Immune defenses of the mammary gland epithelium of dairy ruminants

Abstract

The epithelium of the mammary gland (MG) fulfills three major functions: nutrition of progeny, transfer of immunity from mother to newborn, and its own defense against infection. The defense function of the epithelium requires the cooperation of mammary epithelial cells (MECs) with intraepithelial leucocytes, macrophages, DCs, and resident lymphocytes. The MG is characterized by the secretion of a large amount of a nutrient liquid in which certain bacteria can proliferate and reach a considerable bacterial load, which has conditioned how the udder reacts against bacterial invasions. This review presents how the mammary epithelium perceives bacteria, and how it responds to the main bacterial genera associated with mastitis. MECs are able to detect the presence of actively multiplying bacteria in the lumen of the gland: they express pattern recognition receptors (PRRs) that recognize microbe-associated molecular patterns (MAMPs) released by the growing bacteria. Interactions with intraepithelial leucocytes fine-tune MECs responses. Following the onset of inflammation, new interactions are established with lymphocytes and neutrophils recruited from the blood. The mammary epithelium also identifies and responds to antigens, which supposes an antigen-presenting capacity. Its responses can be manipulated with drugs, plant extracts, probiotics, and immune modifiers, in order to increase its defense capacities or reduce the damage related to inflammation. Numerous studies have established that the mammary epithelium is a genuine effector of both innate and adaptive immunity. However, knowledge gaps remain and newly available tools offer the prospect of exciting research to unravel and exploit the multiple capacities of this particular epithelium.

Keywords: MAMPs; PRR; bacteria; epithelial cells; innate immunity; macrophages; mastitis.

Figure 1

Histological organization of the mammary epithelium. The teat and gland cisterns and large lactiferous ducts are lined with a double layer of non-secretory epithelial cells resting on a basement membrane. Quite a few ductal macrophages lie between the luminal and basal layers of epithelial cells, extending dendrites that contact multiple epithelial cells and can access the lumen. Small lactiferous ducts and acini are lined with a single layer of epithelial cells. This layer is sheathed by a network of myoepithelial cells. Ductal macrophages are also present in the lobular zone, in close contact with luminal epithelial cells. The representation of ductal macrophages dendrites accessing the lumen is speculative only.

Figure 2

Schematic view of the potential capacity of MECs to sense and react to bacteria that invade the MG lumen. At the apical side of the cell, the plasma membrane exposes Toll-like receptors (TLR1, TLR2, TLR6, TLR4) that pair to interact with bacterial lipoproteins and LPS. The TLRs receive help from accessory molecules such as CD36 and MD2. TLR5 has not been documented in relation to MECs. MECs are devoid of membrane CD14, but milk provides soluble CD14 (sCD14). This allows TLR4 to be internalized upon ligation to smooth LPS in endosomes from where the TRAM-TRIF adaptors can be recruited to activate TRAF3. TLR3 from endosomes can also activate TRAF3. The TRIF-dependent signaling pathway induces the translocation of IRF3, resulting in the induction of type 1 IFNs and IFN-inducible genes. Contrary to TLR3, the other TLRs depend on the adaptor molecule Myd88. This triggers a cascade of activating steps involving TRAF6 and allows NF-κB to translocate into the nucleus and activate the transcription of cytokine genes. Another activation pathway mobilizes the MAP kinase cascade and leads to the activation of the transcription factor AP-1, critical in the activation of cytokine genes. MECs can respond to degradation products of bacterial cell wall peptidoglycan via the cytosolic sensors NOD1 and NOD2. The oligomerization of these sensors induces the recruitment of the adaptor protein RIP2, followed by the activation of the NF-κB pathway (93). A number of stimuli induce the formation of molecular platforms called inflammasomes. The NLRP3 inflammasome recruits Caspase 1 that can cleave pro-IL-1β molecules and contribute to the secretion of the mature pro-inflammatory cytokine IL-1β. TRIF, Toll/IL-1 receptor (TIR) domain-containing adaptor protein inducing IFNβ; TRAM, TRIF-related adaptor molecule; IRF3, IFN-regulatory factor 3; Myd88, myeloid differentiation primary-response protein 88; NOD, nucleotide-binding oligomerisation domain; RIP2, Receptor-interacting-serine/threonine-protein kinase 2.

Figure 3

The mammary epithelium reacts differently to different pathogens. Gram-negative bacteria such as E. coli are essentially perceived through the lipid A moiety of LPS (endotoxin), and in combination with lipopeptides they activate the transcription factors NF-κB, AP-1, and TRF3. This leads to the expression of a large number of genes. In particular, there is a high production of chemokines and antimicrobial peptides (AMPs) by MECs, and the pro-inflammatory cytokines TNF-α and IL-1β by macrophages (brown cells). Together with IL-6 which can be produced by MECs, this results in an amplification of the self-defence of MECs and recruits a high number of circulating leucocytes, while strongly reducing their milk secretory activity. Gram-positive bacteria, which lack endotoxin, induce a comparatively lesser reaction, especially from MECs. Staphylococcus aureus is perceived through lipopeptides, lipoteichoic acid, protein A, α-toxin and other components and metabolites. Despite the variety of agonists, the production of IL-1β, and especially of TNF-α, is much lower than in the case of E. coli, presumably in relation to a much weaker activation of NF-κB. Nevertheless, the AP-1 pathway works, and IL-6 is produced. As a result, recruitment of leucocytes by chemokines takes place, but activation of self-defence mechanisms is limited. The case of Streptococcus uberis remains somewhat mysterious since the MECs do not seem to detect them. The late but sometimes intense reaction that these pathogens induce in the udder could result from their phagocytosis by ductal macrophages, which remains to be established.

Figure 4

Interactions of MECs with leucocytes. The main communication activity of MECs with leucocytes is mediated by the secretion of chemokines that target preferentially either neutrophils, mononuclear cells (dendritic cells and macrophages), or lymphocytes. In this way, they fulfill an important alerting role. In turn, leucocytes release pro-inflammatory (TNF-α, IL-1β) or modulatory (IL-17, IFN-γ) cytokines that modify the response of MECs to infection. Epithelial cells may also modulate leucocyte activity via the release of local molecular cues such as serum amyloid A3 (SAA3) or TGF-β.

Figure 5

Manipulating the reactivity of the mammary epithelium. Upon contact with bacteria and bacterial products (MAMPs, metabolites), the mammary epithelium reacts according to the intensity of the stimulus and its degree of alertness (reactivity). A high reactivity will lead to a strong inflammatory reaction characterized by a large influx of neutrophils, to the detriment of the secretory function of MECs but with the advantage of killing the bacteria. A weak reactivity will lead to a reaction of less intensity, recruiting fewer neutrophils and sparing the secretion of milk components (represented here by fat globules and casein micelles). As a result, bacteria can proliferate in milk. In principle, it is possible to manipulate the alertness and reactivity of the mammary epithelium. Nonspecific immunomodulation with immune modifiers could either increase or decrease the epithelial reactivity, whereas vaccination tends to increase the specific immune response to bacteria. Anti-inflammatory drugs (corticosteroid or NSAIDS) would reduce the inflammatory response, an effect also sought with herbal extracts.