Spatial transcriptomic sequencing reveals immune microenvironment features of Mycobacterium tuberculosis granulomas in lung and omentum.

Granulomas are a key pathological feature of tuberculosis (TB), characterized by cell heterogeneity, spatial composition, and cellular interactions, which play crucial roles in granuloma progression and host prognosis. This study aims to analyze the transcriptome profiles of cell populations based on their spatial location and to understand the core transcriptome characteristics of granuloma formation and development. Methods In this study, we collected four clinical biopsy samples including Mycobacterium tuberculosis (Mtb) infected lung (MTB-L) and omentum tissues (MTB-O), as well as two lung and omentum biopsies from non-TB patients. The tissues were analyzed by spatial transcriptomics to create a spatial atlas. Utilizing cell enrichment scores and intercellular communication analysis, we investigated the transcriptome signatures of cell populations in various spatial regions and identified genes that may play a decisive role in the formation of pulmonary and omental tuberculosis granulomas. To validate our major findings, an in vitro TB model based on organoid-macrophage co-culture was established. Results Spatial transcriptomics mapped the cell composition and spatial distribution characteristics of tuberculosis granulomas in lung and omental tissues infected with Mtb. The characteristics and evolutionary relationships of major cell populations in granulomas reveal a shift in the immune microenvironment: from a predominance of B cells and fibroblasts in pulmonary granulomas to a predominance of myeloid cells and fibroblasts in omental granulomas. Furthermore, our data identified key differentially expressed genes across cell clusters and regions, showing that upregulation of collagen genes is a common feature of granulomas. Using an organoid-macrophage co-culture model, we demonstrated the notable efficacy of Thrombospondin-1 (THBS1) in reducing protein expression levels related to extracellular matrix remodeling. Conclusion These results provide insights into the pathogenesis and development of tuberculosis, enhancing our understanding of the composition and interactions of tuberculosis granuloma cells from a spatial perspective, and pave the way for novel adjuvant treatments for tuberculosis.

Histological evidence of MAPK pathway activation across subtypes of adult orbital xanthogranulomatous disease irrespective of the detection of oncogenic mutations.

Adult orbital xanthogranulomatous disease (AOXGD) is a spectrum of histiocytoses with four subtypes. Mitogen-activated protein kinase (MAPK) pathway mutations have been detected in various histiocytic neoplasms, little is known about this in AOXGD. Targeted regions of cancer- and histiocytosis-related genes were analyzed and immunohistochemical staining of phosphorylated ERK (pERK), cyclin D1 and PU.1 was performed in 28 AOXGD and 10 control xanthelasma biopsies to assess MAPK pathway activation. Mutations were detected in 7/28 (25%) patients. Positive staining for pERK and/or cyclin D1 was found across all subtypes in 17/27 (63%) patients of whom 12/17 (71%) did not harbour a mutation. Xanthelasma tissue stained negative for pERK and cyclin D1. Relapse occurred in 5/7 (71%) patients with a MAPK pathway mutation compared to 8/21 (38%) patients in whom no mutation could be detected. Molecular analysis and evaluation for systemic disease is warranted to identify patients at risk of recurrent xanthomatous disease.

Regnase-1 D141N mutation induces CD4+ T cell-mediated lung granuloma formation via upregulation of Pim2.

Regnase-1 is an RNase that plays a critical role in negatively regulating immune responses by destabilizing inflammatory messenger RNAs (mRNAs). Dysfunction of Regnase-1 can be a major cause of various inflammatory diseases with tissue injury and immune cell infiltration into organs. This study focuses on the role of the RNase activity of Regnase-1 in developing inflammatory diseases. We have constructed mice with a single point mutation at the catalytic center of the Regnase-1 RNase domain, which lacks endonuclease activity. D141N mutant mice demonstrated systemic inflammation, immune cell infiltration into various organs, and progressive development of lung granuloma. CD4+ T cells, mainly affected by this mutation, upregulated the mTORC1 pathway and facilitated the autoimmune trait in the D141N mutation. Moreover, serine/threonine kinase Pim2 contributed to lung inflammation in this mutation. Inhibition of Pim2 kinase activity ameliorated granulomatous inflammation, immune cell infiltration, and proliferation in the lungs. Additionally, Pim2 inhibition reduced the expression of adhesion molecules on CD4+ T cells, suggesting a role for Pim2 in facilitating leukocyte adhesion and migration to inflamed tissues. Our findings provide new insights into the role of Regnase-1 RNase activity in controlling immune functions and underscore the therapeutic relevance of targeting Pim2 to modulate abnormal immune responses.

Zebrafish tsc1 and cxcl12a increase susceptibility to mycobacterial infection.

Regulation of host miRNA expression is a contested node that controls the host immune response to mycobacterial infection. The host must counter subversive efforts of pathogenic mycobacteria to launch a protective immune response. Here, we examine the role of miR-126 in the zebrafish-Mycobacterium marinum infection model and identify a protective role for infection-induced miR-126 through multiple effector pathways. We identified a putative link between miR-126 and the tsc1a and cxcl12a/ccl2/ccr2 signalling axes resulting in the suppression of non-tnfa expressing macrophage accumulation at early M. marinum granulomas. Mechanistically, we found a detrimental effect of tsc1a expression that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death via mTOR inhibition. We found that macrophage recruitment driven by the cxcl12a/ccl2/ccr2 signalling axis was at the expense of the recruitment of classically activated tnfa-expressing macrophages and increased cell death around granulomas. Together, our results delineate putative pathways by which infection-induced miR-126 may shape an effective immune response to M. marinum infection in zebrafish embryos.

Exome Sequencing Implicates DGKZ , ESRRA , and GXYLT1 for Modulating Granuloma Formation in Crohn Disease.

Non-caseating granulomas may indicate a more aggressive phenotype of Crohn disease (CD). Genetic associations of granulomatous CD (GCD) may help elucidate disease pathogenesis. Whole-exome sequencing was performed on peripheral blood-derived DNA from 17 pediatric patients with GCD and 19 with non-GCD (NGCD), and from an independent validation cohort of 44 GCD and 19 NGCD cases. PLINK (a tool set for whole-genome association and population-based linkage analyses) analysis was used to identify single nucleotide polymorphisms (SNPs) differentiating between groups, and subgroup allele frequencies were also compared to a public genomic database (gnomAD). The Combined Annotation Dependent Depletion scoring tool was used to predict deleteriousness of SNPs. Human leukocyte antigen (HLA) haplotype findings were compared to a control group (n = 8496). PLINK-based analysis between GCD and NGCD groups did not find consistently significant hits. gnomAD control comparisons, however, showed consistent subgroup associations with DGKZ , ESRRA , and GXYLT1 , genes that have been implicated in mammalian granulomatous inflammation. Our findings may guide future research and precision medicine.

Delayed Diagnosis of Chronic Necrotizing Granulomatous Skin Lesions due to TAP2 Deficiency.

Major histocompatibility complex class I (MHC-I) deficiency, also known as bare lymphocyte syndrome type 1 (BLS-1), is a rare autosomal recessively inherited immunodeficiency disorder with remarkable clinical and biological heterogeneity. Transporter associated with antigen processing (TAP) is a member of the ATP-binding cassette superfamily of transporters and consists of two subunits, TAP1 or TAP2. Any defect resulting from a mutation or deletion of these two subunits may adversely affect the peptide translocation in the endoplasmic reticulum, which is an important process for properly assembling MHC-I molecules. To date, only 12 TAP2-deficient patients were reported in the literature. Herein, we described two Iranian cases with 2 and 3 decades of delayed diagnosis of chronic necrotizing granulomatous skin lesions due to TAP2 deficiency without pulmonary involvement. Segregation analysis in family members identified 3 additional homozygous asymptomatic carriers. In both asymptomatic and symptomatic carriers, HLA-I expression was only 4-15% of the one observed in healthy controls. We performed the first deep immunophenotyping in TAP2-deficient patients. While total CD8 T cell counts were normal as previously reported, the patients showed strongly impaired naïve CD8 T cell counts. Mucosal-associated invariant T (MAIT) cells and invariant natural killer T (iNKT) cell counts were increased.

Case report: Persistent shedding of a live vaccine-derived rubella virus in a young man with severe combined immunodeficiency and cutaneous granuloma.

A young man with X-linked severe combined immunodeficiency developed a persistent vaccine-derived rubella virus (VDRV) infection, with the emergence of cutaneous granulomas more than fifteen years after receipt of two doses of measles-mumps-rubella (MMR) vaccine. Following nasopharyngeal swab (NP) collection, VDRV was detected by real-time polymerase chain reaction (RT-qPCR) and sequencing, and live, replication-competent VDRV was isolated in cell culture. To assess duration and intensity of viral shedding, sequential respiratory samples, one cerebrospinal fluid sample, and two urine samples were collected over 15 months, and VDRV RNA was detected in all samples by RT-qPCR. Live VDRV was cultured from nine of the eleven respiratory specimens and from one urine specimen. To our knowledge, this was the first reported instance of VDRV cultured from respiratory specimens or from urine. To assess potential transmission to close contacts, NP specimens and sera were collected from all household contacts, all of whom were immunocompetent and previously vaccinated with MMR. VDRV RNA was not detected in any NP swabs from the contacts, nor did serologic investigations suggest VDRV transmission to any contacts. This report highlights the need to understand the prevalence and duration of VDRV shedding in granuloma patients and to estimate the risk of VDRV transmission to immune and non-immune contacts.

Granulomatous interstitial nephritis with CTLA-4 haploinsufficiency: a case report.

BACKGROUND: Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential inhibitory regulator of immune activation. CTLA-4 haploinsufficiency is known to be associated with dysregulation of FOXP3+ regulatory T cells, hyperactivation of effector T cells, and lymphocytic infiltration of multiple organs. However, there have only been a few reports of renal involvement with CTLA-4. Herein, we present a case of acute granulomatous tubulointerstitial nephritis (TIN) in a patient with CTLA-4 haploinsufficiency. CASE PRESENTATION: A 44-year-old man presented with a 3-week history of fever and malaise, and subsequently developed acute kidney injury (AKI) a few days after treatment with levofloxacin (LVFX). A kidney biopsy and immunohistochemical staining revealed granulomatous TIN with dominantly infiltrating CD4+ T cells. General symptoms and renal impairment showed improvement after discontinuation of LVFX and initiation of oral steroids. However, they worsened following steroid tapering. Further, a colon biopsy analysis showed similar findings to the renal tissue analysis. We suspected that granulomatous TIN was possibly associated with CTLA-4 haploinsufficiency. Therefore, the patient was transferred to another hospital for further treatment of CTLA-4 haploinsufficiency using immunosuppressive agents. CONCLUSIONS: There have been few reports regarding renal involvement of CTLA-4 haploinsufficiency. In the present case, granulomatous TIN could have arisen due to instability of immune regulatory functions, such as CTLA-4 haploinsufficiency, and treatment with LVFX could have triggered immunologic activation and severe inflammation as well as renal dysfunction.

Gene Silencing of Toll-like Receptor 2 Gene Expression as a Tactic to Control Mycobacterium Tuberculosis and Granuloma Formation.

Mycobacterium tuberculosis (MT) is the causative agent of tuberculosis (TB) in humans. Tuberculosis is one of the top 10 causes of mortality worldwide, resulting in 1.8 million deaths and 10.4 million new cases in 2016. Understanding the fundamental features of MT biology is critical to the eradication of MT in the future. Due to the increasing frequency of antimicrobial treatment resistance and problems in vaccine development, the pathogenesis of TB for its survival and growth is highly dependent on host lipids and stimulated-lipid droplets formation. Toll-like receptor 2 (TLR2) forms heterophilic dimers with TLR1 and TLR6, therefore, recognizing many MT components. Both of these receptors identify the invading antigen and activate downstream protein kinases. Some studies demonstrated that the cyclooxygenase-2 (COX-2) promoter-driven gene expression includes connecting sites for transcription factors, such as nuclear factor-kappa B, CREB, NFAT, and c/EBPß. The current study aimed to investigate the role of the TLR2 receptor in positively regulating prostaglandin E2 production in M. bovis (BCG) infected macrophages in vivo using a human monocytic cell line THP-1. Our results revealed that MT infection triggers a time-dependent increase in COX-2 expression via pathways involving TLR2 receptor activation and enhances COX-2 expression, leading to an increase in lipid droplet formation and suppression of macrophage activation.

Case Report: Rubella Virus-Induced Cutaneous Granulomas in Two Pediatric Patients With DNA Double Strand Breakage Repair Disorders - Outcome After Hematopoietic Stem Cell Transplantation.

We report two patients with DNA repair disorders (Artemis deficiency, Ataxia telangiectasia) with destructive skin granulomas, presumably triggered by live-attenuated rubella vaccinations. Both patients showed reduced naïve T cells. Rapid resolution of skin lesions was observed following hematopoietic stem cell transplantation. However, the patient with AT died due to complications of severe hepatic veno-occlusive disease 6 month after HSCT. Dried blood spots obtained after birth were available from this patient and showed absent T-cell receptor excision circles (TRECs). Therefore, newborn screening may help to prevent patients with moderate T-cell deficiency from receiving live-attenuated rubella vaccine potentially causing granulomas.

The Immunogenetics of Granulomatous Diseases.

Non-infectious granulomatous skin diseases are a wide category of well-defined reactive inflammatory conditions that share main similarities. While cutaneous sarcoidosis is the prototype of non-infectious (sterile) granulomatous dermatitides, there are several other entities in this group including granuloma annulare and necrobiosis lipoidica. Non-infectious granulomatous diseases are caused by complex associations between genetic situations and environmental triggers resulting in a variety of cutaneous and systemic manifestations. The genetic backgrounds of these diseases are the main topic of this manuscript.

Rubella vaccine-induced granulomas are a novel phenotype with incomplete penetrance of genetic defects in cytotoxicity.

BACKGROUND: Rubella virus-induced granulomas have been described in patients with various inborn errors of immunity. Most defects impair T-cell immunity, suggesting a critical role of T cells in rubella elimination. However, the molecular mechanism of virus control remains elusive. OBJECTIVE: This study sought to understand the defective effector mechanism allowing rubella vaccine virus persistence in granulomas. METHODS: Starting from an index case with Griscelli syndrome type 2 and rubella skin granulomas, this study combined an international survey with a literature search to identify patients with cytotoxicity defects and granuloma. The investigators performed rubella virus immunohistochemistry and PCR and T-cell migration assays. RESULTS: This study identified 21 patients with various genetically confirmed cytotoxicity defects, who presented with skin and visceral granulomas. Rubella virus was demonstrated in all 12 accessible biopsies. Granuloma onset was typically before 2 years of age and lesions persisted from months to years. Granulomas were particularly frequent in MUNC13-4 and RAB27A deficiency, where 50% of patients at risk were affected. Although these proteins have also been implicated in lymphocyte migration, 3-dimensional migration assays revealed no evidence of impaired migration of patient T cells. Notably, patients showed no evidence of reduced control of concomitantly given measles, mumps, or varicella live-attenuated vaccine or severe infections with other viruses. CONCLUSIONS: This study identified lymphocyte cytotoxicity as a key effector mechanism for control of rubella vaccine virus, without evidence for its need in control of live measles, mumps, or varicella vaccines. Rubella vaccine-induced granulomas are a novel phenotype with incomplete penetrance of genetic disorders of cytotoxicity.

RAG1 splicing mutation causes enhanced B cell differentiation and autoantibody production.

Hypomorphic RAG1 or RAG2 mutations cause primary immunodeficiencies and can lead to autoimmunity, but the underlying mechanisms are elusive. We report here a patient carrying a c.116+2T>G homozygous splice site mutation in the first intron of RAG1, which led to aberrant splicing and greatly reduced RAG1 protein expression. B cell development was blocked at both the pro-B to pre-B transition and the pre-B to immature B cell differentiation step. The patient B cells had reduced B cell receptor repertoire diversity and decreased complementarity determining region 3 lengths. Despite B cell lymphopenia, the patient had abundant plasma cells in the BM and produced large quantities of IgM and IgG Abs, including autoantibodies. The proportion of naive B cells was reduced while the frequency of IgD-CD27- double-negative (DN) B cells, which quickly differentiated into Ab-secreting plasma cells upon stimulation, was greatly increased. Immune phenotype analysis of 52 patients with primary immunodeficiency revealed a strong association of the increased proportion of DN B and memory B cells with decreased number and proportion of naive B cells. These results suggest that the lymphopenic environment triggered naive B cell differentiation into DN B and memory B cells, leading to increased Ab production.

Tuberculosis lymph node granulomas: using transcriptomics to discover immunopathology paradigms and guide host-directed therapy.

Immunometabolism is a burgeoning field of investigation in tuberculosis host defense, susceptibility, and pathophysiology. Unbiased approaches to studying tuberculosis have, as expected, confirmed that pathways of immunometabolism are crucial in these disease processes. In this issue of the JCI, Reichmann et al. studied carefully controlled human lymph node tuberculosis and uncovered Sphingosine kinase 1 as a druggable target of interest that could support the infected host. Future host-directed therapy research might seek to establish the different cellular consequences of sphingolipid pathway manipulation. Animal models will be especially useful to establish the role of this pathway, which might target diseased organs to improve mycobactericidal effect and limit pathology.

Hypoxia Promotes a Mixed Inflammatory-Fibrotic Macrophages Phenotype in Active Sarcoidosis.

Background: Macrophages are pivotal cells in sarcoidosis. Monocytes-derived (MD) macrophages have recently been demonstrated to play a major role especially in pulmonary sarcoidosis. From inflammatory tissues to granulomas, they may be exposed to low oxygen tension environments. As hypoxia impact on sarcoidosis immune cells has never been addressed, we designed the present study to investigate MD-macrophages from sarcoidosis patients in this context. We hypothesized that hypoxia may induce functional changes on MD-macrophages which could have a potential impact on the course of sarcoidosis. Methods: We studied MD-macrophages, from high active sarcoidosis (AS) (n=26), low active or inactive sarcoidosis (IS) (n=24) and healthy controls (n=34) exposed 24 hours to normoxia (21% O2) or hypoxia (1.5% O2). Different macrophage functions were explored: hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) activation, cytokines secretion, phagocytosis, CD80/CD86/HLA-DR expression, profibrotic response. Results: We observed that hypoxia, with a significantly more pronounced effect in AS compared with controls and IS, increased the HIF-1α trans-activity, promoted a proinflammatory response (TNFα, IL1ß) without activating NF-κB pathway and a profibrotic response (TGFß1, PDGF-BB) with PAI-1 secretion associated with human lung fibroblast migration inhibition. These results were confirmed by immunodetection of HIF-1α and PAI-1 in granulomas observed in pulmonary biopsies from patients with sarcoidosis. Hypoxia also decreased the expression of CD80/CD86 and HLA-DR on MD-macrophages in the three groups while it did not impair phagocytosis and the expression of CD36 expression on cells in AS and IS at variance with controls. Conclusions: Hypoxia had a significant impact on MD-macrophages from sarcoidosis patients, with the strongest effect seen in patients with high active disease. Therefore, hypoxia could play a significant role in sarcoidosis pathogenesis by increasing the macrophage proinflammatory response, maintaining phagocytosis and reducing antigen presentation, leading to a deficient T cell response. In addition, hypoxia could favor fibrosis by promoting profibrotic cytokines response and by sequestering fibroblasts in the vicinity of granulomas.

New Findings of Immunodysregulation, Polyendocrinopathy, and Enteropathy X-linked Syndrome (IPEX); Granulomas in Lung and Duodenum.

Immune dysregulation, polyendocrinopathy and enteropathy, X-linked (IPEX) syndrome is a rare disorder caused by loss-of-function mutations in the gene forkhead box protein 3 (FOXP3). IPEX patients frequently show chronic diarrhea (enteropathy) associated with villous atrophies in the small intestine. Our case is different from this classical information in the literature, since he presented with neonatal onset inflammatory bowel disease within the first months of life accompanied by deep ulcers throughout colonic mucosa. Moreover, he developed chronic lung disease during follow-up and histopathological examinations showed granulomas in both gastrointestinal tract and lung parenchyma. Genetic analysis revealed the diagnosis of IPEX syndrome with a germline mutation in FOXP3. Thus, our study provides an unusual presentation of IPEX syndrome with colitis and granulomas presence in histopathological examinations.

Tim4 recognizes carbon nanotubes and mediates phagocytosis leading to granuloma formation.

Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.

The adapter protein Myd88 plays an important role in limiting mycobacterial growth in a zebrafish model for tuberculosis.

Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells.

Characterization of macrophages, giant cells and granulomas during muscle regeneration after irradiation.

Macrophages play a fundamental role in the different stages of muscle regeneration although the precise mechanisms involved are not entirely understood. Here we investigated the types of macrophages and cytokines that appeared in muscles after local gamma irradiation of mini-pigs that underwent no subsequent treatment or received three successive adipose tissue-derived stem cell (ASC) injections. Although some variability was observed among the three animals included in each study group, a general picture emerged. No macrophages appeared in control muscles from regions that had not been irradiated nor in muscles from irradiated regions derived from two animals. A third irradiated, but untreated animal, with characteristic muscle fibrosis and necrosis due to irradiation, showed invasion of M2 macrophages within small muscle lesions. In contrast, among the three ASC-treated and irradiated animals, one of them had completely recovered normal muscle architecture at the time of sampling with no invading macrophages, muscle from a second one contained mostly M1 macrophages and some M2-like macrophages whereas muscle from a third one displayed granulomas and giant cells. ASC treatment was associated with the presence of similar levels of pro-inflammatory cytokines within the two animals in the process of muscle regeneration whereas the levels of IL-4 and IL-10 expression were distinct from one animal to another. Microspectrofluorimetry and in situ hybridization revealed strong expression of TGF-ß1 and TNFα in regenerating muscle. Overall, the data confirm the critical role of macrophages in muscle regeneration and suggest the involvement of a complex network of cytokine expression for successful recovery.