LATE GESTATION MATERNAL IMMUNE FUNCTION AND CONCURRENT ESCHERICHIA COLI INTRAMAMMARY INFECTION DYNAMICS

Cows are susceptible to new Escherichia coli intramammary infections (IMI) in the non-lactating period of late gestation (known as the ‘dry period’). These IMI often persist up until parturition without inducing highly inflammatory responses and increase the risk of postpartum mastitis in the subsequent lactation. The bovine maternal immune system is hypothesized to be regulated during late gestation to prevent highly inflammatory, cell-mediated responses however the mechanisms in generating such tolerance have not been fully elucidated. In other mammalian species mononuclear phagocytes play a primary role in generating maternal immune tolerance, and although beneficial for the fetus, often negatively impacts immune responses to invading pathogens. The work presented here investigated changes in maternal immunity and its relationship to dry period E. coli IMI dynamics through the use of experimental and mathematical approaches. The objectives were to 1) characterize changes in blood monocyte composition and monocyte-derived dendritic cell (moDC) function over pregnancy to identify unique changes in late gestation, 2) investigate pregnancy-associated factors that regulate these cells, 3) investigate the relationship between maternal immune regulation and dry period E. coli IMI dynamics and risk of postpartum mastitis through mathematical models, and 4) investigate the effect of intramammary immunization with UV-killed E. coli on IMI dynamics and host response. Pregnancy was accompanied with a decrease in inflammatory monocytes and impaired moDC maturation following E. coli stimulation. Aspects of hindered moDC maturation could be induced by in vitro treatment of late gestation levels of progesterone and estradiol. Deviations in prepartum monocyte composition related to risk of postpartum disease. Mathematical model results indicated these shifts in cytokine production alone were not able to recapitulate IMI dynamics in the dry period, but rather it involved an interaction between maternal immune regulation and physiological and immunological changes to the mammary gland that accompany the dry period. Intramammary immunization at dry-off generated protective responses against E. coli challenge later in the dry period. Overall, shifts in maternal immunity both in the periphery and in the mammary gland during the dry period relate to persistent E. coli IMI and postpartum mastitis, and local immunity can be manipulated to generate protection against IMI in the dry period

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

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

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

Counts of bovine monocyte subsets prior to calving are predictive for postpartum occurrence of mastitis and metritis

The heightened susceptibility to infectious diseases in postpartum dairy cows is often attributed to immune dysfunction associated with the transition period. However, the cell populations involved in this immune dysfunction and the dynamics between those populations are not well defined. Monocytes play a crucial role in governing initial immune response in bacterial infections. Bovine monocytes are subdivided in classical (CD14+/CD16−), intermediate (CD14+/CD16+) and non-classical monocytes (CD14−/CD16+) with distinct phenotypic and functional differences. This study investigated the relationship of monocyte subsets counts in blood at 42 and 14 days prior to expected calving date to occurrence of metritis and mastitis within 2 weeks postpartum. In the enrolled prospective cohort of 27 German Holstein cows, housed at the Institute of Animal Nutrition of the Friedrich-Loeffler-Institute Braunschweig, Germany, n = 13 developed metritis and/or mastitis postpartum. A multivariable logistic regression was used to analyze the relationship between prepartum cell counts of monocyte subsets and neutrophils with postpartum disease. Our model revealed that higher counts of the two CD14+ monocyte subsets were predictive of disease. In contrast, higher numbers of the CD14− monocyte subset were negatively associated with disease. Interestingly, the neutrophil count, a common hallmark for inflammatory response, was not associated with the outcome variable at either time point. The results indicate that the number and composition of monocyte subsets before calving are related to the susceptibility to infectious disease within 2 weeks postpartum. Furthermore the oppositional effect of CD14+ and CD14− subsets strengthens the hypothesis that these subsets have different functional roles in the inflammatory response in dairy cows

Counts of bovine monocyte subsets prior to calving are predictive for postpartum occurrence of mastitis and metritis

The heightened susceptibility to infectious diseases in postpartum dairy cows is often attributed to immune dysfunction associated with the transition period. However, the cell populations involved in this immune dysfunction and the dynamics between those populations are not well defined. Monocytes play a crucial role in governing initial immune response in bacterial infections. Bovine monocytes are subdivided in classical (CD14+/CD16−), intermediate (CD14+/CD16+) and non-classical monocytes (CD14−/CD16+) with distinct phenotypic and functional differences. This study investigated the relationship of monocyte subsets counts in blood at 42 and 14 days prior to expected calving date to occurrence of metritis and mastitis within 2 weeks postpartum. In the enrolled prospective cohort of 27 German Holstein cows, housed at the Institute of Animal Nutrition of the Friedrich-Loeffler-Institute Braunschweig, Germany, n = 13 developed metritis and/or mastitis postpartum. A multivariable logistic regression was used to analyze the relationship between prepartum cell counts of monocyte subsets and neutrophils with postpartum disease. Our model revealed that higher counts of the two CD14+ monocyte subsets were predictive of disease. In contrast, higher numbers of the CD14− monocyte subset were negatively associated with disease. Interestingly, the neutrophil count, a common hallmark for inflammatory response, was not associated with the outcome variable at either time point. The results indicate that the number and composition of monocyte subsets before calving are related to the susceptibility to infectious disease within 2 weeks postpartum. Furthermore the oppositional effect of CD14+ and CD14− subsets strengthens the hypothesis that these subsets have different functional roles in the inflammatory response in dairy cows

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