Infection with Mycobacterium avium subsp. paratuberculosis results in rapid interleukin-1β release and macrophage transepithelial migration

Abstract

Pathogen processing by the intestinal epithelium involves a dynamic innate immune response initiated by pathogen-epithelial cell cross talk. Interactions between epithelium and Mycobacterium avium subsp. paratuberculosis have not been intensively studied, and it is currently unknown how the bacterium-epithelial cell cross talk contributes to the course of infection. We hypothesized that M. avium subsp. paratuberculosis harnesses host responses to recruit macrophages to the site of infection to ensure its survival and dissemination. We investigated macrophage recruitment in response to M. avium subsp. paratuberculosis using a MAC-T bovine macrophage coculture system. We show that M. avium subsp. paratuberculosis infection led to phagosome acidification within bovine epithelial (MAC-T) cells as early as 10 min, which resulted in upregulation of interleukin-1β (IL-1β) at transcript and protein levels. Within 10 min of infection, macrophages were recruited to the apical side of MAC-T cells. Inhibition of phagosome acidification or IL-1β abrogated this response, while MCP-1/CCL-2 blocking had no effect. IL-1β processing was dependent upon Ca(2+) uptake from the extracellular medium and intracellular Ca(2+) oscillations, as determined by EGTA and BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester)] treatments. Thus, M. avium subsp. paratuberculosis is an opportunist that takes advantage of extracellular Ca(2+)-dependent phagosome acidification and IL-1β processing in order to efficiently transverse the epithelium and enter its niche--the macrophage.

Fig 1

M. avium subsp. paratuberculosis (MAP) induces phagosome acidification and IL-1β processing at the epithelium interface. (A) Confocal microscopy of phagosome acidification in MAC-T cells. MAC-T cells were assessed for phagosome acidification at 10, 30, and 60 min after infection with M. avium subsp. paratuberculosis using LysoTracker blue. Approximately 60% of MAC-T cells contained M. avium subsp. paratuberculosis. M. avium subsp. paratuberculosis induced phagosome acidification at 10 and 30 min postinfection but ceased at 60 min postinfection. (B) Rab7 stain of MAC-T cells infected with M. avium subsp. paratuberculosis 30 min postinfection. LysoTracker staining was validated with indirect staining for the late endosomal marker Rab7. Infected MAC-T cells were positive for Rab7. (C) qRT-PCR of uninfected and infected MAC-T cells at 10 min postinfection. In stark contrast to uninfected cells, M. avium subsp. paratuberculosis-infected MAC-T cells showed a 50-fold upregulation of IL-1β. (D) IL-1β protein levels in uninfected and infected MAC-T cells. Infected MAC-T cells reached peak IL-1β expression at 30 min postinfection. **, P < 0.01; ***, P < 0.001.

Fig 2

M. avium subsp. paratuberculosis (MAP) enlistment of IL-1β-recruited macrophages to the initial site of infection. (A) FACS analysis of macrophage recruitment in response to M. avium subsp. paratuberculosis infection in the epithelium-bovine macrophage coculture system. M. avium subsp. paratuberculosis infection readily recruits macrophages to the apical chamber. Macrophage recruitment was abolished when phagosome acidification was blocked with bafilomycin A1 or IL-1β expression was prevented. Recruitment was rescued in bafilomycin A1 treatment with the addition of recombinant IL-1β protein. MCP-1 blocking did not impact macrophage recruitment. (B and C) Confocal microscopy of a coculture infection with M. avium subsp. paratuberculosis. (B) Macrophages are readily recruited to the apical chamber, as the Transwell contained only MAC-T cells. (C) Macrophages were found only in the apical chamber. Recruited macrophages contained M. avium subsp. paratuberculosis. (D) Bafilomycin A1 treatment abrogated phagosome acidification and IL-1β transcription (qRT-PCR). Confocal microscopy of bafilomycin A1 treated cells and vehicle control (DMSO) (top). Bafilomycin A1 treatment blocked IL-1β transcription, and results are indistinguishable from those of uninfected controls (bottom). (E) qRT-PCR of infected MAC-T cells treated with IL-1β blocking antibody or MCP-1 blocking antibody. MCP-1 blocking does not impact IL-1β expression. (F) IL-1β dot blot. Cell supernatants were collected during M. avium subsp. paratuberculosis infection of MAC-T cells. The addition of the MCP-1 blocking antibody did not influence IL-1β protein levels. ***, P < 0.001.

Fig 3

M. avium subsp. paratuberculosis-induced phagosome acidification and IL-1β processing is dependent upon an extracellular calcium flux. (A) Confocal microscopy of BAPTA-AM-treated MAC-T cells during M. avium subsp. paratuberculosis infection. BAPTA-AM abolished LysoTracker staining. (B) qRT-PCR of infected MAC-T cells treated with BAPTA-AM and calcium-free EGTA medium. BAPTA-AM and calcium-free medium prevented IL-1β transcription. (C) Confocal microscopy of infected MAC-T cells treated with calcium-free EGTA medium 10 and 30 min postinfection. Calcium-free EGTA medium abrogated LysoTracker staining. (D) IL-1β dot blot of infected MAC-T cells treated with calcium-free EGTA medium. The addition of calcium-free EGTA medium prevented IL-1β expression at all postinfection time points. (E) Confocal microscopy of combined UTP and calcium-free treatment in infected MAC-T cells 10 min and 30 min p.i. The addition of UTP to calcium-free treatment restored phagosome acidification. Therefore, intracellular calcium stores remained functional. ***, P < 0.001.