Josef G. Heuer

Adoptive Transfer of In Vitro-Stimulated CD4+CD25+ Regulatory T Cells Increases Bacterial Clearance and Improves Survival in Polymicrobial Sepsis

Regulatory CD4+CD25+ T cells (Tregs) suppress autoimmune and inflammatory diseases through mechanisms that are only partly understood. Previous studies suggest that Tregs can suppress bacterially triggered intestinal inflammation and respond to LPS through TLRs with enhanced suppressive activity. In this study, we have used murine cecal ligation and puncture as a model of polymicrobial sepsis to explore the effects of adoptive transfer of Tregs on septic outcome. Adoptive transfer of in vitro-stimulated Tregs in both prevention and therapeutic modes significantly improved survival of cecal ligation and puncture mice. Furthermore, the effect was dependent on both the number of Tregs adoptively transferred and the presence of host T cells. Animals that received stimulated Tregs had significantly increased peritoneal mast cells and peritoneal TNF-α production. More importantly, adoptive transfer of in vitro-stimulated Tregs significantly improved bacterial clearance, which resulted in improved survival. Our results suggest a novel role for Tregs in sepsis.

FGF21 Is Increased by Inflammatory Stimuli and Protects Leptin-Deficient ob/ob Mice from the Toxicity of Sepsis

The acute phase response (APR) produces marked alterations in lipid and carbohydrate metabolism including decreasing plasma ketone levels. Fibroblast growth factor 21 (FGF21) is a recently discovered hormone that regulates lipid and glucose metabolism and stimulates ketogenesis. Here we demonstrate that lipopolysaccharide (LPS), zymosan, and turpentine, which induce the APR, increase serum FGF21 levels 2-fold. Although LPS, zymosan, and turpentine decrease the hepatic expression of FGF21, they increase FGF21 expression in adipose tissue and muscle, suggesting that extrahepatic tissues account for the increase in serum FGF21. After LPS administration, the characteristic decrease in plasma ketone levels is accentuated in FGF21-/- mice, but this is not due to differences in expression of carnitine palmitoyltransferase 1α or hydroxymethyglutaryl-CoA synthase 2 in liver, because LPS induces similar decreases in the expression of these genes in FGF21-/- and control mice. However, in FGF21-/- mice, the ability of LPS to increase plasma free fatty acid levels is blunted. This failure to increase plasma free fatty acid could contribute to the accentuated decrease in plasma ketone levels because the transport of fatty acids from adipose tissue to liver provides the substrate for ketogenesis. Treatment with exogenous FGF21 reduced the number of animals that die and the rapidity of death after LPS administration in leptin-deficient ob/ob mice and to a lesser extent in control mice. FGF21 also protected from the toxic effects of cecal ligation and puncture-induced sepsis. Thus, FGF21 is a positive APR protein that protects animals from the toxic effects of LPS and sepsis.

Evaluation of protein C and other biomarkers as predictors of mortality in a rat cecal ligation and puncture model of sepsis.

Objective:To evaluate protein C and other factors associated with the septic response as predictors of mortality in a clinically relevant animal model of sepsis. Design:Laboratory investigation. Setting:Eli Lilly and Company discovery research laboratory. Subjects:Forty female Sprague Dawley Rats weighing 245–265 g. Interventions:Polyethylene catheters were surgically implanted into the femoral vein and sepsis was induced by cecal ligation and puncture (CLP). A solution of 5% dextrose in 0.9% saline was continuously infused via femoral catheters immediately following surgery. Blood sampling was done before surgery and at 6 and 20 hrs after surgery. Rats were then monitored for survival out to 4 days. Measurements and Main Results:Blood collections were used to measure blood glucose, bacteremia, plasma protein C, D-dimer, hormones, chemokines, cytokines, and myoglobin (as a marker of organ damage). Mortality was categorized into three groups: early death (before 30 hrs post-CLP), late death (after 30 hrs post-CLP), and survivors (96 hrs post-CLP). Compared with survivors, early death rats had statistically significant differences in 30 variables indicative of severe inflammation, coagulopathy, and muscle damage including less bacterial clearance, hypoglycemia, lower plasma protein C, higher plasma D dimer, higher plasma cytokine/ chemokines, and higher plasma myoglobin concentrations. Twenty variables had a moderate to strong correlation with time of death. Receiver operator characteristic curves generated from a simple logistic regression model indicated that KC and macrophage inflammatory protein-2, rodent homologues of the human growth related oncogene CXC chemokine family, and protein C were the best predictors of mortality in this model. Conclusions:The data from this study indicate that an early decrease in protein C concentration predicts poor outcome in a rat sepsis model. The data further indicate that increases in the CXC chemokines macrophage inflammatory protein-2 and KC precede poor outcome.

Role of Protein C in Renal Dysfunction after Polymicrobial Sepsis

Protein C (PC) plays an important role in vascular function, and acquired deficiency during sepsis is associated with increased mortality in both animal models and in clinical studies. This study explored the consequences of PC suppression on the kidney in a cecal ligation and puncture model of polymicrobial sepsis. This study shows that a rapid drop in PC after sepsis is strongly associated with an increase in blood urea nitrogen, renal pathology, and expression of known markers of renal injury, including neutrophil gelatinase-associated lipocalin, CXCL1, and CXCL2. The endothelial PC receptor, which is required for the anti-inflammatory and antiapoptotic activity of activated PC (APC), was significantly increased after cecal ligation and puncture as well as in the microvasculature of human kidneys after injury. Treatment of septic animals with APC reduced blood urea nitrogen, renal pathology, and chemokine expression and dramatically reduced the induction of inducible nitric oxide synthase and caspase-3 activation in the kidney. The data demonstrate a clear link between acquired PC deficiency and renal dysfunction in sepsis and suggest a compensatory upregulation of the signaling receptor. Moreover, these data suggest that APC treatment may be effective in reducing inflammatory and apoptotic insult during sepsis-induced acute renal failure.

Treatment of sepsis-induced acquired protein C deficiency reverses Angiotensin-converting enzyme-2 inhibition and decreases pulmonary inflammatory response.

The protein C (PC) pathway plays an important role in vascular and immune function, and acquired deficiency during sepsis is associated with increased mortality in both animal models and in clinical studies. However, the association of acquired PC deficiency with the pathophysiology of lung injury is unclear. We hypothesized that low PC induced by sepsis would associate with increased pulmonary injury and that replacement with activated protein C (APC) would reverse the activation of pathways associated with injury. Using a cecal ligation and puncture (CLP) model of polymicrobial sepsis, we examined the role of acquired PC deficiency on acute lung injury assessed by analyzing changes in pulmonary pathology, chemokine response, inducible nitric-oxide synthase (iNOS), and the angiotensin pathway. Acquired PC deficiency was strongly associated with an increase in lung inflammation and drivers of pulmonary injury, including angiotensin (Ang) II, thymus and activation-regulated chemokine, plasminogen activator inhibitor (PAI)-1, and iNOS. In contrast, the protective factor angiotensin-converting enzyme (ACE)-2 was significantly suppressed in animals with acquired PC deficiency. The endothelial protein C receptor, required for the cytoprotective signaling of APC, was significantly increased post-CLP, suggesting a compensatory up-regulation of the signaling receptor. Treatment of septic animals with APC reduced pulmonary pathology, suppressed the macrophage inflammatory protein family chemokine response, iNOS expression, and PAI-1 activity and up-regulated ACE-2 expression with concomitant reduction in AngII peptide. These data demonstrate a clear link between acquired PC deficiency and pulmonary inflammatory response in the rat sepsis model and provide support for the concept of APC as a replacement therapy in acute lung injury associated with acquired PC deficiency.

Myostatin Neutralization Results in Preservation of Muscle Mass and Strength in Preclinical Models of Tumor Induced Muscle Wasting

Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non–tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass– and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non–tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting. Mol Cancer Ther; 14(7); 1661–70. ©2015 AACR.

Generation and activity of a humanized monoclonal antibody that selectively neutralizes the epidermal growth factor receptor ligands transforming growth factor-α and epiregulin.

At least seven distinct epidermal growth factor (EGF) ligands bind to and activate the EGF receptor (EGFR). This activation plays an important role in the embryo and in the maintenance of adult tissues. Importantly, pharmacologic EGFR inhibition also plays a critical role in the pathophysiology of diverse disease states, especially cancer. The roles of specific EGFR ligands are poorly defined in these disease states. Accumulating evidence suggests a role for transforming growth factor α (TGFα) in skin, lung, and kidney disease. To explore the role of Tgfa, we generated a monoclonal antibody (mAb41) that binds to and neutralizes human Tgfa with high affinity (KD = 36.5 pM). The antibody also binds human epiregulin (Ereg) (KD = 346.6 pM) and inhibits ligand induced myofibroblast cell proliferation (IC50 values of 0.52 and 1.12 nM for human Tgfa and Ereg, respectively). In vivo, a single administration of the antibody to pregnant mice (30 mg/kg s.c. at day 14 after plug) or weekly administration to neonate mice (20 mg/kg s.c. for 4 weeks) phenocopy Tgfa knockout mice with curly whiskers, stunted growth, and expansion of the hypertrophic zone of growth plate cartilage. Humanization of this monoclonal antibody to a human IgG4 antibody (LY3016859) enables clinical development. Importantly, administration of the humanized antibody to cynomolgus monkeys is absent of the skin toxicity observed with current EGFR inhibitors used clinically and no other pathologies were noted, indicating that neutralization of Tgfa could provide a relatively safe profile as it advances in clinical development.

Accelerated Onset and Increased Severity of Acute Graft-Versus-Host Disease Following Adoptive Transfer of DR6-Deficient T Cells

DR6 is a recently identified member of the TNFR family. In a previous study, we have shown that DR6 KO mice have enhanced CD4+ T cell proliferation and Th2 cytokine production. Acute graft-vs-host disease (GVHD) results from the activation and expansion of alloreactive donor T cells following bone marrow transplantation. In this article, we demonstrate that the transfer of donor T cells from DR6 KO mice into allogeneic recipient mice in a parent into an F1 model of acute GVHD results in a more rapid onset of GVHD with increased severity. Recipients of DR6 KO T cells exhibit earlier systemic symptoms of GVHD, more rapid weight loss, earlier histopathological organ damage in the thymus, spleen, and intestines, and earlier mortality. The rapid onset of GVHD in these mice may be attributable to the enhanced activation and expansion of DR6 KO CD4+ and CD8+ T cells. Our findings support the hypothesis that DR6 serves as an important regulatory molecule in T cell immune responses. The identification and use of DR6 ligands and/or agonistic Abs to DR6 may represent useful therapeutics in the treatment of T cell-mediated diseases such as GVHD.

Activated Protein C Modulates Chemokine Response and Tissue Injury in Experimental Sepsis

The protein C (PC) pathway plays an important role in vascular function, and acquired deficiency during sepsis is associated with increased mortality. We have explored the role of PC suppression in modulating early inflammatory events in a model of polymicrobial sepsis. We show that increased levels of organ damage and dysfunction are associated with decreased levels of endogenous PC. Notably, animals with low PC had correspondingly high levels of pulmonary iNOS expression, which correlated with chemokines KC/Gro and MIP2, previously shown to predict outcome in this model. Treatment with activated protein C (aPC) not only reduced the pathology score, leukocyte infiltration and markers of organ dysfunction, but also suppressed the induction of iNOS, and the chemokine response (including KC/Gro, MIP2, IP-10, RANTES, GCP-2 and lymphotactin), and increased apoA1. aPC treatment also suppressed the induction of VEGF, a marker recently suggested to play a pathophysiological role in sepsis. These data demonstrate a clear link between low protein C and degree of organ damage and dysfunction in sepsis, as well as the early reversal with aPC treatment. Moreover, our data show a direct role of aPC in broadly modulating monocyte and T-cell chemokines following systemic inflammatory response.

FoxA2 Involvement in Suppression of Protein C, an Outcome Predictor in Experimental Sepsis

ABSTRACT Low levels of protein C (PC) predict outcome as early as 10 h after insult in a rat polymicrobial sepsis model and were associated with suppression of PC mRNA, upstream transcription factor FoxA2, and cofactor hepatocyte nuclear factor 6 (HNF6). Small interfering RNA suppression of FoxA2 in isolated hepatocytes demonstrated regulation of both its cofactor HNF6 and PC. Our data suggest that reduced FoxA2 may be important in the suppression of PC and resulting poor outcome in sepsis.