MicroRNA Maneuvers in Osteoclastogenesis See article p.4977

Universal Flu Vaccine Potential in Pigs See article p.5014

Slaying Pneumonia with M-CSF See article p.5047

Fyn Foils LPS Activity in Mast Cells See article p.5075

Gram-negative bacilli, including Klebsiella pneumoniae, can cause respiratory infections that colonize the lower respiratory tract and lead to life-threatening cases of pneumonia. Macrophage-CSF (M-CSF) is a cytokine known to influence the proliferation, differentiation, and effector functions of multiple lineages of mononuclear phagocytes. In this issue, Bettina et al. (p. 5047) examine the role of M-CSF in mononuclear phagocyte-mediated host defense using a mouse model of K. pneumoniae pulmonary infection. Elevated levels of M-CSF were detected in the lungs of infected WT mice compared with uninfected controls. Infected mice carrying a homozygous inactivating mutation in the M-CSF locus or treated with Abs that block the M-CSF signaling axis had greater mortality and bacterial lung burdens compared with WT control mice or mice treated with Ab isotype controls. Further analysis showed that M-CSF facilitated the expansion of monocyte subsets in the lung during infection and was required for the survival and antimicrobial function of these cell types. M-CSF was also needed to control infection and prevent tissue injury in the livers of infected mice. Together these results suggest that M-CSF is critical to the survival and host defense functions of mononuclear phagocytes in the lung and liver during K. pneumoniae pulmonary infection.

A thorough understanding of influenza A virus (IAV) infection in pigs, animals that play a central role in the emergence of novel IAV such as the 2009 H1N1 pandemic strain, would aid the health of both livestock and humans. Because IAV evolves rapidly, development of a universal vaccine against this virus is sought to replace the current strain-specific inactivated IAV vaccines. In this issue, Morgan et al. (p. 5014) assessed the potential of the universal vaccine candidate S-FLU to protect pigs from homologous or heterologous IAV infection. S-FLU, which has been shown to protect mice and ferrets against IAV, is an attenuated viral vaccine that cannot replicate and allows optimal presentation of conserved viral Ags to T cells, resulting in strong cell-mediated immunity but no neutralizing Abs. An S-FLU vaccine expressing hemagglutinin (HA) from H1N1 was delivered to the lower respiratory tract of groups of pigs via intratracheal administration (H1 i.t.) or by aerosol (H1 aer), and a vaccine expressing HA from H5N1 was delivered i.t. (H5 i.t.). Following challenge with H1N1 virus, pigs in the H1 aer group showed the greatest reduction in viral titers in both nasal swabs and lungs. Interestingly, of the vaccinated animals, the H5 i.t. group had the strongest H1N1-specific T cell responses in their peripheral blood and tracheobronchial lymph nodes; however, anti-H1N1 T cell responses in the bronchoalveolar lavage were strongest among the H1 aer group. Antiviral T cell responses, measured by patterns of cytokine production, varied between the two vaccines and the different routes of immunization. Principal component analysis supported the utility of H1 aer immunization for the reduction of viral titers but indicated that the strongest anti-H1N1 response was seen following immunization with the heterologous H5N1 vaccine. This study suggests the possibility of a universal IAV vaccine for pigs and supports using immunization via aerosol for efficient vaccine delivery.

Inflammatory diseases such as rheumatoid arthritis can cause osteoclastogenesis and subsequent inflammatory bone resorption. Immune mechanisms to curb the differentiation of osteoclasts under inflammatory conditions are being studied, and one candidate regulator is RBP-J, a transcription factor that negatively regulates TNF-α–driven osteoclastogenesis. Miller et al. (p. 4977) examined the genome-wide profile of microRNAs (miRNAs) in TNF-α–treated bone marrow–derived macrophages (BMMs)/osteoclast precursors from wild-type (WT) mice or those with a targeted deletion of RBP-J in myeloid macrophages. This analysis revealed that only miRNA-182-5p (miR-182) was expressed significantly higher in TNF-α–treated RBP-J–deficient cells than in WT cells. Inhibition of miRNA-182 expression with an antagomir significantly reduced TNF-α–driven osteoclastogenesis in RBP-J–deficient BMMs. In contrast, increased expression of miR-182 promoted osteoclastogenesis in TNF-α–treated WT BMMs, and miR-182 positively regulated NFATc1 and Blimp1, which are transcription factors critical to driving osteoclast differentiation. In addition, miR-182 was shown to target Foxo3 and Maml1 to relieve their inhibition of osteoclastogenesis induced by TNF-α treatment. Together these results show that RBP-J suppresses miR-182 expression, which in turn inhibits TNF-α–driven osteoclastogenesis. These findings may provide the means to identify novel therapeutic targets to help reverse inflammatory bone resorption.

In addition to their roles in allergy, mast cells (MCs) also modulate inflammatory reactions following infection. Stimulation of TLR4 on the surface of MCs results in cytokine production through signaling pathways that have not yet been fully elucidated. To better understand MC responses to LPS, Martín-Ávila et al. (p. 5075) analyzed the involvement of the Src kinase Fyn in TLR4 signaling in MCs. Relative to their wild-type (WT) counterparts, LPS-treated Fyn−/− MCs secreted significantly more TNF, both from preformed stores and via de novo synthesis. TNF was found in cellular compartments associated with the SNARE VAMP3, and LPS treatment induced an association between VAMP3, SNAP-23, and Stx4 that was augmented in MCs lacking Fyn, supporting enhanced secretion of preformed TNF in Fyn−/− MCs. Analysis of de novo production of TNF indicated that LPS treatment of Fyn−/− MCs increased both the production and stability of mRNA encoding TNF, relative to LPS treatment of WT MCs. In Fyn−/− MCs, LPS augmented TAK1 phosphorylation more than in WT MCs, and IκB kinase (IKK)-dependent transcription factors localized to the nuclei of these cells even in the absence of LPS. Further mechanistic analysis linked these observations to inhibition of protein phosphatase 2A (PP2A) activity and resultant increased activity of classical isoforms of protein kinase C (PKC α/β) in Fyn−/− MCs. The observed activation of PKC α/β in Fyn−/− MCs was connected to dissociation of these kinases from PP2A and association instead with the adaptor molecule AHNAK (desmoyokin) following MC treatment with LPS, and PKC α/β activation was also linked to the secretion of preformed TNF via translocation of VAMP3-containing vesicles to the plasma membrane. Confirming the in vivo relevance of these studies, LPS-induced TNF production was significantly increased in both Fyn−/− mice and MC-deficient mice reconstituted with Fyn−/− MCs, relative to WT mice and MC-deficient mice reconstituted with WT MCs. Taken together, these data demonstrate a detailed mechanism by which Fyn dampens TLR4-induced TNF production in MCs.