Mesenchymal stem cell exosomes differentially regulate gene expression of mast cells.

Emerging evidence indicates that the immunomodulatory effect of mesenchymal stem cells (MSCs) is primarily attributed to the paracrine pathway. As a key paracrine effector, MSC-derived exosomes are small vesicles that play an important role in cell-to-cell communication by carrying bioactive substances. We previously found that exosomes derived from tonsil-derived mesenchymal stem cells (T-MSCs) were able to effectively attenuate inflammatory responses in mast cells. Here we investigated how T-MSC exosomes impact mast cells in steady state, and how exposure of T-MSCs to Toll-like receptors (TLRs) ligands changes this impact. Transcriptomic analysis of HMC-1 cells, a human mast cell line, using DNA microarrays showed that T-MSC exosomes broadly regulate genes involved in the normal physiology of mast cells. TLR3 or TLR4 primed T-MSC exosomes impacted fewer genes involved in specific functions in mast cells. This distinguishable regulation also was apparent in the analysis of related gene interactions. Our results suggest that MSC exosomes maintain immune homeostasis in normal physiology and impact the inflammatory state by modulating mast cell transcription.

Identification of novel Carnobacterium maltaromaticum strains in bone marrow samples of patients with acute myeloid leukemia using a metagenomic binning approach.

The aim of this study aimed to examine the existence of a bacterial metagenome in the bone marrow of patients with acute myeloid leukemia (AML). We re-examined whole-genome sequencing data from the bone marrow samples of seven patients with AML, four of whom were remitted after treatment, for metagenomic analysis. After the removal of human reads, unmapped reads were used to profile the species-level composition. We used the metagenomic binning approach to confirm whether the identified taxon was a complete genome of known or novel strains. We observed a unique and novel microbial signature in which Carnobacterium maltaromaticum was the most abundant species in five patients with AML or remission. The complete genome of C. maltaromaticum "BMAML_KR01," which was observed in all samples, was 100% complete with 8.5% contamination and closely clustered with C. maltaromaticum strains DSM20730 and SF668 based on single nucleotide polymorphism variations. We identified five unique proteins that could contribute to cancer progression and 104 virulent factor proteins in the BMAML_KR01 genome. To our knowledge, this is the first report of a new strain of C. maltaromaticum in patients with AML. The presence of C. maltaromaticum and its new strain in patients indicates an urgent need to validate the existence of this bacterium and evaluate its pathophysiological role.

Lymphatic endothelial cells promote T lymphocyte migration into lymph nodes under hyperlipidemic conditions.

Lymphatic vessels serve as conduits through which immune cells traffic. Because lymphatic vessels are also involved in lipid transport, their function is vulnerable to abnormal metabolic conditions such as obesity and hyperlipidemia. Exactly how these conditions impact immune cell trafficking, however, is not well understood. Here, we found higher numbers of LYVE-1-positive lymphatic endothelial cells and CD3-positive T cells in the lymph nodes of mice fed high-cholesterol or high-fat diets compared with those of mice fed a normal chow diet. To confirm the effect of fat content on immune cell trafficking, the lymphatic endothelial SVEC4-10 cell line was treated with palmitic acid at a 100 µM concentration. After 24 h, palmitic acid-treated cells exhibited increased expression of podoplanin and vascular growth-associated molecules (VEGFC, VEGFD, VEGFR3, and NRP2) and enhanced tube formation. Microarray analysis showed an increase in pro-inflammatory cytokine and chemokine transcription after palmitic acid treatment. Finally, transwell migration assay confirmed that T cell line moved toward medium previously cultured with palmitic acid-treated SVEC4-10 cells. Together, our results suggest that hyperlipidemia drives lymphatic vessel remodeling and T cell migration toward lymphatic endothelial cells.

Application of Tonsil-Derived Mesenchymal Stem Cells in Tissue Regeneration: Concise Review.

Since the discovery of stem cells and multipotency characteristics of mesenchymal stem cells (MSCs), there has been tremendous development in regenerative medicine. MSCs derived from bone marrow have been widely used in various research applications, yet there are limitations such as invasiveness of obtaining samples, low yield and proliferation rate, and questions regarding their practicality in clinical applications. Some have suggested that MSCs from other sources, specifically those derived from palatine tonsil tissues, that is, tonsil-derived MSCs (TMSCs), could be considered as a new potential therapeutic tool in regenerative medicine due to their superior proliferation rate and differentiation capabilities with low immunogenicity and ease of obtaining. Several studies have determined that TMSCs have differentiation potential not only into the mesodermal lineage but also into the endodermal as well as ectodermal lineages, expanding their potential usage and placing them as an appealing option to consider for future studies in regenerative medicine. In this review, the differentiation capacities of TMSCs and their therapeutic competencies from past studies are addressed. Stem Cells 2019;37:1252-1260.

Dexamethasone Promotes Keratinocyte Proliferation by Triggering Keratinocyte Growth Factor in Mast Cells.

BACKGROUND: The skin is a dynamic body organ that can be activated by both central and local hypothalamic-pituitary-adrenal axis systems. This phenomenon might be the crucial explanation why stress can cause relapse of chronic inflammatory skin diseases, such as psoriasis. Here, we determined the effects of mast cells on keratinocyte proliferation under stress hormone stimulation. METHODS: We subcutaneously injected dexamethasone on the shaved back of mice and evaluated histological changes and keratinocyte growth factor (KGF) expression on dermal mast cells. Further, human mast cell line (HMC-1) and keratinocyte cell line (HaCaT) cells were treated with dexamethasone in vitro to observe the extent of proliferation and the expression of KGF. Finally, the supernatants of HMC-1 cells treated with dexamethasone were used for the culture of HaCaT cells to investigate the effect on proliferation. RESULTS: We observed epidermal thickening in dexamethasone-injected mice, accompanied by an increase in the number of KGF-expressing dermal mast cells. Similar to mouse dermal mast cells, KGF was highly expressed in the human mast cell line HMC-1 following stimulation with dexamethasone. Further, dexamethasone-treated mast cells promoted keratinocyte proliferation in vitro. However, the effects of mast cells on keratinocytes were significantly diminished in the presence of anti-KGF-blocking antibodies. CONCLUSION: Taken together, our results show that a stressful environment may disturb skin barrier homeostasis through mast cell-derived KGF expression.

Inhibiting Sphingosine Kinase 2 Derived-sphingosine-1-phosphate Ameliorates Psoriasis-like Skin Disease via Blocking Th17 Differentiation of Naïve CD4 T Lymphocytes in Mice.

Sphingosine-1-phosphate (S1P) is a signalling sphingolipid metabolite that regulates important cell processes, including cell proliferation and apoptosis. Circulating S1P levels have been reported to be increased in patients with psoriasis relative to healthy patients. The aim of this study was to examine the potency of S1P inhibition using an imiquimod-induced psoriasis mouse model. Both topical ceramidase and sphingosine kinase 1/2 inhibition, which blocks S1P generation, alleviated imiquimod-induced skin lesions and reduced the serum interleukin 17-A levels induced by application of imiquimod. These treatments also normalized skin mRNA levels of genes associated with inflammation and keratinocyte differentiation. Inhibition of sphingosine kinase 2, but not sphingosine kinase 1, diminished levels of suppressor of cytokine signalling 1 and blocked T helper type 17 differentiation of naïve CD4+ T cells; imiquimod-induced psoriasis-like skin symptoms were also ameliorated. These results indicate the distinct effects of sphingosine kinase 1 and sphingosine kinase 2 inhibition on T helper type 17 generation and suggest molecules that inhibit S1P formation, including ceramidase and sphingosine kinase 2 inhibitors, as novel therapeutic candidates for psoriasis.

Comparison of Immune Responses to Two Quadrivalent Meningococcal Conjugate Vaccines (CRM197 and Diphtheria Toxoid) in Healthy Adults.

BACKGROUND: After the introduction of the meningococcal ACWY-CRM197 conjugate vaccine (MenACWY-CRM) in 2012 and the meningococcal ACWY-diphtheria toxoid conjugate vaccine (MenACWY-DT) in 2014, immunization was recommended for certain high-risk groups including new military recruits in Korea. However, comparative immunogenicity studies for these vaccines have not been performed in Korea. Here, we compared the immunogenicity of these two vaccines in healthy adults. METHODS: A total of 64 adults, 20-49 years of age, were randomly divided into two groups (1:1) to receive either of the two vaccines. The sera were obtained before and 1 month after vaccination and tested for serogroup-specific serum bactericidal activity using baby rabbit complement. RESULTS: There were no significant differences post-vaccination in the geometric mean indices and the seropositive rate to all serogroups between the vaccines. The proportion of seropositive subjects after vaccination ranged from 88% to 100%. CONCLUSION: Both meningococcal conjugate vaccines showed good immunogenicity in healthy Korean adults without statistically significant differences. Further investigations for serotype distribution of circulating meningococci and the immune interference between other diphtheria toxin-containing vaccines concomitantly used for military recruits are needed to optimize immunization policies. TRIAL REGISTRATION: Clinical Research Information Service Identifier: KCT0002460.

Conditioned media from human palatine tonsil mesenchymal stem cells regulates the interaction between myotubes and fibroblasts by IL-1Ra activity.

Saturated free fatty acids (FFAs) act as lipid mediators and induce insulin resistance in skeletal muscle. Specifically, in obesity-related diseases such as type 2 diabetes, FFAs directly reduce insulin sensitivity and glucose uptake in skeletal muscle. However, the knowledge of how FFAs mediate inflammation and subsequent tissue disorders, including fibrosis in skeletal muscle, is limited. FFAs are a natural ligand for toll-like receptor 2 (TLR2) and TLR4, and induce chronic low-grade inflammation that directly stimulates skeletal muscle tissue. However, persistent inflammatory stimulation in tissues could induce pro-fibrogenic processes that ultimately lead to perturbation of the tissue architecture and dysfunction. Therefore, blocking the link between inflammatory primed skeletal muscle tissue and connective tissue might be an efficient therapeutic option for treating obesity-induced muscle inactivity. In this study, we investigated the impact of conditioned medium obtained from human palatine tonsil-derived mesenchymal stem cells (T-MSCs) on the interaction between skeletal muscle cells stimulated with palmitic acid (PA) and fibroblasts. We found that PA-treated skeletal muscle cells actively secreted interleukin-1ß (IL-1ß) and augmented the migration, proliferation and expression of fibronectin in L929 fibroblasts. Furthermore, T-CM inhibited the skeletal muscle cell-derived pro-fibrogenic effect via the production of the interleukin-1 receptor antagonist (IL-1Ra), which is an inhibitor of IL-1 signalling. Taken together, our data provide novel insights into the therapeutic potential of T-MSC-mediated therapy for the treatment of pathophysiological processes that occur in skeletal muscle tissues under chronic inflammatory conditions.

Th17 cell-mediated immune responses promote mast cell proliferation by triggering stem cell factor in keratinocytes.

Although mast cells are traditionally thought to function as effector cells in allergic responses, they have increasingly been recognized as important regulators of various immune responses. Mast cells mature locally; thus, tissue-specific influences are important for promoting mast cell accumulation and survival in the skin and the gastrointestinal tract. In this study, we determined the effects of keratinocytes on mast cell accumulation during Th17-mediated skin inflammation. We observed increases in dermal mast cells in imiquimod-induced psoriatic dermatitis in mice accompanied by the expression of epidermal stem cell factor (SCF), a critical mast cell growth factor. Similar to mouse epidermal keratinocytes, SCF was highly expressed in the human HaCaT keratinocyte cell line following stimulation with IL-17. Further, keratinocytes promoted mast cell proliferation following stimulation with IL-17 in vitro. However, the effects of keratinocytes on mast cells were significantly diminished in the presence of anti-CD117 (stem cell factor receptor) blocking antibodies. Taken together, our results revealed that the Th17-mediated inflammatory environment promotes mast cell accumulation through keratinocyte-derived SCF.

TRPV3 Channel in Keratinocytes in Scars with Post-Burn Pruritus.

Post-burn pruritus is a common and distressing sequela of burn scars. Empirical antipruritic treatments usually fail to have a satisfactory outcome because of their limited selectivity and possible side effects. Therefore, novel drug targets need to be identified. Here, we aimed to investigate the possible role of protease-activated receptor 2 (PAR2) and transient receptor potential vanniloid 3 (TRPV3), along with the relation of TRPV3 to thymic stromal lymphopoietin (TSLP). Specimens from normal (unscarred) or burn-scarred (with or without pruritus) tissue were obtained from burn patients for this study. In each sample, the keratinocytes were isolated and cultured, and the intracellular Ca2+ level at the time of stimulation of each factor was quantified and the interaction was screened. PAR2 function was reduced by antagonism of TRPV3. Inhibiting protein kinase A (PKA) and protein kinase C (PKC) reduced TRPV3 function. TSLP mRNA and protein, and TSLPR protein expressions, increased in scars with post-burn pruritus, compared to scars without it or to normal tissues. In addition, TRPV1 or TRPV3 activation induced increased TSLP expression. Conclusively, TRPV3 may contribute to pruritus in burn scars through TSLP, and can be considered a potential therapeutic target for post-burn pruritus.

Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on ß-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus.

Tonsil-derived mesenchymal stromal cells produce CXCR2-binding chemokines and acquire follicular dendritic cell-like phenotypes under TLR3 stimulation.

We previously isolated mesenchymal stromal cells from human tonsils (T-MSCs) and showed the potential of these cells to differentiate into the mesodermal lineage and acquire a follicular dendritic cell (FDC) phenotype under cytokine stimulation. Because these T-MSCs were originally isolated from inflamed tonsillar tissues, we were curious about their activation status in response to innate immune stimuli, such as Toll-like receptors (TLRs). Therefore, we analyzed the expression profile of TLRs in T-MSCs and stimulated the T-MSCs with TLR agonists. TLR3 stimuli induced C-C chemokine receptor type 6 expression in T-MSCs after 24h. Furthermore, results from cytokine arrays showed increases in epithelial neutrophil-activating peptide-78/C-X-C motif chemokine (CXCL) 5, granulocyte chemotactic protein-2/CXCL6, growth-related oncogene-α/CXCL1, interleukin-8/CXCL8, and interferon gamma-induced protein-10/CXCL10. CD54 expression was also increased after TLR3 stimulation. However, co-culturing T-MSCs with human B cells did not induce B-cell proliferation. This suggests that TLR3 stimulates the differentiation of T-MSCs into FDC-like cells and induces chemokine secretion, possibly by recruiting C-X-C chemokine receptor 2-expressing immune cells. In addition, T-MSCs also appeared to exert immunomodulatory effects by inhibiting B-cell proliferation, possibly by down-regulating CD18.

Overexpression of mutant HSP27 causes axonal neuropathy in mice.

BACKGROUND: Mutations in heat shock 27 kDa protein 1 (HSP27 or HSPB1) cause distal hereditary motor neuropathy (dHMN) or Charcot-Marie-Tooth disease type 2 F (CMT2F) according to unknown factors. Mutant HSP27 proteins affect axonal transport by reducing acetylated tubulin. RESULTS: We generated a transgenic mouse model overexpressing HSP27-S135F mutant protein driven by Cytomegalovirus (CMV) immediate early promoter. The mouse phenotype was similar to dHMN patients in that they exhibit motor neuropathy. To determine the phenotypic aberration of transgenic mice, behavior test, magnetic resonance imaging (MRI), electrophysiological study, and pathology were performed. Rotarod test showed that founder mice exhibited lowered motor performance. MRI also revealed marked fatty infiltration in the anterior and posterior compartments at calf level. Electrophysiologically, compound muscle action potential (CMAP) but not motor nerve conduction velocity (MNCV) was reduced in the transgenic mice. Toluidine staining with semi-thin section of sciatic nerve showed the ratio of large myelinated axon fiber was reduced, which might cause reduced locomotion in the transgenic mice. Electron microscopy also revealed abundant aberrant myelination. Immunohistochemically, neuronal dysfunctions included elevated level of phosphorylated neurofilament and reduced level of acetylated tubulin in the sural nerve of transgenic mice. There was no additional phenotype besides motor neuronal defects. CONCLUSIONS: Overexpression of HSP27-S135F protein causes peripheral neuropathy. The mouse model can be applied to future development of therapeutic strategies for dHMN or CMT2F.

A novel homozygous MPV17 mutation in two families with axonal sensorimotor polyneuropathy.

BACKGROUND: Mutations in MPV17 cause the autosomal recessive disorder mitochondrial DNA depletion syndrome 6 (MTDPS6), also called Navajo neurohepatopathy (NNH). Clinical features of MTDPS6 is infantile onset of progressive liver failure with seldom development of progressive neurologic involvement. METHODS: Whole exome sequencing (WES) was performed to isolate the causative gene of two unrelated neuropathy patients (9 and 13 years of age) with onset of the syndrome. Clinical assessments and biochemical analysis were performed. RESULTS: A novel homozygous mutation (p.R41Q) in MPV17 was found by WES in both patients. Both showed axonal sensorimotor polyneuropathy without liver and brain involvement, which is neurophysiologically similar to axonal Charcot-Marie-Tooth disease (CMT). A distal sural nerve biopsy showed an almost complete loss of the large and medium-sized myelinated fibers compatible with axonal neuropathy. An in vitro assay using mouse motor neuronal cells demonstrated that the abrogation of MPV17 significantly affected cell integrity. In addition, the expression of the mutant protein affected cell proliferation. These results imply that both the loss of normal function of MPV17 and the gain of detrimental effects of the mutant protein might affect neuronal function. CONCLUSION: We report a novel homozygous mutation in MPV17 from two unrelated patients harboring axonal sensorimotor polyneuropathy without hepatoencephalopathy. This report expands the clinical spectrum of diseases caused by mutations of MPV17, and we recommend MPV17 gene screening for axonal peripheral neuropathies.

Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury.

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.

RhoA/phosphatidylinositol 3-kinase/protein kinase B/mitogen-activated protein kinase signaling after growth arrest-specific protein 6/mer receptor tyrosine kinase engagement promotes epithelial cell growth and wound repair via upregulation of hepatocyte growth factor in macrophages.

Growth arrest-specific protein 6 (Gas6)/Mer receptor tyrosine kinase (Mer) signaling modulates cytokine secretion and helps to regulate the immune response and apoptotic cell clearance. Signaling pathways that activate an epithelial growth program in macrophages are still poorly defined. We report that Gas6/Mer/RhoA signaling can induce the production of epithelial growth factor hepatic growth factor (HGF) in macrophages, which ultimately promotes epithelial cell proliferation and wound repair. The RhoA/protein kinase B (Akt)/mitogen-activated protein (MAP) kinases, including p38 MAP kinase, extracellular signal-regulated protein kinase, and Jun NH2-terminal kinase axis in RAW 264.7 cells, was identified as Gas6/Mer downstream signaling pathway for the upregulation of HGF mRNA and protein. Conditioned medium from RAW 264.7 cells that had been exposed to Gas6 or apoptotic cells enhanced epithelial cell proliferation of the epithelial cell line LA-4 and wound closure. Cotreatment with an HGF receptor-blocking antibody or c-Met antagonist downregulated this enhancement. Inhibition of Mer with small interfering RNA (siRNA) or the RhoA/Rho kinase pathway by RhoA siRNA or Rho kinase pharmacologic inhibitor suppressed Gas6-induced HGF mRNA and protein expression in macrophages and blocked epithelial cell proliferation and wound closure induced by the conditioned medium. Our data provide evidence that macrophages can be reprogrammed by Gas6 to promote epithelial proliferation and wound repair via HGF, which is induced by the Mer/RhoA/Akt/MAP kinase pathway. Thus, defects in Gas6/Mer/RhoA signaling in macrophages may delay tissue repair after injury to the alveolar epithelium.

Combination of phytochemicals, including ginsenoside and curcumin, shows a synergistic effect on the recovery of radiation-induced toxicity.

Radiotherapy is commonly used to treat solid cancers located in the pelvis. A considerable number of patients experience proctitis of varying severity, even for a considerable period after radiotherapy. These side effects are often long-lasting or progressively worsen despite multiple therapeutic efforts and are a primary cause of an unexpectedly low quality of life, even after successful cancer treatment. Therefore, this study evaluated the individual and combined efficacy of ginsenoside, curcumin, butyric acid, and sucralfate compounds in treating radiation-induced proctitis. While the candidate compounds did not affect the proliferation and migration of cancer cells, they promoted the recovery of cell activity, including motility. They exhibited anti-inflammatory effects on human dermal fibroblasts or human umbilical vein endothelial cells within in vitro disease models. When each compound was tested, curcumin and ginsenoside were the most effective in cell recovery and promoted the migration of human dermal fibroblasts and cell restoration of human umbilical vein endothelial cells. The combination of ginsenoside and curcumin resulted in cell migration recovery of approximately 54%. In addition, there was a significant improvement in the length of the endothelial tube, with an increase of approximately 25%, suggesting that the ginsenoside-curcumin-containing combination was the most effective against radiation-induced damage. Furthermore, studies evaluating the effects of combined treatments on activated macrophages indicated that the compounds effectively reduced the secretion of inflammatory cytokines, including chemokines, and alleviated radiation-induced inflammation. In conclusion, our study provides valuable insights into using curcumin and ginsenoside as potential compounds for the effective treatment of radiation-induced injuries and highlights the promising therapeutic benefits of combining these two compounds.

Upregulation of Mer receptor tyrosine kinase signaling attenuated lipopolysaccharide-induced lung inflammation.

Mer receptor tyrosine kinase (Mer) signaling plays a central role in the intrinsic inhibition of the inflammatory response to Toll-like receptor activation. Previously, we found that lung Mer protein expression decreased after lipopolysaccharide (LPS) treatment due to enhanced Mer cleavage. The purpose of the present study was to examine whether pharmacologically restored membrane-bound Mer expression upregulates the Mer signaling pathways and suppresses lung inflammatory responses. Pretreatment with the ADAM17 (a disintegrin and metalloproteinase-17) inhibitor TAPI-0 (tumor necrosis factor alpha protease inhibitor-0) reduced LPS-induced production of soluble Mer protein in bronchoalveolar lavage (BAL) fluid, restored membrane-bound Mer expression, and increased Mer activation in alveolar macrophages and lungs after LPS treatment. TAPI-0 also enhanced Mer downstream signaling, including phosphorylation of protein kinase b, focal adhesion kinase, and signal transducer and activator of transcription 1. As expected from enhanced Mer signaling, TAPI-0 also augmented suppressor of cytokine signaling-1 and -3 mRNA and protein levels and inhibited nuclear factor κB activation at 4 and 24 hours after LPS treatment. TAPI-0 suppressed LPS-induced inflammatory cell accumulation, total protein level elevation in BAL fluid, and production of inflammatory mediators, including tumor necrosis factor-α, interleukin-1ß, and macrophage inflammatory protein-2. Additionally, the effects of TAPI-0 on the activation of Mer signaling and the production of inflammatory responses could be reversed by cotreatment with specific Mer-neutralizing antibody. Restored Mer protein expression by treatment with TAPI-0 efficiently prevents the inflammatory cascade during acute lung injury.

IL-17 and IL-22 enhance skin inflammation by stimulating the secretion of IL-1ß by keratinocytes via the ROS-NLRP3-caspase-1 pathway.

BACKGROUND: The pathogenesis of inflammatory skin disease involves the release of cytokines from keratinocytes, and one of these, IL-1ß, has been previously implicated in inflammatory skin disease. T(h)17 cells, a subset of T(h) cells involved in autoimmunity and inflammation, possess IL-1ß receptors and secrete cytokines such as IL-17 and IL-22 in response to IL-1ß stimulation. A mutation in the inflammasome protein NLRP3 (NACHT, LRR and PYD domains-containing protein 3) causes excess production of IL-1ß, resulting in an augmentation of T(h)17-dominant pathology. METHODS: To determine the feedback effect, if any, of IL-17 and/or IL-22 on the secretion of IL-1ß from keratinocytes, we stimulated the human keratinocyte cell line HaCaT, as well as caspase-1-deficient mice, with IL-17 or IL-22. RESULTS: We found that treatment with IL-17 and IL-22 causes an increase in IL-1ß via the activation of NLRP3 by a process that involves the generation of reactive oxygen species. Moreover, skin inflammation induced by IL-17 and IL-22 was lower in caspase-1 knockout (KO) mice relative to that induced by IL-1ß treatment. Additionally, skin inflammation induced by the drug imiquimod was lower in caspase-1 KO mice than in wild-type mice. CONCLUSION: These results indicate that cytokines from T(h)17 cells may potentiate IL-1ß-mediated skin inflammation and result in phenotypic alterations of keratinocytes via a feedback mechanism.

SIRPα/CD172a regulates eosinophil homeostasis.

Eosinophils are abundant in the lamina propria of the small intestine, but they rarely show degranulation in situ under steady-state conditions. In this study, using two novel mAbs, we found that intestinal eosinophils constitutively expressed a high level of an inhibitory receptor signal regulatory protein α (SIRPα)/CD172a and a low, but significant, level of a tetraspanin CD63, whose upregulation is closely associated with degranulation. Cross-linking SIRPα/CD172a on the surface of wild-type eosinophils significantly inhibited the release of eosinophil peroxidase induced by the calcium ionophore A23187, whereas this cross-linking effect was not observed in eosinophils isolated from mice expressing a mutated SIRPα/CD172a that lacks most of its cytoplasmic domain (SIRPα Cyto(-/-)). The SIRPα Cyto(-/-) eosinophils showed reduced viability, increased CD63 expression, and increased eosinophil peroxidase release with or without A23187 stimulation in vitro. In addition, SIRPα Cyto(-/-) mice showed increased frequencies of Annexin V-binding eosinophils and free MBP(+)CD63(+) extracellular granules, as well as increased tissue remodeling in the small intestine under steady-state conditions. Mice deficient in CD47, which is a ligand for SIRPα/CD172a, recapitulated these phenomena. Moreover, during Th2-biased inflammation, increased eosinophil cell death and degranulation were obvious in a number of tissues, including the small intestine, in the SIRPα Cyto(-/-) mice compared with wild-type mice. Collectively, our results indicated that SIRPα/CD172a regulates eosinophil homeostasis, probably by interacting with CD47, with substantial effects on eosinophil survival. Thus, SIRPα/CD172a is a potential therapeutic target for eosinophil-associated diseases.