Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jul 25;14(8):901.
doi: 10.3390/biom14080901.

Activation of ARP2/3 and HSP70 Expression by Lipoteichoic Acid: Potential Bidirectional Regulation of Apoptosis in a Mastitis Inflammation Model

Bo Fang  1   2 , Tingji Yang  1   2 , Yan Chen  1   2 , Zhiwei Duan  1   2 , Junjie Hu  1   2 , Qi Wang  1   2 , Yuxuan He  1   2 , Yong Zhang  1   2 , Weitao Dong  1   2 , Quanwei Zhang  2   3 , Xingxu Zhao  1   2
Affiliations

Activation of ARP2/3 and HSP70 Expression by Lipoteichoic Acid: Potential Bidirectional Regulation of Apoptosis in a Mastitis Inflammation Model

Bo Fang et al. Biomolecules. .

Abstract

Mastitis typically arises from bacterial invasion, where host cell apoptosis significantly contributes to the inflammatory response. Gram-positive bacteria predominantly utilize the virulence factor lipoteichoic acid (LTA), which frequently leads to chronic breast infections, thereby impacting dairy production and animal husbandry adversely. This study employed LTA to develop models of mastitis in cow mammary gland cells and mice. Transcriptomic analysis identified 120 mRNAs associated with endocytosis and apoptosis pathways that were enriched in the LTA-induced inflammation of the Mammary Alveolar Cells-large T antigen (MAC-T), with numerous differential proteins also concentrated in the endocytosis pathway. Notably, actin-related protein 2/3 complex subunit 3 (ARPC3), actin-related protein 2/3 complex subunit 4 (ARPC4), and the heat shock protein 70 (HSP70) are closely related. STRING analysis revealed interactions among ARPC3, ARPC4, and HSP70 with components of the apoptosis pathway. Histological and molecular biological assessments confirmed that ARPC3, ARPC4, and HSP70 were mainly localized to the cell membrane of mammary epithelial cells. ARPC3 and ARPC4 are implicated in the mechanisms of bacterial invasion and the initiation of inflammation. Compared to the control group, the expression levels of these proteins were markedly increased, alongside the significant upregulation of apoptosis-related factors. While HSP70 appears to inhibit apoptosis and alleviate inflammation, its upregulation presents novel research opportunities. In conclusion, we deduced the development mechanism of ARPC3, ARPC4, and HSP70 in breast inflammation, laying the foundation for further exploring the interaction mechanism between the actin-related protein 2/3 (ARP2/3) complex and HSP70.

Keywords: ARPC3/4; HSP70; apoptosis; lipoteichoic acid; mastitis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
High expression of TNF-α/IL-1β/IL-6 accelerates the inflammation process in vivo and in vitro. (A) The neutrophil infiltration in HE staining and TNF-α/IL-1β/IL-6 IHC staining. FB, fibroblast; MEC, mammary epithelial cell; NEUT, neutrophil. The IHC panel is a high magnification (400×, scale 50 microns) image that corresponds to the HE panel (100×, scale 200 microns). (B) IHC scores of each group. (C,D) Quantification of relative mRNA expression of TNF-α, IL-1β, and IL-6, in mouse mammary tissue (C) and MAC-T (D). (E) Relative expression of TNF-α, IL-1β, and IL-6 protein in mouse mammary tissue. Data are means ± SEM (n = 3 per group). **, p < 0.01. Please see the original Western blot image in the Supplementary Material.
Figure 1
Figure 1
High expression of TNF-α/IL-1β/IL-6 accelerates the inflammation process in vivo and in vitro. (A) The neutrophil infiltration in HE staining and TNF-α/IL-1β/IL-6 IHC staining. FB, fibroblast; MEC, mammary epithelial cell; NEUT, neutrophil. The IHC panel is a high magnification (400×, scale 50 microns) image that corresponds to the HE panel (100×, scale 200 microns). (B) IHC scores of each group. (C,D) Quantification of relative mRNA expression of TNF-α, IL-1β, and IL-6, in mouse mammary tissue (C) and MAC-T (D). (E) Relative expression of TNF-α, IL-1β, and IL-6 protein in mouse mammary tissue. Data are means ± SEM (n = 3 per group). **, p < 0.01. Please see the original Western blot image in the Supplementary Material.
Figure 2
Figure 2
Analysis of transcriptomic data processed by LTA. (A) Partial pathway of GSEA. (B) Endocytosis, apoptosis, and S. aureus infection were enriched by GSEA. (C) The relationship predicted by STRING.
Figure 3
Figure 3
Inflammation enhanced the expression of ARPC3/ARPC4/HSP70. (A,B) Relative expressions of mRNA (A) and protein (B) of ARPC3, ARPC4, and HSP70 in mouse mammary tissue. (C) Colocation of ARPC3, ARPC4, and HSP70 in mouse mammary tissue. (D,E) Relative expression of mRNA (D) and protein (E) of ARPC3, ARPC4, and HSP70 in MAC-T. (F) Colocation of ARPC3, ARPC4, and HSP70 in MAC-T. Data are means ± SEM (n = 3 per group). *, p < 0.05; **, p < 0.01. Please see the original Western blot image in the Supplementary Material.
Figure 4
Figure 4
Apoptosis detection by TUNEL and FC. (A) Apoptosis detection by TUNEL in MAC-T. (B) Apoptosis assay by FC in MAC-T. **, p < 0.01.
Figure 5
Figure 5
Apoptosis was increased under the effect of LTA. (A) Quantification of relative mRNA expression of Caspase3, Caspase7, Caspase8, Bax, and Bcl-2 in mouse mammary tissue. (B) The relative expression of Caspase3, Caspase7, Caspase8, Bax, and Bcl-2 protein in mouse mammary tissue. (C) Quantification of relative mRNA expression of Caspase3, Caspase7, Caspase8, Bax, and Bcl-2 in MAC-T. (D) Relative expression of Caspase3, Caspase7, Caspase8, Bax, and Bcl-2 protein in MAC-T. Data are means ± SEM (n = 3 per group). *, p < 0.05; **, p < 0.01. Please see the original Western blot image in the Supplementary Material.
Figure 5
Figure 5
Apoptosis was increased under the effect of LTA. (A) Quantification of relative mRNA expression of Caspase3, Caspase7, Caspase8, Bax, and Bcl-2 in mouse mammary tissue. (B) The relative expression of Caspase3, Caspase7, Caspase8, Bax, and Bcl-2 protein in mouse mammary tissue. (C) Quantification of relative mRNA expression of Caspase3, Caspase7, Caspase8, Bax, and Bcl-2 in MAC-T. (D) Relative expression of Caspase3, Caspase7, Caspase8, Bax, and Bcl-2 protein in MAC-T. Data are means ± SEM (n = 3 per group). *, p < 0.05; **, p < 0.01. Please see the original Western blot image in the Supplementary Material.
Figure 6
Figure 6
Model pattern of LTA-induced apoptosis via ARPC3/ARPC4/HSP70. LTA modulated ARPC3 and ARPC4 expression in the endocytic pathway inducing cell apoptosis and activating HSP70 to mitigate sustained host cell innate immunity. In addition, LTA can also induce the inflammatory response by activating the TLR2/PI3K-AKT/NF-κB pathway.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Cobirka M., Tancin V., Slama P. Epidemiology and Classification of Mastitis. Animals. 2020;10:2212. doi: 10.3390/ani10122212. - DOI - PMC - PubMed
    1. Magro G., Biffani S., Minozzi G., Ehricht R., Monecke S., Luini M., Piccinini R. Virulence Genes of S. aureus from Dairy Cow Mastitis and Contagiousness Risk. Toxins. 2017;9:195. doi: 10.3390/toxins9060195. - DOI - PMC - PubMed
    1. Günther J., Czabanska A., Bauer I., Leigh J.A., Holst O., Seyfert H.-M. Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages. Vet. Res. 2016;47:13–27. doi: 10.1186/s13567-015-0287-8. - DOI - PMC - PubMed
    1. Evans M.D., McDowell S.A. Pleiotropic effects of statins: New therapeutic approaches to chronic, recurrent infection by Staphylococcus aureus. Pharmaceutics. 2021;13:2047. doi: 10.3390/pharmaceutics13122047. - DOI - PMC - PubMed
    1. Ireton K., Payrastre B., Chap H., Ogawa W., Sakaue H., Kasuga M., Cossart P. A role for phosphoinositide 3-kinase in bacterial invasion. Science. 1996;274:780–782. doi: 10.1126/science.274.5288.780. - DOI - PubMed

MeSH terms

LinkOut - more resources