Accumulation of metals in GOLD4 COPD lungs is associated with decreased CFTR levels

Abstract Background 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. Methods Expression of CFTR and the cigarette smoke metal content were assessed in lung samples of controls and COPD patients with established GOLD stage 4. CFTR protein and mRNA were quantified by immunohistochemistry and quantitative RT-PCR, respectively. Metals present in lung samples were quantified by ICP-AES. The effect of cigarette smoke on down-regulation of CFTR expression and function was assessed using primary human airway epithelial cells. The role of leading metal(s) found in lung samples of GOLD 4 COPD patients involved in the alteration of CFTR was confirmed by exposing human bronchial epithelial cells 16HBE14o- to metal-depleted cigarette smoke extracts. Results We found that CFTR expression is reduced in the lungs of GOLD 4 COPD patients, especially in bronchial epithelial cells. Assessment of metals present in lung samples revealed that cadmium and manganese were significantly higher in GOLD 4 COPD patients when compared to control smokers (GOLD 0). Primary human airway epithelial cells exposed to cigarette smoke resulted in decreased expression of CFTR protein and reduced airway surface liquid height. 16HBE14o-cells exposed to cigarette smoke also exhibited reduced levels of CFTR protein and mRNA. Removal and/or addition of metals to cigarette smoke extracts before exposure established their role in decrease of CFTR in airway epithelial cells. Conclusions CFTR expression is reduced in the lungs of patients with severe COPD. This effect is associated with the accumulation of cadmium and manganese suggesting a role for these metals in the pathogenesis of COPD

High Prevalence of Natural Chlamydophila Species Infection in Calves

We investigated the acquisition and prevalence of Chlamydophila sp. infection in calves. Specimens were collected at weekly intervals from birth to week 12 postpartum from 40 female Holstein calf-dam pairs in a dairy herd. Real-time PCR detected, quantified, and differentiated Chlamydophila 23S rRNA gene DNA from vaginal cytobrush swabs and milk samples. Chemiluminescence enzyme-linked immunosorbent assay with lysed Chlamydophila abortus or Chlamydophila pecorum elementary body antigens quantified antibodies against Chlamydophila spp. in sera. Chlamydophila sp. DNA was found in 61% of calves and 20% of dams in at least one positive quantitative PCR. In calves, clinically inapparent C. pecorum infection with low organism loads was fivefold more prevalent than C. abortus infection and was most frequently detected by vaginal swabs compared to rectal or nasal swabs. In dams, C. abortus dominated in milk and C. pecorum dominated in the vagina. The group size of calves correlated positively (P < 0.01) with Chlamydophila infection in quadratic, but not linear, regression. Thus, a doubling of the group size was associated with a fourfold increase in frequency and intensity of Chlamydophila infection. For groups of 14 or 28 calves, respectively, logistic regression predicted a 9 or 52% probability of infection of an individual calf and a 52 or 99.99% probability of infection of the group. Anti-Chlamydophila immunoglobulin M antibodies in Chlamydophila PCR-positive calves and dams and in dams that gave birth to calves that later became positive were significantly higher than in PCR-negative animals (P ≤ 0.02). Collectively, crowding strongly enhances the frequency and intensity of highly prevalent Chlamydophila infections in cattle

Number of neonatal mice with bacterial colonization in the colon at 3 and 10 days of age.

<p>Number of neonatal mice with bacterial colonization in the colon at 3 and 10 days of age.</p

Microbiome changes in Cxcr2^-/- mice with symptoms (diseased) and without symptoms (resistant) 14 days after RRV infection.

<p>(A) Relative abundance of three genera in the colon of diseased and resistant Cxcr2^-/- mice exposed to SMZ/TMP diet (N = 4 per group). (B) CART analysis of all three genera identifies Anaerococcus genus most clearly differentiating the high from low survivability (N = 43). (C) Nested-PCR shows only A. lactolyticus in colons of Cxcr2^-/- mice exposed to SMZ/TMP (N = 8). Median with interquartile range. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.</p

Cxcr2 signaling and the microbiome suppress inflammation, bile duct injury, and the phenotype of experimental biliary atresia

<div><p>Biliary atresia is progressive fibro-inflammatory cholangiopathy of young children. Central to pathogenic mechanisms of injury is the tissue targeting by the innate and adaptive immune cells. Among these cells, neutrophils and the IL-8/Cxcl-8 signaling via its Cxcr2 receptor have been linked to bile duct injury. Here, we aimed to investigate whether the intestinal microbiome modulates Cxcr2-dependent bile duct injury and obstruction. Adult wild-type (WT) and <i>Cxcr2</i>^<i>-/-</i> mice were fed a diet supplemented with sulfamethoxazole/trimethoprim (SMZ/TMP) during pregnancy and lactation, and their pups were injected intraperitoneally with rhesus rotavirus (RRV) within 24 hours of life to induce experimental biliary atresia. The maternal exposure to SMZ/TMP significantly lowered the incidence of jaundice and bile duct obstruction and resulted in improved survival, especially in <i>Cxcr2</i>^<i>-/-</i> mice. Analyses of the microbiome by deep sequencing of 16S rRNA of the neonatal colon showed a delay in bacterial colonization of WT mice induced by SMZ/TMP, with a notable switch from <i>Proteobacteria</i> to <i>Firmicutes</i>. Interestingly, the genetic inactivation of <i>Cxcr2</i> alone produced a similar bacterial shift. When treated with SMZ/TMP, <i>Cxcr2</i>^<i>-/-</i> mice infected with RRV to induce experimental biliary atresia showed further enrichment of <i>Corynebacterium</i>, <i>Anaerococcus</i> and <i>Streptococcus</i>. Among these, <i>Anaerococcus lactolyticus</i> was significantly associated with a suppression of biliary injury, cholestasis, and survivability. These results suggest that the postnatal development of the intestinal microbiota is an important susceptibility factor for experimental biliary atresia.</p></div

Gut-Microbiome changes induced by SMZ/TMP in Cxcr2^-/- mice after RRV infection.

<p>(A) Bacterial taxa representing significantly different abundances between the regular (green) and SMZ/TMP (red) diets in Cxcr2^-/- mice according to their LDA scores. (B) Cladograms were generated on the basis of the LDA scores, showing significantly different abundance between regular and SMZ/TMP diet. Green-highlighted regions are indicative of taxa more abundant in regular diet, and red-highlighted regions, more abundant in SMZ/TMP diet (N = 8–13 per group).</p

Hepatic expression for Cxcr2-related chemokines and cytokines.

<p>mRNA expression levels quantified by real-time PCR using RNA from livers of neonatal Cxcr2^-/- mice with the biliary atresia phenotype (diseased) or asymptomatic (resistant) mice 14 days after RRV infection. mRNA is expressed as a ratio to Gapdh (N = 4 per group). Median with interquartile range. * P<0.05.</p

Changes in the intestinal microbiome induced by SMZ/TMP in Cxcr2^-/- mice after RRV infection.

<p>(A) Phylum analyses show a switch from Proteobacteria to Firmicutes upon exposure to SMZ/TMP or mutation of Cxcr2 (N = 8–13 per group). (B) Colons from the SMZ/TMP-treated Cxcr2^-/- mice had less Proteobacteria, more Firmicutes and Actinobacteria (n = 8–13 per group). Median with interquartile ranges in microbiome changes in WT and Cxcr2^-/- mice in relation to regular and SMZ/TMP-containing diets (N = 8–13 per group). Solid and dotted lines are defined as statistically significant and non-significant, respectively, between regular and SMZ/TMP diet in each mouse type. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.</p

Bacteria diversity in SMZ/TMP-treated WT and Cxcr2^-/- mice after RRV infection.

<p>(A) Microbial alpha diversity was measured by observed taxa and Chao1 for richness, and by Dominance and Shannon diversity index for evenness (N = 8–13 per group). SMZ/TMP exposure and/or mutation in Cxcr2 significantly increased bacterial diversity without affecting richness. (B) Non-metric dimensional scaling (NMDS) ordinations based upon Bray-Curtis similarity using all OTUs significantly separated regular diet-treated WT mice from all other groups (N = 8–13 per group, P<0.001). Median with interquartile range. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.</p