Impact of in vitro treatments of physiological levels of estradiol and progesterone observed in pregnancy on bovine monocyte-derived dendritic cell differentiation and maturation

The specific factors which regulate differentiation and maturation of dendritic cells in bovine pregnancy remain unclear. We evaluated the influence of physiologically relevant in vitro treatments of progesterone (PG) and estradiol (E2) observed in late pregnancy on the differentiation and maturation of CD14+ monocyte-derived dendritic cell (moDC) from non-pregnant, lactating dairy cows (n = 7). We found that moDC differentiated in the presence of both E2 and PG had impaired E. coli-induced phenotypic maturation, specifically a significant reduction in CD80 and MHC II expression. Contrary to our previous work characterizing moDC from late gestating dairy cattle, we did not observe an increase in CD14 expression relative to the untreated control; this increase was only observed in the current data in the dexamethasone-treated moDC. The moDC treated with a combination of both E2 and PG had significantly greater upregulation of anti-inflammatory cytokine IL-10 relative to the untreated control, but TNFα production was not suppressed; only dexamethasone-treated moDC showed abrogated TNFα production. These data suggest moDC may be regulated by E2 and PG to hinder phenotypic maturation and regulate inflammatory responses. Pregnancy-associated hormone profiles appear to be involved in the generation of maternal immune tolerance in pregnancy. These hormone-facilitated changes to moDC in pregnancy may also impede optimal immune responses to both invading pathogens and routine vaccinations administered in late gestation through limited antigen presentation and increased anti-inflammatory cytokine production. These results provide insight into maternal immune modulation and elucidate potential immune changes necessary to facilitate bovine pregnancy

Longitudinal characterization of bovine monocyte-derived dendritic cells from mid-gestation into subsequent lactation reveals nadir in phenotypic maturation and macrophage-like cytokine profile in late gestation

Changes in monocyte and dendritic cell populations during bovine pregnancy and lactation remain poorly described despite the key roles these cells play in immune tolerance and activation. Using a prospective longitudinal study, we characterized CD14+ monocyte-derived dendritic cell (moDC) differentiation and maturation and captured monocyte composition dynamics from mid-gestation through calving and into the subsequent lactation in dairy cows (n = 7). First, we measured absolute counts of classical (CD14 + CD16−, cM), intermediate (CD14 + CD16+, intM), and nonclassical (CD14-CD16+, ncM) monocytes in the blood and determined proportions of individual subsets within the total monocyte population. We found the proportion of cM decreased and intM increased significantly by early lactation, whereas there was a nadir in the proportion of ncM in late gestation, two weeks prepartum. Monocyte composition appears to be regulated in pregnancy, possibly to limit the proportion of highly inflammatory monocytes i.e. intM. Ultimately, we found that moDC differentiated from CD14+ monocytes isolated in the early dry period of late gestation had impaired E. coli-induced maturation, with nadirs in upregulation of CD80 and MHC II, and downregulation of CD14. The moDC from late gestation also had altered cytokine profiles with greatest production of pro-inflammatory IL-1β and anti-inflammatory IL-10. These data suggest monocytes in late gestation, in contrast to other stages of pregnancy and lactation, differentiate and maturate into moDC less capable of eliciting strong T cell activation, and have macrophage-like cytokine profiles. These results provide insight into maternal immune modulation and elucidate potential immune changes necessary to facilitate bovine pregnancy

Longitudinal characterization of bovine monocyte-derived dendritic cells from mid-gestation into subsequent lactation reveals nadir in phenotypic maturation and macrophage-like cytokine profile in late gestation

Changes in monocyte and dendritic cell populations during bovine pregnancy and lactation remain poorly described despite the key roles these cells play in immune tolerance and activation. Using a prospective longitudinal study, we characterized CD14+ monocyte-derived dendritic cell (moDC) differentiation and maturation and captured monocyte composition dynamics from mid-gestation through calving and into the subsequent lactation in dairy cows (n = 7). First, we measured absolute counts of classical (CD14 + CD16−, cM), intermediate (CD14 + CD16+, intM), and nonclassical (CD14-CD16+, ncM) monocytes in the blood and determined proportions of individual subsets within the total monocyte population. We found the proportion of cM decreased and intM increased significantly by early lactation, whereas there was a nadir in the proportion of ncM in late gestation, two weeks prepartum. Monocyte composition appears to be regulated in pregnancy, possibly to limit the proportion of highly inflammatory monocytes i.e. intM. Ultimately, we found that moDC differentiated from CD14+ monocytes isolated in the early dry period of late gestation had impaired E. coli-induced maturation, with nadirs in upregulation of CD80 and MHC II, and downregulation of CD14. The moDC from late gestation also had altered cytokine profiles with greatest production of pro-inflammatory IL-1β and anti-inflammatory IL-10. These data suggest monocytes in late gestation, in contrast to other stages of pregnancy and lactation, differentiate and maturate into moDC less capable of eliciting strong T cell activation, and have macrophage-like cytokine profiles. These results provide insight into maternal immune modulation and elucidate potential immune changes necessary to facilitate bovine pregnancy

Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46+/CD3−) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2 h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2 h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis

Longitudinal characterization of bovine monocyte-derived dendritic cells from mid-gestation into subsequent lactation reveals nadir in phenotypic maturation and macrophage-like cytokine profile in late gestation

Changes in monocyte and dendritic cell populations during bovine pregnancy and lactation remain poorly described despite the key roles these cells play in immune tolerance and activation. Using a prospective longitudinal study, we characterized CD14+ monocyte-derived dendritic cell (moDC) differentiation and maturation and captured monocyte composition dynamics from mid-gestation through calving and into the subsequent lactation in dairy cows (n = 7). First, we measured absolute counts of classical (CD14 + CD16−, cM), intermediate (CD14 + CD16+, intM), and nonclassical (CD14-CD16+, ncM) monocytes in the blood and determined proportions of individual subsets within the total monocyte population. We found the proportion of cM decreased and intM increased significantly by early lactation, whereas there was a nadir in the proportion of ncM in late gestation, two weeks prepartum. Monocyte composition appears to be regulated in pregnancy, possibly to limit the proportion of highly inflammatory monocytes i.e. intM. Ultimately, we found that moDC differentiated from CD14+ monocytes isolated in the early dry period of late gestation had impaired E. coli-induced maturation, with nadirs in upregulation of CD80 and MHC II, and downregulation of CD14. The moDC from late gestation also had altered cytokine profiles with greatest production of pro-inflammatory IL-1β and anti-inflammatory IL-10. These data suggest monocytes in late gestation, in contrast to other stages of pregnancy and lactation, differentiate and maturate into moDC less capable of eliciting strong T cell activation, and have macrophage-like cytokine profiles. These results provide insight into maternal immune modulation and elucidate potential immune changes necessary to facilitate bovine pregnancy

Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46+/CD3−) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2 h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2 h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis

Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46+/CD3−) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2 h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2 h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis

Impact of intramammary treatment on gene expression profiles in bovine Escherichia coli mastitis.

Clinical mastitis caused by E. coli accounts for significant production losses and animal welfare concerns on dairy farms worldwide. The benefits of therapeutic intervention in mild to moderate cases are incompletely understood. We investigated the effect of intramammary treatment with cefapirin alone or in combination with prednisolone on gene expression profiles in experimentally-induced E. coli mastitis in six mid-lactating Holstein Friesian cows. Cows were challenged with E. coli in 3 quarters and received 4 doses of 300 mg cefapirin in one quarter and 4 doses of 300 mg cefapirin together with 20 mg prednisolone in another quarter. At 24 h (n = 3) or 48 h (n = 3) post-challenge, tissue samples from control and treated quarters were collected for microarray analysis. Gene expression analysis of challenged, un-treated quarters revealed an up-regulation of transcripts associated with immune response functions compared to un-challenged quarters. Both treatments resulted in down-regulation of these transcripts compared to challenged, un-treated quarters most prominently for genes representing Chemokine and TLR-signaling pathways. Gene expression of Lipopolysaccharide Binding Protein (LBP), CCL2 and CXCL2 were only significantly down-regulated in cefapirin-prednisolone-treated quarters compared to un-treated controls. Down-regulation of chemokines was further confirmed on the basis of protein levels in milk whey for CXCL1, CXCL2 and CXCL8 in both treatments with a greater decrease in cefapirin-prednisolone-treated quarters. The data reveal a significant effect of treatment on cell recruitment with a more pronounced effect in cefapirin-prednisolone treated quarters. Provided a rapid bacteriological clearance, combination therapy may prevent neutrophil-induced tissue damage and promote recovery of the gland

Levels of chemokines in milk whey.

<p>A) Levels (pg/ml) of chemokines (CXCL1, CXCL2 and CXCL8) in milk whey were determined by ELISA at time of (0), 6, 12, 18, 24 (n = 6) and 48 h (n = 3) post challenge with 100 cfu <i>E. coli</i>. Per cow one quarter was left unchallenged (open circles) and one quarter was challenged but not treated (filled circles), one quarter was challenged and cefapirin treated (open triangles) and one quarter was challenged and treated with cefapirin plus prednisolone (filled squares) at 4, 12 and 24 h post challenge. Data are shown as LSM of chemokine levels plus standard errors. In the case of CXCL8 LSM of the log10 transformed levels are shown. Statistically significant differences (P<0.05) are indicated by <b>*</b> (significantly different to all other groups), <b>†</b> (significantly different to not infected, not treated) and <b>‡</b> (significantly different to infected, cefapirin treated). B) Example of H&E stained sections from four quarters of one animal at 24 h post challenge. Arrows indicate PMN in tissue.</p