Kinetics of T helper subsets and associated cytokines correlate well with the clinical activity of graft-versus-host disease

Background

CD4⁺interferon (IFN)-γ⁺ T cell (Th1) and CD4⁺interleukin (IL)-4⁺ T cell (Th2) polarizations are crucial in the pathogenesis of graft-versus-host disease (GVHD). However, this hypothesis is largely based on animal experiments of Parent-into-F1 GVHD model. The causal relationship between kinetics of Th1, Th2 and associated cytokines and the clinical activity of GVHD in a real world situation remains unknown.

Methodology

Peripheral blood was collected every week prospectively from Day 0 to Day 210 (patients without GVHD) or Day 300 (patients with chronic GVHD) after allogeneic peripheral blood stem cell transplantation in consecutive 27 patients. The frequencies of Th1 and Th2 within CD4⁺ T cells were determined by flow cytometry and pplasma IFN-γ, IL-12, IL-4, and IL-10 were determined by ELISA.

Principal Findings

Kinetics of Th1, Th2 frequency, and the plasma IL-10 and IFN-γ more commonly coincided with, rather than predicted, the activity of GVHD. These markers are significantly higher when acute or chronic GVHD developed. The kinetics of IL-10 is especially correlated well with the activity of GVHD during clinical course of immunosuppressive treatment. For patients with hepatic GVHD, there is a positive correlation between plasma IL-10 levels and the severity of hepatic injury. The frequency of Th2 is also significant higher in acute GVHD and tends to be higher in chronic GVHD. Interestingly, there is a very good positive correlation between the frequency of Th1 and Th2 (r = 0.951, p<0.001). The plasma level of IL-4 and IL-12 are not associated with the activity of GVHD.

Conclusions

The frequency of Th1, Th2 within CD4⁺ T cells and plasma IL-10 and IFN-γ are good biomarkers of GVHD. Plasma IL-10 can also be used to monitor the therapeutic responsiveness. Furthermore, both Th1 and Th2 likely contribute to the pathogenesis of GVHD.     

Genetic Variation in the NOC Gene Is Associated with Body Mass Index in Chinese Subjects

Circadian clock genes are critical regulators of energy homeostasis and metabolism. However, whether variation in the circadian genes is associated with metabolic phenotypes in humans remains to be explored. In this study, we systemically genotyped 20 tag single nucleotide polymorphisms (SNPs) in 8 candidate genes involved in circadian clock, including CLOCK, BMAL1(ARNTL), PER1, PER2, CRY1, CRY2, CSNK1E,, and NOC(CCRN4L) in 1,510 non-diabetic Chinese subjects in Taipei and Yunlin populations in Taiwan. Their associations with metabolic phenotypes were analyzed. We found that genetic variation in the NOC gene, rs9684900 was associated with body mass index (BMI) (P = 0.0016, Bonferroni corrected P = 0.032). Another variant, rs135764 in the CSNK1E gene was associated with fasting glucose (P = 0.0023, Bonferroni corrected P = 0.046). These associations were consistent in both Taipei and Yunlin populations. Significant epistatic and joint effects between SNPs on BMI and related phenotypes were observed. Furthermore, NOC mRNA levels in human abdominal adipose tissue were significantly increased in obese subjects compared to non-obese controls.

Conclusion

Genetic variation in the NOC gene is associated with BMI in Chinese subjects.     

Effects of Cystamine on antioxidant activities and regulatory T cells in lupus‐prone mice

Attenuated antioxidant activities, irregular cytokines expressions and reduced regulatory T cells, are strongly associated with the pathogenesis of systemic lupus erythematosus (SLE). Despite the well‐established beneficial effects of cystamine on lupus‐prone mice, the extent to which cystamine contributes to antioxidant activity and the reduction of regulatory T cells has seldom been investigated. Therefore, this study elucidates how cystamine affects anti‐oxidant activities in NZB/W F1 mice by performing assays of Glutathione (GSH), 1,1‐diphenyl‐2‐ picryl‐hydrazyl (DPPH) and malondialdehyde thiobarbituric acid (MDA). In addition, investigations of the effects of cystamine on CD4⁺/CD25⁺ regulatory T cells and interleukin‐6 (IL6)/STAT‐3 signalling were performed with flow cytometry and immunoblots. Experimental results reveal more significantly reduced MDA and increased GSH and DPPH in NZB/W F1 mice receiving cystamine than in those mice receiving PBS. Meanwhile, CD4⁺/CD25⁺ regulatory T cells more significantly increase in NZB/W F1 mice receiving cystamine than in those mice receiving PBS, accompanied by significantly reduced IL‐6/phosphorylated STAT‐3 expression. The above findings suggest the beneficial effects of cystamine in terms of increasing antioxidant activities and CD4⁺/CD25⁺ regulatory T cells in lupus‐prone mice by suppressing IL‐6/STAT3 signalling.     

Involvement of 15-lipoxygenase in the inflammatory arthritis

15-Lipoxygenase (15-LOX) is involved in many pathological processes. The aim of this study is to examine the role of 15-LOX in the matrix metalloproteinase (MMP) expression and inflammatory arthritis. It was found that treatment of 15-LOX downstream product of 15-(S)-HETE (15-S-hydroxyeicosatetraenoic acid) increased the mRNA and protein levels of MMP-2 in rheumatoid arthritis synovial fibroblast (RASF) derived from rheumatoid arthritis patients. The enhancement effect of 15-(S)-HETE was antagonized by the addition of LY294002 (PI3K inhibitor) and PDTC (NF-κB inhibitor). Treatment of 15-(S)-HETE increased the phosphorylation of AKT, nuclear translocation of p65 and the breakdown of IκBα. TNF-α and IL-1β are the key cytokines involved in arthritis and also increase the activity of MMP-2 in RASF, which was antagonized by pretreatment with 15-LOX inhibitor PD146176 or knockdown of 15-LOX. It was also found that these two cytokines increased the expression of 15-LOX in RASF. Treatment of glucocorticoid but not NSAIDs inhibited 15-(S)-HETE-induced expression of MMP-2. In comparison with wild-type mice, adjuvant-induced arthritis and MMP-2 expression in synovial membrane were markedly inhibited in 15-LOX knockout (KO) mice. These results indicate that 15-LOX plays an important role in the disease progression of arthritis and may be involved in the inflammatory action induced by TNF-α and IL-1β. 15-LOX is thus a good target for developing drugs in the treatment of inflammatory arthritis.     

Soft substrate induces apoptosis by the disturbance of Ca2+ homeostasis in renal epithelial LLC-PK1 cells

Different rigidities of adhesive collagen substrate affect cellular functions with unclear mechanisms. Here, we cultured a renal epithelial cell line (LLC-PK1) and a tumor cell line (HeLa) on substrates of different rigidities and compared the cell type-specific responses. The culture dish was coated with a very thin layer of collagen gel (control group) or overlaid with collagen gel (soft substrate). LLC-PK1 cells contracted as they grew on collagen gel and the apoptotic bodies obviously appeared with time. The protein levels of procaspase-12 and its downstream target procaspase-3 were decreased when LLC-PK1 cells cultured on collagen gel. Mu-calpain was activated on collagen gel. Collage gel also induced the cleavage of alpha-spectrin which resulted in the disorganization of actin cytoskeleton. In contrast, there was no significant change in cytochrome c revelation, mitochondrial membrane potential, and the protein levels of procaspase-8 and procaspase-9. Moreover, soft substrate caused elevated cytosolic Ca(2+), Ca(2+) overload in ER and upregulation of capacitative calcium entry. Ca(2+) chelator or channel blocker partially rescued the collagen-gel induced apoptosis by inhibiting mu-calpain activation. In contrast, for HeLa cells cultured either on collagen gel or on gel-coated dish, there was no significant change in positive Annexin V staining, no activation of procaspase-12 and no cleavage of mu-calpain. Thus, soft substrate induces apoptosis in LLC-PK1 cells by the disturbance of Ca(2+) homeostasis.