Neuronally expressed PDL1, not PD1, suppresses acute nociception.

PDL1 is a protein that induces immunosuppression by binding to PD1 expressed on immune cells. In line with historical studies, we found that membrane-bound PD1 expression was largely restricted to immune cells; PD1 was not detectable at either the mRNA or protein level in peripheral neurons using single neuron qPCR, immunolabeling and flow cytometry. However, we observed widespread expression of PDL1 in both sensory and sympathetic neurons that could have important implications for patients receiving immunotherapies targeting this pathway that include unexpected autonomic and sensory related effects. While signaling pathways downstream of PD1 are well established, little to no information is available regarding the intracellular signaling downstream of membrane-bound PDL1 (also known as reverse signaling). Here, we administered soluble PD1 to engage neuronally expressed PDL1 and found that PD1 significantly reduced nocifensive behaviors evoked by algogenic capsaicin. We used calcium imaging to examine the underlying neural mechanism of this reduction and found that exogenous PD1 diminished TRPV1-dependent calcium transients in dissociated sensory neurons. Furthermore, we observed a reduction in membrane expression of TRPV1 following administration of PD1. Exogenous PD1 had no effect on pain-related behaviors in sensory neuron specific PDL1 knockout mice. These data indicate that neuronal PDL1 activation is sufficient to modulate sensitivity to noxious stimuli and as such, may be an important homeostatic mechanism for regulating acute nociception.

Decoding the secreted inflammatory response of primary human hepatocytes to hypoxic stress in vitro.

BACKGROUND: The cellular and molecular response of liver cells to hypoxic stress is not fully understood. We used computational modeling to gain insights into the inflammatory response of primary human hepatocytes (HC) to hypoxic stress in vitro. METHODS: Primary HC from cancer patients were exposed to hypoxia (1% O2) or normoxia (21% O2) for 1-48 h, and the cell supernatants were assayed for 21 inflammatory mediators. Data were analyzed by Two-Way ANOVA, Dynamic Bayesian Network (DBN) inference, Dynamic Network Analysis (DyNA), and Time-interval Principal Component Analysis (TI-PCA). RESULTS: The chemokines MCP-1/CCL2 and IP-10/CXCL10, along with the cytokines interleukin (IL)-2 and IL-15 were altered significantly over time in hypoxic vs. normoxic HC. DBN inference suggested central, coordinating roles for MCP-1 and IL-8 in regulating a largely conserved inflammatory program in both hypoxic and normoxic HC. DyNA likewise suggested similar network trajectories of decreasing complexity over time in both hypoxic and normoxic HC, though with differential connectivity of MCP-1, IP-10, IL-8, and Eotaxin. TI-PCA pointed to IL-1ß as a central characteristic of inflammation in hypoxic HC across all time intervals, along with IL-15 and IL-10, vs. Eotaxin, IL-7, IL-10, IL-15, and IL-17A in normoxic HC. CONCLUSIONS: Thus, diverse human HC appear to respond in a largely conserved fashion to cell culture stress, with distinct characteristics based on the presence or absence of hypoxia.

"Thinking" vs. "Talking": Differential Autocrine Inflammatory Networks in Isolated Primary Hepatic Stellate Cells and Hepatocytes under Hypoxic Stress.

We hypothesized that isolated primary mouse hepatic stellate cells (HSC) and hepatocytes (HC) would elaborate different inflammatory responses to hypoxia with or without reoxygenation. We further hypothesized that intracellular information processing ("thinking") differs from extracellular information transfer ("talking") in each of these two liver cell types. Finally, we hypothesized that the complexity of these autocrine responses might only be defined in the absence of other non-parenchymal cells or trafficking leukocytes. Accordingly, we assayed 19 inflammatory mediators in the cell culture media (CCM) and whole cell lysates (WCLs) of HSC and HC during hypoxia with and without reoxygenation. We applied a unique set of statistical and data-driven modeling techniques including Two-Way ANOVA, hierarchical clustering, Principal Component Analysis (PCA) and Network Analysis to define the inflammatory responses of these isolated cells to stress. HSC, under hypoxic and reoxygenation stresses, both expressed and secreted larger quantities of nearly all inflammatory mediators as compared to HC. These differential responses allowed for segregation of HSC from HC by hierarchical clustering. PCA suggested, and network analysis supported, the hypothesis that above a certain threshold of cellular stress, the inflammatory response becomes focused on a limited number of functions in both HSC and HC, but with distinct characteristics in each cell type. Network analysis of separate extracellular and intracellular inflammatory responses, as well as analysis of the combined data, also suggested the presence of more complex inflammatory "talking" (but not "thinking") networks in HSC than in HC. This combined network analysis also suggested an interplay between intracellular and extracellular mediators in HSC under more conditions than that observed in HC, though both cell types exhibited a qualitatively similar phenotype under hypoxia/reoxygenation. Our results thus suggest that a stepwise series of computational and statistical analyses may help decipher how cells respond to environmental stresses, both within the cell and in its secretory products, even in the absence of cooperation from other cells in the liver.

Cardiac Arrest Disrupts Caspase-1 and Patterns of Inflammatory Mediators Differently in Skin and Muscle Following Localized Tissue Injury in Rats: Insights from Data-Driven Modeling.

BACKGROUND: Trauma often cooccurs with cardiac arrest and hemorrhagic shock. Skin and muscle injuries often lead to significant inflammation in the affected tissue. The primary mechanism by which inflammation is initiated, sustained, and terminated is cytokine-mediated immune signaling, but this signaling can be altered by cardiac arrest. The complexity and context sensitivity of immune signaling in general has stymied a clear understanding of these signaling dynamics. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that advanced numerical and biological function analysis methods would help elucidate the inflammatory response to skin and muscle wounds in rats, both with and without concomitant shock. Based on the multiplexed analysis of inflammatory mediators, we discerned a differential interleukin (IL)-1α and IL-18 signature in skin vs. muscle, which was suggestive of inflammasome activation in the skin. Immunoblotting revealed caspase-1 activation in skin but not muscle. Notably, IL-1α and IL-18, along with caspase-1, were greatly elevated in the skin following cardiac arrest, consistent with differential inflammasome activation. CONCLUSION/SIGNIFICANCE: Tissue-specific activation of caspase-1 and the NLRP3 inflammasome appear to be key factors in determining the type and severity of the inflammatory response to tissue injury, especially in the presence of severe shock, as suggested via data-driven modeling.

Central role for MCP-1/CCL2 in injury-induced inflammation revealed by in vitro, in silico, and clinical studies.

The translation of in vitro findings to clinical outcomes is often elusive. Trauma/hemorrhagic shock (T/HS) results in hepatic hypoxia that drives inflammation. We hypothesize that in silico methods would help bridge in vitro hepatocyte data and clinical T/HS, in which the liver is a primary site of inflammation. Primary mouse hepatocytes were cultured under hypoxia (1% O2) or normoxia (21% O2) for 1-72 h, and both the cell supernatants and protein lysates were assayed for 18 inflammatory mediators by Luminex™ technology. Statistical analysis and data-driven modeling were employed to characterize the main components of the cellular response. Statistical analyses, hierarchical and k-means clustering, Principal Component Analysis, and Dynamic Network Analysis suggested MCP-1/CCL2 and IL-1α as central coordinators of hepatocyte-mediated inflammation in C57BL/6 mouse hepatocytes. Hepatocytes from MCP-1-null mice had altered dynamic inflammatory networks. Circulating MCP-1 levels segregated human T/HS survivors from non-survivors. Furthermore, T/HS survivors with elevated early levels of plasma MCP-1 post-injury had longer total lengths of stay, longer intensive care unit lengths of stay, and prolonged requirement for mechanical ventilation vs. those with low plasma MCP-1. This study identifies MCP-1 as a main driver of the response of hepatocytes in vitro and as a biomarker for clinical outcomes in T/HS, and suggests an experimental and computational framework for discovery of novel clinical biomarkers in inflammatory diseases.

Hypoxia-induced overexpression of BNIP3 is not dependent on hypoxia-inducible factor 1α in mouse hepatocytes.

We sought to investigate the expression of the cell death protein BNIP3 in hypoxic hepatocytes, as well as the role that hypoxia-inducible factor 1 (HIF-1α) plays in the upregulation of BNIP3 in hypoxic primary mouse hepatocytes and in the livers of mice subjected to ischemia-reperfusion. Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 1, 3, 6, 24, and 48 h, and the RNA and protein were isolated for reverse transcriptase-polymerase chain reaction and Western blot analysis. Similarly, livers from mice subjected to segmental (70%) hepatic warm ischemia for 30 min or 1 h, or to 1-h ischemia followed by 0.5- to 4-h reperfusion, were collected and subjected to Western blot analysis for HIF-1α protein. We showed that hypoxic stress increases the formation of the BNIP3 homodimer while decreasing the amount of the monomeric form of BNIP3 in primary mouse hepatocytes. In contrast to RAW264.7 macrophages, there is a basal expression of HIF-α protein in normoxic primary mouse hepatocytes that does not change significantly upon exposure to hypoxia. Using siRNA technology, we demonstrated that reduced HIF-1α protein levels did not block the hypoxia-induced overexpression of BNIP3. In contrast to the effect on BNIP3 expression reported previously, livers from ischemic animals demonstrated only a modest increase in HIF-1α protein as compared with resting livers from control animals; and this expression was not statistically different from sham controls. These results suggest that HIF-1α does not mediate the hypoxia-induced upregulation of BNIP3 in mouse hepatocytes in vitro and possibly in the liver in vivo.

Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress.

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1-4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

Matrix metalloproteinase-1 promotes muscle cell migration and differentiation.

Injured skeletal muscle has the capacity to regenerate through a highly coordinated sequence of events that involves both myoblast migration and differentiation into myofibers. Fibrosis may impede muscle regeneration by posing as a mechanical barrier to cell migration and fusion, providing inappropriate signals for cell differentiation, and limiting vascular perfusion of the injury site, subsequently leading to incomplete functional recovery. Our previous studies demonstrated that matrix metalloproteinase-1 (MMP-1) is able to digest fibrous scar tissue and improve muscle healing after injury. The goal of this study is to investigate whether MMP-1 could further enhance muscle regeneration by improving myoblast migration and differentiation. In vitro wound healing assays, flow cytometry, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blot analyses demonstrated that MMP-1 enhances myoblast migration but is not chemoattractive. We discovered that MMP-1 also enhances myoblast differentiation, which is a critical step in the sequence of muscle regeneration. In addition, RT-PCR and Western blot analyses demonstrated the up-regulation of myogenic factors after MMP-1 treatment. In vivo, we observed that myoblast transplantation was greatly improved after MMP-1 treatment within the dystrophic skeletal muscles of MDX mice. MMP-1 may therefore be able to improve muscle function recovery after injury or disease by increasing both the number of myofibers that are generated by activated myoblasts and the size of myoblast coverage area by promoting migration, thus fostering a greater degree of engraftment.