Higher TNFα responses in young males compared to females are associated with attenuation of monocyte adenylyl cyclase expression.

Tumor necrosis factor α (TNFα) expression is strongly attenuated by the intracellular signaling mediator cyclic adenosine monophosphate (cAMP), which is synthesized by adenylyl cyclase (AC) enzymes. We have compared AC regulation and TNFα production in male and female monocytes, and characterized the role of monocyte AC isoforms in TNFα regulation. Males and females, age groups 20-30 years and 50-70 years donated blood for this study. In lipopolysaccharide-stimulated blood from young male donors, we observed significantly higher TNFα responses (6h, p=0.03) compared to females of the same age, a difference not observed in the older donors. Rapid down-regulation of the monocyte AC isoforms AC4, AC7 and AC9 were observed in young males. AC-directed siRNA experiments in the human monocyte cell line THP-1 demonstrated that AC7 and AC9 knock-down significantly induced TNFα release (p=0.01 for both isoforms). These data indicate that the stronger TNFα-responses in young males may be partly associated with male-specific down-regulation of adenylyl cyclases.

Human monocyte responses to lipopolysaccharide and 9-cis retinoic acid after laparoscopic surgery for colon cancer.

Surgery, even modern minimal invasive laparoscopic surgery, induces an initial inflammatory and acute phase response which is followed by a period of immunosuppression rendering surgical patients more susceptible to infection. Here, we aimed to study changes in monocyte inflammatory responses and inflammatory modulation mechanisms following laparoscopic colorectal surgery for colon cancer. Blood samples were collected from 19 colon cancer patients before, directly after and daily for 3 days following surgery. Blood cells were exposed ex vivo to bacterial lipopolysaccharide (LPS) or the inflammatory modulator 9-cis retinoic acid (9cisRA). In blood samples taken prior to surgery, we found significant pro-inflammatory responses to LPS, indicating classical monocyte activation. Directly after surgery, LPS induced significantly less early pro-inflammatory cytokines and monocyte/granulocyte-attracting chemokines. The LPS-mediated release of interleukin (IL)-1ß was still significantly attenuated 3 days after surgery. In patient monocytes collected after surgery, we found increased levels of suppressors of cytokine signaling (SOCS)1 and SOCS3 mRNA, reported to be associated with polarization towards resolving macrophages. The retinoic acid isomer 9cisRA, reported to attenuate LPS-mediated inflammatory responses and alter chemokine responses in cultured monocytes, had a similar effect in patient blood. Three days after surgery, 9cisRA still attenuated pro-inflammatory responses, but the induction of monocyte chemoattractive protein (MCP)-1/CCL2 mRNA in monocytes was reduced. This study indicates changes in monocyte responses that last for at least 3 days after laparoscopic surgery.

Cecal ligation and puncture sepsis is associated with attenuated expression of adenylyl cyclase 9 and increased miR142-3p.

The host inflammatory response in sepsis may be resolved by endogenous anti-inflammatory immune cell responses, avoiding fatal pathogenesis, organ injury, and death. The intracellular signaling mediator cyclic 3'5'-adenosine monophosphate is a potent modulator of inflammatory responses and initiates the polarization of immune cells in a direction that suppresses inflammatory activation. Cyclic 3'5'-adenosine monophosphate is enzymatically produced by adenylyl cyclases (ACs). The expression of ACs is previously shown to be reduced in rat organs after in vivo endotoxemia, concurrent with the progressing systemic inflammation. In the present study, tissue AC gene expression and regulation are explored in a rat model of cecal ligation and puncture (CLP) sepsis. Eighteen hours after CLP operation, expression of several AC isoforms in the liver, spleen, and kidney was reduced, significantly so for AC9 in all tissues. AC9 expression is regulated by the microRNA miR142-3p in T cells. When microRNA was extracted and amplified for miR142-3p expression, it was increasingly expressed 18 h after CLP. A correlation between increased miR142-3p and decreased AC9 expression was found in the liver, kidney, and spleen, and when hepatocytes, Kupffer cells (KCs), and liver sinusoidal endothelial cells were isolated after CLP, reduced AC expression and increased miR142-3p expression were found in KCs and liver sinusoidal endothelial cells. Transfecting a miR142-3p inhibitor probe in rat KCs abolished LPS-mediated AC9 inhibition in vitro. These results indicate that CLP leads to miR142-3p-mediated AC9 reduction in liver macrophages, which may further limit cyclic 3'5'-adenosine monophosphate signaling and the ability of macrophages to resolve the proinflammatory response.