Epithelial processed *Mycobacterium avium* subsp. *paratuberculosis* induced prolonged Th17 response and suppression of phagocytic maturation in bovine peripheral blood mononuclear cells

The study investigates how epithelial processing of Mycobacterium avium subsp. paratuberculosis (MAP) influences the early host immune response, with a focus on mechanisms underlying MAP persistence. Johne’s disease has a long subclinical period with intermittent shedding, complicating diagnosis; persistence is linked to immune evasion and shifts in host responses. Prior work suggests that early Th17 responses may be critical in MAP infection, yet initial events after epithelial traversal are not well characterized. The authors hypothesize that MAP processed by epithelial cells modulates immune responses in peripheral blood mononuclear cells (PBMCs), potentially inducing prolonged Th17 activity and suppressing phagocyte maturation, thereby contributing to pathogen persistence.

Previous studies have highlighted limitations of single-cell in vitro models and the complexity of in vivo infection, where MAP first crosses the intestinal epithelium via M cells or enterocytes before encountering macrophages and dendritic cells. Traditional Th1-to-Th2 shifts do not fully explain MAP persistence; accumulating evidence emphasizes early Th17-mediated responses in MAP infection. Epithelial processing of MAP has been shown to alter bacterial phenotypes, including increased invasiveness and changes in lipid biosynthesis and iron assimilation pathways during epithelial infection. Transcriptomic studies in cattle and cell models identified dysregulation of chemokine/cytokine pathways and host lipid metabolism during MAP infection. These observations support examining epithelial passage effects on subsequent immune cell responses.

- Study design: An in vitro epithelial passage model was used to compare bovine PBMC responses to native MAP (T1) versus MAP after infection and recovery from bovine epithelial cells (MDBK; T2). Time points: 24 h, 72 h, and 120 h post-infection (p.i.). RNA-seq was conducted at 24 h and 72 h; qPCR and ELISA extended to 120 h. - Bacteria: MAP ATCC19698 cultured in Middlebrook 7H9 with Mycobactin J, Casitone, glycerol, and OADC at 37 °C. Aliquoted at OD600≈1.0 and stored at −80 °C. - Cells: MDBK epithelial cells cultured in DMEM + 10% heat-inactivated FBS. PBMCs isolated from a JD-negative Holstein cow by density gradient centrifugation, then cultured in RPMI 1640 + 20% FBS. - Epithelial passage: MDBK (5×10^5 cells/well) infected with MAP for 4 h (MOI 20:1). Intracellular bacteria recovered by Triton X-100 lysis and differential centrifugation to remove cellular debris. PBMCs (5×10^6 cells/well) infected with native MAP (T1) or MDBK-processed MAP (T2) at MOI 0.1:1 for 24, 72, and 120 h. - RNA extraction: RNeasy Mini Kit with bead beating to disrupt MAP. Purity assessed by A260/230 and A260/280. - RNA-seq: Libraries (RIN>7.0) prepared with TruSeq Stranded mRNA kit; NovaSeq 6000 sequencing. Reads mapped with TopHat; expression quantified with Cufflinks v2.1.1 using multiread-correction and frag-bias-correction. - Differential expression and pathway analysis: DEGs defined vs time-matched uninfected controls (fold-change ≥2 or ≤0.5; p<0.05). Canonical pathway analyses performed using Ingenuity Pathway Analysis (IPA), including p-value and z-score assessments. - qPCR: Targets IL-17A, IL-17F, IL-23p19, IFNG, IL-6, RORC at 24, 72, 120 h; normalization to GAPDH; 2^−ΔΔCT method. MAP viability verified via sigA qPCR. - ELISA: IL-17a quantified in PBMC supernatants at 72 h and 120 h. - Statistics: ANOVA with Tukey’s multiple comparisons (p<0.05) for qPCR and ELISA; biological triplicates. - Ethics: Approved by institutional committees in compliance with Korean regulations. - Data availability: GEO GSE149494.

- Epithelial processing of MAP induces prolonged Th17 responses in PBMCs: - IL-17A and IL-17F were among the most upregulated T cell-related genes and remained elevated longer in T2 (MDBK-processed MAP) than T1 (native MAP), with persistence up to 120 h p.i. by qPCR. - RORC, a Th17 lineage transcription factor, was upregulated at 72 h in both groups but maintained or further increased at 120 h in T2 while decreasing in T1. - ELISA confirmed higher IL-17a secretion in T2 versus T1 at 120 h p.i., with both groups elevated over controls at 72 h and 120 h. - Some upstream cytokines typically associated with Th17 polarization (e.g., IL-6) were downregulated at 72 h, whereas IL-1B and IL-23A did not increase until later, suggesting complex regulation. - Global suppression of immune pathways at 72 h p.i.: - Canonical pathway z-scores indicated broad suppression at 72 h in both T1 and T2, despite early activation (e.g., HMGB1 signaling activated at 24 h then suppressed at 72 h). - Pathways such as Th17 activation and Role of IL-17F remained activated across time points, while TREM1 signaling, Dendritic cell maturation, and pattern recognition receptor pathways trended toward suppression. - Downregulation of phagocytic cell surface receptors at 72 h p.i.: - Reduced expression of TLR2, TLR4, MHC class II (e.g., HLA-DQ/DR bovine orthologs), CD40, Fcγ receptors, TREM1/TREM2, DAP12/TYROBP. - IPA predicted downstream suppression of dendritic cell maturation and TREM1 signaling. - Manipulation of lipid/cholesterol metabolism: - LXR/RXR signaling pathway was downregulated in both T1 and T2 at 72 h p.i. - Cholesterol efflux transporters ABCA1, ABCG1, and APOE were downregulated; additional lipid metabolism genes (CD36, CYP27A1, LDLR) decreased, consistent with cholesterol accumulation and altered lipid handling. - Between-group analysis suggested T2 may engage relatively more protective LXR/RXR-related responses than T1, despite overall suppression versus controls. - DEG dynamics: - More genes were downregulated than upregulated at 72 h p.i. in both groups; divergence between T1 and T2 increased over time, indicating epithelial processing shapes temporal immune transcriptomics.

The findings support the hypothesis that epithelial processing of MAP modifies subsequent immune interactions, leading to a prolonged Th17-skewed response in PBMCs. Sustained IL-17A/F and RORC expression and increased IL-17a secretion in T2 imply that epithelial passage imprints MAP to trigger extended Th17 activity, a response implicated in granuloma formation and containment of mycobacteria. Concurrently, broad downregulation of phagocyte surface receptors (TLRs, MHC II, TREM, FcγR) and maturation pathways at 72 h p.i. indicates impaired antigen recognition, processing, and presentation, potentially dampening Th1 responses (e.g., reduced IL-1β, IL-6, IL-12, IL-18, IFNG) and facilitating persistence. Suppression of LXR/RXR signaling and cholesterol efflux transporters suggests MAP-driven reprogramming of host lipid metabolism, favoring intracellular survival via cholesterol accumulation and disrupted homeostasis. Collectively, these adjustments provide a mechanistic framework for early MAP persistence: epithelial processing prolongs Th17 signaling while MAP concurrently suppresses phagocytic maturation and lipid regulatory pathways, balancing pathogen survival with moderated host inflammation.

This study introduces an epithelial passage infection model demonstrating that epithelial-processed MAP elicits prolonged Th17 responses in bovine PBMCs, as evidenced by sustained IL-17A/F and RORC expression and elevated IL-17a secretion. In parallel, MAP broadly suppresses phagocyte surface receptors and maturation pathways and downregulates LXR/RXR-mediated cholesterol efflux, mechanisms that likely facilitate intracellular persistence. These insights clarify early host–pathogen interactions relevant to Johne’s disease pathogenesis and may inform biomarkers or interventions targeting Th17 dynamics and lipid metabolism. Future research should: (1) include earlier time points (e.g., 4–8 h p.i.) to capture initial Th17 polarization triggers; (2) dissect bacterial determinants altered by epithelial processing, including antigenic modifications; (3) validate findings in vivo and across diverse hosts; and (4) assess therapeutic modulation of LXR/RXR and phagocytic receptor pathways.

- In vitro model may not fully recapitulate in vivo intestinal microenvironments and multicellular granulomatous contexts. - PBMCs were sourced from a single JD-negative Holstein cow, limiting generalizability and not accounting for inter-animal variability. - RNA-seq was performed only at 24 h and 72 h p.i.; 120 h assessments relied on qPCR/ELISA, potentially missing transcriptomic changes beyond 72 h. - Early polarization events (prior to 24 h) were not captured, limiting mechanistic resolution of Th17 induction. - The study focused on host transcriptomics; direct profiling of MAP phenotypic changes after epithelial processing was not performed.