Toll-Like Receptor 4 on both Myeloid Cells and Dendritic Cells Is Required for Systemic Inflammation and Organ Damage after Hemorrhagic Shock with Tissue Trauma in Mice

Abstract

Trauma combined with hemorrhagic shock (HS/T) leads to systemic inflammation, which results in organ injury. Toll-like Receptor 4 (TLR4)-signaling activation contributes to the initiation of inflammatory pathways following HS/T but its cell-specific roles in this setting are not known. We assessed the importance of TLR4 on leukocytes of myeloid lineage and dendritic cells (DCs) to the early systemic inflammatory response following HS/T. Mice were subjected to HS/T and 20 inflammatory mediators were measured in plasma followed by Dynamic Bayesian Network (DBN) Analysis. Organ damage was assessed by histology and plasma ALT levels. The role of TLR4 was determined using TLR4^-/-, MyD88^-/-, and Trif^-/- C57BL/6 (B6) mice, and by in vivo administration of a TLR4-specific neutralizing monoclonal antibody (mAb). The contribution of TLR4 expressed by myeloid leukocytes and DC was determined by generating cell-specific TLR4^-/- B6 mice, including Lyz-Cre × TLR4^loxP/loxP, and CD11c-Cre × TLR4^loxP/loxP B6 mice. Adoptive transfer of bone marrow-derived TLR4^+/+ or TLR4^-/- DC into TLR4^-/- mice confirmed the contribution of TLR4 on DC to the systemic inflammatory response after HS/T. Using both global knockout mice and the TLR4-blocking mAb 1A6 we established a central role for TLR4 in driving systemic inflammation. Using cell-selective TLR4^-/- B6 mice, we found that TLR4 expression on both myeloid cells and CD11c^high DC is required for increases in systemic cytokine levels and organ damage after HS/T. We confirmed the capacity of TLR4 on CD11c^high DC to promote inflammation and liver damage using adoptive transfer of TLR4^+/+ conventional (CD11c^high) DC into TLR4^-/- mice. DBN inference identified CXC chemokines as proximal drivers of dynamic changes in the circulating levels of cytokines/chemokines after HS/T. TLR4 on DC was found to contribute selectively to the elevations in these proximal drivers. TLR4 on both myeloid cells and conventional DC is required for the initial systemic inflammation and organ damage in a mouse model of HS/T. This includes a role for TLR4 on DC in promoting increases in the early inflammatory networks identified in HS/T. These data establish DC along with macrophages as essential to the recognition of tissue damage and stress following tissue trauma with HS.

Keywords: dendritic cells; hemorrhagic shock; myeloid cells; toll-like receptor 4; trauma.

Figure 1

Toll-like receptor 4 (TLR4) drives systemic inflammation and end-organ damage in a mouse model of hemorrhagic shock (HS) and trauma (HS/T). Wild-type (WT), TLR4^−/−, MyD88^−/−, or Trif^−/− B6 mice were subjected to HS/T. IL-6 and ALT plasma levels were measured 6 h after the onset of HS/T as described in Section “Materials and Methods” (A,B). In separate experiments, the selective anti-mouse TLR4 Ab 1A6 or its isotype control (W6/32 Ab) was administered intravenously 30 min prior to the initiation of the HS and the levels of IL-6 and ALT were measured at 3, 6, and 20 h after the onset of HS/T (C,D). Panels (A,C) show IL-6 plasma levels, (B,D) show ALT plasma levels, (E,F) show IL-6 and ALT plasma levels after 6 h following the initiation of HS/T in the following cell-specific TLR4^−/− B6 mouse strains: Albumin-Cre × TLR4^loxP/loxP (Alb-Cre), CD11c-Cre × TLR4^loxP/loxP (CD11c-Cre), and Lyz-Cre × TLR4^loxP/loxP (Lyz-Cre). WT and TLR4^loxP/loxP (FloxP) mice were used as controls. Panels (G,H) show IL-6 and ALT plasma levels in lethally irradiated WT B6 mice reconstituted with bone marrow cells from CD11c-diphtheria toxin (DT) receptor (DTR) B6 mice or CD11c-TLR4^−/− B6 mice at 6 h after HS/T. WT B6 mice were used as controls [(A,B,E,F) *P < 0.05, analyzed by Mann–Whitney Rank Sum Test; (C) P = 0.050, (D) *P < 0.001, analyzed by two-way ANOVA; (G,H) *P < 0.001, analyzed by Student’s t-test (n = 6–15 animals/experimental condition)].

Figure 2

Histopathological analysis of liver in experimental bone marrow (BM) chimeras after HS/T. Liver sections from wild-type and experimental BM chimeras [TLR4^−/− and dendritic cell (DC)-TLR4^−/−], left untreated (control) or 6 h after HS/T were stained with H&E (A) as described in Section “Materials and Methods.” Images are representative of 7–10 mice per treatment group. Arrows indicate areas of necrosis in liver. Panel (B) shows the quantification of organ damage (*P < 0.001, analyzed by Student’s t-test).

Figure 3

Histopathological analysis of lung in experimental bone marrow (BM) chimeras after HS/T. Lung sections from wild-type (WT) and experimental BM chimeras [TLR4^−/− and dendritic cell (DC)-TLR4^−/−], left untreated (control) or 6 h after HS/T were stained with H&E (A) as described in Section “Materials and Methods.” Images are representative of 7–10 mice per treatment group. Arrow heads point to inflammatory cell infiltration in the lung. Panel (B) shows the quantification of organ damage (*P < 0.001, analyzed by Student’s t-test).

Figure 4

CD11c-dendritic cell (DC) contribute to systemic inflammation and liver damage after HS/T. (A) Flow cytometric analysis of TLR4^−/− DC generated from bone marrow precursors (day 6), purified by sorting with CD11c paramagnetic beads (Myltenyi), and adoptively transferred (i.v.) to TLR4^−/− [or wild-type (WT), control] B6 mice. The injected inoculum consisted mostly of immature DC (purity >95%). (B,C) TLR4^−/− B6 mice adoptively transferred (i.v.) with TLR4^−/− DC exhibited significantly lower concentrations of IL-6 and ALT in plasma measured 6 h after HS/T, as compared to TLR4^−/− mice injected with WT DCs. There were no significant differences in plasma concentrations of IL-6 and ALT following HS/T in WT B6 mice reconstituted (i.v.) with either WT or TLR4^−/− DCs (B) *P < 0.05, analyzed by Student’s t-test; (C) *P < 0.05, analyzed by Mann–Whitney Rank Sum Test (n = 8–12 independent experiments).

Figure 5

MIG and KC are early drivers of inflammation in a mouse model of HS/T. Dynamic changes in 20 cytokines and chemokines were measured in wild-type (WT) B6 mice subjected to HS/T at 0, 2 (end of shock), 3, 6, and 24 h as described in Section “Materials and Methods.” Using the time courses of circulating inflammatory mediators, Dynamic Bayesian Network (DBN) inference was used to determine possible network structures that exhibit self-feedback as central nodes. DBN inference suggested a primary network driven with core motifs consisting of MIG and KC, each of which drives its own expression and were inferred to affect the downstream production of IP-10, MCP-1, and IL-10 (n = 6 independent experiments).

Figure 6

Toll-like receptor 4 (TLR4) on CD11^high dendritic cell (DC) and myeloid cells is required for KC, MIG, and IP-10 increases following HS/T. Mice where TLR4 was selectively deleted from CD11c⁺ or Lyz⁺ cells were subjected to HS/T and plasma levels of KC (A), MIG (B), and IP-10 (C) were measured by Luminex as described in Section “Materials and Methods.” All three chemokines levels were significantly lower in mice subjected to HS/T where TLR4 was selectively deleted from CD11c⁺ or Lyz⁺ cells. MIG and IL-10, but not KC, were lower in DC-TLR4^−/− mice subjected HS/T [*P < 0.05, analyzed by Student’s t-test (n = 7–13 independent experiments)]. Comparison of the basal levels (control) showed, with one exception, no statistical significant differences between the different strains as compared to wild-type (WT) [(A) WT vs. TLR4^−/− (P = 0.228), DC-TLR4^−/− (P = 0.207), Lyz^−/− (P = 0.760); (B) WT vs. TLR4^−/− (P = 0.067), DC-TLR4^−/− (P = 0.013), Lyz^−/− (P = 0.059); (C) WT vs. TLR4^−/− (P = 0.057), DC-TLR4^−/− (P = 0.079), Lyz^−/− (P = 0.145)].