**Overview of Research Development on the Role of NF-κB Signaling in Mastitis**

#### **Muhammad Zahoor Khan <sup>1</sup> , Adnan Khan <sup>2</sup> , Jianxin Xiao <sup>1</sup> , Jiaying Ma <sup>1</sup> , Yulin Ma <sup>1</sup> , Tianyu Chen <sup>1</sup> , Dafu Shao <sup>3</sup> and Zhijun Cao 1, \***


Received: 17 August 2020; Accepted: 28 August 2020; Published: 10 September 2020

**Simple Summary:** NF-κB signaling has been widely studied for its role in inflammatory and immunity-related diseases. Mastitis is considered one of the inflammatory and immunity associated diseases which are a serious threat to the global dairy industry. Having such a critical role in immunity and inflammation, NF-κB signaling is currently under target for therapeutic purposes in mastitis control research. The virulent factor, lipopolysaccharides (LPS), of bacteria after attachment with relevant Toll-like receptors (TLRs) on mammary epithelial cells starts its pathogenesis by using NF-κB signaling to cause mastitis. Several studies have proved that the blocking of NF-κB signaling could be a useful strategy for mastitis control.

**Abstract:** Mastitis is the inflammation of the mammary gland. *Escherichia coli* and *Staphylococcus aureus* are the most common bacteria responsible for mastitis. When mammary epithelial cells are infected by microorganisms, this activates an inflammatory response. The bacterial infection is recognized by innate pattern recognition receptors (PRRs) in the mammary epithelial cells, with the help of Toll-like receptors (TLRs). Upon activation by lipopolysaccharides, a virulent agent of bacteria, the TLRs further trigger nuclear factor-κB (NF-κB) signaling to accelerate its pathogenesis. The NF-κB has an essential role in many biological processes, such as cell survival, immune response, inflammation and development. Therefore, the NF-κB signaling triggered by the TLRs then regulates the transcriptional expression of specific inflammatory mediators to initiate inflammation of the mammary epithelial cells. Thus, any aberrant regulation of NF-κB signaling may lead to many inflammatory diseases, including mastitis. Hence, the inhibiting of NF-κB signaling has potential therapeutic applications in mastitis control strategies. In this review, we highlighted the regulation and function of NF-κB signaling in mastitis. Furthermore, the role of NF-κB signaling for therapeutic purposes in mastitis control has been explored in the current review.

**Keywords:** mastitis; bovine mammary epithelial cells; inflammatory cytokines; NF-κB signaling; PRRs; TLRs

#### **1. Introduction**

Mastitis is the inflammation of the mammary gland, which is associated with pathological changes in udder tissue and decreases in the quantity and quality of milk [1,2]. Based on its duration and symptoms, mastitis might be acute or chronic [3,4]. Udder swelling, reduced milk yield, clots and increase somatic cell counts in milk are the most common clinical signs of mastitis [5]. All these factors are associated with pathogenic invasion, which is followed by the involvement of neutrophils under a specific stimulus. The inflammatory conditions may lead to chronic inflammation if not properly controlled and treated [6,7]. Different types of etiological invading bacterial pathogens are involved in bovine mastitis, of which *Coliforms, Escherichia coli*, *Streptococci* and *Staphylococcus aureus* are the most common bacteria [8–11]. Gram-negative bacteria, such as *E. coli*, can often cause clinical mastitis, and Gram-positive bacteria, such as *S. aureus*, are involved in subclinical mastitis infection [12–14].

Previous reports have documented that mammary epithelial cells work as the first line of defense of the mammary gland by generating multiple inflammatory cytokines against bacteria invading the epithelial cells [15,16]. Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) expressed by many cell types, including mammary epithelial and immune cells [17]. In addition, it has been reported that innate immune systems recognize pathogens through TLRs [18–20].

The TLRs are distributed on the host cell surface that regulates the initial sensation of infection [21,22]. Every pathogen uses specific receptors on host cells—for example, *S. aureus* uses TLR2 and TLR6 [23], while *E. coli* utilizes TLR2 and TLR4—to transmit their signals inside the cell [21]. This specificity to TLRs depends on the virulent factor of pathogens. The cell wall of *S. aureus* is composed of lipoteichoic acid and peptidoglycan [24], while Gram-negative bacteria, such as *E. coli*, have lipopolysaccharides (LPS) in their cell wall [25]. The binding of pathogenic virulent factors to TLRs leads to the activation of several signaling components, including nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) [26], which is considered one of the key players associated with inflammatory action. Besides, NF-κB signaling has been widely studied for its role in regulation of immunity and inflammation. Keeping in view the versatile functions of NF-κB signaling, the current review has specifically concentrated on summarizing possible research development on the role of NF-κB signaling activation and regulation of immunity and inflammation in bovine mastitis.

#### **2. Materials and Methods**

All studies which have discussed the role of NF-κB signaling in mammary gland infection, mainly bovine mastitis, were screened through authentic sources, such as PubMed, ScienceDirect, Web of Science, SpringerLink, Scopus and Google Scholar. The major keywords used for the search of literature were milk production, mastitis, NF-κB signaling, TLRs, MYD88, PPRs, cytokines, *E. coli-* and *S. aureus-*mastitis. The related data published in the English language in well-reputed peer-reviewed journals have been included for discussion in the current review. Furthermore, we excluded all content available in the form of conference abstracts, books, book chapters and unpublished findings.

#### **3. General Regulatory Pattern of NF-**κ**B Signaling**

NF-κB is a common term used for inducible dimeric transcription factors. It is composed of a Rel family DNA binding protein which distinguishes common sequence motifs. Mammals express 5 Rel (NF-κB) proteins which are composed of two classes including Rel A (p65), c-Rel and Rel-B proteins which do not need proteolytic processing as the class is composed of NF-κB1 and NF-κB2 genes, encoded for p105 and p100, respectively, which do not require proteolytic processing to synthesize mature p50 and p52 NF-κB proteins [27]. The NF-κB protein was first found in murine B-lymphocytes, but currently, it has been identified in many cell types, including mammary epithelial cells [28]. Different external stimuli, such as tumor necrosis factor Alpha (TNF-α) [29], interleukin 1-beta (IL-1β) [30], LPS and reactive oxygen species (ROS) [31] after attachment with TLRs, activate NF-κB [32]. NF-κB signaling has an essential role in the regulation of immunity and inflammation [33], cell apoptosis, cell survival and proliferation (Figure 1) [34,35].

**Figure 1.** The key inducers of the NF-κB pathway and regulation of immunity, inflammation, cell survival and apoptosis by NF-κB signaling.

In addition, NF-κB signaling plays a vital role in the regulation of inflammatory cytokines, adhesion molecules, chemokines and growth factors involved in mammary gland inflammation [36]. Adhesion molecules are important proteins of tight junctions [37], which are closely related to the link between cell membranes and are required for normal lactation in mammals [38]. Song et al. has shown that LPS disrupt the permeability of the blood–milk barrier by activating the NF-κB signaling pathway. The pro-inflammatory cytokines regulated by the NF-κB signaling pathway promote the process of inflammation and interrupt the integrity of tight junction structures in the mammary epithelial cells [39]. The disruption in the blood–milk barrier has been reported during mastitis, which might be due to damage of the tight junctions responsible for normal lactation [40]. The disruption of tight junctions also may lead to loss of milk which is one of the common signs of mastitis in dairy cattle. Having such a critical role in inflammation and immunity, the NF-κB pathway has been widely targeted in mastitis research [41–46].

#### **4. Role of NF-**κ**B Signaling in Normal Physiology of Mammary Gland Development**

A regulated pattern of activation of NF-κB during the various stages of the development of mammary glands has been demonstrated [46]. NF-κB activation rises during pregnancy and decreases during lactation, followed by elevation during the mammary gland involution, again [47,48]. This change in pattern suggests that NF-κB plays a significant role during pregnancy and involution. Mammary gland involution is associated with apoptosis of the secretory alveolar epithelium [49], and NF-κB has been explored to mediate the anti-apoptotic proteins [50]. These findings revealed the role of NF-κB in promoting the survival of epithelial cells [51]. It has been demonstrated that NF-κB activates the two essential lactogenic hormones, namely prolactin and oxytocin [52,53]. In addition to playing a role in the developmental process of normal mammary glands, NF-κB activation was found to be associated with mammary gland infections.

#### **5. Role of NF-**κ**B Signaling in Mastitis**

The murine model and bovine reports have shown the link of NF-κB regulation with mastitis [43]. Most of the studies investigated the role of NF-κB in mastitis as a regulator of inflammatory cytokines [54,55]. Considerable losses of milk have been observed during mastitis and mammary gland involution which showed the link of both with the up-regulation of NF-κB during a time of milk loss and mammary gland remodeling.

#### *5.1. Mechanism of NF-*κ*B Signaling Activation by Bacteria during Mastitis*

LPS, a bacterial virulence factor, interacts with TLRs which are residing on surface mammary epithelial cells [56]. Upon activation, the TLRs further engage myeloid differentiation factor 88 (MyD88) [57] and c-Jun N-terminal kinase (JNK) [58], which triggers NF-κB [32] and mitogen-activated protein kinase (MAPK) signaling. The translocation of NF-κB and MAPK signaling further regulates the production of target inflammatory genes [59–62]. The mechanism of NF-κB signaling activated by *S. aureus* and *E. coli* during mastitis is shown in Figure 2.

**Figure 2.** The interactive mechanism of *E. coli* and *S. aureus* with TLR2, TLR4 and TLR6, and the regulation of NF-κB signaling to activate the inflammatory genes.

#### *5.2. Mechanism of NF-*κ*B Signaling Activation by Inflammatory Cytokines*

Besides *S. aureus* and *E. coli,* various inflammatory cytokines activate NF-κB signaling regulation in mammary epithelial cells. The NF-κB and MAPK pathways activate pro-inflammatory cytokines interleukin 6 (IL-6), IL-1β and TNF-α [63]. Nuclear factor-κB is a nuclear transcription factor that exists in an inactive form in the cytoplasm and is bound to its inhibitor IκB [64,65]. Once activated, the NF-κB unit p65 separates from IκB and translocates from the cytoplasm to the nucleus, where it regulates inflammatory gene expression [66]. The pathogenic message usually causes the liberation of NF-κB from IκB [65]. The regulation of the inflammation through NF-κB by pro-inflammatory cytokines is shown in Figure 3. The promoter of the inflammatory genes contains binding sites for NF-κB, and thus mostly depends on NF-κB for its regulation [67]. It has been reported that active NF-κB complexes cannot be detected in healthy cow milk cells, while the NF-κB elevated level was noticed in the milk cells of cows with acute mastitis. In addition, the activity of NF-κB in milk cells varies from low to high in chronic mastitis [67]. Stimulation of LPS causes mammary epithelial cells to produce cytokines TNF-α, IL-6 and IL-1β [68]. The increased levels of TNF-α, IL-6 and IL-1β have been observed in LPS-infused mammary glands [69]. Furthermore, Blum et al. reported the high level of cytokines (TNF-α, IL-6 and IL-17), somatic cell count (SCC), and up-regulation of TLR4 expression in leukocytes of the milk of an *E. coli*-induced mastitic cow [70]. In the mammary glands, inflammation is associated with an increased level of neutrophil chemo-attractants and the cytokines IL-1ß, IL-6, IL8 and TNF-α [71,72]. The expression level reported for IL8 and TNF-α in *E. coli* induced-mastitis in bovine mammary epithelial cells (BMECs) was much higher than for *S. aureus,* which is due to the weak Lipoteichoic acid (LTA) induction of TNF-α, or inactivation of NF-κB signaling [73]. Boulanger et al. observed that NF-κB was highly associated with the level of the expression of interleukin-8 and granulocyte/macrophage colony-stimulating factors, two NF-κB-dependent cytokines critically linked to the regulation and continuation of neutrophilic inflammation. Altogether, these findings suggested the crucial role of NF-κB in the pathogenesis of mastitis.

**Figure 3.** The regulation of the inflammation process by pro-inflammatory cytokines through NF-κB signaling; the cytokines, after attachment with receptors, cause the degradation of IKB from NF-κB. Upon activation, NF-κB directly binds to the promoters of target genes on DNA in the nucleus and regulates the specific inflammatory proteins.

#### *5.3. Bovine Myeloid Di*ff*erentiation Primary Response 88 (MYD88), NFKBIA and TRAPPC9 Role as a Regulator of Lipopolysaccharide (LPS)-Induced NF-*κ*B Signaling Pathways*

MYD88 is the main adopter molecule for TLR2, 4, 5, 7, 8 and 9 signaling [74]. The TLRs, when activated by mastitis-induced bacteria, pass the signal to MYD88, which is considered the critical immune regulator adapter molecule against various pathogens [75,76]. MYD88 acts as the key regulator of NF-κB by causing the degradation of IKB. Wang and his co-authors compared the expression level of MYD88 in healthy and mastitic cows. It was observed that MYD88 expression, which works as a bridge between TLRs and NF-κB, was elevated in mastitic cows compared to healthy ones [69,77,78]. It was noticed in a study that inhibition of MYD88, along with its inhibitor, Pepinh-MYD, significantly reduced the level of NF-κB [63].

Another essential protein is the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA), which encodes IκB and is responsible for the negative activation of NF-κB transcription factors. It has been shown in a report that LPS cause the degradation of IκBα; they facilitate the translocation of NF-κB in the nucleus, which in response accelerates the re-synthesis of IκBα [79]. Fang et al. noticed the up-regulation of NFKBIA in *S. aureus*-induced mastitis [80]. The trafficking protein particle complex 9 (TRAPPC9), also called NIK-and-IKK2-binding protein (NIBP), is a key regulator of NF-κB signaling [72,81,82]. An in-vitro study revealed NIBP low expression results in the down-regulation of TNF-α-induced NF-κB [83]. Wang et al. noticed through a genome-wide association study (GWAS) that the mutation in TRAPPC9 is associated with milk SCS [84]. The high expression level of the TRAPPC9 gene was reported in mammary epithelial cells infected with *S. aureus*. Furthermore, it was revealed that the TRAPPC9 gene might be considered a potential marker against mastitis [85]. The above-published studies showed that MYD88, NFKBIA and TRAPPC9 might work as a bridge between cell surface receptors and NF-κB. Thus, any change in these genes may disturb NF-κB signaling, which facilitates the pathogenesis of mastitis.

#### *5.4. NF-*κ*B Regulates the Immunity and Inflammatory Linked Genes during Mastitis*

When NF-κB signaling is activated by external stimuli, such as bacteria or cytokines, it starts to regulate the production of inflammatory chemokines (IL-8, CXCL1, CXCL10, etc.), cytokines (IL-6, TNF-α, IFN-gamma and IL-1β), adhesion molecules (ICAM-1 and MMPs), growth factors (CSF) and apoptotic associated genes [76,86,87]. For the site of infection, many proteins are required; these

proteins are: adhesion factors, such as ICAM-1 and VCAM-1, which facilitate neutrophil margination, diapedesis and transepithelial migration; chemokines, such as interleukin (IL)-8, which are responsible for chemotactic of neutrophils; IL-1β and TNF-α, which regulate neutrophils [67]. When bacteria enter the teat, the mammary epithelial cells secrete chemokines (CXCL8 and CXCL20) and cytokines (TNF-α and IL-1β). Production of cytokines and chemokines in the milk of the mastitic mammary gland is considered the key player of inflammation [72,88]. The TNF-α and fatty acid synthetase (FAS) mRNA expression was significantly up-regulated in LPS-challenged quarters [89]. A study reported the up-regulation of CXCL8 and TNF-α in *E. coli* induced mastitis in mammary epithelial cells [88]. In addition, the high expression of CXCL10, CCL2, CCL5 *and* CCL20 was noticed in bovine mammary epithelial cells in *E. coli* induced mastitis, which is essential for the recruitment of leucocytes [90]. The expression levels of IL-6, complement factor 3 (C3), NFKBIA and MMP9 were also elevated during mammary gland infection [90]. It has been reported that monocytes, natural killer cells and activated lymphocytes are majorly regulated by the chemokines CXCL10 and CCL5 [91]. Apart from the above functions, CXCL10 directs the recruitment and activation of neutrophils towards LPS-infection spots in mice and humans [83,92,93]. In addition, CXCL10 was reported as a highly expressed gene in response to *E. coli* infection in mammary glands [94]. The levels of CXCL10 and ICAM1 were noticed to be significantly elevated in the *S. aureus*-mastitic mammary glands of cows [95]. Similarly, the high expression of CCl5 has also reported in *E. coli*-induced mastitis in BMECs [96]. The expression levels of CXCL8, IL6 and CSF3 were higher in *S. aureus* challenged BMECs [80]. Additionally, many other immunity and inflammatory associated genes, such as SAA3, CCL5, C3 and CSF3, were also documented in mastitis-infected mammary glands [69]. Furthermore, the high expressions of CXCL10, IL6, CXCL8, IFN-gamma and IL-1β induced by LPS in BMECs are able to regulate inflammation [97]. It has been demonstrated in previous reports that inflammatory cytokines and chemokines create protection against foreign invading pathogens in bovine mammary glands, by increasing the movement of leucocytes from the blood into the mammary tissue [98]. Similarly, a study reported the protecting role of IL-1β by recruiting neutrophils into the mammary gland [99].

#### *5.5. Research Progress on Target of NF-*κ*B Signaling as a Therapeutic in Mastitis Control*

It is well known that TLRs, upon recognition of external stimuli, activate NF-κB regulation to produce inflammatory linked genes to eliminate the cause of infection in mammary epithelial cells. TLR4, a pro-inflammatory cytokine, and LPS, a component of the cell wall of bacteria, are common inducers of NF-κB signaling. The LPS-induced inflammation in mammary epithelial cells causes the up-regulation of TLR4 [100,101]. Recently, NF-κB signaling is being widely targeted as a therapeutic choice against mastitis resistance. A study proved, experimentally, that selenium restricts *S. aureus*-induced mastitis through inhibition of the MAPK and NF-κB pathways and TLR2 [102]. Cytokines, an important group of inflammatory mediators, play a major role in the process of inflammation [103]. Stimulation by LPS causes mammary epithelial cells to produce the cytokines TNF-α, IL-6 and IL-1β[60]. Increased levels of TNF-α, IL-6 and IL-1βhave been observed in LPS-infused mammary glands. Similarly, Akhter et al. [104] noticed the up-regulation of pro-inflammatory cytokines in *S. aureus*-induced mammary epithelial cells. Further, they proved that the expression levels of genes associated with TLR2/TLR4-mediated NF-κB/MAPKs pathways were higher in *S. aureus*-infected mammary epithelial cells. The excessive expression of pro-inflammatory IL1β may lead to pathological conditions [105]. Dai et al. noticed that methionine and arginine attenuated the proinflammatory action by preventing the regulation of NF-κB. Furthermore, methionine and arginine down-regulated the levels of TLR4 and IL1β in LPS-induced mastitis, which caused the excessive regulation of inflammatory changes, and thus damaged the cells [106]. Taken together, it has been concluded here that methionine and arginine, being blockers of NF-κB, can be considered as prophylactic agents of mastitis.

Exogenous hydrogen sulfide has the ability to suppress inflammatory cytokine production, ROS [107–109], and promotes anti-inflammatory proteins [110]. The high level of ROS is associated with the imbalance between cellular redox states and oxidative stress, which has a significant role

in the promotion of inflammation [111]. It was noticed that LPS alone diminished cell viability and caused inflammatory changes in mammary epithelial cells. However, it was found that the hydrogen sulfide (H2S) combined with LPS restored the viability of the cells [112]. Sun et al. revealed that H2S, after entry into the cells, first blocked the TLR4 and ROS, and thereby no signal was given for NF-κB to produce a high level of inflammatory proteins in mammary epithelial cells [113]. In addition, the mRNA expression of TNF-α, IL-1β, IL-8 and IL-6 was also very low in H2S-treated mammary epithelial cells.

Morin has anti-inflammatory properties [114] and inhibits the release of the inflammatory cytokines IL-6 and IL-8 and tumor necrosis factor (TNF) from mast cells [115]. It was experimentally proved that morin is associated with inhibition of TNF-α, IL-6 and IL-1β in LPS-induced bovine mammary epithelial cells (bMECs). To suppress the level of cytokines, morin down-regulates the levels of MAPK and NF-κB pathways in LPS-induced mammary epithelial cells [78]. NLRP3 inflammasome is the key regulator of IL-1β, while a recent study noticed that morin significantly down-regulated the level of IL-1β [116] in LPS-induced bovine mammary epithelial cells by suppression of NF-κB and nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain containing 3 (NLRP3) inflammasome [117]. Furthermore, it has been noticed that morin also maintained the integrity of the tight junction from the action of the inflammatory cytokines regulated by NF-κB [63]. Likewise, polydatin has anti-inflammatory efficiency and can be used to control *S. aureus*-induced mastitis. The most in-depth mechanism showed that polydatin decreased the expression of TLR2 and MyD88, which further suppressed the level of NF-κB in mammary epithelial cells of *S. aureus-* induced mastitis [60].

Tea tree oil (TTO) is an essential oil which has antibacterial and anti-inflammatory properties and promotes the movement of polymorphonuclear leukocytes towards the infection. TTO also inhibits NFKBIA and TNF-α [118]. In addition, TTO act as an inhibitor of the NF-κB pathway, which is essential for the regulation of immunity and inflammatory responses in mammary glands. Nucleotide-binding oligomerization domain (NOD) is a type of PRR that plays an important role in the regulation of innate immunity [119]. Recently, it was documented that by blocking NOD1/NF-κB signaling, LPS stimulation reduced neutrophil migration and phagocytic killing ability. Further, it was proved that the activation of NOD1/NF-κB in vitro restricted the action of LPS by promoting the functional capacity of neutrophil [60]. Chlorogenic acid has anti-inflammatory and antibacterial effects [120,121]. A study reported that chlorogenic acid inhibits cytokine production in LPS-stimulated RAW264.7 cells by suppressing the phosphorylation of NF-κB [122]. Similarly, chlorogenic acid was noticed to reduce the level of cytokines followed by inhibition of TLR4 and phosphorylation of NF-κB in LPS-induced mastitic mammary epithelial cells [123]. Thymol was found to be very effective in mastitis treatment. The mechanism for the association of thymol was tested in BMECs. The western blot result showed that thymol treatment significantly inhibited the production of IL-6 and TNF-α, followed by suppression of the NF-κB pathway [124]. In Table 1, we summarized those studies which targeted the NF-κB signaling to control mastitis.



From the above discussion, it has been cleared that NF-κB signaling plays a role of backbone in the pathogenesis of mastitis by promoting cytokine production. Thus, by targeting NF-κB, mastitis can be effectively controlled [124].

#### **6. Conclusions**

Overall, the current review, based on published studies, revealed that activation of NF-κB resulted in decreased of milk and apoptotic signaling, which could be minimized through selective modulation of NF-κB signals. Furthermore, the review suggested that NF-κB is a vital regulator of milk loss during mammary gland involution and infection, and recognized the NF-κB signaling pathway as a possible target for preventing mastitis-induced milk loss in dairy cattle. In addition, based on published literature, we concluded that TLR4, IL-1β, IL-6, TNF-α and MYD88 are key players in NF-κB signaling and also have an essential role in mastitis development. From the literature studies, it was revealed that *S. aureus* and *E. coli,* after attachment with TLRs, used NF-κB pathway for pathogenesis. Thus, the utilization of NF-κB as a therapeutic target in mastitis control showed successful outcomes. In addition, TLR4, IL-1β, IL-6, TNF-α, MYD88 and NF-κB might be a useful addition as markers in mastitis control strategies.

**Author Contributions:** Conceptualization, M.Z.K. and Z.C.; writing—original draft preparation, M.Z.K. and Z.C.; editing and technical review, A.K., J.M., Y.M., T.C., D.S., J.X. and Z.C.; visualization, Z.C.; supervision, Z.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** The review was supported by the National Key Research and Development Program of China (2018YFD0501600). The funder had no role in the study design, data collection, analysis, decision to publish, and preparation of the manuscript.

**Acknowledgments:** We acknowledge the financial support of the National Key Research and Development Program of China (2018YFD0501600). We also acknowledge the China Agricultural University, Beijing, China, for providing us with an environment of learning. Without this platform, the completion of this work would not have been an easy task.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Role of the JAK-STAT Pathway in Bovine Mastitis and Milk Production**

#### **Muhammad Zahoor Khan 1, Adnan Khan 2, Jianxin Xiao 1, Yulin Ma 1, Jiaying Ma 1, Jian Gao <sup>3</sup> and Zhijun Cao 1,\***


Received: 23 September 2020; Accepted: 5 November 2020; Published: 13 November 2020

**Simple Summary:** The cytokine-activated Janus kinase (JAK)—signal transducer and activator of transcription (STAT) pathway has an important role in the regulation of immunity and inflammation. In addition, the signaling of this pathway has been reported to be associated with mammary gland development and milk production. Because of such important functions, the JAK-STAT pathway has been widely targeted in both human and animal diseases as a therapeutic agent. Recently, the *JAK2*, *STATs*, and inhibitors of the JAK-STAT pathway, especially cytokine signaling suppressors (SOCSs), have been reported to be associated with milk production and mastitis-resistance phenotypic traits in dairy cattle. Thus, in the current review, we attempt to overview the development of the JAK-STAT pathway role in bovine mastitis and milk production.

**Abstract:** The cytokine-activated Janus kinase (JAK)—signal transducer and activator of transcription (STAT) pathway is a sequence of communications between proteins in a cell, and it is associated with various processes such as cell division, apoptosis, mammary gland development, lactation, anti-inflammation, and immunity. The pathway is involved in transferring information from receptors on the cell surface to the cell nucleus, resulting in the regulation of genes through transcription. The Janus kinase 2 (*JAK2*), signal transducer and activator of transcription A and B (STAT5 A & B), STAT1, and cytokine signaling suppressor 3 (*SOCS3*) are the key members of the JAK-STAT pathway. Interestingly, prolactin (Prl) also uses the JAK-STAT pathway to regulate milk production traits in dairy cattle. The activation of *JAK2* and *STATs* genes has a critical role in milk production and mastitis resistance. The upregulation of *SOCS3* in bovine mammary epithelial cells inhibits the activation of *JAK2* and *STATs* genes, which promotes mastitis development and reduces the lactational performance of dairy cattle. In the current review, we highlight the recent development in the knowledge of JAK-STAT, which will enhance our ability to devise therapeutic strategies for bovine mastitis control. Furthermore, the review also explores the role of the JAK-STAT pathway in the regulation of milk production in dairy cattle.

**Keywords:** bovine mastitis; JAK-STAT pathway; *JAK2*; *STATs*; *SOCS3*; immunity; milk production

#### **1. Introduction**

Bovine mastitis is a seriously infectious and contagious disease, which is a massive threat to the dairy industry throughout the globe [1]. Mastitis is the inflammation of the mammary gland, which is characterized by physical, chemical, and microbiological alterations in milk, following pathological changes in udder tissue [2]. Bovine mastitis is described as acute or chronic based on inflammation, redness, and localized heat at the infected area, with more severe symptoms, such as fever, leading to septicemia, and the formation of abscesses [3,4]. There are two types of mastitis: clinical and subclinical mastitis. In most cases, infection with Gram-negative bacteria such as *Escherichia coli* (*E. coli*) can often cause clinical mastitis, and Gram-positive bacteria such as *Staphylococcus aureus* (*S. aureus*) are involved in subclinical mastitis infection [5,6].

Bovine mastitis is considered one of the costly diseases of dairy cattle because of milk losses, treatment costs, and rare death [7,8]. In China, the annual losses of 15–45 billion Chinese Yuan (CNY) have been documented [7], while in the US and India, the dairy industry has experienced losses of 2 billion and 526 million dollars, respectively [9]. In Europe, collectively, the cost due to mastitis has reached 1.55 billion euros per year [10]. This increased frequency was linked to public concerns for animal welfare and has made mastitis the key disease of the dairy sector [11]. In addition, bovine mastitis has a major zoonotic risk, correlated with the shedding of bacteria and their toxins into milk [12].

Mammary epithelial cells are the first line of defense of the mammary gland to invading bacteria. They not only act as physical barriers but also are capable of producing inflammatory mediators. While interacting with invading bacteria, mammary epithelial cells generate multiple inflammatory cytokines [13,14]. Several genes and pathways have been reported to be associated with the regulation of bovine mastitis [15]. It is well known that the innate immune system recognizes the presence of pathogens ligands through a membrane receptor family known as Toll-like receptors (TLRs) [16]. TLRs are pattern recognition receptors (PRRs) on the host cell surface that recognize bacterial-pathogen-associated molecular patterns [17]. Upon activation, TLRs further mediate different important signaling, such as that of the JAK-STAT pathway.

Any disruption in the JAK-STAT pathway may lead to various diseases, including bovine mastitis that compromises the immune system of the host. Furthermore, it has also been documented that *STAT5A* works as a mediator for extracellular prolactin receptors. At the same time, *JAK2* plays a role as a bridge between *STAT5A* and prolactin receptor (PrlR), which is essential for milk production and mammary gland development. Keeping in view the vital role of JAK-STAT signaling in immunity, inflammation, and milk production, the current review paper is designed with aims to summarize the role of the JAK-STAT pathway in bovine mastitis and milk production.

#### **2. General Mechanism of the JAK-STAT Pathway Regulation**

There are three main components of the JAK-STAT pathway: receptors, Janus kinases (JAKs), signal transducers, and activators of transcription proteins (*STATs*) [18]. The mammalian JAK family consists of *JAK1*, *JAK2*, *JAK3*, and tyrosine kinase 2 (*TYK2*), which are linked to the cytoplasmic domains of diverse cytokine receptors [19]. Among the seven members of *STATs* (*STAT1*-4, 5a, 5b, and 6) in mammalian cells, *STAT5A* and *STAT5B* show high sequence identity and lie closest in a head-to-head pattern next to *STAT3* [19,20]. The members of the STAT family are involved in cell growth, differentiation, cell survival and apoptosis, and mammary gland development. The cytokines, after attachment with receptors on the cell surface, activate JAKs. The two JAKs come close through receptor oligomerization. Furthermore, these JAKs phosphorylate the receptor complex's intracellular tyrosines, generating the docking sites for STATs. Consequently, the activated *STATs* form hetero- or homodimers, where the Src-homology 2 (SH2) domain of each STAT binds the phosphorylated tyrosine of the opposite STAT, and the dimers then translocate to the cell nucleus to induce transcription of the target genes. JAK-STAT has been revealed to operate downstream of several peptide hormones and cytokines that are necessary for the development of the postnatal and secretory

function of the mammary gland [21]. The phosphorylated *STAT5A* and *STAT5B* form homodimers and heterodimers in mammary epithelial cells in order to regulate the process of differentiation, survival, and proliferation through the modification in cellular gene expression [22]. The rapamycin target phosphatidylinositol 3-kinase-protein kinase B/mammalian signaling pathway (PI3K-Akt/mTOR) mediates many cellular processes such as cell proliferation, growth, survival, and metastasis [23], and it is necessary for the development of the mammary gland [24]. A conditional knockout of Akt1 prevents the extensive survival of mammary epithelial cells, which express hyperactive *STAT5,* indicating that the PI3K-Akt/mTOR pathway is a crucial downstream signaling effector of JAK-STAT signaling [25]. To find out the interconnection between different genes and their biological functions in the JAK-STAT pathway, we exploited an online software database for annotation, visualization, and integrated discovery (DAVID; https://david.ncifcrf.gov/) [26], which are summarized in Figure 1.

**Figure 1.** The regulation of the cytokine-activated Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway by cytokines, hormones, and growth factors; engagement of the JAK-STAT pathway in the process of differentiation, survival, and proliferation through the modification in cellular gene expression.

*STAT5*, being the main gene of the JAK-STAT inflammatory signaling pathway, has an essential role in prolactin-induced mammary gland factor and is assumed to be associated with mammary gland development in transgenic mice [27]. Consequently, upon activation, JAK regulates the cellular mechanisms such as cell migration, apoptosis, cell proliferation, and differentiation, which are essential for hematopoietic responses, immune development, mammary gland development, and the lactation process [28]. Cytokines play a vital role in the regulation of the JAK-STAT pathway, which further facilitates immunity and inflammation. Consequently, the JAK-STAT pathway has been widely studied for its critical role in immunity and inflammation [29,30], and evidence indicates that persistent activation of this pathway might lead to many immune- and inflammatory-related diseases [31,32]. Performing a critical role in immunity, cell proliferation, cell differentiation, and inflammation, the JAK-STAT pathway has been widely targeted for therapeutic purposes in several inflammatory diseases [33].

#### **3. The JAK-STAT Pathway Role in Milk Production in Dairy Cattle**

The JAK-STAT pathway regulates lactation [34], while PI3K/Akt within the JAK-STAT pathway shows overexpression in lactating cows [35]. Gene deletion analysis in mice has documented an important role of the JAK-STAT signaling pathway in the lactation and development of the mammary gland [36,37]. In the mammary gland, the JAK-STAT pathway, along with *SOCS* signaling, plays a critical role in controlling cytokine signals and has shown an association with mammary gland development and milk production [38]. Moreover, studies have documented the essential role of the JAK-STAT pathway in blood cell differentiation and casein gene regulation during milk production [39,40]. It has been shown that some JAK-STAT-associated proteins are regulated by PrlR, which may establish a balance between growth hormone and milk protein yield [41]. It has been illustrated that by using the JAK-STAT pathway, the lactogenic hormones, through their receptors on cell membranes, regulate milk proteins [42]. Prolactin also uses JAK-STAT signaling and regulates the processes of lactation and reproduction in mammals [43]. It has been documented that a higher concentration of Prl in blood circulation is associated with an increased level of milk production in dairy cattle [44]. During hypothyroidism, a severe decrease in milk production has been documented. Furthermore, it has been found that hypothyroidism decreases the level of prolactin, resulting in lower expression of the JAK-STAT pathway, which is responsible for lower milk production in hypothyroidized rats [45].

#### *3.1. Role of JAKs in Milk Production in Dairy Cattle*

JAK2 is the tyrosine kinase responsible for phosphorylation of both PrlR and Stat5, based on tissue culture cell studies. According to one report, in the absence of the *JAK2* gene, mammary epithelium proliferation and differentiation were reduced by 95% around parturition [46]. The endocrine factor prolactin attaches to the PrlRs and causes their dimerization. JAK protein kinases are linked to these receptors and these JAK proteins alter a receptor into a tyrosine kinase receptor. The regulated receptors may specifically phosphorylate inactive *STATs*, which result in dimerization. These dimers are further translocated into the nucleus. The *STATs* attach to the upstream promoter elements of the casein gene and cause their transcription. Growth hormones (GHs) control the growth and development of the mammary gland and regulate milk production and milk protein levels in cattle [41,47]. *STAT5* passes on messages from cytokines and growth factors outside the cell to the nucleus of the mammary gland epithelial cells and thereby mediates the transcription of the gene during pregnancy, lactation, and weaning [48].

It has been consistently reported that the polymorphisms T-C39652459 and T-C39645396, at intron 15 and exon 20, respectively, in the *JAK2* gene, are significantly associated with milk lactose production in dairy cows [49]. Furthermore, the variant *JAK2*/*RsaI* is involved in the regulation of milk and milk protein and can be considered a milk production marker in dairy cattle [50]. The variants 39630048C/T and 39631175T/C in the *JAK2* gene significantly influence milk fat and milk proteins, respectively, in Chinese Holsteins [51]. PrlR uses *STAT5A* and *JAK2* as mediators to activate the proteins associated with milk production traits [52].

#### *3.2. Role of STATs in Milk Production in Dairy Cattle*

STATs are activated by specific ligands, i.e., *STAT5A* is regulated by Prl, while STAT5B regulation is mediated through growth hormones (GHs) [53]. STAT5 is an important intracellular mediator of prolactin signaling and can activate transcription of milk proteins in response to Prl. *STAT5* has been suggested to be candidate marker genes for milk protein yield and composition in dairy cattle [54]. During pregnancy, *STAT5A* and PrlR play essential roles in mammary epithelium proliferation and differentiation [55,56]. Consequently, it has been found that PrlR has a positive impact on lactation performance in cows, possibly due to its involvement in steroid synthesis and cholesterol regulation [57]. During pregnancy and lactation, STAT5A and STAT5B are the essential proteins required for the synthesis of luminal progenitor cells from mammary stem cells and the differentiation of milk-producing alveolar cells [58]. *STAT5A* and *STAT5B* have been linked with the development of the mammary gland during pregnancy [59]. It was previously found that *STATs* promote the mammary gland cells' survival by mediating the promoters of genes essential for milk proteins [34,60]. *STATs* facilitate various peptide hormones and cytokines in targeted cells such as Prl and GH and are linked to milk production. Whey acidic protein (WAP) is expressed in the mammary gland and is associated with

the improvement of milk protein. *STAT5* has been considered an important transcription factor that is responsible for the regulations of Prl at 5 flanking regions of *WAP* [61]. It has been observed that the downregulation of Prl in hypothyroidized rats causes the inhibition of the transcriptional activity of *STAT5.* Consequently, any abnormality in the thyroid gland severely affects milk production efficiency in rats because of the low level of Prl [45]. In addition, GH also regulates the STAT1 gene and its expression has been reported during mammary gland development [62,63]. Furthermore, a study has reported the combination effect of STAT1 with other JAK-STAT signaling members on milk production traits [38]. Keeping in view the important role of *STATs* as a mediator of prolactin signaling, the polymorphisms in these genes were further studied for its role in milk production.

The mutations in the *STAT5A* gene have been reported for their effect on milk yield [64]. Consistently, the *STAT5A*/*AvaI* polymorphism at position C-T 6853/exon7 was documented to be associated with milk production and could be used as a significant marker for milk improvement [65]. In addition, the *STAT5A*/*MslI* locus has been found to be correlated with milk yield, milk fat, and protein [65–67]. The polymorphic site A14217G and 17266indelCCT in *STAT5A* have shown significant associations with milk protein percentage and milk yield, respectively [68]. Consequently, Schennink et al. documented that single nucleotide polymorphism (SNP) 9501G>A in *STAT5A* significantly influenced milk fat composition [69]. Khatib et al. noticed that variant 12195T/C in *STAT5A* was significantly linked to a decrease in milk fat and protein percentage in dairy cattle [70]. The variant 31562 T>C in *STAT5B* was reported to be associated with milk yield and milk protein [71]. The association of *CD4* and *STAT5B* with milk traits might be due to their role in the regulation of prolactin-induced mammary gland factor [72]. Moreover, the variant in the STAT 1 gene has been documented to be linked with milk fat, milk protein, and milk yield in dairy cattle [73]. Consequently, the polymorphism STAT1/BspHI has been reported to be associated with milk production traits in Jersey cows [74]. Similarly, Deng et al. reported that SNPs in STAT1 have a significant association with milk production traits and could be a useful addition to the marker-assisted selection for milk production [75].

The above findings reveal that the JAK-STAT pathway plays a central role in the regulation of milk production traits.

#### **4. The JAK-STAT Signaling Role in Bovine Mastitis**

As mastitis is an immunity- and inflammatory-related disease, scientists have widely targeted the JAK-STAT pathway in bovine mastitis control research. Besides having a critical role in mammary gland development, any abnormal regulation may disturb the normal function of the JAK-STAT pathway, resulting in impairment of mammary gland development and exposure to mammary infections. Buitenhuis et al. found the altered expression of the JAK-STAT pathway in the mammary gland tissue of cows challenged with *E. coli* [76]. It is well known that the JAK-STAT pathway is regulated by IFN, LPS, or growth factors. In its turn, JAK-STAT signaling mediates proinflammatory cytokines. Tiezzi et al. documented the JAK-STAT pathway as a key pathway that regulates clinical mastitis [77]. Recently, it has been reported that cirsimarin (an extract of *Cirsium japonicum* var. ussuriense) treatment suppressed the expression of inflammatory cytokines by downregulating the phosphorylation of the JAK-STAT pathway in the mammary gland. Thus, this substance can be targeted as a therapeutic agent in many inflammatory diseases, including bovine mastitis [78]. It has been shown that 8-methoxypsoralen treatment protects bovine mammary epithelial cells against lipopolysaccharide-induced inflammatory injury by inhibition of the JAK/STAT and NF-κB pathways [79]. JAK-STAT suppression by xanthotoxin resulted in the downregulation of IL-6, nitric oxide (NO), and tumor necrosis factor (TNF-α) induced by LPS in bovine mammary epithelial cells [80]. This mechanism is essential for regulating udder reactions to infection as it controls the chronic accumulation of neutrophils in the bovine mammary gland [81], whereas JAK also serves as a signaling agent for hormones and interleukin receptors [82] and *JAK2* is considered one of the top-rated genes of bovine mastitis tolerance [83].

#### *4.1. Association of JAK2 Activity with Bovine Mastitis*

Any dysfunctions of the JAK-STAT pathway may expose cattle to mastitis because of abnormal activation of the proliferation and apoptosis of cells. From this point of view, it can be expected that mutations in genes involved in the JAK-STAT pathway might be a target in bovine mastitis research. The inflammatory- and immunity-associated diseases are polygenic traits [71], and polymorphisms in immunity-linked genes can regulate the immune responses of the host to pathogens [84]. Two major approaches are dominantly targeted by animal scientists to control mastitis. The first approach is to look for major genes associated with mastitis resistance, while the second one is to target the polymorphisms within genes and their links with mastitis resistance traits.

Many types of mutations in the JAK-STAT pathway have been identified; most of them are related to *JAK2*.

As demonstrated in Table 1, the polymorphism 39630048C/T in *JAK2* is associated with interleukin-17 (IL-17) [85], IL-6, and interferon-gamma (IFN-γ) expression [51]. Furthermore, the SNPs (39652267A/G, 39631175T/C) in the *JAK2* gene have been documented for their significant links with milk somatic cell counts (SCCs), IL-6, and IFN-γ [51,85]. Mutation 39631044G/A in the *JAK2* gene was noticed to be significantly associated with milk somatic cell scores (SCSs) in Chinese Holsteins [85]. Moreover, the polymorphism 39645396C/T in the *JAK2* gene was linked to milk SCCs, IL-6, and IFN-γ [86], while SNP-39631044G/A in *JAK2* was associated with milk SCSs [85]. SCCs and SCSs are widely targeted as early mastitis indicators [7]. Increased SCCs in early lactation can signify the presence of intramammary infection, and, in many countries, the indirect selection against mastitis using milk SCCs is practiced [87]. However, in the early phase of infectivity, the neutrophil and inflammatory cytokine levels increase quicker than milk SCCs [88]. That is why, nowadays, more interest is paid to the increase in cells and cytokine levels in milk and blood, respectively, rather than just the overall SCC, which may provide an early status of udder health [89]. A study showed that inflammatory cytokines (IL-6, IL-17, and IFN-γ, TNF-α) could be used as subclinical mastitis indicators, in addition to SCSs and SCCs [51,86,90]. In addition, it is predicted that the 39645396C/T SNP changes lysine to asparagine [86]. The expression of IL-6 was higher in plasma cell mastitis (PCM), which indicated that the IL-6/STAT3 pathway could play a key role in the pathogenesis of PCM [22,91]. The IL-17 family consists of cytokines that participate in acute and chronic inflammation and provoke the host's defense against microbial organisms [92]. T-helper 17 cells are thought to be a significant source of IL-17A; furthermore, IL-17, producing innate immune cells, activate the fast release of IL-17A [93] in response to pathogens or tissue injury [94].

**Table 1.** Association of SNPs in *JAK2* with bovine mastitis resistance phenotypic traits.


IL-17 has been shown to be significantly upregulated in goat milk infected with *E. coli* or *S. aureus* [95]. *IL-17A* production was documented during *S. uberis* mastitis [96], and slightly increased expression was also noticed in the somatic cells of cows infected with *S. aureus* [97]. Furthermore, an in-vitro study illustrated that IL-17A reinforces the ability of mammary epithelial cells (MECs) to resist the consequences produced by *S. aureus* [98]. It has been reported that *IL-17A* and *IL-17F* play a critical role in regulating host–pathogen interactions during the development of mastitis [99]. The SNPs in *IL-17F* and *IL-17A* have been shown to be associated with milk SCCs [90]. Moreover, IL-17 also activates IL-6 with IFN-γ and tumor necrosis factor-alpha (TNF-α) [100]. Usman et al. revealed that

IL-6 is the best indicator of mastitis and can be a target in mastitis control strategies [85]. Altogether, the above-published studies show that IL-17, IL-6, IL-4, IFN-γ, SCS, and SCC are the key indicators of mastitis. The interactions of polymorphisms in *JAK2* with bovine mastitis resistance phenotypic traits (IL-17, IL-6, IL-4, IFN-γ, SCS, and SCC) show that *JAK2* might be considered a useful marker in bovine mastitis resistance strategies.

#### *4.2. Role of STATs in Bovine Mastitis*

A variety of cytokines and growth factors activate *STATs*, which are a family of latent transcription factors. During the process of inflammation, *STAT5B* regulates CD4+ T-cells differentiation [101]. STAT1 raised the expression of *SOCS3* and SOCS1 in *S. aureus*-infected mammary epithelial cells [102]. Furthermore, it was reported that upon treatment with JAK inhibitors, the plasma cells in PCM decreased considerably due to the suppression of IL6/STAT/JAK signaling, resulting in the reversion of pathogenesis [91]. Accordingly, it was found that the inflammatory cytokines regulate the JAK-STAT pathway in the mammary gland; in response, the phosphorylation of STAT takes place. The phosphorylated STAT translocates into the nucleus and mediates the production of proinflammatory genes that facilitate mastitis's pathogenesis [78]. It is known that the inflammatory cells are recruited towards the site of infection, in which T-cells, particularly CD4+ cells, are predominantly observed in bovine mastitis [103]. Rivas et al. revealed that *S. aureus*-infected dairy cows showed a remarkable elevation in the level of CD4+ T-cells at the early stage of infection in the mammary gland [104]. Eder's team recently proved that the CD4+T-cell level was higher in dry cows compared to lactating cows. These findings show that a decrease in the level of CD4+ T-cells in lactating dairy might be one of the reasons for susceptibility to infection during this stage [105]. Usman et al. reported a significant association of variant T104010752C in the *CD4* gene with milk SCCs [90]. In the previous study, it was noticed that polymorphisms in *CD4* and *STAT5B* genes are significantly linked with mastitis-resistance phenotypic traits [83]. Furthermore, the polymorphism in *CD4* at locus g.13598C>T showed a significant association with SCS, which is the crucial indicator of mastitis.

The combination geneotype analysis of CD4 g.13598C>T and STAT5b g.31562 T>C is associated with milk SCSs in Chinese Holsteins. Furthermore, it was reported that cows with combination genotypes of CCTT show the highest estimated breeding value (EBV) for SCSs [71]. Another study documented that the silencing of the *CD4* gene through DNA methylation influences the progress of CD4+ T-cells in inflammatory conditions [106]. These findings demonstrate that CD4 protein and CD4+ T-cells play essential roles in host defense during the development of mastitis.

As demonstrated in Table 2, the polymorphism in *STAT5A* (43046497A/C) is associated with IL-6 and also changes the amino acid isoleucine to valine [85]. Similarly, mutation at point 43673888A>G in the *STAT5B* gene was significantly linked to mastitis-resistance phenotypic traits (IL-4 and SCC) [86]. Bochniarz et al. reported the elevated level of IL-6 and decreased level of IL-4 in the milk and serum of cows infected with *S. aureus* [107]. In addition, the polymorphism STAT5A-AvaI was associated with milk SCCs and electrical conductivity (EC) in the milk of mastitic cows [108]. EC in milk is one of the essential indicators of bovine mastitis because of its association with Na and Cl levels, which increase during mastitis. Cai et al. also reported a STAT5A gene through genomewide association studies (GWAS) as a potential candidate marker for bovine mastitis resistance [109]. Based on the above-published findings, we concluded that *STAT5A* and *STAT5B* might be target mastitis-resistance markers in dairy cattle.

**Table 2.** Association of SNPs in *STAT5A* and *STAT5B* with bovine mastitis resistance phenotypic traits.


#### **5. Inhibitors of the JAK-STAT Pathway: Role in Mastitis and Milk Production**

The protein inhibitors of activated STAT (PIAS) [110], protein tyrosine phosphatases (PTPs) [111], and cytokine signaling suppressors (SOCSs) [112] are three major classes used by cells to control the JAK-STAT pathway [113]. PIAS proteins are considered important transcriptional coregulators of JAK-STAT signaling because of their significant contribution to the control of gene expression [114]. PIAS proteins restrict the regulation of the JAK-STAT pathway in three ways: (1) by adding a small ubiquitin-like modifier (SUMO) group to STAT and blocking its phosphorylation, (2) by preventing the binding of STAT to DNA [115], and (3) by recruiting histone deacetylase to remove acetyl changes to histones by lowering gene expression [116]. Similarly, PIAS3, a member of the PIAS family, has been identified to inhibit STAT3 signaling after regulation by the cytokine IL-6 [117]. Moreover, PIAS1 could inhibit NF-κB and JAK-STAT activity regulated by cytokine TNF and the LPS endotoxin [110]. PIAS has a major role in cell proliferation [118], cell apoptosis, and the immune response [115]. Protein tyrosine phosphatases (PTPs) are a group of enzymes that remove the phosphate group from the JAK-STAT pathway and prevent the action of signaling [119]. The *STATs* are deactivated by PTPS in both the nucleus and cytoplasm. Src homology phosphatase 2 (SHP-2) is one of the members of PTPs that inactivate STAT5 in the cytoplasm. Similarly, SHP1 prevents the phosphorylation of the JAK-STAT pathway and blocks its further action [120,121]. The general role of JAK-STAT inhibitors has been summarized by recently published reviews in more detail [31,122]. Although the two groups of PTPs and PIAS have essential roles in the regulation of the JAK-STAT pathway, their tasks have not been evaluated in milk production or bovine mastitis to date. Therefore, we have only focused on cytokine signaling suppressors (SOCSs) in our current review.

Some SOCS proteins are triggered by cytokines and pathogenic mediators and, thus, function in a classical negative-feedback loop to impede the transduction of cytokine signals. Consequently, they represent an effective mechanism for the negative regulation of the cytokine-mediated JAK-STAT pathway [123]. The DNA binding of STAT protein regulates the mRNA expression of SOCSs [124]. *SOCS3* can inhibit JAK tyrosine kinase activity directly via its kinase-inhibitory region (KIR), which has been proposed to serve as a pseudosubstrate and is essential for cytokine signal suppression [125]. Undeniably, both a KIR and a KIR-mimetic peptide, classified as the tyrosine kinase inhibitor peptide (TKIP), have been described to inhibit *JAK2*-regulated transcription factor STAT1 phosphorylation [126,127]. The SH2 domain of SOCS can also directly bind to the receptors and prevent the signal from passing to JAK-STAT signaling [128]. Moreover, Kimura et al. revealed that LPS could activate *JAK2* and *STAT5*, which participate in the induction of IL-6, while SOCS1 inhibits this process selectively [129].

The suppression of IL-6 and IFN-γ usually occurs around parturition, which depresses immunity and exposes dairy cattle to mastitis [130]. Normal levels of IL-6 and IFN-γ are necessary for the maintenance of bovine immunity. Moreover, *SOCS3* has been reported to be one of the key inhibitors of IL-6 and IFN-gamma. This evidence shows that *SOCS3* might have a potential role in mastitis development in dairy cattle [131]. Moreover, Fang et al. found that *SOCS3* was significantly upregulated after the mammary gland had been infected with *S. aureus*. The authors further supposed that *SOCS3* could negatively regulate the JAK-STAT pathway, which might be one of the reasons for its critical role in mastitis development [132]. Huang et al. also reported that *SOCS3* is a negative regulator of the JAK-STAT pathway. Furthermore, it was demonstrated that overexpression and inhibition of *SOCS3* brought visible changes in milk protein, which might be due to the action of *SOCS3* on the JAK-STAT pathway [133]. The Huang team further suggested that a low level of *SOCS3* is essential for the regulation of milk synthesis. Similarly, a study reported that *SOCS3* inhibits the induction of Prl and activation of STAT5 [134]. Zahoor et al. found that merTK reduces the inflammatory changes induced by *S. aureus* through *STATs*/SOCS3 signaling [102]. Furthermore, it has been revealed that impaired SOCS1/3 has a crucial role in the susceptibility of mammary epithelial cells to *S. aureus* infections. Additionally, a study reported a polymorphism in SOCS2, which was significantly associated with susceptibility to inflammation of the mammary gland [135]. *SOCS3* also has an inhibitory role in STAT5

regulation, which is one of the strong reasons for their influence on lactational performance in dairy cattle. Further study is highly recommended to find out the specific variants in *SOCS3* that interact with *STAT5* and *JAK2* during mastitis development and milk production in dairy cattle.

#### **6. Conclusions**

Altogether, it can be concluded that a delicate equilibrium must be achieved for the effective activation of the JAK/STAT pathway, when the immune system is needed for action against infection, and proper restoration when the infection is diminished. Thus, the JAK-STAT pathway can be considered as a therapeutic option in mastitis control and enhancement of milk production strategies. Furthermore, it is suggested that the interactive mechanism of *SOCS3*, *STATs*, and *JAK2*, *STAT5A*, and *STAT5B* during milk production and mastitis development should be considered in future rodent-knockout research models. It is highly recommended that further polymorphisms in STAT1 and *SOCS3* and their associations with milk production and mastitis resistance traits be found out. Finally, PTPs and PIAS are critical inhibitors of the JAK-STAT pathway, so research on the evaluation of their role in bovine mastitis would be an interesting development.

**Author Contributions:** Conceptualization, M.Z.K. and Z.C.; writing—original draft preparation, M.Z.K. and A.K.; writing—review and editing, M.Z.K., Y.M., J.G., J.X., J.M., and Z.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** The review is supported by the National Key Research and Development Program of China (2018YFD0501600). The funder has no role in study design, data collection, analysis, decision to publish, and preparation of the manuscript.

**Acknowledgments:** We acknowledge the financial support of the National Key Research and Development Program of China (2018YFD0501600). We also acknowledge China Agricultural University, Beijing, China, for providing us an environment of learning. Without this platform, the completion of this work would not have been an easy task.

**Conflicts of Interest:** The authors declare that they have no competing interests.

#### **References**


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