The Lrp of Mycobacterium tuberculosis regulates the innate immune response of macrophages

Mycobacterium tuberculosis (Mtb) is the pathogenic bacteria that causes one-third of tuberculosis infections globally, but the mechanism of its pathogenesis has not been fully elucidated.¹ The main pathway by which Mtb infects humans is via the pneogaster; thus, the lung is the main reservoir where Mtb exists. However, Mtb can also infect other human organs. Mtb is an intracellular bacterium of immune cells and, interestingly, it can escape elimination by the host immune system.² To date, the Bacillus Calmette–Guerin vaccine is the only vaccine that has proved effective and has been approved worldwide. This vaccine was developed by two French scientists, namely, Leon Calmette and Guerin.³

After infecting the human body, Mtb can cause an immune response, including the innate immune response and adaptive immune response.⁴ Many pathogenic bacteria have conserved molecular patterns called pathogen-associated molecular patterns (PAMPs), many of which induce signaling through Toll-like receptors (TLRs) expressed on innate immune cells.⁵ The TLR family belongs to pattern-recognition receptors that recognize the PAMPs of bacteria; such PAMPs include lipopolysaccharide (LPS), peptidoglycan, zymosan and bacterial DNA.⁶ Certain studies have shown that during the early stage of Mtb infection, TLRs have an important function and affect the subsequent activation of immune cells.⁷ Although research has elucidated the means by which Mtb infects humans and the way in which the body's immune response protects against the mycobacterium, how Mtb evades immune clearance by the host and survives in macrophages remains unknown.⁸ Hence, this aspect of Mtb pathology has gained increasing attention. Mtb can survive in both monocytes and macrophages.⁹ There are no symptoms of Mtb bacillus during the incubation period, at which time it is very difficult to treat tuberculosis.¹⁰ The protein family including the leucine-responsive regulatory protein (Lrp/AsnC) has been found to play an important role in the nutritional regulation of Mtb, so Lrp must be studied further with respect to its role in Mtb.¹¹ Lrp serves as the paranuclein of Mtb and, hence, has been highly conserved. Lrp (Rv2779c) exists in Mtb H37Rv, but little has been reported concerning the influence of Lrp on the host immune system.

This issue of Cellular and Molecular Immunology provides novel and interesting data demonstrating that recombinant Lrp (rLrp) interacts with TLR2 directly to exert its inhibitory effects on macrophages (Figure 1). These effects were not evident in macrophages from TLR2^–/– mice, however, so this finding supports the conclusion that the interaction between rLrp and TLR2 is necessary to downregulate TLR-mediated signaling. Deletion of the 19 N-terminal-most residues of rLrp (a region that likely mediates rLrp interaction with other partners) was sufficient to remove its effects. Deletion of the 20 C-terminal-most residues of rLrp did not inhibit TLR signaling.

Figure 1.

Mechanisms by which rLrp inhibits the production of LPS-induced cytokines. In our study, we found that rLrp inhibited the production of LPS-induced TNF-α, IL-12, and IL-6. rLrp blocked the nuclear translocation of the transcription factor NF-κB and attenuated NF-κB transcriptional activity and DNA binding activity, which was accompanied by the degradation of IαBα. We then found that the binding of TRAF6 to TAK-1 induced by LPS could be blocked by rLrp. Furthermore, in Akt protein kinase-silenced macrophages and TLR2^–/– macrophages, rLrp did not attenuate the release of proinflammatory cytokines or the expression of MHC-II molecules and CD86 induced by interferon-γ, indicating that the inhibitory effects of rLrp depend on TLR2 and the activation of the PI3K/Akt pathway. rLrp could also stimulate the rapid phosphorylation/activation of PI3K, Akt and glycogen synthase kinase-3β in macrophages. LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; rLrp, recombinant Lrp; TLR, Toll-like receptor; TNF-α, tumor necrosis factor α; TRAF6, TNF receptor-associated factor 6.

We found that Lrp can regulate the innate immune response that is mediated by the TLR2 pathway.¹² rLrp was found to inhibit the LPS-induced secretion of tumor necrosis factor α (TNF-α), IL-6, and IL-12p40. Further research revealed that rLrp can prevent the ubiquitination of TRAF6 (TNF receptor-associated factor 6) induced by LPS in macrophages via TLR2, and it can prevent TRAF6 from recruiting TAK-1, which prevents the phosphorylation of TAK-1. This process prevents the degradation of NF-κB inhibitory factor IκBα, which inhibits NF-κB activation. We subsequently determined how rLrp inhibits the expression of inflammatory cytokines induced by LPS. Concurrently, we confirmed that the attenuating effects of rLrp that are dependent on Akt extend to the inhibition of the production of inflammatory cytokines, particularly TNF-α and IL-12, because we showed that rLrp can activate the PI3K/Akt pathway by stimulating the rapid phosphorylation/activation of PI3K, Akt and glycogen synthase kinase-3β in macrophages.

Two other important functions of macrophages are autophagy and antigen presentation. We found that treatment of RAW264.7 macrophages with interferon-γ promotes the formation of autophagosomes.^13,14 As expected, treatment with interferon-γ increased the percentage of cells with large vacuoles that stained positively for monodansylcadaverine, a marker of autophagic vacuoles, and this effect was inhibited by rLrp. Moreover, rLrp attenuates interferon-γ-induced expression of MHC-II in mouse macrophages in a TLR2-mediated and PI3K/Akt pathway activation-dependent manner. When we assessed the effects of rLrp on the morphology of Mtb colonies, we found that mutant rLrp caused morphologically variant colonies having a small, smooth and translucent phenotype compared with the rough, glossy, larger and strongly cohesive phenotype of wild-type Mtb. This observation suggests a role for rLrp in Mtb survival and virulence in the context of adaptation to stress conditions.

Taken together, the studies summarized here identify a role for Lrp in the negative regulation of the immune response of macrophages. These findings extend our knowledge of the function of the Mtb paranuclein and will inform future studies concerning the escape mechanism of Mtb and help us study the escape mechanism of Mtb and develop a new vaccine and drugs to reduce the morbidity of tuberculosis.

Conflict of interest

The authors declare no conflict of interest.