Date Published: April 15, 2019
Publisher: Public Library of Science
Author(s): Ning Zhang, Peng Gao, Bao Yin, Jiahe Li, Tong Wu, Yu Kuang, Wenxue Wu, Jinxiang Li, Paulo Lee Ho.
Cathepsin L (CTSL) has been proved to help contain leishmaniasis and mycoplasma infection in mice by supporting cellular immune responses, but the regulatory functions of CTSL on mucosal immune responses haven’t been tested and remain undefined. Here, we investigated the effects of CTSL on SIgA responses and invariant chain (Ii) degradations in the co-cultured swine dendritic cells (DCs) and B cells system in vitro. When the cells system were transfected with vector CTSL-GFP or incubated with recombinant CTSL (rCTSL) before they were infected with Mycoplasma hyopneumoniae (M.hp), SIgA significantly increased and Ii chain was degraded into smaller intermediates, while SIgA decreased when CTSL was knockdown or inhibited with E64. To confirm the SIgA responses promoted by CTSL contribute to the resistance to mycoplasma pneumonia, pigs injected with rCTSL before they were challenged with M.hp, showed milder clinical symptoms and histopathological damage of lungs, less mycoplasma burden together with higher secretion of SIgA, percentages of CD4+ T cells and level of MHC II molecules comparing with the group without rCTSL. Collectively, these results suggested that rCTSL could provide effective protection for piglets against mycoplasma pneumonia by enhancing M.hp-specific mucosal immune responses through its role in antigen presentation by processing the invariant chain.
Mucosal surfaces of the respiratory tract and gastrointestinal tract are the first line of defense to prevent the infection of pathogens and clear the evaded microorganism by excreting active components including secretory immunoglobulin A (SIgA) [1, 2]. Experimental evidence showed that SIgA response provides effective protection especially for the cases of chronic mucosal infections and reinfections of many bacteria, virus, parasites and fungi [3–6]. Mycoplasma, unlike virus or bacteria, leads infection by firmly adhering to respiratory epithelial cells. To clear mycoplasma, mucosal immunity instead of systemic immunity plays a key role during infection . And SIgA is the first sign of mycoplasma infection which raises the strategy considerations about the induction of SIgA by vaccination [8, 9].
It has been proved that the antigen-specific SIgA induced by pathogens or vaccines, constitutes a first-line defense and confers to effective protections against mucosal infectious diseases, primarily by immune exclusion of viral or bacterial pathogens or neutralizing microbial toxins and pathogens [43–45]. In this study, the levels of SIgA in pig BALFs increased significantly at 21 DPI (Fig 5) while almost no IgG and IgM were detected in sera (S3A Fig), indicating mucosal immune was the main immune responses to Mycoplasma infections as reported previously [46, 47]. In airways, antigen-specific SIgA is released by epithelial cells after the association of secretory component (SC) with polymeric IgA, which is produced by IgA-secreting plasma cells differentiated from IgA+ effector B cells. The latter originates from the B cells undergone IgA class-switch recombination (CSR) in response to CD40 ligand (CD40L) and transforming growth factor-β1 (TGFβ1) from activated T cells. Interleukin-5 (IL-5), IL-6 and IL-10, as well as B-cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) from DCs are required for the expression of IgA, and the differentiation could be enhanced by APRIL secreted by epithelial cells and DCs . In vitro studies with human and mouse cells have shown that the interaction between B cells and DCs is required to achieve a successful IgA class switch, DCs play a critical role in B cell development and B lymphocytes have an important regulatory effect on the Ag-presenting function of DCs in vitro and in vivo [10, 49, 50]. CTSL is widespread in macrophages derived from different tissues with cathepsin S proteins in their active forms, but its immune related effect in macrophages was not as striking as in DCs and B cells. In this study, stronger responses of CTSL were present in DCs rather than macrophages after challenged with M.hp, implying DCs+B cells coculturing is a preferred model to study the correlation between CTSL and IgA responses of pigs in vitro when transgenic pigs are unavailable. In this report, SIgA response was induced obviously in the cocultured B cells-DCs after infected with M.hp, and SIgA levels varied in consistent with the changes of CTSL activity by knockdown with CRISPR/Cas9 plasmid, overexpression with CTSL-GFP or inhibited with E64, although E64 could react with some other cysteine proteases such as Cat B, Cat H and Cat S (Fig 2B and 2D). These results revealed that CTSL play a major part in promoting the SIgA response of cocultured DCs +B cells to M.hp infection.
In conclusion, CTSL plays crucial roles in innate and adaptive immune response and may bring about different and even opposite effects on the infections depending on the pathogens. Our results suggested that CTSL provide obvious protection against M.hp for pigs through promoting mucosal immune with definite actions on facilitating MHC class II peptide presentation and CD4+T lymphocyte differentiation, although further studies should be undertaken to clarify the exact mechanisms considering the complexity of mucosal immune system and CTSL involved in many pathologic process.