Immunosuppression in patients who die of sepsis and multiple organ failure

Abstract

Context: Severe sepsis is typically characterized by initial cytokine-mediated hyperinflammation. Whether this hyperinflammatory phase is followed by immunosuppression is controversial. Animal studies suggest that multiple immune defects occur in sepsis, but data from humans remain conflicting.

Objectives: To determine the association of sepsis with changes in host innate and adaptive immunity and to examine potential mechanisms for putative immunosuppression.

Design, setting, and participants: Rapid postmortem spleen and lung tissue harvest was performed at the bedsides of 40 patients who died in intensive care units (ICUs) of academic medical centers with active severe sepsis to characterize their immune status at the time of death (2009-2011). Control spleens (n = 29) were obtained from patients who were declared brain-dead or had emergent splenectomy due to trauma; control lungs (n = 20) were obtained from transplant donors or from lung cancer resections.

Main outcome measures: Cytokine secretion assays and immunophenotyping of cell surface receptor-ligand expression profiles were performed to identify potential mechanisms of immune dysfunction. Immunohistochemical staining was performed to evaluate the loss of immune effector cells.

Results: The mean ages of patients with sepsis and controls were 71.7 (SD, 15.9) and 52.7 (SD, 15.0) years, respectively. The median number of ICU days for patients with sepsis was 8 (range, 1-195 days), while control patients were in ICUs for 4 or fewer days. The median duration of sepsis was 4 days (range, 1-40 days). Compared with controls, anti-CD3/anti-CD28-stimulated splenocytes from sepsis patients had significant reductions in cytokine secretion at 5 hours: tumor necrosis factor, 5361 (95% CI, 3327-7485) pg/mL vs 418 (95% CI, 98-738) pg/mL; interferon γ, 1374 (95% CI, 550-2197) pg/mL vs 37.5 (95% CI, -5 to 80) pg/mL; interleukin 6, 3691 (95% CI, 2313-5070) vs 365 (95% CI, 87-642) pg/mL; and interleukin 10, 633 (95% CI, -269 to 1534) vs 58 (95% CI, -39 to 156) pg/mL; (P < .001 for all). There were similar reductions in 5-hour lipopolysaccharide-stimulated cytokine secretion. Cytokine secretion in sepsis patients was generally less than 10% that in controls, independent of age, duration of sepsis, corticosteroid use, and nutritional status. Although differences existed between spleen and lung, flow cytometric analysis showed increased expression of selected inhibitory receptors and ligands and expansion of suppressor cell populations in both organs. Unique differences in cellular inhibitory molecule expression existed in immune cells isolated from lungs of sepsis patients vs cancer patients and vs transplant donors. Immunohistochemical staining showed extensive depletion of splenic CD4, CD8, and HLA-DR cells and expression of ligands for inhibitory receptors on lung epithelial cells.

Conclusions: Patients who die in the ICU following sepsis compared with patients who die of nonsepsis etiologies have biochemical, flow cytometric, and immunohistochemical findings consistent with immunosuppression. Targeted immune-enhancing therapy may be a valid approach in selected patients with sepsis.

Conflict of interest statement

Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Dr Hotchkiss reports receiving grant support from Pfizer, Bristol-Meyers Squibb, and Aurigene. No other disclosures were reported.

Figures

Figure 1. Cytokine Secretion in Stimulated Splenocytes

Spleens were harvested from patients who died of sepsis (n=24–26) or nonsepsis etiologies (n=20–21). Cells were dissociated and washed and viability determined by trypan blue exclusion. Viable splenocytes (1 × 107) were stimulated with lipopolysaccharide or anti-CD3/anti-CD28 antibody. Supernatants were harvested at 5 and 22 hours and tumor necrosis factor (TNF), interferon γ (IFN-γ), and interleukins (IL) 6 and 10 were measured by enzyme-linked immunosorbent assay. There was a marked decrease in cytokine secretion in sepsis patients vs nonsepsis controls. Data were analyzed by 2-tailed nonparametric t test (Mann-Whitney U test). Each data marker represents an individual patient. Horizontal lines represent mean values. P<.001 for all plots, except P<.01 for TNF with lipopolysaccharide stimulation at 22 hours.

Figure 2. Expression of Cell Surface Receptors on Splenic CD4 and CD8 T Cells

See Figure 3 legend for explanation of geo-mean fluorescence intensity units, laboratory methods and statistical analysis. Compared with nonsepsis controls (n=24–26), sepsis patients (n=28–31) had activated T cells (increased CD69 in CD4 and CD8 T cells as well as increased CD25 [interleukin {IL} 2 receptor α] in CD4 T cells). Despite an activation phenotype, sepsis induced down-regulation of positive costimulatory receptors (CD28 in CD4 and CD8 T cells) as well as increased inhibitory receptors (programmed cell death 1 [PD-1] for CD4 T cells and cytotoxic T-lymphocyte antigen 4 [CTLA-4] for CD8 T cells). The IL-7 receptor α chain (CD127) was decreased in CD4 and CD8 T cells in sepsis.

Figure 3. Expression of Cell Surface Receptors on Splenic Antigen-Presenting Cells and Tissue Macrophages

Splenocytes (2 × 106) were stained with fluorescently conjugated antibodies or isotype-matched control antibodies and analyzed by flow cytometry (eAppendix). A positive gate was established based on isotype control staining. The percentage positive for each marker was defined by subtracting the percentage within the positive gate in the isotype control from the percentage within the positive gate in the specific stain. The geo-mean fluorescence intensity was determined by subtraction of the nonspecific fluorescence of the isotype control antibody from the fluorescence of the specific conjugated antibody and is expressed in units, which are an average of fluorescence intensity of the data collected within the selected gate after subtracting fluorescence intensity of isotope control. Antigen-presenting cells, ie, dendritic cells and macrophages/monocytes, as well as tissue-specific macrophages, showed an immunosuppressive phenotype in sepsis as evidenced by decreased expression of CD86 and HLA-DR. In addition, antigen-presenting cells from sepsis patients had increased expression of PD-L1, the ligand for the inhibitory receptor programmed cell death 1 PD-1 on T cells. Each data marker represents an individual patient. Statistical analysis was performed by 2-tailed nonparametric t test (Mann-Whitney U test). Horizontal lines represent mean values.

Figure 4. Expression of Cell Surface Receptors on Cells Isolated from Lung Tissue

Lung tissue was digested via collagenase followed by isolation of single cells. Lung cells (3 × 106) were stained with fluorescently conjugated antibodies or isotype-matched control antibodies and analyzed via flow cytometry (eAppendix). Lung CD4 and CD8 T cells from sepsis patients had increased programmed cell death 1 (PD-1), as determined by geo-mean fluorescence intensity (MFI), compared with nonsepsis controls. Data for MFI are expressed in units, which are an average of fluorescence intensity of the data collected within the selected gate after subtracting fluorescence intensity of isotope control. The majority of T cells from both sepsis and nonsepsis lung also expressed PD-1 (>50%) and B- and T-lymphocyte attenuator (BTLA) (>60%). Lung CD4 T cells from resection controls had elevated BTLA expression (in percentage and MFI) compared with sepsis patients. Plasmacytoid dendritic cells, a subset of dendritic cells, isolated from sepsis lungs had increased PD-L1 (in percentage and MFI) and PD-L2 (in percentage), ligands for PD-1 expressed on lung T cells, compared with both nonsepsis control groups. For PD-1 analysis, n=22 (CD4) and n=20 (CD8) for sepsis patients, n=9 (CD4) and n=8 (CD8) for transplant donor controls, and n=8 (both CD4 and CD8) for lung resection controls. For BTLA analysis, n=21 (CD4) and n=19 (CD8) for sepsis patients, n=9 (CD4) and n=8 (CD8) for transplant donor controls, and n=8 (both CD4 and CD8) for lung resection controls. For plasmacytoid dendritic cell analysis, n=17 for sepsis patients, n=6 for transplant donor controls, and n=8 for lung resection controls. Statistical analysis was performed using a non-parametric 2-tailed Mann-Whitney U test. Each data marker represents an individual patient. Horizontal lines represent mean values.

Figure 5. Immune Effector Cells in Spleen Tissue

Spleen from sepsis patients (n=22) or nonsepsis controls (n=12) was stained for HLA-DR, CD4, or CD8 and examined by an investigator (P.E.S.) blinded to sample identity (eAppendix). 3,3′-diaminobenzidine 4-HCl was used as a chromogen to stain the cells of interest (brown), and a hematoxylin counterstain (blue) was used for background staining. A (HLA-DR immunostain; 200×), In a representative control sample, HLA-DR immunoreactivity is robust in all periarteriolar T- and B-cell zones, consistent with major histocompatibility complex (MHC) II pattern 2A (see reference and eTable 7). There is marked loss of HLA-DR reactivity in B and T lymphocytes typical of nearly subtotal depletion of HLA-DR–reactive elements in sepsis (MHC II pattern 4). Note that sinusoidal endothelium staining is pronounced, a change seen only in sepsis. B (CD4 immunostain; 400×), Periarteriolar CD4 cells are quantitatively decreased in sepsis (right panel) relative to control (left panel). C (CD8 immunostain; 400×), Peri-arteriolar CD8 cells are also quantitatively decreased in sepsis (right panel) relative to controls (left panel). D, The dot plots are cell counts for CD4 and CD8 T cells, obtained by counting number of cells per field in periarteriolar lymphoid sheaths. Two fields were counted per slide and averaged. Statistical analysis was performed using a non-parametric 2-tailed Mann-Whitney U test. Each data marker represents an individual patient. Horizontal bars represent mean values.

Figure 6. Expression of HVEM in Lung Tissue

Sepsis or nonsepsis lung tissue was fixed in 4% paraformaldehyde and paraffin-embedded sections prepared (eAppendix). Lung sections were incubated with isotype-matched controls (A) or primary antibodies to anti–herpes virus entry mediator (HVEM) (B) followed by visualization of brown staining. Sepsis lung tissue stained positive for HVEM, the ligand for B- and T-lymphocyte attenuator. Lung resections, in particular of the same airway, were photographed using the 200× and 400× (inset) objectives for each antibody stain. C, Slides presented in (B) were evaluated in a blinded fashion and scored (in percentage) based on their positive staining for HVEM. Data were graphed as percentage positive HVEM staining in lung tissue (epithelium, endothelium, and macrophages) of sepsis patients and nonsepsis controls. Data presented in (C) are as follows: n=16 sepsis patients, n=7 transplant donor controls, and n=5 lung resection controls. Each data marker represents an individual patient. Horizontal bars represent mean values. 3,3′-diaminobenzidine 4-HCl was used as a chromogen to stain the cells of interest (brown), and a hematoxylin counterstain (blue) was used for background staining. aComparison of HVEM-positive epithelium greater in sepsis patients vs nonsepsis controls: P=.01.

Figure 7. Expression of PD-L1 and PD-L2 in Lung Tissue

Sepsis or nonsepsis lung tissue was fixed in 4% paraformaldehyde and paraffin-embedded sections prepared (eAppendix). Lung sections were incubated with isotype-matched controls (see Figure 6) or primary antibodies to anti–programmed cell death ligand 1 (PD-L1) (A), or anti–PD-L2 (B), followed by visualization of brown staining. Lung resections, in particular of the same airway, were photographed using the 200× and 400× (inset) objectives for each antibody stain. 3,3′-diaminobenzidine 4-HCl was used as a chromogen to stain the cells of interest (brown), and a hematoxylin counterstain (blue) was used for background staining. Sepsis lung tissue stained positive for PD-L1 and PD-L2, the ligands for PD-1, in lung epithelium compared with transplant donor lung tissue. Lung resection (normal-appearing lung tissue distal to cancerous tissue) stained positive in airway epithelium for PD-L2, like sepsis lung tissue, compared with transplant donor lung tissue.