Research Article31 August 2018Open Access Source DataTransparent process Targeting miR-223 in neutrophils enhances the clearance of Staphylococcus aureus in infected wounds Maiko de Kerckhove Maiko de Kerckhove Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Katsuya Tanaka Katsuya Tanaka Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Department of Plastic and Reconstructive Surgery, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Department of Plastic and Reconstructive Surgery, Ehime Prefectural Center Hospital, Ehime, Japan Search for more papers by this author Takahiro Umehara Takahiro Umehara Department of Forensic Pathology and Science, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Momoko Okamoto Momoko Okamoto Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Department of Immunology and Rheumatology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Sotaro Kanematsu Sotaro Kanematsu Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate of Frontier Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroko Hayashi Hiroko Hayashi Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Hiroki Yano Hiroki Yano Department of Plastic and Reconstructive Surgery, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Soushi Nishiura Soushi Nishiura Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Shiho Tooyama Shiho Tooyama Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Yutaka Matsubayashi Yutaka Matsubayashi Schools of Biochemistry and Physiology, Pharmacology & Neuroscience, Faculty of Biomedical Sciences, University of Bristol, Bristol, UK Randall Division of Cell and Molecular Biophysics, King's College London, London, UK Search for more papers by this author Toshimitsu Komatsu Toshimitsu Komatsu Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Seongjoon Park Seongjoon Park Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Yuka Okada Yuka Okada Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan Search for more papers by this author Rina Takahashi Rina Takahashi Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan Search for more papers by this author Yayoi Kawano Yayoi Kawano Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan Search for more papers by this author Takehisa Hanawa Takehisa Hanawa Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan Search for more papers by this author Keisuke Iwasaki Keisuke Iwasaki Department of Pathology, Sasebo City General Hospital, Sasebo, Nagasaki, Japan Search for more papers by this author Tadashige Nozaki Tadashige Nozaki Department of Pharmacology, Faculty of Dentistry, Osaka Dental University, Hirakata, Osaka, Japan Search for more papers by this author Hidetaka Torigoe Hidetaka Torigoe Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, Japan Search for more papers by this author Kazuya Ikematsu Kazuya Ikematsu Department of Forensic Pathology and Science, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Yutaka Suzuki Yutaka Suzuki Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate of Frontier Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Katsumi Tanaka Katsumi Tanaka Department of Plastic and Reconstructive Surgery, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Paul Martin Paul Martin orcid.org/0000-0002-2665-5086 Schools of Biochemistry and Physiology, Pharmacology & Neuroscience, Faculty of Biomedical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Isao Shimokawa Isao Shimokawa Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Ryoichi Mori Corresponding Author Ryoichi Mori [email protected] orcid.org/0000-0002-7596-9620 Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Maiko de Kerckhove Maiko de Kerckhove Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Katsuya Tanaka Katsuya Tanaka Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Department of Plastic and Reconstructive Surgery, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Department of Plastic and Reconstructive Surgery, Ehime Prefectural Center Hospital, Ehime, Japan Search for more papers by this author Takahiro Umehara Takahiro Umehara Department of Forensic Pathology and Science, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Momoko Okamoto Momoko Okamoto Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Department of Immunology and Rheumatology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Sotaro Kanematsu Sotaro Kanematsu Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate of Frontier Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroko Hayashi Hiroko Hayashi Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Hiroki Yano Hiroki Yano Department of Plastic and Reconstructive Surgery, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Soushi Nishiura Soushi Nishiura Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Shiho Tooyama Shiho Tooyama Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Yutaka Matsubayashi Yutaka Matsubayashi Schools of Biochemistry and Physiology, Pharmacology & Neuroscience, Faculty of Biomedical Sciences, University of Bristol, Bristol, UK Randall Division of Cell and Molecular Biophysics, King's College London, London, UK Search for more papers by this author Toshimitsu Komatsu Toshimitsu Komatsu Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Seongjoon Park Seongjoon Park Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Yuka Okada Yuka Okada Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan Search for more papers by this author Rina Takahashi Rina Takahashi Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan Search for more papers by this author Yayoi Kawano Yayoi Kawano Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan Search for more papers by this author Takehisa Hanawa Takehisa Hanawa Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan Search for more papers by this author Keisuke Iwasaki Keisuke Iwasaki Department of Pathology, Sasebo City General Hospital, Sasebo, Nagasaki, Japan Search for more papers by this author Tadashige Nozaki Tadashige Nozaki Department of Pharmacology, Faculty of Dentistry, Osaka Dental University, Hirakata, Osaka, Japan Search for more papers by this author Hidetaka Torigoe Hidetaka Torigoe Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, Japan Search for more papers by this author Kazuya Ikematsu Kazuya Ikematsu Department of Forensic Pathology and Science, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Yutaka Suzuki Yutaka Suzuki Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate of Frontier Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Katsumi Tanaka Katsumi Tanaka Department of Plastic and Reconstructive Surgery, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Paul Martin Paul Martin orcid.org/0000-0002-2665-5086 Schools of Biochemistry and Physiology, Pharmacology & Neuroscience, Faculty of Biomedical Sciences, University of Bristol, Bristol, UK Search for more papers by this author Isao Shimokawa Isao Shimokawa Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Ryoichi Mori Corresponding Author Ryoichi Mori [email protected] orcid.org/0000-0002-7596-9620 Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan Search for more papers by this author Author Information Maiko Kerckhove1, Katsuya Tanaka1,2,3, Takahiro Umehara4, Momoko Okamoto1,5, Sotaro Kanematsu6, Hiroko Hayashi1, Hiroki Yano2, Soushi Nishiura1, Shiho Tooyama1, Yutaka Matsubayashi7,8, Toshimitsu Komatsu1, Seongjoon Park1, Yuka Okada9, Rina Takahashi10, Yayoi Kawano10, Takehisa Hanawa10, Keisuke Iwasaki11, Tadashige Nozaki12, Hidetaka Torigoe13, Kazuya Ikematsu4, Yutaka Suzuki6, Katsumi Tanaka2, Paul Martin7, Isao Shimokawa1 and Ryoichi Mori *,1 1Department of Pathology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan 2Department of Plastic and Reconstructive Surgery, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan 3Department of Plastic and Reconstructive Surgery, Ehime Prefectural Center Hospital, Ehime, Japan 4Department of Forensic Pathology and Science, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan 5Department of Immunology and Rheumatology, Nagasaki University School of Medicine and Graduate School of Biomedical Sciences, Nagasaki, Japan 6Laboratory of Functional Genomics, Department of Medical Genome Science, Graduate of Frontier Science, The University of Tokyo, Tokyo, Japan 7Schools of Biochemistry and Physiology, Pharmacology & Neuroscience, Faculty of Biomedical Sciences, University of Bristol, Bristol, UK 8Randall Division of Cell and Molecular Biophysics, King's College London, London, UK 9Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan 10Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan 11Department of Pathology, Sasebo City General Hospital, Sasebo, Nagasaki, Japan 12Department of Pharmacology, Faculty of Dentistry, Osaka Dental University, Hirakata, Osaka, Japan 13Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, Japan *Corresponding author. Tel: +81 95 819 7051; Fax: +81 95 819 7052; E-mail: [email protected] EMBO Mol Med (2018)10:e9024https://doi.org/10.15252/emmm.201809024 See also: P Hiebert & S Werner (September 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Argonaute 2 bound mature microRNA (Ago2-miRNA) complexes are key regulators of the wound inflammatory response and function in the translational processing of target mRNAs. In this study, we identified four wound inflammation-related Ago2-miRNAs (miR-139-5p, miR-142-3p, miR-142-5p, and miR-223) and show that miR-223 is critical for infection control. miR-223Y/− mice exhibited delayed sterile healing with prolonged neutrophil activation and interleukin-6 expression, and markedly improved repair of Staphylococcus aureus-infected wounds. We also showed that the expression of miR-223 was regulated by CCAAT/enhancer binding protein alpha in human neutrophils after exposure to S. aureus peptides. Treatment with miR-223Y/−-derived neutrophils, or miR-223 antisense oligodeoxynucleotides in S. aureus-infected wild-type wounds markedly improved the healing of these otherwise chronic, slow healing wounds. This study reveals how miR-223 regulates the bactericidal capacity of neutrophils at wound sites and indicates that targeting miR-223 might be of therapeutic benefit for infected wounds in the clinic. Synopsis In this study, miR-223 is identified as a major player for infection control during wound inflammatory response. miR-223 regulates the bactericidal capacity of neutrophils at wound sites, and targeting miR-223 might be of therapeutic benefit for infected wounds in the clinic. miR-223, miR-142-3p, miR-142-5p, and miR-139-5p were expressed upon wound inflammation. miR-223 is critical for neutrophils activation and subsequent resolution of the acute inflammatory response in wound sites. Upon stimulation with S. aureus peptides, IL-6 secretion is regulated by the C/EBPα-miR-223 signaling pathway. Cell transplantation therapy using miR-223-deficient neutrophils or miR-223 antisense oligodeoxynucleotide knockdown in wounds improved the healing of S. aureus-infected WT wounds. Introduction After tissue damage to the adult skin, there always follows a robust recruitment of inflammatory cells, including innate immune cells, neutrophils, and macrophages, into the wounded tissues to kill and phagocytose invading microbes as well as secreting bioactive substances that aid tissue repair (Eming et al, 2017). Healing of the damaged tissue is accomplished by the concerted actions of re-epithelialization and wound angiogenesis and by migration and subsequent contraction of fibroblasts that lay down granulation tissue by deposition of collagen and other matrix components (Eming et al, 2014). These contributing cell behaviors are thought to be partly governed by signals from the inflammatory cell influx (Eming et al, 2007). Following collagen deposition, a scar develops at the healed wound site (Eming et al, 2014). Interestingly, skin wounds of embryos and late gestation fetuses (before embryonic day 15 in mice and end of second trimester in humans) can result in almost perfect repair without scarring and these early wounds are associated with a markedly reduced wound inflammatory response (Hopkinson-Woolley et al, 1994), suggesting that inflammation might cause skin fibrosis (Martin & Leibovich, 2005). Indeed, neonatal PU.1-deficient (PU.1−/−) mice, which possess no neutrophils, macrophages, mast cells, or T cells, exhibit rapid repair and scarless healing in contrast to wild-type (WT) siblings (Martin et al, 2003; Cooper et al, 2005). We previously showed that knockdown of osteopontin, a wound upregulated inflammation-dependent gene, by antisense oligodeoxynucleotides (AS ODNs) at wound sites reduced scarring and improved healing in vivo (Mori et al, 2008). A similar effect was observed for wounds by knockdown of the transcription factor Foxo1 (Mori et al, 2014). In general, these findings suggest that gene therapies based on dampening wound inflammatory responses (reducing inflammatory cell influx, blocking pro-fibrotic signals from inflammatory cells once they arrive at the wound site, or enhancing the resolution of inflammation; Cash et al, 2014) might provide novel molecular therapeutic targets. MicroRNAs (miRNAs) of the small, non-coding RNA family are approximately 20–25 nucleotides of single-stranded RNA that regulate translational processing of target mRNAs (Winter et al, 2009). miRNAs are degraded by selective loading into an RNA-induced silencing complex (RISC) with Argonaute (Ago) as its core. Ago family members (Ago1-4) are ubiquitously expressed, but Ago2 is the most highly expressed and exhibits miRNA silencer activity in mice (Liu et al, 2004). Many studies have now indicated that a number of diverse miRNAs are involved with, and regulate, inflammatory responses in humans and mice (O'Connell et al, 2012). Furthermore, the specific miRNAs miR-21 (Wang et al, 2012), miR-130a (Pastar et al, 2012), and miR-132 (Li et al, 2015) contribute to the healing of skin wounds. miRNA profiling using next-generation sequencing (NGS) has already proved successful in identifying novel miRNAs from several tissues and organisms (Tam et al, 2014; Ma et al, 2015). However, methods to purify Ago2-miRNA complexes from wound sites and miRNA library construction to perform NGS from these tissues have not previously been reported. miR-223Y/− mice have an expanded granulocytic compartment resulting from a cell-autonomous increase in the number of granulocyte progenitors (Johnnidis et al, 2008). Moreover, miR-223 is overexpressed in rheumatoid arthritis patients (Fulci et al, 2010). Together, these studies suggest that inflammation-related miRNAs, particularly miR-223, might be key regulators of the inflammatory response and/or its subsequent resolution during skin tissue repair; however, the pathways involved have not been comprehensively characterized. In this study, we developed a unique purification system to isolate functional miRNA-Ago2 complexes from wounded skin tissues by immunoprecipitation (IP) using an anti-Ago2 antibody (Ab), followed by the construction of libraries to perform NGS and identify candidate wound inflammation-related miRNAs. Using this approach, we identified several inflammation-dependent miRNAs including miR-139-5p, miR-142-3p, miR-142-5p, and miR-223. Of these, miR-223 was the most highly expressed in wound sites during the inflammatory phase and so the present study focused on the molecular mechanisms of miR-223 in skin wound healing. Our wound healing studies showed the delayed repair of aseptic wounds in miR-223Y/− mice was associated with enhanced neutrophil activation and interleukin-6 (Il6) expression. However, if wounds were infected with Staphylococcus aureus, then miR-223Y/− mice showed considerably enhanced repair compared with WT infected wounds, and either transplanting miR-223Y/− neutrophils or the delivery of miR-223 AS ODNs to infected WT wounds rescued the impaired wound healing phenotype. The expression level of miR-223 in human neutrophils was regulated by CCAAT/enhancer binding protein alpha (C/EBPα) after exposure to S. aureus peptides. Thus, miR-223 is a potential therapeutic target for the treatment of skin wounds infected with S. aureus. Results Over 300 Ago2-miRNAs are expressed during skin wound healing To comprehensively identify wound-induced mature miRNAs involved in skin wound healing, a 4-mm-diameter wound was made in WT mouse dorsal skin, and 6-mm-diameter unwounded and wound sites were harvested on day 1, 3, 7, 10, and 14 after injury as described previously (Mori et al, 2008, 2014). Because mature Ago2-miRNA complexes bind to target mRNAs and inhibit their translation, we isolated Ago2-miRNA complexes rather than total miRNAs. We purified Ago2-miRNA complexes from skin wound tissues by IP with Ago2 Ab, followed by library generation and NGS with the Illumina platform (Appendix Fig S1). Known murine miRNA sequencing reads accounted for 76.6–91.0% of all sequence reads in our small RNA libraries that were highly enriched for mature miRNA sequences (Appendix Table S1). We identified over 300 known murine miRNA categories expressed during skin wound healing (Dataset EV1), demonstrating the high efficiency of our NGS procedure for identifying functional miRNAs from skin wound tissues. miR-139-5p, miR-142 family members, and miR-223 are wound inflammation-related miRNAs As a partial filter to identify inflammation-related miRNAs in skin wound healing, miRNAs in the inflammatory phase (day 1 after injury) were arranged by rank of upregulation and compared with intact skin (Appendix Table S2). miRNA expression levels were confirmed by qPCR (Fig 1). miR-147, miR-223 (miR-223-3p), miR-129-3p, miR-129-5p, miR-139-5p, miR-21* (miR-21-3p), miR-340-5p, miR-142-3p, and miR-142-5p expressions at wound sites on day 1 after injury were significantly increased compared with intact skin. Figure 1. Identification and expression of nine candidate inflammation-related miRNAs in skin wound healingTemporal expression of murine miR-147, miR-223, miR-129-3p, miR-139-5p, miR-21*, miR-340-5p, miR-142-3p, miR-142-5p, and miR-486 in skin wound healing measured by qPCR relative to snoRNA202 or 5S rRNA (n = 4–6). All values represent the mean ± SD. Tukey's multiple comparison tests were used to generate the P-values indicated in the Figure. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint In WT neonatal mice, miR-223 and miR-142-3p expression levels were markedly increased, peaking at 12 h after injury compared with intact skin, and were subsequently significantly decreased by 24 h (Fig 2A) indicating a temporal association with the inflammatory phase of healing. To confirm that these were inflammation-related miRNAs expressed during skin wound healing, we made a 1-cm incisional wound in the dorsal skin of WT neonatal mice and compared miR expression with heterozygous PU.1-deficient (PU.1+/−) and PU.1−/− mice. qPCR analysis showed miR-223, miR-142-3p, miR-142-5p, and miR-139-5p expression levels at wound sites in PU.1−/− mice were significantly decreased compared with WT mice (Fig 2B), indicating that these four miRs are inflammation-related miRNAs expressed during skin wound healing. Figure 2. miR-139-5p, miR-142-3p, miR-142-5p, and miR-223 are independently expressed by wound inflammation A. Temporal expression of miR-223, miR-142-3p, and miR-340-5p in skin wound healing of neonate mice using qPCR (n = 6). B. qPCR analysis indicates the expression levels of inflammation-related miRNAs at 3 h in wound sites of WT (n = 5), PU.1+/− (n = 6), and PU.1−/− (n = 6) neonatal mice. Data information: Results shown are the mean ± SD. Tukey's multiple comparison tests were used to generate the P-values indicated in the Figure. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint Skin wound healing is delayed in miR-223Y/− mice Of all inflammation-related miRNAs, miR-223 was the most highly expressed during the inflammatory phase at wound sites and so the present study focused on the molecular mechanisms of miR-223 in skin wound healing (Appendix Fig S2). To determine which cells expressed miR-223 during skin wound healing, we performed immunohistochemistry (IHC) and in situ hybridization (ISH). Wound-infiltrated neutrophils at day 1 in the wound site of WT mice predominantly expressed miR-223 (Fig 3A and B), and this was similar in human skin-inflamed sites (Fig 3C). We used qPCR to confirm that murine wound-infiltrating neutrophils (1 day after wounding) and macrophages (3 days after wounding) isolated by immunoaffinity selection with anti-Ly-6G (neutrophil marker) and anti-CD11b (macrophage marker) Abs (Tanaka et al, 2017) expressed miR-223 at the wound sites (Fig 3D). Thus, miR-223 is predominantly expressed in neutrophils and macrophages at skin wound sites. Figure 3. miR-223Y/− mice show delayed aseptic skin wound healing A. IHC of neutrophils (Ly-6G and Ly-6C) at day 1 wound sites. Arrowhead indicates the wound margin. Area indicated with a rectangle is shown at a higher magnification below. Scale bars: 50 μm (bottom) and 500 μm (top). B. ISH of Mus musculus (mmu) miR-223 showing that wound-infiltrated neutrophils were predominantly present in the wound sites of adult WT mice at day 1 after injury. Arrowhead indicates the wound margin. Area indicated with a rectangle is shown at a higher magnification below. Scale bars: 50 μm (bottom) and 500 μm (top). C. Neutrophils express Homo sapiens (hsa) miR-223 in human skin-inflamed sites. Representative H&E staining (top) and ISH of miR-223 (bottom) showing that miR-223-expressing neutrophils were predominantly present in patient skin-inflamed sites (n = 15, Appendix Table S3). Scale bars: 50 μm. D. qPCR analysis shows miR-223 was expressed by wound-infiltrating neutrophils (day 1) and macrophages (day 3) (n = 3). E. Representative photo images of the gross appearance of excisional wounds in WT (left) and miR-223Y/− (right) mice. F. Proportion of the wound area remaining open relative to initial wound area at each time point in WT (n = 14) and miR-223Y/− (n = 13) mice. G. H&E staining of re-epithelialization at day 3 after injury (wound margin (arrowheads) and the leading edge of epithelia (arrows)). Scale bar: 500 μm. H. Measurement of epithelial tongue at day 3 after injury in WT (n = 8) and miR-223Y/− (n = 5) mice. Data information: All values represent the mean ± SD. Two-way ANOVA followed by Sidak multiple comparisons test (F) and unpaired t-test (H) was used to generate P-values indicated in the Figure. *P < 0.05, ***P < 0.001. Download figure Download PowerPoint To clarify the pathophysiological role of miR-223 in skin wound healing, we made dorsal skin wounds in miR-223Y/− mice. Gross examination showed that wound closure was significantly delayed in miR-223Y/− mice compared with WT mice (Fig 3E and F). We investigated re-epithelialization using histological analysis and observed that epithelial wound tongues in miR-223Y/− mice at day 3 after injury were significantly shorter (188.3 ± 24.11 μm, P = 0.0115) than in WT mice (380.1 ± 47.08 μm; Fig 3G and H) confirming that miR-223Y/− mice showed delayed wound re-epithelialization. We investigated the area of granulation tissue at day 7 and day 14 at wound sites in miR-223Y/− mice and found them to be significantly increased (day 7; 0.60 ± 0.16 mm2, day 14; 0.18 ± 0.064 mm2) compared with WT mice (day 7; 0.36 ± 0.16 mm2, day 14; 0.11 ± 0.036 mm2; Fig EV1A–C). We examined the localization and expression level of α-smooth muscle actin (αSMA), a marker of contractile myofibroblasts (Gabbiani et al, 1971), using IHC. The expression of αSMA at day 7 in granulation tissues of miR-223Y/− mice was markedly decreased (58%) compared with WT mice, even though the localization of αSMA-expressing cells at day 7 in granulation tissues of miR-223Y/− mice was not altered when compared with WT mice (Fig EV1D and E). Collectively, miR-223Y/− mice showed delayed skin wound healing and an increased scar area. Click here to expand this figure. Figure EV1. Attenuation of wound contraction and increased scar area in miR-223Y/− mice A. Representative images using H&E staining of wound sites at day 7 in WT (n = 8) and miR-223Y/− (n = 6) mice. Scale bars: 500 μm. B. Representative images of Masson's Trichrome staining of collagen fibers (blue) in skin wound tissues of WT (n = 7) and miR-223Y/− (n = 8) mice at day 14 after injury. Scar sites were visualized at the mid-point of the wound (indicated by dotted line). Nuclei are stained black, and cytosol is stained red. Scar formation is clearly recognizable by day 14 after injury. Scale bars: 500 μm. C. Measurement of granulation tissue area at wound sites from WT (day 7; n = 8, day 14; n = 7) and miR-223Y/− (day 7; n = 6, day 14; n = 8) mice. D. Representative images of αSMA-positive wound-infiltrated myofibroblasts at day 7 in wound sites from WT and miR-223Y/− mice (n = 7). Scale bars: 50 μm. E. Quantification of expression of αSMA at day 7 in wound sites from WT and miR-223Y/− mice (n = 7). Data information: All values represent the mean ± SD. Unpaired t-tests were used to generate P-values indicated in the Figure. *P < 0.05, **P < 0.01. Download figure Download PowerPoint MiR-223 regulates neutrophils in the acute inflammatory responses at wound sites Because we found miR-223 was expressed in neutrophils at wound sites and miR-223Y/− mice exhibited significantly delayed skin repair, we investigated whether miR-223 regulates neutrophil functions in acute inflammatory responses, in ways which might impact on the recruitment of macrophages lea
Abstract Background Previous studies have shown conflicting evidence regarding the incidence of cancer in patients with systemic lupus erythematosus (SLE) compared with that in healthy individuals. Calcineurin inhibitors (CNIs) such as cyclosporine and tacrolimus have been widely used to treat SLE; however, their effects on cancer risk remain unclear. We aimed to investigate the incidence of cancer in patients with SLE and determine the potential association between CNI use and cancer risk. Methods The standardized incidence ratio (SIR) of cancer among patients with lupus in the Lupus Registry of Nationwide Institutions (LUNA) was calculated based on the age-standardized incidence rate of cancer reported by Japan’s Ministry of Health, Labour and Welfare. We also examined the association between CNI exposure and cancer risk, while considering potential confounding factors. The analysis accounted for confounding variables such as age, sex, smoking history, maximum glucocorticoid dose, treatment history with cyclophosphamide, ongoing hydroxychloroquine, Systemic Lupus International Collaboration Clinics/American College of Rheumatology Damage Index (SDI) value (excluding cancer occurrence), comorbidity of diabetes mellitus, and smoking history. Results The study included 704 patients with SLE (625 females; 88.8%) with a median age of 44 years [interquartile range (IQR) = 34–55] years. The median past maximum glucocorticoid dose was 40 mg/day [IQR = 30–60 mg/day], and the SDI at registration was 1 [IQR = 0–2]. Among the patients, 246 (35.1%) had smoking histories, and 38 (5.4%) experienced cancer complications. Gynecological malignancies accounted for 63.2% of all cancers. The SIR of cancer in the LUNA cohort was 1.08 (95% confidence interval [CI] = 0.74–1.43). No statistically significant risks of cancer were found in relation to CNI treatment history; the odds ratio using multiple logistic regression was 1.12 (95% CI = 0.42–3.00), the risk ratio using standardization was 1.18 (95% CI = 0.47–2.16), and the risk ratio using inverse probability weighting was 1.8 (95% CI = 0.41–4.66). Conclusions The incidence of cancer in patients with SLE in the LUNA cohort did not significantly differ from that in the general population. These findings suggest that CNI treatment in this cohort did not pose a risk factor for cancer development.
To compare therapeutic efficacy of tumour necrosis factor inhibitor (TNFi) cyclers and non-TNFi switchers in patients with rheumatoid arthritis (RA) having inadequate response to previous TNFis (TNF-IR patients) using composite measures including imaging assessment with power Doppler ultrasonography (PDUS). Patients with RA who had inadequate response to one or more previous TNFi agents with moderate or higher disease activity were enrolled. The outcomes of 56 TNF-IR patients were analysed. Patients were divided into 19 TNFi cyclers and 37 non-TNFi switchers (16 abatacept [ABT] and 21 tocilizumab [TCZ] switchers). Retention ratio at 6 months was significantly higher in non-TNFi switchers than in TNFi cyclers (p < .05). Although there was no significant difference, non-TNFi switchers tended to have a larger decrease than TNFi cyclers in efficacy indicators based on clinical disease activity index and PDUS. Multivariate logistic regression analysis identified a following independent factor associated with both EULAR good response and retention of a biologic agent: non-TNFi switch (p < .05 for both). Non-TNFi switchers were shown to have significantly higher percentage of EULAR good response and higher retention than TNFi cyclers. A non-TNFi biologic agent may hence be a preferential next-line treatment for TNF-IR patients.
Abstract Rationale: Mucormycosis is a rare opportunistic fungal infection with poor prognosis. The incidence of mucormycosis has been increasing, and it is a threat to immunocompromised hosts. We present a case of gastric mucormycosis complicated by a gastropleural fistula during immunosuppressive treatment for adult-onset Still disease (AOSD). Patient concerns: An 82-year-old woman diagnosed with AOSD who developed gastric ulcers during the administration of an immunosuppressive therapy with corticosteroids, cyclosporine, and tocilizumab complained of melena and epigastralgia. Esophagogastroduodenoscopy showed multiple ulcers covered with grayish or greenish exudates. Diagnoses: The patient diagnosed with mucormycosis based on culture and biopsy of the ulcers, which showed nonseptate hyphae branching at wide angles. Mucor indicus was identified using polymerase chain reaction. Interventions and outcomes: Although liposomal amphotericin B was administered, gastric mucormycosis was found to be complicated by a gastropleural fistula. The patient died because of pneumonia due to cytomegalovirus infection, and autopsy revealed the presence of Mucorales around the fistula connecting the stomach and diaphragm. Lessons: Gastric mucormycosis is refractory to treatment and fatal. Surgical resection, if possible, along with antifungal drugs can result in better outcomes.
To compare the efficacy and safety of tofacitinib and baricitinib in patients with RA in a real-world setting. A total of 242 patients with RA who were treated with tofacitinib (n = 161) or baricitinib (n = 81) were enrolled. We evaluated efficacy and safety between tofacitinib and baricitinib using multivariable analyses to avoid confounding. Their clinical disease activity and AEs were evaluated for 24 weeks. The mean (SD) DAS28-ESR change from baseline to 24 weeks was 1.57 (1.55) (tofacitinib) and 1.46 (1.36) (baricitinib). There was no significant difference in the clinical response between the two groups (adjusted mean difference, 0.04; 95% CI, -0.35 to 0.28). The efficacy was not significantly changed in the patients without concomitant MTX use in both groups, but the concomitant MTX use showed better clinical efficacy in the cases of baricitinib treatment. In both groups, the most common AE was herpes zoster infection, and the AE rates were similar between the two groups. However, the predictive factors contributing to clinical response as revealed by a multivariable logistic analysis differed. The concomitant oral steroid use was independently associated with the achievement of DAS-low disease activity in the tofacitinib group, whereas in the baricitinib group, the number of biological and/or targeted synthetic DMARDs previously used was associated. Our findings indicate that tofacitinib and baricitinib had comparable continuing efficacies and safety profiles. However, there is a possibility that the influence of clinical characteristics on the treatment response differs. The comparison provides useful information to the optimal use of JAK inhibitors in real-world settings.
Abstract Rationale: Severe fever with thrombocytopenia syndrome (SFTS) is a recently recognized fatal infectious disease caused by the SFTS virus, and severe cases are complicated by the presence of hemophagocytic lymphohistiocytosis (HLH) associated with a cytokine storm. Herein, we report on serial changes of serum cytokine levels and viral load in a severe case of SFTS. Patient concerns: A 63-year-old Japanese woman presented with high-grade fever, abdominal pain, diarrhea, impaired consciousness, leukocytopenia, and thrombocytopenia. Diagnosis: SFTS was diagnosed based on a positive serum test for SFTS virus RNA and electroencephalogram (EEG) findings of encephalopathy. Interventions: The patient was treated with supportive therapy, including steroid pulse therapy (intravenous methylprednisolone 1 g/d for 3 days) for HLH and intravenous recombinant thrombomodulin 19200 U/d for 7 days for disseminated intravascular coagulation. Outcomes: Treatment for 7 days improved both symptoms and abnormal EEG findings, and SFTS virus RNA disappeared from the serum at day 10 from the onset of symptoms. The serum cytokines and chemokines analysis during the clinical course revealed 2 distinct phases: the acute phase and the recovery phase. The cytokines and chemokines elevated in the acute phase included interleukin (IL)-6, IL-10, interferon (IFN)-α2, IFN-γ, tumor necrosis factor-α, interferon-γ-induced protein-10, and fractalkine, while the IL-1β, IL-12p40, IL-17, and vascular endothelial growth factor levels were higher in the recovery phase. Conclusion: These observations suggest that the cytokines and chemokines elevated in the acute phase may reflect the disease severity resulted in a cytokine storm, while those in the recovery phase may be attributed to T-cell activation and differentiation.
A 53-year-old man with recurrent episodes of large joint pain and a low-grade fever at irregular intervals for 16 years developed right knee and ankle arthralgia, watery diarrhea, and abdominal pain. Following an ileum and colon biopsy, he was diagnosed with gastrointestinal amyloidosis. We suspected familial Mediterranean fever (FMF) based on his history and administered colchicine; his symptoms subsequently improved. Thus, he was diagnosed with atypical FMF. After tocilizumab administration, the amyloid deposits disappeared. This case suggests that physicians should consider FMF even in cases with atypical symptoms in order to prevent the progression of amyloidosis and that amyloid deposits can be eliminated by interleukin (IL)-6 inhibition.
