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.

Divalent Metal Uptake and the Role of ZIP8 in Host Defense Against Pathogens.

Manganese (Mn) and Zinc (Zn) are essential micronutrients whose concentration and location within cells are tightly regulated at the onset of infection. Two families of Zn transporters (ZIPs and ZnTs) are largely responsible for regulation of cytosolic Zn levels and to a certain extent, Mn levels, although much less is known regarding Mn. The capacity of pathogens to persevere also depends on access to micronutrients, yet a fundamental gap in knowledge remains regarding the importance of metal exchange at the host interface, often referred to as nutritional immunity. ZIP8, one of 14 ZIPs, is a pivotal importer of both Zn and Mn, yet much remains to be known. Dietary Zn deficiency is common and commonly occurring polymorphic variants of ZIP8 that decrease cellular metal uptake (Zn and Mn), are associated with increased susceptibility to infection. Strikingly, ZIP8 is the only Zn transporter that is highly induced following bacterial exposure in key immune cells involved with host defense against leading pathogens. We postulate that mobilization of Zn and Mn into key cells orchestrates the innate immune response through regulation of fundamental defense mechanisms that include phagocytosis, signal transduction, and production of soluble host defense factors including cytokines and chemokines. New evidence also suggests that host metal uptake may have long-term consequences by influencing the adaptive immune response. Given that activation of ZIP8 expression by pathogens has been shown to influence parenchymal, myeloid, and lymphoid cells, the impact applies to all mucosal surfaces and tissue compartments that are vulnerable to infection. We also predict that perturbations in metal homeostasis, either genetic- or dietary-induced, has the potential to impact bacterial communities in the host thereby adversely impacting microbiome composition. This review will focus on Zn and Mn transport via ZIP8, and how this vital metal transporter serves as a "go to" conductor of metal uptake that bolsters host defense against pathogens. We will also leverage past studies to underscore areas for future research to better understand the Zn-, Mn- and ZIP8-dependent host response to infection to foster new micronutrient-based intervention strategies to improve our ability to prevent or treat commonly occurring infectious disease.

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.

Perfluorocarbon Nanoemulsions Enhance Therapeutic siRNA Delivery in the Treatment of Pulmonary Fibrosis.

Local pulmonary administration of therapeutic siRNA represents a promising approach to the treatment of lung fibrosis, which is currently hampered by inefficient delivery. Development of perfluorooctylbromide (PFOB) nanoemulsions as a way of improving the efficiency of pulmonary polycation-based delivery of siRNA is reported. The results show that the polycation/siRNA/PFOB nanoemulsions are capable of efficiently silencing the expression of STAT3 and inhibiting chemokine receptor CXCR4-two validated targets in pulmonary fibrosis. Both in vitro and in vivo results demonstrate that the nanoemulsions improve mucus penetration and facilitate effective cellular delivery of siRNA. Pulmonary treatment of mice with bleomycin-induced pulmonary fibrosis shows strong inhibition of the progression of the disease and significant prolongation of animal survival. Overall, the study points to a promising local treatment strategy of pulmonary fibrosis.

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.

The adverse impact of cadmium on immune function and lung host defense.

Cadmium (Cd) is a transition metal, also referred to as a heavy metal, that is naturally abundant in the earth's crust. It has no known benefit to humans. It is primarily released into our environment through mining and smelting in industrial processes and enters the food chain through uptake by plants from contaminated soil and water. In humans, Cd primarily enters the body through ingestion of foods and cigarette smoke and has an extremely long resident half-life in the body compared to other transition metals. Environmental workplace exposure is also a source through inhalation, although much less common. The principal organs adversely affected by Cd following acute and chronic exposure are the kidneys, bone, vasculature and lung. Cd adversely impacts cell function through changes in gene expression and signal transduction and is recognized as a carcinogen. Despite a substantial body of mechanistic studies in cells and animal models, the overall impact of Cd on innate immune function in humans remains poorly understood. The best evidence is perhaps alteration of reactive oxygen species balance and signaling in cells that regulate innate immunity causing alteration of the inflammatory response that is postulated to contribute to chronic diseases. Epidemiologic studies support this possibility since increased tissue levels in humans are strongly associated with leading chronic diseases including chronic obstructive pulmonary disease (COPD), which will be discussed in depth. Additional studies are required to understand how chronic exposure and accumulation of this leading environmental toxicant in vital organs negatively impact innate immune function and host defense leading to chronic disease in humans.

Pulmonary siRNA delivery for lung disease: Review of recent progress and challenges.

Lung diseases are a leading cause of mortality worldwide and there exists urgent need for new therapies. Approval of the first siRNA treatments in humans has opened the door for further exploration of this therapeutic strategy for other disease states. Pulmonary delivery of siRNA-based biopharmaceuticals offers the potential to address multiple unmet medical needs in lung-related diseases because of the specific physiology of the lung and characteristic properties of siRNA. Inhalation-based siRNA delivery designed for efficient, targeted delivery to specific cells within the lung holds great promise. Efficient delivery of siRNA directly to the lung, however, is relatively complex. This review focuses on the barriers that impact pulmonary siRNA delivery and successful recent approaches to advance this field forward. We focus on the pulmonary barriers that affect siRNA delivery, the disease-dependent pathological changes and their role in pulmonary disease and impact on siRNA delivery, as well as the recent development on the pulmonary siRNA delivery systems.

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.

Essential Role of Zinc and Zinc Transporters in Myeloid Cell Function and Host Defense against Infection.

Zinc is an essential micronutrient known to play a vital role in host defense against pathogens. Diets that are deficient in zinc lead to impaired immunity and delayed recovery from and worse outcomes following infection. Sustained insufficient zinc intake leads to dysregulation of the innate immune response and increases susceptibility to infection whereas zinc supplementation in at-risk populations has been shown to restore host defense and reduce pathogen-related morbidity and mortality. Upon infection, zinc deficiency leads to increased pathology due to imbalance in key signaling networks that result in excessive inflammation and collateral tissue damage. In particular, zinc impacts macrophage function, a critical front-line cell in host defense, in addition to other immune cells. Deficits in zinc adversely impact macrophage function resulting in dysregulation of phagocytosis, intracellular killing, and cytokine production. An additional work in this field has revealed a vital role for several zinc transporter proteins that are required for proper bioredistribution of zinc within mononuclear cells to achieve an optimal immune response against invading microorganisms. In this review, we will discuss the most recent developments regarding zinc's role in innate immunity and protection against pathogen invasion.

Elemental Ingredients in the Macrophage Cocktail: Role of ZIP8 in Host Response to Mycobacterium tuberculosis.

Tuberculosis (TB) is a global epidemic caused by the infection of human macrophages with the world's most deadly single bacterial pathogen, Mycobacterium tuberculosis (M.tb). M.tb resides in a phagosomal niche within macrophages, where trace element concentrations impact the immune response, bacterial metal metabolism, and bacterial survival. The manipulation of micronutrients is a critical mechanism of host defense against infection. In particular, the human zinc transporter Zrt-/Irt-like protein 8 (ZIP8), one of 14 ZIP family members, is important in the flux of divalent cations, including zinc, into the cytoplasm of macrophages. It also has been observed to exist on the membrane of cellular organelles, where it can serve as an efflux pump that transports zinc into the cytosol. ZIP8 is highly inducible in response to M.tb infection of macrophages, and we have observed its localization to the M.tb phagosome. The expression, localization, and function of ZIP8 and other divalent cation transporters within macrophages have important implications for TB prevention and dissemination and warrant further study. In particular, given the importance of zinc as an essential nutrient required for humans and M.tb, it is not yet clear whether ZIP-guided zinc transport serves as a host protective factor or, rather, is targeted by M.tb to enable its phagosomal survival.

Zinc Modulates Endotoxin-Induced Human Macrophage Inflammation through ZIP8 Induction and C/EBPß Inhibition.

Two vital functions of the innate immune system are to initiate inflammation and redistribute micronutrients in favor of the host. Zinc is an essential micronutrient used in host defense. The zinc importer ZIP8 is uniquely induced through stimulation of the NF-κB pathway by LPS in monocytes and functions to regulate inflammation in a zinc-dependent manner. Herein we determined the impact of zinc metabolism following LPS-induced inflammation in human macrophages. We observed that ZIP8 is constitutively expressed in resting macrophages and strikingly elevated following LPS exposure, a response that is unique compared to the 13 other known zinc import proteins. During LPS exposure, extracellular zinc concentrations within the physiological range markedly reduced IL-10 mRNA expression and protein release but increased mRNA expression of TNFα, IL-8, and IL-6. ZIP8 knockdown inhibited LPS-driven cellular accumulation of zinc and prevented zinc-dependent reduction of IL-10 release. Further, zinc supplementation reduced nuclear localization and activity of C/EBPß, a transcription factor known to drive IL-10 expression. These studies demonstrate for the first time that zinc regulates LPS-mediated immune activation of human macrophages in a ZIP8-dependent manner, reducing IL-10. Based on these findings we predict that macrophage zinc metabolism is important in host defense against pathogens.

IκBζ Regulates Human Monocyte Pro-Inflammatory Responses Induced by Streptococcus pneumoniae.

Pneumococcal lung infections represent a major cause of death worldwide. Single nucleotide polymorphisms (SNPs) in the NFKBIZ gene, encoding the transcription factor IκBζ, are associated with increased susceptibility to invasive pneumococcal disease. We hence analyzed how IκBζ might regulate inflammatory responses to pneumococcal infection. We first demonstrate that IκBζ is expressed in human blood monocytes but not in bronchial epithelial cells, in response to wild type pneumococcal strain D39. D39 transiently induced IκBζ in a dose dependent manner, with subsequent induction of downstream molecules involved in host defense. Of these molecules, IκBζ knockdown reduced the expression of IL-6 and GMCSF. Furthermore, IκBζ overexpression increased the activity of IL-6 and GMCSF promoters, supporting the knockdown findings. Pneumococci lacking either pneumolysin or capsule still induced IκBζ. While inhibition of TLR1/TLR2 blocked D39 induced IκBζ expression, TLR4 inhibition did not. Blockade of p38 MAP kinase and NFκB suppressed D39 induced IκBζ. Overall, our data demonstrates that IκBζ regulates monocyte inflammatory responses to Streptococcus pneumoniae by promoting the production of IL-6 and GMCSF.

Cadmium attenuates the macrophage response to LPS through inhibition of the NF-κB pathway.

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 IKKß 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.

Alveolar Epithelial Cells in Mycobacterium tuberculosis Infection: Active Players or Innocent Bystanders?

Tuberculosis (TB) is a disease that kills one person every 18 s. TB remains a global threat due to the emergence of drug-resistant Mycobacterium tuberculosis (M.tb) strains and the lack of an efficient vaccine. The ability of M.tb to persist in latency, evade recognition following seroconversion, and establish resistance in vulnerable populations warrants closer examination. Past and current research has primarily focused on examination of the role of alveolar macrophages and dendritic cells during M.tb infection, which are critical in the establishment of the host response during infection. However, emerging evidence indicates that the alveolar epithelium is a harbor for M.tb and critical during progression to active disease. Here we evaluate the relatively unexplored role of the alveolar epithelium as a reservoir and also its capacity to secrete soluble mediators upon M.tb exposure, which influence the extent of infection. We further discuss how the M.tb-alveolar epithelium interaction instigates cell-to-cell crosstalk that regulates the immune balance between a proinflammatory and an immunoregulatory state, thereby prohibiting or allowing the establishment of infection. We propose that consideration of alveolar epithelia provides a more comprehensive understanding of the lung environment in vivo in the context of host defense against M.tb.

Zinc regulates the acute phase response and serum amyloid A production in response to sepsis through JAK-STAT3 signaling.

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.

Tissue-Specific Induction of Mouse ZIP8 and ZIP14 Divalent Cation/Bicarbonate Symporters by, and Cytokine Response to, Inflammatory Signals.

Mouse Slc39a8 and Slc39a14 genes encode ZIP8 and ZIP14, respectively, which are ubiquitous divalent cation/(HCO3-)2 symporters responsible for uptake of Zn2+, Fe2+, and Mn2+ into cells. Cd2+ and other toxic nonessential metals can displace essential cations, thereby entering vertebrate cells. Whereas Slc39a8 encodes a single protein, Slc39a14 has 2 exons 4 which, via alternative splicing, give rise to ZIP14A and ZIP14B; why differences exist in cell type-specific expression of ZIP14A and ZIP14B remains unknown. Inflammatory stimuli have been associated with upregulation of ZIP8 and ZIP14, but a systematic study of many tissues simultaneously in a laboratory animal following inflammatory cytokine exposure has not yet been reported. Herein, we show that C57BL/6J male mice--treated intraperitoneally with lipopolysaccharide or the proinflammatory cytokines tumor necrosis factor (TNF) or interleukin-6 (IL6)--exhibited quantatively very different, highly tissue-specific, and markedly time-dependent up- and downregulation of ZIP8, ZIP14A, and ZIP14B messenger RNA (mRNA) levels in 12 tissues. The magnitude of inflammatory response was confirmed by measuring the proinflammatory cytokine TNF, IL6, and interleukin-1ß mRNA levels in the same tissues of these animals. Our data suggest that most if not all tissues use ZIP8, ZIP14A, and/or ZIP14B for Zn2+ uptake, some tissues under basal conditions and others moreso when inflammatory stressors are present; collectively, this might lead to substantial alterations in plasma Zn2+ levels due to Zn2+ redistribution not just in liver but across many vital organs. In the context of cadmium-mediated toxicity, our data suggest that tissues other than liver, kidney, and lung should also be considered.

Zinc deficiency augments leptin production and exacerbates macrophage infiltration into adipose tissue in mice fed a high-fat diet.

Zinc (Zn) deficiency and obesity are global public health problems. Zn deficiency is associated with obesity and comorbid conditions that include insulin resistance and type 2 diabetes. However, the function of Zn in obesity remains unclear. Using a mouse model of combined high-fat and low-Zn intake (0.5-1.5 mg/kg), we investigated whether Zn deficiency exacerbates the extent of adiposity as well as perturbations in metabolic and immune function. C57BL/6 mice were randomly assigned to receive either a high-fat diet (HFD) or a control (C) diet for 6 wk, followed by further subdivision into 2 additional groups fed Zn-deficient diets (C-Zn, HFD-Zn), along with a C diet and an HFD, for 3 wk (n = 8-9 mice/group). The extent of visceral fat, insulin resistance, or systemic inflammation was unaffected by Zn deficiency. Strikingly, Zn deficiency significantly augmented circulating leptin concentrations (HFD-Zn vs. HFD: 3.15 ± 0.16 vs. 2.59 ± 0.12 µg/L, respectively) and leptin signaling in the liver of obese mice. Furthermore, gene expression of macrophage-specific markers ADAM8 (A disintegrin and metalloproteinase domain-containing protein 8) and CD68 (cluster of differentiation 68) was significantly greater in adipose tissue in the HFD-Zn group than in the HFD group, as confirmed by CD68 protein analysis, indicative of increased macrophage infiltration. Inspection of Zn content and mRNA profiles of all Zn transporters in the adipose tissue revealed alterations of Zn metabolism to obesity and Zn deficiency. Our results demonstrate that Zn deficiency increases leptin production and exacerbates macrophage infiltration into adipose tissue in obese mice, indicating the importance of Zn in metabolic and immune dysregulation in obesity.