Chandrashekhar Charavaryamath

Multiple exposures to swine barn air induce lung inflammation and airway hyper-responsiveness

BackgroundSwine farmers repeatedly exposed to the barn air suffer from respiratory diseases. However the mechanisms of lung dysfunction following repeated exposures to the barn air are still largely unknown. Therefore, we tested a hypothesis in a rat model that multiple interrupted exposures to the barn air will cause chronic lung inflammation and decline in lung function.MethodsRats were exposed either to swine barn (8 hours/day for either one or five or 20 days) or ambient air. After the exposure periods, airway hyper-responsiveness (AHR) to methacholine (Mch) was measured and rats were euthanized to collect bronchoalveolar lavage fluid (BALF), blood and lung tissues. Barn air was sampled to determine endotoxin levels and microbial load.ResultsThe air in the barn used in this study had a very high concentration of endotoxin (15361.75 ± 7712.16 EU/m3). Rats exposed to barn air for one and five days showed increase in AHR compared to the 20-day exposed and controls. Lungs from the exposed groups were inflamed as indicated by recruitment of neutrophils in all three exposed groups and eosinophils and an increase in numbers of airway epithelial goblet cells in 5- and 20-day exposure groups. Rats exposed to the barn air for one day or 20 days had more total leukocytes in the BALF and 20-day exposed rats had more airway epithelial goblet cells compared to the controls and those subjected to 1 and 5 exposures (P < 0.05). Bronchus-associated lymphoid tissue (BALT) in the lungs of rats exposed for 20 days contained germinal centers and mitotic cells suggesting activation. There were no differences in the airway smooth muscle cell volume or septal macrophage recruitment among the groups.ConclusionWe conclude that multiple exposures to endotoxin-containing swine barn air induce AHR, increase in mucus-containing airway epithelial cells and lung inflammation. The data also show that prolonged multiple exposures may also induce adaptation in AHR response in the exposed subjects.

ROLE OF TOLL-LIKE RECEPTOR 4 IN LUNG INFLAMMATION FOLLOWING EXPOSURE TO SWINE BARN AIR

The authors tested a hypothesis that lung inflammation and airway hyperresponsiveness (AHR) induced following barn air exposure are dependent on Toll-like receptor 4 (TLR4) by exposing C3HeB/FeJ (intact TLR4, wild type [WT]) and C3H/HeJ (defective TLR4, mutant) mice either to the barn air (8 hours/day for 1, 5, or 20 days) or ambient air. Both strains of mice, compared to their respective controls, showed increased AHR following 5 exposures but dampened AHR after 20 exposures to show lack of effect of TLR4 on AHR. However, swine barn air induced lung inflammation with recruitment of inflammatory cells and cytokine expression was observed in WT but not in mutant mice. These data show different roles of TLR4 in lung inflammation and AHR in mice exposed to swine barn air.

Pulmonary effects of exposure to pig barn air

Swine production has undergone rapid transformation from family owned operation to a large scale industrial enterprise. Since increasing number of pigs are reared on a large scale in confined buildings, some of the swine barn workers may be employed to work eight hours per day. Swine barn workers suffer from higher incidences of impaired air flow and lung inflammation, which is attributed to high intensity and interrupted exposures to pig barn air. The air in these barns contains gases, dust, microbes and endotoxin with endotoxin being the major suspect as the cause of lung dysfunction. This review attempts to describe the current state of knowledge of incidences and mechanisms of pulmonary dysfunction following exposure to the barn air.

Mucosal changes in a long-term bovine intestinal segment model following removal of ingesta and microflora.

The intestinal immune system influences responses to both enteric pathogens and commensal microflora but few models are available to analyze mucosal immune responses to either enteric pathogens or commensal microflora. We surgically isolated ileal segments in 2-3 week old calves, infused antibiotics, and subdivided each segment into three compartments. Following a 6-8 week period the isolated ileal segments appeared grossly normal in 4 of 5 calves, retained compartmentalization, and contents were culture positive for either Enterococcus spp. or Escherichia coli. In a second experiment, isolated ileal segments were examined following a 9-11 month period and appeared grossly normal with compartmentalization retained in 8 of 11 animals. Streptococcus spp or Escherichia coli were cultured from segment contents collected from 3 of these 8 animals. Histology revealed a marked reduction in villus height in isolated ileal segments despite sustained crypt epithelium proliferation. Lymphoid follicles in ileal Peyers patches were reduced in size but remained sites of active lymphoproliferation within segments. Significant mucosal T cell, macrophage, and dendritic cell depletion was observed in isolated ileal segments and T cell and NK cell depletion increased significantly in the absence of culturable bacteria. Finally, Toll-like receptor (TLR)-4 expression was decreased but TLR-5 and -6 expression increased in ileal segments. Thus, isolated ileal segments remained relatively stable for prolonged periods and significant changes in mucosal leukocyte populations were correlated with the presence or absence of culturable microflora. Stable, as opposed to sterile, isolated ileal segments provide an opportunity to analyze bovine mucosal immune responses in the presence or absence of commensal microflora.

Host responses to persistent Mycobacterium avium subspecies paratuberculosis infection in surgically isolated bovine ileal segments.

ABSTRACT A lack of appropriate disease models has limited our understanding of the pathogenesis of persistent enteric infections with Mycobacterium avium subsp. paratuberculosis. A model was developed for the controlled delivery of a defined dose of M. avium subsp. paratuberculosis to surgically isolated ileal segments in newborn calves. The stable intestinal segments enabled the characterization of host responses to persistent M. avium subsp. paratuberculosis infections after a 9-month period, including an analysis of local mucosal immune responses relative to an adjacent uninfected intestinal compartment. M. avium subsp. paratuberculosis remained localized at the initial site of intestinal infection and was not detected by PCR in the mesenteric lymph node. M. avium subsp. paratuberculosis-specific T cell proliferative responses included both CD4 and γδ T cell receptor (γδTcR) T cell responses in the draining mesenteric lymph node. The levels of CD8+ and γδTcR+ T cells increased significantly (P < 0.05) in the lamina propria, and M. avium subsp. paratuberculosis-specific tumor necrosis factor alpha (TNF-α) and gamma interferon secretion by lamina propria leukocytes was also significantly (P < 0.05) increased. There was a significant (P < 0.05) accumulation of macrophages and dendritic cells (DCs) in the lamina propria, but the expression of mucosal toll-like receptors 1 through 10 was not significantly changed by M. avium subsp. paratuberculosis infection. In conclusion, surgically isolated ileal segments provided a model system for the establishment of a persistent and localized enteric M. avium subsp. paratuberculosis infection in cattle and facilitated the analysis of M. avium subsp. paratuberculosis-specific changes in mucosal leukocyte phenotype and function. The accumulation of DC subpopulations in the lamina propria suggests that further investigation of mucosal DCs may provide insight into host responses to M. avium subsp. paratuberculosis infection and improve vaccine strategies to prevent M. avium subsp. paratuberculosis infection.

Lung inflammation following a single exposure to swine barn air

BackgroundExposure to swine barn air is an occupational hazard. Barn workers following an eight-hour work shift develop many signs of lung dysfunction including lung inflammation. However, the in situ cellular and molecular mechanisms responsible for lung dysfunction induced following exposure to the barn air remain largely unknown. Specifically, the recruitment and role of pulmonary intravascular monocytes/macrophages (PIMMs), which increase host susceptibility for acute lung inflammation, remain unknown in barn air induced lung inflammation. We hypothesized that barn exposure induces recruitment of PIMMs and increases susceptibility for acute lung inflammation with a secondary challenge.MethodsSprague-Dawley rats were exposed either to the barn or ambient air for eight hours and were euthanized at various time intervals to collect blood, broncho-alveolar lavage fluid (BALF) and lung tissue. Subsequently, following an eight hour barn or ambient air exposure, rats were challenged either with Escherichia coli (E. coli) lipopolysaccharide (LPS) or saline and euthanized 6 hours post-LPS or saline treatment. We used ANOVA (P < 0.05 means significant) to compare group differences.ResultsAn eight-hour exposure to barn air induced acute lung inflammation with recruitment of granulocytes and PIMMs. Granulocyte and PIMM numbers peaked at one and 48 hour post-exposure, respectively.Secondary challenge with E. coli LPS at 48 hour following barn exposure resulted in intense lung inflammation, greater numbers of granulocytes, increased number of cells positive for TNF-α and decreased amounts of TGF-β2 in lung tissues. We also localized TNF-α, IL-1β and TGF-β2 in PIMMs.ConclusionA single exposure to barn air induces lung inflammation with recruitment of PIMMs and granulocytes. Recruited PIMMs may be linked to more robust lung inflammation in barn-exposed rats exposed to LPS. These data may have implications of workers exposed to the barn air who may encounter secondary microbial challenge.

Expression of calcineurin and its interacting proteins in epileptic fowl.

Calcineurin (CaN), a Ca2+–calmodulin (CaM)‐dependent protein phosphatase, is important for Ca2+‐mediated signal transduction. The main objective of this study was to examine the potential role of CaN in epileptic brain and its involvement in neuronal apoptosis. We investigated CaN expression and its interaction with various signaling molecules in normal, carrier and epileptic brain tissues of chicken. Our results revealed higher Ca2+–CaM‐dependent phosphatase activity of CaN and a correspondingly strong immunoreactive band of CaN A in epileptic and carrier brain samples compared with normal brain. Furthermore, immunohistochemical analysis showed a higher level of expression of CaN in epileptic brain tissue. However, the intensity of immunoreactivity was less in carrier than epileptic brain. We observed that the interaction of CaN with m‐calpain and µ‐calpain was strong in carrier and epileptic chickens compared with that in normal birds. In addition, the interaction of CaN with Bcl‐2, caspase‐3 and p53 was greater in carrier and epileptic fowl than in normal chickens. The greater interaction of CaN with various apoptotic factors in epileptic chickens adds to our understanding of the mechanism of CaN signaling in neuronal apoptosis.

Pulmonary innate inflammatory responses to agricultural occupational contaminants

Agricultural workers are exposed to many contaminants and suffer from respiratory and other symptoms. Dusts, gases, microbial products and pesticide residues from farms have been linked to effects on the health of agricultural workers. Growing sets of data from in vitro and in vivo models demonstrate the role of the innate immune system, especially Toll-like receptor 4 (TLR4) and TLR9, in lung inflammation induced following exposure to contaminants in agricultural environments. Interestingly, inflammation and lung function changes appear to be discordant indicating the complexity of inflammatory responses to exposures. Whereas the recent development of rodent models and exposure systems have yielded valuable data, we need new systems to examine the combined effects of multiple contaminants in order to increase our understanding of farm-exposure-induced negative health effects.

Evaluation of inhibition of F4ac positive Escherichia coli attachment with xanthine dehydrogenase, butyrophilin, lactadherin and fatty acid binding protein

BackgroundNeonatal and post-weaning colibacillosis caused by enterotoxigenic E. coli is responsible for substantial economic losses encountered by the pork industry. Intestinal colonization of young piglets by E. coli depends on the efficiency of bacterial attachment to host gastrointestinal epithelium that is mediated by fimbriae. We tested the effect of porcine individual milk fat globule membrane (MFGM) proteins on F4ac positive E. coli attachment to porcine enterocytes in vitro.ResultsButyrophilin, lactadherin and fatty acid binding protein inhibited fimbriae-dependent adherence of E. coli to enterocytes in vitro, while xanthine dehydrogenase did not. The inhibiting activity was dose-dependent for all three proteins, but the inhibiting efficiency was different.ConclusionsThe results indicate that MFGM proteins may interfere with attachment of E. coli to porcine neonatal intestinal mucosa.

Kinome analyses of inflammatory responses to swine barn dust extract in human bronchial epithelial and monocyte cell lines

Exacerbated inflammation upon persistent barn organic dust exposure is a key contributor to the pathogenesis of lung inflammation and lung function decline. Barn dust constituents and the mechanisms contributing to the exacerbated inflammation are not clearly known. We set out to understand the inflammatory effects of Swine Barn Dust Extracts (SBDE) on human lung epithelial (BEAS2B) and macrophage (THP-1 monocyte derived) cell lines on a kinome array to determine phosphorylation events in the inflammatory signaling pathways. Upon identifying events unique to SBDE or those induced by innate immune ligands in each cell line, we validated the signaling pathway activation by transcriptional analyses of downstream inflammatory cytokines. Our findings indicate that SBDE-mediated pro-inflammatory effects are predominantly due to the induction of neutrophilic chemokine IL-8. Differentially phosphorylated peptides implicated in IL-8 induction in BEAS2B cell line include, TLR2, 4, 5, 7, 8, 9, PKC, MAP kinases (p38, JNK), inflammasomes (NLRP1, NLRP3), NF-κB and AP-1. In the THP-1 cell line, in addition to the aforementioned peptides, peptides corresponding to RIG-I-like receptors (RIG-I, MDA5) were found. This is the first report to demonstrate the application of a kinome array to delineate key inflammatory signaling pathways activated upon SBDE exposure in vitro.