Korean J Ophthalmol.  2014 Jun;28(3):246-256. 10.3341/kjo.2014.28.3.246.

Bone Marrow-derived Mesenchymal Stem Cells Affect Immunologic Profiling of Interleukin-17-secreting Cells in a Chemical Burn Mouse Model

• Lee JY
• Jeong HJ
• Kim MK
• Wee WR

• KMID: 2133324
• DOI: http://doi.org/10.3341/kjo.2014.28.3.246

Abstract

PURPOSE
This study investigated interleukin (IL)-17-secreting cell involvement in sterile inflammation, and evaluated the effect of mesenchymal stem cells (MSCs) on IL-17-secreting cell immunologic profiling.
METHODS
Twenty mice were sacrificed at time points of 6 hours, 1 day, 1 week, and 3 weeks (each group, n = 5) after the cornea was chemically injured with 0.5N NaOH; IL-17 changes in the cornea were evaluated using enzyme-linked immunosorbent assay. Further, IL-17 secreting cells were assessed in the cervical lymph nodes by a flow cytometer. Rat MSCs were applied intraperitoneally in a burn model (n = 10), IL-17-secreting T helper 17 (Th17) cell and non-Th17 cell changes were checked using a flow cytometer in both cornea and cervical lymph nodes at 1week, and compared with those in the positive control (n = 10).
RESULTS
IL-17 was highest in the cornea at 1 week, while, in the cervical lymph nodes, IL-17-secreting cells showed early increase at 6 hours, and maintained the increase through 1 day to 1 week, and levels returned to the basal level at 3 weeks. Specifically, the non-Th17 cells secreted IL-17 earlier than the Th17 cells. When the MSCs were applied, IL-17 secretion was reduced in CD3(+)CD4(-)CD8(-), CD3(+)CD4(+)CD8(-), and CD3(+) CD4(-)CD8(+) cells of the cervical lymph nodes by 53.7%, 43.8%, and 50.8%, respectively. However, in the cornea, IL-17 secretion of CD3(+)CD4(-)CD8(-) cells was completely blocked.
CONCLUSIONS
The results indicated that both IL-17-secreting non-Th17 and Th17 cells were involved in the chemical burn model, and MSCs appeared to mainly modulate non-Th17 cells and also partially suppress the Th17 cells.

Keyword

Chemical burns; Interleukin-17; Mesenchymal stromal cells; Th17 cells

MeSH Terms

Animals
Burns, Chemical/*immunology/metabolism/pathology
Cells, Cultured
Disease Models, Animal
Enzyme-Linked Immunosorbent Assay
Eye Burns/*immunology/metabolism/pathology
Flow Cytometry
*Immunity, Cellular
Interleukin-17/*secretion
Male
Mesenchymal Stromal Cells/immunology/pathology/*secretion
Mice
Mice, Inbred C57BL
Interleukin-17

Figure

• 

  Fig. 1 Enzyme-linked immunosorbent assay analysis of the corneal lysate after chemical injury. The bar charts show the mean concentrations of pro-inflammatory cytokines. (A) Interleukin (IL)-17. (B) IL-6. (C) Tumor necrosis factor alpha (TNF-α). Control represents the normal cornea. Note that the production of cytokines IL-17 and TNF-α peaked at 1 week post-injury, and in contrast, peak IL-6 levels increased immediately after injury. The experiments were performed in duplicate.

• 

  Fig. 2 This photo represented a sort-out analyses of the interleukin (IL)-17/interferon gamma (IFN-γ) secreting cells in the cervical lymph nodes, following corneal chemical injury. The non-Th-cells (CD3(-)CD4(-) cells or CD3(+)CD4(-) cells) and CD3(+)CD4(+) T helper cells were separated and were analyzed.

• 

  Fig. 3 The bar charts show the mean (A) percentages and (B) cell numbers of the interferon gamma (IFN-γ) secreting cells in the cervical lymph nodes of the 4 groups (each group, n = 5), divided over the time course of 6 hours, 1 day, 1 week, and 3 weeks after the onset of chemical injury. Note that IFN-γ-secreting non-Th-cells (CD3(-)CD4(-) T-cells) reached the highest peak at day 1 after injury, and the Th-cells also increased at 1 day before the levels returned to baseline. *p < 0.05 (Moses extreem reactions test).

• 

  Fig. 4 The bar charts show the mean (A) percentages and (B) cell numbers of the interleukin-17-secreting cells in the cervical lymph nodes in the 4 groups (each group, n = 5), divided over the time course of 6 hours, 1 day, 1 week, and 3 weeks after the onset of chemical injury. Note that both the non-T helper 17 (Th17) cells and Th17 cells increased 1 week after injury, and then gradually decreased. *p < 0.05 (Moses extreem reactions test).

• 

  Fig. 5 Fluorescent-activated cell sorter analysis of cervical lymph nodes on day 7 (A), following corneal chemical injury in the group treated with mesenchymal stem cells (MSCs) and the control group (each group, n = 10). The bar charts show the (B) percentages and (C) cell numbers of interleukin (IL)-17=secreting cells in cervical lymph nodes. Both non-T helper 17 (Th17) cells (CD3(+)CD4(-)CD8(-) and CD3(+)CD4(-)CD8(+)) and Th17 cells were effectively reduced in the group treated with MSCs. All of the cervical lymph nodes for each group were pooled to perform the experiment.

• 

  Fig. 6 Fluorescent-activated cell sorter analysis of corneas on day 7, following corneal chemical injury in the group treated with mesenchymal stem cells (MSCs) and the control group. The bar charts show the (A) percentages and (B) cell numbers of the interleukin (IL)-17-secreting cells in the corneas. Complete blockage of IL-17-secreting and the non-T helper 17 cells (CD3(+)/CD4(-)/CD8(-) cells) was shown in the MSC treated group. All of the cornea and lymph nodes for each group were pooled to perform the experiment.

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