Mannose-capped Lipoarabinomannan from Mycobacterium tuberculosis induces soluble tumor necrosis factor receptor production through tumor necrosis factor alpha-converting enzyme activation

Abstract

Primary Mycobacterium tuberculosis infection results in granuloma formation in lung tissue. A granuloma encapsulates mycobacterium-containing cells, thereby preventing dissemination and further infection. Tumor necrosis factor alpha (TNF-α) is a host-protective cytokine during M. tuberculosis infection due to its role in promoting and sustaining granuloma formation. TNF activity is regulated through the production of soluble TNF receptors (sTNFRI and sTNFRII). Therefore, we examined the potential production of endogenous sTNFRs during M. tuberculosis infection. Using the murine model of aerosol M. tuberculosis infection, we determined that levels of sTNFR production were elevated in bronchoalveolar lavage fluid 1 month following infection. An investigation of M. tuberculosis cell wall components identified that the known virulence factor mannose-capped lipoarabinomannan (ManLAM) was sufficient to induce sTNFR production, with sTNFRII being produced preferentially compared with sTNFRI. ManLAM stimulated the release of sTNFRs without TNF production, which corresponded to an increase in TNF-α-converting enzyme (TACE) activity. To determine the relevance of these findings, serum samples from M. tuberculosis-infected patients were tested and found to have an increase in the sTNFRII/sTNFRI ratio. These data identify a mechanism by which M. tuberculosis infection can promote the neutralization of TNF and furthermore suggest the potential use of the sTNFRII/sTNFRI ratio as an indicator of tuberculosis disease.

Fig 1

Production of TNF-α, sTNFRI, and sTNFRII in BALF and serum samples of naïve mice versus M. tuberculosis Erdman-infected mice at 1 month postinfection. (a to c) M. tuberculosis Erdman-infected mice had dramatically increased levels of TNF (a), sTNFRI (b), and sTNFRII (c) in their BALF compared to levels in BALF of naïve mice. (d to f) The level of TNF was significantly elevated in the sera of M. tuberculosis-infected mice (d); however, there was no statistically significant change in either sTNFRI (e) or sTNFRII (f) levels. Samples were measured in quadruplicate by using an in-house multiplex ELISA system (n = 6 to 8 mice per group for panels a to c; n = 3 to 5 mice per group for panels d to f) (*, P < 0.05; **, P < 0.01 [determined by Student's t test]).

Fig 2

In vivo and in vitro ManLAM treatment induces sTNFR production. (a to c) Six- to eight-week-old female C57BL/6J mice were treated intratracheally with 25 μg of ManLAM and were sacrificed at 24 h postinstillation. ManLAM treatment significantly induced sTNFRII in BALF compared to controls (*, P < 0.05 by t test [panel c]) (n = 7 or 8 mice per group). (d to f) Mouse spleen and lymph node preparations were incubated with ManLAM for 24 h. (d) ManLAM treatment did not induce the mouse spleen and lymph node preparations to make significant amounts of TNF-α. (e and f) However, in vitro, ManLAM treatment did significantly induce the production of soluble TNFRI (e) and TNFRII (f) over the levels of controls (**, P < 0.01 by t test) (n = 2 experiments run in duplicate). ns, not significant.

Fig 3

Treatment of human whole blood with ManLAM induces sTNFR production. Human whole blood was stimulated ex vivo with ManLAM, PIM6, or AraLAM, and levels of TNF family members in plasma were measured via a multiplex ELISA at 24 h. (a) ManLAM was not able to induce TNF production. (b) ManLAM treatment seemed to induce sTNFRI, but this increase was not statistically significant. (c) Similar to mouse in vivo data, ManLAM was able to significantly increase sTNFRII levels (*, P < 0.05 by t test) (n = 7 donors). (d and e) Flow cytometric analysis of cells at 24 h post-ManLAM treatment shows that TNFRI (d) and TNFRII (e) are lost from the cell surface (gray, isotype; solid line, PBS; dashed line, ManLAM) (n = 2; data for representative donors are shown). FL2-H designates fluorescence channel intensity.

Fig 4

ManLAM treatment does not induce mRNA synthesis. Human whole blood was stimulated ex vivo with ManLAM, and cell pellets were harvested at the indicated time points. Pellets were lysed, mRNA samples from at least two donors were analyzed in triplicate, and ΔΔCT values for TNF (a), TNFRI (b), TNFRII (c), or TACE (d) were normalized to 18S rRNA values. No statistically significant modulation of mRNA was observed at any time point.

Fig 5

ManLAM treatment lowers TIMP3 levels and elevates TACE activity. (a) Cell pellets from ex vivo stimulations of human whole blood were lysed, and TACE enzymatic activity levels were measured with an Innozyme TACE activity kit. The levels of enzymatic activity were elevated in ManLAM-treated samples at 2 to 6 h posttreatment and returned to baseline levels by 12 h. Data are expressed as raw fluorescence units per mg of protein (n = 3 to 5 donors). No tx, no treatment. (b) Percent changes in TIMP3 levels in plasma from ex vivo stimulations of human whole blood were measured with singleplex ELISAs (n = 6 to 12 donors) (*, P < 0.05 by ANOVA with Tukey's posttests). (c) Western blotting of samples treated with the vehicle control or ManLAM demonstrated that ManLAM is able to induce an increase in ERK1/2 phosphorylation (shown is a representative blot from 2 separate experiments; densitometry is shown at right).

Fig 6

In vitro infection with M. tuberculosis results in unique cytokine, TIMP3, and TACE activation patterns. Mouse bone marrow-derived macrophages were infected with M. tuberculosis Erdman or M. smegmatis at an MOI of 5, and culture supernatants and cell lysates were harvested at the indicated time points. Triplicate samples were assayed in duplicate, and results from a representative experiment are shown. (a) Only M. smegmatis induced TNF production. nd, not determined. (b) M. smegmatis and M. tuberculosis induced decreases in the amount of sTNFRI released (P < 0.01 for the control versus M. tuberculosis infection at 24 and 48 h and for M. smegmatis versus M. tuberculosis infection at 48 h, determined by ANOVAs with Bonferroni posttests). (c) M. smegmatis and M. tuberculosis induced increases in the amount of sTNFRII released compared to the amount of sTNFRII released in uninfected control cells. All posttests were significant (P < 0.05 or lower), as determined by ANOVAs with Bonferroni posttests, except for sTNFRII for M. tuberculosis versus M. smegmatis infection at 6 h. (d) M. smegmatis induced a significant amount of IL-10. All posttests were significant (P < 0.05 or lower), as determined by ANOVAs with Bonferroni posttests, except for the control versus M. tuberculosis infection at 6 and 24 h. (e) Both M. tuberculosis and M. smegmatis infection resulted in a decrease in TIMP3 levels at 6 h postinfection (*, P < 0.05 by Student's unpaired t test). TIMP3 levels in culture supernatants were below the detection limit at 24 and 48 h. (f) Both M. smegmatis and M. tuberculosis induced TACE activation at 6 h, but only M. tuberculosis infection induced sustained TACE activation above baseline levels (**, P < 0.004 by Student's unpaired t test).

Fig 7

Serum from M. tuberculosis-infected patients shows higher ratios of sTNFRII/sTNFRI than does serum from COPD patients or healthy controls. M. tuberculosis-infected patient serum samples were analyzed by using singleplex ELISA kits, and data for samples are outlined in Table 1. There was a statistically significant difference in sTNFRII/sTNFRI levels compared to those in sera from COPD patients or healthy controls (*, P < 0.05 by Student's t test) (n = 20 for TB patient sera, n = 15 for COPD patient sera, and n = 5 for normal donor sera).