Daren L. Knoell

Chronic obstructive pulmonary disease (COPD) in the U.S. is primarily caused by cigarette smoking. COPD patients are highly susceptible to respiratory infections in part due to alveolar macrophage dysfunction despite a substantial increase in macrophages in the lung. Cadmium (Cd) is a toxic metal that is concentrated within tobacco and accumulates in the lung of smokers. We hypothesized that Cd uptake into macrophages alters immune function thereby impairing the macrophage response to invading pathogens. Our hypothesis was tested by comparing primary human monocytes and macrophages, primary mouse bronchoalveolar lavage myeloid cells, and related cell lines. Strikingly, Cd exposure followed by LPS stimulation resulted in a dose-dependent, significant decrease in nuclear p65 activity in macrophages that was not observed in monocytes. This corresponded with Cd-mediated inhibition of IKKβ and an impaired ability to transcribe and release cytokines in response to LPS challenge in vivo. These findings provide novel evidence that Cd has the capacity to disrupt macrophage immune function compared with monocytes. Importantly, Cd results in immune dysfunction in macrophages through inhibition of the NF-κB signaling pathway. Based on these findings, we provide new evidence that Cd contributes to immune dysfunction in the lung of COPD subjects and may increase susceptibility to infection.

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that primarily resides in airway epithelial cells. Decreased CFTR expression and/or function lead to impaired airway surface liquid (ASL) volume homeostasis, resulting in accumulation of mucus, reduced clearance of bacteria, and chronic infection and inflammation.

Sepsis rapidly activates the host inflammatory response and acute phase response. Severe sepsis, complicated by multiple organ failure, is associated with overwhelming inflammation and high mortality. We previously observed that zinc (Zn) deficiency significantly increases mortality in a mouse model of polymicrobial sepsis due to over-activation of the inflammatory response. In order to identify potential mechanisms that account for Zn-responsive effects, we generated whole exome expression profiles from the lung tissue of septic mice that were maintained on Zn modified diets. Based on systems analysis, we observed that Zn deficiency enhances the acute phase response and particularly the JAK-STAT3 pathway, resulting in increased serum amyloid A production. In vitro studies of primary hepatocytes and HepG2 cells substantiated that Zn-deficiency augments serum amyloid A production through up-regulation of the JAK-STAT3 and NF-κB pathways. In contrast, Zn inhibited STAT3 activation through the up-regulation of SHP1 activity. Collectively, these findings demonstrate that Zn deficiency enhances the acute phase response through up-regulation of the JAK-STAT3 pathway, thereby perpetuating increased inflammation that may lead to increased morbidity and mortality in response to sepsis.

Zinc (Zn) deficiency and obesity are global public health problems. Zn deficiency is associated with obesity and comorbid conditions that include ins

Cadmium (Cd), a toxic heavy metal and carcinogen that is abundantly present in cigarette smoke, is a cause of smoking-induced lung disease. SLC39A8 (ZIP8), a zinc transporter, is a major portal for Cd uptake into cells. We have recently identified that ZIP8 expression is under the transcriptional control of the NF-κB pathway. On the basis of this, we hypothesized that cigarette-smoke induced inflammation would increase ZIP8 expression in lung epithelia, thereby enhancing Cd uptake and cell toxicity. Herein we report that ZIP8 is a central mediator of Cd-mediated toxicity. TNF-α treatment of primary human lung epithelia and A549 cells induced ZIP8 expression, resulting in significantly higher cell death attributable to both apoptosis and necrosis following Cd exposure. Inhibition of the NF-κB pathway and ZIP8 expression significantly reduced cell toxicity. Zinc (Zn), a known cytoprotectant, prevented Cd-mediated cell toxicity via ZIP8 uptake. Consistent with cell culture findings, a significant increase in ZIP8 mRNA and protein expression was observed in the lung of chronic smokers compared with nonsmokers. From these studies, we conclude that ZIP8 expression is induced in lung epithelia in an NF-κB-dependent manner, thereby resulting in increased cell death in the presence of Cd. From this we contend that ZIP8 plays a critical role at the interface between micronutrient (Zn) metabolism and toxic metal exposure (Cd) in the lung microenvironment following cigarette smoke exposure. Furthermore, dietary Zn intake, or a lack thereof, may be a contributing factor in smoking-induced lung disease.

The phosphoinositide-3 kinase/Akt pathway is a vital survival axis in lung epithelia. We previously reported that inhibition of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major suppressor of this pathway, results in enhanced wound repair following injury. However, the precise cellular and biomechanical mechanisms responsible for increased wound repair during PTEN inhibition are not yet well established. Using primary human lung epithelia and a related lung epithelial cell line, we first determined whether changes in migration or proliferation account for wound closure. Strikingly, we observed that cell migration accounts for the majority of wound recovery following PTEN inhibition in conjunction with activation of the Akt and ERK signaling pathways. We then used fluorescence and atomic force microscopy to investigate how PTEN inhibition alters the cytoskeletal and mechanical properties of the epithelial cell. PTEN inhibition did not significantly alter cytoskeletal structure but did result in large spatial variations in cell stiffness and in particular a decrease in cell stiffness near the wound edge. Biomechanical changes, as well as migration rates, were mediated by both the Akt and ERK pathways. Our results indicate that PTEN inhibition rapidly alters biochemical signaling events that in turn provoke alterations in biomechanical properties that enhance cell migration. Specifically, the reduced stiffness of PTEN-inhibited cells promotes larger deformations, resulting in a more migratory phenotype. We therefore conclude that increased wound closure consequent to PTEN inhibition occurs through enhancement of cell migration that is due to specific changes in the biomechanical properties of the cell.

Individuals at highest risk of zinc deficiency (children, elderly, pregnant and lactating women, morbidly ill, alcoholics) have a higher risk of infection. Whereas the essential role of zinc in maintaining adaptive immunity is well recognized, much less is known regarding the innate immune system. We recently reported that zinc deficiency significantly increases mortality in an animal model of sepsis. In particular, zinc-deficient mice had a decreased capacity to clear bacteria and a concomitant increase in NF-kappaB-mediated signaling across multiple vital organs. This occurred in tandem with exaggeration of the acute phase and innate immune response. Strikingly, sepsis patients revealed similar findings in that lower plasma zinc levels were associated with more inflammation and increased severity of illness. Through these investigations we have consistently observed that SLC39 A8 (ZIP8) is unique, relative to other zinc transporters, in that its expression is significantly induced at the onset of infection. Moreover, induction of ZIP8-mediated zinc transport into innate immune cells is vital for proper immune function. Whether ZIP8 functions beyond the conventional role of a zinc transporter remains a work in progress, although new evidence has revealed that ZIP8 expression itself is regulated by NF-kappaB. Taken together, these findings indicate that zinc is vital for proper innate immune function and that hZIP8 is intricately involved in maintaining innate immune defense.

Zinc is an essential element that facilitates coordination of immune activation during the host response to infection. We recently reported that zinc deficiency increases systemic inflammation, vital organ damage, and mortality in a small animal model of sepsis. To investigate potential mechanisms that cause these phenomena, we used the same animal model and observed that zinc deficiency increases bacterial burden and enhances NF-κB activity in vital organs including the lung. We conducted further studies in the lung to determine the overall impact of zinc deficiency. At the molecular level, NF-κB p65 DNA-binding activity was enhanced by zinc deficiency in response to polymicrobial sepsis. Furthermore, expression of the NF-κB-targeted genes IL-1β, TNFα, ICAM-1, and the acute phase response gene SAA1/2 were elevated by zinc deficiency. Unexpectedly, the amount of NF-κB p65 mRNA and protein was increased in the lung including alveolar epithelia of zinc-deficient mice. These events occurred with a significant and concomitant increase in caspase-3 activity within 24 h of sepsis onset in zinc-deficient mice relative to control group. Short-term zinc supplementation reversed these effects. Reconstitution of zinc deficiency in lung epithelial cultures resulted in similar findings in response to TNFα. Taken together, zinc deficiency systemically enhances the spread of infection and NF-κB activation in vivo in response to polymicrobial sepsis, leading to enhanced inflammation, lung injury, and, as reported previously, mortality. Zinc supplementation immediately before initiation of sepsis reversed these effects thereby supporting the plausibility of future studies that explore zinc supplementation strategies to prevent sepsis-mediated morbidity and mortality.