Single-cell analysis reveals the subthreshold TLR response to Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb) infection remains one of society's greatest human health challenges. Recognition of pathogens such as Mtb by phagocytes via pattern recognition receptors (PRRs) is critical for immune defense. Phagocytes and other innate immune cells must successfully discriminate between pathogen infection or noisy signals derived from transient non-pathogenic exposures to pattern recognition receptor ligands. Previous studies have underscored the importance of toll-like receptor 2 (TLR2) in the recognition of Mtb, and other studies have demonstrated that toll-like receptor 4 (TLR4) exhibits sigmoidal dynamics in inflammatory protein production in response to soluble ligands. How these dynamics extend to TLR2 in response to Mtb or soluble TLR2 ligands is not known. Here, we address a challenging question essential to the pathogenesis of tuberculosis (TB) disease: What is the quality of the early innate response to Mtb infection at low multiplicity of infection and how does the response to soluble TLR2 ligand stimulation reflect the intact pathogen response? Using soluble TLR2 ligands, we demonstrate that inflammatory protein production in response to TLR2 stimulation similarly follows sigmoidal activation. We further show that inflammatory protein production following Mtb infection operates in this subthreshold regime and cannot be attributed to irreversible disruption of inflammatory signaling pathways in Mtb-infected cells. Using highly sensitive assays to interrogate signaling in Mtb-infected primary human macrophages, we further show that Mtb infection results in limited phosphorylation of p38, a key regulator of inflammatory pathways. We further demonstrate that this poor activation is not driven by irreversible inhibition of p38 signaling by Mtb. Lastly, we demonstrate that the quality of canonical inflammatory signal input is decoupled from the ability to control Mtb growth. Together, these data demonstrate that pathways downstream of TLR signaling are differentially activated following Mtb infection and that barriers to activation of these pathways may contribute to functional partitioning of the inflammatory state of Mtb infected cells. Importantly, these studies provide a roadmap to dissect heterogeneous innate responses to Mtb infection as well as provide a tractable experimental system to simulate the early innate immune response to paucibacillary infection in humans.