Mammary gland involution comprises the remodelling of the gland at the end of the lactation period. In humans, mastitis may potentially be associated with weaning particularly if the process is insufficiently gradual and the breast becomes engorged, with the pathogenesis potentially similar to that described to account for the elevated Na/K ratio used as a diagnostic modality in subclinical mastitis.
In ruminants, the start of involution, or the dry period, is well-recognised as a time when there is an increased likelihood of acquisition of new mammary infections, especially in cows with high milk yields prior to drying off [
103]. ‘Summer mastitis’ is mastitis of dairy cows occurring during the summer months and therefore, in traditional systems, is associated with the dry period. This condition is usually caused by mixed bacterial species including
Trueperella pyogenes and
Streptococcus dysgalactiae. The route of infection is thought to be the papillary ostium and duct, and flies attracted to pre-existing teat lesions are implicated in the pathogenesis of the disease. In traditional systems, some of the affected animals at pasture would be dry, whilst others might be immature heifers [
21].
Given the dramatic molecular and structural changes which occur in the mammary gland during post-lactational remodelling, we suggest that when considering episodes of dry period- or involution-associated mastitis, it may be informative to consider changes occurring in the mammary microenvironment during involution and to speculate on how these changes may influence susceptibility of the gland to mastitis-causing pathogens at this time. We will adopt this approach in the following section.
Species Differences in the Mammary Microenvironment During Involution
Excitingly, a recent study used magnetic resonance imaging to describe changes seen in the breast during the first year post-weaning, and the investigators noted that the gland returns to a state similar to that observed pre-conception. Both breast area and fibroglandular fraction decreased significantly between women in the lactation and post weaning periods and measurements for the latter group were comparable to those of a premenopausal control group [
36]. Such a striking degree of glandular remodelling is the result of highly regulated, interconnecting, networks of cellular signals which orchestrate the involution process [
106]. Set at the hub of this molecular system is the transcription factor Signal Transducer and Activation of Transcription 3 (STAT3) [
107,
108]. STAT3 activation is fundamental to the normal progression of involution [
60,
109‐
111].
Mammary gland involution is considered to progress in two distinct phases, which have been well-defined in the mouse. The first reversible phase is proteinase independent and is characterised by dramatic mammary epithelial cell death co-ordinated by Stat3 [
60,
109‐
111]. Mice with a mammary-specific conditional deletion of Stat3 exhibit a pronounced retardation of involution [
109,
110]. Unilateral teat sealing, in which mice have sealant unilaterally applied to the inguinal mammary gland teat, such that pups sucking the contralateral open gland provide a continued systemic suckling stimulus, has demonstrated that local factors, attributed to milk accumulation, are sufficient to induce phosphorylation of Stat3 and consequent cell death at the onset of involution [
112]. Leukemia inhibitory factor (LIF) exhibits a rapid increase in expression, which is independent of systemic factors, and is accordingly observed even in teat-sealed glands [
113]. LIF deficient mice exhibit delayed mammary regression and absence of Stat3 activation during involution [
114], demonstrating that the initial activator for Stat3 in the mammary gland is LIF, and that the up-regulation of pStat3 at the onset of involution is independent of the decrease in circulating lactogenic hormones seen after weaning. However, those glands which are sealed with the contralateral gland left open (thus maintaining systemic hormone stimulation) do not progress to the second phase of involution [
112]. Thus exogenous administration of hydrocortisone, or systemic factors such as endogenous glucocorticoid release, can inhibit progression to the second phase [
115].
When progression of involution is unimpeded, the second phase is accompanied by irreversible degradation of the mammary basement membrane, coinciding with expression, by fibroblasts and other mesenchymal components, of the matrix metalloproteinases (MMPs) MMP2 (gelatinase A), and MMP3 (stromelysin 1), the serine proteinase urokinase-type plasminogen activator [
115], and MMP9 [
53].
During the early phase of involution in mice, there is dramatic up-regulation of genes associated with the acute phase response and innate immunity, including serum amyloid A3 [
52]. Stat3 regulates expression of a subset of these genes, including orosomucoids 1 and 2, secretory leukocyte protease inhibitor, CD14 and leucine-rich α2-glycoprotein 1 [
53].
The later stages of involution are characterised by the mammary microenvironment acquiring an immunomodulatory ‘wound healing’ phenotype [
116‐
118], which we have also demonstrated is dependent on Stat3 [
53]. Factors implicated in acquisition of a ‘wound healing’ phenotype include deposition of fibrillar collagen, high levels of COX-2 expression, itself promoting lymphangiogenesis, and mammary epithelial cell efferocytosis [
58,
119‐
121]. There is an influx of immune cells during the second phase of involution, including mast cells, lymphocytes, and predominantly alternatively activated macrophages [
52,
53,
116,
122‐
124], and postlactational human breast tissue exhibits a transient infiltrate of high IL-10 (+) macrophages and Foxp3 (+) regulatory T cells [
124]. It is easy to speculate that such an immunomodulatory and ‘wound healing’ microenvironment may favour proliferation of bacteria during involution-associated mastitis, particularly when coupled with the presence of milk deposits, which may provide a nidus for bacterial infections.
Intriguingly, Stat3 also regulates expression of members of the chloride channel regulators, calcium activated, (CLCA) family, of proteins during involution. A positive association is observed between murine CLCA1 and CLCA2 and Stat3 activity, whilst Stat3 negatively regulates murine CLCA5, the murine orthologue of human CLCA2 [
125]. The exact functions of CLCA family members within the mammary gland are yet to be determined, but their regulation by Stat3 during the involution period may be pertinent to the pathogenesis of involution-associated mastitis given their postulated modulation of the innate immune response and/or potential activity as signalling molecules [
126,
127].
It is important to note that cattle are usually in the final trimester of pregnancy during the dry period, and some dairy goats may be pregnant, depending on the production system [
128]. Thus, the involution process may be markedly modulated by what we will term a ‘parallel pregnancy signature’. Although some bovine mammary epithelial cells undergo cell death during bovine mammary involution [
129], tissue regression is not notable [
130]. This particular aspect of bovine involution is therefore an important species difference which needs to be carefully considered when adopting a One Health viewpoint before making inter-species comparisons.
In spite of the differences in progression of involution in cattle, high levels of serum amyloid A3 expression are also observed in bovine mammary epithelial cells during mid to late involution and in inflammatory states [
131] indicating that the inflammatory profile of the bovine involution mammary gland may be similar to rodent models. Experiments in which serum amyloid A3 was infused into the mammary gland via the teat canal suggest that serum amyloid A3 may enhance MMP9 activity and may also reduce
Staphylococcus aureus infection [
132].
Abrupt cessation of lactation in sheep heralds a transient increase in gland cistern volume as measured ultrasonographically. Interestingly, approximately one week after weaning, milk within the gland cistern exhibits ultrasonographic evidence of clotting and is interpreted to be gradually resorbed, resulting in a reduction in gland cistern volume [
133]. Again, it is possible that accumulation of milk within the gland cistern may represent a potential nidus for infection, particularly if there is compromise to the innate immune defences of the teat canal such as through mechanical injury. Similar to murine models, the ovine mammary gland also exhibits involution-associated mammary epithelial cell death, efferocytosis mediated by macrophages and mammary epithelial cells, and ultrasonographic evidence of matrix remodelling [
59,
133].