Severe sepsis exacerbates cell-mediated immunity in the lung due to an altered dendritic cell cytokine profile

Abstract

Severe sepsis leads to long-term alterations in the immune response of surviving individuals. We have modeled this alteration in host immunity by studying the survivors of severe experimental sepsis (murine cecal ligation and puncture), which were subsequently challenged with lung granuloma-inducing Schistosoma mansoni eggs. This granulomatous response is a well-studied cell-mediated immune reaction characterized by elevated levels of type-2 cytokines. Pulmonary granulomas induced by S. mansoni eggs in cecal ligation and puncture survivors were significantly larger and contained more eosinophils than granulomas in sham-operated mice. Significantly lower interleukin (IL)-12p40 mRNA and IL-12p70 protein levels were observed in the lungs of postseptic mice with developing granulomas, compared with controls. Postseptic mice had significantly fewer dendritic cells in the lungs during the granulomatous response. Isolated lung dendritic cells from postseptic mice at days 8 and 16 after S. mansoni egg challenge exhibited defective IL-12 synthesis but enhanced IL-10 synthesis after Toll-like receptor agonist challenge. Pulmonary transfection with an IL-12-expressing adenovirus in postseptic mice reversed the skewing of the pulmonary cytokine profile and normalized the lung granulomatous response. Our data indicate that severe sepsis shifts the pulmonary cytokine environment, presumably via effects on pulmonary dendritic cells, which in turn alters the lung cell-mediated immune response.

Figure 1

The histology of pulmonary granulomas induced by S. mansoni eggs. A: Sham and CLP groups were challenged with 5000 live S. mansoni eggs at day 3 after surgery. The left lung lobe from both groups was collected at different time points for histological analysis. HE-stained lung sections from sham or CLP mice are shown. B: Quantification of granuloma size at a series of indicated time points after egg challenge. *, P ≤ 0.05 compared with granuloma size measured in sham mice. C: Quantification of eosinophil number in pulmonary granulomas. Each group: n = 5∼6/time point. The results shown are representative of three experiments and are expressed as mean ± SEM. τ, P ≤ 0.05 compared with eosinophil number in the pulmonary granulomas from sham mice.

Figure 2

Cytokine transcript expression in the lungs from sham and CLP mice. After egg challenge at day 3 after surgery, mice from the sham or CLP groups were anesthetized, and the upper right lobe was collected to investigate the gene expression level of IL-4, IL-5, IL-10, IL-12p40, and IL-13 in the whole lungs at days 1, 2, 4, 6, 8, 16, and 30 after egg challenge. Total RNA was isolated and reverse transcribed to cDNA. Quantitative real-time PCR (Taqman) was performed to measure the transcript level of each cytokine. The mRNA level of each sample was normalized by mRNA level in naïve mice, and the data are shown as the fold of increase [2^(−ΔΔCT)]. *P ≤ 0.05 compared with IL-12p40 mRNA level measured in the lungs of sham mice, n = 4∼6.

Figure 3

Cytokine protein level in the whole lungs from mice of sham and CLP group. To investigate the production of IL-4, IL-5, IL-10, IL-12p40, and IL-13 in the whole lungs of sham and CLP mice, the lower right lobes from sham or CLP mice were analyzed by ELISA and normalized to the total protein level. *, P ≤ 0.05 compared with IL-12p70 protein level in the lungs of sham mice. ϕ, P ≤ 0.05 compared with IL-4 level in the lungs of sham mice at day-1; τ, P ≤ 0.05 compared with IL-10 level in the lungs of sham mice at day 2; γ, P ≤ 0.05 compared with IL-13 level in the lungs of sham mice at day 1.

Figure 4

Severe sepsis impaired DC recruitment to pulmonary granulomas induced by S. mansoni eggs. Mice from sham or CLP group (n = 5∼7/group/time point) were sacrificed at days 2, 8, and 16. Mice killed at day 0 did not receive eggs and were used as baseline values. Lungs were digested by collagenase IV, and the dispersed cells were stained as follows: CD11c⁺CD11b⁺ and CD11c⁺B220⁺. These cell surface markers were assessed by flow cytometry analysis. The results shown are representative of two experiments and are expressed as mean ± SEM. * and τ, P ≤ 0.05 compared with CD11c⁺CD11b⁺ DCs and CD11c⁺B220⁺ DCs in the lungs of sham mice at days 2 and 8, respectively.

Figure 5

Alteration of gene expression of IL-12p40 and IL-10 in DCs from postseptic mice. Sham and CLP-operated mice (n = 6–8/group/time point) were subsequently intravenously challenged with 5000 live S. mansoni eggs. Eight and 16 days later, mice were sacrificed, and lung DCs were purified by positive selection with CD11c⁺ microbeads. After overnight culture, purified DCs (as shown in A) were stimulated with medium, 80 μg/ml SEA (TLR2 agonist), 1 μg/ml LPS (TLR4 agonist), or 2.5 μg/ml Pam3cys (TLR2 agonist). Quantitative real-time PCR was performed to measure the mRNA levels of IL-10 and IL-12p40 in cultured DCs after 6 hours of stimulation. * and α, P ≤ 0.05 compared with IL-12p40 mRNA level in the DCs from sham mice at days 8 and 16, respectively. τ and β, P ≤ 0.05 compared with IL-10 mRNA level in the DCs from sham mice at days 8 and 16, respectively, after egg challenge.

Figure 6

DCs from postseptic mice showed the defective IL-12p70 production and enhanced IL-10 production. Sham and CLP-operated mice were sacrificed at days 8 and 16 after egg challenge. Lung DCs were purified by positive selection with CD11c⁺ microbeads from each animal. After overnight culture, purified DCs were stimulated with medium, 80 μg/ml SEA (TLR2 agonist), 1 μg/ml LPS (TLR4 agonist), or 2.5 μg/ml Pam3cys (TLR2 agonist). ELISA was performed to measure the protein levels of IL-10 and IL-12p70 in the supernatant of cultured DCs after 48 hours of stimulation. * and α, P ≤ 0.05 compared with IL-12p70 level in the DCs from sham mice at days 8 and 16, respectively. τ and β, P ≤ 0.05 compared with IL-10 level in the DCs from sham mice at days 8 and 16, respectively.

Figure 7

Introduction of IL-12-expressing adenovirus reversed the type-2-biased cytokine profile in the postseptic lung. Sham or postseptic mice (n = 4–6/group/time point) were intratracheally administrated with 3 × 10⁸ colony-forming unit (CFU) IL-12-expressing adenovirus (AdmIL-12) or control adenovirus (Ad70) immediately followed by 5000 S. mansoni eggs given intravenously. At 6, 8, and 16 days after virus and egg challenge, mice were killed, and the lower right lobes were collected from each mouse for cytokine analysis by ELISA. The results shown are representative of two experiments and are expressed as mean ± SEM. *, P ≤ 0.05 compared with IL-12p70 level in the lungs of sham mice receiving control virus at days 6 and 8. α, P ≤ 0.05 compared with IL-12p70 level in the lungs of CLP mice receiving Ad70 at days 6 and 8. β, P ≤ 0.05 compared with IL-4 protein level in the lungs of CLP mice receiving control virus at day 6.

Figure 8

Introduction of IL-12-expressing adenovirus attenuates pulmonary granuloma pathology. Sham or postseptic mice (n = 4–6/group/time point) were intratracheally administrated with 3 × 10⁸ PFU IL-12-expressing adenovirus (AdmIL-12) or control adenovirus (Ad70) immediately followed by 5000 S. mansoni eggs given intravenously. At 6, 8, and 16 days after virus and egg challenge, mice were killed, and the left lobe was collected from each mouse for histological analysis. A: Quantification of granuloma size. *, P ≤ 0.05 compared with granuloma size measured in sham mice receiving Ad70 at day 16 after egg challenge. α, P ≤ 0.05 compared with CLP mice receiving Ad70 at all of three time points. B: Eosinophil number. **, P ≤ 0.05 compared with intragranulomatous eosinophil count in sham mice receiving Ad70 at days 8 and 16 after egg challenge. β, P ≤ 0.05 compared with intragranulomatous eosinophil count in CLP mice receiving Ad70.

Figure 9

Administration of IL-12-expressing adenovirus fails to enhance DC recruitment to postseptic lung. Postseptic mice (n = 5/group) were intratracheally administrated with 3 × 10⁸ PFU AdmIL-12 or Ad70 immediately followed by 5000 S. mansoni eggs given intravenously. Eight days later, lungs were harvested from both group and digested by collagenase IV. The dispersed lung cells were stained as follows: CD11c⁺ in combination with CD11b⁺ or B220⁺, followed by flow cytometry analysis.