Upregulation of HSP72 attenuates tendon adhesion by regulating fibroblast proliferation and collagen production via blockade of the STAT3 signaling pathway.

The proliferation of fibroblasts creates an environment favoring post-operative tendon adhesion, but targeted therapy of this pathology remains in its infancy. In this study, we explored the effect of heat shock protein 72 (HSP72), a major inducible member of the heat shock protein family that can protect cells against many cellular stresses including heat shock, on fibroblast proliferation in tendon adhesion, with its underlying mechanisms investigated. HSP72 expression was examined in an established rat model of tendon injury using RT-qPCR and immunoblot analysis. After conducting ectopic expression and depletion experiments in fibroblast NIH3T3 cells, we determined the effects of HSP72 on the expression of α-SMA and STAT3 signaling pathway-related genes, fibroblast proliferation, as well as collagen production. The mRNA (65.46%) and protein (63.65%) expression of HSP72 was downregulated in the rat model of tendon injury. The in vitro experiments revealed that overexpression of HSP72 inhibited fibroblast proliferation (42.57%) and collagen production (45.60%), as well as reducing α-SMA expression (42.49%) and the extent of STAT3 phosphorylation (55.46%). Moreover, we observed that HSP72 overexpression reduced inflammation as well as the number of inflammatory cell infiltration and fibroblasts in vivo. Furthermore, the inhibited extent of STAT3 phosphorylation contributed to the impaired fibroblast proliferation and collagen production evoked by upregulated HSP72. In summary, the present study unveils an inhibitory role of HSP72 in tendon adhesion via inactivation of the STAT3 signaling pathway. This finding may enable the development of new therapeutic strategies for the prevention against tendon adhesion.

Thermal Preconditioning May Prevent Tendon Adhesion by Up-Regulating HSP72 in Rats.

BACKGROUND/AIMS: The study aims to determine the effects of thermal preconditioning on tendon adhesion by regulating the expression of heat shock protein 72 (HSP72) in rat models. METHODS: Sixty male Wistar rats were collected and randomly assigned into the thermal preconditioning and control groups. During the 4th and 8th weeks following surgery, 15 rats were sacrificed in each period respectively, and their tendon adhesion was observed and evaluated. Biomechanical testing was performed to measure the tensile strength and gliding distance of tendons. Hematoxylin-eosin (HE) was used to observe the morphological structure of the tendons. Immunohistochemical staining, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the HSP72, fibroblast growth factor-2 (FGF-2), fibroblast growth factor receptor-1 (FGFR-1), ß-catenin, epithelial cell adhesion molecule (EPCAM), Tenomodulin and scleraxis protein expressions. Pearson correlation analysis was applied to analyze the correlation between HSP72 expression and tendon adhesion. RESULTS: At the 4th week after surgery, we found no differences in the tendon adhesion scores or mRNA and protein expressions of HSP72 between the thermal preconditioning and control groups. However, after the 8th week after surgery, the thermal preconditioning group had a lower tendon adhesion score and higher mRNA and protein expressions of HSP72 than the control group. During the same period, we found longer gliding distance and higher expression levels of FGF-2, FGFR-1, ß-catenin, Tenomodulin and scleraxis, but lower EPCAM expression in the thermal preconditioning group. Pearson correlation analysis indicated that HSP72 mRNA and protein expression levels were negatively correlated with tendon adhesion. CONCLUSIONS: These findings provide evidence that thermal preconditioning may alleviate tendon adhesions via upregulation of HSP72 expression.

Correlations Between COL2A and Aggrecan Genetic Polymorphisms and the Risk and Clinicopathological Features of Intervertebral Disc Degeneration in a Chinese Han Population: A Case-Control Study.

OBJECTIVES: This case-control study was designed to evaluate the association of three COL2A1 single nucleotide polymorphism (SNPs) (rs1793953, rs2276454, and rs1793937) and Aggrecan variable number of tandem repeat (VNTR) polymorphisms with the risk and clinicopathological features of intervertebral disc degeneration (IVDD) in a Chinese Han population. MATERIALS AND METHODS: Data from 295 IVDD patients (case group) and 324 healthy volunteers (control group) were collected between January 2012 and December 2014. Magnetic resonance examinations were conducted on all included subjects. The frequency distributions of the COL2A1 and Aggrecan polymorphisms were detected using direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, respectively. RESULTS: The genotype and allele frequencies of the COL2A1 genetic polymorphisms (rs1793953 and rs2276454) and the Aggrecan VNTR polymorphisms differed significantly between the case group and the control group (all p < 0.05). The haplotype analysis indicated that the frequencies of ACGL (L, long) and GTCL haplotypes were lower in the case group than in the control group (both p < 0.05). In the case group, the genotype and allele frequencies of the COL2A1 genes, rs1793953 and rs2276454, and Aggrecan VNTR significantly differed in terms of Pfirrmann grades III, IV, and V (all p < 0.05). Personal history of spine sprain or crush injury, history of IVDD in a first-degree relative, and COL2A1 rs2276454 and Aggrecan VNTR presence may be independent risk factors of IVDD (all p < 0.05, odds ratio [OR] >1), whereas tea drinking habit, part-time sports participation, and COL2A1 rs1793953 presence may be protective factors of IVDD (all p < 0.05, OR <1). CONCLUSION: Our study provides evidence that COL2A1 and Aggrecan genetic polymorphisms may be correlated with the risk and clinicopathological features of IVDD in a Chinese Han population, and ACGL and GTCL haplotypes may be protective factors of IVDD.

Elevated expression of pleiotrophin in lymphocytic leukemia CD19+ B cells.

Pleiotrophin (PTN) has been demonstrated to be strongly expressed in many fetal tissues, but seldom in healthy adult tissues. While PTN has been reported to be expressed in many types of tumors as well as at high serum concentrations in patients with many types of cancer, to date, there has been no report that PTN is expressed in leukemia, especially in lymphocytic leukemia. We isolated the CD19(+) subset of B cells from peripheral blood from healthy adults, B-cell acute lymphocytic leukemia (B-ALL) patients, and B-cell chronic lymphocytic leukemia (B-CLL) patients and examined these cells for PTN mRNA and protein expression. We used immunocytochemistry, western blotting, and enzyme-linked immunosorbent assay to show that PTN protein is highly expressed in CD19(+) B cells from B-ALL and B-CLL patients, but barely expressed in B cells from healthy adults. We also examined PTN expression at the nucleic acid level using reverse transcription polymerase chain reaction (RT-PCR) and northern blotting and detected a high levels of PTN transcripts in the CD19(+) B cells from both groups of leukemia patients, but very few in the CD19(+) B cells from the healthy controls. Interestingly, the quantity of the PTN transcripts correlated with the severity of disease. Moreover, suppression of PTN activity with an anti-PTN antibody promoted apoptosis of cells from leukemia patients and cell lines SMS-SB and JVM-2. This effect of the anti-PTN antibody suggests that PTN may be a new target for the treatment of lymphocytic leukemia.

High dose lipopolysaccharide triggers polarization of mouse thymic Th17 cells in vitro in the presence of mature dendritic cells.

Lipopolysaccharide (LPS) plays an important role in the activation of innate immune cells, leading to secretion of proinflammatory factors and bridging the adaptive immune system. Exposing total mouse thymic cells culture to LPS induced a unique expression profile of cytokines (IL-17A, IL-17F, and IL-22) and the essential ROR-γt master transcription factor, which suggested a preferential differentiation of thymocytes towards the Th17 cell phenotype. Th17-polarizing molecules (IL-23, IL-23R, IL-6, and TGF-ß) and IL-17A(+)CD4(+) thymocytes were also specifically produced by the in vitro LPS-stimulation of thymic cells. Furthermore, both the expression of Th17 differentiation-related molecules and the frequency of Th17 cells were significantly up-regulated with increasing doses of LPS, as evidenced by quantitative RT-PCR and flow cytometric analysis, respectively. The expressions and frequency reached maximum levels when LPS exposure had been maintained at an extremely high concentration (100 µg/mL) for 48 h. On the other hand, depletion of thymic dendritic cells (DCs) blocked the LPS-induced polarization of thymus-derived Th17 cell lineage. Addition of bone marrow-derived DCs (BMDCs) to the purified immature CD4(+) CD62L(low) thymocytes culture recovered the switch towards Th17 cells, which synergistically prompted the cytotoxic activity of CD8(+) T cells. Taken together, our data indicates that high doses of LPS can promote the differentiation of mouse thymus-derived Th17 cells by a mechanism involving components associated with mature DCs.

EBV promotes human CD8 NKT cell development.

The reports on the origin of human CD8(+) Valpha24(+) T-cell receptor (TCR) natural killer T (NKT) cells are controversial. The underlying mechanism that controls human CD4 versus CD8 NKT cell development is not well-characterized. In the present study, we have studied total 177 eligible patients and subjects including 128 healthy latent Epstein-Barr-virus(EBV)-infected subjects, 17 newly-onset acute infectious mononucleosis patients, 16 newly-diagnosed EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. We have established human-thymus/liver-SCID chimera, reaggregated thymic organ culture, and fetal thymic organ culture. We here show that the average frequency of total and CD8(+) NKT cells in PBMCs from 128 healthy latent EBV-infected subjects is significantly higher than in 17 acute EBV infectious mononucleosis patients, 16 EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. However, the frequency of total and CD8(+) NKT cells is remarkably increased in the acute EBV infectious mononucleosis patients at year 1 post-onset. EBV-challenge promotes CD8(+) NKT cell development in the thymus of human-thymus/liver-SCID chimeras. The frequency of total (3% of thymic cells) and CD8(+) NKT cells ( approximately 25% of NKT cells) is significantly increased in EBV-challenged chimeras, compared to those in the unchallenged chimeras (<0.01% of thymic cells, CD8(+) NKT cells undetectable, respectively). The EBV-induced increase in thymic NKT cells is also reflected in the periphery, where there is an increase in total and CD8(+) NKT cells in liver and peripheral blood in EBV-challenged chimeras. EBV-induced thymic CD8(+) NKT cells display an activated memory phenotype (CD69(+)CD45RO(hi)CD161(+)CD62L(lo)). After EBV-challenge, a proportion of NKT precursors diverges from DP thymocytes, develops and differentiates into mature CD8(+) NKT cells in thymus in EBV-challenged human-thymus/liver-SCID chimeras or reaggregated thymic organ cultures. Thymic antigen-presenting EBV-infected dendritic cells are required for this process. IL-7, produced mainly by thymic dendritic cells, is a major and essential factor for CD8(+) NKT cell differentiation in EBV-challenged human-thymus/liver-SCID chimeras and fetal thymic organ cultures. Additionally, these EBV-induced CD8(+) NKT cells produce remarkably more perforin than that in counterpart CD4(+) NKT cells, and predominately express CD8alphaalpha homodimer in their co-receptor. Thus, upon interaction with certain viruses, CD8 lineage-specific NKT cells are developed, differentiated and matured intrathymically, a finding with potential therapeutic importance against viral infections and tumors.

EBV-induced human CD8(+) NKT cells synergise CD4(+) NKT cells suppressing EBV-associated tumours upon induction of Th1-bias.

CD8(+) natural killer T (NKT) cells from EBV-associated tumour patients are quantitatively and functionally impaired. EBV-induced CD8(+) NKT cells drive syngeneic T cells into a Th1-bias response to suppress EBV-associated malignancies. IL-4-biased CD4(+) NKT cells do not affect either syngeneic T cell cytotoxicity or Th cytokine secretion. Circulating mDC1 cells from patients with EBV-associated malignancies impair the production of IFN-gamma by CD8(+) NKT cells. In this study, we have established a human-thymus-SCID chimaera model to further investigate the underlying mechanism of EBV-induced CD8(+) NKT cells in suppressing EBV-associated malignancies. In the human-thymus-SCID chimera, EBV-induced CD8(+) NKT cells suppress EBV-associated malignancies in a manner dependent on the Th1-bias response and syngeneic CD3(+) T cells. However, adoptive transfer with CD4(+) NKT cells alone inhibits T cell immunity. Interestingly, CD4(+) NKT cells themselves secrete high levels of IL-2, enhancing the persistence of adoptively transferred CD8(+) NKT cells and T cells, thereby leading to a more pronounced T cell anti-tumour response in chimaeras co-transferred with CD4(+) and CD8(+) NKT cells. Thus, immune reconstitution with EBV-induced CD4(+) and CD8(+) NKT cells synergistically enhances T cell tumour immunity, providing a potential prophylactic and therapeutic treatment for EBV-associated malignancies.