The Tuberculous Granuloma and Preexisting Immunity.

Pulmonary granulomas are widely considered the epicenters of the immune response to Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Recent animal studies have revealed factors that either promote or restrict TB immunity within granulomas. These models, however, typically ignore the impact of preexisting immunity on cellular organization and function, an important consideration because most TB probably occurs through reinfection of previously exposed individuals. Human postmortem research from the pre-antibiotic era showed that infections in Mtb-naïve individuals (primary TB) versus those with prior Mtb exposure (postprimary TB) have distinct pathologic features. We review recent animal findings in TB granuloma biology, which largely reflect primary TB. We also discuss our current understanding of postprimary TB lesions, about which much less is known. Many knowledge gaps remain, particularly regarding how preexisting immunity shapes granuloma structure and local immune responses at Mtb infection sites.

The Formation and Function of Granulomas.

Granulomas are organized aggregates of macrophages, often with characteristic morphological changes, and other immune cells. These evolutionarily ancient structures form in response to persistent particulate stimuli-infectious or noninfectious-that individual macrophages cannot eradicate. Granulomas evolved as protective responses to destroy or sequester particles but are frequently pathological in the context of foreign bodies, infections, and inflammatory diseases. We summarize recent findings that suggest that the granulomatous response unfolds in a stepwise program characterized by a series of macrophage activations and transformations that in turn recruit additional cells and produce structural changes. We explore why different granulomas vary and the reasons that granulomas are protective and pathogenic. Understanding the mechanisms and role of granuloma formation may uncover new therapies for the multitude of granulomatous diseases that constitute serious medical problems while enhancing the protective function of granulomas in infections.

A non-canonical type 2 immune response coordinates tuberculous granuloma formation and epithelialization.

The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.

The immunoregulatory landscape of human tuberculosis granulomas.

Tuberculosis (TB) in humans is characterized by formation of immune-rich granulomas in infected tissues, the architecture and composition of which are thought to affect disease outcome. However, our understanding of the spatial relationships that control human granulomas is limited. Here, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) to image 37 proteins in tissues from patients with active TB. We constructed a comprehensive atlas that maps 19 cell subsets across 8 spatial microenvironments. This atlas shows an IFN-γ-depleted microenvironment enriched for TGF-ß, regulatory T cells and IDO1+ PD-L1+ myeloid cells. In a further transcriptomic meta-analysis of peripheral blood from patients with TB, immunoregulatory trends mirror those identified by granuloma imaging. Notably, PD-L1 expression is associated with progression to active TB and treatment response. These data indicate that in TB granulomas, there are local spatially coordinated immunoregulatory programs with systemic manifestations that define active TB.

Single-cell and spatial transcriptomics reveal aberrant lymphoid developmental programs driving granuloma formation.

Granulomas are lumps of immune cells that can form in various organs. Most granulomas appear unstructured, yet they have some resemblance to lymphoid organs. To better understand granuloma formation, we performed single-cell sequencing and spatial transcriptomics on granulomas from patients with sarcoidosis and bioinformatically reconstructed the underlying gene regulatory networks. We discovered an immune stimulatory environment in granulomas that repurposes transcriptional programs associated with lymphoid organ development. Granuloma formation followed characteristic spatial patterns and involved genes linked to immunometabolism, cytokine and chemokine signaling, and extracellular matrix remodeling. Three cell types emerged as key players in granuloma formation: metabolically reprogrammed macrophages, cytokine-producing Th17.1 cells, and fibroblasts with inflammatory and tissue-remodeling phenotypes. Pharmacological inhibition of one of the identified processes attenuated granuloma formation in a sarcoidosis mouse model. We show that human granulomas adopt characteristic aspects of normal lymphoid organ development in aberrant combinations, indicating that granulomas constitute aberrant lymphoid organs.

Decoding the tuberculous granuloma.

In this issue of Immunity,Gideon et al. (2022) couple sophisticated single-cell analyses with detailed in vivo measurements of Mycobacterium tuberculosis granulomas to define the cellular and transcriptional properties of a successful host immune response during tuberculosis.

Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration.

A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers' susceptibility to tuberculosis.

Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression.

The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.

Vitamin A mediates conversion of monocyte-derived macrophages into tissue-resident macrophages during alternative activation.

It remains unclear whether activated inflammatory macrophages can adopt features of tissue-resident macrophages, or what mechanisms might mediate such a phenotypic conversion. Here we show that vitamin A is required for the phenotypic conversion of interleukin 4 (IL-4)-activated monocyte-derived F4/80intCD206+PD-L2+MHCII+ macrophages into macrophages with a tissue-resident F4/80hiCD206-PD-L2-MHCII-UCP1+ phenotype in the peritoneal cavity of mice and during the formation of liver granulomas in mice infected with Schistosoma mansoni. The phenotypic conversion of F4/80intCD206+ macrophages into F4/80hiCD206- macrophages was associated with almost complete remodeling of the chromatin landscape, as well as alteration of the transcriptional profiles. Vitamin A-deficient mice infected with S. mansoni had disrupted liver granuloma architecture and increased mortality, which indicates that failure to convert macrophages from the F4/80intCD206+ phenotype to F4/80hiCD206- may lead to dysregulated inflammation during helminth infection.

Host evasion and exploitation schemes of Mycobacterium tuberculosis.

Tuberculosis, an ancient disease of mankind, remains one of the major infectious causes of human death. We examine newly discovered facets of tuberculosis pathogenesis and explore the evolution of its causative organism Mycobacterium tuberculosis from soil dweller to human pathogen. M. tuberculosis has coevolved with the human host to evade and exploit host macrophages and other immune cells in multiple ways. Though the host can often clear infection, the organism can cause transmissible disease in enough individuals to sustain itself. Tuberculosis is a near-perfect paradigm of a host-pathogen relationship, and that may be the challenge to the development of new therapies for its eradication.

Interleukin 1α and the inflammatory process.

Inflammation occurs after disruption of tissue homeostasis by cell stress, injury or infection and ultimately involves the recruitment and retention of cells of hematopoietic origin, which arrive at the affected sites to resolve damage and initiate repair. Interleukin 1α (IL-1α) and IL-1ß are equally potent inflammatory cytokines that activate the inflammatory process, and their deregulated signaling causes devastating diseases manifested by severe acute or chronic inflammation. Although much attention has been given to understanding the biogenesis of IL-1ß, the biogenesis of IL-1α and its distinctive role in the inflammatory process remain poorly defined. In this review we examine key aspects of IL-1α biology and regulation and discuss its emerging importance in the initiation and maintenance of inflammation that underlie the pathology of many human diseases.

Reprogramming adult Schwann cells to stem cell-like cells by leprosy bacilli promotes dissemination of infection.

Differentiated cells possess a remarkable genomic plasticity that can be manipulated to reverse or change developmental commitments. Here, we show that the leprosy bacterium hijacks this property to reprogram adult Schwann cells, its preferred host niche, to a stage of progenitor/stem-like cells (pSLC) of mesenchymal trait by downregulating Schwann cell lineage/differentiation-associated genes and upregulating genes mostly of mesoderm development. Reprogramming accompanies epigenetic changes and renders infected cells highly plastic, migratory, and immunomodulatory. We provide evidence that acquisition of these properties by pSLC promotes bacterial spread by two distinct mechanisms: direct differentiation to mesenchymal tissues, including skeletal and smooth muscles, and formation of granuloma-like structures and subsequent release of bacteria-laden macrophages. These findings support a model of host cell reprogramming in which a bacterial pathogen uses the plasticity of its cellular niche for promoting dissemination of infection and provide an unexpected link between cellular reprogramming and host-pathogen interaction.

A COVID-19 peptide vaccine for the induction of SARS-CoV-2 T cell immunity.

T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins1,2, combined with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18-80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4+ and CD8+ T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency.

Humoral pathways of innate immune regulation in granuloma formation.

The humoral arm of mammalian innate immunity regulates several molecular mechanisms involved in resistance to pathogens, inflammation, and tissue repair. Recent studies highlight the crucial role played by humoral mediators in granulomatous inflammation. However the molecular mechanisms linking the function of these soluble molecules to the initiation and maintenance of granulomas remain elusive. We propose that humoral innate immunity coordinates fundamental physiological processes in macrophages which, in turn, initiate activation and transformation events that enable granuloma formation. We discuss the involvement of humoral mediators in processes such as immune activation, phagocytosis, metabolism, and tissue remodeling, and how these can dictate macrophage functionality during granuloma formation. These advances present opportunities for discovering novel disease factors and developing targeted, more effective treatments for granulomatous diseases.

Sarcoidosis immunopathogenesis - a new concept of maladaptive trained immunity.

Sarcoidosis is a chronic immune disease of unknown origin for which we still lack an immunological framework unifying causal agents, host factors, and natural history of disease. Here, we discuss the initial triggers of disease, and how myeloid cells drive granuloma formation and contribute to immunopathogenesis. We highlight recent advances in our understanding of innate immune memory and propose the hypothesis that maladaptive innate immune training connects previous environmental exposure to granuloma maintenance and expansion. Lastly, we consider how this hypothesis may open novel therapeutic avenues, while corticosteroids remain the front-line treatment.

Salmonella Persist in Activated Macrophages in T Cell-Sparse Granulomas but Are Contained by Surrounding CXCR3 Ligand-Positioned Th1 Cells.

Salmonella enterica (Se) bacteria cause persistent intracellular infections while stimulating a robust interferon-γ-producing CD4+ T (Th1) cell response. We addressed this paradox of concomitant infection and immunity by tracking fluorescent Se organisms in mice. Se bacteria persisted in nitric oxide synthase (iNOS)-producing resident and recruited macrophages while inducing genes related to protection from nitric oxide. Se-infected cells occupied iNOS+ splenic granulomas that excluded T cells but were surrounded by mononuclear phagocytes producing the chemokines CXCL9 and CXCL10, and Se epitope-specific Th1 cells expressing CXCR3, the receptor for these chemokines. Blockade of CXCR3 inhibited Th1 occupancy of CXCL9/10-dense regions, reduced activation of the Th1 cells, and led to increased Se growth. Thus, intracellular Se bacteria survive in their hosts by counteracting toxic products of the innate immune response and by residing in T cell-sparse granulomas, away from abundant Th1 cells positioned via CXCR3 in a bordering region that act to limit infection.

Normalizing granuloma vasculature and matrix improves drug delivery and reduces bacterial burden in tuberculosis-infected rabbits.

Host-directed therapies (HDTs) represent an emerging approach for bacterial clearance during tuberculosis (TB) infection. While most HDTs are designed and implemented for immuno-modulation, other host targets-such as nonimmune stromal components found in pulmonary granulomas-may prove equally viable. Building on our previous work characterizing and normalizing the aberrant granuloma-associated vasculature, here we demonstrate that FDA-approved therapies (bevacizumab and losartan, respectively) can be repurposed as HDTs to normalize blood vessels and extracellular matrix (ECM), improve drug delivery, and reduce bacterial loads in TB granulomas. Granulomas feature an overabundance of ECM and compressed blood vessels, both of which are effectively reduced by losartan treatment in the rabbit model of TB. Combining both HDTs promotes secretion of proinflammatory cytokines and improves anti-TB drug delivery. Finally, alone and in combination with second-line antitubercular agents (moxifloxacin or bedaquiline), these HDTs significantly reduce bacterial burden. RNA sequencing analysis of HDT-treated lung and granuloma tissues implicates up-regulated antimicrobial peptide and proinflammatory gene expression by ciliated epithelial airway cells as a putative mechanism of the observed antitubercular benefits in the absence of chemotherapy. These findings demonstrate that bevacizumab and losartan are well-tolerated stroma-targeting HDTs, normalize the granuloma microenvironment, and improve TB outcomes, providing the rationale to clinically test this combination in TB patients.