Granuloma Macrophages Research Articles

Aberrant monocytopoiesis drives granuloma development in sarcoidosis.

In sarcoidosis, granulomas develop in multiple organs including the liver and lungs. Although mechanistic target of rapamycin complex 1 (mTORC1) activation in macrophages drives granuloma development in sarcoidosis by enhancing macrophage proliferation, little is known about the macrophage subsets that proliferate and mature into granuloma macrophages. Here, we show that aberrantly increased monocytopoiesis gives rise to granulomas in a sarcoidosis model, in which Tsc2, a negative regulator of mTORC1, is conditionally deleted in CSF1R-expressing macrophages (Tsc2csf1rΔ mice). In Tsc2csf1rΔ mice, common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), common monocyte progenitors / monocyte progenitors (cMoPs / MPs), inducible monocyte progenitors (iMoPs), and Ly6Cint CX3CR1low CD14- immature monocytes (iMOs), but not monocyte-dendritic cell progenitors (MDPs) and common dendritic cell progenitors (CDPs), accumulated and proliferated in the spleen. Consistent with this, monocytes, neutrophils, and neutrophil-like monocytes increased in the spleens of Tsc2csf1rΔ mice, whereas dendritic cells did not. The adoptive transfer of splenic iMOs into wild-type mice gave rise to granulomas in the liver and lungs. In these target organs, iMOs matured into Ly6Chi classical monocytes/macrophages (cMOs). Giant macrophages (gMAs) also accumulated in the liver and lungs, which were similar to granuloma macrophages in expression of cell surface markers such as MerTK and SLAMF7. Furthermore, the gMA-specific genes were expressed in human macrophages from sarcoidosis skin lesions. These results suggest that mTORC1 drives granuloma development by promoting the proliferation of monocyte/neutrophil progenitors and iMOs predominantly in the spleen, and that proliferating iMOs mature into cMOs and then gMAs to give rise to granuloma after migration into the liver and lungs in sarcoidosis.

Temporal and cellular analysis of granuloma development in mycobacterial infected adult zebrafish.

Because granulomas are a hallmark of tuberculosis pathogenesis, the study of the dynamic changes in their cellular composition and morphological character can facilitate our understanding of tuberculosis pathogenicity. Adult zebrafish infected with Mycobacterium marinum form granulomas that are similar to the granulomas in human patients with tuberculosis and therefore have been used to study host-mycobacterium interactions. Most studies of zebrafish granulomas, however, have focused on necrotic granulomas, while a systematic description of the different stages of granuloma formation in the zebrafish model is lacking. Here, we characterized the stages of granulomas in M. marinum-infected zebrafish, including early immune cell infiltration, nonnecrotizing granulomas, and necrotizing granulomas, using corresponding samples from patients with pulmonary tuberculosis as references. We combined hematoxylin and eosin staining and in situ hybridization to identify the different immune cell types and follow their spatial distribution in the different stages of granuloma development. The macrophages in zebrafish granulomas were shown to belong to distinct subtypes: epithelioid macrophages, foamy macrophages, and multinucleated giant cells. By defining the developmental stages of zebrafish granulomas and the spatial distribution of the different immune cells they contain, this work provides a reference for future studies of mycobacterial granulomas and their immune microenvironments.

High expression of interleukin-17A in cutaneous sarcoidosis.

Increasing evidence of Th17 pathways is implicated in sarcoidosis granulomatous inflammation and maintenance. Our study evaluated the presence of IL-17A, a downstream proinflammatory cytokine, in cutaneous sarcoidosis biopsies. We found that all 26 biopsy samples had increased expression of IL-17A, predominantly located in macrophages within granulomas, in the cytoplasm of multinucleated histiocytes surrounding the granulomas and within lymphocytic infiltrates surrounding the inflammation.

Monitoring the immune response of macrophages in tuberculous granuloma through the expression of CD68, iNOS and HLA-DR in naturally infected beef cattle

Bovine tuberculosis still represents a universal threat that creates a wider range of public and animal health impacts. One of the most important steps in the pathogenesis of this disease and granuloma formation is the phagocytosis of tuberculous bacilli by macrophages. Mycobacteria replicate in macrophages, which are crucial to the pathophysiology of mycobacterial infections; however, scarce information is available about the dynamics of the granuloma-stage immunological response. Therefore, immunohistochemistry was used in this work to evaluate the expression of CD68, iNOS, and HLA-DR in different stages of TB granulomas from naturally infected cattle with tuberculosis. Two thousand, one hundred and fifty slaughtered beef cattle were examined during the period from September 2020 to March 2022. Sixty of them showed gross tuberculous pulmonary lesions and samples were collected from all of them for histopathological examination, Ziehl-Neelsen (ZN) staining, and bacteriological culturing. Selected samples that yielded a positive result for ZN and mycobacterial culturing were subjected to an immunohistochemical study of CD68, iNOS, and HLA-DR expression by macrophages according to granuloma stages. Immunohistochemical analysis revealed that the immunolabeling of CD68+, iNOS+, and HLA-DR+ macrophages significantly reduced as the stage of granuloma increased from stage I to stage IV (P < 0.003, P < 0.002, and P < 0.002, respectively). The distribution of immunolabeled macrophages was similar for the three markers, with immunolabeled macrophages distributed throughout early-stage granulomas (I, II), and surrounding the necrotic core in late-stage granulomas (III, IV). Our results suggest a polarization to the pro-inflammatory environment and increased expression of CD68+, iNOS+, and HLA-DR+ macrophages in the early stages of granulomas (I, II), which may play a protective role in the immune response of naturally infected beef cattle with tuberculosis.

SHP2 promotes sarcoidosis severity by inhibiting SKP2-targeted ubiquitination of TBET in CD8+ T cells.

Sarcoidosis is an interstitial lung disease (ILD) characterized by interferon-γ (IFN-γ) and T-box expressed in T cells (TBET) dysregulation. Although one-third of patients progress from granulomatous inflammation to severe lung damage, the molecular mechanisms underlying this process remain unclear. Here, we found that pharmacological inhibition of phosphorylated SH2-containing protein tyrosine phosphatase-2 (pSHP2), a facilitator of aberrant IFN-γ abundance, decreased large granuloma formation and macrophage infiltration in the lungs of mice with sarcoidosis-like disease. Positive treatment outcomes were dependent on the effective enhancement of TBET ubiquitination within CD8+ T cells. Mechanistically, we identified a posttranslational modification pathway in which the E3 F-box protein S-phase kinase-associated protein 2 (SKP2) targets TBET for ubiquitination in T cells under normal conditions. However, this pathway was disrupted by aberrant pSHP2 signaling in CD8+ T cells from patients with progressive pulmonary sarcoidosis and end-stage disease. Ex vivo inhibition of pSHP2 in CD8+ T cells from patients with end-stage sarcoidosis enhanced TBET ubiquitination and suppressed IFN-γ and collagen synthesis. Therefore, these studies provided new mechanistic insights into the SHP2-dependent posttranslational regulation of TBET and identified SHP2 inhibition as a potential therapeutic intervention against severe sarcoidosis. Furthermore, these studies also suggest that the small-molecule SHP2 inhibitor SHP099 might be used as a therapeutic measure against human diseases linked to TBET or ubiquitination.

Comparative Immunohistochemical Analysis of Macrophage Phenotypes in Cutaneous Sarcoid Granuloma, Suture Granuloma, and Lipogranuloma.

Granulomas are composed of a heterogeneous population of resident and recruited macrophages according to the type of lesion, extent of injury, and local tissue environment (eg, involved site and interaction with infiltrating lymphocytes). Although macrophage phenotypes in various types of granulomas have been previously described, the experimental conditions varied across studies, precluding a comparative and comprehensive understanding of granulomas. This study was conducted to comparatively analyze the expression of markers of the M1 and M2 phenotypes in macrophages that compose various types of granulomas, including epithelioid lesions, under strict conditions. Surgical specimens of cutaneous sarcoidosis (11 lesions), suture granuloma (10 lesions), and subcutaneous lipogranuloma (12 lesions) were immunohistochemically stained for CD11c, CD206, CD163, and CD10. The expression of these markers in macrophages composing each type of granuloma was scored and statistically analyzed. Granuloma macrophages were mostly immunoreactive for CD11c and CD206 in all the examined cases, although many intermingling CD206-negative cells were observed in 5 cases of lipogranuloma. CD163 and CD10 were diffusely expressed in macrophages composing suture granuloma and lipogranuloma, whereas they were not expressed in epithelioid cells in cutaneous sarcoidosis. Meanwhile, "interstitial" macrophages around epithelioid granulomas revealed moderate to marked CD163 expression in 7 lesions of cutaneous sarcoidosis. These results indicate significant differences of expression of CD163 and CD10 between cutaneous sarcoidosis and suture granuloma/lipogranuloma; CD163 and CD10 are downregulated after the epithelioid transformation of macrophages in cutaneous sarcoidosis.

Macrophages and neutrophils in lymph node granulomas from Mycobacterium tuberculosis-infected macaques have immunoregulatory phenotypes

Abstract Tuberculosis (TB) is caused by infection with the Mycobacterium tuberculosis (Mtb). Lung granulomas are the defining pathologic feature of TB but Mtb can infect other organs, including lymph nodes, and causes granulomas to form in these sites. Lymph node granulomas have received less attention than lung granulomas but granulomas in these organs are sites of bacterial persistence and have implications for TB progression. Macrophage subsets and neutrophils in lymph node granulomas have not been extensively studied but the biology of these cells may yield clues that underlie the pathobiology of lymph node infection. Our objective was to describe expression of functional markers and cytokine expression by macrophage subsets and neutrophils in lymph node granulomas and to compare these myeloid cells in lymph node and lung granulomas. We used thoracic lymph node and lung granulomas from cynomolgus macaques, a nonhuman primate that experiences human-like TB pathology and course of disease, to do this work. We found that lymph node epithelioid macrophages were distinct from lymph node dendritic cells but resembled epithelioid macrophages in lung granulomas suggesting these cells share a common progenitor or exposure to common stimuli leads to a phenotypic convergence in different tissues. Macrophages expressed pro- and anti-inflammatory cytokines including type 1 interferon, a cytokine family associated with exacerbated TB. Neutrophils were present in low numbers in non-granulomatous lymph node regions but were selectively recruited to granulomas. Our data suggest that macrophages and neutrophils in lymph node granulomas have immunoregulatory properties that may contribute to local and systemic outcomes in Mtb infection. This work is supported by NIH grants R21 AI167710, R01AI164970, and the Wellcome Trust DELTA tissue program.

Adaptation to the intracellular environment of primary human macrophages influences drug susceptibility of Mycobacterium tuberculosis

As a facultative intracellular pathogen, M. tuberculosis (Mtb) is highly adapted to evading antibacterial mechanisms in phagocytic cells. Both the macrophage and pathogen experience transcriptional and metabolic changes from the onset of phagocytosis. To account for this interaction in the assessment of intracellular drug susceptibility, we allowed a 3-day preadaptation phase post-macrophage infection prior to drug treatment. We found that intracellular Mtb in human monocyte-derived macrophages (MDM) presents dramatic alterations in susceptibility to isoniazid, sutezolid, rifampicin and rifapentine when compared to axenic culture. Infected MDM gradually accumulate lipid bodies, adopting a characteristic appearance reminiscent of foamy macrophages in granulomas. Furthermore, TB granulomas in vivo develop hypoxic cores with decreasing oxygen tension gradients across their radii. Accordingly, we evaluated the effects of hypoxia on preadapted intracellular Mtb in our MDM model. We observed that hypoxia induced greater lipid body formation and no additional shifts in drug tolerance, suggesting that the adaptation of intracellular Mtb to baseline host cell conditions under normoxia dominates changes to intracellular drug susceptibility. Using unbound plasma concentrations in patients as surrogates for free drug concentrations in lung interstitial fluid, we estimate that intramacrophage Mtb in granulomas are exposed to bacteriostatic concentrations of most study drugs.

Single-cell profiling identifies ACE+ granuloma macrophages as a nonpermissive niche for intracellular bacteria during persistent Salmonella infection.

Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica. Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures composed of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent S. enterica serovar Typhimurium (STm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using an STm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin-converting enzyme (ACE) defines a granuloma macrophage population that is nonpermissive for intracellular bacteria, and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked to preferential depletion of ACE+ macrophages in infected tissues. Thus, ACE+ macrophages have limited capacity to serve as cellular niche for intracellular bacteria to establish persistent infection.

Spatial multiomic profiling reveals the novel polarization of foamy macrophages within necrotic granulomatous lesions developed in lungs of C3HeB/FeJ mice infected with Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis leads to the development of tuberculosis (TB) with the formation of granulomatous lesions. Foamy macrophages (FM) are a hallmark of TB granulomas, because they provide the primary platform of M. tuberculosis proliferation and the main source of caseous necrosis. In this study, we applied spatial multiomic profiling to identify the signatures of FM within the necrotic granulomas developed in a mouse model resembling human TB histopathology. C3HeB/FeJ mice were infected with M. tuberculosis to induce the formation of necrotic granulomas in the lungs. Using laser microdissection, necrotic granulomas were fractionated into three distinct regions, including the central caseous necrosis, the rim containing FM, and the peripheral layer of macrophages and lymphocytes, and subjected to proteomic and transcriptomic analyses. Comparison of proteomic and transcriptomic analyses of three distinct granulomatous regions revealed that four proteins/genes are commonly enriched in the rim region. Immunohistochemistry confirmed the localization of identified signatures to the rim of necrotic granulomas. We also investigated the localization of the representative markers for M1 macrophages in granulomas because the signatures of the rim included M2 macrophage markers. The localization of both macrophage markers suggests that FM in necrotic granulomas possessed the features of M1 or M2 macrophages. Gene set enrichment analysis of transcriptomic profiling revealed the upregulation of genes related to M2 macrophage activation and mTORC1 signaling in the rim. These results will provide new insights into the process of FM biogenesis, leading to further understanding of the pathophysiology of TB granulomas.

The role of the response to DNA damage in granulomatous diseases

Granulomas are organized aggregates of immune cells, which are formed in response to a persistent stimulus and are found in various rheumatic diseases, including sarcoidosis, rheumatoid arthritis and granulomatosis with polyangiitis. The core of granulomas contains a multitude of different macrophage subtypes, including multinucleated macrophages and foam cells. The mechanisms which induce the formation of granulomas are not well understood; however, recent data show that the DNA damage response regulates granuloma macrophage differentiation.

Distribution of M1 and M2 macrophages in cerebral granulomas caused by Encephalitozoon cuniculi

Encephalitozoon cuniculi spores cause severe granulomatous inflammation in the brain where mononuclear cells and macrophages infiltrate. Here, we orally infected New Zealand white rabbits with 1 × 106E. cuniculi viable spores to study the recruitment and localization of macrophages in brain granulomas. At day 30 post-infection, the positive phenotype markers iNOS (M1) and Arg-1 (M2) were located in the periphery and center of granulomas, respectively. Live intracytoplasmic spores were found only in positive Arg-1 cells. This is the first work to describe the recruitment and distribution of M1 and M2 macrophages in the brain granulomas of rabbits infected with E. cuniculi.

MTOR-associated Mitochondrial Energy Metabolism Limits Mycobacterium ESX-1-induced Cytotoxicity

Abstract Necrosis of macrophages in the tuberculous granuloma represents a major pathogenic event in tuberculosis. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations were required to prevent mitochondrial damage and death caused specifically by the mycobacterial ESX-1 secretion system. Our finding that the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism may help explain why Mycobacterium tuberculosis, albeit humanity’s most lethal pathogen, only causes disease in a minority of infected individuals. This work was supported by a Wellcome Trust Principal Research Fellowship (103950/Z/14) and an NIH MERIT award (R37 AI054503) (L.R.).

The Role of Lymphocyte Cytosolic Protein 1 in a Mycobacterium Marinum Infection

Mycobacterium tuberculosis is an issue worldwide with multidrug‐resistant forms becoming prevalent due to the lack of effective treatments. The goal of this project is to identify if there are host genes that affect the disease outcome. We decided to study the influence of the Lymphocyte Cytosolic Protein 1 (LCP1) gene in zebrafish larvae infected with mycobacterium marinum. To understand the immune response in the fish we observed the bacterial burden in fish that had LCP1 (heterozygotes) and in fish that did not (knockout mutants). M. marinum was injected into the caudal vein of the zebrafish larvae and the fish were infected. Once the fish were fixed and euthanized, images of the bacterial infection were taken using a fluorescent microscope. The bacteria was tagged with tdTomato, a red fluorescent protein which allows for the bacteria to glow red under the microscope. The images were then analyzed, and the bacterial burden was quantified using ImageJ to count the number of glowing pixels. Following the imaging of the infected larvae, the fish were genotyped. The DNA from the larvae was extracted through a hotSHOT PCR protocol, then LCP1 Exon 2 was amplified through PCR amplification. The PCR product was then cut with the Bsl‐I enzyme to show where the gene was expressed, gel electrophoresis was then run to analyze where the Bsl‐I cut and identify whether the fish was a heterozygote or knockout mutant. We then matched the suitable images of the fish to the genotype and found that fish with LCP1 may have a higher rate of infection. Understanding why fish with LCP1 have a higher bacterial burden could provide an insight to the role LCP1 plays in the human body’s immune response. The future directions of this project are to observe more fish in order to replicate the findings and to test other phenotypes including granuloma size, macrophage motility, and extracellular growth in order to have a better understanding of LCP1's role in the disease outcome. Mycobacterium tuberculosis is an issue worldwide with multidrug‐resistant forms becoming prevalent.

A Blunted GPR183/Oxysterol Axis During Dysglycemia Results in Delayed Recruitment of Macrophages to the Lung During Mycobacterium tuberculosis Infection.

BackgroundWe previously reported that reduced GPR183 expression in blood from tuberculosis (TB) patients with diabetes is associated with more severe TB. MethodsTo further elucidate the role of GPR183 and its oxysterol ligands in the lung, we studied dysglycemic mice infected with Mycobacterium tuberculosis (Mtb).ResultsWe found upregulation of the oxysterol-producing enzymes CH25H and CYP7B1 and increased concentrations of 25-hydroxycholesterol upon Mtb infection in the lungs of mice. This was associated with increased expression of GPR183 indicative of oxysterol-mediated recruitment of GPR183-expressing immune cells to the lung. CYP7B1 was predominantly expressed by macrophages in TB granulomas. CYP7B1 expression was significantly blunted in lungs from dysglycemic animals, which coincided with delayed macrophage infiltration. GPR183-deficient mice similarly had reduced macrophage recruitment during early infection.ConclusionsTaken together, we demonstrate a requirement of the GPR183/oxysterol axis for positioning of macrophages to the site of infection and add an explanation to more severe TB in diabetes patients.

Clofazimine induced pigmentation in leprosy patches.

Leprosy is a neurocutaneous infectious disease with a highest prevalence in African region in the world. With the introduction of newer monitoring strategies of identifying the cases and multidrug therapy the overall global burden of leprosy has reduced. However, early detection and treatment is still important in leprosy due to the rapidly developing nerve damage by the bacteria and the associated disability. Here, presenting a case of 65-year-old male patient of leprosy in borderline pole, with complaint of hyperpigmentation over back, chest and arm in a ring like pattern from past 4 months. Patient has been on WHO multibacillary multidrug regimen (Rifampicin=600mg once a month, Clofazimine= 100mg once a month + 50mg daily dose, Dapsone= 100mg daily). On starting the treatment patient started noticing resolution in thickness of the lesion along with appearance of reddish-brown color of the lesion. On cutaneous examination, multiple geographic and annular patches with central clearing and shiny coppery reddish brown pigmentation in the peripheral margin of the lesion is seen. This case exemplifies the lesional hyperpigmentation, secondary to accumulation of ceroid lipofuscin pigment and Clofazimine inside the macrophage phagolysosome. Also, only marginal pigmentation with central clearing attributes to the punched-out lesion of the borderline lepromatous leprosy primarily which on histopathology typically shows presence of macrophage granuloma. Clofazimine induced pigmentation is reversible but takes months to years to clear after stopping the drug. As a dermatologist with leprosy moving towards elimination monitoring and managing the adverse effect of treatment and course of the disease is important.

ATHEROSCLEROSIS: IS INFLAMMATION IN THE ARTERIES IMMUNE?

In the literature there are no clinical and morphological data that would prove the participation of hypersensitivity reactions, autoimmune and immunocomplex processes in the development of granulomatosis in the arteries in atherosclerosis. In cholesterol mature macrophage granulomas (plaques), markers of immune inflammation are not detected - epithelioid and giant multinuclear Pirogov-Langhans cells; in xanthogranulomas there are giant multinucleated cells of foreign bodies. Granulomatosis in the arteries is induced by endogenous foreign bodies – lipid foci containing non-antigenic, non-hydrolysable cholesterol by macrophage lysosomal enzymes. The properties of its molecule, crystallization and insolubility in the blood and interstitial fluid are the cause of the unresolved nature of the developing inflammation in the arteries.

On-Slide Heat Sterilization Enables Mass Spectrometry Imaging of Tissue Infected with High-Threat Pathogens Outside of Biocontainment: A Study Directed at Mycobacterium tuberculosis.

Mass spectrometry imaging investigations of tissues infected with agents that require high-security biocontainment, such as Mycobacterium tuberculosis, have been limited due to incompatible sterilization techniques. Here we describe an on-slide heat sterilization method that enables mass spectrometry imaging investigations of pharmaceuticals, lipids, and metabolites in infected tissue samples outside of biocontainment. An evaluation of different temperatures and incubation times determined that 100 °C for 1 h was essential to sterilize 5 times the bacterial burden observed in tuberculosis (TB) cavity sections. Laser-capture microdissection combined with liquid chromatography with tandem mass spectrometry quantitation, in addition to mass spectrometry imaging, showed that no degradation was observed following the on-slide heat sterilization protocol for a variety of drug classes covering a range of physicochemical properties. Utilizing the tissue mimetic model, we demonstrated that the detection of lipid and metabolite ions was not impacted by heat sterilization and that, for several metabolites, the on-slide heat sterilization method improved the sensitivity when compared to control samples. An application of the on-slide heat sterilization to M. tuberculosis infected tissue enabled the first detection and spatial distribution of lipids indicative of a lysosomal storage disease phenotype within TB granuloma macrophages, in addition to the differential distribution of metabolites central to the fatty acid oxidation pathway. These initial investigations detected a pronounced heterogeneity within the cellular regions and necrotic cores of individual TB granulomas and across different evolving granulomas. This study provides the framework for mass spectrometry imaging investigations of high-threat pathogens outside of biocontainment.

Immunohistochemical expression of macrophages in chronic periapical lesions

Objective: To analyze the expression of macrophages in periapical granulomas (PGs) and radicular cysts (RCs). Methodology: We selected 264 cases of chronic periapical lesions stored at the Laboratory of Pathology, Dental School of Pernambuco (FOP)/UPE, including 89 PGs and 175 RCs. Seventy-nine cases, 23 PGs and 56 RCs, were submitted to immunohistochemical analysis by the streptavidin-biotin method using anti-CD68 antibody. The chi-square and Fisher’s exact tests were used to determine the existence of associations with categorical and clinical variables, adopting a 95% confidence level (p≤0.05). Results: We observed immunoexpression of CD68 in 83% of cases. The number of CD68+ cells (score 3 or 4) was higher in PGs, with predominantly intense staining. Significant differences in the number of CD68+ cells and staining intensity were observed between the chronic periapical lesions analyzed. Conclusion: The presence of CD68+ cells in periapical tissues may indicate the development, maintenance and/or severity of the inflammation-mediated immune response, which is more intense in PG.

Refractory hypercalcaemia associated with disseminated Cryptococcus neoformans infection.

SummaryHypercalcaemia is a very common endocrine condition, yet severe hypercalcaemia as a result of fungal infection is rarely described. There are have only been two reported cases in the literature of hypercalcaemia associated with Cryptococcus infection. Although the mechanism of hypercalcaemia in these infections is not clear, it has been suggested that it could be driven by the extra-renal production of 1-alpha-hydroxylase by macrophages in granulomas. We describe the case of a 55-year-old woman with a 1,25-OH D-mediated refractory hypercalcaemia in the context of a Cryptococcus neoformans infection. She required treatment with antifungals, pamidronate, calcitonin, denosumab and high-dose glucocorticoids. A disseminated fungal infection should be suspected in immunosuppressed individuals presenting with hypercalcaemia.Learning pointIn immunocompromised patients with unexplained hypercalcaemia, fungal infections should be considered as the differential diagnoses;Glucocorticoids may be considered to treat 1,25-OH D-driven hypercalcaemia; however, the benefits of lowering the calcium need to be balanced against the risk of exacerbating an underlying infection;Fluconazole might be an effective therapy for both treatment of the hypercalcaemia by lowering 1,25-OH D levels as well as of the fungal infection.