Multiple mechanisms allow Mycobacterium tuberculosis to continuously inhibit MHC class II-mediated antigen presentation by macrophages

Abstract

Previous experimental studies suggest that Mycobacterium tuberculosis inhibits a number of macrophage intracellular processes associated with antigen presentation, including antigen processing, MHC class II expression, trafficking of MHC class II molecules, and peptide-MHC class II binding. In this study, we investigate why multiple mechanisms have been observed. Specifically, we consider what purpose multiple mechanisms may serve, whether experimental protocols favor the detection of some mechanisms over others, and whether alternative mechanisms exist. By using a mathematical model of antigen presentation in macrophages that tracks levels of various molecules, including peptide-MHC class II complexes on the cell surface, we show that mechanisms targeting MHC class II expression are effective at inhibiting antigen presentation, but only after a delay of at least 10 h. By comparison, the effectiveness of mechanisms targeting other cellular processes is immediate, but may be attenuated under certain conditions. Therefore, targeting multiple cellular processes may represent an optimal strategy for M. tuberculosis (and other pathogens with relatively long doubling times) to maintain continuous inhibition of antigen presentation. In addition, based on a sensitivity analysis of the model, we identify other cellular processes that may be targeted by such pathogens to accomplish the same effect, representing potentially novel mechanisms.

Fig. 1.

Model schematic. Molecular species represented in the model include extracellular IFN-γ (G), IFN-γ receptors (R, free; C, bound), CIITA (T₁, mRNA; P, protein), MHC class II mRNA (T₂), exogenous antigen (A*, extracellular; A, intracellular; E, peptide), self-peptide (S), free MHC class II molecules (M, intracellular; M*, surface), self peptide-bound MHC class II molecules (M_s, intracellular; M_s*, surface), and exogenous peptide-bound MHC class II molecules (M_e, intracellular; M_e*, surface). Solid arrows indicate one-step reactions and dashed arrows indicate regulatory interactions. Degradation is represented in the model for the following molecules but not shown: G, T₁, P, T₂, A*, M, M*, M_s, M_s*, M_e, M_e*. Up-regulation of M by C directly and contribution of M_s and M_s* to S are also included in the model but are not shown.

Fig. 2.

Model testing using various controls. (A and B), Simulation results and experimental data for levels of CIITA mRNA (solid lines) and MHC class II mRNA (dashed lines) in IFN-γ-treated macrophages from Pai et al. (20). (C and D) Simulation results and experimental data for levels of MHC class II mRNA (solid lines) and MHC class II protein (dashed lines) in IFN-γ-treated macrophages from Cullell-Young et al. (21). (E and F) Simulation results for surface pMHC levels (in arbitrary units) and experimental data for T cell response in non-IFN-γ-treated macrophages exposed to antigen from Buus and Werdelin (22). (G and H) Simulation results for surface pMHC levels (in arbitrary units) and experimental data for T cell response in non-IFN-γ-treated macrophages (solid lines) and IFN-γ-treated macrophages (dashed lines) exposed to antigen from Delvig et al. (25). Pretreatment (16 h) with medium or IFN-γ is not shown; hence, the x axis is enumerated from 16 h onward (i.e., when antigen is present).

Fig. 3.

Simulation results of two in vitro experimental protocols using four published hypotheses. (A and B) Large circles represent macrophages, highlighted circles represent IFN-γ-treated macrophages, and small circles represent T cell hybridomas. (A) Protocol of Hmama et al. (14). A total of 10⁵ monocytes was infected with Mtb at a moi of 50 for 24 h, treated with 200 units/ml IFN-γ for 36 h, pulsed with 1 mg/ml BSA for 0.5 h, and chased for 0.5, 1, or 4 h. (B) Protocol of Noss et al. (10). A total of 5 × 10⁴ macrophages was treated with 2 ng/ml IFN-γ for 20–24 h, infected with Mtb at a moi of 40 for 2 h, treated with 2 ng/ml IFN-γ for an additional 18–26 h, and pulsed with 0–100 μg/ml hen egg lysozyme or 0–1,000 μg/ml RNase for 1–3 h. (C–E) Simulation results using the protocol of Hmama et al. (14) for levels of CIITA mRNA, MHC class II mRNA, and surface MHC class II protein, respectively. (F–H) Simulation results using the protocol of Noss et al. (10) for levels of MHC class II mRNA, total MHC class II protein, and surface pMHC, respectively.

Fig. 4.

Proposed experimental protocol to determine the contribution of different mechanisms to inhibition of antigen presentation by Mtb. (A) Protocol schematic using representations of Fig. 3 A and B. (B) Surface pMHC levels expected in uninfected macrophages, Mtb-infected macrophages if mechanisms target primarily MHC class II expression (in this case, MHC class II transcription), and Mtb-infected macrophages if mechanisms target primarily other processes (in this case, antigen processing). Percentage reductions in infected macrophages (relative to uninfected controls) are also shown.