Johne's disease (JD), caused by *Mycobacterium avium* subsp. *paratuberculosis* (MAP), is a chronic granulomatous enteropathy in ruminants, resulting in significant economic losses. The long incubation period (over two years) and intermittent fecal shedding of MAP make diagnosis challenging. Current diagnostic methods, such as serum ELISA and fecal PCR, have limitations in sensitivity and accuracy. The persistence of MAP, evading host immune mechanisms during early infection, is a key factor in the disease's chronic nature. While Th1/Th2 immune response shifts have been implicated, recent studies suggest a more significant role for Th17 responses. Previous in vitro studies using single cell types have been unable to capture the complexity of the infection process. In reality, MAP enters the host intestine via M cells or enterocytes, before encountering immune cells. An epithelial passage model can better mimic the early stages of MAP infection. Studies have already shown that MAP infection in bovine epithelial cells (MAC-T cells) show an increased invasive phenotype during secondary infection, highlighting the importance of the epithelial barrier. Other research demonstrated that MAP that has passed through epithelial cells activates different iron assimilation pathways and upregulates genes associated with lipid uptake. This study aims to identify pathogenic mechanisms by comparing gene expression in bovine PBMCs infected with MAP directly versus after passage through epithelial cells (MDBK cells).

Numerous studies have explored the host immune response to MAP infection. These studies used various approaches, including in vitro and in vivo models, to investigate the immune mechanisms involved in MAP persistence. In vivo studies often face challenges in elucidating the early stages of infection before granuloma formation. In contrast, in vitro studies often simplify the infection process by utilizing single cell types, failing to account for the complex interactions between different host cell populations, like epithelial cells and immune cells. Research has shown that MAP can survive within non-activated phagocytes by inhibiting phagosome-lysosome maturation. Activated macrophages and DCs produce cytokines that recruit other immune cells, leading to granuloma formation and persistence of MAP. The shift from Th1 to Th2 responses has been associated with disease progression, but recent focus has been on the early Th17 response. Previous work revealed that downregulation of Th17-related cytokines (IL-17A, IL-17F, IL-26) and alterations in granuloma integrity contribute to fecal shedding and pathogen dissemination. The role of epithelial cells in early MAP infection has been increasingly recognized, with evidence suggesting that epithelial-processed MAP has altered virulence and elicits distinct immune responses.

This study employed an epithelial passage model to mimic the in vivo infection process. MDBK cells were infected with MAP (MOI 20:1) for 4 hours. Live MAP was then isolated after lysing the MDBK cells. Bovine PBMCs were isolated from a JD-negative Holstein cow and infected with either native MAP (group T1) or MDBK-processed MAP (group T2) at an MOI of 0.1:1 for 24, 72, and 120 hours. RNA was extracted from PBMCs at 24 and 72 hours post-infection and subjected to RNA-seq analysis. Differential gene expression analysis was performed comparing infected samples to non-infected controls. Canonical pathway analysis was conducted using the Ingenuity Pathway Analysis (IPA) tool to identify significant pathways affected by MAP infection. Quantitative PCR (qPCR) was used to validate RNA-seq findings for Th17-related genes (IL-17A, IL-17F, IL-23p19, IFN-γ, IL-6, RORC) at 24, 72, and 120 hours post-infection. An ELISA was performed to quantify IL-17a secretion in culture supernatants at 72 and 120 hours post-infection. Statistical significance was determined using ANOVA with Tukey's multiple comparisons test.

RNA-seq analysis revealed that more genes were downregulated than upregulated in both groups (T1 and T2) at 72 hours post-infection. Canonical pathway analysis identified "Granulocyte Adhesion and Diapedesis," "T helper cell differentiation," and "Role of pattern recognition receptors" as significantly affected pathways. The Th17 activation pathway was activated at all time points. Notably, epithelial-processed MAP (group T2) induced a prolonged Th17 response, with significantly higher expression of IL-17A and IL-17F at 120 hours post-infection compared to direct MAP infection (group T1). This prolonged Th17 response was confirmed by qPCR and ELISA. Downregulation of cell surface receptors (MHC class II, TLR2, TLR4, CD40, FcyR, TREM2) was observed in both groups, particularly at 72 hours post-infection, suggesting a mechanism for MAP immune evasion. Suppression of the LXR/RXR pathway, involved in cholesterol metabolism, was also observed in both groups, indicating a potential mechanism for MAP survival by altering host lipid metabolism. Genes involved in cholesterol transport (ABCA1, ABCG1, APOE) were downregulated.

This study's findings highlight the importance of the epithelial barrier in MAP infection and its impact on host immune responses. The prolonged Th17 response induced by epithelial-processed MAP suggests a potential role for this pathway in both MAP persistence and host defense against excessive inflammation. The observed downregulation of cell surface receptors in phagocytes suggests a mechanism by which MAP evades immune recognition and killing. The suppression of the LXR/RXR pathway, influencing cholesterol metabolism, likely contributes to MAP intracellular survival and replication. These findings provide valuable insights into the complex interplay between MAP and the bovine immune system, emphasizing the role of epithelial processing in shaping the infection course.

This study demonstrates that epithelial processing of MAP significantly influences the host immune response, leading to a prolonged Th17 response and suppression of phagocytic maturation. These findings suggest dual mechanisms for MAP persistence: prolonged Th17-mediated inflammation and evasion of the host's innate immune system. Future research should focus on identifying the specific bacterial factors involved in the altered immune response to epithelial-processed MAP and clarifying whether the observed Th17 response is primarily a protective or pathogenic mechanism.

This study was conducted using an in vitro model, which might not fully replicate the complexities of in vivo infection. The use of a single bovine source for PBMCs limits the generalizability of the results. Further research with a larger sample size and multiple animal sources is warranted. The precise mechanism by which epithelial processing alters MAP's ability to induce a prolonged Th17 response remains to be fully elucidated. More detailed investigation into the bacterial factors contributing to this phenomenon is necessary.