Melatonin ameliorates bleomycin-induced pulmonary fibrosis via activating NRF2 and inhibiting galectin-3 expression.

Pulmonary fibrosis (PF) is a chronic interstitial lung disease with no effective therapies. Galectin-3 (Gal-3), a marker of oxidative stress, plays a key role in the pathogenesis of PF. Fibroblast-myofibroblast differentiation (FMD) is an important source of fibrotic cells in PF. Previous studies showed that melatonin (MT) exerted anti-fibrotic effect in many diseases including PF through its antioxidant activity. In the present study we investigated the relationships among Gal-3, NRF2, ROS in FMD and their regulation by MT. We established an in vitro model of FMD in TGF-ß1-treated human fetal lung fibroblast1 (HFL1) cells and a PF mouse model via bleomycin (BLM) intratracheal instillation. We found that Gal-3 expression was significantly increased both in vitro and in vivo. Knockdown of Gal-3 in HFL1 cells markedly attenuated TGF-ß1-induced FMD process and ROS accumulation. In TGF-ß1-treated HFL1 cells, pretreatment with NRF2-specific inhibitor ML385 (5 µM) significantly increased the levels of Gal-3, α-SMA and ROS, suggesting that the expression of Gal-3 was regulated by NRF2. Treatment with NRF2-activator MT (250 µM) blocked α-SMA and ROS accumulation accompanied by reduced Gal-3 expression. In BLM-induced PF model, administration of MT (5 mg·kg-1·d-1, ip for 14 or 28 days) significantly attenuated the progression of lung fibrosis through up-regulating NRF2 and down-regulating Gal-3 expression in lung tissues. These results suggest that Gal-3 regulates TGF-ß1-induced pro-fibrogenic responses and ROS production in FMD, and MT activates NRF2 to block FMD process by down-regulating Gal-3 expression. This study provides a useful clue for a clinical strategy to prevent PF. Graphic abstract of the mechanisms. MT attenuated BLM-induced PF via activating NRF2 and inhibiting Gal-3 expression.

Enhanced Mechanical Stability and Proton Conductivity Performance from the Dense Mn(II)-Metal-Organic Framework to Porous Mn(II)-Fe(III)-Metal-Organic Framework.

Postsynthetic modification (PSM) of the metal-organic framework (MOF) has been demonstrated to be an effective strategy to enhance performance. In this particular work, the anion framework Mn-MOF {[Mn3O(H2O)3(HTC)]2-} (HTC6- = (5'-(3,5-dicarboxyphenyl)-[1,1':3',1″-terphenyl]-3,3″,5,5″-tetracarboxylate] was obtained, and NH2(CH3)2+ ions were filled within the pores to balance the charge. In order to release the internal pores of Mn-MOF, the trivalent Fe(III) was introduced instead of Mn(II) nodes, resulting in the porous Mn1-xFex-MOF, and the NH2(CH3)2+ ions were simultaneously deported from the pores. The content of Fe(III) in Mn1-xFex-MOF was highly dependent on the concentration of Fe(III) solution, and the maximum could be up to Mn0.05Fe0.95-MOF with a BET surface area of 1209.457 m2 g-1. Compared to the amorphization of dense Mn-MOF at 0.8 GPa in a diamond anvil cell, the mechanical stability of porous Mn0.05Fe0.95-MOF has been dramatically enhanced, and the framework integrity could be maintained up to 16.5 GPa. The proton conductivity for the Mn1-xFex-MOF series was also investigated, where Mn0.93Fe0.07-MOF showed the best performance of 1.47 × 10-2 S cm-1 under 70 °C and 98% RH due to the onset of reversed charge from the anionic framework to cationic framework and the formation of the most compact hydrogen bonding net. This work has not only provided an example for the PSM strategy but also illustrated that the versatile functionalities of MOF materials were mainly ascribed to the tunable porosity.

M860, a Monoclonal Antibody against Human Lactoferrin, Enhances Tumoricidal Activity of Low Dosage Lactoferrin via Granzyme B Induction.

Lactoferrin (LF) is a soluble glycoprotein of the transferring family found in most biological fluids, functioning as a major first line defense molecule against infection in mammals. It also shows certain anti-tumor activity, but its clinical application in tumor therapy is limited because high dosage is required. In this study, we demonstrate that M860, a monoclonal antibody against human LF (hLF), could significantly increase the anti-tumor potential of low dosage hLF by forming LF-containing immune complex (IC). Human monocytes primed with LF-IC, but not hLF or M860 alone, or control ICs, showed strong tumoricidal activity on leukemia cell lines Jurkat and Raji through induction of secreted Granzyme B (GzB). LF-IC is able to colligate membrane-bound CD14 (a TLR4 co-receptor) and FcγRIIa (a low affinity activating Fcγ receptor) on the surface of human monocytes, thereby triggering the Syk-PI3K-AKT-mTOR pathway leading to GzB production. Our work identifies a novel pathway for LF-mediated tumoricidal activity and may extend the clinical application of LF in tumor therapy.

Fingerprint Analysis and Quantitative Determination of Fourteen Active Components in the Traditional Chinese Medicinal Preparation Feiyangchangweiyan Capsule by HPLC-DAD-ESI-MS/MS.

FeiYangchangweiyan capsule (FY capsule), a traditional Chinese medicinal preparation consisting of three medicinal herbs, has been used to treat bacterial dysentery, acute, and chronic gastroenteritis for several decades. In this study, a novel, convenient, accurate, and valid method was developed by using high-performance liquid chromatography (HPLC) coupled with diode array detection (DAD) to obtain a chromatographic fingerprint of FeiYangchangweiyan capsule (FY capsule). Then, fourteen peaks were identified according to MS/MS fragmentation behavior of the reference standards by using HPLC-DAD-ESI-MS/MS analysis. At the same time, the fingerprint similarity was calculated and the contents of known ingredients were also determined simultaneously. The result demonstrated that the HPLC fingerprint combining similarity evaluation and quantification analysis can be successfully applied to control the quality of FY capsule.

GSP-2, a polysaccharide extracted from Ganoderma sinense, is a novel toll-like receptor 4 agonist.

Ganoderma sinense is a Chinese unique medicinal fungus that has been used in folk medicine for thousands of years. Polysaccharides are considered to be biologically active ingredients due to their immune-modulating functions. Previously we found that GSP-2, a new polysaccharide isolated from Ganoderma sinense, exerts an immunomodulatory effect in human peripheral blood mononuclear cells but the underlying mechanism is unclear. The present study aimed to investigate how GSP-2 triggers immunologic responses and the implicated signaling pathways. GSP-2 effects were investigated both in a macrophagic cell line, RAW264.7, and in primary macrophages. Moreover, the molecular basis of GSP-2 recognition by immune cells, and the consequent activation of signaling cascades, were explored by employing recombinant human HEK293-TLR-Blue clones, individually overexpressing various Toll-like receptors. GSP-2 dose-dependently induced the overexpression of Toll-like receptor 4 (TLR4) but did not affect the expression of other TLRs. Moreover, GSP-2 induced TNFα secretion in primary macrophages from wild-type, but not TLR4-knockout mice. In addition, GSP-2 upregulated TLR4 protein expression and activated the MAPK pathway in RAW246.7 macrophages. Finally, GSP-2 induced the production of the cytokines TNFα, IL1ß, and IL6. Our data demonstrated that GSP-2 was specifically recognized by TLR4, promoting cytokine secretion and immune modulation in macrophages.

ZBTB24 regulates the apoptosis of human T cells via CDCA7/TRAIL-receptor axis.

Mutations in ZBTB24 and CDCA7 cause the Immunodeficiency, Centromeric Instability and Facial Anomalies syndrome type 2 and 3 (ICF2/3), respectively. Most ICF2 patients carry ZBTB24 nonsense mutations and are thus ZBTB24-deficient. Although the immune deficiency in ICF2 patients is primarily regarded as a B-cell defect due to the greatly reduced serum antibodies and circulating memory B cells, the reduced expansions of PBMCs stimulated by mitogens or recall antigens suggest a T-cell defect in these patients as well. However, the molecular mechanisms behind this T-cell dysfunction remain unknown. In the present study, we demonstrated that ZBTB24-deficiency significantly represses the proliferation of human T cells by promoting TRAIL-induced cell death. Downregulation of ZBTB24 in both Jurkat and human primary T cells upregulates the expression of TRAIL and/or its death receptors (TRAIL-R1/2), and induces significant amount of cells to undergo apoptosis. The profound survival defects of ZBTB24-deficient cells are largely reversed by blocking TRAIL/TRAIL-R interactions with exogenous recombinant TRAIL-R2. Moreover, ZBTB24-downregulation reduces the expression of CDCA7, and knockdown of the latter in human T cells results in a phenotype resembling that caused by ZBTB24-depletion. Functionally, overexpression of CDCA7 abrogates the increased apoptosis in ZBTB24-depleted Jurkat T cells. Together, these data indicated that ZBTB24 regulates human T-cell apoptosis via CDCA7/TRAIL-R axis. Our study thus not only provides a molecular explanation for the T-cell defects in ZBTB24-deficient ICF2 patients, but also highlights a convergence between ZBTB24 and CDCA7, the two ICF genes, in modulating the functions of T cells.

Author Correction: Extraordinarily potent proinflammatory properties of lactoferrin-containing immunocomplexes against human monocytes and macrophages.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Lactoferrin-Containing Immunocomplexes Drive the Conversion of Human Macrophages from M2- into M1-like Phenotype.

Macrophages are multifunctional cells that perform diverse roles in health and disease and considered the main source of inflammatory cytokines in affected joints of patients with rheumatoid arthritis (RA). M2 macrophages are well known as anti-inflammation and wound-healing cells; however, recent evidence suggests that they can also promote inflammation in RA, although the underlying mechanism remains to be clarified. Based upon our recent finding that lactoferrin (LTF)-containing IgG immunocomplex (LTF-IC), found elevated in RA sera, potent activators of human monocytes/macrophages, we herein demonstrate that LTF-IC was able to elicit immediate proinflammatory cytokine production by M2-polarized human macrophages through coligation with CD14/toll-like receptor (TLR) 4 and FcγRIIa (CD32a). The LTF-IC-treated M2 cells adopted surface maker expression profile similar to that of M1 phenotype and became functionally hyperactive to subsequent stimuli such as lipopolysaccharide, zymosan and IL-1ß, which could provide a positive feedback signal to promote excessive inflammation in RA. They also acquired the ability to facilitate activation of Th17 cells that are known to play critical roles in RA pathology. We propose that IgG ICs containing TLR agonizing autoantigens are able to directly switch human macrophages from M2 into M1-like phenotype, thereby promoting excessive inflammation in autoimmune diseases such as RA.

Extraordinarily potent proinflammatory properties of lactoferrin-containing immunocomplexes against human monocytes and macrophages.

Lactoferrin (LTF), an important first line defense molecule against infection, is a common target for humoral autoimmune reactions in humans. Since LTF is a multifunctional protein capable of activating innate immune cells via various surface receptors, we hypothesized that LTF-containing immune complexes (ICs) (LTF-ICs), likely formed in patients with high titer anti-LTF autoantibodies, could possess unique monocyte/macrophage-activating properties compared with other ICs. ELISA analysis on serum samples from rheumatoid arthritis (RA) patients (n = 80) and healthy controls (n = 35) for anti-LTF autoantibodies confirmed a positive correlation between circulating LTF-specific IgG and RA. ICs between human LTF and LTF-specific IgG purified from patient sera or immunized rabbits and mice, but not control ICs, LTF or Abs alone, elicited strong production of TNF-α and IL-1ß by freshly fractionated human peripheral blood monocytes and monocytes-derived macrophages. Furthermore, LTF-ICs utilized both membrane-anchored CD14 and CD32a (FcγRIIa) to trigger monocyte activation in an internalization-, Toll-like receptor (TLR)4- and TLR9-dependent manner, and also that LTF-IC-induced cytokine production was blocked by specific inhibitors of caspase-1, NF-κB and MAPK. These results uncover a possible pathway for LTF-ICs perpetuating local inflammation and contributing to the pathogenesis of autoimmune diseases by triggering activation of infiltrating monocytes or tissue macrophages in vivo.

Expression, purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe.

Lactoferrin (LTF), a multifunctional glycoprotein of the transferrin family mainly found in exotic secretions in mammals, is an important defense molecule against not only microbial invasion but also tumors. It folds into two globular domains (N- and C-lobes) each containing an iron-binding site. The cationic antimicrobial peptide in N-lobe is known to exert anti-tumor effect via a non-receptor-mediated pathway. However, whether LTF C-lobe also contributes to its anti-tumor activity remains to be investigated. In this study, a human LTF fragment (amino acid residues 343-682) covering the C-lobe was expressed with a histidine tag in E. coli and the purified polypeptide refolded through a series of buffer changing procedure. The resultant recombinant protein caused significant growth arrest of breast carcinoma cells MDA-MB-231 in a dose- and time-dependent manner, evidently via induction of apoptosis of the cell. Our data suggest a positive role for the C-lobe of human LTF in controlling tumors in vitro.

A Novel Murine Anti-Lactoferrin Monoclonal Antibody Activates Human Polymorphonuclear Leukocytes through Membrane-Bound Lactoferrin and TLR4.

Soluble lactoferrin (LTF) is a versatile molecule that not only regulates the iron homeostasis, but also harbors direct microbicidal and immunomodulating abilities in mammalian body fluids. In contrast, little is known about the function of membrane-bound LTF (mbLTF), although its expression on human polymorphonuclear leukocytes (huPMNs) has been reported for decades. Given that LTF/anti-LTF antibodies represent a potential diagnostic/prognostic biomarker and a therapeutic target in patients with immune disorders, we wished, in the present study, to generate a novel human LTF- (huLTF-) specific mAb suitable for detailed analyses on the expression and function of mbLTF as well as for deciphering the underlying mechanisms. By using the traditional hybridoma cell fusion technology, we obtained a murine IgG1 (kappa) mAb, M-860, against huLTF. M-860 recognizes a conformational epitope of huLTF as it binds to natural, but not denatured, huLTF in ELISA. Moreover, M-860 detects mbLTF by FACS and captures endogenous huLTF in total cell lysates of huPMNs. Functionally, M-860 induces the activation of huPMNs partially through TLR4 but independently of phagocytosis. M-860 is thus a powerful tool to analyze the expression and function of human mbLTF, which will further our understanding of the roles of LTF in health and disease.

Disease association and arthritogenic potential of circulating antibodies against the α1,4-polygalacturonic acid moiety.

Much progress has been made in recent years on the diagnostic value, Ag specificity, and pathogenic roles of autoantibodies correlated to the development of rheumatoid arthritis (RA) in humans. However, carbohydrate Ag-specific autoantibodies that may also play important roles in RA have largely been ignored. In this article, we report that serum levels of Abs capable of recognizing α1,4-polygalacturonic acid [(PGA); major structural component of pectin] strongly correlate with RA in humans. The measurements of PGA-specific Abs (PGA-Abs) in sera are comparable to rheumatoid factors and anti-cyclic citrullinated peptide Abs as serological diagnostic markers for RA in terms of sensitivity and specificity. Immunohistochemical staining results indicate that the PGA-Abs selectively bound synovial membrane cells and chondrocytes in the joints of both humans and rabbits (but not rodents). Induction of PGA-Abs by s.c. immunization of rabbits with carrier protein-conjugated synthetic PGA led to severe inflammatory reactions (synovial hyperplasia, small vessel proliferation, and inflammatory cell infiltration) in the joints. Injection of affinity purified anti-PGA IgG into the synovial cavity of rabbits resulted in accumulation of proinflammatory cytokines such as TNF-α, IL-8, and IL-1ß in synovial fluid, as well as local pathological damage. We conclude that the PGA-cross-reactive moiety represents a major autoantigen in the joints and can be targeted by autoantibodies capable of triggering arthritogenic responses in vivo.

Adjuvanticity of a recombinant calreticulin fragment in assisting anti-ß-glucan IgG responses in T cell-deficient mice.

Polysaccharide-encapsulated fungi are the chief source of diseases in immunocompromised hosts such as those infected with human immunodeficiency virus or neutropenia patients. Currently available polysaccharide-protein conjugate vaccines are mainly T cell dependent and are usually ineffective in weakened immune systems. In this study, laminarin, a well-characterized ß-1,3-glucan, was conjugated with a prokaryotically expressed recombinant fragment (amino acids [aa] 39 to 272) of calreticulin (rCRT/39-272), which exhibits extraordinarily potent immunogenicity and adjuvanticity in experimental animals. The resultant conjugate reserves the immunostimulatory effect of rCRT/39-272 on naïve murine B cells and is capable of eliciting anti-ß-glucan IgG (mostly IgG1) responses in not only BALB/c mice but also athymic nude mice. Laminarin-CRT-induced mouse antibodies (Abs) are able to bind with Candida albicans and inhibit its growth in vitro. In addition, vaccination with laminarin-CRT partially protects mice from lethal C. albicans challenge. These results imply that rCRT/39-272 could be used as an ideal carrier or adjuvant for carbohydrate vaccines aimed at inducing or boosting IgG responses to fungal infections in immunodeficient hosts.