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Basic Science Aspects

Sepsis-Induced Hypercytokinemia and Lymphocyte Apoptosis in Aging-Accelerated Klotho Knockout Mice

Inoue, Shigeaki*†; Sato, Takehito; Suzuki-Utsunomiya, Kyoko*; Komori, Yukako*; Hozumi, Katsuto; Chiba, Tomoki; Yahata, Takashi§; Nakai, Kozo; Inokuchi, Sadaki

Author Information
doi: 10.1097/SHK.0b013e3182845445

Abstract

INTRODUCTION

Sepsis, a systemic inflammatory response to infection, is responsible for more than 210,000 deaths in the United States annually (1) and is one of the most challenging clinical problems worldwide, constituting the leading cause of death in noncoronary intensive care units (2). Sepsis is also known as a disease of the aged, because nearly 60% of cases occur in patients older than 65 years, a group that makes up only 12% of the population (3, 4). The average age of patients with sepsis is increasing over time, and age is a known independent predictor of mortality in septic patients (5). According to the most recent estimates, the number of people in the world older than 60 years will approximately double from 756 million to 1,400 million by the year 2030 (6), which is likely to cause an increase in the incidence and prevalence of sepsis.

The incidence of sepsis appears to increase with increasing age because of the associated increase in comorbidities, malnutrition, institutionalization, exposure to instrumentation, and altered immune function (7), thereby leading to increased susceptibility to infection (8). As in humans, aging plays an important role in the murine response to sepsis. Aged mice subjected to cecal ligation and puncture (CLP) or endotoxemia have been found to have increased systemic levels of inflammatory cytokines (9) and increased splenic apoptosis (10) with increased mortality (11). In addition, aged animals subjected to CLP are resistant to antibiotic therapy (11) and have increased pulmonary inflammation after endotoxemia (12). As these reports suggested, a murine model of aging is essential to improve the treatment of sepsis in the elderly. However, the average life span of a laboratory mouse is approximately 2 years, making aging studies time consuming and costly.

Accelerated aging phenotypes are observed in various mouse models and are frequently used in experimental aging studies (13, 14). One such model is the Klotho knockout mouse (Klotho mouse). The product of the Klotho gene, a 130-kd single-pass transmembrane protein with a short cytoplasmic domain (10 amino acids), is expressed predominantly in the kidney. The Klotho mice develop a syndrome resembling human aging, including symptoms, such as skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis, and pulmonary emphysema, and have a short life span of approximately 8 weeks (15). Conversely, overexpression of the Klotho gene in mice extends the life span (17). It is suggested that Klotho functions as an aging suppressor gene and that the secreted form of the Klotho protein primary works as a humoral antiaging protein (18). Recently, Klotho was reported to function as a putative antiaging protein in cardiovascular and renal disease (19). The total amount of urinary excreted Klotho has been studied in early diabetic nephropathy in humans (20) and may be a potential biomarker for assessing the residual renal function among peritoneal dialysis patients (21). However, although much is known about the aging process in this model system, details regarding the immunity of and immunological changes in Klotho mice during sepsis are still unclear. The purpose of this study was to elucidate immunological changes in septic Klotho mice, with the goal of finding therapeutic targets for sepsis in the aged population.

MATERIALS AND METHODS

Mice

Five-week-old female homozygous Klotho knockout mice (Klotho) and their homogeneous wild-type (WT) littermates were purchased from CLEA Japan (Tokyo, Japan). The mice were housed for at least 1 week before use.

Antibodies

The following surface marker antibodies were used: anti-CD3-FITC (clone 17A2) for T cells, anti–CD8a-APC/Cy5 (clone 53–6.7) for CD8 T cells, anti–CD4-Pacific Blue (clone GK1.5) for CD4+ T cells, anti–CD49b-PerCP/Cy5.5 (clone DX5) for natural killer (NK) cells, anti–CD19-APC (clone 6D5) for B cells, anti–CD11c-PE (clone N418) and FITC I-A/I-E (clone M5/114) for activated dendritic cells, anti–F4/80-PE/Cy7 (clone BN8) and anti–CD11b-APC (clone M1/70) for macrophages, and anti–Ly-6G/Ly-6C (clone Gr-1)-PE (RB6-8C5) for neutrophils. All the antibodies were purchased from Bio-Legend (San Diego, Calif).

For detection of apoptosis, anti–cleaved caspase 3 (Asp175)-PE (clone 5A1E) antibody for flow cytometric analysis and anti–cleaved caspase 3 (Asp175) polyclonal antibody and anti–Bcl-xL antibody (clone 54H6) for immunohistochemistry were purchased from Cell Signaling (Danvers, Mass).

CLP sepsis model

All the animal studies were approved by the Tokai University Animal Studies Committee. The CLP model as developed by Chaudry et al. (22) was used to induce intra-abdominal peritonitis. Mice were anesthetized with isoflurane, and a midline abdominal incision was made. The cecum was mobilized, ligated below the ileocecal valve, and punctured once with a 27-gauge needle. The abdomen was closed in 2 layers, and the mice were injected subcutaneously with 1.0 mL of 0.9% NaCl. The mice used to determine absolute cell counts, apoptosis, and cytokine production were killed at 8 h after surgery. For survival studies, mice who underwent CLP as described above were monitored every 2 h, and the study ended at 7 days after surgery. Another set of CLP mice was used for the cytokine assay, and 1 drop of blood was collected from the cheek plexus of each of these animals at 0, 3, 6, 12, 18, and 24 h after CLP.

Quantification of neutrophils and monocyte/macrophages in and analysis of bacterial colony counts for the peritoneal cavity

Approximately 8 h after surgery, the mice were anesthetized with isoflurane, and the peritoneal cavities were lavaged with 3 mL of prewarmed 0.9% NaCl. The peritoneal fluid was harvested, and neutrophils and monocyte/macrophages were identified by flow cytometric analysis (LSR Fortessa; BD Pharmingen, Franklin Lakes, NJ) by using cell surface markers. Sample data were acquired using the CellQuest-Pro software (BD Biosciences, Franklin Lakes, NJ) and analyzed with the FlowJo software version 7.5.5 for Microsoft (TreeStar, San Carlos, Calif). Peritoneal fluid was also placed in sterile vials, and equal volumes were used for culture. Ten-fold serial dilutions of the homogenate were prepared and plated, and colony counts were determined after 24 h of incubation.

Spleen and thymus harvest

Next, mice were gently killed by cervical dislocation under sedation, and the spleen and thymus were surgically removed. Isolated splenocytes and thymocytes were prepared by gently pressing the organ through a 70-µm filter; the cells were then washed, and the red blood cells were lysed.

Quantification of absolute cell counts and apoptosis

Total cell counts per spleen and thymus were determined via the CDA-1000 kit (Sysmex, Kobe, Japan). The percentages of individual cell phenotypes (CD4, CD8, and B cells) were determined via flow cytometric analysis. The absolute cell count for each splenic and thymus population subset was calculated by the following formula: cell counts of cell subpopulations = total cell counts multiplied by the subset population percentage. Apoptosis was quantified by flow cytometry using an antibody against cleaved caspase 3. Harvested splenocytes were fixed in 1% paraformaldehyde, washed with Perm/Wash solution (BD Biosciences), and incubated with cleaved caspase 3 antibody for 1 h at 4°C.

Bright-field microscopy of tissue sections stained with hematoxylin and eosin

Splenic and thymic tissue sections were obtained at 8 h after surgery and fixed overnight in 10% buffered formalin. Tissue sections were then processed and stained with hematoxylin and eosin (H&E). The degree of splenocyte and thymocyte apoptosis was evaluated via bright-field microscopy using a Nikon Eclipse E600 (Tokyo, Japan). Microscopic evaluation of apoptosis was used to confirm the presence of sepsis-induced apoptosis in the Klotho-CLP group throughout the architecturally distinct regions of the spleen. Apoptotic splenocytes exhibit the characteristic findings of nuclear compaction (pyknosis) and nuclear fragmentation (karyorrhexis). Tissue sections from four to five samples were examined, and a minimum of five to seven random fields were evaluated for each organ section (200× magnification). Higher magnifications (400×–600×) were used to visualize the finer details of cellular apoptotic changes.

Immunohistochemistry staining

Immunolocalization was performed on paraffin-embedded formalin-fixed spleen and thymus samples. Briefly, after paraffin removal in xylene, the sections were rehydrated and submitted to microwave treatment (800 W/10 min) in 0.01 M EDTA. After quenching of endogenous peroxidase with 3% H2O2 for 30 min, the sections were exposed to anti–caspase 3 polyclonal antibody (1:200 dilution) and anti–Bcl-xL polyclonal antibody (1:200 dilution) for 30 min. After incubation with the primary antibody, immunodetection was performed using biotinylated anti–rabbit or anti–mouse immunoglobulin G. Peroxidase-conjugated streptavidin (Vector Laboratories, Burlingame, Calif) with diaminobenzidine as the substrate completed the immunostaining. Negative controls for nonspecific binding included normal rabbit or mouse serum.

Quantitative real-time polymerase chain reaction

T cells were enriched from disaggregated thymocytes by using positive magnetic bead selection; the purity of Thy-1.2–positive T cells was greater than 85% as determined by flow cytometry. Quantitative real-time polymerase chain reaction (PCR) was performed to identify transcriptionally regulated candidate genes involved in mediating apoptosis. The relative abundance of each gene was determined using β-actin as a reference. Polymerase chain reaction products were evaluated by dissociation curves to confirm the presence of single amplicons and the absence of significant primer-dimer contamination. Quantitative real-time PCR results for each gene have been reported as fold change in gene expression versus the expression in WT-sham mice.

Cytokine analysis

Serum cytokines were quantitated using the BD FACS Array and the Inflammation Kit per the manufacturer’s recommendations as previously described (23). The lower limits of detection were as follows: interleukin 6 (IL-6), 1.4 pg/mL; tumor necrosis factor α (TNF-α), 2.8 pg/mL; monocyte chemotactic protein 1 (MCP1), 2.7 pg/mL; and IL-10, 9.6 pg/mL.

Statistical methods

Data were analyzed with IBM SPSS Statistics version 20 (SPSS Inc, Chicago, Ill). Data are presented as mean ± SEM values. Two-way analysis of variance was performed to determine the main effects of surgery (CLP vs. sham) and the type of mouse (WT vs. Klotho) as well as the interaction between these 2 effects. The Mann-Whitney U test was performed for cytokine analysis and peritoneal lavage bacterial colony counts to compare nonparametric values between two independent groups. For survival studies, a log-rank test was used. Significance was reported at P < 0.05.

RESULTS

Decreased survival in Klotho mice compared with WT mice after CLP

The survival of Klotho mice after CLP (Klotho-CLP) was significantly lower than that of WT mice after CLP (WT-CLP; 0% vs. 100%, P < 0.01). Most Klotho-CLP mice started to die 8 to 12 h after surgery, and all of them died within 36 h (Fig. 1).

F1-13
Fig. 1:
Decreased survival in Klotho mice compared with WT mice after CLP. The survival of Klotho mice after CLP was significantly lower than that of WT mice after CLP (0% vs. 100%, P < 0.01). Klotho-CLP mice started to die within 8 to 12 h after CLP, and all the Klotho-CLP mice died within 36 h after CLP. **P < 0.01.

Impaired bacterial clearance with decreased recruitment of innate immune cells in Klotho septic mice

The number of colony-forming units from peritoneal lavage at 8 h after surgery was significantly increased in peritoneal lavage fluid samples from Klotho-CLP mice than in WT-CLP mice after 24 h of incubation (P < 0.05; Fig. 2A). To detect the recruitment of neutrophils and macrophages into the peritoneal cavity, the cells in the peritoneal lavage were analyzed by flow cytometry. Two-way analysis of variance showed that CLP significantly increased the number of neutrophils, macrophages, and activated dendritic cells in the lavage fluid (P < 0.01) but that the lack of the Klotho gene significantly decreased the recruitment of these cells to the peritoneal cavity (P < 0.05; Fig. 2B).

F2-13
Fig. 2:
Impaired bacterial clearance with decreased recruitment of neutrophils, macrophages, and dendritic cells in Klotho septic mice. A, Increased bacterial count in the peritoneal cavity of Klotho septic mice. The number of colony-forming units in peritoneal lavage fluid samples at 8 h after surgery obtained from the Klotho-CLP group was significantly higher than that from the WT-CLP group (P < 0.05). The horizontal line indicates the mean value. B, Decreased recruitment of neutrophils, macrophages, and dendritic cells to the peritoneal cavity of Klotho mice with sepsis. Significant differences were observed in this regard for both surgery (sham vs. CLP, P < 0.01) and the type of mouse (WT vs. Klotho, P < 0.05). No significant interaction was observed between surgery and the type of mouse for all the groups. n = 10–12 in each group. *P < 0.05. **P < 0.01.

Reduction in lymphocytes and increased apoptosis of lymphocytes in Klotho septic mice

In the spleen obtained at 8 h after surgery, we observed significantly smaller lymphocyte populations (CD4+ T cells, CD8+ T cells, B cells, and NK cells) in Klotho mice than in WT mice (P < 0.01; Fig. 3A). Significant interactions were observed between surgery (CLP vs. sham) and the type of mouse (WT vs. Klotho) in terms of the number of NK and B cells, suggesting a difference between sham and CLP operations with respect to the impact on NK and B cell fates in Klotho mice. The thymus of the Klotho mice had a significantly lesser number of CD4+/CD8+ double-positive T cells than the thymus of the WT mice (P < 0.01; Fig. 3B).

F3-13
Fig. 3:
Decreased number of lymphocytes in the spleen and thymus in normal and septic Klotho mice. A, The numbers of total cells, NK cells, B cells, CD4+ T cells, and CD8+ T cells in the spleen at 8 h after surgery were lower in Klotho mice than in WT mice (P < 0.01). A statistically significant interaction was observed between surgery and the type of mouse in terms of the total number of NK and B cells (P < 0.01). B, The numbers of CD4+/CD8+ cells, CD4+/CD8 cells and CD8+/CD4 cells were lower in Klotho mice than in WT mice. A and B, n = 10–12 in each group. *P < 0.05, **P < 0.01. Pint; P value of the interaction between the main effects of surgery and the type of mouse.

In addition to the reduction in lymphocytes in Klotho mice, we found that sepsis induced extensive apoptosis in both the spleen and the thymus obtained at 8 h after surgery of these mice. Hematoxylin-eosin staining of the spleen demonstrated extensive classic features of apoptosis, including pyknosis and karyorrhexis in Klotho-CLP mice. The number of caspase 3–positive cells in the spleen was significantly increased by CLP in Klotho mice (P < 0.01) and according to the type of mouse (WT vs. Klotho; P < 0.01; Fig. 4A). These results are consistent with the flow cytometry data that show that sepsis induced a significant increase in the number of caspase 3–positive and a significant decrease in the number of Bcl-xL–positive B cells in the Klotho mice (P < 0.01; Fig. 4B). The results obtained for the spleen were similar to those obtained for the thymus. Histological and flow cytometric analyses showed that the rate of apoptosis in the thymus was significantly higher in Klotho-CLP mice than in the WT-CLP mice (P < 0.01; Supplemental Fig. 1A, B, https://links.lww.com/SHK/A156). Immunohistochemical and quantitative real-time PCR analyses showed that Bcl-xL expression was significantly lower in the thymocytes from Klotho-CLP mice than in the thymocytes from WT-CLP mice (P < 0.05; Supplemental Fig. 1A, C, https://links.lww.com/SHK/A156).

F4-13
Fig. 4:
Sepsis induces apoptosis of spleen cells in Klotho mice. A, Representative fields from H&E- and caspase 3 antibody–stained spleen sections obtained 8 h after surgery (magnification ×400). H&E staining of the spleen at 8 h after surgery demonstrated that the Klotho-CLP groups showed the classic features of apoptosis, including pyknosis and karyorrhexis, to a greater extent than the WT-CLP groups. The number of caspase 3–positive cells in the spleen was significantly increased by the CLP operation in Klotho mice (P < 0.01) and showed differences according to the type of mouse (WT vs. Klotho) (P < 0.01). B, Flow cytometric analysis showed that sepsis induced a significant increase in apoptosis in the caspase 3–positive cells and a significant decrease in apoptosis in the Bcl-xL–positive B cells in Klotho mice (P < 0.01). n = 4–5 in each group. **P < 0.01

Hypercytokinemia in Klotho septic mice

Serum concentrations of TNF-α, IL-6, MCP-1, and IL-10 in the Klotho-CLP group were significantly higher than those in the WT-CLP group from 6 to 12 h after CLP (P < 0.01; Fig. 5).

F5-13
Fig. 5:
Prolonged hypercytokinemia in Klotho septic mice. The levels of both proinflammatory and anti-inflammatory cytokines, i.e., TNF-α, IL-6, MCP-1, and IL-10, were significantly higher in the Klotho-CLP groups than in the WT-CLP groups from 6 to 12 h after CLP. The levels of baseline at 0 h (before CLP) were 0 to 10 ng/mL in each cytokine. n = 10–12 in each group. **P < 0.01.

DISCUSSION

This study demonstrated the decreased survival of Klotho mice during sepsis, as well as impaired bacterial clearance, hypercytokinemia, and lymphocyte apoptosis in these mice. Although Klotho mice have been widely used in the study of aging and age-related diseases (15–17), immunity against infection in Klotho mice had not been previously examined. Besides the impaired B cell development (24), details regarding the immune functions of Klotho mice had not been reported. Our study might be the first to investigate the immune cells of Klotho mice and the changes in these cells during septic insult.

We speculate that hypercytokinemia plays a key role in understanding various phenomena in Klotho septic mice. In humans, elevated proinflammatory cytokines are a known indicator and prognostic factor of severe sepsis (25). The persistently high levels of IL-6 and IL-10, key markers of proinflammatory and anti-inflammatory responses, respectively, have been associated with the development of multiple organ failure and fatal outcome in sepsis (26). For example, Yende et al. (27) recently showed that serum concentrations of IL-6 and IL-10 are elevated in patients after hospitalization for pneumonia and that these increased cytokine levels are associated with increased mortality over the subsequent 3 months (25). In a murine model, circulating IL-6 is one of the best prognostic markers for sepsis induced by CLP because of its high sensitivity and specificity (28). In aged mice, elevated proinflammatory and anti-inflammatory cytokines were significantly higher than those of young mice (29). In our study, we observed persistently higher proinflammatory and anti-inflammatory cytokine levels in Klotho septic mice than in WT septic mice.

One possible reason for this hypercytokinemia might be incomplete bacteria clearance, leading to sustained activation of innate immune cells, thereby producing proinflammatory and anti-inflammatory cytokines. We showed that the recruitment of neutrophils and macrophages from the bone marrow to the peritoneal cavity, which plays an essential role in the defense against microbial infection, decreased in Klotho septic mice. The recruitment of dendritic cells, a critical link between innate and adaptive immunity, also decreased in Klotho septic mice. These results suggest that the impaired innate immunity in Klotho mice leads to incomplete bacterial clearance, immune system perturbation, systematic organ failure, and acute death. Alternatively, we can assume that the innate immune cells in aged septic mice have increased potential to produce inflammatory cytokines. Indeed, bone marrow–derived macrophages from Klotho-CLP mice produced higher amounts of proinflammatory and anti-inflammatory cytokines than those from WT-CLP mice (data not shown). This aspect will be further investigated in the future.

In addition to the importance of lymphocytes in the innate immune response, these cells also play important roles during septic insult. Adaptive immunity severely deteriorates with age, causing many health problems in the elderly (30). Age-related thymopoietic insufficiency results in a reduced peripheral T-cell pool and the clonal expansion of preexisting memory T cells to maintain the overall T-cell pool (31). In Klotho mice, extensive thymus atrophy has also been demonstrated (15). Aged mice and rats have shown an increase in both basal and activation-induced lymphocyte apoptosis (10), and aged human lymphocytes are more prone to apoptosis than young lymphocytes (32). Our data also showed a decreased T-cell population and increased sepsis-induced apoptosis in the spleen and thymus of Klotho mice.

There are several limitations to this study. First, Klotho mice are smaller than WT mice, even though the outward physiological appearance of Klotho mice resembles that of the WT mice. Body weight and spleen weight of Klotho mice were 50% and 60% that of WT mice, respectively. We adjusted the splenocyte subfraction populations by body weight and confirmed that the relative numbers of splenocytes in each subfraction were also smaller in WT mice than in Klotho mice. These findings suggest that baseline differences between Klotho and WT mice are more important than the impact of CLP. Second, it has been reported that Klotho-deficient mice and FGF23-deficient mice develop many common phenotypes, including shortened life span, growth retardation, infertility, muscle atrophy, hypoglycemia, and vascular calcification in the kidneys (33), but we did not examine the relevance of FGF23 in our murine sepsis model. Furthermore, it has been reported that Klotho induces IFG-1 and insulin resistance, which is crucial for the development and proliferation of lymphocytes (34, 35); however, our experiments did not examine circulating levels of IGF-1 or insulin.

Third, Klotho mice undergo accelerated aging, which may be different from the normal process of aging. The product of the Klotho gene is mainly expressed in the kidney; however, it has been reported that renal function is unaffected because creatinine levels in Klotho mice are normal (15). We also find no specific pathological changes between WT and Klotho mice in both sham and CLP model (data not shown), suggesting to us that the Klotho gene does not affect renal injury after sepsis. Similar to the kidney, both the liver and lung showed no specific pathological change after sepsis, except for the enlargement of the air spaces distal to the terminal bronchiole, which was identified as emphysema as previously reported (15). Septic Klotho mice also had demonstrated conventional necrotic hepatocytes in the liver, with infiltration and edema in the lung that was similar to that of septic WT mice. These results suggest that impaired immunity, not dysfunction of major organs, may affect decreased survival after sepsis in Klotho mice.

Finally, our study revealed similarities and differences between Klotho and normal aged mice. Similarities, including increased circulating cytokines and apoptosis of lymphocytes with poor survival, were observed in both Klotho and aged mice. However, Klotho mice die earlier after CLP (within 8–12 h), whereas aged mice usually die around 24 h or more after CLP (11). Furthermore, decreased bacterial clearance after sepsis, presumably associated with decreased recruitment of neutrophils and macrophages, was observed in Klotho mice, but not in aged mice (29). Therefore, Klotho mice serve as a useful experimental tool for the study of aging immunity in sepsis, although care should be taken before applying these findings to aged mice.

CONCLUSIONS

Hypercytokinemia was observed in Klotho septic mice together with impaired bacterial clearance and increased lymphocyte apoptosis. These findings may be related to poor survival in Klotho mice after sepsis. We believe that the use of Klotho mice in this study will shed light on aging immunity during sepsis.

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Keywords:

Aging; hypercytokinemia; immunosuppression; Klotho; lymphocyte; sepsis

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