Published papers

RATIONALE: Rheumatoid arthritis (RA) causes inflammation in various organs including the lungs. Pulmonary manifestations include inflammation of the pleura, vasculature, airway, and parenchyma, including interstitial lung disease (ILD). RA-organizing pneumonia (OP) is the third most common cause of RA-ILD. Cases of OP coexisting/complicated with lung cancer have been reported. Therefore, lung cancer can represent a diagnostic challenge, especially in patients with underlying pulmonary diseases including OP. PATIENT CONCERNS: An 81-year-old woman with a 12-year history of RA-OP underwent multiple transbronchial lung biopsies (TBLBs), all of which resulted in no malignant findings. She was treated with prednisolone (PSL) depending on the deteriorated infiltrations. At admission, chest computed tomography (CT) images showed exacerbation of left S8 consolidation on chest CT. Additionally, her RA activity was exacerbated, and PSL dose was increased to 30 mg/day, which resulted in improved dyspnea and consolidation. Accordingly, PSL dose was gradually decreased. However, 6 months later, when PSL dose was 11 mg/d, due to a worsening of consolidation and the joint symptoms of RA, PSL dose was increased to 20 mg/d and tacrolimus 2 mg/d was administered. 3 months after the increase in PSL dose, dyspnea improved and PSL dose was reduced to 15 mg/d; however, she was admitted to our hospital because of low back pain. DIAGNOSIS: Spinal magnetic resonance imaging showed bone metastases in the third and fifth lumbar vertebrae, and lung cancer was suspected as the primary tumor on CT. INTERVENTIONS: TBLB was performed on the left B8 infiltrate, which showed no evidence of malignancy in the previous TBLB. OUTCOMES: Pathological examination of TBLB on the left B8 revealed an adenocarcinoma that was positive for anaplastic lymphoma kinase. LESSONS: Physicians should be aware of the development of lung cancer in regions with OP, even after a partial response to corticosteroid therapy.

Elevated levels of cytokines IL-1β and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1β released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1β release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1β release. After release, IL-1β stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1β secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1β and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.

An association between coronavirus disease 2019 (COVID-19) and the ABO blood group has been reported. However, such an association has not been studied in the Japanese population on a large scale. Little is known about the association between COVID-19 and ABO genotype. This study investigated the association between COVID-19 and ABO blood group/genotype in a large Japanese population. All Japanese patients diagnosed with COVID-19 were recruited through the Japan COVID-19 Task Force between February 2020 and October 2021. We conducted a retrospective cohort study involving 1790 Japanese COVID-19 patients whose DNA was used for a genome-wide association study. We compared the ABO blood group/genotype in a healthy population (n = 611, control) and COVID-19 patients and then analyzed their associations and clinical outcomes. Blood group A was significantly more prevalent (41.6% vs. 36.8%; P = 0.038), and group O was significantly less prevalent (26.2% vs. 30.8%; P = 0.028) in the COVID-19 group than in the control group. Moreover, genotype OO was significantly less common in the COVID-19 group. Furthermore, blood group AB was identified as an independent risk factor for most severe diseases compared with blood group O [aOR (95% CI) = 1.84 (1.00-3.37)]. In ABO genotype analysis, only genotype AB was an independent risk factor for most severe diseases compared with genotype OO. Blood group O is protective, whereas group A is associated with the risk of infection. Moreover, blood group AB is associated with the risk of the "most" severe disease.

BACKGROUND: Multiple prolonged symptoms are observed in patients who recover from an acute COVID-19 infection, which is defined as long COVID. General fatigue is frequently observed in patients with long COVID during acute and post-acute phases. This study aimed to identify the specific risk factors for general fatigue in long COVID. METHODS: Hospitalized patients with COVID-19 aged over 18 years were enrolled in a multicenter cohort study at 26 medical institutions. Clinical data during hospitalization and patient-reported outcomes after discharge were collected from medical records, paper-based questionnaires, and smartphone apps. RESULTS: Among prolonged symptoms through 1-year follow-ups, general fatigue was the most interfering symptom in daily life. Patients with protracted fatigue at all follow-up periods had lower quality of life scores at the 12-month follow-up. Univariate logistic regression analysis of the presence or absence of general fatigue at the 3-month, 6-month, and 12-month follow-ups identified asthma, younger age, and female sex as risk factors for prolonged fatigue. Multivariable logistic regression analysis revealed that asthma was an independent risk factor for persistent fatigue during the 12-month follow-up period. Longitudinal changes in the symptoms of patients with or without asthma demonstrated that general fatigue, not cough and dyspnea, was significantly prolonged in patients with asthma. CONCLUSIONS: In a Japanese population with long COVID, prolonged general fatigue was closely linked to asthma. A preventive approach against COVID-19 is necessary to avoid sustained fatigue and minimize social and economic losses in patients with asthma.

With the rising numbers of patients infected with severe acute respiratory syndrome coronavirus 2, long coronavirus disease 2019 (COVID-19)-a sequelae of COVID-19-has become a major problem. Different sexes and age groups develop different long COVID symptoms, and the risk factors for long COVID remain unclear. Therefore, we performed subgroup analyses of patients with COVID-19, classifying them into different groups. In this multicenter cohort study, using an original questionnaire, we examined patients (≥18 years old) diagnosed with COVID-19 from November 2020 to March 2022 and hospitalized at participating medical facilities. In total, 1066 patients were registered (361 female, 620 male). Hypertension was the most common comorbidity (n = 344; 32.5%). Females with hypertension were significantly less likely to develop long COVID symptoms than those without hypertension (odds ratio [OR] 0.51, 95% confidence interval [CI] 0.27-0.98; p = 0.043). In females, Ca channel blocker administration, rather than having hypertension, was significantly associated with reductions in the frequency of alopecia (OR 0.14, 95% CI 0.03-0.67, p = 0.015), memory impairment (OR 0.14, 95% CI 0.02-0.82, p = 0.029), sleeping disorders (OR 0.17, 95% CI 0.04-0.67, p = 0.012), tinnitus (OR 0.23, 95% CI 0.05-0.98, p = 0.047), sputum (OR 0.31, 95% CI 0.10-0.92, p = 0.035), and fever (OR 0.33, 95% CI 0.12-0.93, p = 0.036). Several long COVID symptoms, including alopecia, were significantly negatively associated with Ca channel-blocker administration in female patients with long COVID. Calcium channel blockers may reduce the development of long COVID in females.

OBJECTIVES: Coronavirus disease (COVID-19) was severe in the Delta variant-dominated epidemic wave (fifth wave) in Japan. The clinical characteristics and effectiveness of COVID-19 vaccination are not fully understood in the Omicron variant-dominated wave (sixth and seventh waves), especially in hospitalized patients. We investigated the relationship between vaccination and disease severity in the Omicron-dominated wave and compared these variant-dominated waves. METHODS: The nationwide COVID-19 database (Japan COVID-19 Task Force) was used to compare clinical characteristics and critical outcome in patients hospitalized from Delta (5th, N=735) vs Omicron-dominated waves (6th, N=495; 7th, N=128). RESULTS: Patients in the sixth and seventh waves had a lower incidence of critical outcomes and respiratory outcomes, and a higher incidence of bacterial infection, although the mortality rate did not differ significantly between waves. In the sixth and seventh waves, 138 (27.9%) and 29 (22.7%) COVID-19 patients were unvaccinated, respectively. Multivariable analysis adjusted with previously reported factors revealed that the proportion of 1) critical outcomes and 2) respiratory outcomes decreased in a frequency-dependent manner; 1) (the number of vaccinations): 1-2 times, aOR 0.37 (95% CI; 0.20-0.69); 3-4 times: aOR 0.25 (95% CI; 0.11-0.58), 2) 1-2 times, aOR 0.43 (95% CI; 0.27-0.66); 3-4 times: aOR 0.36 (95% CI; 0.21-0.60). CONCLUSIONS: COVID-19 hospitalized patients with Omicron infections showed a lower incidence of critical outcomes than those with Delta infections, and COVID-19 vaccination may contribute to preventing respiratory failure.

The severity of chest X-ray (CXR) findings is a prognostic factor in patients with coronavirus disease 2019 (COVID-19). We investigated the clinical and genetic characteristics and prognosis of patients with worsening CXR findings during early hospitalization. We retrospectively included 1656 consecutive Japanese patients with COVID-19 recruited through the Japan COVID-19 Task Force. Rapid deterioration of CXR findings was defined as increased pulmonary infiltrates in ≥ 50% of the lung fields within 48 h of admission. Rapid deterioration of CXR findings was an independent risk factor for death, most severe illness, tracheal intubation, and intensive care unit admission. The presence of consolidation on CXR, comorbid cardiovascular and chronic obstructive pulmonary diseases, high body temperature, and increased serum aspartate aminotransferase, potassium, and C-reactive protein levels were independent risk factors for rapid deterioration of CXR findings. Risk variant at the ABO locus (rs529565-C) was associated with rapid deterioration of CXR findings in all patients. This study revealed the clinical features, genetic features, and risk factors associated with rapid deterioration of CXR findings, a poor prognostic factor in patients with COVID-19.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O 2 , airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na + and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of β and γENaC (epithelial Na + channel) subunit expression. The combination of increased Na + absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA–in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

BACKGROUND: Patients often experience multiple prolonged symptoms following acute coronavirus disease 2019 (COVID-19) recovery, defined as long coronavirus disease (COVID). Patients with long COVID may experience dyspnea during acute and post-acute phases. Therefore, this study aimed to identify specific risk factors for dyspnea in patients with long COVID. METHODS: Hospitalized patients with COVID-19, aged ≥18 years, were enrolled in this multicenter cohort study conducted at 26 medical institutions across Japan. Clinical data during hospitalization and patient-reported outcomes after discharge at the 3, 6, and 12-month follow-ups were retrieved from medical records and paper-based or smartphone application-based questionnaires, respectively. RESULTS: Generalized linear mixed model (GLMM) analysis of prolonged dyspnea at each time point during follow-up showed that this symptom was associated with chronic obstructive pulmonary disease (COPD) (odds ratio [OR], 2.74; 95% confidence interval [CI], 1.31-5.74), asthma (OR, 2.21; 95%CI, 1.17-4.16), and ventilator management (OR, 3.10; 95%CI, 1.65-5.83). In addition, patients with COPD (44.4%) and ventilator management (25.0%) were more frequently associated with delayed dyspnea onset. The generalized estimating equations analysis results with multiple imputed datasets, conducted as a sensitivity analysis, confirmed the adjusted GLMM analysis results. CONCLUSIONS: Prolonged dyspnea was associated with COPD, asthma, and severe infection that required mechanical ventilation in the Japanese population with long COVID. Further investigation is needed to clarify its mechanism and develop prophylactic and therapeutic strategies for dyspnea in patients with long COVID.

RATIONALE: The clinical features and prognosis of nontuberculous mycobacterial (NTM) pleuritis and pleural effusion combined with NTM lung disease (NTM-LD) remain unclear. OBJECTIVES: To investigate the clinical features and prognosis of NTM pleuritis. METHODS: This retrospective observational study included patients with NTM pleuritis from January 2001 to June 2018 across eight hospitals in Japan. NTM pleuritis was defined by a positive culture of NTM from pleural effusion. We matched patients with Mycobacterium complex lung disease (MAC-LD) without pleuritis by sex and age to obtain comparative data and investigated the association between clinical parameters and prognosis. RESULTS: We identified 64 patients with NTM pleuritis (median age, 73 years; 37 female). The median follow-up duration was 11 months, and 27 patients died. Patients with MAC pleuritis had a significantly worse survival compared to matched patients with MAC-LD without pleuritis. Multivariate analysis revealed that pleuritis (adjusted hazard ratio [aHR], 6.99; 95% confidence interval [CI], 2.58-19.00) and underlying pulmonary diseases (aHR, 3.01; 95% CI, 1.44-6.28) were independently associated with all-cause mortality in MAC-LD patients. CONCLUSIONS: The prognosis of MAC pleuritis is poorer than that of MAC-LD without pleuritis. Pleuritis is an independent prognostic factor in patients with MAC-LD.

Gastrointestinal (GI) long-COVID symptoms, including diarrhea and abdominal pain, have been reported in patients with long-COVID. However, the clinical features of patients with GI long-COVID symptoms remain unclear. We conducted a large-scale prospective cohort study focusing on the clinical characteristics of patients with GI long-COVID symptoms in Japan. Among 943 COVID-19 patients, 58 patients (6.2%) had GI long-COVID symptoms. The health-related quality of life (QOL) parameters (the Short Form-8 [SF-8] and Euro Quality of Life 5 Dimensions 5 level [EQ-5D-5L]) at 12 months after diagnosis in patients with GI long-COVID symptoms were significantly lower than in those without GI long-COVID symptoms (P < 0.0001). Moreover, patients with GI long-COVID symptoms had more varied long-COVID symptoms compared to patients without GI long-COVID symptoms.

AIM: Diabetes mellitus (DM) is a known risk factor for severe coronavirus disease 2019 (COVID-19), but the clinical impact of undiagnosed diabetes and prediabetes in COVID-19 are unclear particularly in Japan. We clarify the difference in clinical characteristics, including age, sex, body mass index and co-morbidities, laboratory findings and critical outcomes, in a large Japanese COVID-19 cohort without diabetes, with prediabetes, undiagnosed diabetes and diagnosed diabetes, and to identify associated risk factors. MATERIALS AND METHODS: This multicentre, retrospective cohort study used the Japan COVID-19 Task Force database, which included data on 2430 hospitalized COVID-19 patients from over 70 hospitals from February 2020 to October 2021. The prevalence of prediabetes, undiagnosed diabetes and diagnosed diabetes were estimated based on HbA1c levels or a clinical diabetes history. Critical outcomes were defined as the use of high-flow oxygen, invasive positive-pressure ventilation or extracorporeal membrane oxygenation, or death during hospitalization. RESULTS: Prediabetes, undiagnosed diabetes and diagnosed diabetes were observed in 40.9%, 10.0% and 23.0%, respectively. Similar to diagnosed diabetes, prediabetes and undiagnosed diabetes were risk factors for critical COVID-19 outcomes (adjusted odds ratio [aOR] [95% CI]: 2.13 [1.31-3.48] and 4.00 [2.19-7.28], respectively). HbA1c was associated with COVID-19 severity in prediabetes patients (aOR [95% CI]: 11.2 [3.49-36.3]), but not other groups. CONCLUSIONS: We documented the clinical characteristics and outcomes of Japanese COVID-19 patients according to HbA1c levels or diabetes co-morbidity. As well as undiagnosed and diagnosed diabetes, physicians should be aware of prediabetes related to COVID-19 severity.

BACKGROUND: Cat scratch disease (CSD) is an infectious disease caused by Bartonella henselae. CSD follows a typical course, characterized by regional lymphadenopathy. In atypical CSD, the lesions spread to systemic organs and can cause fever of unknown origin (FUO). A previous study showed the usefulness of whole-body magnetic resonance imaging (WB-MRI) without diffusion-weighted imaging (DWI) in the diagnosis of FUO, but there are no studies on the clinical utility of whole-body DWI (WB-DWI). We herein report the case of an immunocompetent young man in whom contrast-enhanced CT-unidentifiable multiple liver abscess and osteomyelitis were successfully detected by WB-DWI. Follow up with a liver biopsy helped confirm an atypical CSD diagnosis. CASE PRESENTATION: A 23-year-old previously healthy man was admitted for a 19-day history of high fever despite 3-day treatment by azithromycin. His physical examination was unremarkable and contrast-enhanced CT showed only a low attenuated area in the right lobe of the liver, indicating a cyst. WB-DWI revealed multiple nodular lesions of hypo-diffusion in the liver, spine, and pelvic region. The biopsy specimens of the liver abscess showed no evidence of tuberculosis/malignancy and the polymerase chain reaction (PCR) test of liver abscess aspirate showed positive findings for B. henselae, confirming the diagnosis of CSD. He completed minocycline monotherapy for a total of 60 days without any deterioration. CONCLUSIONS: WB-DWI can be useful for the diagnosis of atypical CSD with hepatic and bone involvement, which can cause FUO in young immunocompetent adults.

BACKGROUND: Computed tomography (CT) imaging and artificial intelligence (AI)-based analyses have aided in the diagnosis and prediction of the severity of COVID-19. However, the potential of AI-based CT quantification of pneumonia in assessing patients with COVID-19 has not yet been fully explored. This study aimed to investigate the potential of AI-based CT quantification of COVID-19 pneumonia to predict the critical outcomes and clinical characteristics of patients with residual lung lesions. METHODS: This retrospective cohort study included 1,200 hospitalized patients with COVID-19 from four hospitals. The incidence of critical outcomes (requiring the support of high-flow oxygen or invasive mechanical ventilation or death) and complications during hospitalization (bacterial infection, renal failure, heart failure, thromboembolism, and liver dysfunction) was compared between the groups of pneumonia with high/low-percentage lung lesions, based on AI-based CT quantification. Additionally, 198 patients underwent CT scans 3 months after admission to analyze prognostic factors for residual lung lesions. RESULTS: The pneumonia group with a high percentage of lung lesions (N = 400) had a higher incidence of critical outcomes and complications during hospitalization than the low percentage group (N = 800). Multivariable analysis demonstrated that AI-based CT quantification of pneumonia was independently associated with critical outcomes (adjusted odds ratio [aOR] 10.5, 95% confidence interval [CI] 5.59-19.7), as well as with oxygen requirement (aOR 6.35, 95% CI 4.60-8.76), IMV requirement (aOR 7.73, 95% CI 2.52-23.7), and mortality rate (aOR 6.46, 95% CI 1.87-22.3). Among patients with follow-up CT scans (N = 198), the multivariable analysis revealed that the pneumonia group with a high percentage of lung lesions on admission (aOR 4.74, 95% CI 2.36-9.52), older age (aOR 2.53, 95% CI 1.16-5.51), female sex (aOR 2.41, 95% CI 1.13-5.11), and medical history of hypertension (aOR 2.22, 95% CI 1.09-4.50) independently predicted persistent residual lung lesions. CONCLUSIONS: AI-based CT quantification of pneumonia provides valuable information beyond qualitative evaluation by physicians, enabling the prediction of critical outcomes and residual lung lesions in patients with COVID-19.

BACKGROUND: Multiple prolonged symptoms observed in patients who recovered from COVID-19 are defined as long COVID. Although diverse phenotypic combinations are possible, they remain unclear. This study aimed to perform a cluster analysis of long COVID in Japan and clarify the association between its characteristics and background factors and quality of life (QOL). METHODS: This multicentre prospective cohort study collected various symptoms and QOL after COVID-19 from January 2020 to February 2021. This study included 935 patients aged ≥18 years with COVID-19 at 26 participating medical facilities. Hierarchical cluster analysis was performed using 24 long COVID symptom at 3 months after diagnosis. RESULTS: Participants were divided into the following five clusters: numerous symptoms across multiple organs (cluster 1, n=54); no or minor symptoms (cluster 2, n=546); taste and olfactory disorders (cluster 3, n=76); fatigue, psychoneurotic symptoms and dyspnoea (low prevalence of cough and sputum) (cluster 4, n=207) and fatigue and dyspnoea (high prevalence of cough and sputum) (cluster 5, n=52). Cluster 1 included elderly patients with severe symptoms, while cluster 3 included young female with mild symptoms. No significant differences were observed in the comorbidities. Cluster 1 showed the most impaired QOL, followed by clusters 4 and 5; these changes as well as the composition of symptoms were observed over 1 year. CONCLUSIONS: We identified patients with long COVID with diverse characteristics into five clusters. Future analysis of these different pathologies could result in individualised treatment of long COVID. TRIAL REGISTRATION NUMBER: The study protocol is registered at UMIN clinical trials registry (UMIN000042299).

OBJECTIVES: To assess the value of serum ferritin and Krebs von den Lungen-6 (KL-6) levels for predicting severe COVID-19 (death or requirement for invasive mechanical ventilation [IMV]/high-flow oxygen). METHODS: Data were analyzed on 2495 patients with COVID-19 from February 2020 to November 2022 using data from a nationwide COVID-19 database. RESULTS: Patients with high KL-6 and low ferritin levels were older with more comorbidities and higher mortality rates, whereas those with high ferritin and low KL-6 levels were younger, predominantly male, and more likely to need IMV. A high level of both markers was strongly associated with critical outcomes (adjusted odds ratio: 13.6, 95% confidence interval: 8.58-21.5). The combination of both markers had higher predictive value than either marker alone (area under the curve: 0.709, 0.745, and 0.781 for KL-6, ferritin, and KL-6 + ferritin, respectively). CONCLUSIONS: The combination of both markers accurately predicted COVID-19 severity.

Mycobacterium avium complex (MAC) is considered a paramount microbe, especially in East Asia, including Japan. The commonly used commercial Minimum Inhibitory Concentrations (MIC) assay using Middlebrook 7H9 (7H9) medium deviates from the latest Clinical and Laboratory Standards Institute (CLSI) guidelines. Alternatively, measurement with cation-adjusted Mueller-Hinton broth (CAMHB) that conforms to CLSI standards is not yet widely available. Following the approval and commercialization of amikacin liposome inhalation suspension (ALIS) in 2021, a more precise evaluation of amikacin (AMK) susceptibility in MAC is necessary for treatment decisions. In the present study, 33 sputum samples were extracted from 27 patients, and MICs of AMK were compared between the frequently used 7H9 and the recommended CAMHB of the isolated MAC strains. The history of exposure to aminoglycosides for each sample was also added as clinical information. The findings indicated that there was only an 18% concordance rate in MIC between the two media, with 19 samples (58%) indicating lower MICs in 7H9 relative to CAMHB. The 17 samples had a history of exposure to aminoglycosides for periods ranging from 1.5 to 28 months. Specifically, 10 samples were exposed to amikacin by inhalation and intravenous injection, and the remaining seven samples had a history of ALIS inhalation. Samples with a prior utilization of aminoglycosides were significantly predisposed to developing resistance to ALIS compared to those without such a history (P = 0.046). Physicians are encouraged to scrutinize the findings of susceptibility testing utilizing CLSI-endorsed MIC assay using CAMHB medium to ascertain the optimal therapeutic approach.

Microfold (M) cells residing in the follicle-associated epithelium of mucosa-associated lymphoid tissues are specialized for sampling luminal antigens to initiate mucosal immune responses. In the past decade, glycoprotein 2 (GP2) and Tnfaip2 were identified as reliable markers for M cells in the Peyer's patches of the intestine. Furthermore, RANKL-RANK signaling, as well as the canonical and non-canonical NFκB pathways downstream, is essential for M-cell differentiation from the intestinal stem cells. However, the molecular characterization and differentiation mechanisms of M cells in the lower respiratory tract, where organized lymphoid tissues exist rarely, remain to be fully elucidated. Therefore, this study aimed to explore M cells in the lower respiratory tract in terms of their specific molecular markers, differentiation mechanism, and functions. Immunofluorescence analysis revealed a small number of M cells expressing GP2, Tnfaip2, and RANK is present in the lower respiratory tract of healthy mice. The intraperitoneal administration of RANKL in mice effectively induced M cells, which have a high capacity to take up luminal substrates, in the lower respiratory epithelium. The airway M cells associated with lymphoid follicles were frequently detected in the pathologically induced bronchus-associated lymphoid tissue (iBALT) in the murine models of autoimmune disease as well as pulmonary emphysema. These findings demonstrate that RANKL is a common inducer of M cells in the airway and digestive tracts and that M cells are associated with the respiratory disease. We also established a two-dimensional culture method for airway M cells from the tracheal epithelium in the presence of RANKL successfully. This model may be useful for functional studies of M cells in the sampling of antigens at airway mucosal surfaces.

BACKGROUND: Pneumonia is the fourth leading cause of death worldwide, and Streptococcus pneumoniae is the most commonly associated pathogen. Increasing evidence suggests that mesenchymal stromal cells (MSCs) have anti-inflammatory roles during innate immune responses such as sepsis. However, little is known about the effect of MSCs on pneumococcal pneumonia. METHODS: Bone marrow-derived macrophages (BMDMs) were stimulated with various ligands in the presence or absence of MSC-conditioned medium. For in vivo studies, mice intranasally-inoculated with S. pneumoniae were intravenously treated with MSCs or vehicle, and various parameters were assessed. RESULTS: After stimulation with toll-like receptor (TLR) 2, TLR9 or TLR4 ligands, or live S. pneumoniae, TNF-α and interleukin (IL)-6 levels were significantly decreased, whereas IL-10 was significantly increased in BMDMs cultured in MSC-conditioned medium. In mice, MSC treatment decreased the number of neutrophils in bronchoalveolar lavage fluid (BALF) after pneumococcal infection, and this was associated with a decrease in myeloperoxidase activity in the lungs. Levels of proinflammatory cytokines, including TNF-α, IL-6, GM-CSF and IFN-γ, were significantly lower in MSC-treated mice, and the bacterial load in the lung after pneumococcal infection was significantly reduced. In addition, histopathologic analysis confirmed a decrease in the number of cells recruited to the lungs; however, lung edema, protein leakage into the BALF and levels of the antibacterial protein lipocalin 2 in the BALF were comparable between the groups. CONCLUSIONS: These results indicate that MSCs could represent a potential therapeutic application for the treatment of pneumonia caused by S. pneumoniae.