The Potential of Metalloproteinase-9 Administration to Accelerate Mammary Involution and Boost the Immune System at Dry-Off

The dry period is decisive for the milking performance of dairy cows. The promptness of mammary gland involution at dry-off affects not only the productivity in the next lactation, but also the risk of new intra-mammary infections since it is closely related with the activity of the immune system. Matrix metalloproteinase-9 (MMP-9) is an enzyme present in the mammary gland and has an active role during involution by disrupting the extracellular matrix, mediating cell survival and the recruitment of immune cells. The objective of this study was to determine the potential of exogenous administration of a soluble and recombinant version of a truncated MMP-9 (rtMMP-9) to accelerate mammary involution and boost the immune system at dry-off, avoiding the use of antibiotics. Twelve Holstein cows were dried abruptly, and two quarters of each cow received an intra-mammary infusion of either soluble rtMMP-9 or a positive control based on immunostimulant inclusion bodies (IBs). The contralateral quarters were infused with saline solution as negative control. Samples of mammary secretion were collected during the week following dry-off to determine SCC, metalloproteinase activity, bovine serum albumin, lactoferrin, sodium, and potassium concentrations. The soluble form of rtMMP-9 increased endogenous metalloproteinase activity in the mammary gland compared with saline quarters but did not accelerate either the immune response or involution in comparison with control quarters. The results demonstrated that the strategy to increase the mammary gland immunocompetence by recombinant infusion of rtMMP-9 was unsuccessful.

Estrogen markedly reduces circulating low-density neutrophils and enhances pro-tumoral gene expression in neutrophil of tumour-bearing mice

Background Neutrophils are important for immune surveillance of tumour cells. Neutrophils may also be epigenetically programmed in the tumour microenvironment to promote tumour progression. In addition to the commonly known high-density neutrophils (HDN) based on their separation on density gradient, recent studies have reported the presence of high levels of low-density neutrophils (LDN) in tumour-bearing mice and cancer patients. We reported previously that estrogen promotes the growth of estrogen receptor α-negative mammary tumours in mice undergoing mammary involution through stimulating pro-tumoral activities of neutrophils in the mammary tissue. Methods Female BALB/cAnNTac mice at 7–8 weeks old were mated and bilateral ovariectomy was performed 2 days post-partum. At 24 h after forced-weaning of pups to induce mammary involution, post-partum female mice were injected with either E2V, or vehicle control on alternative days for 2-weeks. On 48 h post-weaning, treated female mice were inoculated subcutaneously with 4 T1-Luc2 cells into the 9th abdominal mammary gland. Age-matched nulliparous female was treated similarly. Animals were euthanized on day 14 post-tumour inoculation for analysis. To evaluate the short-term effect of estrogen, post-partum females were treated with only one dose of E2V on day 12 post-tumour inoculation. Results Estrogen treatment for 2-weeks reduces the number of blood LDN by more than 10-fold in tumour-bearing nulliparous and involuting mice, whilst it had no significant effect on blood HDN. The effect on tumour-bearing mice is associated with reduced number of mitotic neutrophils in the bone marrow and increased apoptosis in blood neutrophils. Since estrogen enhanced tumour growth in involuting mice, but not in nulliparous mice, we assessed the effect of estrogen on the gene expression associated with pro-tumoral activities of neutrophils. Whilst 48 h treatment with estrogen had no effect, 2-weeks treatment significantly increased the expression of Arg1, Il1b and Tgfb1 in both HDN and LDN of involuting mice. In contrast, estrogen increased the expression of Arg1 and Ccl5 in HDN and LDN of nulliparous mice. Conclusions Prolonged estrogenic stimulation in tumour-bearing mice markedly hampered tumour-associated increase of LDN plausibly by inhibiting their output from the bone marrow and by shortening their life span. Estrogen also alters the gene expression in neutrophils that is not seen in tumour-free mice. The results imply that estrogen may significantly influence the tumour-modulating activity of blood neutrophils.

Comparison of Bovine Mammary Involution and Intramammary Infections Following Intramammary Treatment with Casein Hydrolysate and Other Conventional Treatments at Dry-Off

Alternatives to routine antibiotic treatment of dairy cattle during the dry period before their next calving are of interest. This was a preliminary study of whether intramammary infusion of casein hydrolysate, administered alone or combined with standard dry treatment, accelerated the rate of mammary involution early in the dry period. Four treatments were studied in a split udder design. One udder half was assigned a treatment, and the contralateral half was administered dry cow treatment + internal teat sealant as a control. Treatments were casein hydrolysate, casein hydrolysate + dry cow treatment, casein hydrolysate + teat sealant and casein hydrolysate + dry cow treatment + teat sealant. Cows (n = 16) were blocked by a number of intramammary infections per udder half (0 or 1+) and randomized to treatments. Milk production was not different between control or treated udder halves post-calving. A generalized linear mixed model tested for differences between the treatment groups in the concentration of mammary involution indicators in milk: somatic cell count, bovine lactoferrin and bovine serum albumin. At 7 days, dry udder halves treated with casein hydrolysate had higher milk concentrations of lactoferrin than those treated with casein hydrolysate + dry cow treatment, casein hydrolysate + teat sealant and control. At 10 days dry, bovine serum albumin was higher in udder halves treated with casein hydrolysate than in those treated with casein hydrolysate + dry cow treatment, casein hydrolysate + dry cow treatment + teat sealant and control. Post-calving, casein hydrolysate-treated udder halves produced 51% of total milk, unchanged from before dry-off. There were seven total intramammary infections entering the dry period, all caused by coagulase-negative staphylococci. Cure rates (3/7, 43%) were not different among all treatments and control, partly because of the small sample size. Intramammary infusion of casein hydrolysate at the end of lactation may be an alternative or possible adjunct to antibiotic dry cow therapy.

Estrogen exacerbates mammary involution through neutrophil-dependent and -independent mechanism

There is strong evidence that the pro-inflammatory microenvironment during post-partum mammary involution promotes parity-associated breast cancer. Estrogen exposure during mammary involution drives tumor growth through neutrophils’ activity. However, how estrogen and neutrophils influence mammary involution are unknown. Combined analysis of transcriptomic, protein, and immunohistochemical data in BALB/c mice showed that estrogen promotes involution by exacerbating inflammation, cell death and adipocytes repopulation. Remarkably, 88% of estrogen-regulated genes in mammary tissue were mediated through neutrophils, which were recruited through estrogen-induced CXCR2 signalling in an autocrine fashion. While neutrophils mediate estrogen-induced inflammation and adipocytes repopulation, estrogen-induced mammary cell death was via lysosome-mediated programmed cell death through upregulation of cathepsin B, Tnf and Bid in a neutrophil-independent manner. Notably, these multifaceted effects of estrogen are mostly mediated by ERα and unique to the phase of mammary involution. These findings are important for the development of intervention strategies for parity-associated breast cancer.

Estrogen exacerbates mammary involution through neutrophil dependent and independent mechanism

There is strong evidence that the pro-inflammatory microenvironment during post-partum mammary involution promotes parity-associated breast cancer. Estrogen exposure during mammary involution drives tumour growth through the activity of neutrophils. However, how estrogen and neutrophils influence mammary involution are unknown. Combined analysis of transcriptomic, protein, and immunohistochemical data in Balb/c mice with and without neutrophil depletion showed that estrogen promotes involution by exacerbating inflammation, cell death and adipocytes repopulation through neutrophil-dependent and neutrophil-independent mechanisms. Remarkably, 88% of estrogen-regulated genes in mammary tissue were mediated through neutrophils, which were recruited through estrogen-induced CXCL2-CXCR2 signalling. While neutrophils mediate estrogen-induced inflammation and adipocytes repopulation, estrogen-induced mammary cell death was mediated by neutrophils-independent upsurges of cathepsins and their lysosomal leakages that are critical for lysosome-mediated cell death. Notably, these multifaceted effects of estrogen are unique to the phase of mammary involution. These findings are important for the development of intervention strategies for parity-associated breast cancer.