Impaired blood dendritic cell numbers and functions after aneurysmal subarachnoid hemorrhage

Abstract

Previous presentation: Portions of this study were presented at the Annual Congress of Société Française d'Anesthésie et de Réanimation in Paris, September 2012.

Background: Toll-like receptor (TLR) agonists are promising therapy for the prevention of nosocomial infections in critical ill patients. We aimed to analyze the TLR-reactivity of circulating dendritic cells (DC) as assessed by cytokine production after an ex vivo challenge with TLR agonists in aneurysmal subarachnoid hemorrhage (SAH) patients.

Methods and findings: A single-center prospective observational study took place in one intensive care unit of a teaching hospital. Blood samples were harvested on days 2, 5 and 10 in 21 severe SAH patients requiring mechanical ventilation and 17 healthy controls. DC production of cytokines (Tumour Necrosis Factor, TNF-α; Interleukin, IL-12; and Interferon, IFN-α) was assessed by intracellular immunostaining on TLR-3, 4, 7/8 and 9 stimulations. SAH patients had decreased numbers of blood myeloid (mDCs) and plasmacytoid DCs (pDCs) on days 2, 5 and 10. Compared with the healthy controls, the frequency of mDCs producing TNF-α after TLR-3 stimulation was decreased in the SAH patients. The frequency of myeloid DCs producing IL-12 after TLR-3 and 4 stimulations was also decreased in the SAH patients. In contrast, the mDCs response to TLR-7/8 was not impaired in the SAH patients. The frequency of pDCs producing TNF-α(+) and IFN-α(+) on TLR-7/8 stimulation were reduced at all of the tested times in the SAH patients, whereas reactivity to TLR-9 was preserved. On day 2, the pDCs from non-survivor patients (n=8) had a decreased ability to produce IFN-α on TLR-9 stimulation compared with the survivors.

Conclusions: These data suggest functional abnormalities of circulating pDCs and mDCs that could be important for immunomodulation after SAH.

Figure 1. Time course of circulating cytokines in patients with aneurysmal subarachnoid hemorrhage.

Blood samples were drawn from SAH patients (N = 18) and from HC (N = 11). The level of IL-2, -4, -6, -8, -10, -12, IFN-α, IFN-γ, TNF-α and GM-CSF were assessed in plasma by luminex on days 2, 5 and 10. Plots represent median (interquartile range). HC: healthy controls. SAH: aneurysmal subarachnoid hemorrhage. Dashed line indicates the lower limit of detection. * P<0.05, ** P<0.01.

Figure 2. Gating strategy used to identify blood DC subsets and intracellular cytokines production in DCs in whole blood stimulated with TLR ligands.

Whole blood samples were incubated with TLR3, 4, 7/8 or 9 ligands for 3.5-hour and then stained for identification of myeloid DC (HLA-DR+, Lin-, CD11c+, CD123-) and plasmacytoid DC (HLA-DR+, lin−, CD11c−, CD123+) together with intracellular cytokine production (TNFα, IL-12, IFNα).

Figure 3. Time course of circulating mDCs and pDCs numbers in patients with aneurysmal subarachnoid hemorrhage.

Comparison of circulating (A) myeloid DC and (B) plasmacytoid DC counts in SAH patients (N = 21) on days 2, 5 and 10 compared with HC (N = 11). Plots represent median (Interquartile ranges). HC: healthy controls. SAH: aneurysmal subarachnoid hemorrhage. DCs: dendritic cells. * P<0.05, ** P<0.01.

Figure 4. Time course of TLR-induced production of TNF-α and IL-12 in circulating mDCs from patients with aneurysmal subarachnoid hemorrhage.

Intracellular cytokine measurement was performed in circulating mDCs from SAH patients (N = 21) on days 2, 5 and 10 and from HC (N = 11). The percentages of mDCs expressing TNF-α or IL-12 were assessed after a 3.5-hour ex vivo stimulation with (A) polyIC (TLR-3 agonist), (B) lipopolysaccharide (TLR-4 agonist) or (C) CL097 (TLR-7/8 agonist). The percentage of positive DCs without TLR-stimulation was below 1% (data not shown). The results are presented as percentages of mDCs expressing TNF-α (%TNF-α⁺) or IL-12 (% IL-12⁺). Plots represent median (Interquartile ranges). HC: healthy controls. mDCs: myeloid dendritic cells. SAH: aneurysmal subarachnoid hemorrhage. TNF-α: tumour necrosis factor -α. IL-12: intreleukin-12. * P<0.05.

Figure 5. Time course of TLR-induced productions of TNF-α and IFN-α in circulating pDCs from patients with aneurysmal subarachnoid hemorrhage.

Intracellular cytokine measurement was performed in circulating pDCs from SAH patients (N = 21) on days 2, 5 and 10 after brain injury and from HC (N = 11). The percentages of pDCs expressing TNF-α or IFN-α were assessed after a 3.5-hour ex vivo stimulation with (A) CL097 (TLR-7/8 agonist) or (B) CpG (TLR-9 agonist). The percentage of positive pDCs without TLR-stimulation was below 1% (data not shown). The results are presented as percentages of pDCs expressing TNF-α (%TNF-α⁺) or IFN-α (%IFN-α⁺). Plots represent median (Interquartile ranges). HC: healthy controls. IFN-α: interferon. pDCs: plasmacytoid dendritic cells. SAH: aneurysmal subarachnoid hemorrhage. TNF-α: tumour necrosis factor -α. *P<0.05.

Figure 6. Exploratory comparison of mDC and pDC status on day 2 in survivors and non-survivors.

(A) The number of circulating myeloid DCs and plasmacytoid DCs were compared on day 2 between 13 survivors and 8 non-survivors. On day 2, mDCs and pDCs were stimulated ex vivo with polyIC (TLR-3 agonist), lipopolysaccharide (TLR-4 agonist), CL097 (TLR-7/8 agonist) and CpG (TLR-9 agonist) for 3.5 hours. (B) The percentages of mDCs expressing TNF-α or IL-12 and (C) the percentages of pDCs expressing TNF-α or IFN-α were compared between survivors and non-survivors. The percentage of positive DCs without TLR stimulation was below 1% (data not shown). The results are presented as percentages of DCs expressing TNF-α (%TNF-α⁺), IL-12 (%IL-12⁺) or IFN-α (%IFN-α⁺). Plots represent median (Interquartile ranges). * P<0.05.