Tranexamic acid modulates the immune response and reduces postsurgical infection rates

Abstract

Tranexamic acid (TXA) is an antifibrinolytic agent that blocks plasmin formation. Because plasmin is known to promote inflammatory and immunosuppressive responses, we explored the possibility that plasmin-mediated immunosuppression in patients undergoing cardiac surgery can be directly reversed by TXA and decrease postoperative infection rates. The modulatory effect of TXA on inflammatory cytokine levels and on innate immune cell activation were evaluated with multiplex enzyme-linked immunosorbent assay and flow cytometry, respectively. Postoperative infection rates were determined in patients undergoing cardiac surgery and randomized to TXA (ACTRN12605000557639; http://www.anzca.edu.au). We demonstrate that TXA-mediated plasmin blockade modulates the immune system and reduces surgery-induced immunosuppression in patients following cardiac surgery. TXA enhanced the expression of immune-activating markers while reducing the expression of immunosuppressive markers on multiple myeloid and lymphoid cell populations in peripheral blood. TXA administration significantly reduced postoperative infection rates, despite the fact that patients were being administered prophylactic antibiotics. This effect was independent of the effect of TXA at reducing blood loss. TXA was also shown to exert an immune-modulatory effect in healthy volunteers, further supporting the fibrin-independent effect of TXA on immune function and indicating that baseline plasmin levels contribute to the regulation of the immune system in the absence of any comorbidity or surgical trauma. Finally, the capacity of TXA to reduce infection rates, modulate the innate immune cell profile, and generate an antifibrinolytic effect overall was markedly reduced in patients with diabetes, demonstrating for the first time that the diabetic condition renders patients partially refractory to TXA.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

© 2019 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

TXA modulates myeloid and lymphoid cells, as well as plasma cytokines, in the immune response after cardiac surgery. Patients undergoing cardiac surgery were treated with placebo or TXA, and the cellular immune response was characterized using flow cytometry before drug administration, as well as on POD-1 and POD-3. (A) CD83 expression was significantly increased on nonclassical monocytes in the TXA group at POD-3. (B) CD83 on CD1c+ cDCs was downregulated to a significantly lower extent in TXA-treated patients at POD-3. (C) TXA treatment also resulted in a significant relative increase in CCR7-expressing CD1c+ cDCs. (D) Moreover, TXA reduced expression of the programmed cell death inducing PD-L1 on nonclassical monocytes at POD-3. (E) CD4+ T cells were reduced significantly after cardiac surgery, whereby the decrease was significantly greater in the TXA group at POD-1. (F) CD8+ Tmem counts were also reduced at POD-1, but they remained reduced only in the TXA-treated patients at POD-3. Expression of the latent TGF-β releasing LAP (G) and CCR7 (H) relative to baseline was significantly lower in TXA-treated patients at POD-3. (I) In contrast, CCR7 expression on NK cells was significantly increased in the TXA group on POD-1. (J) Plasma levels of the proinflammatory cytokine IL-1β were significantly reduced in the TXA group compared with placebo at POD-1. Data represent fold-change from preoperative (preOP) levels and are expressed as mean ± standard error of the mean. Placebo: n = 19, TXA: n = 22. #P < .05, ##P < .01, ###P < .001, ####P < .0001 vs preOP, 1-way ANOVA with Dunnett’s correction test for multiple comparisons; *P < .05, placebo vs TXA, 2-tailed Student t test.

Figure 2.

TXA induces changes to the cellular immune profile in cardiac surgery patients, consistent with enhanced immune activation, selectively in patients without diabetes. Results obtained with flow cytometry were analyzed separately for diabetic and nondiabetic patients. Consistent with our findings on infection outcome, the activation marker CD83 was enhanced by TXA on several myeloid subsets (classical [A] and intermediate [B] monocytes and CD1c+ cDCs [C]) at POD-3 in patients without diabetes, whereas the immunosuppressive marker PD-L1 was downregulated on CD1c+ cDCs (D) and nonclassical monocytes (E). (G-L) In contrast, these effects were not seen in the diabetic cohort. In fact, PD-L1 even exhibited increased expression (significant in nonclassical monocytes [K], nonsignificant in CD1c+ cDCs [J]) at POD-1. In addition, expression of the immunosuppressive marker LAP on Tregs was significantly reduced by TXA on POD-3 in patients without diabetes (F) but not in patients with diabetes (L). Data represent fold-change from preoperative (preOP) levels and are expressed as mean ± standard error of the mean. Placebo: n = 11, TXA: n = 16 (patients without diabetes); placebo: n = 8, TXA: n = 6 (patients with diabetes). *P < .05, **P < .01, 2-tailed Student t test. ns, not significant.

Figure 3.

TXA changes plasma cytokine levels and functional marker expression on myeloid cell populations in healthy volunteers. Plasma cytokine levels and expression of functional markers on myeloid cells were evaluated in healthy volunteers at various time points after administration of TXA. TXA significantly reduced plasma levels of the proinflammatory cytokine TNF-α at 4 hours, as well as IL-6 between 2 hours and 24 hours. (Ai) Levels of the type 1 T helper cell cytokine IFN-γ were significantly reduced at 2 hours and 4 hours, and the levels of the type 2 T helper cell cytokine IL-10 were significantly reduced at 2 hours (n = 9). (Aii) sTNFR2 levels were significantly enhanced by TXA at 2 hours and 4 hours but returned to baseline at 24 hours (n = 9). (B) Levels of circulating nonclassical monocytes were significantly enhanced by TXA after 24 hours, whereas pDC levels were reduced after 4 hours, yet returned to baseline levels after 24 hours (n = 6). (C) TXA significantly increased expression of the activation marker CD83 on classical and intermediate monocytes, as well as on CD1c+ cDCs (n = 6). (D) The maturation marker HLA-DR was reduced at 24 hours on intermediate monocytes (already at 4 hours), classical monocytes, and pDCs (n = 6). (E) CCR7, indicating migration to secondary lymphatic organs, was significantly reduced by TXA in classical and intermediate monocytes (n = 6). (F) TNFR2, mediating TNF-α signaling, was increased at 4 hours and 24 hours in classical and intermediate monocytes and CD1c+ cDCs, as well as in pDCs at 24 hours after TXA intake (n = 6). (G) PD-L1, which induces programmed cell death in effector cells of the immune system, was significantly downregulated by TXA after 4 hours and even further after 24 hours in classical monocytes and intermediate monocytes, as well as in CD1c+ cDCs and the immunosuppressive monocytic myeloid-derived suppressor cell (MO-MDSC) at 24 hours (n = 6). Data are expressed as mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001, repeated measures 1-way ANOVA with Dunnett’s correction test for multiple comparisons.

Figure 4.

TXA changes levels of T cell subsets and functional marker expression on lymphoid cell populations in healthy volunteers. Expression of functional markers on lymphoid subsets was evaluated in healthy volunteers at various time points after administration of TXA. (A) A marked increase was observed in the levels of CD4+ T cells, including Tregs and CD8+ T cells (n = 7). (B) CCR4, which mediates migration to sites of inflammation, was upregulated after 24 hours in NKT cells, CD4+ T cells, CD8+ T cells, and the CD8+ Teff and Tmem subsets (n = 7). (C) TNFR2 expression was significantly reduced on CD8+ Teffs after 4 hours, yet it returned to normal levels after 24 hours (n = 7). (D) Expression of PD-1, the receptor of PD-L1, which mediates programmed cell death upon activation, was enhanced on Tregs and CD8+ Tmems (n = 7). (E) CD95, another death receptor, was rapidly upregulated by TXA on NKT cells and CD4+ T cells after 4 hours but had returned to normal levels at the 24-hour time point (n = 7). (F) Augmented CTLA4 expression was evident on CD4+ T cells at 4 hours and on NK cells at 24 hours post-TXA intake (n = 7). (G) The TGF-β releasing LAP was expressed significantly more weakly on NK and NKT cells, as well as CD4+ and CD8+ T cells, including CD8+ Teffs, 24 hours after TXA intake (n = 7). Data are expressed as mean ± standard error of the mean. *P < .05, **P < .01, repeated-measures 1-way ANOVA with Dunnett’s correction test for multiple comparisons.

Figure 5.

Summary of the effects of TXA treatment in healthy volunteers and surgery patients. TXA administration causes marked alteration in a variety of plasma cytokine levels, numbers of immune cells, and/or expression of key markers associated with immune activation and immunosuppression.