Novel anti-inflammatory compounds from endophyte fungus in inhibitions of cytokine responses in macrophage RAW 264.7 cells

Cytokines are pivotal proteins in regulating the homeostasis of immune system. Up- or down-regulation of cytokine production indicates modulation of the immune system in response to the environment. Many diseases are caused by excessive production of inflammatory cytokines in local or systemic tissues or organs. Here, two indole diterpenes, lolitrem B, produced by endophyte fungus, and its isomer. 31-epilolitrem B, were used to investigate their effects on pro-inflammatory cytokine interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-a)responses in in vitro cultured murine macrophage RAW 264.7 cells. Our studies showed that both compounds have significant effects on inhibiting both IL-6 and TNF-a production at 24 hr in the presence of lipopolysaccharide (LPS) from the lowest concentration of 20 nm and up to 2 uM. In the absence of LPS, there were no significant increases in production of either cytokine. Statistical analysis showed that the effects we observed were not caused by the presence of the dimethyl sulfoxide used to dissolve both toxins. Paxilline, the precursor of lolitrems and known big conductance potassium channel activated (BK) blocker, also inhibited the cytokine responses but at a much higher concentration of 1 uM. Cell proliferation assays showed that the lolitrems alone did not inhibit the growth of the cells at concentrations as high as 1 uM. Cells treated with both toxins in the presence and absence of LPS did not undergo apoptosis with FACS analysis. This further demonstrated that inhibition was not through the apoptotic pathway, suggesting alternative cascades involved in the regulation of the anti-inflammatory response. The ability of these compounds to inhibit cytokine responses may have potential as immunotherapeutic drugs for inflammation diseases in the future