Distinct IFN-Gamma Dependent Effect of the TLR5 Agonist, Flagellin On NK and CD4+ T Cell Immunity Against CMV

Opportunistic CMV infection remains a major clinical complication in allogeneic BMT (allo-BMT) recipients. We previously published that peri-transplant treatment of highly purified flagellin, a TLR5 agonist extracted from S. typhimurium reduced GvHD in lethally irradiated allo-BMT recipients by reducing early activation and proliferation of donor T cells. Flagellin-treated recipients had enhanced lymphoid immune reconstitution and were protected from lethal MCMV infection. CMV initiates innate immunity through the activation of Toll-like receptors (TLR) 9, 3 and 2 but direct enhancement of anti-CMV immunity through TLR5 has not been studied.

To further explore the effects of a TLR5 agonist on anti-CMV immunity, we infected flagellin-treated C57BL/6 (B6), TLR5KO and Myd88KO mice (B6 background) with sub lethal dose (1×10⁵pfu/mouse i.p) of MCMV. Anti-CMV immunity of NK and T-cells were determined by measuring surface activation markers on the NK and T-cells. Flagellin-treated and MCMV infected IL-12KO, IFN-γRKO and IDO KO mice were used to study the role of IL-12, IFN-γ and IDO on anti-CMV immunity. The relationship between the MCMV lethality and flagellin signaling in hematopoietic or epithelial tissues expressing TLR5 was further investigated by generating radiation chimeras, using WT mice engrafted with TLR5 KO bone marrow (BM) and TLR5 KO mice engrafted with WT B6 BM. Chimeric recipients with WT or TLR5KO hematopoietic cells (or the reverse) were then treated with flagellin or PBS and infected with a sub lethal dose of MCMV infection.

Without MCMV infection, flagellin treatment significantly increased numbers of NK cells (p= 0.001) and KLRG1+ NK cells (p=0.009) in WT B6 mice compared with the PBS-treated control mice. Following MCMV infection (day 3), there were significantly increased numbers of splenic NK cells (p= 0.01) and KLRG1+ NK cells (p=0.0008) in flagellin-treated mice compared with the control mice. Flagellin-treatment also enhanced viral clearance, tending to decrease liver viral loads in WT B6 mice. In contrast, MCMV-infected and flagellin-treated TLR5 KO or Myd88KO mice had significantly increased liver viral loads (p=00001) compared with the flagellin- and PBS-treated B6 mice on day 3 post MCMV infection. While flagellin-treatment increased the numbers and activation status of NK cells, it decreased the numbers of activated (CD69+, ICOS+ and KLRG1+) CD4+ T cells on day 3 post MCMV infection. On day 10 post MCMV infection, enhanced numbers of splenic CD8+ T cells and CMV-specific tetramer+ CD8+ T cells (p<0.05) were detected in flagellin-treated mice compared with controls. The numbers of splenic CD4+ T cells increased >2 fold (p= 0.008) associated with significantly increased numbers of splenic CD4+CD25+ Foxp3+ regulatory T cells (p<0.05) on day 10 post MCMV infection in flagellin-treated mice compared with the control mice. Flagellin-induced anti-CMV immunity required IFN-γ signaling as flagellin-treated IFN-γRKO mice had markedly increased susceptibility to MCMV infection whereas flagellin-treated IL-12 KO and IDO KO mice had significantly reduced liver viral loads compared to the PBS-treated IL-12 KO and IDO KO mice on day 10 post MCMV infection. To explore interaction of flagellin with TLR5⁺ host hematopoietic cells and TLR5⁺host gut epithelium on CMV immunity, we next infected radiation chimeras expressing either TLR5 on hematopoietic cells or on gut epithelium with sub lethal i.p dose of MCMV. Flagellin-treated TLR5 KO BM → B6 radiation chimeras (TLR5+ host epithelium) had increased mortality compared with other groups following MCMV infection indicating that TLR5+ hematopoietic cells are required for protective immunity against CMV infection.

In summary, flagellin caused selective early increased activation of NK cells and reduced activation of CD4+ T cells in presence of MCMV infection. The protective effect of flagellin on anti-CMV immune responses are IFN-γ dependent but independent of IL-12 and IDO. The TLR5+ hematopoietic cells are required for the protective immunity against CMV infection. These data suggest the clinical importance of flagellin in modulating both innate and adaptive immunity against CMV infection and also as an alternative GvHD prophylaxis in allo-BMT recipients.

No relevant conflicts of interest to declare.