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

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 Cxcr2-/- 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 Cxcr2-/- 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 Proteobacteria to Firmicutes. Interestingly, the genetic inactivation of Cxcr2 alone produced a similar bacterial shift. When treated with SMZ/TMP, Cxcr2-/- mice infected with RRV to induce experimental biliary atresia showed further enrichment of Corynebacterium, Anaerococcus and Streptococcus. Among these, Anaerococcus lactolyticus 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.

Sublingual targeting of STING with 3'3'-cGAMP promotes systemic and mucosal immunity against anthrax toxins.

Anthrax is caused by Bacillus anthracis, a zoonotic bacterial pathogen affecting humans and livestock worldwide. The current human anthrax vaccine, anthrax vaccine adsorbed (AVA), is an injected vaccine with a cumbersome administration schedule and fails to promote mucosal immunity. Bacterial enterotoxins, which stimulate production of the cyclic nucleotide cAMP are effective experimental mucosal vaccine adjuvants, but their inherent toxicity has precluded their use in humans. We investigated whether cyclic dinucleotides that target Stimulator of Interferon Gamma Genes (STING) in mammalian cells could represent an alternative to bacterial enterotoxins as adjuvant for sublingual immunization and promotion of mucosal immunity and secretory IgA responses in addition to systemic immunity. We found that sublingual immunization of mice with Bacillus anthracis protective antigen (PA) and the STING ligand 3'3'-cGAMP promotes PA-specific serum IgG Ab responses of the same magnitude as those induced after immunization with PA and the experimental adjuvant cholera toxin (CT). Interestingly, this STING ligand also promoted serum anti-PA IgA and IgA-producing cells in the bone marrow. Furthermore, the saliva of mice immunized with the STING ligand exhibited similar levels of PA-specific IgA Abs as groups immunized with CT as adjuvant. The adjuvant activity of 3'3'-cGAMP was associated with mixed Th1, Th2, and Th17 responses. This STING ligand also induced rapid IFN-ß and IL-10 responses in sublingual tissues and cervical lymph nodes, and TGF-ß responses in the cervical lymph nodes, which could contribute to promoting IgA responses after sublingual immunization.

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

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.

Routes of allergic sensitization and myeloid cell IKKß differentially regulate antibody responses and allergic airway inflammation in male and female mice.

Gender influences the incidence and/or the severity of several diseases and evidence suggests a higher rate of allergy and asthma among women. Most experimental models of allergy use mice sensitized via the parenteral route despite the fact that the mucosal tissues of the gastrointestinal and respiratory tracts are major sites of allergic sensitization and/or allergic responses. We analyzed allergen-specific Ab responses in mice sensitized either by gavage or intraperitoneal injection of ovalbumin together with cholera toxin as adjuvant, as well as allergic inflammation and lung functions following subsequent nasal challenge with the allergen. Female mice sensitized intraperitoneally exhibited higher levels of serum IgE than their male counterparts. After nasal allergen challenge, these female mice expressed higher Th2 responses and associated inflammation in the lung than males. On the other hand, male and female mice sensitized orally developed the same levels of allergen-specific Ab responses and similar levels of lung inflammation after allergen challenge. Interestingly, the difference in allergen-specific Ab responses between male and female mice sensitized by the intraperitoneal route was abolished in IKKßΔMye mice, which lack IKKß in myeloid cells. In summary, the oral or systemic route of allergic sensitization and IKKß signaling in myeloid cells regulate how the gender influences allergen-specific responses and lung allergic inflammation.

Effects of dietary vitamin A content on antibody responses of feedlot calves inoculated intramuscularly with an inactivated bovine coronavirus vaccine.

OBJECTIVE: To investigate effects of low dietary vitamin A content on antibody responses in feedlot calves inoculated with an inactivated bovine coronavirus (BCoV) vaccine. ANIMALS: 40 feedlot calves. PROCEDURES: Calves were fed diets containing high (3,300 U/kg) or low (1,100 U/kg) amounts of vitamin A beginning on the day of arrival at a feedlot (day 0) and continuing daily until the end of the study (day 140). Serum retinol concentrations were evaluated in blood samples obtained throughout the study. Calves were inoculated IM with an inactivated BCoV vaccine on days 112 and 126. Blood samples obtained on days 112 and 140 were used for assessment of BCoV-specific serum IgG1, IgG2, IgM, and IgA titers via an ELISA. RESULTS: The low vitamin A diet reduced serum retinol concentrations between days 112 and 140. After the BCoV inoculation and booster injections, predominantly serum IgG1 antibodies were induced in calves fed the high vitamin A diet; however, IgG1 titers were compromised at day 140 in calves fed the low vitamin A diet. Other isotype antibodies specific for BCoV were not affected by the low vitamin A diet. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary vitamin A restriction increases marbling in feedlot cattle; however, its effect on antibody responses to vaccines is unknown. A low vitamin A diet compromised the serum IgG1 responses against inactivated BCoV vaccine, which suggested suppressed T-helper 2-associated antibody (IgG1) responses. Thus, low vitamin A diets may compromise the effectiveness of viral vaccines and render calves more susceptible to infectious disease.

Contributions of edema factor and protective antigen to the induction of protective immunity by Bacillus anthracis edema toxin as an intranasal adjuvant.

We have shown that intranasal coapplication of Bacillus anthracis protective Ag (PA) together with a B. anthracis edema factor (EF) mutant having reduced adenylate cyclase activity (i.e., EF-S414N) enhances anti-PA Ab responses, but also acts as a mucosal adjuvant for coadministered unrelated Ags. To elucidate the role of edema toxin (EdTx) components in its adjuvanticity, we examined how a PA mutant lacking the ability to bind EF (PA-U7) or another mutant that allows the cellular uptake of EF, but fails to efficiently mediate its translocation into the cytosol (PA-dFF), would affect EdTx-induced adaptive immunity. Native EdTx promotes costimulatory molecule expression by macrophages and B lymphocytes, and a broad spectrum of cytokine responses by cervical lymph node cells in vitro. These effects were reduced or abrogated when cells were treated with EF plus PA-dFF, or PA-U7 instead of PA. We also intranasally immunized groups of mice with a recombinant fusion protein of Yersinia pestis F1 and LcrV Ags (F1-V) together with EdTx variants consisting of wild-type or mutants PA and EF. Analysis of serum and mucosal Ab responses against F1-V or EdTx components (i.e., PA and EF) revealed no adjuvant activity in mice that received PA-U7 instead of PA. In contrast, coimmunization with PA-dFF enhanced serum Ab responses. Finally, immunization with native PA and an EF mutant lacking adenylate cyclase activity (EF-K346R) failed to enhance Ab responses. In summary, a fully functional PA and a minimum of adenylate cyclase activity are needed for EdTx to act as a mucosal adjuvant.

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

Reinfection with Chlamydophila abortus by uterine and indirect cohort routes reduces fertility in cattle preexposed to Chlamydophila.

This study investigated the effects of controlled reinfection on fertility of cattle naturally preexposed to Chlamydophila abortus. All animals had high prechallenge levels of immunoglobulin M (IgM), IgG, IgG1, and IgG2 serum antibodies against ruminant C. abortus in a chemiluminescent enzyme-linked immunosorbent assay. Twenty virgin heifers were estrus synchronized with prostaglandin F2, artificially inseminated 2 to 3 days later, and challenged immediately by intrauterine administration of 0, 10(4), 10(5), 10(6), or 10(8) inclusion-forming units (IFU) of C. abortus. Ten heifers were estrus synchronized, inseminated, and uterine challenged 2 weeks later. These animals were also indirectly exposed to C. abortus infection (cohort challenged) by contact with their previously challenged cohorts. Pregnancy was determined by rectal palpation 42 days after insemination. All anti-C. abortus antibody isotypes increased in heifers following uterine challenge with 10(8) IFU. A total of 11, 83, 50, 66, and 0% of heifers were pregnant after uterine challenge with 0, 10(4), 10(5), 10(6), and 10(8) IFU of C. abortus, respectively. A total of 50 and 65% of heifers were pregnant with and without cohort challenge, respectively. Uterine inoculum dose and cohort challenge (or, alternatively, a negative pregnancy outcome [infertility]) correlated highly significantly with a rise in postchallenge anti-C. abortus IgM levels over prechallenge levels. Logistic regression modeled fertility, with uterine challenge dose and cohort challenge or prechallenge IgM as predictors (P < 0.05). The models predict that the uterine C. abortus inoculum causing infertility is 8.5-fold higher for heifers without cohort exposure and 17-fold higher for heifers with high IgM levels than for heifers with cohort exposure or with low IgM levels.