Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages.

Cystic fibrosis (CF), one of the most common human genetic diseases worldwide, is caused by a defect in the CF transmembrane conductance regulator (CFTR). Patients with CF are highly susceptible to infections caused by opportunistic pathogens (including ), which induce excessive lung inflammation and lead to the eventual loss of pulmonary function. Abundant neutrophil recruitment into the lung is a key characteristic of bacterial infections in CF patients. In response to infection, inflammatory neutrophils release reactive oxygen species and toxic proteins, leading to aggravated lung tissue damage in patients with CF. The present study shows a defect in reactive oxygen species production by mouse , human F508del-CFTR, and CF neutrophils; this results in reduced antimicrobial activity against Furthermore, dysregulated Ca homeostasis led to increased intracellular concentrations of Ca that correlated with significantly diminished NADPH oxidase response and impaired secretion of neutrophil extracellular traps in human CF neutrophils. Functionally deficient human CF neutrophils recovered their antimicrobial killing capacity following treatment with pharmacological inhibitors of Ca channels and CFTR channel potentiators. Our findings suggest that regulation of neutrophil Ca homeostasis (via CFTR potentiation or by the regulation of Ca channels) can be used as a new therapeutic approach for reestablishing immune function in patients with CF.

Stenosis is a critical problem in the long-term efficacy of tissue-engineered vascular grafts (TEVGs). We previously showed that host monocyte infiltration and activation within the graft drives stenosis and that TGF-b receptor 1 (TGF-bR1) inhibition can prevent it, but the latter effect was attributed primarily to inhibition of mesenchymal cell expansion. In this study, we assessed the effects of TGF-bR1 inhibition on the host monocytes. Biodegradable TEVGs were implanted as inferior vena cava interposition conduits in 2 groups of C57BL/6 mice (n = 25/group): unseeded grafts and unseeded grafts with TGF-bR1 inhibitor systemic treatment for the first 2 wk. The TGF-bR1 inhibitor treatment effectively improved TEVG patency at 6 mo compared to the untreated control group (91.7 vs. 48%, P < 0.001), which is associated with a reduction in classic activation of mononuclear phagocytes. Consistent with these findings, the addition of rTGF-b to LPS/IFN-g-stimulated monocytes enhanced secretion of inflammatory cytokines TNF-a, IL-12, and IL-6; this effect was blocked by TGF-bR1 inhibition (P < 0.0001). These findings suggest that the TGF-b signaling pathway contributes to TEVG stenosis by inducing classic activation of host monocytes. Furthermore, blocking monocyte activation by TGF-bR1 inhibition provides a viable strategy for preventing TEVG stenosis while maintaining neotissue formation.-Lee, Y.-U., de Dios Ruiz-Rosado, J., Mahler, N., Best, C. A., Tara, S., Yi, T., Shoji, T., Sugiura, T., Lee, A. Y., Robledo-Avila, F., Hibino, N., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. TGF-b receptor 1 inhibition prevents stenosis of tissue-engineered vascular grafts by reducing host mononuclear phagocyte activation. FASEB J. 30, 2627FASEB J. 30, -2636FASEB J. 30, (2016. www.fasebj.orgPrevious data from our laboratory demonstrated that bone marrow-derived mononuclear cell (BM-MNC) seeding of tissue-engineered vascular grafts (TEVGs) promoted the process of neovessel formation (1). In contrast to our original expectation that the seeded cells differentiated into mature vascular cells, we found that the seeded BMMNCs disappeared within a few days of implantation. Instead, the seeded cells exerted their effect by controlling recruitment of host mononuclear phagocytes in early time point, which recruit smooth muscle cells and endothelial cells (;3 wk) (1). While BM-MNC seeding also significantly improved patency of the TEVGs, both in a clinical study and in mouse models, the occurrence of graft stenosis remained a major complication in 25% of TEVG recipients (2, 3). Our mouse models revealed that the formation of TEVG stenosis is also driven by paracrine functions of infiltrating host mononuclear phagocytes. A significant proportion of cells that form stenotic lesions in the mouse model appear to arise from the endothelialmesenchymal transition (Endo-MT) (1,3,4). Because TGFb signaling is the key to multiple vascular disorders, including Endo-MT (5-8), we investigated if inhibition of this cytokine coul...

To characterize early events in neotissue formation during the first 2 weeks after vascular scaffold implantation. Materials & methods: Biodegradable polymeric scaffolds were implanted as abdominal inferior vena cava interposition grafts in wild-type mice. Results: All scaffolds explanted at day 1 contained a platelet-rich mural thrombus. Within the first few days, the majority of cell infiltration appeared to be from myeloid cells at the peritoneal surface with modest infiltration along the lumen. Host reaction to the graft was distinct between the scaffold and mural thrombus; the scaffold stimulated an escalating foreign body reaction, whereas the thrombus was quickly remodeled into collagen-rich neotissue. Conclusion: Mural thrombi remodel into neotissue that persistently occludes the lumen of vascular grafts.

Helminths cause chronic infections and affect the immune response to unrelated inflammatory diseases. Although helminths have been used therapeutically to ameliorate inflammatory conditions, their anti-inflammatory properties are poorly understood. Alternatively activated macrophages (AAMϕs) have been suggested as the anti-inflammatory effector cells during helminth infections. Here, we define the origin of AAMϕs during infection with Taenia crassiceps, and their disease-modulating activity on the Experimental Autoimmune Encephalomyelitis (EAE). Our data show two distinct populations of AAMϕs, based on the expression of PD-L1 and PD-L2 molecules, resulting upon T. crassiceps infection. Adoptive transfer of Ly6C+ monocytes gave rise to PD-L1+/PD-L2+, but not PD-L1+/PD-L2− cells in T. crassiceps-infected mice, demonstrating that the PD-L1+/PD-L2+ subpopulation of AAMϕs originates from blood monocytes. Furthermore, adoptive transfer of PD-L1+/PD-L2+ AAMϕs into EAE induced mice reduced disease incidence, delayed disease onset, and diminished the clinical disability, indicating the critical role of these cells in the regulation of autoimmune disorders.