n.) administration of mice with c-di-GMP induces recruitment of monocytes and granulocytes [20] and activates the host immune response [21] and [22]. In one study, the lungs and draining lymph nodes from mice intranasally check details treated either with c-di-GMP

or phosphate buffered saline (PBS) were examined 24 or 48 h after treatment for differences in cell number or composition. Results showed that the draining lymph nodes of c-di-GMP-treated mice had significantly higher total cell numbers as well as higher percentages of CD44low cells and CD86 positive cells. In the lung, however, the picture was less clear with no difference in total numbers of monocytes or neutrophils or pulmonary DCs as determined by flow cytometry [21]. However, there was some indication that c-di-GMP did affect lung parenchymal cells in that the lungs from c-di-GMP-treated mice had a larger proportion of alveolar macrophages which were newly recruited (CD11chiMHCIIlowCD11b+). Also, DCs (CD11chiMHCIIhi), although not significantly increased in number, expressed higher levels of CD40 and CD86 than PBS-treated control mice [21]. Work from our own laboratories has indicated that 24 h after a single i.n. administration of c-di-GMP, there is a significant increase in the number of pulmonary DCs with higher expression

of CD40 and CD80 but not CD86 or MHCII [23]. The treatment also induced a rapid but transient recruitment of neutrophils and other inflammatory and cells into the bronchoalveolar space [23] and increased levels of Galunisertib cost proinflammatory cytokines and chemokines IL-12p40, IL-1β, IL-6, keratinocyte derived chemokine (KC), MCP-1, macrophage inflammatory protein (MIP)-1β, RANTES and tumor necrosis factor (TNF)-α in a dose-dependent manner [22]. A number of recent studies have shown that the innate immune response elicited by c-di-GMP is a potent immunomodulator for the treatment of bacterial infections. In this regard, studies of the effect of c-di-GMP on the course of bacterial infection have clearly shown a striking protective

effect of c-di-GMP administration against a number of serious bacterial infections. Using a mouse model of mastitis, Karaolis and co-workers showed that, despite no direct bactericidal activity, c-di-GMP co-administered with S. aureus directly into the mammary glands significantly decreases bacterial burdens [24]. Previous work by the same group had shown that c-di-GMP inhibits biofilm formation of the same S. aureus strain as well as its adherence to HeLa cells [25]. To rule out the possibility that the c-di-GMP-mediated protection is solely due to its role in the inhibition of biofilm formation, subsequent work showed that pretreatment with c-di-GMP 12 and 6 h before intramammary infection with S. aureus also results in a 1.5 log and a 3.