3- and 6-month (M) old heterozygous Tg mice and their Non-Tg (NTg) littermates were harvested and compared. The heart weight/body weight (HW/BW) ratio in Tg group was significantly smaller (overall: 14%↓; 1.5-M: 10%↓; 3-M: 16%↓; 6-M: 12%↓) vs. the NTg group ( p < 0.01, n=23). This result was confirmed by 2D echocardiography (2D-echo) measuring the left ventricular myocardial area (LVMA) determined by subtracting endocardial area from epicardial area in short axis view at the end diastole. The LVMA (mm 2 ) in 3-M and 6-M mice was significantly ( p < 0.05, n=21) smaller in Tg (0.44±0.02) than the NTg (0.50±0.02) with equal average BW in both groups. LV internal dimensions and systolic function were preserved without difference in Tg vs. NTg. The LV wall thickness is smaller in Tg vs. NTg by both 2D-echo and hematoxylin/eosin staining of LV cross-sections, but no change in fibrosis was detected in both groups by trichrome staining (n=4). Interestingly, the expression of NOX catalytic subunit gp91 phox in Tg hearts was significantly decreased (~3-fold↓, n=5) vs. NTg by Western blot, whereas the cardiac expression of other NOX subunits p47 phox and p22 phox remained the same in both groups. In summary, we have generated a viable Tg mouse model with cardiac-specific overexpression of a DN p67 phox , which shows significant reduction in both myocardial mass/size and gp91 phox expression with normal cardiac function and development. Our results first show the novel and critical role of NOX/p67 phox in normal cardiac growth and heart development. This research has received full or partial funding support from the American Heart Association, AHA National Center.