Kiyoharu Fukushima

Identification of an atypical monocyte and committed progenitor involved in fibrosis

Monocytes and macrophages comprise a variety of subsets with diverse functions. It is thought that these cells play a crucial role in homeostasis of peripheral organs, key immunological processes and development of various diseases. Among these diseases, fibrosis is a life-threatening disease of unknown aetiology. Its pathogenesis is poorly understood, and there are few effective therapies. The development of fibrosis is associated with activation of monocytes and macrophages. However, the specific subtypes of monocytes and macrophages that are involved in fibrosis have not yet been identified. Here we show that Ceacam1+Msr1+Ly6C−F4/80−Mac1+ monocytes, which we term segregated-nucleus-containing atypical monocytes (SatM), share granulocyte characteristics, are regulated by CCAAT/enhancer binding protein β (C/EBPβ), and are critical for fibrosis. Cebpb deficiency results in a complete lack of SatM. Furthermore, the development of bleomycin-induced fibrosis, but not inflammation, was prevented in chimaeric mice with Cebpb−/− haematopoietic cells. Adoptive transfer of SatM into Cebpb−/− mice resulted in fibrosis. Notably, SatM are derived from Ly6C−FcεRI+ granulocyte/macrophage progenitors, and a newly identified SatM progenitor downstream of Ly6C−FcεRI+ granulocyte/macrophage progenitors, but not from macrophage/dendritic-cell progenitors. Our results show that SatM are critical for fibrosis and that C/EBPβ licenses differentiation of SatM from their committed progenitor.

Development of microscopic polyangiitis-related pulmonary fibrosis in a patient with autoimmune pulmonary alveolar proteinosis

BackgroundAutoimmune pulmonary alveolar proteinosis (aPAP) is a rare lung disease caused by the autoantibody against granulocyte-macrophage colony stimulating factor (GM-CSF). The clinical course of aPAP is variable; in severe cases, patients develop lethal respiratory failure due to pulmonary fibrosis. However, the pathogenesis of pulmonary fibrosis in aPAP has never been delineated.Case presentationHere, we describe a rare case of aPAP that was subsequently complicated by microscopic polyangiitis-related pulmonary fibrosis. The patient was a 75-year-old Japanese man diagnosed with aPAP based on the crazy-paving appearance on high-resolution computed tomography (HRCT), “milky” appearance of broncho-alveolar lavage fluid (BALF), and elevated serum levels of the anti-GM-CSF antibody. The patient was followed-up without aPAP-specific treatment for 3 years. During this period, both hematuria and proteinuria appeared; in addition, serum myeloperoxidase (MPO)-anti-neutrophil cytoplasmic antibody (ANCA) turned positive and increased markedly. The second BAL performed one year after the diagnosis, showed that the “milky” appearance had resolved. The HRCT showed that fibrotic changes had developed and that the crazy-paving appearance had disappeared. These data suggest an association between pulmonary fibrosis that developed during the natural course of aPAP and ANCA-related systemic vasculitis.ConclusionThis is the first case report that suggests the existence of a pathogenetic relationship between ANCA-associated systemic vasculitis and aPAP-related pulmonary fibrosis. The link between ANCA-associated systemic vasculitis and aPAP-related pulmonary fibrosis requires further investigation.

Favorable response to trastuzumab plus irinotecan combination therapy in two patients with HER2-positive relapsed small-cell lung cancer

Small-cell lung cancer (SCLC) easily recurs with multidrug resistance phenotype. However, standard therapeutic strategies for relapsed-SCLC remain unestablished. Human epidermal growth factor receptor 2 (HER2) expression correlates with poor prognosis in extensive disease-SCLC. We have reported previously that HER2 expression is upregulated when HER2-positive SCLC cells acquire chemoresistance, and also demonstrated that trastuzumab exerts significant antitumor activity toward HER2-upregulated chemoresistant SCLC, mainly via antibody-dependent cell-mediated cytotoxicity mechanism. Based on these preclinical data, we treated two patients with HER2-positive SCLC by combination of trastuzumab (6 mg/kg, day 1) and irinotecan (80 mg/m(2), days 1 and 8) every 21 days as the third-line chemotherapy following two prior regimens, first-line carboplatin plus etoposide and second-line amrubicin. One patient achieved partial response after the first cycle and received 6 cycles in total without disease progression for 4.5 months. The other also received 4 cycles and kept stable disease for 3.5 months. This treatment can be continued safely at an outpatient clinic without any severe adverse event. In conclusion, trastuzumab plus irinotecan chemotherapy is promising and feasible against HER2-positive relapsed SCLC. Further clinical studies are encouraged to confirm the antitumor efficacy of trastuzumab in SCLC.

Dapsone Hypersensitivity Syndrome-related Lung Injury without Eosinophilia in the Bronchoalveolar Lavage Fluid

A 73-year-old man was admitted in respiratory failure that had subacutely progressed after five weeks of dapsone treatment for a skin rash. He also presented with fever, systemic erythroderma and liver dysfunction. Chest computed tomography showed diffuse reticular shadows with ground-glass opacity and bilateral mediastinal lymphadenopathy. Lymphocytes, but not eosinophils, were increased in the bronchoalveolar lavage fluid. Moreover, reactivation of human herpes virus-6 was confirmed on a paired serum test. Finally, we diagnosed the patient with dapsone hypersensitivity syndrome (DHS), a rare adverse event of this drug. Lung injury unaccompanied by eosinophilia in the bronchoalveolar lavage fluid is even more rare as a DHS-related lung manifestation.

Antitumor effect of Batf2 through IL-12 p40 up-regulation in tumor-associated macrophages

Significance The therapeutic activity of checkpoint blockers and toll-like receptor (TLR) agonists, which show some efficacy against malignancies, appears to at least partially result from the secretion of type-I IFNs. Thus, we hypothesized that type-I IFN-inducible transcription factors, such as basic leucine zipper transcription factor ATF-like 2 (Batf2), might play a role in tumor immunity. Here, we investigated the role of Batf2, especially its positive transcriptional activities, and evaluated its antitumor effect. This study shows that Batf2 has an antitumor effect through the up-regulation of IL-12 p40 in tumor-associated macrophages, which eventually induces the activation of CD8+ T cells and their accumulation within the tumor. Batf2 may be an important target in anticancer treatment with immune checkpoint blockers and TLR agonists. The development of effective treatments against cancers is urgently needed, and the accumulation of CD8+ T cells within tumors is especially important for cancer prognosis. Although their mechanisms are still largely unknown, growing evidence has indicated that innate immune cells have important effects on cancer progression through the production of various cytokines. Here, we found that basic leucine zipper transcription factor ATF-like 2 (Batf2) has an antitumor effect. An s.c. inoculated tumor model produced fewer IL-12 p40+ macrophages and activated CD8+ T cells within the tumors of Batf2−/− mice compared with WT mice. In vitro studies also revealed that the IL-12 p40 expression was significantly lower in Batf2−/− macrophages following their stimulation by toll-like receptor ligands, such as R848. Additionally, we found that BATF2 interacts with p50/p65 and promotes IL-12 p40 expression. In conclusion, Batf2 has an antitumor effect through the up-regulation of IL-12 p40 in tumor-associated macrophages, which eventually induces CD8+ T-cell activation and accumulation within the tumor.

Schlafen-8 is essential for lymphatic endothelial cell activation in experimental autoimmune encephalomyelitis

Schlafen-8 (Slfn8) is a member of the Schlafen family of proteins, which harbor helicase domains and are induced by LPS and interferons. It has been reported that the Schlafen family are involved in various cellular functions, including proliferation, differentiation and regulation of virus replication. Slfn8 has been implicated in T-cell differentiation in the thymus. However, the roles of Slfn8 in the immune system remains unclear. In this study, we generated Slfn8 knockout mice (Slfn8-/-) and investigated the immunological role of Slfn8 using the T-cell-mediated autoimmune model experimental autoimmune encephalomyelitis (EAE). We found that the clinical score was reduced in Slfn8-/- mice. IL-6 and IL-17A cytokine production, which are associated with EAE onset and progression, were decreased in the lymph nodes of Slfn8-/- mice. Immune cell populations in Slfn8-/- mice, including macrophages, neutrophils, T cells and B cells, did not reveal significant differences compared with wild-type mice. In vitro activation of Slfn8-/- T cells in response to TCR stimulation also did not reveal significant differences. To confirm the involvement of non-hematopoietic cells, we isolated CD45- CD31+ endothelial cells and CD45-CD31- gp38+ fibroblastic reticular cells by FACS sorting. We showed that the levels of IL-6 and Slfn8 mRNA in CD45- CD31+ endothelial cells were increased after EAE induction. In contrast, the level of IL-6 mRNA after EAE induction was markedly decreased in CD31+ endothelial cells from Slfn8-/- mice. These results indicate that Slfn8 may play a role in EAE by regulating inflammation in endothelial cells.

Abstract 4506: Targeting stepwise HER2 and VEGF can overcome multidrug resistance in small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Small-cell lung cancer (SCLC) accounts for approximately 15% of primary lung cancer and has the poorest outcome of all its histological types. One of the major reasons of extreme aggressiveness of SCLC is that it recurs shortly after initial therapy with multidrug resistance (MDR) phenotype. However, standard therapeutic strategy for relapsed-SCLC has not been established yet. As one of the targetable receptor tyrosine kinases, human epidermal growth factor receptor 2 (HER2) was reported to be a negative prognostic factor in extensive-disease SCLC (Micke et al. Int J Cancer. 2001;92:474-9). We found that HER2 was more frequently overexpressed in SCLC cell lines of Japanese origin (6/10) compared to those of Caucasian origin (0/3). We also detected HER2 expression in SCLC tissues in 7 out of 25 patients tested by our highly sensitive immunohistochemistry system. Moreover, we found that HER2 was upregulated when HER2-positive SCLC cells acquired MDR. Trastuzumab, a humanized monoclonal antibody against HER2, exerted differential levels of killing effect on HER2-positive parental and chemoresistant SCLC cells only when Fcγ receptor-positive natural killer (NK) cells coexisted. This result suggests that trastuzumab-induced SCLC cell-killing effect was caused mainly via antibody-dependent cell-mediated cytotoxicity (ADCC) but not via direct inhibition of HER2 signal. Among these cell lines, etoposide-resistant SCLC cells were most susceptible to trastuzumab in vitro and in vivo, and the antitumor effects of trastuzumab were not only dependent on the amount of HER2 expression on SCLC cells. We focused on cell-cell contact between SCLC cells and NK cells to determine the molecule affecting trastuzumab-mediated ADCC other than HER2. We found that intercellular adhesion molecule (ICAM)-1 was abundantly expressed on etoposide-resistant SCLC cells, and trastuzumab-mediated ADCC was canceled in the presence of an ICAM-1 functional blocking antibody. These results indicate that ICAM-1 expression on SCLC cell surface is indispensable to augment trastuzumab-mediated ADCC. Thus, trastuzumab could overcome etoposide-resistance in SCLC. On the contrary, irinotecan-resistant SCLC cells were still refractory to trastuzumab. The reason for this was thought that they not only lacked ICAM-1 expression but also came to produce abundant vascular endothelial growth factor (VEGF). Bevacizumab, a humanized monoclonal antibody against VEGF, treatment could significantly inhibit the in vivo growth of irinotecan-resistant xenografts through decreasing microvesseles in mice. These results suggest that bevacizumab-mediated antiangiogenesis is a promising therapeutic strategy to salvage irinotecan-resistance. Collectively, targeting stepwise HER2 and VEGF could overcome MDR in SCLC and bring about a favorable outcome for patients with relapsed-SCLC. Citation Format: Toshiyuki Minami, Takashi Kijima, Osamu Morimura, Yuhei Kinehara, Masayoshi Higashiguchi, Kotaro Miyake, Haruhiko Hirata, Yoshiko Takeuchi, Kiyoharu Fukushima, Yoshitomo Hayama, Koji Inoue, Izumi Nagatomo, Yoshito Takeda, Hiroshi Kida, Atsushi Kumanogoh. Targeting stepwise HER2 and VEGF can overcome multidrug resistance in small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4506. doi:10.1158/1538-7445.AM2014-4506