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Accumulating evidence suggests that two chronic respiratory diseases, nontuberculous mycobacterium (NTM)-pulmonary disease (PD) and allergic asthma, are frequently present together and that they likely influence the disease development and progression of each other. However, their precise interactions regarding the pathogenesis of comorbid diseases versus that of individual diseases are not well understood. In this study, comorbid diseases (i.e., Mycobacteria avium (Mav) pulmonary infection (PI) (Mav-PI) and ovalbumin-induced allergic asthma) were established in mice in different orders and at different time periods. Individual disease-specific characteristics, including alterations in immune cell populations and antigen-specific immune responses, were analyzed and compared. To assess Mav-PI pathogenesis, lung inflammation and bacterial burden levels were also determined. Allergic asthma induction in the presence of Mav-PI markedly aggravated Mav-PI pathogenesis by increasing the bacterial burden and the severity of lung inflammation. Interestingly, the general outcome of allergic asthma with goblet cell hyperplasia was alleviated at a chronic stage in the comorbid mouse model. Overall, the increase in the number of Mav CFUs was inversely correlated with the Mav-specific Th17 response, as confirmed by comparing BALB/c and C57BL/6J mice. Overall, the pathogenesis of existing Mav-PI is more severely affected by allergen exposure than vice versa. This Mav-PI exacerbation is associated with disruption of Mav-specific Th17 responses. This study provides the first evidence that the Mav-specific Th17 response plays an important role in the control of Mav pathogenesis in the presence of allergic asthma, indicating that targeting the Th17 response has therapeutic potential for NTM-PD accompanied by allergic asthma.

In the wake of the COVID-19 pandemic, lung disorders have become a major health concern for humans. Allergic asthma is the most prevalent form of asthma, and its treatments target the inflammation process. Despite significant developments in the diagnosis and management of allergic asthma, side effects are a major concern. Additionally, its extreme heterogeneity impedes the efficacy of the majority of treatments. Thus, newer, safer therapeutic substances, such as natural products, are desired. Citrus junos Tanaka has traditionally been utilized as an anti-inflammatory, sedative, antipyretic, and antitoxic substance. In this study, the protective effects of Citrus junos Tanaka peel extract (B215) against lung inflammation were examined, and efforts were made to understand the underlying protective mechanism using an HDM-induced lung inflammation murine model. The administration of B215 reduced immune cell infiltration in the lungs, plasma IgE levels, airway resistance, mucus hypersecretions, and cytokine production. These favorable effects alleviated HDM-induced lung inflammation by modulating the NF-κB signaling pathway. Hence, B215 might be a promising functional food to treat lung inflammation without adverse effects.

Background: Macrophages produce many inflammation-associated molecules, released by matrix metalloproteinases, such as adhesion molecules, and cytokines, as well, which play a crucial role in atherosclerosis. In this context, we investigated the relationship between Ninjurin-1 (Ninj1 [nerve injury-induced protein]), a novel matrix metalloproteinase 9 substrate, expression, and atherosclerosis progression. Methods: Ninj1 expression and atherosclerosis progression were assessed in atherosclerotic aortic tissue and serum samples from patients with coronary artery disease and healthy controls, and atheroprone apolipoprotein e–deficient ( Apoe −/− ) and wild-type mice, as well. Apoe −/− mice lacking systemic Ninj1 expression ( Ninj1 −/− Apoe −/− ) were generated to assess the functional effects of Ninj1. Bone marrow transplantation was also used to generate low-density lipoprotein receptor–deficient ( Ldlr −/− ) mice that lack Ninj1 specifically in bone marrow–derived cells. Mice were fed a Western diet for 5 to 23 weeks, and atherosclerotic lesions were investigated. The anti-inflammatory role of Ninj1 was verified by treating macrophages and mice with the peptides Ninj1 1 –56 (ML56) and Ninj1 26 –37 (PN12), which mimic the soluble form of Ninj1 (sNinj1). Results: Our in vivo results conclusively showed a correlation between Ninj1 expression in aortic macrophages and the extent of human and mouse atherosclerotic lesions. Ninj1 -deficient macrophages promoted proinflammatory gene expression by activating mitogen-activated protein kinase and inhibiting the phosphoinositide 3-kinase/Akt signaling pathway. Whole-body and bone marrow–specific Ninj1 deficiencies significantly increased monocyte recruitment and macrophage accumulation in atherosclerotic lesions through elevated macrophage-mediated inflammation. Macrophage Ninj1 was directly cleaved by matrix metalloproteinase 9 to generate a soluble form that exhibited antiatherosclerotic effects, as assessed in vitro and in vivo. Treatment with the sNinj1-mimetic peptides, ML56 and PN12, reduced proinflammatory gene expression in human and mouse classically activated macrophages, thereby attenuating monocyte transendothelial migration. Moreover, continuous administration of mPN12 alleviated atherosclerosis by inhibiting the enhanced monocyte recruitment and inflammation characteristics of this disorder in mice, regardless of the presence of Ninj1. Conclusions: Ninj1 is a novel matrix metalloproteinase 9 substrate in macrophages, and sNinj1 is a secreted atheroprotective protein that regulates macrophage inflammation and monocyte recruitment in atherosclerosis. Moreover, sNinj1-mediated anti-inflammatory effects are conserved in human macrophages and likely contribute to human atherosclerosis.

Valvular inflammation triggered by hyperlipidemia has been considered as an important initial process of aortic valve disease; however, cellular and molecular evidence remains unclear. Here, we assess the relationship between plasma lipids and valvular inflammation, and identify association of low-density lipoprotein with increased valvular lipid and macrophage accumulation. Single-cell RNA sequencing analysis reveals the cellular heterogeneity of leukocytes, valvular interstitial cells, and valvular endothelial cells, and their phenotypic changes during hyperlipidemia leading to recruitment of monocyte-derived MHC-IIhi macrophages. Interestingly, we find activated PPARγ pathway in Cd36+ valvular endothelial cells increased in hyperlipidemic mice, and the conservation of PPARγ activation in non-calcified human aortic valves. While the PPARγ inhibition promotes inflammation, PPARγ activation using pioglitazone reduces valvular inflammation in hyperlipidemic mice. These results show that low-density lipoprotein is the main lipoprotein accumulated in the aortic valve during hyperlipidemia, leading to early-stage aortic valve disease, and PPARγ activation protects the aortic valve against inflammation.

Dendritic cells (DCs) are the main mediators of Th2 immune responses in allergic asthma, and Fms-like tyrosine kinase 3 ligand (Flt3L) is an important growth factor for the development and homeostasis of DCs. This study identified the DC populations that primarily cause the initiation and development of allergic lung inflammation using Fms-like tyrosine kinase 3 (Flt3) knockout (KO) mice with allergen-induced allergic asthma. We observed type 2 allergic lung inflammation with goblet cell hyperplasia in Flt3 KO mice, despite a significant reduction in total DCs, particularly CD103+ DCs, which was barely detected. In addition, bone marrow-derived dendritic cells (BMDCs) from Flt3 KO mice directed Th2 immune responses in vitro, and the adoptive transfer of these BMDCs exacerbated allergic asthma with more marked Th2 responses than that of BMDCs from wild-type (WT) mice. Furthermore, we found that Flt3L regulated the in vitro expression of OX40 ligand (OX40L) in DCs, which is correlated with DC phenotype in in vivo models. In conclusion, we revealed that Flt3-independent CD11b+ DCs direct Th2 responses with the elevated OX40L and are the primary cause of allergic asthma. Our findings suggest that Flt3 is required to control type 2 allergic inflammation.

In this study, a cyclic isotropic plasma atomic layer etching (ALE) process was developed for aluminum oxide that involves fluorination with NF3 plasma and ligand exchange with trimethylaluminum (TMA). The isotropic plasma ALE consists of two steps: fluorination and removal. During the fluorination step, the Al2O3 surface was fluorinated to AlOFx with NF3 plasma at 100 °C. The formation of the AlOFx layer was confirmed by x-ray photoelectron spectroscopy analysis, and the atomic fraction of fluorine on the surface was saturated at 25% after 50 s of plasma fluorination. The depths of the fluorinated layers were in the range of 0.79–1.14 nm at different plasma powers. In the removal step, the fluorinated layer was removed by a ligand exchange reaction with TMA at an elevated temperature range of 250–480 °C. The etch per cycle (EPC) was 0.20–0.30 nm/cycle and saturated after 30 s in the temperature range of 290–330 °C. No etching was observed below 250 °C, and the EPC increased in the temperature range of 250–300 °C during the removal step with the ligand exchange reaction and reached the maximum at 300 °C. Then, the EPC was significantly reduced at high temperatures, possibly due to TMA decomposition. The fluorine atomic fraction on the surface was reduced to 14% after the removal. In conclusion, Al2O3 was successfully etched at the atomic scale by the cyclic plasma ALE process. The average surface roughness of Al2O3 was reduced from 8.6 to 5.3 Å after 20 cycles of etching.

Rationale: Monocyte infiltration into the subintimal space and its intracellular lipid accumulation are the most prominent features of atherosclerosis. To understand the pathophysiology of atherosclerotic disease, we need to understand the characteristics of lipid-laden foamy macrophages in the subintimal space during atherosclerosis. Objective: We sought to examine the transcriptomic profiles of foamy and nonfoamy macrophages isolated from atherosclerotic intima. Methods and Results: Single-cell RNA sequencing analysis of CD45 + leukocytes from murine atherosclerotic aorta revealed that there are macrophage subpopulations with distinct differentially expressed genes involved in various functional pathways. To specifically characterize the intimal foamy macrophages of plaque, we developed a lipid staining–based flow cytometric method for analyzing the lipid-laden foam cells of atherosclerotic aortas. We used the fluorescent lipid probe BODIPY493/503 and assessed side-scattered light as an indication of cellular granularity. BODIPY hi SSC hi foamy macrophages were found residing in intima and expressing CD11c. Foamy macrophage accumulation determined by flow cytometry was positively correlated with the severity of atherosclerosis. Bulk RNA sequencing analysis showed that compared with nonfoamy macrophages, foamy macrophages expressed few inflammatory genes but many lipid-processing genes. Intimal nonfoamy macrophages formed the major population expressing IL (interleukin)-1β and many other inflammatory transcripts in atherosclerotic aorta. Conclusions: RNA sequencing analysis of intimal macrophages from atherosclerotic aorta revealed that lipid-loaded plaque macrophages are not likely the plaque macrophages that drive lesional inflammation.

Purpose Simple and reliable animal models of human diseases contribute to the understanding of disease pathogenesis as well as the development of therapeutic interventions. Although several murine models to mimic human asthma have been established, most of them require anesthesia, resulting in variability among test individuals, and do not mimic asthmatic responses accompanied by T-helper (Th) 17 and neutrophils. As dendritic cells (DCs) are known to play an important role in initiating and maintaining asthmatic inflammation, we developed an asthma model via adoptive transfer of allergen-loaded DCs. Methods Ovalbumin (OVA)-loaded bone marrow-derived DCs (BMDCs) (OVA-BMDCs) were injected intravenously 3 times into non-anesthetized C57BL/6 mice after intraperitoneal OVA-sensitization. Results OVA-BMDC-transferred mice developed severe asthmatic immune responses when compared with mice receiving conventional OVA challenge intranasally. Notably, remarkable increases in systemic immunoglobulin (Ig) E and IgG1 responses, Th2/Th17-associated cytokines (interleukin [IL]-5, IL-13 and IL-17), Th2/Th17-skewed T-cell responses, and cellular components, including eosinophils, neutrophils, and goblet cells, were observed in the lungs of OVA-BMDC-transferred mice. Moreover, the asthmatic immune responses and severity of inflammation were correlated with the number of OVA-BMDCs transferred, indicating that the disease severity and asthma type may be adjusted according to the experimental purpose by this method. Furthermore, this model exhibited less variation among the test individuals than the conventional model. In addition, this DCs-based asthma model was partially resistant to steroid treatment. Conclusions A reliable murine model of asthma by intravenous (i.v.) transfer of OVA-BMDCs was successfully established without anesthesia. This model more accurately reflects heterogeneous human asthma, exhibiting a robust Th2/Th17-skewed response and eosinophilic/neutrophilic infiltration with good reproducibility and low variation among individuals. This model will be useful for understanding the pathogenesis of asthma and would serve as an alternative tool for immunological studies on the function of DCs, T-cell responses and new drugs.

Plasmacytoid dendritic cells (pDCs) are unique bone-marrow-derived cells that produce large amounts of type I interferon in response to microbial stimulation. Furthermore, pDCs also promote T cell tolerance in sterile-inflammation conditions. However, the immunomodulatory role of aortic pDCs in atherosclerosis has been poorly understood. Here, we identified functional mouse and human pDCs in the aortic intima and showed that selective, inducible pDC depletion in mice exacerbates atherosclerosis. Aortic pDCs expressed CCR9 and indoleamine 2,3-dioxygenase 1 (IDO-1), an enzyme involved in driving the generation of regulatory T cells (Tregs). As a consequence, loss of pDCs resulted in decreased numbers of Tregs and reduced IL-10 levels in the aorta. Moreover, antigen presentation by pDCs expanded antigen-specific Tregs in the atherosclerotic aorta. Notably, Tregs ablation affected pDC homeostasis in diseased aorta. Accordingly, pDCs in human atherosclerotic aortas colocalized with Tregs. Collectively, we identified a mechanism of atheroprotection mediated by tolerogenic aortic pDCs.

To this date, the criteria to distinguish peritoneal macrophages and dendritic cells (DCs) are not clear. Here we delineate the subsets of myeloid mononuclear cells in the mouse peritoneal cavity. Considering phenotypical, functional, and ontogenic features, peritoneal myeloid mononuclear cells are divided into 5 subsets: large peritoneal macrophages (LPMs), small peritoneal macrophages (SPMs), DCs, and 2 MHCII+CD11c+CD115+ subpopulations (i.e., MHCII+CD11c+CD115+CD14−CD206− and MHCII+CD11c+CD115+CD14+CD206+). Among them, 2 subsets of competent Ag presenting cells are demonstrated with distinct functional characteristics, one being DCs and the other being MHCII+CD11c+CD115+CD14−CD206− cells. DCs are able to promote fully activated T cells and superior in expanding cytokine producing inflammatory T cells, whereas MHCII+CD11c+CD115+CD14−CD206− cells generate partially activated T cells and possess a greater ability to induce Treg under TGF-β and retinoic acid conditions. While the development of DCs and MHCII+CD11c+CD115+CD14−CD206− cells are responsive to the treatment of FLT3 ligand and GM-CSF, the number of LPMs, SPMs, and MHCII+CD11c+CD115+CD14+CD206+ cells are only influenced by the injection of GM-CSF. In addition, the analysis of gene expression profiles among MHCII+ peritoneal myeloid mononuclear cells reveals that MHCII+CD11c+CD115+CD14+CD206+ cells share high similarity with SPMs, whereas MHCII+CD11c+CD115+CD14−CD206− cells are related to peritoneal DC2s. Collectively, our study identifies 2 distinct subpopulations of MHCII+CD11c+CD115+ cells, 1) MHCII+CD11c+CD115+CD14−CD206− cells closely related to peritoneal DC2s and 2) MHCII+CD11c+CD115+CD14+CD206+ cells to SPMs.

Dendritic cells (DCs) are central to initiate antigen-specific immunity and tolerance. The in vivo development and distribution of DCs are now better understood even in nonlymphoid tissues [1]. Atherosclerosis is a chronic inflammatory disease of blood vessels and DCs are highly enriched in the intimal area of the aorta, which is predisposed to develop atherosclerosis. Previously, we were the first to show antigen presenting DCs and their subsets in the aorta [2, 3]. Here, we discuss several useful methods to characterize not only DCs but also other immune cells in steady state and atherosclerotic aorta. These comprise multiparameter flow cytometry strategies including intracellular staining and cell sorting, en face immunohistochemistry of DCs and regulatory T cells (Tregs), and Oil Red O staining of atherosclerotic lesions in the aorta.

Dendritic cells (DCs) are central to initiate antigen-specific immunity and tolerance. The in vivo development and distribution of DCs are now better understood even in nonlymphoid tissues [1]. Atherosclerosis is a chronic inflammatory disease of blood vessels and DCs are highly enriched in the intimal area of the aorta, which is predisposed to develop atherosclerosis. Previously, we were the first to show antigen presenting DCs and their subsets in the aorta [2, 3]. Here, we discuss several useful methods to characterize not only DCs but also other immune cells in steady state and atherosclerotic aorta. These comprise multiparameter flow cytometry strategies including intracellular staining and cell sorting, en face immunohistochemistry of DCs and regulatory T cells (Tregs), and Oil Red O staining of atherosclerotic lesions in the aorta.

Background Allergic rhinitis (AR) is the most common allergic disease but little is known about the difference of local immune responses in children and adults with AR. Objective To compare local immune responses between children and adults with AR and nonallergic rhinitis (NAR), and to investigate whether the association of local and systemic immune responses is different between the two age groups. Methods Fifty-one patients with chronic rhinitis were enrolled and grouped into children (N = 27, mean age 7.2 years) and adults (N = 24, mean age 29.9 years). Diagnosis of AR was based on symptoms, skin prick tests and serum specific IgEs. Nasal lavage (NAL) fluids were collected from all subjects and used to measure the levels of total IgE, specific IgEs to house dust mites (Dp and Df), and cytokines (TNF-α, IL-4, IL-10, IL-17A and IFN-γ). Flow cytometry was used to measure inflammatory cell types in NAL fluids. Results AR had significantly increased local levels of total IgE and specific IgEs to Dp and Df compared with NAR in both age groups (P < 0.05). Nasal eosinophils % (P = 0.01) was significantly increased only in children with AR. Local-systemic correlations of total IgE (r = 0.662, P = 0.000) and eosinophil % (r = 0.461, P = 0.015) between the peripheral blood and NAL fluids were found only in children. Moreover, children had correlations between total IgE and eosinophil % in the peripheral blood (r = 0.629, P = 0.001) and in NAL fluids (r = 0.373, P = 0.061). Conclusion Elevated local IgE is a common feature of AR in children and adults. Local measures in NAR showed naive state of immune response which disagree with the hypothesis of local allergic rhinitis. Children showed intense local inflammation and close local-systemic interactions compared to adults supporting pediatric AR as a distinct feature.

Resistin-like molecule alpha (Retnla), also known as ` Found in inflammatory zone 1', is a secreted protein that has been found in bronchoalveolar lavage (BAL) fluid of ovalbumin (OVA)-induced asthmatic mice and plays a role as a regulator of T helper (Th) 2-driven inflammation. However, the role of Retnla in the progress of Th2-driven airway inflammation is not yet clear. To better understand the function of Retnla in Th2driven airway inflammation, we generated Retnla-overexpressing (Retnla-Tg) mice. Retnla-Tg mice showed increased expression of Retnla protein in BAL fluid and airway epithelial cells. Retnla overexpression itself did not induce any alteration in lung histology or lung function compared to non-Tg controls. However, OVA-sensitized/ challenged Retnla-Tg mice had decreased numbers of cells in BAL and inflammatory cells accumulating in the lung. They also showed a reduction in mucus production in the airway epithelium, concomitant with a decreased Muc5ac level. These results were accompanied by reduced levels of Th2 cytokines, including interleukin (IL)-4, IL5, and IL-13, with no effect on levels of OVA-specific immunoglobulin isotypes. Furthermore, phosphorylation of ERK was markedly reduced in the lungs of OVAchallenged Retnla-Tg mice. Taken together, these results indicates that Retnla protects against Th2-mediated inflammation in an experimental mouse model of asthma, suggesting that therapeutic approaches to enhance the production of Retnla or Retnla-like molecules could be valuable for preventing allergic lung inflammation.