Toll-like receptor 4 (TLR4) signaling at the plasma membrane and in endosomes results in distinct contributions to inflammation and host defence. Current understanding indicates that endocytosis of cell surface-activated TLR4 is required to enable subsequent signaling from endosomes. Contrary to this prevailing model, our data show that endosomal TLR4 signaling is not reliant on cell surface-expressed TLR4 or ligand-induced TLR4 endocytosis. Moreover, previously recognized requirements for the accessory molecule CD14 in TLR4 endocytosis and endosomal signaling are likely attributable to CD14 binding as well as trafficking and transferring lipopolysaccharide (LPS) to TLR4 at different subcellular localizations. TLR4 endocytosis requires the TLR4 intracellular signaling domain, contributions by phospholipase C gamma 2, spleen tyrosine kinase, E1/E2 ubiquitination enzymes, but not canonical TLR signaling adaptors and cascades. Thus, our study identifies independently operating TLR4 signaling modes that control TLR4 endocytosis, pro-inflammatory cell surface-derived, as well as endosomal TLR4 signaling. This revised understanding of how TLR4 functions within cells might be harnessed to selectively amplify or restrict TLR4 activation for the development of adjuvants, vaccines and therapeutics.

Toll-like receptors (TLRs) are innate immune sensors for the presence of pathogens and endogenous danger signals. TLR activation results in conserved intracellular signaling events that orchestrate inflammation and antimicrobial defense. While the identity and interplay of key TLR signaling components are well established, how these largely cytosolic proteins are physically connected is not well understood. For the activation of conserved intracellular signaling events, most TLRs engage the adapter MyD88 (myeloid differentiation primary response 88), which assembles into higher-order protein complexes, myddosomes. In their recent publication, Fisch et al. present evidence that oligomeric myddosomes detach from initiating TLRs and evolve into larger scaffolds that dynamically assemble not only proximal but also distal cytosolic elements required to execute the entire cascade of the TLR-MyD88 signaling pathway. Coinciding with decline in TLR signaling over time, myddosomes progressively recruit autophagy machinery that mediates myddosome clearance. These findings expand the current understanding of TLR signaling by positioning myddosomes as the central structural element that physically assembles the key executors and regulators of TLR-MyD88-dependent intracellular signaling cascades. A recent study by Fisch et al. revealed that upon release from seeding Toll-like receptors (TLRs), myddosomes dynamically assemble into larger cytoplasmic scaffolds that recruit proximal and distal elements of TLR-signaling cascades. image

Granulomas are key histopathological features of Mycobacterium tuberculosis (Mtb) infection, with complex roles in pathogen control and dissemination. Thus, understanding drivers and regulators of granuloma formation is important for improving tuberculosis diagnosis, treatment, and prevention. Yet, molecular mechanisms underpinning granuloma formation and dynamics remain poorly understood. Here we used low-dose Mtb infection of C57BL/6 mice, which elicits structured lung granulomas composed of central macrophage clusters encased by a lymphocyte mantle, alongside the disorganized lymphocyte and macrophage clusters commonly observed in Mtb-infected mice. Using gene-deficient mice, we observed that Toll-like receptor (TLR) 2 and the TLR-related Radioprotective 105 kDa protein (RP105) contributed to the extent and spatial positioning of pathology in infected lung tissues, consistent with functional cooperation between TLR2 and RP105 in the innate immune recognition of Mtb. In mice infected with the highly virulent Mtb clinical isolate HN878, TLR2, but not RP105, positively regulated the extent of central macrophage regions within structured granulomas. Moreover, RP105, but not TLR2, promoted the formation of structured lung granulomas, suggesting that the functions of RP105 as an innate immune sensor for Mtb reach beyond its roles as TLR2 co-receptor. TLR2 and RP105 contributions to lung pathology are governed by Mtb biology, as neither receptor affected the frequency or architecture of structured granulomas in mice infected with the reference strain Mtb H37Rv. Thus, by revealing distinctive as well as cooperative functions of TLR2 and RP105 in lung pathology, our data identify TLRs as molecular determinants of TB granuloma formation and architecture, and expand understanding of how interactions between innate immune receptors and Mtb shape TB disease manifestation.

The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in the host control of mycobacterial infections. Expression and release of TNF are tightly regulated, yet the molecular mechanisms that control the release of TNF by mycobacteria-infected host cells, in particular macrophages, are incompletely understood. Rab GTPases direct the transport of intracellular membrane-enclosed vesicles and are important regulators of macrophage cytokine secretion. Rab6b is known to be predominantly expressed in the brain where it functions in retrograde transport and anterograde vesicle transport for exocytosis. Whether it executes similar functions in the context of immune responses is unknown. Here we show that Rab6b is expressed by primary mouse macrophages, where it localized to the Golgi complex. Infection with Mycobacterium bovis bacille Calmette–Guérin (BCG) resulted in dynamic changes in Rab6b expression in primary mouse macrophages in vitro as well as in organs from infected mice in vivo . We further show that Rab6b facilitated TNF release by M. bovis BCG-infected macrophages, in the absence of discernible impact on Tnf messenger RNA and intracellular TNF protein expression. Our observations identify Rab6b as a positive regulator of M. bovis BCG-induced TNF trafficking and secretion by macrophages and positions Rab6b among the molecular machinery that orchestrates inflammatory cytokine responses by macrophages.

The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in the host control of mycobacterial infections. Expression and release of TNF are tightly regulated, yet the molecular mechanisms that control the release of TNF by mycobacteria-infected host cells, in particular macrophages, are incompletely understood. Rab GTPases direct the transport of intracellular membrane-enclosed vesicles and are important regulators of macrophage cytokine secretion. Rab6b is known to be predominantly expressed in the brain where it functions in retrograde transport and anterograde vesicle transport for exocytosis. Whether it executes similar functions in the context of immune responses is unknown. Here we show that Rab6b is expressed by primary mouse macrophages, where it localized to the Golgi complex. Infection with Mycobacterium bovis bacille Calmette-Guerin (BCG) resulted in dynamic changes in Rab6b expression in primary mouse macrophages in vitro as well as in organs from infected mice in vivo. We further show that Rab6b facilitated TNF release by M. bovis BCG-infected macrophages, in the absence of discernible impact on Tnf messenger RNA and intracellular TNF protein expression. Our observations identify Rab6b as a positive regulator of M. bovis BCG-induced TNF trafficking and secretion by macrophages and positions Rab6b among the molecular machinery that orchestrates inflammatory cytokine responses by macrophages.

Radioprotective 105 kDa (RP105, CD180) is a member of the Toll-like receptor (TLR) family that interacts with TLR2 and facilitates recognition of mature lipoproteins expressed by Mycobacterium tuberculosis and Mycobacterium bovis BCG. In this study, we used synthetic lipopeptide analogs of the M. tuberculosis 19 kDa lipoprotein to define structural characteristics that promote RP105-mediated host cell responses. A tripalmitoylated lipopeptide composed of the first 16 N-terminal amino acids of the M. tuberculosis 19 kDa lipoprotein induced RP105-dependent TNF and IL-6 production by macrophages. Di- and tripalmitoylated variants of this lipopeptide elicited an equivalent RP105-dependent response, indicating that while the lipid moiety is required for macrophage activation, it is not a determinant of RP105 dependency. Instead, substitution of two polar threonine residues at positions 7 and 8 with nonpolar alanine residues resulted in reduced RP105 dependency. These results strongly suggest that the amino acid composition of the M. tuberculosis 19 kDa lipoprotein, and likely other mycobacterial lipoproteins, is a key determinant of RP105 agonism.

Tuberculosis (TB) remains a major global health threat. Urgent needs in the fight against TB include improved and innovative treatment options for drug-sensitive and -resistant TB as well as reliable biological indicators that discriminate active from latent disease and enable monitoring of treatment success or failure. Prominent interferon (IFN) inducible gene signatures in TB patients and animal models of Mycobacterium tuberculosis infection have drawn significant attention to the roles of type I IFNs in the host response to mycobacterial infections. Here, we review recent developments in the understanding of the innate immune pathways that drive type I IFN responses in mycobacteria-infected host cells and the functional consequences for the host defense against M. tuberculosis, with a view that such insights might be exploited for the development of targeted host-directed immunotherapies and development of reliable biomarkers.

Radioprotective 105 kDa (CD180) is an unusual TLR that lacks an intracellular Toll-IL-1R signaling domain and exhibits unconventional homodimerization behavior. Differential expression and functions of radioprotective 105 kDa have been associated with immune-mediated pathologies, including infection, chronic inflammation, and autoimmune disorders. Radioprotective 105 kDa activates macrophages and B cells independently of canonical TLR signaling. Current understanding of the functional consequences of radioprotective 105 kDa signaling in B cells, macrophages, and dendritic cells indicates overlapping, but also some apparent opposing, cell-specific roles for radioprotective 105 kDa in shaping cellular functions. This review compares interactions of radioprotective 105 kDa with its coreceptors, CD19, TLR4, and TLR2; integrates recent, novel findings on radioprotective 105 kDa-mediated molecular signaling mechanisms; and summarizes current understanding of its contributions to infectious, inflammatory, and autoimmune disease.

Radioprotective 105 kDa (CD180) is an unusual TLR that lacks an intracellular Toll-IL-1R signaling domain and exhibits unconventional homodimerization behavior. Differential expression and functions of radioprotective 105 kDa have been associated with immune-mediated pathologies, including infection, chronic inflammation, and autoimmune disorders. Radioprotective 105 kDa activates macrophages and B cells independently of canonical TLR signaling. Current understanding of the functional consequences of radioprotective 105 kDa signaling in B cells, macrophages, and dendritic cells indicates overlapping, but also some apparent opposing, cell-specific roles for radioprotective 105 kDa in shaping cellular functions. This review compares interactions of radioprotective 105 kDa with its coreceptors, CD19, TLR4, and TLR2; integrates recent, novel findings on radioprotective 105 kDa-mediated molecular signaling mechanisms; and summarizes current understanding of its contributions to infectious, inflammatory, and autoimmune disease.

Cytokines are key regulators of adequate immune responses to infection with Mycobacterium tuberculosis. We demonstrate that the p110δ catalytic subunit of PI3K acts as a downstream effector of the TLR family member RP105 (CD180) in promoting mycobacteria-induced cytokine production by macrophages. Our data show that the significantly reduced release of TNF and IL-6 by RP105−/− macrophages during mycobacterial infection was not accompanied by diminished mRNA or protein expression. Mycobacteria induced comparable activation of NF-κB and p38 MAPK signaling in wild-type (WT) and RP105−/− macrophages. In contrast, mycobacteria-induced phosphorylation of Akt was abrogated in RP105−/− macrophages. The p110δ-specific inhibitor, Cal-101, and small interfering RNA–mediated knockdown of p110δ diminished mycobacteria-induced TNF secretion by WT but not RP105−/− macrophages. Such interference with p110δ activity led to reduced surface-expressed TNF in WT but not RP105−/− macrophages, while leaving TNF mRNA and protein expression unaffected. Activity of Bruton’s tyrosine kinase was required for RP105-mediated activation of Akt phosphorylation and TNF release by mycobacteria-infected macrophages. These data unveil a novel innate immune signaling axis that orchestrates key cytokine responses of macrophages and provide molecular insight into the functions of RP105 as an innate immune receptor for mycobacteria.