Transplantation of mesenchymal stem cells genetically engineered to overexpress interleukin-10 promotes alternative inflammatory response in rat model of traumatic brain injury

Abstract

Background: Traumatic brain injury (TBI) is a major cause for long-term disability, yet the treatments available that improve outcomes after TBI limited. Neuroinflammatory responses are key contributors to determining patient outcomes after TBI. Transplantation of mesenchymal stem cells (MSCs), which release trophic and pro-repair cytokines, represents an effective strategy to reduce inflammation after TBI. One such pro-repair cytokine is interleukin-10 (IL-10), which reduces pro-inflammatory markers and trigger alternative inflammatory markers, such as CD163. In this study, we tested the therapeutic effects of MSCs that were engineered to overexpress IL-10 when transplanted into rats following TBI in the medial frontal cortex.

Methods: Thirty-six hours following TBI, rats were transplanted with MSCs and then assessed for 3 weeks on a battery of behavioral tests that measured motor and cognitive abilities. Histological evaluation was then done to measure the activation of the inflammatory response. Additionally, immunomodulatory effects were evaluated by immunohistochemistry and Western blot analyses.

Results: A significant improvement in fine motor function was observed in rats that received transplants of MSCs engineered to overexpress IL-10 (MSCs + IL-10) or MSCs alone compared to TBI + vehicle-treated rats. Although tissue spared was unchanged, anti-inflammatory effects were revealed by a reduction in the number of glial fibrillary acidic protein cells and CD86 cells in both TBI + MSCs + IL-10 and TBI + MSC groups compared to TBI + vehicle rats. Microglial activation was significantly increased in the TBI + MSC group when compared to the sham + vehicle group. Western blot data suggested a reduction in tumor necrosis factor-alpha in the TBI + MSCs + IL-10 group compared to TBI + MSC group. Immunomodulatory effects were demonstrated by a shift from classical inflammation expression (CD86) to an alternative inflammation state (CD163) in both treatments with MSCs and MSCs + IL-10. Furthermore, co-labeling of both CD86 and CD163 was detected in the same cells, suggesting a temporal change in macrophage expression.

Conclusions: Overall, our findings suggest that transplantation of MSCs that were engineered to overexpress IL-10 can improve functional outcomes by providing a beneficial perilesion environment. This improvement may be explained by the shifting of macrophage expression to a more pro-repair state, thereby providing a possible new therapy for treating TBI.

Keywords: CD163; Interleukin-10; Mesenchymal stem cells; Neuroinflammation; Traumatic brain injury.

Fig. 1

MSCs transduced with IL-10 lentivirus. a PCR product confirmed that IL-10 sequence was successfully integrated in the plasmid and detectable in transduced MSCs and absent in control virus plasmid and MSCs. b MSCs infected with lentivirus expressed GFP (green). MSCs were characterized by staining with the following antibodies: CD11b, CD45, CD34, CD44, CD90, and CD105 using appropriate secondary AlexaFlour 594 shown in the red. MSCs were negative for CD11b, CD45, and CD34. MSCs were positive for CD44, CD90, and CD105 (scale bar = 50 μm)

Fig. 2

In vitro expression of IL-10 in transduced MSCs. a Representative immunocytochemistry images of MSCs with and without IL-10 overexpression. MSCs + IL10-GFP group appeared to have higher IL-10 immunofluorescent signal than MSCs + GFP group. b Mean integrated density of IL-10 showed that MSCs + IL-10 had significantly higher expression of IL-10 than MSCs + GFP (***p < 0.001). c RT-qPCR resulted in significant mean fold changes of IL-10 expression in MSCs + IL-10 group in comparison to MSCs + GFP (**p < 0.01). d Western blot IL-10 levels were compared between the two MSC groups. e MSCs + IL-10-GFP cells expressed a significantly higher amount of IL-10 level in comparison to MSCs + GFP (*p < 0.05; scale bar = 50 μm). Error bars represent standard error of the mean (± SEM)

Fig. 3

Behavioral outcomes after treatment with MSCs + IL-10. a Mean latency to find the platform in the MWM task across 4 days with platform in the northeast (NE) quadrant, all TBI groups took a significantly longer time to find the target platform than the sham + vehicle group (***p < 0.001). b Reversal trials with the platform in the southwest (SW) quadrant (mean latency across 3 days) indicated sham + vehicle group was significantly faster in finding the platform than TBI + vehicle (*p < 0.05) and TBI + MSCs (**p < 0.01) but not TBI + MSCs + IL-10. c Mean number of foot faults in the ladder rung walking task. Ladder rung test was run once a week for 3 weeks. TBI + vehicle group had significantly more foot faults than all other groups (*p < 0.05, ***p < 0.001), and the sham + vehicle group had significantly fewer foot faults than all other groups (##p < 0.01). d Mean latency (sec) to remain on the rotating rod in the rotarod test. The rotarod test was performed for 5 days, 4 trials per day. An injury effect was observed in TBI groups, whereas sham + vehicle group stayed on the rod significantly longer time than all other groups (**p < 0.01, ***p < 0.001). Error bars represent ± SEM

Fig. 4

Mean volume of tissue spared were analyzed in rat brain sections at bregma + 3, + 2, + 1, and 0 mm. a Representative photomicrograph of lesions in the frontal cortex of each group. (Scale bar = 1 mm). b A significant reduction in tissue remaining was seen in TBI + vehicle group compared to sham + vehicle (*p < 0.05). Error bars represent ± SEM

Fig. 5

Astrocytes labeled with GFAP antibody in the frontal cortex and hippocampus. a Representive images of GFAP-postive cells in the frontal cortex and CA1 and CA3 region of the hippocampus. b A significant reduction in GFAP-positive cells was seen in TBI + MSCs (***p < 0.001) and TBI + MSCs + IL-10 (***p < 0.001) groups in the frontal cortex in comparison to TBI + vehicle group. c In the CA1 region of the hippocampus, a significant reduction of GFAP-positive cells were found in the TBI + MSCs + IL-10 (***p < 0.001) and sham + vehicle group (*p < 0.05) in comparison to TBI + vehicle group. d In the CA3 region of the hippocampus, TBI + vehicle group had a significant increase in GFAP-positive cells when compared to all other groups (*p < 0.05, **p < 0.01; scale bar = 50 μm). Error bars represent ± SEM

Fig. 6

Macrophages/microglia labeled with Iba1 antibody in the frontal cortex and hippocampus. a Representive images of Iba1-postive cells in the frontal cortex and CA1 and CA3 region of the hippocampus. b In the frontal cortex, an injury effect was seen with an increased number of Iba1-positive cells in all TBI groups (*p < 0.05, ***p < 0.001). c No significant differences were seen among the groups in the CA1 region of the hippocampus. d No significant differences were seen among the groups in the CA3 region of the hippocampus (scale bar = 50 μm). Error bars represent ± SEM

Fig. 7

Microglia activation state in the frontal cortex. Microglia were more activated in TBI + MSC group compared to sham + vehicle group (***p < 0.001). Error bars represent ± SEM

Fig. 8

CD86 and CD163-labeled cells in the frontal cortex and hippocampus. a–c The images in the frontal cortex, CA1 region of the hippocampus, and CA3 region of the hippocampus are as follows: CD86 is in red, CD163 is green, and merge in yellow images show co-labeling of CD163 and CD86, arrows show an example of a co-labeled cell. Hoechst 33258 was used as a counterstain in blue (scale bar = 50 μm). d In the frontal cortex, a significant increase in the number of CD163-expressing cells was seen in all TBI groups compared to sham + vehicle group (*p < 0.05, **p < 0.01). e The mean number of CD163-labeled cells in the CA1 region of the hippocampus was significantly increased in the TBI + vehicle (*p < 0.05) and the TBI + MSCs + IL-10 (*p < 0.05) groups compared to sham + vehicle. f In the CA3 region of the hippocampus, CD163-positive cells were significantly increased in TBI + vehicle group compared to sham + vehicle (**p < 0.01). g Mean number of CD86-positive cells in the frontal cortex was significantly increased in TBI + vehicle group compared to all other groups (**p < 0.01, ***p < 0.001). h, i In the CA1 and CA3 region of the hippocampus, no significant differences in number of CD86-positive cells between groups were observed. j–l Cell counts for CD163 and CD86 were used to find the ratio of CD163:CD86 and multiplied by 100 to get the percent. j A significant shift to alternative macrophage/microglia state was seen in the frontal cortex (*p < 0.05). k–l No significant differences were observed among the groups in the CA1 and CA3 region of the hippocampus. Error bars represent ± SEM

Fig. 9

Western blots showed the levels of IL-10, TNF-α, and CD163 in the cortex. a–b IL-10 levels in TBI + MSCs + IL-10 group was elevated compared to TBI + vehicle (*p < 0.05). c, d TNF-α levels in the cortex was significantly elevated in TBI + MSC group when compared to all other groups (*p < 0.05). e, f CD163 levels were significantly elevated in TBI + MSCs and TBI + MSCs + IL-10 compared to sham + vehicle group (*p < 0.05). Error bars represent ± SEM