Spatial transcriptomics uncovers hybrid, pro-inflammatory and pro-fibrotic cellular niches in pulmonary granuloma of patients with chronic sarcoidosis

Spatial transcriptomics uncovers hybrid, pro-inflammatory and pro-fibrotic cellular niches in pulmonary granuloma of patients with chronic sarcoidosis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Spatial transcriptomics uncovers hybrid, pro-inflammatory and pro-fibrotic cellular niches in pulmonary granuloma of patients with chronic sarcoidosis Leonard Christian , Hande Yilmaz , Jannik Ruwisch , Leon Giercke , Benjamin Seeliger , Jan C. Kamp , Sirvan Bayraktar , Raphael Ewen , Theresa Graalmann , Jan Fuge , Mark Greer , Fabio Ius , Tobias Welte , Jens M. Hohlfeld , Marius M. Hoeper , Jens Gottlieb , Naftali Kaminski , Antje Prasse , Danny Jonigk , Yang Li , View ORCID Profile Christine Falk , Lavinia Neubert , Jonas C. Schupp doi: https://doi.org/10.1101/2025.02.11.636632 Leonard Christian 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hande Yilmaz 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jannik Ruwisch 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Leon Giercke 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Benjamin Seeliger 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jan C. Kamp 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sirvan Bayraktar 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Raphael Ewen 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Theresa Graalmann 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany 3 Junior Research Group Translational Immunology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research (HZI) and the Hannover Medical School (MHH) , Hannover, Germany 4 Department for Rheumatology and Immunology, Hannover Medical School (MHH) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jan Fuge 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mark Greer 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Fabio Ius 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany 5 Clinic for cardiac, thoracic, transplant and vascular surgery, Hannover Medical School (MHH) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tobias Welte 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jens M. Hohlfeld 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany 6 Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marius M. Hoeper 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jens Gottlieb 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Naftali Kaminski 7 Pulmonary, Critical Care and Sleep Medicine, Yale University , New Haven, CT, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Antje Prasse 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany 6 Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM) , Hannover, Germany 8 Clinics of Respiratory Medicine, University Hospital Basel, University of Basel , Hebelstrasse 20, 4031, Basel, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Danny Jonigk 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany 9 Institute of Pathology, Hannover Medical School (MHH) , Hannover, Germany 10 University Clinic of the RWTH Aachen , Aachen, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yang Li 11 Computational Biology for Individualised Medicine, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research (HZI) and the Hannover Medical School (MHH) , Hannover, Germany 12 Centre for Individualised Infection Medicine (CiiM), a joint venture between the Helmholtz-Centre for Infection Research (HZI) and the Hannover Medical School (MHH) , Hannover, Germany 13 Lower Saxony center for Artificial intelligence and causal Methods in Medicine (CAIMed) , Hannover, Germany 14 Department of internal Medicine and Radboud center for infectious diseases, Radboud University Medical center , Nijmegen, Netherlands 15 Cluster of excellence Resolving Infection Susceptibility (RESIST) Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christine Falk 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany 16 Institute of Transplant Immunology, Hannover Medical School (MHH) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christine Falk Lavinia Neubert 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany 9 Institute of Pathology, Hannover Medical School (MHH) , Hannover, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jonas C. Schupp 1 Department of Respiratory Medicine and Infectious Diseases, Hannover Medical School (MHH) , Hannover, Germany 2 Biomedical Research in End-Stage and Obstructive Lung Disease (BREATH), Hannover Medical School (MHH), German Center for Lung Research (DZL) , Hannover, Germany 6 Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM) , Hannover, Germany 7 Pulmonary, Critical Care and Sleep Medicine, Yale University , New Haven, CT, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: schupp.jonas{at}mh-hannover.de Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Background Sarcoidosis is a disease of unknown etiology characterized by the formation of immune cell accumulation (granuloma) in the lung and other tissues. Chronic sarcoidosis may lead to pulmonary fibrosis. Aim To unravel cellular niches within pulmonary granuloma of chronic sarcoidosis patients using spatial transcriptomics. Methods Spatial transcriptomics using the Visium platform (10x Genomics) was performed on nine granuloma-containing lung explants from sarcoidosis patients. Validation of gene expression was performed through immunohistofluorescence protein staining and RNA in situ hybridization. Results Spatial gene expression covered 30,587 gene expression spots and 173 granulomas. A CD68 + macrophage niche was localized in the center of the granuloma, with a CD3 + T and CD20 + B cell niche in close proximity, surrounded by a COL3A1 + fibroblast niche. In the central granuloma macrophage niche, expression of the pro-fibrotic macrophage genes SPP1 , CHIT1 and CHI3L1 was observed, genes whose expression has recently been described for macrophages in idiopathic pulmonary fibrosis. Additionally, pro-inflammatory macrophage genes were expressed in the central granuloma niche: macrophages appear armed for lysosomal degradation and ready for phagocytosis. Inner granuloma niches showed high responsiveness to interferon gamma (IFN-γ), expressing a multitude of IFN-γ-induced genes. High collagen and CTHRC1 expression were observed in granuloma fibroblasts niches, characteristics of pro-fibrotic lung remodeling. Ligand-receptor analysis identified pro-inflammatory and pro-fibrotic interactions between granuloma niches. Conclusion Taken together, macrophages in the center of the sarcoidosis granuloma form an armed-and-ready, hybrid pro-inflammatory and pro-fibrotic niche, supporting granuloma persistence through continuous IFN-γ-stimulation and fibrotic remodeling conducted by fibrotic fibroblasts surrounding the granuloma. Introduction Sarcoidosis is a multi-organ immune disease characterized by non-caseating granuloma and local inflammation. Lungs and intrathoracic lymph nodes are the most often affected organs ( 1 ). In many patients, these granulomas resolve without specific treatment, while in around 20% of cases, chronic sarcoidosis may lead to lung fibrosis or organ failure ( 2 , 3 ). As granulomas are the histological hallmark of sarcoidosis, their formation, structure, and significance have been studied over the last decades ( 4 , 5 ). However, the precise interactions on a cellular level and factors playing a role in granuloma persistence remain obscure. The first step towards granuloma formation is carried out by sessile alveolar macrophages, reacting to a specific, yet unknown antigen in the lung ( 6 ). Then, monocytes are attracted to the inflammatory region, forming the core of the granuloma, where they acquire an epithelioid phenotype or fuse to multinucleated giant cells ( 7 ). The middle layer of the granuloma consists of recruited lymphocytes, including CD8 + T cells, T-helper (Th) Th1 cells, and Th17/Th17.1 cells, and B cells ( 8 ). In patients with chronic sarcoidosis, fibroblasts accumulate in the periphery of the granuloma, forming the outer granuloma layer. Although the formation of a fibroblast layer may be an important step of fibrotic tissue remodeling, which drastically worsens the prognosis for sarcoidosis patients, only little advancements were made towards characterizing these fibroblasts ( 9 – 11 ). In this study, we employed spatial transcriptomics to explore the expression profiles, potential origins and interactions between cellular niches within the pulmonary granuloma of patients with chronic sarcoidosis, which may ultimately contribute to pulmonary fibrosis. Methods Methods are detailed in the supplement and only briefly summarized here. This study was approved by the local Institutional Review Board (10142_BO_K_2022). Basic patient characteristics can be found in supplemental table E1. Processing of human lung tissue samples Biobanked lung specimens of nine sarcoidosis patients, who underwent lung transplantation and had provided informed consent, were studied. Formalin-fixed and paraffin-embedded (FFPE) tissue blocks were sectioned at a microtome. Sections were stained with hematoxylin and eosin (H&E) to confirm the presence of granuloma. RNA quality was assessed using the RNeasy FFPE kit (Qiagen) for RNA isolation and DV200 measurement on a bioanalyzer. Spatial transcriptomics Spatial transcriptomics and library generation were performed using the Visium platform from 10x Genomics according to manufacturer’s instructions. DNA library sequencing was carried out on the NovaSeq 6000 (Illumina) using associated reagents and standard procedure. Visium data acquisition, processing and analysis Processing and visualization of spatial expression data were done with the ‘Seurat’ package in R software. Nine Visium samples passed the quality checks. Cluster of cells were identified through a clustering algorithm based on shared nearest neighbor (SNN) modularity optimization. Marker genes for each niche were generated with the Wilcoxon rank-sum test. FDR adjusted P values less than 0.05 were considered statistically significant in this study. Downstream analyses included gene set enrichment analysis and ligand-receptor analysis to explore niche-niche signaling networks. Validation Multicolor immunofluorescence protein stains and RNA in situ hybridization (ISH) were performed for validation genes identified from the spatial gene expression analysis. Results To characterize the cellular niches of the chronic pulmonary sarcoidosis granuloma, we performed spatial transcriptomics using the Visium platform (10x Genomics) on nine samples featuring granuloma of adequate number and RNA quality ( Figure 1A and B , supplemental figure E1). In total, 30.587 tissue-covered gene expression spots were analyzed covering 173 granulomas. Cell type annotation based on the expression of distinct marker genes identified 13 niches, including four granuloma associated niches ( Figure 1C-E ). Download figure Open in new tab Figure 1: A) Biobanked FFPE tissue samples of nine chronic sarcoidosis patients undergoing lung transplantation were used. Spatial transcriptomics was performed on these samples using the Visium platform (10x Genomics). DNA libraries were sequenced and the data was analyzed. Based on spatial transcriptomics findings, validation via immunohistofluorescence protein staining (IHF) and RNA in situ hybridization (ISH) was performed. B) Exemplary Hematoxylin & Eosin (H&E) staining of a lung section containing granuloma. C) Spatial dimension plot showing the distribution of the cell type niches with each niche represented by a color-coded dot and niches identified based on marker gene expression in spatial transcriptomics. D) UMAP of spatial transcriptomics niches from all samples, with each niche color-coded and every spot representing a dot on a spatial plot. E) Heatmap displaying exemplary marker genes used to define the cell type niches in spatial transcriptomics. F) Spatial gene expression plot shows expression of macrophage marker CD68 in the granuloma center. G) Spatial gene expression plot shows prominent expression of fibroblast marker COL3A1 around the granuloma. H) Spatial gene expression plot shows weak expression of T cell marker CD3E around the granuloma. I) Representative immunohistofluorescence protein staining of macrophage marker CD68 (green) and fibroblast marker COL3A1 (red) and DAPI (blue) confirm the presence of macrophages in the granuloma and fibroblasts surrounding it (scale bar = 100 µm). J) Representative immunohistofluorescence protein staining of T cell markers CD3 (green) and CD4 (red), B cell marker CD20 (pink) and DAPI (blue) reveal the presence of T cells and B cells around the granuloma core (scale bar = 100 µm). The main cell populations within the granuloma were uncovered based on marker gene expression, showing CD68 + macrophages in the granuloma center ( Figure 1F ) with a dense layer of COL3A1 + fibroblasts around it ( Figure 1G ). CD3 + T cells were loosely scattered in between the macrophage core and the fibroblast layer ( Figure 1H ). Low expression of dendritic cell marker genes (Supplemental figure E2) and B cell marker genes such as MS4A1 (CD20, figure 1E ) were detected in predominantly in the T cell layer. The observed cellular distribution was confirmed through immunohistofluorescence protein staining (IHF) of CD68 and COL1A1 ( Figure 1I ) alongside staining for CD3, CD4 and CD20 ( Figure 1J ). Central granuloma macrophages occupy a hybrid, pro-inflammatory and pro-fibrotic niche First, we explored the gene expression profile of granuloma macrophages, the main cells in the central granuloma niche. We observed high expression of SPP1 , CHIT1 and CHI3L1 in the granuloma center ( Figure 2A ), genes associated with pro-fibrotic macrophages in idiopathic pulmonary fibrosis (IPF) ( 12 ), and markers of alternatively-activated (M2) macrophages ( 13 , 14 ). In parallel, high expression of LYZ , IL1B , IL18 and CYBB , pro-inflammatory genes associated with conventionally-activated (M1) macrophages, was observed in the central granuloma niche ( Figure 2B ), suggesting that central granuloma macrophages exhibit both pro-fibrotic and pro-inflammatory characteristics. GPNMB , a novel marker for giant cells in sarcoidosis displaying characteristics of M1 and M2 macrophage polarization ( 15 ), was detected in the granuloma center ( Figure 2A ). Validation through IHF stains confirmed the presence of SPP1 + CHIT1 + macrophages in the central granuloma ( Figure 2C ). While SPP1 was distributed across the central granuloma, CHIT1 was colocalized specifically with CD68. Download figure Open in new tab Figure 2: A) Spatial gene expression plot displaying genes associated with pro-inflammatory conventional macrophages ( LYZ and CYBB ), monocyte-derived macrophages ( CD14 ) and pro-fibrotic alternatively-activated macrophages ( CHI3L1 , GPNMB , CHIT1 , SPP1 ) in a granuloma. B) Heatmap featuring the gene expression across all subjects of pan-macrophage marker genes, macrophage origin marker genes (alveolar and monocyte), as well as markers for macrophage activation types alternative (M2) and conventional (M1). C) Representative immunohistofluorescence protein staining of the pro-fibrotic macrophage markers osteopontin (SPP1, green) and chitinase 1 (CHIT1, red), as well as macrophage marker CD68 (violet) and DAPI (blue) shows their presence in the center of the granuloma, but not in the surrounding tissue (scale bar = 100 µm). D) RNA in situ hybridization stains show the presence of SPP1 (green) and CD14 (red) in the center of the granuloma, while alveolar macrophage marker gene FABP4 (violet) is expressed by tissue resident macrophages (scale bar = 100 µm). E) UMAP feature plot displaying the expression of macrophage marker genes across all niches. Alveolar macrophage marker genes FABP4 and RBP4 are highly expressed in the alveolar niche, while monocyte, alternative (M2) and conventional (M1) marker genes are expressed in the central granuloma niche. To uncover the potential origin of granuloma macrophages, we examined the expression of alveolar and monocyte markers ( Figure 2B and E )( 16 ). While alveolar macrophage markers like FABP4 were expressed in the alveolar niche, monocyte markers like CD14 were expressed in the granuloma center. The expression of FABP4 in alveolar, but not granuloma macrophages, and CD14 in central granuloma macrophages alongside SPP1 , was confirmed through ISH ( Figure 2D ). Taken together, this data suggests that central granuloma macrophages are monocyte-derived, forming a hybrid, pro-inflammatory and pro-fibrotic niche. Central granuloma macrophages are primed to eliminate pathogens To expand upon the inflammatory signaling within the central granuloma niche, we performed a pathway enrichment analysis using EnrichR, revealing associations with phagocytosis and endocytosis (GO:0050764, GO:0045807, and GO:0006896) as well as Toll-like receptor (TLR) signaling, phagosome, and lysosome pathways ( Figure 3A and B ). Expression of TLR2 and its coreceptor TLR1 was highest in the central granuloma niche ( Figure 3C and F ). Although TLR4 expression was detected in the central granuloma, its expression was low compared to its coreceptors LY96 and CD14 and downstream TLR2/4 signaling genes, like MYD88 and IRAK1 ( Figure 3C ). Besides pathogen recognition, central granuloma macrophages displayed activity in pathogen uptake through expression of endocytosis-related genes such as RAB5C . Lysosomal degradation was especially prominent in the central granuloma, highlighted by expression of several lysosomal genes including CTSB , CTSS , DNASE2 , LIPA , and LYZ ( Figure 3C , D, and F). A shift to glycolysis and pentose phosphate pathway metabolism, facilitating quick immune cell proliferation and providing NADPH for oxidative stress response, was observed in granuloma macrophages through HK3 , GALM , TKT and FBP1 expression ( Figure 3B and D ), with confirmed protein expression of HK3 by CD14 + macrophages ( Figure 3E ). Download figure Open in new tab Figure 3: A) Gene ontology (GO) analysis of the top 200 genes expressed in the central granuloma niche by average log 2 (fold-change(FC)). Reference database: Biological Processes. B) Pathway analysis using the top 200 genes expressed in the central granuloma niche by log 2 (FC). Reference database: KEGG. C) Heatmap displaying the expression of genes involved in inflammatory pathways in the granuloma center. D) Spatial gene expression plot of lysosomal genes ( CTSB , CTSS ) and pentose phosphate pathway gene FBP1 in a granuloma. E) Representative immunohistofluorescence protein staining of CD14 (green), HK3 (red), and DAPI (blue) shows that monocyte-derived macrophages in the center of the granuloma express glycolysis related genes (scale bar = 100 µm). F) Expression of genes involved in pathogen detection ( TLR2 ), elimination ( LIPA , DNASE2 ), and energy metabolism ( HK3 ) is high in macrophage containing granuloma niches. In summary, high expression of genes involved in pathogen recognition, uptake, and clearance alongside shifted energy metabolism suggests that macrophages in the central granuloma niche are primed to eliminate – potentially longe gone - pathogens. Interferon-γ signaling sustains a pro-inflammatory environment in the granuloma Next, we examined how lymphocytes are involved in maintaining this pro-inflammatory and pro-fibrotic niche. Pathway analysis of the granuloma T cell, dendritic cell (DC) and macrophage (ΜΦ) niche revealed high responsiveness to IFN-γ by granuloma-associated cells ( Figure 4A-C ). These results are in line with high gene expression of IFN-γ-induced genes CD74 , CXCL9 and IFI30 in the granuloma center ( Figure 4D ). Evaluating the expression of 50 genes induced by IFN-γ revealed specific spatial distribution of these genes across three different niches: i) central granuloma; ii) granuloma T cells, DCs, and ΜΦ; iii) B cell ( Figure 4E ). The IFN-γ-inducible chemokines CCL5 , CCL22 and CXCL10 were prominently expressed in granuloma niches ( Figure 4F ), facilitating the recruitment of activated T cells and regulatory T cells ( 17 , 18 ). CXCL9/CXCL10 and CCL5/CCL22 expression were similar in the central granuloma and the granuloma T cells, DCs, and ΜΦ niche, underlining the ongoing lymphocyte recruitment to the granuloma. The highest expression of CXCL16 and IRF8 was observed in the central granuloma and the B cell niche, respectively. Taken together, pro-inflammatory IFN-γ signaling is maintained within pulmonary granuloma of chronic sarcoidosis patients. Download figure Open in new tab Figure 4: A) Gene ontology (GO) analysis of the top 200 genes expressed in the granuloma T cells, DCs, and ΜΦ granuloma niche by log 2 (FC). Reference database: KEGG. B) Gene ontology (GO) analysis of the top 200 genes expressed in the granuloma T cells, DCs, and ΜΦ granuloma niche by log 2 (FC). Reference database: Biological Processes. C) Pathway analysis of the top 200 genes expressed in the granuloma T cells, DCs, and ΜΦ granuloma niche by log 2 (FC). Reference database: MSigDB Hallmark. D) Spatial gene expression plot of interferon gamma (IFN-γ)-response-related genes in a granuloma. E) Heatmap displaying the expression of genes related to IFN-γ-response. F) Expression of genes involved in IFN-γ-response is high in granuloma macrophages, T cells, and DCs compared to surrounding tissue. The outer granuloma fibroblast niches contain CTHRC1 + fibrotic fibroblasts Spatial transcriptomics revealed three fibroblast niches that were identified and classified by their location and gene expression: fibroblasts near small alveolar vessels (“adventitial fibroblasts”), fibroblasts in fibrotic lesions near granulomas (“fibrotic granuloma fibroblasts”), and fibroblasts around granulomas near immune cells (“granuloma fibroblasts and macrophages”). Granuloma-associated fibroblasts showed no clear expression of adventitial and alveolar fibroblast markers, but expressed general fibroblast and myofibroblast markers ( Figure 5A and C ), while displaying prominent expression of the fibroblast marker genes COL1A1 , COL6A3 , LUM , and SPARC ( Figure 5B ). Fibrotic granuloma fibroblasts expressed high amounts of CTHRC1 , which has recently been associated with IPF myofibroblasts ( 19 , 20 ). The granuloma fibroblasts and macrophages niche was characterized by its expression of THY1 and TNC ( Figure 5A ). Additionally, all granuloma related niches showed high expression of FAP , a marker for collagen-producing fibroblasts in IPF ( 21 , 22 ). Pathway enrichment analysis of the fibrotic granuloma fibroblast niche revealed high levels of extracellular matrix (ECM) remodeling (GO:0030198, Figure 5D ). ISH confirmed the expression of COL1A1 , CTHRC1 , and TNC in fibroblasts surrounding the sarcoid granuloma ( Figure 5E ). In summary, ECM remodeling was very active around the sarcoid granuloma and granuloma-associated fibroblasts show similarities to fibroblasts in IPF. Download figure Open in new tab Figure 5: A) Heatmap displaying the expression of marker genes for adventitial fibroblasts, alveolar fibroblasts, and myofibroblasts, as well as pan-fibroblast markers and genes expressed primarily by granuloma fibroblasts and macrophages. B) Spatial gene expression plot of fibroblast-associated genes around a granuloma. C) UMAP feature plot displaying the expression of fibroblast marker genes across all niches. Adventitial fibroblast markers ( FBLN2 , SFRP2 ) are expressed in the adventitial fibroblast niche, alveolar fibroblast markers ( NPNT , GPC3 ) are expressed in the alveolar niche, and fibrotic myofibroblast associated genes CTHRC1 and COL14A1 are expressed in the fibrotic granuloma fibroblast niche. D) Gene ontology (GO) analysis of the top 200 genes expressed in the fibrotic granuloma fibroblast niche by log 2 (FC). Reference database: Biological Processes. E) RNA in situ hybridization stains show the presence of COL1A1 (green), CTHRC1 (red), and TNC (violet) surrounding the granuloma. Nuclei are stained with DAPI (blue); scale bar = 100 µm. Pro-inflammatory and pro-fibrotic ligand-receptor interactions support granuloma integrity Previous results demonstrated the expression and role of pro-inflammatory and pro-fibrotic genes in the granuloma, however, their importance in the crosstalk between granuloma niches and involvement in homeostasis and maintenance of the granuloma remained inconclusive. To bridge this gap, we aimed to further characterize this crosstalk employing ligand receptor analysis of the four granuloma associated niches distinguished in this study. This approach confirmed pro-inflammatory signaling of the central granuloma niche via CCL5 alongside pro-fibrotic signaling through TGFB1 , SPP1 , and MMP9 ( Figure 6A ). SPP1 and MIF signaling were found to be initiated by the central granuloma niche as the main sender, with interaction partners like CD44 , CD74 , and CXCR4 being expressed in all granuloma niches ( Figure 6B ). Complement signaling via C3 was sent by all niches, as highlighted by the strong expression of C3 in and around the granuloma, with its receptors ITGAX and ITGB2 being expressed predominantly in the central granuloma niche ( Figure 6C ). Signaling via CXC ligands showed strong involvement of CXCL12 , whose receptor CXCR4 was expressed in all granuloma-associated niches except the fibrotic fibroblast niche (Supplemental figure E5). Collagen and CXC ligand interactions were especially prominent in fibroblast containing outer niches, due to the strong gene expression of the associated ligands COL1A1 , COL1A2 , and CXCL12 ( Figure 6B ). Spatial gene expression showed overlapping expression of ligand receptor pairs like SPP1 and CD44 within the boundaries of the granuloma ( Figure 6C ). Taken together, active pro-inflammatory and pro-fibrotic signaling persists within chronic sarcoid granuloma, with SPP1, CXCL chemokine, and collagen interactions playing a central role in this microenvironment. Download figure Open in new tab Figure 6: A) Circos plot showing the main ligand-receptor interactions within a granuloma. B) Heatmaps featuring ligand-receptor interactions within a granuloma. Depicted are pro-inflammatory (CXCL, Complement) and pro/fibrotic (SPP1, MIF, Collagen) signaling networks. C) Spatial gene expression of a ligand (red), its receptor (blue), the co-expression of ligand and receptor (gold) and neither ligand or receptor (gray). Discussion In this study, we employed spatial transcriptomics on lung tissue samples from nine chronic pulmonary sarcoidosis patients to examine the gene expression of key niches in the granuloma, aiming to understand the mechanisms driving fibrotic remodeling and granuloma maintenance. Spatial transcriptomics revealed that macrophages in the central granuloma niche display hybrid characteristics of profibrotic and proinflammatory macrophages. These macrophages express pro-fibrotic genes like SPP1 , CHI3L1 , and CHIT1 , while additionally expressing genes involved in pathogen detection and clearance, like TLRs, LYZ , and LIPA . Pathway analysis revealed that granuloma associated immune cells remain constantly stimulated by IFN-γ. Granulomas were surrounded by a prominent fibroblast layer, featuring high expression of collagens and CTHRC1 with high ECM remodeling activity. Although the signal of T cells was weak compared to macrophages and fibroblasts, we were able to detect a distinct expression of receptors binding T cell derived chemokines in granuloma niches. While the presence of pro-inflammatory and pro-fibrotic macrophages in the sarcoid granuloma have been observed before, their significance remains uncertain. A possible explanation is that a transition from pro-inflammatory to pro-fibrotic macrophages is concomitant with disease progression ( 23 , 24 ), as progressive, peri-granulomatous fibrosis is a typical characteristic of chronic sarcoidosis ( 3 ). Through this study, we found that genes associated with pro-inflammatory macrophages are still expressed within chronic sarcoid granuloma. In parallel, we observed that central granuloma macrophages resemble SPP1 + macrophages first described in IPF ( 12 , 19 ) and systemic sclerosis-associated interstitial lung disease (SSc-ILD)( 25 ). SPP1 + macrophages in IPF similarly showed expression of GPNMB ( 26 ), alongside CHIT1 and CHI3L1 , which have been associated with development and progression of fibrosis in different kinds of interstitial lung disease (ILD) ( 27 ). High serum levels of chitinase 1 and YKL-40, encoded by the CHIT1 and CHI3L1 genes, respectively, have been shown to correlate with disease activity and progression in sarcoidosis ( 28 – 31 ). The expression of SPP1 , CHIT1 and MMP9 by granuloma macrophages was recently shown on a single cell level ( 32 ) and SPP1 , CHIT1 and CHI3L1 were detected in the center of dermal granuloma from sarcoidosis patients ( 33 ). Ligand-receptor analysis demonstrated the expression of SPP1 receptors involved in ECM remodeling in granuloma proximity, like CD44 , supporting the role of SPP1 in maintaining the granuloma structure. The expression of pathogen recognition receptors by granuloma macrophages supports the hypothesis that stimulation of innate immune receptors triggers granuloma formation ( 8 ), while their expression in chronic sarcoidosis suggests a role in granuloma maintenance. Central granuloma macrophages expressed TLR2 , which is involved in the detection of mycobacteria ( 34 ). Granuloma macrophages were not only able to detect mycobacteria, they also exerted high lysosomal activity, demonstrated by LYZ , DNASE2 , and LIPA expression. Besides their function in lysosomal degradation, these genes are involved in granuloma formation through mTORc1/S6/STAT3 signaling ( 35 – 37 ). Additionally, we found expression of genes involved in glycolysis, a hallmark of disease progression in sarcoidosis ( 38 ), which also supports phagocytic and bactericidal activity management ( 39 ). Consistent with a recent report from a single cell sequencing sarcoidosis study ( 23 ), we observed high expression of pentose phosphate pathway-related genes in macrophages. As high pentose phosphate pathway activity is often observed during macrophage activation, this metabolic shift might promote granuloma formation in sarcoidosis. Whether the complement, MIF and CXC ligand interactions observed through ligand-receptor analysis might contribute to this pro-inflammatory environment remains unclear. Despite the reported involvement of alveolar macrophages in the initiation of the granuloma formation ( 8 ), granuloma macrophages did not express alveolar macrophage marker genes. Instead, granuloma macrophages expressed monocyte marker CD14 . This is in line with the findings that SPP1 + recruited macrophages were demonstrated to be derived from monocytes ( 40 ) and that monocytes are able to promote fibrosis in IPF ( 41 ). Considering the above, the dysregulated expression of pro-inflammatory genes in the central granuloma niche throughout granuloma maturation combined with pro-fibrotic genes might be the key to understanding the upkeeping of the granuloma. Previous studies showed the importance of T cell-derived IFN-γ in sarcoidosis granuloma formation ( 42 , 43 ). While our assay did not detect IFN-γ itself, we found that CXCL9 , CXCL10 , and other genes involved in IFN-γ signaling pathways were expressed in both the central granuloma and the granuloma lymphocyte niches, indicating that IFN-γ signaling continues to play a role in sustaining established granulomas, supporting the potential use of drugs intercepting the IFN-γ pathway also in chronic patients. The importance of ongoing IFN-γ stimulation of these macrophages is underscored by evidence showing that elevated serum levels of CXCL9 and CXCL10 are linked to disease severity and help to regulate regulatory T cell recruitment ( 44 , 45 ) as well as to attract Th1 lymphocytes to the sarcoid lungs ( 46 ). One of the most prominent complications associated with chronic sarcoidosis is loss of functional lung parenchyma due to fibrotic remodeling, which has been proposed to originate from the granuloma ( 47 ). Granuloma in patients with chronic pulmonary sarcoidosis were surrounded by fibroblasts, which displayed high expression of CTHRC1 , a gene expressed in fibrotic lungs of IPF patients, but not in healthy people ( 20 ). CTHRC1 + fibroblasts have not been described for sarcoidosis yet, but may play a similar role in the development of fibrosis around pulmonary granuloma as their counterparts in IPF. CTHRC1 has been proposed to be both an activator and inhibitor of Wnt/β-catenin signaling ( 15 , 48 ), suggesting a role in the differentiation of myofibroblasts participating in pulmonary fibrosis. This underlines the finding that granuloma-associated fibroblasts express myofibroblast associated genes, but only low amounts of alveolar or adventitial fibroblast associated genes. While TNC was described as an inducer of collagen expression in patients with SSc-ILD ( 49 ), is also an endogenous activator of TLR4 signaling ( 50 ), indicating that granuloma fibroblasts may harbor an undiscovered role in pro-inflammatory signaling within the granuloma. This study is not without limitation. Statistically, our findings are mainly limited by the small sample size. Clinically, pulmonary manifestations are most common amongst sarcoidosis patients, granuloma forming in different organs, such as lymph nodes, skin, and heart, may differ from pulmonary ones. Additionally, we focused solely on chronic sarcoidosis cases that required lung transplantation due to disease severity. Technologically, the limited resolution of the Visium assay only allowed the characterization of cell type niches, not of single cells types, necessitating further in-depth studies on a larger scale. Taken together, the sarcoid granuloma is a complex structure whose maintenance involves the crosstalk of diverse immune cells and fibroblasts. Using spatial transcriptomics, we explored the expression profile of the main niches within the chronic pulmonary sarcoid granuloma to elucidate their role within the granuloma. Our data suggests that macrophages present in the central granuloma niche possess seemingly contradictory functions, as they express genes involved in pathogen clearance and fibrotic ECM remodeling. While it is unclear if macrophages exhibiting a pro-inflammatory M1 phenotype later acquire the pro-fibrotic M2 phenotype or whether M2 macrophages accumulate in the central granuloma next to M1 macrophages forming an armed-and-ready M1/M2 hybrid niche, a fine balance of pro-inflammatory and pro-fibrotic factors appears to be the key of granuloma maintenance. Both SPP1 + macrophages and CTHRC1 + fibroblasts previously discovered in IPF emerged in our chronic sarcoidosis dataset. Translation from IPF research to sarcoidosis might alleviate chronic sarcoidosis research or open new avenues for sarcoidosis treatment in the future. Acknowledgments The authors thank the Research Core Unit for Laser Microscopy and the Research Core Unit Genomics (RCUG) at Hannover Medical School for their support. The authors thank all study participants for their permission to use their respective tissue specimens for research. 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Hoeper , Jens Gottlieb , Naftali Kaminski , Antje Prasse , Danny Jonigk , Yang Li , Christine Falk , Lavinia Neubert , Jonas C. Schupp bioRxiv 2025.02.11.636632; doi: https://doi.org/10.1101/2025.02.11.636632 Share This Article: Copy Citation Tools Spatial transcriptomics uncovers hybrid, pro-inflammatory and pro-fibrotic cellular niches in pulmonary granuloma of patients with chronic sarcoidosis Leonard Christian , Hande Yilmaz , Jannik Ruwisch , Leon Giercke , Benjamin Seeliger , Jan C. Kamp , Sirvan Bayraktar , Raphael Ewen , Theresa Graalmann , Jan Fuge , Mark Greer , Fabio Ius , Tobias Welte , Jens M. Hohlfeld , Marius M. Hoeper , Jens Gottlieb , Naftali Kaminski , Antje Prasse , Danny Jonigk , Yang Li , Christine Falk , Lavinia Neubert , Jonas C. 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