A Biohybrid Device for the Systemic Control of Acute Inflammation

Abstract

Properly regulated inflammation facilitates recognition and reaction to injury or infection, but inadequate or overly robust inflammation can lead to disease. Sepsis is an inflammatory disease that accounts for nearly 10% of total U.S. deaths, costing more than $17 billion. Acute inflammation in sepsis may evolve too rapidly to be modulated appropriately, and we suggest that therapies should focus not on abolishing inflammation, but rather on attenuating the positive feedback cycle of inflammation/damage/inflammation. In Gram-negative sepsis, bacterial endotoxin causes inflammation and is driven and regulated by the cytokine tumor necrosis factor-α (TNF-α), which is, in turn, negatively regulated via its endogenous inhibitor, soluble TNF-α receptor (sTNFR). We generated stably gene-modified variants of human HepG2 hepatocytes, using lentiviral constructs coding for mouse sTNFR driven by the constitutive cytomegalovirus promoter, and seeded them in a scaled-down, experimental liver bioreactor. When connected to anesthetized, cannulated rats subjected to endotoxin infusion and maintained solely by the animals' circulation, this biohybrid device elevated circulating sTNFR, reduced the levels of TNF-α and other key inflammatory mediators, alleviated hypotension, and reduced circulating markers of organ damage. This novel class of biohybrid devices may bemodified for patient- and disease-specific application, and, thus, may represent a disruptive strategy that offers the potential for rational inflammation reprogramming.

Keywords: biohybrid device; endotoxin; multiple organ dysfunction syndrome; sepsis; soluble tumor necrosis factor-alpha receptor 1; tumor necrosis factor alpha.

FIG. 1

Conceptual diagram of a biohybrid device for the systemic control of acute inflammation. Lentiviral vectors consisting of constitutive promoters upstream of endogenous cytokine inhibitors are constructed using standard molecular biology techniques. The vectors are used to create stably transduced variants of hepatocyte cell lines, as these cells can resist inflammation. The cells are seeded into liver bioreactors. The resultant biohybrid devices are connected extra-corporeally to affect systemic inflammation.

FIG. 2

Design and implementation of a biohybrid device for systemic control of acute inflammation. Lentiviral vectors were produced as described in the Materials and Methods section, which were used as a negative control (A) or for constitutive production and delivery of mouse sTNFR (B). (C) HepG2 cells were transduced with either the mouse sTNFR gene driven by the constitutive CMV promoter (filled symbols) or with a control HepG2 variant that, though transduced with a lentivirus coding for mouse sTNFR driven by the mouse NF-jB promoter, did not produce any sTNFR protein (open symbols). These gene-modified HepG2 variants were seeded in a 0.8 mL liver bioreactor, and the cells were maintained on the bioreactor for 3-day periods, at which time the bioreactor was removed from the perfusion equipment, flushed, and connected to cannulated rats for the studies depicted in Figures 2D, 3, and 4. At the conclusion of each in vivo experiment, the bioreactors were returned to the perfusion laboratory and maintained for a further 3 days, until they were utilized for a subsequent in vivo experiment. (D) liver bioreactors seeded with the cell lines depicted in (C) were attached to anesthetized, cannulated rats undergoing endotoxemia. Black arrows indicate the various components of the experimental setup, which is detailed in the Materials and Methods section. Red arrows indicate the direction of blood flow through the bioreactor. White arrows indicate the inlet and outlet ports of the bioreactor.

FIG. 3

Schematic of endotoxemia study. Rats were anesthetized and cannulated. Intravenous infusion of LPS was initiated immediately after establishing the extracorporeal circuit with the bioreactor connected, and continued to 4 h of bioreactor treatment. At 4 h, the LPS infusion was discontinued while continuing treatment with the bioreactor for an additional 2 h; thus, the total bioreactor treatment time was 6 h. Blood samples were collected at the indicated times and assayed as described. Mean arterial pressure was monitored throughout the experiment. See Materials and Methods section for experimental details. LPS, Gram-negative bacterial lipopolysaccharide; AST, Aspartate transaminase; ALT, alanine aminotransferase; ABG, arterial blood gases; HCT, hematocrit; Hgb, hemoglobin.

FIG. 4

Elevated sTNFR and suppression of endotoxin-induced systemic inflammation by a biohybrid device that produces sTNFR. Rats were subjected to endotoxemia as depicted in Figure 3, in the presence of 0.8 mL liver bioreactors seeded with either HepG2 cells expressing mouse sTNFR constitutively (filled symbols; n = 7 rats) or with a control HepG2 variant that did not produce any sTNFR (open symbols; n = 4 rats). A single sTNFR-producing and a single control bioreactor was used to treat all the animals in each experimental group, respectively. In between each in vivo study, the cells were maintained on the bioreactor for 3-day periods, at which time the bioreactor was removed from the perfusion equipment, flushed, and connected to the subsequent cannulated rat. (A) plasma sTNFR concentrations as a function of time and treatment. (B) plasma TNF-α concentration as a function of time and treatment. (C) plasma IL-1β concentration as a function of time and treatment. (D) plasma IL-6 concentration as a function of time and treatment. (E) plasma IL-10 concentration as a function of time and treatment. (F) plasma MCP-1 concentration as a function of time and treatment. *statistically significant differences from the control bioreactor (p < 0.05 by 2-way ANOVA followed by Holm–Sidak post hoc test). sTNFR, soluble tumor necrosis factor-alpha receptor I; TNF-α, tumor necrosis factor alpha; IL, interleukin; ANOVA, analysis of variance.

FIG. 5

Amelioration of endotoxin-induced pathophysiology by a biohybrid device that produces sTNFR. Various indices of pathophysiology were assessed in the rats described in Figure 4. (A) plasma ALT concentrations as a function of time and treatment. (B) MAP as a function of time and treatment. (C) plasma NO₂⁻/NO₃⁻ concentration as a function of time and treatment. *statistically significant differences from the control bioreactor (p < 0.05 by 2-way ANOVA followed by Holm–Sidak post hoc test). MAP, mean arterial pressure.