In poultry respiratory system, trachea acts as the air channel for gas exchange and composes the first line of defense against pathogenic microorganisms[21], factors that make it a target of pathogenic attack such as MG. MG infection mainly causes tracheal mucosal epithelial injury, which destroys the permeability and stability of the mucosa, leading to the entry of a large amount of tissue fluid into the tracheal and alveolar spaces, ultimately causing pulmonary ventilation/ventilation dysfunction in chicks and the phenomenon of respiratory distress[22, 23]. Thus, intranasal infection of MG was used in this study. The effects of MG infection on the structural integrity of the tracheal mucosal epithelium and the function of intercellular tight junctions in chickens were studied in detail.
During the animal experiment, by bacterial counting analysis the bacterial load in the trachea of chickens revealed that MG proliferated in the trachea of chicks after inoculation, and the bacterial load in the tracheal tissue samples showed an increasing trend with the extension of infection time during the 15 day post-infection, and caused air sac lesions in chickens. In addition, MG infection impaired the structural integrity of the tracheal mucosal epithelium in chickens. Histopathological examination showed that the mucosal epithelium of the infected group had different degrees of shedding, hyperplasia, and thickening of the submucosal tissue, and the infiltration of inflammatory cells led to the expansion of the lamina propria, which was consistent with the results of previous studies on MG. MohammedAetal. ( 2005), found that challenge of the trachea of chicks using the Mycoplasma gallisepticum R low strain resulted in infiltration of a large number of lymphoid cells (CD4 + and CD8 +) as well as plasma cells secreted by immunoglobulins (IgA and IgG), resulting in expansion of the lamina propria[24]. Based on these findings, it can be speculated that MG infection can cause damage to the tracheal mucosal epithelium and disrupt the normal function of the tracheal mucosal barrier. However, the underlying mechanism of host inflammatory responses to this bacterial infection is still elusive. Thus, the DEGs in the ctracheal of MG- infected chickens are explored in this study with RNA-seq to understand its pathogenic mechanism.
In the RNAseq data analysis, a total of 3112 significant (p < 0.05) DEGs were selected between MG-infection group and control group, including 1646 up-regulated genes and 1466 down-regulated genes. From the results of the GO enrichment analysis, there were more genes in the subcategories involved in the defense response and mechanical barrier of tracheal mucosa. In parallel, cytokine and chemokine activities were enhanced. A previous study using high-throughput sequencing to study the effect of MG on the immune pathway in chicks, it was found that the expression of genes encoding proteins with immune-related functions increased after Mycoplasma gallisepticum R low strain infection, causing high levels of cellular stress and further promoting immune cell infiltration. In this study, the expression of various inflammatory factors, including collagen alpha-2 (XI) chain, fibrinogen alpha chain (FGA), dermatan sulfate proteoglycan (EPYC), folate receptor 1 (FOLR1), TNF-α, IL-6, IL-1β, and CCL20, was increased in the trachea of MG-infected chicks. Collagen alpha-2 (XI) is the most abundant fibrous protein in the extracellular matrix and has a role in regulating inflammatory cell in inflammation models[25]. Which was consistent with the results that was observed through the histochemical analysis. EPYC and FGA play a vital role in a variety of pathophysiological processes, such as cell migration and inflammation, which increased during the inflammatory response and participate in persistent inflammation[26]. FOLR1 plays a crucial role in inflammation and its level is reported increased during the inflammatory response. TNF-α is a cytokine mainly secreted by monocytes and macrophages after activation by inflammation. By binding to its specific receptor, it regulates the metabolic activity of other tissues and impairs the function of endothelial cells[27]. IL-6 is a multipotent cytokine with a wide range of functions, which has the function of regulating immune response, and plays an important role in the anti-infective immune response of the body[28]. IL-1β is an important inflammatory factor, which participates in the body's immune response, induces apoptosis and directly kills target cells, etc[29]. CCL20 is able to control immune cell-directed chemotaxis by interacting with G protein-linked transmembrane receptors and is involved in the inflammatory pathology of many diseases. Therefore, the up-regulated pro-inflammatory factors and cytokines after exposure to MG may play a role in the pathogenesis of MG-induced tracheal mucosal epithelial injury.
Previous studies reported that invasion of the body by external pathogens activates local immunity in the tracheal mucosa, leading to the development of inflammation and disrupting the respiratory mucosal barrier[30]. In this study, we found that several genes involved in constituting the structural basis of the mucosal barrier were down-regulated, including CRNN, claudins, ZO-1, and occludin. CRNN has been shown to play an important role in mucosal epithelial immune response and epidermal differentiation[31]. Occludin is an integral type II transmembrane protein that plays an important role in maintaining the integrity of tight junction structures as well as the barrier function of the intestinal epithelium[32]. Claudins, another class of transmembrane proteins involved in tight junctions discovered after occludin, constituting the major skeleton proteins of tight junctions[33]. ZO-1, as a peripheral membrane protein, has the functions of connecting transmembrane proteins with the cytoskeleton and transmitting signaling molecules, regulating and transporting intracellular substances, maintaining the polarity of epithelial cells, and is committed to constituting a stable junctional system[34]. These findings suggest that MG infection may lead to mucosal mechanical barrier dysfunction in chicks, decreased defense, and make chicks more susceptible to infection by other respiratory diseases.
The mechanical barrier of tracheal mucosa is composed of the connection between tracheal mucosal epithelial cells and adjacent cells, which is the most important link in tracheal mucosal immunity and can effectively prevent the passage of macromolecules, especially inhibit the route of bacterial and toxin diffusion through the tracheal mucosa into the body[35]. MG infection stimulates the respiratory mucosal immune system and increases the permeability of the tight junctions between tracheal mucosal epithelial cells. This is a key pathological process that causes chronic respiratory diseases in chickens[36, 37]. From the results of KEGG pathway analysis of the two samples, it can be seen that the regulatory mechanism of tight junction proteins can explain MG-induced mechanical barrier damage in the tracheal mucosa of chicks. Lipschutz, J.H. (Fig. 2005), a survey conducted observed that MAPK signaling pathways are able to achieve modulation of tight junction-selective barrier function by up-regulating or decreasing the expression of key proteins of tight junctions[38]. As found, stimulation of MDCK cells with cytokines (TNFα and IFNγ) significantly activated ERK1/2 and led to decreased expression of the tight junction proteins occludin, ZO-1, and claudin-1. In the presence of TNFα/IFNγ Treatment with ERK1/2 inhibitors enhances the expression of occludin and claudin-1 at the junction interface. The expression of TNFα was significantly up-regulated after MG infection, which activated the ERK-MLCK signal transduction pathway, and then the phosphorylation level of ERK1/2 was up-regulated, which translocated from the cytoplasm into the nucleus. The activated transcription factor c-fos then up-regulates the expression and activity of MLCK, regulates the distribution of TJ in tracheal epithelial cells and significantly down-regulates the major structural proteins Claudin-1, Occludin, ZO-1 and JAM4. The process may be the mechanism by which MG infection damages the tracheal mucosal epithelium.
Also, FISH with specific probe of LmCq showed a large amount of red fluorescent signals presence in the chicken embryo tracheal ring tissue. LmCq was detected widely in the mucosal epithelium, submucosa, and adventitial regions. This indicates that MG infection reduces the selective permeability of tracheal mucosal epithelium and its barrier function, provides a channel for harmful substances such as bacteria, histamine and endotoxin to enter the tissue, forms a cascade amplification effect, which further strengthens the damage to the tracheal mucosa.
To further confirm whether the transcriptome data in this study is reliable, and to explore the effects of MG infection on the mechanical barrier function of chicken tracheal mucosal epithelium, we used qRT-PCR to detect the activation of ERK-MLCK signaling pathway in tracheal epithelial cells by MG infection, and immunohistochemical analysis to assess the levels of claudin-1, occludin, and ZO-1 in each group. Results showed that the expression of related genes in the ERK-MCLK signaling pathway was significantly increased after MG infection of the trachea in chickens. And the levels of tight junction proteins (claudin-1, occludin, ZO-1) in vivo was significantly reduced after activation of the ERK-MCLK signaling pathway. The results were consistent with the transcriptome data obtained. Ras, as an upstream protein of the ERK-MLCK signaling pathway, is activated by cytokines (TNF-α), which increases the level of ERK1/2 phosphorylation and activates MLCK. The activated MLCK can cause phosphorylation of MLC, which is closely related to the increase of permeability of epithelial cells. Therefore, we speculated that MG infection would induce the secretion of cytokines (TNF-α) to activate the ERK-MCLK signaling pathway, which is the cause of tracheal mucosal epithelial injury in MG-infected chicks. Our results suggest that the regulation of TJ protein in tracheal epithelial cells by TNF-α-ERK-MLCK signal transduction pathway after MG infection caused damage to the chicken tracheal mucosal epithelium. Nevertheless, the underlying mechanism of tight junction protein regulation induced by Mycoplasma gallisepticum infection need to be further studied.