ZIP8 Zinc Transporter: Indispensable Role for Both Multiple-Organ Organogenesis and Hematopoiesis In Utero

Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn2+/(HCO3–)2 symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects–proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis

TLR and NKG2D signaling pathways mediate CS-induced pulmonary pathologies.

Long-term exposure to cigarette smoke (CS) can have deleterious effects on lung epithelial cells including cell death and the initiation of inflammatory responses. CS-induced cell injury can elaborate cell surface signals and cellular byproducts that stimulate immune system surveillance. Our previous work has shown that the expression of ligands for the cytotoxic lymphocyte activating receptor NKG2D is enhanced in patients with COPD and that the induction of these ligands in a mouse model can replicate COPD pathologies. Here, we extend these findings to demonstrate a role for the NKG2D receptor in CS-induced pathophysiology and provide evidence linking nucleic acid-sensing endosomal toll-like receptor (TLR) signaling to COPD pathology through NKG2D activation. Specifically, we show that mice deficient in NKG2D exhibit attenuated pulmonary inflammation and airspace enlargement in a model of CS-induced emphysema. Additionally, we show that CS exposure induces the release of free nucleic acids in the bronchoalveolar lavage and that direct exposure of mouse lung epithelial cells to cigarette smoke extract similarly induces functional nucleic acids as assessed by TLR3, 7, and 9 reporter cell lines. We demonstrate that exposure of mouse lung epithelial cells to TLR ligands stimulates the surface expression of RAET1, a ligand for NKG2D, and that mice deficient in TLR3/7/9 receptor signaling do not exhibit CS-induced NK cell hyperresponsiveness and airspace enlargement. The findings indicate that CS-induced airway injury stimulates TLR signaling by endogenous nucleic acids leading to elevated NKG2D ligand expression. Activation of these pathways plays a major role in the altered NK cell function, pulmonary inflammation and remodeling related to long-term CS exposure

Chronic Cigarette Smoke Exposure Generates Pathogenic T Cells Capable of Driving COPD-like Disease in Rag2−/− Mice

Rationale: Pathogenic T cells drive, or sustain, a number of inflammatory diseases. Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with the accumulation of activated T cells. We previously demonstrated that chronic cigarette smoke (CS) exposure causes oligoclonal expansion of lung CD4+ T cells and CD8+ T cells in a mouse model of COPD, thus implicating these cells in disease pathogenesis

CCR7 Deficiency Leads to Leukocyte Activation and Increased Clearance in Response to Pulmonary Pseudomonas aeruginosa Infection▿

CCR7 is a chemokine receptor expressed on the surfaces of T cells, B cells, and mature dendritic cells that controls cell migration in response to the cognate ligands CCL19 and CCL21. CCR7 is critical for the generation of an adaptive T cell response. However, the roles of CCR7 in the host defense against pulmonary infection and innate immunity are not well understood. We investigated the role of CCR7 in the host defense against acute pulmonary infection with Pseudomonas aeruginosa. We intranasally infected C57BL/6 mice with P. aeruginosa and characterized the expression of CCR7 ligands and the surface expression of CCR7 on pulmonary leukocytes. In response to infection, expression of CCL19 and expression of CCL21 were oppositely regulated, and myeloid dendritic cells upregulated CCR7 expression. We further examined the effects of CCR7 deficiency on the inflammatory response to P. aeruginosa infection. We infected Ccr7−/− and wild-type mice with P. aeruginosa and characterized the accumulation of pulmonary leukocytes, production of proinflammatory mediators, neutrophil activation, and bacterial clearance. CCR7 deficiency led to an accumulation of myeloid dendritic cells and T cells in the lung in response to infection. CCR7 deficiency resulted in higher expression of CD80 and CD86 on dendritic cells; increased production of interleukin-12/23p40 (IL-12/23p40), gamma interferon (IFN-γ), and IL-1α; increased neutrophil respiratory burst; and, ultimately, increased clearance of acute P. aeruginosa infection. In conclusion, our results suggest that CCR7 deficiency results in a heightened proinflammatory environment in response to acute pulmonary P. aeruginosa infection and contributes to more efficient clearance

NKG2D is required for CS-induced pulmonary inflammation and alveolar destruction.

<p>WT and NKG2-deficient deficient mice were exposed to CS or FA for 6 months. (A) BAL was performed and cells were enumerated. > 95% of cells were macrophage and no differences observed in other cell types (data not shown). (B) Inflammatory aggregates surrounding the airways and vasculature were scored by number of occurrences and severity in H%E-stained lung sections. Inflammation scores were transformed by taking the square root of the raw scores, and the total numbers of cells were transformed using Log₁₀ of the raw counts. * Significantly different than FA strain matched mice determined by Student t test. n = -6 mice. (C) Mean linear intercept was quantitated to define the average alveolar diameter. * Significantly different than FA strain matched mice determined by Student t test. ** Significantly different than CS-exposed wild type mice determined by Student t test. n = 5-6 mice.</p

TLR3/7/9 mediates CS-induced NK cell hyperresponsiveness.

<p>Lung leukocytes were isolated from FA- and CS-exposed mice, NK cells were purified, and stimulated with 10 ng/ml IL-12 or IL-18 alone, or in combination for 20 h. NK cells were also cultured with TLR7/8 agonist R848 (100ng/ml) as a negative control for TLR7 mutation and LPS (10ng/ml) for a positive control for TLR4 responsiveness. IFN- γ was measured by ELISA. Values are presented as means ± SEM. # Denotes values significantly different from wild-type FA. *Denotes values that are significantly different from wild-type CS exposure determined by Student t test. <i>p</i> < 0.05. Data are representative of three independent experiments. n= 4 mice per group.</p

Induction of NKG2D ligands by TLR ligands on mouse pulmonary epithelial cells.

<p>(A) MLE-15 cells were treated with synthetic RNA (50 µg/ml poly(I:C)), DNA CpG (1 µM ODN1826) or PBS for 24 h. Cell surface Raet1 expression was assessed by flow cytometry. Histograms are representative of three independent experiments. (B) Quantification of Raet1 induction on MLE-15 cells. Data are shown as the mean ± SEM of three independent experiments. * Significantly different than PBS-treated cells as determined by Student t test. (C) CSE-induces Raet1 expression on MLE-15 cells which can be inhibited with TLR3 antagonists [(R)-2-(3-Chloro-6-fluorobenzo[b]thiophene-2-carboxamido)-3-phenylpropanoic acid] and the TLR9 antagonist (50 nM of ODN 2088), and degradation of nucleic acids using an endonuclease (50 U/mL Benzonase). Data are shown as the mean ± SEM of three independent experiments. # Significantly different than PBS-treated cells as determined by Student t test. * Significantly different than 20% CSE-treated cells as determined by Student t test.</p