NLRP12-PANoptosome activates PANoptosis and pathology in response to heme and PAMPs.

Cytosolic innate immune sensors are critical for host defense and form complexes, such as inflammasomes and PANoptosomes, that induce inflammatory cell death. The sensor NLRP12 is associated with infectious and inflammatory diseases, but its activating triggers and roles in cell death and inflammation remain unclear. Here, we discovered that NLRP12 drives inflammasome and PANoptosome activation, cell death, and inflammation in response to heme plus PAMPs or TNF. TLR2/4-mediated signaling through IRF1 induced Nlrp12 expression, which led to inflammasome formation to induce maturation of IL-1ß and IL-18. The inflammasome also served as an integral component of a larger NLRP12-PANoptosome that drove inflammatory cell death through caspase-8/RIPK3. Deletion of Nlrp12 protected mice from acute kidney injury and lethality in a hemolytic model. Overall, we identified NLRP12 as an essential cytosolic sensor for heme plus PAMPs-mediated PANoptosis, inflammation, and pathology, suggesting that NLRP12 and molecules in this pathway are potential drug targets for hemolytic and inflammatory diseases.

Serine synthesis sustains macrophage IL-1ß production via NAD+-dependent protein acetylation.

Serine metabolism is involved in the fate decisions of immune cells; however, whether and how de novo serine synthesis shapes innate immune cell function remain unknown. Here, we first demonstrated that inflammatory macrophages have high expression of phosphoglycerate dehydrogenase (PHGDH, the rate-limiting enzyme of de novo serine synthesis) via nuclear factor κB signaling. Notably, the pharmacological inhibition or genetic modulation of PHGDH limits macrophage interleukin (IL)-1ß production through NAD+ accumulation and subsequent NAD+-dependent SIRT1 and SIRT3 expression and activity. Mechanistically, PHGDH not only sustains IL-1ß expression through H3K9/27 acetylation-mediated transcriptional activation of Toll-like receptor 4 but also supports IL-1ß maturation via NLRP3-K21/22/24/ASC-K21/22/24 acetylation-mediated activation of the NLRP3 inflammasome. Moreover, mice with myeloid-specific depletion of Phgdh show alleviated inflammatory responses in lipopolysaccharide-induced systemic inflammation. This study reveals a network by which a metabolic enzyme, involved in de novo serine synthesis, mediates post-translational modifications and epigenetic regulation to orchestrate IL-1ß production, providing a potential inflammatory disease target.

The matricellular protein SPARC induces inflammatory interferon-response in macrophages during aging.

The risk of chronic diseases caused by aging is reduced by caloric restriction (CR)-induced immunometabolic adaptation. Here, we found that the matricellular protein, secreted protein acidic and rich in cysteine (SPARC), was inhibited by 2 years of 14% sustained CR in humans and elevated by obesity. SPARC converted anti-inflammatory macrophages into a pro-inflammatory phenotype with induction of interferon-stimulated gene (ISG) expression via the transcription factors IRF3/7. Mechanistically, SPARC-induced ISGs were dependent on toll-like receptor-4 (TLR4)-mediated TBK1, IRF3, IFN-ß, and STAT1 signaling without engaging the Myd88 pathway. Metabolically, SPARC dampened mitochondrial respiration, and inhibition of glycolysis abrogated ISG induction by SPARC in macrophages. Furthermore, the N-terminal acidic domain of SPARC was required for ISG induction, while adipocyte-specific deletion of SPARC reduced inflammation and extended health span during aging. Collectively, SPARC, a CR-mimetic adipokine, is an immunometabolic checkpoint of inflammation and interferon response that may be targeted to delay age-related metabolic and functional decline.

Exploiting dynamic enhancer landscapes to decode macrophage and microglia phenotypes in health and disease.

The development and functional potential of metazoan cells is dependent on combinatorial roles of transcriptional enhancers and promoters. Macrophages provide exceptionally powerful model systems for investigation of mechanisms underlying the activation of cell-specific enhancers that drive transitions in cell fate and cell state. Here, we review recent advances that have expanded appreciation of the diversity of macrophage phenotypes in health and disease, emphasizing studies of liver, adipose tissue, and brain macrophages as paradigms for other tissue macrophages and cell types. Studies of normal tissue-resident macrophages and macrophages associated with cirrhosis, obese adipose tissue, and neurodegenerative disease illustrate the major roles of tissue environment in remodeling enhancer landscapes to specify the development and functions of distinct macrophage phenotypes. We discuss the utility of quantitative analysis of environment-dependent changes in enhancer activity states as an approach to discovery of regulatory transcription factors and upstream signaling pathways.

The ATP-Binding Cassette Gene ABCF1 Functions as an E2 Ubiquitin-Conjugating Enzyme Controlling Macrophage Polarization to Dampen Lethal Septic Shock.

Sepsis is a bi-phasic inflammatory disease that threatens approximately 30 million lives and claims over 14 million annually, yet little is known regarding the molecular switches and pathways that regulate this disease. Here, we have described ABCF1, an ATP-Binding Cassette (ABC) family member protein, which possesses an E2 ubiquitin enzyme activity, through which it controls the Lipopolysaccharide (LPS)- Toll-like Receptor-4 (TLR4) mediated gram-negative insult by targeting key proteins for K63-polyubiquitination. Ubiquitination by ABCF1 shifts the inflammatory profile from an early phase MyD88-dependent to a late phase TRIF-dependent signaling pathway, thereby regulating TLR4 endocytosis and modulating macrophage polarization from M1 to M2 phase. Physiologically, ABCF1 regulates the shift from the inflammatory phase of sepsis to the endotoxin tolerance phase, and modulates cytokine storm and interferon-ß (IFN-ß)-dependent production by the immunotherapeutic mediator, SIRT1. Consequently, ABCF1 controls sepsis induced mortality by repressing hypotension-induced renal circulatory dysfunction.

Immunity against Moraxella catarrhalis requires guanylate-binding proteins and caspase-11-NLRP3 inflammasomes.

Moraxella catarrhalis is an important human respiratory pathogen and a major causative agent of otitis media and chronic obstructive pulmonary disease. Toll-like receptors contribute to, but cannot fully account for, the complexity of the immune response seen in M. catarrhalis infection. Using primary mouse bone marrow-derived macrophages to examine the host response to M. catarrhalis infection, our global transcriptomic and targeted cytokine analyses revealed activation of immune signalling pathways by both membrane-bound and cytosolic pattern-recognition receptors. We show that M. catarrhalis and its outer membrane vesicles or lipooligosaccharide (LOS) can activate the cytosolic innate immune sensor caspase-4/11, gasdermin-D-dependent pyroptosis, and the NLRP3 inflammasome in human and mouse macrophages. This pathway is initiated by type I interferon signalling and guanylate-binding proteins (GBPs). We also show that inflammasomes and GBPs, particularly GBP2, are required for the host defence against M. catarrhalis in mice. Overall, our results reveal an essential role for the interferon-inflammasome axis in cytosolic recognition and immunity against M. catarrhalis, providing new molecular targets that may be used to mitigate pathological inflammation triggered by this pathogen.

Chanzyme TRPM7 Mediates the Ca2+ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation.

Toll-like receptors (TLRs) sense pathogen-associated molecular patterns to activate the production of inflammatory mediators. TLR4 recognizes lipopolysaccharide (LPS) and drives the secretion of inflammatory cytokines, often contributing to sepsis. We report that transient receptor potential melastatin-like 7 (TRPM7), a non-selective but Ca2+-conducting ion channel, mediates the cytosolic Ca2+ elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca2+ elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca2+ signaling initiated by TRPM7 was also essential for the nuclear translocation of NFκB. Consequently, TRPM7-deficient macrophages exhibited major deficits in the LPS-induced transcriptional programs in that they failed to produce IL-1ß and other key pro-inflammatory cytokines. In accord with these defects, mice with myeloid-specific deletion of Trpm7 are protected from LPS-induced peritonitis. Our study highlights the importance of Ca2+ signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling.

A TLR4/TRAF6-dependent signaling pathway mediates NCoR coactivator complex formation for inflammatory gene activation.

The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1ß complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1ß coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1ß complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.

Reconstruction of LPS Transfer Cascade Reveals Structural Determinants within LBP, CD14, and TLR4-MD2 for Efficient LPS Recognition and Transfer.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, binds Toll-like receptor 4 (TLR4)-MD2 complex and activates innate immune responses. LPS transfer to TLR4-MD2 is catalyzed by both LPS binding protein (LBP) and CD14. To define the sequential molecular interactions underlying this transfer, we reconstituted in vitro the entire LPS transfer process from LPS micelles to TLR4-MD2. Using electron microscopy and single-molecule approaches, we characterized the dynamic intermediate complexes for LPS transfer: LBP-LPS micelles, CD14-LBP-LPS micelle, and CD14-LPS-TLR4-MD2 complex. A single LBP molecule bound longitudinally to LPS micelles catalyzed multi-rounds of LPS transfer to CD14s that rapidly dissociated from LPB-LPS complex upon LPS transfer via electrostatic interactions. Subsequently, the single LPS molecule bound to CD14 was transferred to TLR4-MD2 in a TLR4-dependent manner. The definition of the structural determinants of the LPS transfer cascade to TLR4 may enable the development of targeted therapeutics for intervention in LPS-induced sepsis.

Genetic and immune determinants of E. coli liver abscess formation.

Systemic infections can yield distinct outcomes in different tissues. In mice, intravenous inoculation of Escherichia coli leads to bacterial replication within liver abscesses, while other organs such as the spleen clear the pathogen. Abscesses are macroscopic necrotic regions that comprise the vast majority of the bacterial burden in the animal, yet little is known about the processes underlying their formation. Here, we characterize E. coli liver abscesses and identify host determinants of abscess susceptibility. Spatial transcriptomics revealed that liver abscesses are associated with heterogenous immune cell clusters comprised of macrophages, neutrophils, dendritic cells, innate lymphoid cells, and T-cells that surround necrotic regions of the liver. Abscess susceptibility is heightened in the C57BL lineage, particularly in C57BL/6N females. Backcross analyses demonstrated that abscess susceptibility is a polygenic trait inherited in a sex-dependent manner without direct linkage to sex chromosomes. As early as 1 d post infection, the magnitude of E. coli replication in the liver distinguishes abscess-susceptible and abscess-resistant strains of mice, suggesting that the immune pathways that regulate abscess formation are induced within hours. We characterized the early hepatic response with single-cell RNA sequencing and found that mice with reduced activation of early inflammatory responses, such as those lacking the LPS receptor TLR4 (Toll-like receptor 4), are resistant to abscess formation. Experiments with barcoded E. coli revealed that TLR4 mediates a tradeoff between abscess formation and bacterial clearance. Together, our findings define hallmarks of E. coli liver abscess formation and suggest that hyperactivation of the hepatic innate immune response drives liver abscess susceptibility.

Disulfiram blocks inflammatory TLR4 signaling by targeting MD-2.

Toll-like receptor 4 (TLR4) sensing of lipopolysaccharide (LPS), the most potent pathogen-associated molecular pattern of gram-negative bacteria, activates NF-κB and Irf3, which induces inflammatory cytokines and interferons that trigger an intense inflammatory response, which is critical for host defense but can also cause serious inflammatory pathology, including sepsis. Although TLR4 inhibition is an attractive therapeutic approach for suppressing overexuberant inflammatory signaling, previously identified TLR4 antagonists have not shown any clinical benefit. Here, we identify disulfiram (DSF), an FDA-approved drug for alcoholism, as a specific inhibitor of TLR4-mediated inflammatory signaling. TLR4 cell surface expression, LPS sensing, dimerization and signaling depend on TLR4 binding to MD-2. DSF and other cysteine-reactive drugs, previously shown to block LPS-triggered inflammatory cell death (pyroptosis), inhibit TLR4 signaling by covalently modifying Cys133 of MD-2, a key conserved residue that mediates TLR4 sensing and signaling. DSF blocks LPS-triggered inflammatory cytokine, chemokine, and interferon production by macrophages in vitro. In the aggressive N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease (PD) in which TLR4 plays an important role, DSF markedly suppresses neuroinflammation and dopaminergic neuron loss, and restores motor function. Our findings identify a role for DSF in curbing TLR4-mediated inflammation and suggest that DSF and other drugs that target MD-2 might be useful for treating PD and other diseases in which inflammation contributes importantly to pathogenesis.

A novel 12-membered ring non-antibiotic macrolide EM982 attenuates cytokine production by inhibiting IKKß and IκBα phosphorylation.

Antimicrobial resistance poses a serious threat to human health worldwide and its incidence continues to increase owing to the overuse of antibiotics and other factors. Macrolide antibiotics such as erythromycin (EM) have immunomodulatory effects in addition to their antibacterial activity. Long-term, low-dose administration of macrolides has shown clinical benefits in treating non-infectious inflammatory respiratory diseases. However, this practice may also increase the emergence of drug-resistant bacteria. In this study, we synthesized a series of EM derivatives, and screened them for two criteria: (i) lack of antibacterial activity and (ii) ability to suppress tumor necrosis factor-α (TNF-α) production in THP-1 cells stimulated with lipopolysaccharide. Among the 37 synthesized derivatives, we identified a novel 12-membered ring macrolide EM982 that lacked antibacterial activity against Staphylococcus aureus and suppressed the production of TNF-α and other cytokines. The effects of EM982 on Toll-like receptor 4 (TLR4) signaling were analyzed using a reporter assay and Western blotting. The reporter assay showed that EM982 suppressed the activation of transcription factors, NF-κB and/or activator protein 1 (AP-1), in HEK293 cells expressing human TLR4. Western blotting showed that EM982 inhibited the phosphorylation of both IκB kinase (IKK) ß and IκBα, which function upstream of NF-κB, whereas it did not affect the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase, which act upstream of AP-1. These results suggest that EM982 suppresses cytokine production by inhibiting phosphorylation of IKKß and IκBα, resulting in the inactivation of NF-κB.

Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells.

HIV-1 has a broad range of nuanced interactions with the immune system, and the incorporation of cellular proteins by nascent virions continues to redefine our understanding of the virus-host relationship. Proteins located at the sites of viral egress can be selectively incorporated into the HIV-1 envelope, imparting new functions and phenotypes onto virions, and impacting viral spread and disease. Using virion capture assays and western blot, we show that HIV-1 can incorporate the myeloid antigen CD14 into its viral envelope. Virion-incorporated CD14 remained biologically active and able to bind its natural ligand, bacterial lipopolysaccharide (LPS), as demonstrated by flow virometry and immunoprecipitation assays. Using a Toll-like receptor 4 (TLR4) reporter cell line, we also demonstrated that virions with bound LPS can trigger TLR4 signaling to activate transcription factors that regulate inflammatory gene expression. Complementary assays with THP-1 monocytes demonstrated enhanced secretion of inflammatory cytokines like tumor necrosis factor alpha (TNF-α) and the C-C chemokine ligand 5 (CCL5), when exposed to LPS-loaded virus. These data highlight a new type of interplay between HIV-1 and the myeloid cell compartment, a previously well-established cellular contributor to HIV-1 pathogenesis and inflammation. Persistent gut inflammation is a hallmark of chronic HIV-1 infection, and contributing to this effect is the translocation of microbes across the gut epithelium. Our data herein provide proof of principle that virion-incorporated CD14 could be a novel mechanism through which HIV-1 can drive chronic inflammation, facilitated by HIV-1 particles binding bacterial LPS and initiating inflammatory signaling in TLR4-expressing cells.IMPORTANCEHIV-1 establishes a lifelong infection accompanied by numerous immunological changes. Inflammation of the gut epithelia, exacerbated by the loss of mucosal T cells and cytokine dysregulation, persists during HIV-1 infection. Feeding back into this loop of inflammation is the translocation of intestinal microbes across the gut epithelia, resulting in the systemic dissemination of bacterial antigens, like lipopolysaccharide (LPS). Our group previously demonstrated that the LPS receptor, CD14, can be readily incorporated by HIV-1 particles, supporting previous clinical observations of viruses derived from patient plasma. We now show that CD14 can be incorporated by several primary HIV-1 isolates and that this virion-incorporated CD14 can remain functional, enabling HIV-1 to bind to LPS. This subsequently allowed CD14+ virions to transfer LPS to monocytic cells, eliciting pro-inflammatory signaling and cytokine secretion. We posit here that virion-incorporated CD14 is a potential contributor to the dysregulated immune responses present in the setting of HIV-1 infection.

Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

Exogenous L-Alanine promotes phagocytosis of multidrug-resistant bacterial pathogens.

Multidrug-resistant bacteria present a major threat to public health that urgently requires new drugs or treatment approaches. Here, we conduct integrated proteomic and metabolomics analyses to screen for molecular candidates improving survival of mice infected with Vibrio parahaemolyticus, which indicate that L-Alanine metabolism and phagocytosis are strongly correlated with mouse survival. We also assess the role of L-Alanine in improving mouse survival by in vivo bacterial challenge experiments using various bacteria species, including V. parahaemolyticus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Functional studies demonstrate that exogenous L-Alanine promotes phagocytosis of these multidrug-resistant pathogen species. We reveal that the underlying mechanism involves two events boosted by L-Alanine: TLR4 expression and L-Alanine-enhanced TLR4 signaling via increased biosynthesis and secretion of fatty acids, including palmitate. Palmitate enhances binding of lipopolysaccharide to TLR4, thereby promoting TLR4 dimer formation and endocytosis for subsequent activation of the PI3K/Akt and NF-κB pathways and bacteria phagocytosis. Our data suggest that modulation of the metabolic environment is a plausible approach for combating multidrug-resistant bacteria infection.

Role of pattern recognition receptors in chemotherapy-induced neuropathic pain.

Progress in the development of effective chemotherapy is producing a growing population of patients with acute and chronic painful chemotherapy-induced peripheral neuropathy (CIPN), a serious treatment-limiting side effect for which there is currently no US Food and Drug Administration-approved treatment. CIPNs induced by diverse classes of chemotherapy drugs have remarkably similar clinical presentations, leading to the suggestion they share underlying mechanisms. Sensory neurons share with immune cells the ability to detect damage associated molecular patterns (DAMPs), molecules produced by diverse cell types in response to cellular stress and injury, including by chemotherapy drugs. DAMPs, in turn, are ligands for pattern recognition receptors (PRRs), several of which are found on sensory neurons, as well as satellite cells, and cells of the immune system. In the present experiments, we evaluated the role of two PRRs, TLR4 and RAGE, present in dorsal root ganglion (DRG), in CIPN. Antisense (AS)-oligodeoxynucleotides (ODN) against TLR4 and RAGE mRNA were administered intrathecally before ('prevention protocol') or 3 days after ('reversal protocol') the last administration of each of three chemotherapy drugs that treat cancer by different mechanisms (oxaliplatin, paclitaxel and bortezomib). TLR4 and RAGE AS-ODN prevented the development of CIPN induced by all three chemotherapy drugs. In the reversal protocol, however, while TLR4 AS-ODN completely reversed oxaliplatin- and paclitaxel-induced CIPN, in rats with bortezomib-induced CIPN it only produced a temporary attenuation. RAGE AS-ODN, in contrast, reversed CIPN induced by all three chemotherapy drugs. When a TLR4 antagonist was administered intradermally to the peripheral nociceptor terminal, it did not affect CIPN induced by any of the chemotherapy drugs. However, when administered intrathecally, to the central terminal, it attenuated hyperalgesia induced by all three chemotherapy drugs, compatible with a role of TLR4 in neurotransmission at the central terminal but not sensory transduction at the peripheral terminal. Finally, since it has been established that cultured DRG neurons can be used to study direct effects of chemotherapy on nociceptors, we also evaluated the role of TLR4 in CIPN at the cellular level, using patch-clamp electrophysiology in DRG neurons cultured from control and chemotherapy-treated rats. We found that increased excitability of small-diameter DRG neurons induced by in vivo and in vitro exposure to oxaliplatin is TLR4-dependent. Our findings suggest that in addition to the established contribution of PRR-dependent neuroimmune mechanisms, PRRs in DRG cells also have an important role in CIPN.

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense.

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and ß-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Heparin-binding hemagglutinin of Mycobacterium tuberculosis inhibits autophagy via TLR4 and drives M2 polarization in macrophages.

BACKGROUND: Tuberculosis (TB), predominantly caused by Mycobacterium tuberculosis (MTB) infection, remains a prominent global health challenge. Macrophages are the frontline defense against MTB, relying on autophagy for intracellular bacterial clearance. However, MTB can combat and evade autophagy, and it influences macrophage polarization, facilitating immune evasion and promoting infection. We previously found that heparin-binding hemagglutinin (HBHA) inhibits autophagy in A549 cells; however, its role in macrophage autophagy and polarization remains unclear. METHODS: Bacterial cultures, cell cultures, western blotting, immunofluorescence, macrophage infection assays, siRNA knockdown, and ELISA were used to investigate HBHA's impact on macrophages and its relevance in Mycobacterium infection. RESULTS: HBHA inhibited macrophage autophagy. Expression of recombinant HBHA in Mycobacterium smegmatis (rMS-HBHA) inhibited autophagy, promoting bacterial survival within macrophages. Conversely, HBHA knockout in the Mycobacterium bovis Bacillus Calmette-Guérin (BCG) mutant (BCG-ΔHBHA) activated autophagy and reduced bacterial survival. Mechanistic investigations revealed that HBHA may inhibit macrophage autophagy through the TLR4-dependent PI3K-AKT-mTOR signaling pathway. Furthermore, HBHA induced macrophage M2 polarization. CONCLUSIONS: Mycobacterium may exploit HBHA to suppress the antimicrobial immune response in macrophages, facilitating intracellular survival, and immune evasion through autophagy inhibition and M2 polarization induction. Our findings may help identify novel therapeutic targets and develop more effective treatments against MTB infection.

Homodimeric Granzyme A Opsonizes Mycobacterium tuberculosis and Inhibits Its Intracellular Growth in Human Monocytes via Toll-Like Receptor 4 and CD14.

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete granzyme A (GzmA) protective against intracellular Mtb growth. However, GzmA-enzymatic activity is unnecessary for pathogen inhibition, and the mechanisms of GzmA-mediated protection remain unknown. We show that GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes, and subsequent pathogen clearance within the phagolysosome. Although monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Without access to surface-expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1 was associated with inhibitory activity. Furthermore, GzmA colocalized with and was regulated by protein disulfide isomerase AI (PDIA1), which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify a previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis, and elimination of Mtb in human monocytes, and they highlight PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

CD14 recycling modulates LPS-induced inflammatory responses of murine macrophages.

TLR4 is activated by the bacterial endotoxin lipopolysaccharide (LPS) and triggers two proinflammatory signaling cascades: a MyD88-dependent one in the plasma membrane, and the following TRIF-dependent one in endosomes. An inadequate inflammatory reaction can be detrimental for the organism by leading to sepsis. Therefore, novel approaches to therapeutic modulation of TLR4 signaling are being sought after. The TLR4 activity is tightly connected with the presence of CD14, a GPI-anchored protein that transfers LPS monomers to the receptor and controls its endocytosis. In this study we focused on CD14 trafficking as a still poorly understood factor affecting TLR4 activity. Two independent assays were used to show that after endocytosis CD14 can recycle back to the plasma membrane in both unstimulated and stimulated cells. This route of CD14 trafficking can be controlled by sorting nexins (SNX) 1, 2 and 6, and is important for maintaining the surface level and the total level of CD14, but can also affect the amount of TLR4. Silencing of these SNXs attenuated especially the CD14-dependent endosomal signaling of TLR4, making them a new target for therapeutic regulation of the inflammatory response of macrophages to LPS.