Simultaneous Expression of Th1- and Treg-Associated Chemokine Genes and CD4 +, CD8 +, and Foxp3 + Cells in the Premalignant Lesions of 4NQO-Induced Mouse Tongue Tumorigenesis
Chemokines and cytokines in the tumor microenvironment influence immune cell infiltration and activation. To elucidate their role in immune cell recruitment during oral cancer development, we generated a mouse tongue cancer model using the carcinogen 4-nitroquinoline 1-oxide (4NQO) and investigated the carcinogenetic process and chemokine/cytokine gene expression kinetics in the mouse tongue. C57/BL6 mice were administered 4NQO in drinking water, after which tongues were dissected at 16 and 28 weeks and subjected to analysis using the RT2 Profiler PCR Array, qRT-PCR, and pathologic and immunohistochemical analyses. We found that Th1-associated chemokine/cytokine (Cxcl9, Cxcl10, Ccl5, and Ifng) and Treg-associated chemokine/cytokine (Ccl17, Ccl22, and Il10) mRNA levels were simultaneously increased in premalignant lesions of 4NQO-treated mice at 16 weeks.
Additionally, although levels of Gata3, a Th2 marker, were not upregulated, those of Cxcr3, Ccr4, and Foxp3 were upregulated in the tongue tissue. Furthermore, immunohistochemical analysis confirmed the infiltration of CD4+, CD8+, and Foxp3+ cells in the tongue tissue of 4NQO-treated mice, as well as significant correlations between Th1- or Treg-associated chemokine/cytokine mRNA expression and T cell infiltration. These results indicate that CD4+, CD8+, and Foxp3+ cells were simultaneously recruited through the expression of Th1- and Treg-associated chemokines in premalignant lesions of 4NQO-induced mouse tongue tissue.
CXCL4 promoted the production of CD4 + CD25 + FOXP3 + treg cells in mouse sepsis model through regulating STAT5/FOXP3 pathway
CXCL4 plays an essential role in the regulation of multiple immune diseases. However, the underlying role of CXCL4 is still not clear in sepsis. Aim: In the present study, we aimed to investigate the function of CXCL4 in sepsis.
Sepsis model was constructed on mouse. Flow cytometry was used to determine the ratio of CD4+CD25+FOXP3+Treg cells. ELISA assays were used to determine the levels of CXCL4, IL-6, IL-10, and TNF-α respectively. Western blot was used to examine protein contents.
Our results suggested that the serum level of CXCL4 was upregulated in patients with sepsis and positively associated with the ratio of human CD4+CD25+FOXP3+Treg cells. To further examine the role of CXCL4 in sepsis, we constructed the mouse sepsis model. Our results indicated that the mouse antibody of CXCL4 treatment reduced the expression of urine creatinine and urea nitrogen in sepsis model. Moreover, the frequency of CD25+FOXP3+ mouse regulatory T cells (Tregs) cells was decreased in mouse CD4+ T cells in the presence of mouse CXCL4 antibody. Further, the mouse recombinant protein CXCL4 was used to culture normal mouse CD4+ T cells in vitro. Our finding indicated that the recombinant protein CXCL4 promoted the percentage of mouse CD25+FOXP3+Treg cells and enhanced the phosphorylation of STAT5 in mouse CD4+ T cells in a dose-dependent manner. However, these effects were significantly reversed by the STAT5 inhibitor (p < .001). Conclusion: our findings not only indicated the function and signalling pathway of CXCL4 in CD4+ T cells but also provided novel insight and target in sepsis treatment.
Brain Foxp3+ regulatory T cells can be expanded by Interleukin-33 in mouse ischemic stroke
Regulatory T (Treg) cells are known as immune regulators to decrease infarct volume and improve outcomes after ischemic stroke. Thus, the strategies for increasing Treg cells in the ischemic brain may have beneficial effects on stroke. In this study, we aim to examine the effect of Interleukin-33 (IL-33) on Treg cell expansion in a mouse model of ischemic stroke. Mice were subjected to 30 min of middle cerebral artery occlusion (MCAO) followed by 24 h, 48 h of 72 h of reperfusion. Recombinant mouse IL-33 (2 μg) was pre-treated intracerebroventricularly at 30 min prior to MCAO. The percentage of Treg cells in the ischemic brain, related cytokines, and transcription factors, the levels of ST2 receptor, amphiregulin (AREG), and epidermal growth factor receptor (EGFR) were measured.
IL-33 treatment can increase the number of Foxp3+ Treg cells in the ischemic brain and the levels of IL-10 and TGF- β1 in serum and brain tissues at MCAO 48 h and 72 h, but not at MCAO 24 h. In the Treg cells separated from ischemic brain tissue following MCAO treated by IL-33, the expression level of the ST2 receptor was up-regulated. In addition, IL-33 may increase the mRNA level of transcription factor Foxp3. Correspondingly, IL-33 treatment also elevated the levels of AREG and EGFR at MCAO 48 h and 72 h.
We speculated that intracerebroventricular IL-33 can activate the downstream Foxp3 via ST2 receptor to increase Treg proportions in the ischemic brain. The elevated Treg cells produce AREG to activate EGFR located in neurons, which contribute to better outcomes.
Ectopic germline recombination activity of the widely-used Foxp3-YFP-Cre mouse: a case-report
Regulatory T cell (Treg)-specific deletion of a gene of interest is a procedure widely used to study mechanisms controlling Treg development, homeostasis, and function. Accordingly, several transgenic mouse lines have been generated that bear the Cre-recombinase under control of the Foxp3 promoter either as a random transgene insertion or knocked into the endogenous Foxp3 locus, with the Foxp3YFP-Cre strain of mice being one of the most widely used. In an attempt to generate Tregs which lacked expression of the insulin receptor (Inst), we crossed Foxp3YFP-Cre mice to Insrfl/fl mice. Using a conventional two-band PCR genotyping method we found that offspring genotypes did not correspond to the expected Mendelian ratios.
We thus developed a quantitative PCR-based genotyping method to investigate possible ectopic recombination outside of the Treg lineage. With this method, we found that ~50% of the F1-generation mice showed evidence of ectopic recombination and that ~10% of the F2-generation mice had germline Cre recombination activity leading to a high frequency of offspring with global Insr deletion.
The use of the quantitative PCR genotyping method enabled accurate selection of mice without ectopic recombination and only the desired Treg-specific Insr deletion. Our data highlight the need to use genotyping methods which allow for the assessment of possible ectopic recombination driven by the Foxp3YFP-Cre allele, particularly when studying genes that are systemically expressed.
Thymically-derived Foxp3+ regulatory T cells are the primary regulators of type 1 diabetes in the non-obese diabetic mouse model
Regulatory T cells (Tregs) are an immunosuppressive population that are identified based on the stable expression of the fate-determining transcription factor forkhead box P3 (Foxp3). Tregs can be divided into distinct subsets based on whether they developed in the thymus (tTregs) or in the periphery (pTregs). Whether there are unique functional roles that distinguish pTregs and tTregs remains largely unclear. To elucidate these functions, efforts have been made to specifically identify and modify individual Treg subsets.
Deletion of the conserved non-coding sequence (CNS)1 in the Foxp3 locus leads to selective impairment of pTreg generation without disrupting tTreg generation in the C57BL/6J background. Using CRISPR-Cas9 genome editing technology, we removed the Foxp3 CNS1 region in the non-obese diabetic (NOD) mouse model of spontaneous type 1 diabetes mellitus (T1D) to determine if pTregs contribute to autoimmune regulation.
Mouse Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RD-FOXP3-Mu-48Tests | Reddot Biotech | 48 Tests | 511 EUR | |
Mouse Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RD-FOXP3-Mu-96Tests | Reddot Biotech | 96 Tests | 709 EUR | |
Mouse Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| DLR-FOXP3-Mu-48T | DL Develop | 48T | 508 EUR | |
Mouse Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| DLR-FOXP3-Mu-96T | DL Develop | 96T | 661 EUR | |
Mouse Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RDR-FOXP3-Mu-48Tests | Reddot Biotech | 48 Tests | 534 EUR | |
Mouse Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RDR-FOXP3-Mu-96Tests | Reddot Biotech | 96 Tests | 742 EUR | |
CuO-Mouse Foxp3-IRES-GFP-EF1-CymR-T2A-Puro Cumate inducible lentivector plasmid | ||||
| TCL500A-1 | SBI | 10ug | 1362 EUR | |
Human Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RD-FOXP3-Hu-48Tests | Reddot Biotech | 48 Tests | 500 EUR | |
Human Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RD-FOXP3-Hu-96Tests | Reddot Biotech | 96 Tests | 692 EUR | |
Rat Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RD-FOXP3-Ra-48Tests | Reddot Biotech | 48 Tests | 534 EUR | |
Rat Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RD-FOXP3-Ra-96Tests | Reddot Biotech | 96 Tests | 742 EUR | |
Human Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| DLR-FOXP3-Hu-48T | DL Develop | 48T | 498 EUR | |
Human Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| DLR-FOXP3-Hu-96T | DL Develop | 96T | 647 EUR | |
Rat Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| DLR-FOXP3-Ra-48T | DL Develop | 48T | 528 EUR | |
Rat Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| DLR-FOXP3-Ra-96T | DL Develop | 96T | 690 EUR | |
Human Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RDR-FOXP3-Hu-48Tests | Reddot Biotech | 48 Tests | 522 EUR | |
Human Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RDR-FOXP3-Hu-96Tests | Reddot Biotech | 96 Tests | 724 EUR | |
Rat Forkhead Box Protein P3 (FOXP3) ELISA Kit | ||||
| RDR-FOXP3-Ra-48Tests | Reddot Biotech | 48 Tests | 558 EUR | |
Deletion of CNS1 impaired in vitro induction of Foxp3 in naïve NOD CD4+ T cells, but it did not alter Tregs in most lymphoid and non-lymphoid tissues analyzed except for the large intestine lamina propria, where a small but significant decrease in RORγt+ Tregs and corresponding increase in Helios+ Tregs was observed in NOD CNS1-/- mice. CNS1 deletion also did not alter the development of T1D or glucose tolerance despite increased pancreatic insulitis in pre-diabetic female NOD CNS1-/- mice. Furthermore, the proportions of autoreactive Tregs and conventional T cells (Tconvs) within pancreatic islets were unchanged. These results suggest that pTregs dependent on the Foxp3 CNS1 region are not the dominant regulatory population controlling T1D in the NOD mouse model.