Dual Inhibition of the MEK/ERK and PI3K/AKT Pathways Prevents Pulmonary Graft-Versus-Host Disease through Suppression of Arteriovenous Inflammation and Bronchiolitis

Introduction Despite clinical development of novel immunosuppressants, the prognosis of pulmonary graft-versus-host disease (pGVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still poor. It is clinically classified into obstructive and constrictive subtypes, but its pathophysiology is not fully elucidated due to difficulty in collecting human lung specimens. We have shown that the MEK inhibitors that target the MAP kinase pathway suppress gastrointestinal and cutaneous GVHD in mice. Therefore, other kinase inhibitors may have potentials to suppress pGVHD. We here explored key molecules in pGVHD patients who underwent lung transplantation and verified the potency of dual inhibition of the MEK/ERK and PI3K/AKT pathways. Methods For human histopathological analysis, we reviewed 45 lung specimens from the allo-HSCT recipients who underwent lung transplantation at our institute for pGVHD from 2008 through 2018. Single-cell level imaging mass cytometry of human pGVHD specimens was performed to visualize interactions of lymphocytes and monocytes around bronchioles. As for a murine pGVHD model, eight-week-old B10.BR (H2K k) mice were pretreated with 240 mg/kg cyclophosphamide and 7 Gy total body irradiation, followed by infusion of C57BL/6J (H2K b) bone marrow cells and splenocytes. Vehicles, the MEK inhibitors trametinib (tra) 0.1 mg/kg/day, cobimetinib (cobi) 0.1 mg/kg/day, the PI3K inhibitor taselisib (tase) 5 mg/kg/day and tacrolimus (tac) 1 mg/kg were given from day 0 through 28. Evaluation was made at day 42 following transplant: primary endpoint was survival rate and secondary endpoints included development of bronchiolitis and values of airway resistance and lung compliance. Immunosuppressive effects of those reagents for human cells were evaluated in vitro: CSFE dilution of T cells stimulated with allogeneic dendritic cells; expression of CD23/CD69 on B cells activated with IL-4/CD40L; TNF-α production of monocytes stimulated with lipopolysaccharide. Results Three pathological findings characterize human pGVHD: bronchiolitis obliterans, lymphocyte infiltration and wall thickening of bronchioles; pleuroparenchymal fibroelastosis (PPFE), proliferation of subpleural elastic fibers; peribronchitis, macrophage aggregation around the bronchi. Most cases with PPFE had subpleural arteriovenous inflammation, and immunostaining revealed that B cells and macrophages were abundantly infiltrated around the bronchioles. These results indicated that suppressing B cells and macrophages may be crucial to prevent pGVHD through avoiding vascular inflammation and bronchiolitis. In the murine pGVHD, cobi but not tra/tac improved survival rate (vehicle 31%; cobi 62%, tra 7%, tac 10% at day 42; p < 0.01). Cobi attenuated development of bronchiolitis, venulitis and peribronchial macrophage aggregation. Cobi improved respiratory functions, such as airway resistance (vehicle 1.65 ± 0.19, cobi 2.86 ± 0.75 cmH 2O.s/mL, p < 0.001) and lung compliance (vehicle 13.14 ± 0.71 cobi 8.99 ± 1.08 μL/cmH 2O, p < 0.05). Notably, T cell infiltration around bronchioles was inhibited by cobi and tra, but B cell infiltration was inhibited only by cobi. Whereas cobi inhibited phosphorylation of AKT, tra compensatory upregulated it. Cobi inhibited B cell activation more than tra (%CD23 +CD69 + B cells: 32.8% with cobi 1μM, 53.0% with tra 1μM, p < 0.05). In addition, cobi suppressed TNF-α production by monocytes more strongly than tra (%TNF-α + monocytes: 53.1% with cobi 1μM vs 71.6% with tra 1μM, p < 0.05). Dual inhibition of the MEK/ERK and PI3K/AKT pathways with combination of tra and tase strongly suppressed B cells in vitro, and improved survival rate compared with vehicle and monotherapy of tra or tase in mice (Figure). Finally, imaging mass cytometry of human pGVHD specimens revealed that lymphocytes around bronchioles and venules were positive for phospho-ERK1/2 and phospho-AKT. Conclusions pGVHD is associated with vascular inflammation and bronchiolitis in humans and mice. Furthermore, phosphorylation of ERK1/2 and AKT is upregulated in human pGVHD. Given that dual inhibition of MEK/ERK and PI3K/AKT signaling suppresses vascular inflammation and bronchiolitis thorough inhibition of B cells and monocytes in mice, this strategy may provide a novel treatment option against human pGVHD. Figure 1 Figure 1. Disclosures Takaori-Kondo: ONO PHARMACEUTICAL CO., LTD.: Research Funding; Bristol-Myers K.K.: Honoraria; Celgene: Research Funding.

NPY and CGRP Inhibitor Influence on ERK Pathway and Macrophage Aggregation during Fracture Healing

Aim: The aims of this study are to investigate the effects of neurotransmitters NPY and CGRP on ERK signaling in fracture healing, and to identify the correlation between macrophage aggregation and fracture healing. Methods: Male Sprague-Dawley rats were used to build a fracture model. The neurotransmitter receptor inhibitors were injected intraperitoneally into the rats. Immunofluorescence staining and ELISA were employed to determine the expression of NPY and CGRP in fracture area and the peripheral blood, respectively. Micro-CT together with histological staining were utilized to assess the fracture healing conditions. Relative protein expression was determined using western blot. Immunofluorescence staining was used to detect the aggregation of macrophages in the injury area. Results: During fracture healing, the serum NPY and CGRP significantly increased. The levels of NPY and CGRP reached a peak in the 8th week and reduced significantly thereafter. NPY and CGRP inhibitors could inhibit fracture healing and down-regulate the phosphorylated ERK. Macrophages (NPY+ and CGRP+) aggregated in the injury area. Conclusion: NPY and CGRP participated in fracture healing, in which they were also shown to influence phosphorylated ERK expression. In addition, macrophages are involved in the fracture healing process.

Myxosporidia and macrophage centres in chub (Leuciscus cephalus)–quantitative interactions focus on Myxobolus cyprini

Six myxosporidian species were found in chub (Leuciscus cephalus) originating from Lower Austrian rivers. The frequency of the parasites and their localization was recorded. In all chub, independent of size and origin, Myxobolus cyprini occurred predominantly in the macrophage centres (MCs) of the haematopoietic organs, spleen and kidney. Exclusively in the head kidney of young fish not yet described vermicular plasmodia containing spores of M. cyprini were found. In muscle tissue the prevalence of M. cyprini was comparatively low. Other species of Myxobolus characterized by plasmodial cysts frequently occurred in gills and swimbladder but were rarely detected, and only in small numbers, in the haematopoietic organs. The number of M. cyprini spores and the relative volume of MCs in the haematopoietic organs were estimated in order to examine possible correlations. Significant interrelated changes were found only in juvenile fish up to a size of 15cm. In bigger fish, the number and size of macrophage aggregates were highly variable and independent of infection intensity and fish size, but the number of spores never exceeded that of the aggregated macrophages. The data suggest that due to an early date of infection M. cyprini is the only species which is closely associated with macrophage aggregation.

Smoking Decreases Alveolar Macrophage Function during Anesthesia and Surgery

Background Smoking changes numerous alveolar macrophage functions and is one of the most important risk factors for postoperative pulmonary complications. The current study tested the hypothesis that smoking impairs antimicrobial and proinflammatory responses in alveolar macrophages during anesthesia and surgery. Method The authors studied 30 smoking and 30 nonsmoking patients during propofol-fentanyl general anesthesia. Alveolar immune cells were harvested by bronchoalveolar lavage immediately and 2, 4, and 6 h after induction of anesthesia and at the end of surgery. The types of alveolar immune cell and macrophage aggregation were determined. The authors measured opsonized and unopsonized phagocytosis. Microbicidal activity was determined as the ability of the macrophages to kill Listeriamonocytogenes directly. Finally, RNA was extracted from harvested cells and cDNA was synthesized by reverse transcription. The expression of interleukin 1beta, 6, and 8, interferon gamma, and tumor necrosis factor alpha were measured by semiquantitative polymerase chain reaction using beta-actin as an internal standard. Results The fraction of aggregated macrophages increased significantly over time in both groups, whereas phagocytosis of opsonized and nonopsonized particles and microbicidal activity of alveolar macrophages decreased significantly. The changes, though, were nearly twice as great as in patients who smoked. Gene expression of all proinflammatory cytokines in alveolar immune cells except interleukin 6 increased 2- to 20-fold over time in both groups. The expression of interleukin 1beta, interferon gamma, and tumor necrosis factor alpha, however, increased only half as much in smokers as in nonsmokers. Conclusion Smoking was associated with macrophage aggregation but markedly reduced phagocytic and microbicidal activity-possibly because expression of proinflammatory cytokines was reduced in these patients. Our data thus suggest that smokers may have a limited ability to mount an effective pulmonary immune defense after anesthesia and surgery.