Fig 1.
Study design of an acute lung injury model in pigs. Pig were randomly assigned into 5 groups and received LPS (n=11), LPS + CDCs at 3 different doses (25M n=6, 50M n=6, 100M n=5 cells total), or naïve pigs (n=4) were given PBS alone. Diagnostics were repeated at different intervals, baseline, 1 hour after LPS or saline infusion, and at the 48 hour endpoint. Diangostics included arterial blood gases, measures of respiratory phyisology (PA pressure, P/F ratio, compliance, plateau pressure, peak pressure, PCO2) peripheral venous blood sampling, bronchoalveolar lavage. Following euthanasia, endpoint lung tissue was harvested for histologic analysis.
Fig 2.
A- CDC injection attenuated a decrease in P/F ratio following LPS injury, which was most evident at the 100M dose.
(LPS-CDC25 -46.67±71.39, LPS-CDC50 -54.67±31.16 LPS-CDC100 -28.8±38.43 vs. LPS -109.4±52.97, p=0.03). B- Following suit, acute changes in compliance were preserved in CDC and saline injected pigs, but not in LPS only animals. (LPS-CDC25 -1.8±3.89ml/cmH2O, LPS-CDC50 -0.600±2.70ml/cmH2O, LPS-CDC100 -2.00±5.64ml/cmH2O vs. LPS -14.77±8.79ml/cmH20, p=0.002. C- We observed differences in peak pressure in 2 of the 3 CDC injected groups (LPS-CDC25 0.33±2.65cm/H2O, LPS-CDC50 -0.20±1.64cm/H2O, LPS-CDC100 3.20±2.77cm/H2O vs LPS 4.08±3.37cm/H2O, p=0.01) D- while changes in plateau pressure were preferable in the CDC injected groups (LPS-CDC25 0.20± 1.09cm/H2O, LPS-CDC50 -2.0±0.83cm/H2O, LPS-CDC100 0.400±1.14cm/H2O, LPS 3.81±4.35cm/H20, p=0.049) E- Pulmonary pressure was held closer to baseline or reduced in most groups except for LPS only (LPS-CDC25 2.2±6.14mmhg, LPS-CDC50, 7.33±±5.50mmhg, LPS-CDC100 0.10±1.24mmhg, LPS 10.91±6.71mmhg, p=0.0008). F- Gas exchange was more efficient in all groups as changes in CO2 were improved compared to LPS (LPS-CDC25 -2.7±2.1mmhg, LPS-CDC50 -0.92±3.25mmhg LPS-CDC100 -2.24±4.74mmhg vs. LPS 7.11±7.42mmhg, p=0.003. (D values represent baseline D0, and endpoint, D2).
Fig 3.
Automated MATLAB analysis of H&E stained lung sections.
A- Representative H&E sections of randomly selected slides from the porcine lung. B- Mask of control (left) LPS (middle) and LPS+CDC (right). Pigs suffering from LPS induced lung injury had the severity of atelectic injury greatly attenuated by administration of CDCs. Healthy animals with no lung injury demonstrated an alveolar area of 1.665±1.120area/total, LPS injured animals demonstrated collapsed alveoli 1.043±0.543area/total, while CDC treated pigs demonstrated the most preserved alveolar space 1.825±1.330area/total.(p=0.02) C- Mask analysis of hemorrhagic damage at endpoint. LPS injury alone caused significant bleeding within the lung parenchyma 0.0329±0.019area/total where this was rescued in LPS injured pigs treated with CDC’s, 0.014±0.001area/total (p=0.001).D- Next, cellular and proteinaceous debris was evaluated. Although there was a non statistical difference between groups, LPS pigs treated with CDC’s demonstrated a healthier trend when compared to LPS injury alone, CDC 0.008±0.007area/total vs. LPS 0.01134±0.01138, p=ns . E- Lastly, Alveolar wall thickness in CDC pigs more closely reflected healthy alveoli of naïve pigs, Naïve 0.4070±0.1427area/total, LPS 0.4847±0.1213area/total, CDC 0.3825±0.1116area/total (p=0.01). Magnification 10x, scale bar 200um.
Fig 4.
Peripheral blood mononuclear cell concentration over the course of the study at 3 timepoints (T1-T3).
A- Neutrophil concentration at baseline was similar in all groups. In LPS pigs, there was a shift out of peripheral circulation immediately following LPS administration, however this rebounded by the 48 hour endpoint (T3). B-Lavage samples demonstrated an overall increase in neutrophil population in LPS pigs from T1-T3. This was attenuated at endpoint in the LPS-CDC100 group compared to LPS pigs (LPS-CDC100 9.5±6.87%,LPS 30.58±10.43%, p=0.03). C-D Lymphocyte activity was similar between all groups in both blood and lavage samples, p=ns. E- A peripheral monocytosis can be observed in all LPS injured groups over the course of the study, however there were no differences at endpoint. F- Alveolar lavage samples showed a progressive decrease in macrophage population in LPS injured pigs, however this was rescued in the LPS-CDC100 group`(LPS-CDC100 88.7±6.87%, LPS 62.00±23.61%, p=0.037).
Fig 5.
Cytokine expression in alveolar lavage fluid.
Multiplex array for cytokine quantification lung lavage samples. A- IFNγ was increased in BAL samples of LPS only pigs (LPS, baseline, 36.25±67.62pg/ml, endpoint 129.6±234.3pg/ml, p=ns) . There was no difference over the course of the experiment in healthy animals (baseline, 32.50±14.5pg/ml, endpoint, 37.00±17.45pg/ml, p=ns), or LPS+CDC pigs (baseline, 39±31.44pg/ml, endpoint, 38.8±27.03pg/ml, p=ns) B- Inflammatory cytokine IL-1α significantly increased in BAL samples from LPS only pigs (baseline, 5.27±5.39pg/ml, endpoint 37.93±46.20pg/ml, p=0.02). Small changes in IL-1α were observed in CDC injected pigs (baseline, 6.00±17.28pg/ml, endpoint 17.28±19.63pg/ml p=ns. C- IL-1β was found elevated in LPS only injured pigs (baseline, 2.89±3.43pg/ml, endpoint 130.3±152.0pg/ml, p=0.0083). D- In our LPS injured pigs, there was an increase in IL-1ra (baseline, 63.13±55.15pg/ml, endpoint, 301.3±330.6pg/ml, p=0.021. Lavage samples showed an attenuated IL-1ra response in healthy pigs and LPS+CDC group. E- Inflammatory cytokine IL-6 was increased in LPS lavage fluid (baseline, 59.82±.39.07pg/ml, endpoint, 125.6±66.02, p=0.0071). Injection of CDCs to LPS injured pigs reduced IL-6 concentration in alveolar samples . F- In alveolar lavage fluid, LPS injured pigs showed a large increase in IL-8 concentration (baseline,46.63± 12.34pg/ml, endpoint 432.9±640.8pg/ml, p=0.048), while CDC treated pigs demonstrated the opposite G- 48 hours after LPS injury IL-18 increases substantially in alveolar fluid (baseline, 239.3±152.4pg/ml, endpoint, 676.7±406.5pg/ml, p=0.0021). This occurrence is present in a small percentage of CDC pigs (baseline, 93±126.9pg/ml, endpoint 211±.306.6pg/ml, p=ns).
Fig 6.
A- IFNγ was increased in serum of LPS pigs (LPS, Δ1540±1829pg/ml, vs LPS+ CDC, -Δ1987±2219pg/ml p=0.003). B- Inflammatory cytokine IL-1α was significantly increased in LPS serum samples (LPS, Δ5.61±7.65pg/ml, vs LPS+CDC -Δ10.41±13.5pg/ml, p=0.014). C- Following suit, IL-1β increased in LPS only serum samples, (LPS, Δ67.5±39.92pg/ml, vs LPS+CDC -Δ58±121.3pg/ml, p=0.0048). D- In our LPS injury model we observed an increase in IL-1ra LPS pigs in serum samples. In serum LPS, (LPS, Δ2401±2049pg/ml, vs LPS+CDC ±1063±1031pg/ml, p=0.19). E- Inflammatory cytokine IL-6 was increased in serum in LPS pigs and was reduced in LPS+CDC pigs (LPS, Δ75.93±69.48pg/ml, vs LPS +CDC -Δ19.87±109.9pg/ml, p=0.04). F- Both groups demonstrated a decrease in IL-8 from baseline to endpoint in serum (LPS, -Δ50.17±113pg/ml, vs LPS+CDC 43.3±44.32, p=0.8). G- 48 hours after LPS injury IL-18 increases substantially in systemic circulation. This phenomenon is opposite in CDC pigs as there is either no change, or a decrease in IL-18 from baseline to endpoint (LPS, Δ389.5±163.1pg/ml, vs LPS+CDC -Δ277±520pg/ml p=0.0014).
Fig 7.
Clinical evaluation of renal azotemia.
A-Peripheral blood samples were obtained and serum isolated. Across the study, there was no increase in BUN exept for in the LPS only group (T1 9.92±2.36mg/dl, vs, T3 31.18±26.47mg/dl, p=0.008. B-serum creatinine followed suit with a significant increase in the LPS only group between baseline and the 48hr endpoint (T1 1.83±0.24mg/dl, vs, T3 5.12±4.80mg/dl, p=0.02).