Regulation of Natural Killer Cell TGF-ß and AhR Signaling Pathways Via the Intestinal Microbiota is Critical for Host Defense Against Alcohol-Associated Bacterial Pneumonia.

Alcohol use is an independent risk factor for the development of bacterial pneumonia due, in part, to impaired mucus-facilitated clearance, macrophage phagocytosis, and recruitment of neutrophils. Alcohol consumption is also known to reduce peripheral natural killer (NK) cell numbers and compromises NK cell cytolytic activity, especially NK cells with a mature phenotype. However, the role of innate lymphocytes, such as NK cells during host defense against alcohol-associated bacterial pneumonia is essentially unknown. We have previously shown that indole supplementation mitigates increases in pulmonary bacterial burden and improves pulmonary NK cell recruitment in alcohol-fed mice, which were dependent of aryl hydrocarbon receptor (AhR) signaling. Employing a binge-on-chronic alcohol-feeding model we sought to define the role and interaction of indole and NK cells during pulmonary host defense against alcohol-associated pneumonia. We demonstrate that alcohol dysregulates NK cell effector function and pulmonary recruitment via alterations in two key signaling pathways. We found that alcohol increases transforming growth factor beta (TGF-ß) signaling, while suppressing AhR signaling. We further demonstrated that NK cells isolated from alcohol-fed mice have a reduced ability to kill Klebsiella pneumoniae. NK cell migratory capacity to chemokines was also significantly altered by alcohol, as NK cells isolated from alcohol-fed mice exhibited preferential migration in response to CXCR3 chemokines but exhibited reduced migration in response to CCR2, CXCR4, and CX3CR1 chemokines. Together this data suggests that alcohol disrupts NK cell specific TGF-ß and AhR signaling pathways leading to decreased pulmonary recruitment and cytolytic activity thereby increasing susceptibility to alcohol-associated bacterial pneumonia.

Human Alcohol-Microbiota Mice have Increased Susceptibility to Bacterial Pneumonia.

Preclinical studies have shown that chronic alcohol abuse leads to alterations in the gastrointestinal microbiota that are associated with behavior changes, physiological alterations, and immunological effects. However, such studies have been limited in their ability to evaluate the direct effects of alcohol-associated dysbiosis. To address this, we developed a humanized alcohol-microbiota mouse model to systematically evaluate the immunological effects of chronic alcohol abuse mediated by intestinal dysbiosis. Germ-free mice were colonized with human fecal microbiota from individuals with high and low Alcohol Use Disorders Identification Test (AUDIT) scores and bred to produce human alcohol-associated microbiota or human control-microbiota F1 progenies. F1 offspring colonized with fecal microbiota from individuals with high AUDIT scores had increased susceptibility to Klebsiella pneumoniae and Streptococcus pneumoniae pneumonia, as determined by increased mortality rates, pulmonary bacterial burden, and post-infection lung damage. These findings highlight the importance of considering both the direct effects of alcohol and alcohol-induced dysbiosis when investigating the mechanisms behind alcohol-related disorders and treatment strategies.

The Inherited Intestinal Microbiota from Myeloid-Specific ZIP8KO Mice Impairs Pulmonary Host Defense against Pneumococcal Pneumonia.

Intestinal dysbiosis increases susceptibility to infection through the alteration of metabolic profiles, which increases morbidity. Zinc (Zn) homeostasis in mammals is tightly regulated by 24 Zn transporters. ZIP8 is unique in that it is required by myeloid cells to maintain proper host defense against bacterial pneumonia. In addition, a frequently occurring ZIP8 defective variant (SLC39A8 rs13107325) is strongly associated with inflammation-based disorders and bacterial infection. In this study, we developed a novel model to study the effects of ZIP8-mediated intestinal dysbiosis on pulmonary host defense independent of the genetic effects. Cecal microbial communities from a myeloid-specific Zip8 knockout mouse model were transplanted into germ-free mice. Conventionalized ZIP8KO-microbiota mice were then bred to produce F1 and F2 generations of ZIP8KO-microbiota mice. F1 ZIP8KO-microbiota mice were also infected with S. pneumoniae, and pulmonary host defense was assessed. Strikingly, the instillation of pneumococcus into the lung of F1 ZIP8KO-microbiota mice resulted in a significant increase in weight loss, inflammation, and mortality when compared to F1 wild-type (WT)-microbiota recipients. Similar defects in pulmonary host defense were observed in both genders, although consistently greater in females. From these results, we conclude that myeloid Zn homeostasis is not only critical for myeloid function but also plays a significant role in the maintenance and control of gut microbiota composition. Further, these data demonstrate that the intestinal microbiota, independent of host genetics, play a critical role in governing host defense in the lung against infection. Finally, these data strongly support future microbiome-based interventional studies, given the high incidence of zinc deficiency and the rs13107325 allele in humans.

ZIP8-Mediated Intestinal Dysbiosis Impairs Pulmonary Host Defense against Bacterial Pneumonia.

Pneumococcal pneumonia is a leading cause of morbidity and mortality worldwide. An increased susceptibility is due, in part, to compromised immune function. Zinc is required for proper immune function, and an insufficient dietary intake increases the risk of pneumonia. Our group was the first to reveal that the Zn transporter, ZIP8, is required for host defense. Furthermore, the gut microbiota that is essential for lung immunity is adversely impacted by a commonly occurring defective ZIP8 allele in humans. Taken together, we hypothesized that loss of the ZIP8 function would lead to intestinal dysbiosis and impaired host defense against pneumonia. To test this, we utilized a novel myeloid-specific Zip8KO mouse model in our studies. The comparison of the cecal microbial composition of wild-type and Zip8KO mice revealed significant differences in microbial community structure. Most strikingly, upon a S. pneumoniae lung infection, mice recolonized with Zip8KO-derived microbiota exhibited an increase in weight loss, bacterial dissemination, and lung inflammation compared to mice recolonized with WT microbiota. For the first time, we reveal the critical role of myeloid-specific ZIP8 on the maintenance of the gut microbiome structure, and that loss of ZIP8 leads to intestinal dysbiosis and impaired host defense in the lung. Given the high incidence of dietary Zn deficiency and the ZIP8 variant allele in the human population, additional investigation is warranted to improve surveillance and treatment strategies.

Critical Role of Zinc Transporter (ZIP8) in Myeloid Innate Immune Cell Function and the Host Response against Bacterial Pneumonia.

Zinc (Zn) is required for proper immune function and host defense. Zn homeostasis is tightly regulated by Zn transporters that coordinate biological processes through Zn mobilization. Zn deficiency is associated with increased susceptibility to bacterial infections, including Streptococcus pneumoniae, the most commonly identified cause of community-acquired pneumonia. Myeloid cells, including macrophages and dendritic cells (DCs), are at the front line of host defense against invading bacterial pathogens in the lung and play a critical role early on in shaping the immune response. Expression of the Zn transporter ZIP8 is rapidly induced following bacterial infection and regulates myeloid cell function in a Zn-dependent manner. To what extent ZIP8 is instrumental in myeloid cell function requires further study. Using a novel, myeloid-specific, Zip8 knockout model, we identified vital roles of ZIP8 in macrophage and DC function upon pneumococcal infection. Administration of S. pneumoniae into the lung resulted in increased inflammation, morbidity, and mortality in Zip8 knockout mice compared with wild-type counterparts. This was associated with increased numbers of myeloid cells, cytokine production, and cell death. In vitro analysis of macrophage and DC function revealed deficits in phagocytosis and increased cytokine production upon bacterial stimulation that was, in part, due to increased NF-κB signaling. Strikingly, alteration of myeloid cell function resulted in an imbalance of Th17/Th2 responses, which is potentially detrimental to host defense. These results (for the first time, to our knowledge) reveal a vital ZIP8- and Zn-mediated axis that alters the lung myeloid cell landscape and the host response against pneumococcus.

Modeling of an immune response: Queuing network analysis of the impact of zinc and cadmium on macrophage activation.

Chronic obstructive pulmonary disease is characterized by progressive, irreversible airflow obstruction resulting from an abnormal inflammatory response to noxious gases and particles. Alveolar macrophages rely on the transcription factors, nuclear factor κB and mitogen-activated protein kinase, among others, to facilitate the production of inflammatory mediators designed to help rid the lung of foreign pathogens and noxious stimuli. Building a kinetic model using queuing networks, provides a quantitative approach incorporating an initial number of individual molecules along with rates of the reactions in any given pathway. Accordingly, this model has been shown useful to model cell behavior including signal transduction, transcription, and metabolic pathways. The aim of this study was to determine whether a queuing theory model that involves lipopolysaccharide-mediated macrophage activation in tandem with changes in intracellular Cd and zinc (Zn) content or a lack thereof, would be useful to predict their impact on immune activation. We then validate our model with biologic cytokine output from human macrophages relative to the timing of innate immune activation. We believe that our results further prove the validity of the queuing theory approach to model intracellular molecular signaling and postulate that it can be useful to predict additional cell signaling pathways and the corresponding biological outcomes.

Imbalance in zinc homeostasis enhances lung Tissue Loss following cigarette smoke exposure.

Cigarette smoke exposure is a major cause of chronic obstructive pulmonary disease. Cadmium is a leading toxic component of cigarette smoke. Cadmium and zinc are highly related metals. Whereas, zinc is an essential metal required for normal health, cadmium is highly toxic. Zrt- and Irt-like protein 8 (ZIP8) is an avid transporter of both zinc and cadmium into cells and is abundantly expressed in the lung of smokers compared to nonsmokers. Our objective was to determine whether disturbed zinc homeostasis through diet or the zinc transporter ZIP8 increase susceptibility to lung damage following prolonged cigarette smoke exposure. METHODS: Cigarette smoke exposure was evaluated in the lungs of mice subject to insufficient and sufficient zinc intakes, in transgenic ZIP8 overexpressing mice, and a novel myeloid-specific ZIP8 knockout strain. RESULTS: Moderate depletion of zinc intakes in adult mice resulted in a significant increase in lung cadmium burden and permanent lung tissue loss following prolonged smoke exposure. Overexpression of ZIP8 resulted in increased lung cadmium burden and more extensive lung damage, whereas cigarette smoke exposure in ZIP8 knockout mice resulted in increased lung tissue loss without a change in lung cadmium content, but a decrease in zinc. CONCLUSIONS: Overall, findings were consistent with past human studies. Imbalance in Zn homeostasis increases susceptibility to permanent lung injury following prolonged cigarette smoke exposure. Based on animal studies, both increased and decreased ZIP8 expression enhanced irreversible tissue damage in response to prolonged tobacco smoke exposure. We believe these findings represent an important advancement in our understanding of how imbalance in zinc homeostasis and cadmium exposure via tobacco smoke may increase susceptibility to smoking-induced lung disease.

Insufficient zinc intake enhances lung inflammation in response to agricultural organic dust exposure.

Organic dust exposure particularly within hog confinement facilities is a significant cause of airway inflammation and lung disease. In a cohort of Midwestern veterans with COPD and agricultural work exposure we observed reduced zinc intakes which were associated with decreased lung function. Because insufficient zinc intake is common within the U.S. and a potent modulator of innate immune function, we sought to determine whether deficits in zinc intake would impact the airway inflammatory response to hog confinement facility dust extract (HDE). Adult male C57BL/6 mice were randomized to zinc deficient or matched zinc sufficient diets for 3 weeks and subsequently treated with intranasal HDE inhalation or saline once or daily for 3 weeks while maintained on specific diets. Lavage fluid and lung tissue was collected. Conditions of zinc deficiency were also studied in macrophages exposed to HDE. Single and repetitive HDE inhalation exposure resulted in increased influx of total cells and neutrophils, increased mediator hyper-responsiveness (TNFα, IL-6, CXCL1, and amphiregulin), and enhanced tissue pathology that was more pronounced in zinc deficient mice compared to normal dietary counterparts. Airway inflammation was most pronounced in zinc deficient mice treated with repetitive HDE for 3 weeks. Similarly, macrophages maintained in a zinc deficient environment exhibited increased CXCL1 and IL-23 production as a result of increased NF-κB activation. Conclusion: Given the relatively high incidence of dietary deficiencies in agriculture workers, we anticipate that zinc intake, or a lack thereof, may play an important role in modulating the host response to organic dust exposure.

Kinase Suppressor of Ras 2 (KSR2) expression in the brain regulates energy balance and glucose homeostasis.

OBJECTIVE: Kinase Suppressor of Ras 2 (KSR2) is a molecular scaffold coordinating Raf/MEK/ERK signaling that is expressed at high levels in the brain. KSR2 disruption in humans and mice causes obesity and insulin resistance. Understanding the anatomical location and mechanism of KSR2 function should lead to a better understanding of physiological regulation over energy balance. METHODS: Mice bearing floxed alleles of KSR2 (KSR2fl/fl) were crossed with mice expressing the Cre recombinase expressed by the Nestin promoter (Nes-Cre) to produce Nes-CreKSR2fl/fl mice. Growth, body composition, food consumption, cold tolerance, insulin and free fatty acid levels, glucose, and AICAR tolerance were measured in gender and age matched KSR2-/- mice. RESULTS: Nes-CreKSR2fl/fl mice lack detectable levels of KSR2 in the brain. The growth and onset of obesity of Nes-CreKSR2fl/fl mice parallel those observed in KSR2-/- mice. As in KSR2-/- mice, Nes-CreKSR2fl/fl are glucose intolerant with elevated fasting and cold intolerance. Male Nes-CreKSR2fl/fl mice are hyperphagic, but female Nes-CreKSR2fl/fl mice are not. Unlike KSR2-/- mice, Nes-CreKSR2fl/fl mice respond normally to leptin and AICAR, which may explain why the degree of obesity of adult Nes-CreKSR2fl/fl mice is not as severe as that observed in KSR2-/- animals. CONCLUSIONS: These observations suggest that, in the brain, KSR2 regulates energy balance via control of feeding behavior and adaptive thermogenesis, while a second KSR2-dependent mechanism, functioning through one or more other tissues, modulates sensitivity to leptin and activators of the energy sensor AMPK.

Cell non-autonomous regulation of hepatic IGF-1 and neonatal growth by Kinase Suppressor of Ras 2 (KSR2).

Individuals with poor postnatal growth are at risk for cardiovascular and metabolic problems as adults. Here we show that disruption of the molecular scaffold Kinase Suppressor of Ras 2 (KSR2) causes selective inhibition of hepatic GH signaling in neonatal mice with impaired expression of IGF-1 and IGFBP3. ksr2(-/-) mice are normal size at birth but show a marked increase in FGF21 accompanied by reduced body mass, shortened body length, and reduced bone mineral density (BMD) and content (BMC) first evident during postnatal development. However, disrupting FGF21 in ksr2(-/-) mice does not normalize mass, length, or bone density and content in fgf21(-/-)ksr2(-/-) mice. Body length, BMC and BMD, but not body mass, are rescued by infection of two-day-old ksr2(-/-) mice with a recombinant adenovirus encoding human IGF-1. Relative to wild-type mice, GH injections reveal a significant reduction in JAK2 and STAT5 phosphorylation in liver, but not in skeletal muscle, of ksr2(-/-) mice. However, primary hepatocytes isolated from ksr2(-/-) mice show no reduction in GH-stimulated STAT5 phosphorylation. These data indicate that KSR2 functions in a cell non-autonomous fashion to regulate GH-stimulated IGF-1 expression in the liver of neonatal mice, which plays a key role in the development of body length.

AMPK Promotes Aberrant PGC1ß Expression To Support Human Colon Tumor Cell Survival.

A major goal of cancer research is the identification of tumor-specific vulnerabilities that can be exploited for the development of therapies that are selectively toxic to the tumor. We show here that the transcriptional coactivators peroxisome proliferator-activated receptor gamma coactivator 1ß (PGC1ß) and estrogen-related receptor α (ERRα) are aberrantly expressed in human colon cell lines and tumors. With kinase suppressor of Ras 1 (KSR1) depletion as a reference standard, we used functional signature ontology (FUSION) analysis to identify the γ1 subunit of AMP-activated protein kinase (AMPK) as an essential contributor to PGC1ß expression and colon tumor cell survival. Subsequent analysis revealed that a subunit composition of AMPK (α2ß2γ1) is preferred for colorectal cancer cell survival, at least in part, by stabilizing the tumor-specific expression of PGC1ß. In contrast, PGC1ß and ERRα are not detectable in nontransformed human colon epithelial cells, and depletion of the AMPKγ1 subunit has no effect on their viability. These data indicate that Ras oncogenesis relies on the aberrant activation of a PGC1ß-dependent transcriptional pathway via a specific AMPK isoform.

Identification of a cytotoxic form of dimeric interleukin-2 in murine tissues.

Interleukin-2 (IL-2) is a multi-faceted cytokine, known for promoting proliferation, survival, and cell death depending on the cell type and state. For example, IL-2 facilitates cell death only in activated T cells when antigen and IL-2 are abundant. The availability of IL-2 clearly impacts this process. Our laboratory recently demonstrated that IL-2 is retained in blood vessels by heparan sulfate, and that biologically active IL-2 is released from vessel tissue by heparanase. We now demonstrate that heparanase digestion also releases a dimeric form of IL-2 that is highly cytotoxic to cells expressing the IL-2 receptor. These cells include "traditional" IL-2 receptor-bearing cells such as lymphocytes, as well as those less well known for IL-2 receptor expression, such as epithelial and smooth muscle cells. The morphologic changes and rapid cell death induced by dimeric IL-2 imply that cell death is mediated by disruption of membrane permeability and subsequent necrosis. These findings suggest that IL-2 has a direct and unexpectedly broad influence on cellular homeostatic mechanisms in both immune and non-immune systems.

Interleukin-2 is present in human blood vessels and released in biologically active form by heparanase.

Interleukin-2 (IL-2) is a multifaceted cytokine with immunostimulatory and immunosuppressive properties. Our laboratory recently demonstrated that the availability of IL-2 is regulated, in part, by association with perlecan, a heparan sulfate proteoglycan. Given the abundance of perlecan in blood vessels, we asked whether IL-2 is present in vessel walls. Our results indicate that IL-2 is associated with endothelial and smooth muscle cells within the human arterial wall. This IL-2 is released by heparanase, and promotes the proliferation of an IL-2-dependent cell line. Given the presence of IL-2 in human arteries, we asked whether the large vessels of IL-2-deficient mice were normal. The aortas of IL-2-deficient mice exhibited a loss of smooth muscle cells, suggesting that IL-2 may contribute to their survival. In their entirety, these results suggest a here-to-fore unrecognized role of IL-2 in vascular biology, and have significant implications for both the immune and cardiovascular systems.

Perlecan: a major IL-2-binding proteoglycan in murine spleen.

Although interleukin-2 (IL-2) is typically considered a soluble cytokine, our laboratory has shown that the availability of IL-2 in lymphoid tissues is regulated, in part, by an association with heparan sulfate glycosaminoglycan. Heparan sulfate is usually found in proteoglycan form, in which the heparan sulfate chains are covalently linked to a specific core protein. We now show that perlecan is one of the major IL-2-binding heparan sulfate proteoglycans in murine spleen. IL-2 binds perlecan via heparan sulfate chains, as enzymatic removal of heparan sulfate from splenic perlecan abolishes its ability to bind IL-2. Furthermore, we demonstrate that perlecan-bound IL-2 supports the proliferation of an IL-2-dependent cell line. Identification of perlecan as a major heparan sulfate proteoglycan that binds IL-2 has implications for both the localization and regulation of IL-2 in vivo.

Influence of interleukin-2 deficiency on the generation of autoimmune B cells.

The production of auto-antibodies is one of the predominant characteristics of autoimmune disorders. Because IL-2 deficient mice develop autoimmunity, we asked how IL-2 deficiency might impair endogenous mechanisms of B cell tolerance. To this end, we mated BALB/c anti-dsDNA H chain knock-in mice, in which B cells producing anti-dsDNA antibodies are properly regulated, with IL-2 deficient mice and assessed the phenotype of their offspring. IL-2 deficient mice expressing the anti-dsDNA H chain knock-in allele developed anti-dsDNA antibodies of both IgM and IgG isotypes. Production of these antibodies occurred through the disruption of several mechanisms of endogenous tolerance, including deletion, maturational arrest, and follicular exclusion. In summary, our results suggest that IL-2 plays an important role in regulating B cell tolerance.

Maternal microchimerism leads to the presence of interleukin-2 in interleukin-2 knock out mice: implications for the role of interleukin-2 in thymic function.

The role of interleukin-2 (IL-2) in thymic development is uncertain. Not surprisingly, IL-2 knockout (KO) mice have been used to address this question. However, as we report here, such mice are chimeric, containing both IL-2 KO cells and IL-2-expressing cells transferred in utero from their heterozygous mothers. These cells produce IL-2 in amounts detectable by conventional means, and their presence in lymphoid tissues confounds efforts to define the true IL-2 KO phenotype. To minimize the amount of IL-2 available to the thymus, we subjected recombinase activating gene-1 KO mice to bone marrow transplantation using IL-2 KO donors, and then followed the reconstitution of the thymus. The thymuses of these mice became increasingly aberrant over time, including abnormalities in both stromal cells and thymocytes. These results demonstrate that IL-2 is critical to several aspects of thymic function, a finding previously obscured by the presence of IL-2 in IL-2 KO mice.