Predictive Effect of Triglyceride-Glucose Index on Adverse Prognostic Events in Patients with Type 2 Diabetes Mellitus and Ischemic Cardiomyopathy.

Background: The triglyceride-glucose (TyG) index is regarded as an independent predictor of cardiovascular (CV) consequences and a reliable surrogate measure of insulin resistance (IR). However, the predictive significance of the TyG index in patients with type 2 diabetes mellitus (T2DM) and ischemic cardiomyopathy (ICM) remains unknown. Methods: This study included 1514 consecutive subjects with ICM and T2DM. The tertile of the TyG index values was used to categorize these patients into three groups. Major adverse cardiac and cerebral events (MACCEs) were also noted. The TyG index was calculated using the [fasting triglycerides (mg/dL) × fasting plasma glucose (mg/dL)/2] equation. Results: After adjusting for age, BMI, and other potential confounders, the scores of multivariate Cox proportional hazards regression models for chest pain [9.056 (4.370 to 18.767), p<0.001], acute myocardial infarction [4.437 (1.420 to 13.869), p=0.010], heart failure [7.334 (3.424 to 15.708), p<0.001], cardiogenic shock [3.707 (1.207 to 11.384), p=0.022], malignant arrhythmia [5.309 (2.367 to 11.908), p<0.001], cerebral infarction [3.127 (1.596 to 6.128), p<0.001], gastrointestinal bleeding [4.326 (1.612 to 11.613), p=0.004], all-cause death [4.502 (3.478 to 5.827), p<0.001] and cumulative incidence of MACCEs [4.856 (3.842 to 6.136), p<0.001] increased significantly with an increase in TyG index levels (all p<0.05). Time-dependent ROC analysis revealed that the area under the TyG index curve (AUC) reached 0.653 in the 3rd year, 0.688 in the 5th year, and 0.764 in the 10th year. The predictive efficiency of this model on MACCEs improved [net reclassification improvement (NRI): 0.361 (0.253 to 0.454); C-index: 0.678 (0.658 to 0.698); integrated discrimination improvement (IDI): 0.138 (0.098 to 0.175), all p<0.05] following the incorporation of the TyG index into the base risk model. Conclusion: TyG index could be useful in predicting MACCEs and initiating preventive measures in subjects with ICM and T2DM.

Membrane Bound CRT Fragment Accelerates Tumor Growth of Melanoma B16 Cell In Vivo through Promoting M2 Polarization via TLR4.

Calreticulin (CRT) is a major calcium-binding luminal resident protein on the endoplasmic reticulum that can also be released extracellular as well as anchored on surface of cells. Previously, we demonstrated that soluble recombinant CRT fragment 39-272 (CRT/39-272) exhibited potent immunostimulatory effects as well as immunoregulation effects on immune cells. Here, we constructed stable B16 melanoma cell lines expressing recombinant CRT/39-272 on the membrane (B16-tmCRT/39-272) to investigate the roles of cell surface CRT on tumor progression. We found that B16-tmCRT/39-272 cells subcutaneously inoculated into C57BL/6 mice exhibited stronger tumorigenicity than the B16-EGFP control cells. The tumor associated macrophages infiltrated in tumors were mainly M2 phenotype. Regulatory T cells (Tregs) were also expanded more in bearing mice. Consistent with the in vivo results, B16-tmCRT/39-272 promoted macrophage polarization toward F4/80+CD206+ M2 macrophages and promoted transforming growth factor beta (TGF-ß) secretion in vitro, which could promote naïve CD4+ T cell differentiation into Tregs. These results imply that the tmCRT/39-272 could accelerate tumor development by enhancing M2 macrophage polarization to induce TGF-ß secretion, and then promoted Treg differentiation in the tumor microenvironment. Our data may provide useful clues for better understanding of the potentiating roles of CRT in tumorigenesis.

Calreticulin as an Adjuvant In Vivo to Promote Dendritic Cell Maturation and Enhance Antigen-Specific T Lymphocyte Responses against Melanoma.

An endoplasmic reticulum resident protein, calreticulin (CRT), participates in many cellular processes. CRT is a tumor-associated antigen with an important role in antitumor immunity. Previously, we reported that the recombinant CRT fragment 39-272 (CRT/39-272) exhibited superior immunobiological activity, activating macrophages to release cytokines and promoting dendritic cell (DC) maturation. However, the effect of CRT/39-272 in vivo, especially its adjuvant effect on in vivo antitumor immune responses, was not fully investigated. In this study, we constructed a fusion protein linking CRT/39-272 to an ovalbumin (OVA) peptide (residues 182-297, OVAp) and used the fusion protein (OVAp-CRT) to examine the adjuvant effect of CRT. We investigated whether CRT/39-272 could induce bone marrow-derived DC maturation and strongly promote the proliferation of OVA-specific T cells in vitro. Compared with OVAp, OVAp-CRT induced stronger antigen-specific T lymphocyte responses, including antigen-specific T cell proliferation, interferon-γ secretion, and cytotoxic T lymphocyte responses. OVAp-CRT-immunized mice generated significantly increased OVAp-specific antibody and CD4+/CD8+ memory T cells, which mediated long-term protective effects. OVAp-CRT upregulated CD40, CD80, and CD86 expressions in splenic conventional DCs. Furthermore, OVAp-CRT protected immunized mice against OVA-expressing B16 melanoma cells in vivo. Moreover, mice that were adoptively transferred with OVAp-CRT-pulsed DCs showed inhibited tumor growth and prolonged mouse survival. Our results demonstrate that CRT/39-272 can be used as a potential new adjuvant for tumor vaccines, and this finding may be useful in tumor vaccine development.

Neutrophils as regulators of macrophage-induced inflammation in a setting of allogeneic bone marrow transplantation.

Clinical data reveal that patients with allogeneic hematopoietic stem cell transplantation (HSCT) are vulnerable to infection and prone to developing severe sepsis, which greatly compromises the success of transplantation, indicating a dysregulation of inflammatory immune response in this clinical setting. Here, by using a mouse model of haploidentical bone marrow transplantation (haplo-BMT), we found that uncontrolled macrophage inflammation underlies the pathogenesis of both LPS- and E.coli-induced sepsis in recipient animals with graft-versus-host disease (GVHD). Deficient neutrophil maturation in GVHD mice post-haplo-BMT diminished modulation of macrophage-induced inflammation, which was mechanistically dependent on MMP9-mediated activation of TGF-ß1. Accordingly, adoptive transfer of mature neutrophils purified from wild-type donor mice inhibited both sterile and infectious sepsis in GVHD mice post-haplo-BMT. Together, our findings identify a novel mature neutrophil-dependent regulation of macrophage inflammatory response in a haplo-BMT setting and provide useful clues for developing clinical strategies for patients suffering from post-HSCT sepsis.

Cytokine Cocktail Promotes Alveolar Macrophage Reconstitution and Functional Maturation in a Murine Model of Haploidentical Bone Marrow Transplantation.

Infectious pneumonia is one of the most common complications after bone marrow transplantation (BMT), which is considered to be associated with poor reconstitution and functional maturation of alveolar macrophages (AMs) post-transplantation. Here, we present evidence showing that lack of IL-13-secreting group 2 innate lymphoid cells (ILC2s) in the lungs may underlay poor AM reconstitution in a mouse model of haploidentical BMT (haplo-BMT). Recombinant murine IL-13 was able to potentiate monocyte-derived AM differentiation in vitro. When intranasally administered, a cocktail of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-13, and CCL2 not only promoted donor monocyte-derived AM reconstitution in haplo-BMT-recipient mice but also enhanced the innate immunity of the recipient animals against pulmonary bacterial infection. These results provide a useful clue for a clinical strategy to prevent pulmonary bacterial infection at the early stage of recipients post-BMT.

Control of axillary bud growth in tobacco through toxin gene expression system.

The control of axillary bud development after removing the terminal buds (topping) of plants is a research hotspot, and the control of gene expression, like switching on and off, allows us to further study biological traits of interest, such as plant branching and fertility. In this study, a toxin gene control system for plants based on dexamethasone (DEX) induction was constructed, and the positive transgenic tobacco exhibited growth retardation in the application area (axillary bud). The expression level of the lethal Diphtheria toxin A (DTA) gene under different DEX concentrations at different application days was analyzed. The highest expression levels appeared at 5 days after the leaf injection of DEX. The DTA transcripts were induced by 5 µM DEX and peaked in response to 50 µM DEX at 5 days after leaf injection. Here, a chemical induction system, combined with a toxin gene, were used to successfully control the growth of tobacco axillary buds after topping. The DTA expression system under DEX induction was sensitive and efficient, therefore, can be used to control axillary bud growth and development in tobacco.

ZBTB Transcription Factors: Key Regulators of the Development, Differentiation and Effector Function of T Cells.

The development and differentiation of T cells represents a long and highly coordinated, yet flexible at some points, pathway, along which the sequential and dynamic expressions of different transcriptional factors play prominent roles at multiple steps. The large ZBTB family comprises a diverse group of transcriptional factors, and many of them have emerged as critical factors that regulate the lineage commitment, differentiation and effector function of hematopoietic-derived cells as well as a variety of other developmental events. Within the T-cell lineage, several ZBTB proteins, including ZBTB1, ZBTB17, ZBTB7B (THPOK) and BCL6 (ZBTB27), mainly regulate the development and/or differentiation of conventional CD4/CD8 αß+ T cells, whereas ZBTB16 (PLZF) is essential for the development and function of innate-like unconventional γδ+ T & invariant NKT cells. Given the critical role of T cells in host defenses against infections/tumors and in the pathogenesis of many inflammatory disorders, we herein summarize the roles of fourteen ZBTB family members in the development, differentiation and effector function of both conventional and unconventional T cells as well as the underlying molecular mechanisms.

IgG Immunocomplexes Drive the Differentiation of a Novel Subset of Osteoclasts Independent of RANKL and Inflammatory Cytokines.

Potentiation of receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis by IgG immunocomplexes (ICs) is generally considered an important pathway leading to cartilage and bone destruction in rheumatoid arthritis (RA). However, whether IgG ICs possess pro-osteoclastogenic potential independent of RANKL and inflammatory cytokines is unclear. Here we demonstrate that by fully cross-linking human FcγRIIa (hFcγRIIa) or co-ligating hFcγRIIa and TLR4, IgG ICs alone could drive the differentiation of human blood monocytes into nuclear factor of activated T cells cytoplasmic 1 (NFATc1-negative nonclassical osteoclasts (NOCs). Surprisingly, IgG ICs could also overrule RANKL-induced classical osteoclast (COC) differentiation in vitro. In mouse model of collagen-induced arthritis, hFcγRIIa-transgenic, but not nontransgenic control, mice suffered from cartilage/bone destruction accompanied by the presence of NFATc1- NOCs lining the eroded cartilage surface in affected joints. Our results not only identify a novel subset of IC-induced NOCs but also provide a possible explanation for the uncoupling of FcγR-mediated cartilage destruction from RANKL-related bone erosion in autoinflammatory arthritis. © 2021 American Society for Bone and Mineral Research (ASBMR)..

NFKB1 gene rs28362491 ins/del variation is associated with higher susceptibility to myocardial infarction in a Chinese Han population.

Myocardial infarction (MI), the leading cause of mortality and disability worldwide, is a disease in which multiple environmental and genetic factors are involved. Recently, researches suggested that insertion/deletion (ins/del) variation of NFKB1 gene rs28362491 is a functional polymorphism. In the present study, we aimed to explore the relation between variation of NFKB1 gene rs28362491 and MI by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 359 MI patients and 1085 control participants. Gensini score was used to evaluate the degree of coronary artery stenosis in MI patients. The plasma levels of interleukin-6 (IL-6), IL-8, malonaldehyde (MDA) and superoxide dismutase (SOD) were randomly measured by ELISA both in MI patients and control participants. We found that the detected frequencies of D allele (41.2% vs. 36.4%, P = 0.021) and DD genotype (17.5% vs. 12.0%, P = 0.022) were significantly higher in MI patients than in control participants. Compared with II or ID genotype carriers, the Gensini score in MI patients with DD genotype was 32-43% higher (both P < 0.001). Moreover, DD genotype carries had more diseased coronary arteries (P = 0.001 vs. II or ID genotype). Of note, IL-6 levels in MI patients carrying DD genotype were significantly higher than that in control participants and other genotype carriers in MI patients (both P < 0.05). In conclusion, NFKB1 gene rs28362491 DD genotype was associated with a higher risk of MI and more severe coronary artery lesion, which also had a potential influence on the level of inflammatory cytokine IL-6.

Disease-Specific Autoantibodies Induce Trained Immunity in RA Synovial Tissues and Its Gene Signature Correlates with the Response to Clinical Therapy.

Much evidence suggests that trained immunity is inappropriately activated in the synovial tissue in rheumatoid arthritis (RA), but the underlying mechanism remains unclear. Here, we describe how RA-specific autoantibody deposits can train human monocytes to exert the hyperactive inflammatory response, particularly via the exacerbated release of tumor necrosis factor α (TNFα). Comparative transcriptomic analysis by plate-bound human IgG (cIgG) or ß-glucan indicated that metabolic shift towards glycolysis is a crucial mechanism for trained immunity. Moreover, the cIgG-trained gene signatures were enriched in synovial tissues from patients with ACPA- (anticitrullinated protein antibody-) positive arthralgia and undifferentiated arthritis, and early RA and established RA bore a great resemblance to the myeloid pathotype, suggesting a historical priming event in vivo. Additionally, the expression of the cIgG-trained signatures is higher in the female, older, and ACPA-positive populations, with a predictive role in the clinical response to infliximab. We conclude that RA-specific autoantibodies can train monocytes in the inflamed lesion as early as the asymptomatic stage, which may not merely improve understanding of disease progression but may also suggest therapeutic and/or preventive strategies for autoimmune diseases.

Overexpression of IκBα in cardiomyocytes alleviates hydrogen peroxide-induced apoptosis and autophagy by inhibiting NF-κB activation.

BACKGROUND: Inflammation and oxidative stress play predominant roles in the initiation and progression of ischaemia/reperfusion (I/R) injury, with nuclear factor kappa B (NF-κB) serving as a crucial mediator. Overexpression of the inhibitor of κB alpha (IκBα) gene is hypothesized to have protective effects against apoptosis and autophagy in cardiomyocytes subjected to hydrogen peroxide (H2O2) by inhibiting the NF-κB pathway. METHODS: The IκBαS32A, S36A gene was transfected via adeno-associated virus serotype 9 (AAV9) delivery into neonatal rat ventricular cardiomyocytes (NRVMs) prior to H2O2 treatment. NRVMs were divided into control, H2O2, GFP + H2O2, IκBα+H2O2, and pyrrolidine dithiocarbamate (PDTC) + H2O2 groups. Nuclear translocation of the NF-κB p65 subunit was evaluated by immunofluorescence and Western blotting. Cell viability was assessed by Cell Counting Kit-8 assay. Supernatant lactate dehydrogenase (LDH) and intracellular malondialdehyde (MDA) were measured to identify H2O2-stimulated cytotoxicity. Apoptosis was determined by Annexin V-PE/7-AAD staining, and the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. Western blotting was used to detect apoptosis- and autophagy-related proteins. RESULTS: IκBα transfection significantly increased cell viability and ΔΨm but decreased the supernatant LDH and cellular MDA levels in cardiomyocytes exposed to H2O2. Meanwhile, IκBα overexpression decreased H2O2-induced apoptosis by upregulating the Bcl-2/Bax ratio and reduced autophagy by downregulating the expression of Beclin-1 and the LC3-II/LC3-I ratio. These effects partly accounted for the ability of IκBα to inhibit the NF-κB signalling pathway, as evidenced by decreases in p65 phosphorylation and nuclear translocation. Indeed, the effects of inactivation of NF-κB signalling with the specific inhibitor PDTC resembled the cardioprotective effects of IκBα during H2O2 stimulation. CONCLUSION: IκBα overexpression can ameliorate H2O2-induced apoptosis, autophagy, oxidative injury, and ΔΨm loss through inhibition of the NF-κB signalling pathway. These findings suggest that IκBα transfection can result in successful resistance to oxidative stress-induced damage by inhibiting NF-κB activation, which may provide a potential therapeutic target for the prevention of myocardial I/R injury.

rAAV9-Mediated MEK1 Gene Expression Restores Post-conditioning Protection Against Ischemia Injury in Hypertrophic Myocardium.

PURPOSE: We investigated whether increased expression of activated mitogen-activated protein kinase (MAPK) kinases 1 (MEK1) restores ischemic post-conditioning (IPostC) protection in hypertrophic myocardium following ischemia/reperfusion (I/R) injury. METHODS: C57Bl/6 mice received recombinant adeno-associated virus type 9 (rAAV9)-mediated activated MEK1 gene delivery systemically, then following the induction of cardiac hypertrophy via transverse aortic constriction for 4 weeks. In a Langendorff model, hypertrophic hearts were subjected to 40 min/60 min I/R or with IPostC intervention consisting of 6 cycles of 10 s reperfusion and 10 s no-flow before a 60-min reperfusion. Hemodynamics, infarct size (IS), myocyte apoptosis and changes in expression of reperfusion injury salvage kinase (RISK) pathway were examined. RESULTS: rAAV9-MEK1 gene delivery led to a 4.3-fold and 2.7-fold increase in MEK1 mRNA and protein expression in the heart versus their control values. I/R resulted in a larger IS in hypertrophic than in non-hypertrophic hearts (52.3 ± 4.7% vs. 40.0 ± 2.5%, P < 0.05). IPostC mediated IS reduction in non-hypertrophic hearts (27.6 ± 2.6%, P < 0.05), while it had no significant effect in hypertrophic hearts (46.5 ± 3.1%, P=NS) compared with the IS in non-hypertrophic or hypertrophic hearts subjected to I/R injury only, respectively. Hemodynamic decline induced by I/R was preserved by IPostC in non-hypertrophic hearts but not in hypertrophic hearts. rAAV9-MEK1 gene delivery restored IPostC protection in hypertrophic hearts evidenced by reduced IS (32.0 ± 2.8% vs. 46.5 ± 3.1%) and cardiac cell apoptosis and largely preserved hemodynamic parameters. These protective effects were associated with significantly increased phosphorylation of ERK1/2 and ribosomal protein S6 kinases (p70S6K), but it had no influence on Akt and glycogen synthase kinase-3ß. CONCLUSION: These results demonstrated that rAAV9-mediated activated MEK1 expression restores IPostC protection in the hypertrophic heart against I/R injury through the activation of ERK pathway.

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.

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.

IgG Immunocomplexes Sensitize Human Monocytes for Inflammatory Hyperactivity via Transcriptomic and Epigenetic Reprogramming in Rheumatoid Arthritis.

Prevalence of circulating immunocomplexes (ICs) strongly correlates with rheumatoid arthritis (RA) in humans. Deposits of IgG-ICs are abundant in affected joints of patients, yet molecular mechanisms for the pathogenic roles of such ICs are not fully understood. In this study, we present evidence that IgG-ICs precipitated from RA sera sensitized human monocytes for a long-lasting inflammatory functional state, characterized by a strong TNF-α response to cellular proteins representing damage-associated molecular patterns and microbe-derived pathogen-associated molecular patterns. Importantly, plate-coated human IgG (a mimic of deposited IC without Ag restriction) exhibited a similarly robust ability of monocyte sensitization in vitro. The plate-coated human IgG-induced functional programming is accompanied by transcriptomic and epigenetic modification of various inflammatory cytokines and negative regulator genes. Moreover, macrophages freshly isolated from synovia of patients with RA, but not sera-negative arthropathy, displayed a signature gene expression profile highly similar to that of IC-sensitized human monocytes, indicative of historical priming events by IgG-ICs in vivo. Thus, the ability of IgG-ICs to drive sustainable functional sensitization/reprogramming of monocytes and macrophages toward inflammation may render them key players in the development of RA.

Aberrant Glycosylation Augments the Immuno-Stimulatory Activities of Soluble Calreticulin.

Calreticulin (CRT), a luminal resident calcium-binding glycoprotein of the cell, is a tumor-associated antigen involved in tumorigenesis and also an autoantigen targeted by autoantibodies found in patients with various autoimmune diseases. We have previously shown that prokaryotically expressed recombinant murine CRT (rCRT) exhibits strong stimulatory activities against monocytes/macrophages in vitro and potent immunogenicity in vivo, which is partially attributable to self-oligomerization of soluble rCRT. However, even in oligomerized form native CRT (nCRT) isolated from mouse liver is much less active than rCRT, arguing against the possibility that self-oligomerization alone would license potent pro-inflammatory properties to nCRT. Since rCRT differs from nCRT in its lack of glycosylation, we wondered if aberrant glycosylation of eukaryotically expressed CRT (eCRT) would significantly enhance its immunological activity. In the present study, tunicamycin, an N-glycosyltransferase inhibitor, was employed to treat CHO cells (CHO-CRT) stably expressing full-length recombinant mouse CRT in secreted form for preparation of aberrantly glycosylated eCRT (tun-eCRT). Our biochemical and immunological analysis results indicate that eCRT produced by CHO-CRT cells is similar to nCRT in terms of glycosylation level, lack of self-oligomerization, relatively poor immunogenicity and weak macrophage-stimulatory activity, while tun-eCRT shows reduced glycosylation yet much enhanced ability to elicit specific humoral responses in mice and TNF-α and nitric oxide production by macrophages in vitro. Given that abberant glycosylation of proteins is a hallmark of cancer cells and also related to the development of autoimmune disorders in humans, our data may provide useful clues for better understanding of potentiating roles of dysregulated glycosylation of molecules such as CRT in tumorigenesis and autoimmunity.

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.

Endogenous Annexin-A1 Regulates Haematopoietic Stem Cell Mobilisation and Inflammatory Response Post Myocardial Infarction in Mice In Vivo.

Endogenous anti-inflammatory annexin-A1 (ANX-A1) plays an important role in preserving left ventricular (LV) viability and function after ischaemic insults in vitro, but its long-term cardioprotective actions in vivo are largely unknown. We tested the hypothesis that ANX-A1-deficiency exaggerates inflammation, haematopoietic stem progenitor cell (HSPC) activity and LV remodelling in response to myocardial ischaemia in vivo. Adult ANX - A1 -/- mice subjected to coronary artery occlusion exhibited increased infarct size and LV macrophage content after 24-48 h reperfusion compared with wildtype (WT) counterparts. In addition, ANX - A1 -/- mice exhibited greater expansion of HSPCs and altered pattern of HSPC mobilisation 8 days post-myocardial infarction, with increased circulating neutrophils and platelets, consistent with increased cardiac inflammation as a result of increased myeloid invading injured myocardium in response to MI. Furthermore, ANX - A1 -/- mice exhibited significantly increased expression of LV pro-inflammatory and pro-fibrotic genes and collagen deposition after MI compared to WT counterparts. ANX-A1-deficiency increased cardiac necrosis, inflammation, hypertrophy and fibrosis following MI, accompanied by exaggerated HSPC activity and impaired macrophage phenotype. These findings suggest that endogenous ANX-A1 regulates mobilisation and differentiation of HSPCs. Limiting excessive monocyte/neutrophil production may limit LV damage in vivo. Our findings support further development of novel ANX-A1-based therapies to improve cardiac outcomes after MI.

Calreticulin Fragment 39-272 Promotes B16 Melanoma Malignancy through Myeloid-Derived Suppressor Cells In Vivo.

Calreticulin (CRT), a multifunctional Ca2+-binding glycoprotein mainly located in the endoplasmic reticulum, is a tumor-associated antigen that has been shown to play protective roles in angiogenesis suppression and anti-tumor immunity. We previously reported that soluble CRT (sCRT) was functionally similar to heat shock proteins or damage-associated molecular patterns in terms of ability to activate myeloid cells and elicit strong inflammatory cytokine production. In the present study, B16 melanoma cell lines expressing recombinant CRT fragment 39-272 (sCRT/39-272) in secreted form (B16-CRT), or recombinant enhanced green fluorescence protein (rEGFP) (B16-EGFP), were constructed for investigation on the roles of sCRT in tumor development. When s.c. inoculated into C57BL/6 mice, the B16-CRT cells were significantly more aggressive (in terms of solid tumor growth rate) than B16-EGFP controls in a TLR4- and myeloid-derived suppressor cells (MDSC)-dependent manner. The B16-CRT-bearing mice showed increased Gr1+ MDSC infiltration in tumor tissues, accelerated proliferation of CD11b+Ly6G+Ly6Clow (G-MDSC) precursors in bone marrow, and higher percentages of G-MDSCs in spleen and blood, which was mirrored by decreased percentage of dendritic cells (DC) in periphery. In in vitro studies, recombinant sCRT/39-272 was able to promote migration and survival of tumor-derived MDSCs via interaction with TLR4, inhibit MDSC differentiation into DC, and also elicit expression of inflammatory proteins S100A8 and S100A9 which are essential for functional maturation and chemotactic migration of MDSCs. Our data provide solid evidence for CRT as a double-edged sword in tumor development.