CRISPR-Cas9-mediated knockout of TLR4 modulates Mycobacterium avium ssp. paratuberculosis cell lysate-induced inflammation in bovine mammary epithelial cells.

Toll-like receptor 4 (TLR4) is a pattern-recognition receptor involved in the recognition of microbial pathogens and host alarmins. Ligation to TLR4 initiates a signaling cascade that leads to inflammation. Polymorphisms in bovine TLR4 have been associated with Mycobacterium avium ssp. paratuberculosis (MAP) susceptibility and resistance, the cause of Johne's disease, and milk somatic cell score, a biomarker of mastitis. Although the contribution of TLR4 to recognition of bacterial lipopolysaccharide (LPS) has been well characterized, its role in MAP recognition is less certain. Clustered regularly interspaced short palindromic repeats-Cas9 mediated gene editing was performed to generate TLR4 knockout (KO) mammary epithelial cells to determine if TLR4 expression is involved in the initiation of the host inflammatory response to MAP cell lysate (5 and 10 µg/mL) and Escherichia coli LPS (5 µg/mL). The absence of TLR4 in KO cells resulted in enhanced expression of key inflammatory genes (TNFA and IL6), anti-inflammatory genes (IL10 and SOCS3), and supernatant cytokine and chemokine levels (TNF-α, IL-6, IL-10, CCL3) in response to the MAP cell lysate (10 µg/mL). However, in response to LPS, the KO cells showed reduced expression of key inflammatory genes (TNFA, IL1A, IL1B, and IL6) and supernatant cytokine levels (TNF-α, IL-6, CCL2, IL-8) as compared with unedited cells. Overall, these results confirm that TLR4 is essential for eliciting inflammation in response to LPS; however, exacerbated gene and protein expression in TLR4 KO cells in response to MAP cell lysate suggests a different mechanism of infection and host response for MAP, at least in terms of how it interacts with TLR4. These novel findings show potential divergent roles for TLR4 in mycobacterial infections, and this may have important consequences for the therapeutic control of inflammation in cattle.

Short communication: Characterizing ovine serum stress biomarkers during endotoxemia.

Breeding stress-resilient livestock is a potential strategy to help mitigate the negative effect of environmental and pathogenic stressors. The hypothalamic-pituitary-adrenal axis and immune system are activated during stress events and release mediators into the circulation that help restore physiological homeostasis. The purpose of this study was to assess a comprehensive set of circulatory mediators released in response to an acute immune stress challenge to identify candidate biomarkers that can be used for the selection of stress-resilient animals. Fifteen female lambs were stress challenged with an intravenous bolus of lipopolysaccharide (LPS; 400 ng/kg), and blood was collected from the jugular vein at 0, 2, 4, and 6 h after LPS challenge to identify and monitor candidate stress biomarkers; temperature was also recorded over time. Biomarker responses were evaluated with a repeated-measures model to compare time points with baseline values. As expected, all sheep had a monophasic febrile response to LPS challenge, and cortisol increased and returned to baseline by 6 h. The cytokines tumor necrosis factor-α, IL-6, IFN-γ (proinflammatory), and IL-10 (anti-inflammatory) increased, but only tumor necrosis factor-α returned to baseline during the monitoring period. The cytokines IL-1α, IL-1ß, IL-17α (proinflammatory), and IL-4 (anti-inflammatory) did not respond to LPS challenge. All chemokines (CCL2, CCL3, CCL4, CXCL10, and IL-8) responded to LPS challenge; however, only CCL2, CCL3, CCL4, and CXCL10 increased over time, and only CCL3, CCL4, and CXCL10 returned to baseline during the monitoring period. MicroRNA (miR-145, miR-233, and miR-1246) also increased and remained elevated during the study. In summary, the LPS challenge induced a strong stress response in Rideau-Dorset sheep that could be monitored with a distinct profile of circulatory biomarkers.

Effect of algae or fish oil supplementation and porcine maternal stress on the adrenal transcriptome of male offspring fed a low-quality protein diet.

Offspring adrenal function may be negatively affected in utero by maternal stressors such as microbial infection. Maternal supplementation with immunomodulatory compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) may help minimize the adverse effects of maternal stress on fetal hypothalamic-pituitary-adrenal development and improve offspring health. Presently, n-3 PUFA sources are primarily fish-based, but n-3 PUFA microalgae (AL) may be an alternative. Previously, it was determined that maternal AL or fish oil (FO) supplementation to sows, in addition to maternal stress induced by Escherichia coli lipopolysaccharide (LPS) challenge appeared to have a greater influence on the stress response of male offspring compared to females. To further elaborate on these findings, this study assessed the effects of maternal AL or FO supplementation combined with a maternal LPS challenge on adrenal gene expression in male offspring fed a nursery diet containing low-quality protein sources. Forty-eight sows were fed gestation diets starting on gestation day (gd) 75 containing either 3.12% AL, 3.1% FO, or a control diet containing 1.89% corn oil. On gd 112, half the sows in each treatment were administered 10 â€‹µg/kg LPS i.m. Piglets were weaned at 21 days of age onto a common low-quality plant-based protein diet, and one week after weaning, four piglets per sow were administered 40 â€‹µg/kg LPS i.m. Two hours later, the piglets were euthanized to obtain adrenal tissue, and total RNA was extracted to carry out transcriptome analysis using the Affymetrix GeneChip WT Plus assay and subsequent validation by real-time PCR. Analysis revealed that adrenal steroidogenesis, fatty acid metabolism and immune function were significantly influenced by maternal diet and stress. Increased expression of immune-related genes including lymphocyte antigen 96, TLR-2 and NF-κB suggests that maternal AL supplementation may increase offspring sensitivity to inflammation after weaning. Decreased expression of lymphocyte antigen 96 in male offspring from sows receiving maternal LPS challenge also suggests a possible role of maternal stress in diminishing the offspring immune response to immune stress challenge. Increased expression of the genes encoding the 11BHSD2 enzyme in offspring from sows fed FO may also reduce the magnitude of the stress response. These data provide insight to the immune and metabolic mechanisms that may be influenced by maternal diet and stress.

In vitro bioassessment of the immunomodulatory activity of Saccharomyces cerevisiae components using bovine macrophages and Mycobacterium avium ssp. paratuberculosis.

The yeast Saccharomyces cerevisiae and its components are used for the prevention and treatment of enteric disease in different species; therefore, they may also be useful for preventing Johne's disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). The objective of this study was to identify potential immunomodulatory S. cerevisiae components using a bovine macrophage cell line (BOMAC). The BOMAC phagocytic activity, reactive oxygen species production, and immune-related gene (IL6, IL10, IL12p40, IL13, IL23), transforming growth factor ß, ARG1, CASP1, and inducible nitric oxide synthase expression were investigated when BOMAC were cocultured with cell wall components from 4 different strains (A, B, C, and D) and 2 forms of dead yeast from strain A. The BOMAC phagocytosis of mCherry-labeled MAP was concentration-dependently attenuated when BOMAC were cocultured with yeast components for 6 h. Each yeast derivative also induced a concentration-dependent increase in BOMAC reactive oxygen species production after a 6-h exposure. In addition, BOMAC mRNA expression of the immune-related genes was investigated after 6 and 24 h of exposure to yeast components. All yeast components were found to regulate the immunomodulatory genes of BOMAC; however, the response varied among components and over time. The in vitro bioassessment studies reported here suggest that dead yeast and its cell wall components may be useful for modulating macrophage function before or during MAP infection.

Bovine PGLYRP1 polymorphisms and their association with resistance to Mycobacterium avium ssp. paratuberculosis.

Mycobacterium avium ssp. paratuberculosis (MAP) causes a chronic, granulomatous inflammatory condition of the intestines in ruminants and wild-type species. It causes significant economic losses to the dairy and beef industries owing to reduced productivity, premature culling and mortality. Bovine peptidoglycan recognition protein 1 is an important pattern recognition molecule that is capable of directly killing microorganisms. The goal of this study was to identify single nucleotide polymorphisms (SNPs) in the gene encoding bovine peptidoglycan recognition protein 1 and to assess their association with susceptibility to MAP infection in dairy cattle. Blood and milk samples were collected from Holsteins in Southwestern and Eastern Ontario and tested for MAP infection using blood and milk ELISAs. A resource population consisting of 197 infected (S/P > 0.25) and 242 healthy (S/P < 0.10) cattle was constructed. Sequencing of pooled DNA was used to identify three SNPs (c.102G>C, c.480G>A and c.625C>A) that were genotyped in the resource population. Statistical analysis was performed using a logistic regression model fitting the additive and dominance effects of each SNP in the model. SNP c.480G>A (P = 0.054) was found to be associated with susceptibility to MAP infection. Cows with a copy of the major allele 'G' at this locus had an odds ratio of 1.51 (95% CI: 0.99-2.31) for being infected with MAP.

Changes in ovine maternal temperature, and serum cortisol and interleukin-6 concentrations after challenge with Escherichia coli lipopolysaccharide during pregnancy and early lactation.

Major changes in maternal physiology during pregnancy and lactation can have a large impact on the immune and neuroendocrine systems. One of the most significant changes, observed in rats and mice, is hyporesponsiveness of the hypothalamic pituitary adrenal axis (HPAA) in response to inflammation, restraint, and other psychological stressors during late pregnancy and lactation. This attenuation, however, has not been well characterized in ruminant animals and may be relevant to their susceptibility to inflammatory diseases during these periods. Thus, the intent of this study was to characterize responsiveness of the ovine HPAA to inflammatory challenge during pregnancy and lactation. Ewes from early (33 d), middle (55 d), and late (138 d) pregnancy, as well as early lactation (10 d), were challenged i.v. with a bolus dose of 400 ng of Escherichia coli lipopolysaccharide (LPS)/kg of BW or saline. A corresponding group of nonpregnant ewes was also challenged with LPS to serve as positive control animals for each pregnancy and lactation study. Responsiveness of the HPAA was assessed by measuring the 4-h change in serum cortisol concentration after LPS challenge. The cortisol increase after LPS challenge was elevated (P < 0.01) in pregnant ewes during late pregnancy over that of nonpregnant animals. In contrast, the characteristic temperature response associated with systemic LPS challenge was decreased (P < 0.01) during early pregnancy and lactation compared with nonpregnant or nonlactating animals. Serum IL-6 concentrations were measured to assess whether changes in HPAA responsiveness during pregnancy or lactation were attributed to changes in proinflammatory signaling to the HPAA. Interestingly, enhanced cortisol responsiveness during late pregnancy was correlated with increased (P < 0.01) serum IL-6 concentrations, indicating that IL-6 may contribute to enhanced HPAA responsiveness during this period. Serum IL-6 concentrations during early and midpregnancy did not increase in response to LPS challenge, indicating that HPAA activation during periods of pregnancy may be independent of IL-6 production.

Oral exposure to atrazine modulates cell-mediated immune function and decreases host resistance to the B16F10 tumor model in female B6C3F1 mice.

Atrazine (ATZ) is used throughout North America to control annual broadleaf weeds and grasses in various crops including; corn, sorghum, and sugar cane. Unfortunately, contamination of surface and ground water has occurred as a result of ATZ's chemical and physical properties, and its widespread use throughout the U.S. Midwest. A study of ATZ's immunomodulatory properties was conducted using female B6C3F1 mice and a panel of immune assays and host resistance models designed to evaluate cell-mediated and antibody-mediated immunity. Mice were administered ATZ by gavage (0, 24, 250, and 500 mg/kg/day) for 14 days then evaluated for immune responsiveness. ATZ treatment significantly increased the number of splenic CD8+ T cells, cytotoxic T cell and mixed leukocyte responses, and dose-dependently reduced host resistance to B16F10 melanoma. Thymus and spleen weights, total spleen cell numbers and fixed macrophage function was also reduced in mice that were exposed to ATZ. These results demonstrate that oral ATZ exposure is sufficient to alter cell-mediated immune function and disease resistance in female B6C3F1 mice.

Nonylphenol alters the activity of splenic NK cells and the numbers of leukocyte subpopulations in Sprague-Dawley rats: a two-generation feeding study.

Nonylphenol (NP) has been identified at low levels in surface waters throughout North America. This industrial chemical is primarily used for the production of certain non-ionic surfactants, and has been reported to have weak estrogen-like activity. As estrogen has immunoregulatory properties and is crucial for normal fetal development, it was hypothesized that adult and developmental exposures to NP had the potential to adversely affect the immune system. Furthermore, developmental exposure to NP might also produce differential immunomodulation in F(1) male and female rats. Thus, a two-generation feeding study was conducted to evaluate the potential for NP to modulate certain immune parameters. Pregnant female Sprague-Dawley rats were exposed to NP (0, 25, 500, and 2000 ppm) in their feed for 65 days, beginning 7 days into gestation. The F(1) generation male and female offspring were exposed in utero at the respective treatment levels, commencing the 7th day of gestation, and continuing through to 64 days of age. Changes in splenic antibody-forming cell response, natural killer cell activity, and leukocyte numbers were used to evaluate NP immunotoxicity. The results from the present study indicate that dietary exposure to NP can increase splenic natural killer (NK) cell activity and splenocyte subpopulation numbers in the F(1) generation rats, without similar changes to the F(0) generation. The immunological changes that were observed in the F(1) generation also appeared to be gender-specific.

Thalidomide modulation of the immune response in female B6C3F1 mice: a host resistance study.

Previously, we have reported that thalidomide (Thd) treatment can modulate the immune responses in female B6C3F1 mice. The present study was designed to evaluate whether or not these immunomodulatory responses were of sufficient magnitude to alter host resistances in a number of pathogen and tumor models. B6C3F1 mice were treated intraperitoneally with Thd (30-150 mg/kg) for 14 or 28 days, then inoculated with either Plasmodium yeolii, PYB6 fibrosarcoma tumor cells, B16F10 melanoma tumor cells, Listeria monocytogenes, or Streptococcus pneumoniae. Significant dose-dependent protection against B16F10 and L. monocytogenes was observed in mice that were treated with Thd. Furthermore, time course study using bacterial colony-forming units per spleen and liver as the endpoints indicated that the protective effect of Thd on host resistance to L. monocytogenes was time-dependent. In contrast, Thd treatment did not affect host resistance to P. yeolii, S. pneumoniae and PYB6 tumor. Additionally, the effect of Thd on the phagocytic function of the mononuclear phagocyte system (MPS) was evaluated following intravenous injection of 51Cr-labeled sRBCs. The overall phagocytic activity of MPS was not significantly altered by Thd treatment. In conclusion, these results demonstrate that Thd immunomodulation altered host resistance to B16F10 and L. monocytogenes; and selective modulation of Thd on the immune system may be responsible for the pathogen or tumor-specific effect of this compound.

Genistein modulates immune responses and increases host resistance to B16F10 tumor in adult female B6C3F1 mice.

The isoflavone genistein (4,7,4'-trihydroxyisoflavone) is a phytoestrogen found in high levels in soy products that has been associated with decreased incidences of breast and prostate cancers. The potential effects of genistein on the immune system were evaluated in adult female B6C3F1 mice. Groups of mice were exposed to vehicle or genistein by gavage for 28 d. The doses of genistein used were 2, 6 and 20 mg/kg body. Consistent with the chemopreventive effect of genistein, exposure to this compound significantly increased host resistance to B16F10 tumor as reflected by a decrease in the number of lung tumor nodules after tumor cell injection at the middle and high dose levels. Inhibition of B16F10 tumor formation was not due to a direct effect of serum genistein and/or its metabolites on the proliferation of B16F10 tumor cells. When innate and acquired immune responses were evaluated, a dose-related increase of cytotoxic T-cell activity was observed in genistein-treated mice with significant changes observed at the middle and high dose levels. Furthermore, in vitro interleukin (IL)-2-stimulated natural killer (NK) cell activity was significantly enhanced in the high genistein dose group, although the basal NK cell activity was not affected. Although no affect on the mixed lymphocyte responses and anti-CD3 antibody-mediated splenocyte proliferation was observed, exposure to genistein significantly increased basal splenocyte proliferation. Exposure to genistein did not alter the activity of the mononuclear phagocyte system and the cytotoxic/cytostatic function of thioglycollate-recruited peritoneal cells on B16F10 tumor cells. Finally, exposure to genistein did not produce biologically meaningful changes in spleen immunoglobulin (Ig)M and IgG antibody-forming cell responses. In conclusion, genistein enhanced host resistance as evaluated in the B16F10 tumor model, which may be related to the increases in the activities of cytotoxic T cells and NK cells.

Immunotoxicity of sodium bromate in female B6C3F1 mice: a 28-day drinking water study.

Bromate is one of the water disinfection by-products (DBPs) produced during the process of ozonation. The purpose of this study was to evaluate the immunotoxic potential of sodium bromate (SB) in female B6C3F1 mice. SB was administered in the drinking water for 28 days at doses of 80-800 mg/l. There was no difference in drinking water consumption between the animals exposed to SB and the tap water controls. Exposure to SB did not produce any signs of overt toxicity. Furthermore, no significant differences were observed in body weight, body weight gain, or the weights of thymus, liver, kidneys or lungs. No gross pathological lesions were observed in SB-treated animals. However, animals exposed to SB had a significant increase in absolute (28%) and relative (26%) spleen weights. The erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), platelet count, total leukocyte count, and counts of differential leukocytes were unaffected by SB. A dose-related increase in reticulocytes was observed following exposure to SB with the greatest increase (78%) observed at the highest dose level. Overall, there were no changes in the absolute number of total T cells, CD4+CD8- T cells, CD4-CD8+ T cells, natural killer (NK) cells and macrophages. Exposure to SB did not affect the percentage of B cells, although a slight increase in absolute number of B cells at the dose of 600 mg/l was observed. There was no alteration in IgM antibody-forming cell (AFC) response, mixed leukocyte reaction (MLR) and NK cell activity after exposure to SB. When the activity of peritoneal macrophages, unstimulated or stimulated with IFN-gamma and LPS, was evaluated using the cytotoxic/cytostatic assay of B16F10 tumor cells, the suppressive effect of macrophages on the proliferation of B16F10 tumor cells was decreased after exposure to SB. In conclusion, SB, when administered in the drinking water at doses from 80 mg/l to 800 mg/l, produced minimal toxicological and immunotoxic effects in female B6C3F1 mice.

Evaluation of the immunomodulatory effects of the macrolide antibiotic, clarithromycin, in female B6C3F1 mice: a 28-day oral gavage study.

The macrolide antibiotic, clarithromycin, is used extensively to treat bacterial infections associated with pneumonia, duodenal ulcers, and the advanced stages of human immunodeficiency viral (HIV) infection. In addition to its antimicrobial properties, several studies have indicated that clarithromycin also has anti-inflammatory and immunomodulatory properties. In this study, clarithromycin's immunomodulatory properties were evaluated using female B6C3F1 mice and a panel of immune assays that were designed to evaluate potential changes in innate, and acquired cellular and humoral immune responses. Female B6C3F1 mice were treated daily by gavage with clarithromycin (0, 125, 250, and 500 mg/kg) for 28 days then evaluated for immunomodulation. Minimal immunological changes were observed after 28 days of treatment. A slight increase in the number of spleen antibody-forming cells was observed at the 250 mg/kg treatment level, but not at other doses. Serum IgM levels were unaffected by the clarithromycin treatment. A significant increase in the number of splenic macrophages was also observed in mice treated with 125 mg/kg of clarithromycin, but this increase was not observed at the other treatment levels. Innate and cell-mediated immunity, as measured by natural killer cell activity, and mixed leukocyte and cytotoxic T cell response, respectively, were unchanged following treatment with clarithromycin. These results suggest that the immune system is not a target for clarithromycin at doses of 500 mg/kg or below.

Dermal exposure to cinnamaldehyde alters lymphocyte subpopulations, number of interferon-gamma-producing cells, and expression of B7 costimulatory molecules and cytokine messenger RNAs in auricular lymph nodes of B6C3F1 mice.

BACKGROUND: Although the Murine Local Lymph Node Assay (LLNA) is efficient in identifying chemicals with sensitizing potential, there is increasing need for alternative end points. Cinnamaldehyde (CIN) was chosen for evaluation based on its moderate potency and extensive use in fragrance materials. OBJECTIVES: The purpose of the present studies is to incorporate some alternative end points, such as phenotypic analysis and cytokine production, into a modified LLNA/irritancy assay (IA) to evaluate the sensitization of female B6C3F1 mice to CIN. METHODS: Several nontraditional end points, including the analysis of lymphocyte subpopulations, B7 costimulatory molecule and cytokine messenger RNA (mRNA) expression, and intracellular interferon-gamma (IFN-gamma) levels, were incorporated into a modified murine local lymph node (LLNA)/irritancy assay (IA) to evaluate the sensitization of female B6C3F1 mice to cinnamaldehyde (CIN). RESULTS: The alternate end points used in these studies support the classification of CIN as a moderately potent sensitizer. Dermal treatment with CIN resulted in an increase in the percentage of B cells in the auricular lymph nodes (ALNs) and expression of the costimulatory molecule, B7-2, on B cells. Lymph node cells also showed increased transforming growth factor-beta1, migration-inhibition factor, and mild increases in IFN-gamma and interleukin-2 cytokine mRNA expression. Although the increase in IFN-gamma mRNA expression did not translate into increased intracellular IFN-gamma levels, the absolute number of T cells producing IFN-gamma in the ALNs increased. Conversely, the MEST did not classify CIN as a contact allergen. CONCLUSION: The nontraditional end points used in the LLNA/IA were not as sensitive as the traditional radioisotope method used to assess cell proliferation. However, they may help identify compounds inappropriately classified as sensitizers or nonsensitizers by the LLNA and MEST.

Oxymetholone modulates cell-mediated immunity in male B6C3F1 mice.

Oxymetholone is a synthetic androgen, structurally related to testosterone. It is currently used to treat anemias, but has also been abused as a performance enhancing anabolic steroid by the sport community. Concern about its suspected immunomodulatory properties provided the incentive for a detailed investigation into its effects on the mammalian immune system. In this study, male B6C3F1 mice were treated for 14 d with oxymetholone (0, 50, 150, and 300 mg/kg) by gastric intubation, then evaluated for immunotoxicity using a panel of immunotoxicity assays. Except for an increasing trend in kidney and liver weights, and a dose-dependent increase in serum blood urea nitrogen levels, no other signs of systemic toxicity were observed. Bone marrow DNA synthesis was reduced, though this did not translate into alterations in myeloid or monocyte colony forming units. Spleen B and T cell numbers, antibody response to sheep red blood cells, proliferative response to both mitogen and immunoglobulin receptor immunogens, and NK cell activity were all unaltered in mice treated with oxymetholone. Peritoneal macrophage activity was also unaffected by oxymetholone treatment. A 38% decrease in the spleen cell mixed leukocyte response, and a 15% decrease in cytotoxic T cell activity, measured in the highest oxymetholone treatment group, indicate that cell-mediated immunity was impaired following exposure. This immunomodulation did not however, translate into a change in host resistance to Listeria monocytogenes.