TEMPORAL CHANGES IN INNATE AND ADAPTIVE IMMUNITY DURING SEPSIS AS DETERMINED BY ELISPOT

Abstract

Background: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The enzyme-linked immunospot (ELISpot) assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis that the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. Methods: Mice were made septic using sublethal cecal ligation and puncture. Blood and spleens were harvested serially, and ex vivo interferon γ and TNF-α production were compared by ELISpot and enzyme-linked immunosorbent assay. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. Results: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example, dexamethasone, arginine, and IL-7, in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and enzyme-linked immunosorbent assay results tended to parallel one another although some differences were noted. Conclusion: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

Figure 1.. Rapid onset of innate and adaptive immune suppression in septic splenocytes.

Mice underwent CLP and splenocytes were harvested 6 and 24 hours after induction of sepsis. Equal numbers of splenocytes from control mice and septic mice were stimulated with anti-CD3/anti-CD28 mAb for IFN-γ or LPS for TNF-α ELISpot assay. Each spot represents an immune cell producing either IFN-γ or TNF-α. The number of spots is depicted at the bottom of each circle and shows the marked loss in the number of cytokine-producing cells after CLP. There is also a decrease in the spot size representing a decrease in the amount of cytokine produced on a per cell basis. Tests were run in duplicate. Results are representative of 6 naïve mice, 6 mice at six hours, and 3 mice at 24 hours. The CLP surgery in these mice was more severe and involved tying off approximately one half of the cecum as opposed to one third of the cecum for all other CLP studies. The more severe CLP model was used to determine how quickly a more fulminant sepsis model would be detectable.

Figure 2.. Time course for effects of sepsis on innate and adaptive immunity measured by both ELISpot and ELISA.

Mice underwent CLP and spleens were harvested at days 1, 2, 3, 4, and 7 (see timeline). IFN-γ and TNF-α production from stimulated splenocytes was quantitated by both ELISpot and ELISA. Each dot represents an individual mouse. The results are the cumulative findings from three experiments.

Figure 3.. Time course for sepsis-induced loss of splenic cells.

The effect of sepsis to cause loss of splenic cells was quantitated serially on days 1, 2, 3, 4, and 7. Sepsis caused a loss in absolute numbers of CD4⁺ and CD8⁺ T cells as well as B cells. The sepsis-induced loss tended to persist for up to seven days. Sepsis did not cause a loss of splenic macrophages but, in contrast, there was a remarkable increase by day 7 after CLP. There was a decrease in splenic dendritic cells that, similar to splenic macrophages, increased dramatically by day 7.

Figure 4.. Time course for effect of sepsis on blood IFN-γ and TNF-α as detected via ELISpot.

Blood was obtained serially after CLP and underwent ELISpot analysis for IFN-γ and TNF-α. Sepsis caused a profound and sustained decrease in the number of IFN-γ producing cells as well as an initial decrease in the number of TNF-α producing cells. TNF-α production recovered and exceeded the naive control group by day 7. Additionally, serum was collected at the indicated time points and TNF-α and IL-6 levels were determined

Figure. 5.. Arginine increases splenocyte IFN-γ and TNF-α production in sepsis.

Mice underwent CLP and were treated with arginine at 3, 24, 48, and 72 hours after surgery. Mice treated with arginine had an increase in both IFN-γ and TNF-α production on day 5. ELISpot photomicrographs show representative images. Tests were performed in duplicate.

Figure 6.. IL-7 increases splenocyte IFN-γ production in sepsis.

Mice underwent CLP and were treated with IL-7 on days 1, 2, and 3. Splenocytes were obtained on day 5 and IFN-γ production assayed by ELISpot. CLP mice treated with IL-7 had increased IFN-γ production compared to mice treated with saline diluent. ELISpot photomicrographs show representative images. Tests were performed in duplicates.

Figure 7.. Dexamethasone and IL-7 effects on blood and spleen ELISpot IFN-γ and TNF-α production.

Mice underwent CLP and were treated with dexamethasone alone or in combination with IL-7 (see timeline). Results show a trend toward decreased numbers of IFN-γ producing immune cells in blood but not spleen of septic mice in response to dexamethasone injections. IL-7 treatment combatted the effects of the dexamethasone and significantly increased the number of IFN-γ producing immune cells in spleen and demonstrated a clear trend toward increased IFN-y production in blood. Representative color microphotographs of ELISpot wells showing effects of dexamethasone and IL-7 are depicted on the right hand of the figure.

Figure 8.. Effect of dexamethasone and IL-7 on splenic cell counts.

Mice underwent CLP and were treated with dexamethasone alone or in combination with IL-7 (see timeline). Sepsis caused a loss in splenic CD3, CD4, and CD8 T cells which was markedly worsened by dexamethasone treatment. Therapy with IL-7 reversed the dexamethasone-induced CD8⁺ T cell loss but did not increase CD4⁺ T cell or B cell numbers within 5 days.