Host responses to persistent Mycobacterium avium subspecies paratuberculosis infection in surgically isolated bovine ileal segments

Abstract

A lack of appropriate disease models has limited our understanding of the pathogenesis of persistent enteric infections with Mycobacterium avium subsp. paratuberculosis. A model was developed for the controlled delivery of a defined dose of M. avium subsp. paratuberculosis to surgically isolated ileal segments in newborn calves. The stable intestinal segments enabled the characterization of host responses to persistent M. avium subsp. paratuberculosis infections after a 9-month period, including an analysis of local mucosal immune responses relative to an adjacent uninfected intestinal compartment. M. avium subsp. paratuberculosis remained localized at the initial site of intestinal infection and was not detected by PCR in the mesenteric lymph node. M. avium subsp. paratuberculosis-specific T cell proliferative responses included both CD4 and γδ T cell receptor (γδTcR) T cell responses in the draining mesenteric lymph node. The levels of CD8(+) and γδTcR(+) T cells increased significantly (P < 0.05) in the lamina propria, and M. avium subsp. paratuberculosis-specific tumor necrosis factor alpha (TNF-α) and gamma interferon secretion by lamina propria leukocytes was also significantly (P < 0.05) increased. There was a significant (P < 0.05) accumulation of macrophages and dendritic cells (DCs) in the lamina propria, but the expression of mucosal toll-like receptors 1 through 10 was not significantly changed by M. avium subsp. paratuberculosis infection. In conclusion, surgically isolated ileal segments provided a model system for the establishment of a persistent and localized enteric M. avium subsp. paratuberculosis infection in cattle and facilitated the analysis of M. avium subsp. paratuberculosis-specific changes in mucosal leukocyte phenotype and function. The accumulation of DC subpopulations in the lamina propria suggests that further investigation of mucosal DCs may provide insight into host responses to M. avium subsp. paratuberculosis infection and improve vaccine strategies to prevent M. avium subsp. paratuberculosis infection.

Fig 1

Gross anatomy and histopathology of noninfected and M. avium subsp. paratuberculosis-infected ileal compartments. (A) Gross appearance of an intestinal segment in situ 9 months after infection with M. avium subsp. paratuberculosis. The surgically isolated intestinal segment was subdivided into three compartments with silk ligatures (red arrows): compartment 1, control (proximal noninfected compartment); compartment 2, interspace (intervening compartment); compartment 3, M. avium subsp. paratuberculosis (distal M. avium subsp. paratuberculosis-infected compartment). The site where intestine proximal and distal to the isolated segment was anastomosed is visible (blue arrow) in the adjacent ileum. The major demarcations on the ruler are 1 cm apart. (B) Hematoxylin-and-eosin-stained tissue section from a noninfected compartment. (C) Hematoxylin-and-eosin-stained tissue section from an M. avium subsp. paratuberculosis-infected compartment with cellular aggregates in the LP (boxed). (D) Cellular aggregate in the LP of an M. avium subsp. paratuberculosis-infected compartment (boxed in panel C), with eosinophils (arrows) surrounding and infiltrating the cellular aggregate.

Fig 2

PCR detection of M. avium subsp. paratuberculosis infection in surgically isolated ileal segments and associated lymph nodes. PCR analysis was performed for all animals (n = 6) by using DNA isolated from formalin-fixed tissue collected from the M. avium subsp. paratuberculosis-infected and noninfected (control) compartments within each surgically isolated ileal segment. Mesenteric lymph nodes located in the mesentery adjacent to each isolated intestinal segment were also sampled.

Fig 3

Comparison of LPL population changes in M. avium subsp. paratuberculosis-infected and noninfected ileal compartments. Enzymatic digestion of 10-cm segments of ileum was performed to isolate LPLs, and the total number of cells isolated was recorded for both M. avium subsp. paratuberculosis-infected and noninfected ileal compartments. The total number of LPLs isolated/10 cm of tissue was calculated by multiplying the total number of cells recovered by the percentage of CD45⁺ cells. (A) Myeloid cells in the LPL populations were identified as CD45⁺ CD11c⁺, and the frequency of individual myeloid cell subpopulations was analyzed for cells coexpressing major histocompatibility complex class II (MHC II), CD11b, CD13, CD14, CD26, CD172a, and CD205. (B) CD45⁺ leukocytes were analyzed for coexpression of CD3, CD4, CD8, γδTcR, and CD335. The data presented are means and 1 SD for values for the M. avium subsp. paratuberculosis-infected (filled bars) and noninfected (open bars) compartments within the same animals (n = 5). *, P < 0.05.

Fig 4

TLR gene expression in M. avium subsp. paratuberculosis-infected and noninfected ileal compartments. Expression of tlr-1 through tlr-10 in ileal tissues collected from M. avium subsp. paratuberculosis-infected and noninfected compartments (n = 3) was quantified by qRT-PCR. TLR expression levels are expressed as the change in the cycle threshold (ΔC_T) relative to the C_T of the housekeeping gene β-actin. The ΔC_T was calculated by subtracting the C_T value for β-actin from the C_T value for each TLR. The data presented are means and 1 SD for values from the three animals. Lower ΔC_T values represent increased expression levels for individual TLR genes.

Fig 5

Comparison of M. avium subsp. paratuberculosis-specific LPRs in PBMCs, mLN cells, and LPLs. Cells were isolated from calves between 9 to 11 months postinfection and were restimulated with the M. avium subsp. paratuberculosis lysate in vitro. Data are shown for cells isolated from mLNs draining each intestinal segment, and LPLs were isolated from the M. avium subsp. paratuberculosis-infected compartment within each intestinal segment. M. avium subsp. paratuberculosis-specific LPRs (SI, ≥2.5) (dashed horizontal line) were observed only in mLNs (4/6) and PBMCs (2/6), not in LPLs.

Fig 6

M. avium subsp. paratuberculosis-specific TNF-α and IFN-γ secretion by LPLs, mLN cells, and PBMCs at 9 to 11 months postinfection. LPLs, mLN cells, and PBMCs (5 × 10⁵ cells/well) were isolated at 9 to 11 months postinfection and were restimulated with the M. avium subsp. paratuberculosis lysate in vitro for 48 h, and culture supernatants were assayed for TNF-α and IFN-γ. (A) LPLs isolated from M. avium subsp. paratuberculosis-infected compartments secreted significantly higher levels of TNF-α (P < 0.05) than did LPLs from adjacent noninfected ileal compartments (Cntrl). (B) LPLs from M. avium subsp. paratuberculosis-infected compartments and cells isolated from mLNs draining each intestinal segment secreted significantly (P < 0.05) higher levels of IFN-γ than did LPLs isolated from noninfected ileal compartments and PBMCs. Data presented are means and 1 SD for values from 6 animals. *, P < 0.05; **, P < 0.01.

Fig 7

mLN T cell subpopulations proliferating in vitro following restimulation with the M. avium subsp. paratuberculosis lysate. mLN leukocytes were labeled with PKH-26 prior to culture in the presence (MAP) or absence (media) of the M. avium subsp. paratuberculosis lysate. Cultured leukocytes were recovered after 72 h of culture and were labeled with MAbs to identify CD4, CD8, and γδTcR T cell subpopulations. Individual T cell subpopulations were gated and were analyzed for changes in PKH-26 fluorescence intensity using ModFit cell cycle analysis. The data presented are representative of results from the four animals displaying positive LPRs in Fig. 5. Cell cycle analysis revealed that both CD4 and γδTcR T cells progressed through multiple cell cycles in the presence of the M. avium subsp. paratuberculosis lysate.