Depicting pathogenesis of osteomyelitis by single cell RNA-sequencing and an involvement of Morrbid in the autoinflammatory disease

Depicting pathogenesis of osteomyelitis by single cell RNA-sequencing and an involvement of Morrbid in the autoinflammatory disease | 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 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https://doi.org/10.1101/2025.01.23.634426 Qingran Huo 1 Tianjin Key Laboratory of Inflammatory Biology, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China 2 National Key Laboratory of Experimental Hematology , Tianjin, China 3 The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jiayu Ding 1 Tianjin Key Laboratory of Inflammatory Biology, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China 2 National Key Laboratory of Experimental Hematology , Tianjin, China 3 The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hongxi Zhou 1 Tianjin Key Laboratory of Inflammatory Biology, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China 2 National Key Laboratory of Experimental Hematology , Tianjin, China 3 The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hang He 1 Tianjin Key Laboratory of Inflammatory Biology, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China 2 National Key Laboratory of Experimental Hematology , Tianjin, China 3 The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lorie Chen Cai 3 The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jingjing Liu 1 Tianjin Key Laboratory of Inflammatory Biology, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China 2 National Key Laboratory of Experimental Hematology , Tianjin, China 3 The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ge Dong 1 Tianjin Key Laboratory of Inflammatory Biology, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China 2 National Key Laboratory of Experimental Hematology , Tianjin, China 3 The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: dongge{at}tmu.edu.cn us36zcai{at}tmu.edu.cn Zhigang Cai 1 Tianjin Key Laboratory of Inflammatory Biology, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China 2 National Key Laboratory of Experimental Hematology , Tianjin, China 3 The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University , Tianjin, China 4 Department of Hematology, Tianjin Medical University Tianjin General Hospital , Tianjin, China 5 Department of Rheumatology and Immunology, Tianjin Medical University Tianjin General Hospital , Tianjin, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zhigang Cai For correspondence: dongge{at}tmu.edu.cn us36zcai{at}tmu.edu.cn Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Autoinflammatory diseases (AIDs) are defined as abnormal activation of the innate immune system leading to spontaneous and uncontrolled inflammation. The AIDs affect bone tissue and lead to chronic recurrent multifocal osteomyelitis (CRMO). However, the etiology and treatment of CRMO remain elusive. A mouse strain, Pstpip2 cmo/cmo ( cmo : chronic multifocal osteomyelitis ), exhibits phenotypic characteristics similar to human CRMO. Morrbid is a long non-coding RNA gene and has been indicated in leukemogenesis in our previous studies. In this study, we demonstrated that Morrbid and Pstpip2 are co-expressed in mature myeloid cells and hypothesized a role of Morrbid in osteomyelitis. The Pstpip2 −/− mice have the same phenotype as Pstpip2 cmo/cmo , mimicking CRMO, while loss of Morrbid in Pstpip2 −/− mice significantly inhibited the initiation and progression of CRMO symptoms, as well as the dysregulated activation of myeloid cells and the excessive release of inflammatory cytokines. Furthermore, single-cell RNA-sequencing (scRNA-seq) analysis from the Pstpip2 −/− mice and the compound mutant mice supports that reduction of osteoclasts and inflammatory cells caused by Morrbid loss. The study systematically profiles the etiology of CRMO by scRNA-seq and warrants that inhibiting the lifespan of inflammatory myeloid cells by targeting Morrbid is an effective therapeutic strategy for osteomyelitis. Highlights We generated a frameshift mutant mouse strain Pstpip2 −/− , which have a classic CRMO-like phenotype as same as Pstpip2 cmo/cmo and could be used for testing various anti-inflammation perturbations. Loss of Morrbid significantly inhibited the autoinflammatory symptoms in Pstpip2 −/− mice, suggesting that Morrbid is a novel target for mitigating CRMO. scRNA-seq analysis of the affected bone marrow cells in Pstpip2 −/− revealed the abnormalities of osteoclasts (OC), neutrophils (NE) and granulocyte macrophage progenitors (GMP) in both their fractions and inflammatory activities. Upon loss of Morrbid , we observed reduced composition and proliferation of OC and decreased activity of Nfkb2 and Rela in the compound mutants. Introduction Inflammatory diseases caused by the instinct dysfunction of the innate immune cells are called autoinflammatory diseases (AIDs) [ 1 , 2 ] . At present, many types of AIDs have been identified in clinic, including colitis, dermatosis, chondritis, osteomyelitis, vasculitis, and a recently reported rheumatoid-hematological VEXAS [ 3 – 5 ] . However, treatments for AIDs are limited [ 4 , 6 ] . It is urgent to have more insightful understanding the pathogenesis of AIDs and develop more accurate and effective treatment methods. Chronic recurrent multifocal osteomyelitis (CRMO) is a non-bacterial autoinflammatory bone disease characterized by multifocal and multiple recurrences [ 7 ] . The typical symptom of CRMO is pain at the bony injury site with or without swelling, which tends to occurs around the metaphysis of femur, tibia and clavicle [ 7 , 8 ] . Patients with CRMO are frequently accompanied by other chronic inflammatory diseases or autoimmune diseases, including arthritis, psoriasis and inflammatory bowel disease (IBD) [ 7 , 9 ] . Recently a mutation in IL1R1 was reported to cause CRMO in a juvenile female with profound inflammatory myeloid cells in peripheral blood [ 10 ] . However, only her peripheral mononuclear blood cells (PBMCs) were profiled by single-cell RNA-sequencing (scRNA-seq). Profiling the immune and stromal cells in the affected region will assist a direct understanding of the pathogenesis of CRMO. A mouse model Pstpip2 cmo/cmo ( cmo : chronic multifocal osteomyelitis ) shows disease characteristics similar to human CRMO and is widely used as an animal model for pathogenesis of AIDs. Pstpip2 cmo/cmo mutant mice spontaneously develop CRMO starting at the 2-month-old age, typically characterized by progressive hind paw swelling and deformity, skin and ear inflammation and inflammatory bone resorption. It is believed that such inflammation is mediated by biased myelogenesis and inflammatory myeloid cells (such as neutrophils and macrophages) of the innate immune system [ 11 – 14 ] . As an adaptor protein, PSTPIP2 (proline-serine-threonine-phosphatase-interacting protein 2) plays a role in regulating membrane curvature and maintaining cytoskeleton stability by binding to its interaction partners, and is highly correlated with the migration and activation of macrophages and osteoclasts (OC, bone-absorbing) [ 15 , 16 ] . Current studies indicate that inflammasome, cysteine aspartate-specific protease (Caspase), cell stress and reactive oxygen species (ROS) are all involved in the pathological development for Pstpip2 cmo/cmo [ 14 , 17 ] . Interestingly, mutations in PSTPIP1 results in PSTPIP1-associated myeloid-related proteinemia inflammatory syndrome (PAMI), a rare autoinflammatory disease [ 18 ] . Nonetheless, mechanisms by which PSTPIP2 regulate myeloid cells especially the osteoclast cells have not been fully elucidated, and the cell populations, cytokines and signaling pathways involved in the occurrence of CRMO for Pstpip2 cmo/cmo are also controversial. Furthermore, effective perturbations or drugs to inhibit the progression of the disease still remains to be explored. Our previous studies have demonstrated that the conserved lncRNA Morrbid (myeloid RNA regulator of Bim-induced death) was specifically expressed in mature eosinophils, neutrophils (NE) and classical monocytes (Mono) and promotes the myeloid-linage leukemogenesis [ 19 ] . In addition, Morrbid was discovered to have an intimate connection with the release of various cytokines and the activity of cell signaling pathways [ 20 – 23 ] . This prompted us to ask whether Morrbid has a role in inhibiting the progression of AIDs including CRMO. In addition, scRNA-seq technologies provide powerful assets as the traditional approaches for pathogenesis of immune diseases. It is believed that single-cell omics are revolutionizing the study of immunology and rheumatology [ 24 ] . Even at the most basic level, single-cell sequencing technologies are able to provide a systematic map (a cell atlas) for affected regions or organs and their microenvironment alteration. As mentioned above, even though a PBMC profile has been described in a CRMO patient [ 10 ] , so far, an extensive understanding of CRMO in the affected tissues in human or even in mice (i.e. in Pstpip2 cmo/cmo ) is still lacking. In the present study, we showed that Pstpip2 and Morrbid were co-expressed in myeloid cells. We generated a new Pstpip2 frameshift mutant mouse strain Pstpip2 −/− and observed its phenotype similar to classical Pstpip2 cmo/cmo . To access the involvement of Morrbid , we generated double knock-out mutant Pstpip2 −/− ; Morbbid −/− (DKO). The deficiency of Morrbid in the DKO mice inhibited the progression of inflammatory symptoms and bone damage, which is very likely mediated by the decrease of myeloid cells and cytokines in the lesion site. The mitigation of CRMO in the DKO were also validated by the scRNA-seq analysis using Pstpip2 −/− and DKO bone marrow cells. Results A 5-bp deletion in Pstpip2 gene leads to chronic osteomyelitis characterized by swelling of the hind paw and bone damage Using the scRNA-seq dataset, we confirmed that Pstpip2 was highly expressed in murine myeloid cells, especially the monocytes in wild type (WT) ( Fig. 1A ). We constructed a mouse strain carrying a novel frameshift mutation ( Pstpip2 −/− ) by knocking out 5-bp in the Pstpip2 exon 2 protein-coding region ( Fig. 1B ). The main lesion of osteomyelitis is located in the bilateral hind paws of mice. Continuously, we observe the hind paws of both WT and Pstpip2 −/− mice of the same age, rigorously documenting the occurrence and progression of the disease. We established a scoring system: each swollen toe counts 1 point, with a total score of 10 points. Through continuous observation and scoring, we found that the hind paw swelling of Pstpip2 −/− mice was characterized by progressive aggravation ( Fig. 1C ). Almost all Pstpip2 −/− mice showed marked swelling in all toes of bilateral hind paws by 12 weeks of age ( Fig. 1C ), and their appearance was dramatically different from that of WT mice ( Fig. 1D ). We isolated popliteal lymph nodes (popLN) from 20-week-old WT and Pstpip2 −/− mice and observed massive lymphomegaly of bilateral popliteal lymph nodes in Pstpip2 −/− mice, suggestive of inflammatory symptoms in nearby tissues ( Fig. 1E ). These results confirm that the newly generated Pstpip2 −/− mice cause a spontaneous, non-bacterial inflammatory disease similar to the classic Pstpip2 cmo/cmo phenotype. Download figure Open in new tab Figure 1 A 5-bp deletion in Pstpip2 leads to chronic multifocal osteomyelitis in mice. ( A ) Expression of Pstpip2 in bone marrow cells (BM) revealed by scRNA-seq. See also Fig. 3 for details. ( B ) Generation of Pstpip2 −/− mice (5-bp deletion in the coding region of the locus, exon 2). The upper panel shows the transcripts by the bulk RNA-seq; the lower panel shows the Sanger sequencing results. The frameshift mutation caused by the 5-bp deletion in Pstpip2 −/− mice is highlighted. ( C ) Disease progression was scored (scale from 0 to 10) by visual inspection of the hind paws of WT and Pstpip2 −/− mice. (n=8 per group; two-way ANOVA, mean±SEM). ( D-E) Representative images of the hind paws and popliteal lymph nodes (popLN, e) of 20-week-old WT and Pstpip2 −/− mice. ( F ) Representative hematoxylin and eosin (H&E) staining images of hind paw sections of WT and Pstpip2 −/− mice (scale bar, 200 μm). ( G ) Frequencies of neutrophils (NE), monocytes (Mono), and lymphocytes (Lymph) in the peripheral blood (PB) of WT and Pstpip2 −/− mice (n≥5 per group). ( H ) Representative IHC images of Ly6G on hind paw sections from WT and Pstpip2 −/− mice. Stain in brown: neutrophil marker Ly6G; scale bar, 200 μm. ( I ) Principal component analysis (PCA) of WT and Pstpip2 −/− mice by bulk RNA-seq. ( J ) Volcano plots of DEGs in BM cells of WT and Pstpip2 −/− mice by bulk RNA-seq. ( K ) Representative micro-CT of hind paw bones from 20-week-old WT and Pstpip2 −/− mice. Gray images show total bone tissue while pseudo-color images provide difference between high-density (in yellow) and low-density (in blue) area. ( L ) Measurement and quantification of superoxide in WT and Pstpip2 −/− mice. The mice were injected (i.p.) with L-012 chemiluminescent probe before imaging. Two-tailed Student’s t test, mean±SEM; * p <0.05, *** p <0.001, **** p <0.0001. To investigate the alteration of immune cells in Pstpip2 −/− osteomyelitis, the number and frequency of immune cells were characterized. The hind paws of WT and Pstpip2 −/− were isolated and examined through hematoxylin and eosin (H&E) staining. Pstpip2 −/− mice showed very high levels of immune cells and granulocyte infiltration in the hind paws, whereas WT mice showed no such infiltration ( Fig. 1F ). By collecting peripheral blood samples from the two groups of mice for hemogram detection, we found that the frequency of neutrophils and monocytes in the peripheral blood of Pstpip2 −/− mice was significantly increased, while the frequency of lymphocytes was inversely decreased ( Fig. 1G ). The results of immunohistochemical (IHC) staining showed that Pstpip2 −/− mice had obviously abundant neutrophils in the hind paw tissue compared to WT ( Fig. 1H ). These data suggest that myeloid cells represented by neutrophils and monocytes were likely the major cell types that drive osteomyelitis caused by Pstpip2 mutation or deficiency. Next, we performed bulk RNA sequencing (RNA-seq) on BM cells from WT and Pstpip2 −/− mice to compare the differences at the transcriptome level ( Fig. 1I ). Analysis of differential expressed genes (DEGs) showed that Pstpip2 −/− mice had significantly increased mRNA expression levels of cytokines compared to WT, including Il1b and Ccl3 ( Fig. 1J ). There were studies showing that cytokines such as IL-1β and CCL3 (MIP-1α) acted as osteoclasts-activating factors and promoted the dissolution of bone matrix in a variety of diseases [ 25 – 30 ] . The classic inflammatory phenotype of CRMO is accompanied by severe bone damage. Therefore, we focused on the differences in bone tissue structure between the two groups of mice. Microscopic CT (micro-CT) results showed that compared with WT mice, there were multiple low-density areas in the hind paw bone tissue of Pstpip2 −/− mice, suggesting severe damage to the bone tissue ( Fig. 1K ). Phagocytes (especially neutrophils) are the central of inflammatory response, and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) present in them is one of the major sources of reactive oxygen species (ROS) [ 31 , 32 ] . ROS not only involve in regulating cell growth, differentiation, apoptosis, and activation of cell signaling cascades as intracellular second messengers, but also mediate inflammation by oxidizing protein and lipid cellular components and damaging DNA [ 32 , 33 ] . Moreover, ROS are also important components in regulating OC differentiation and activation [ 33 ] . We therefore visualize ROS production in living mice by using luminol derivative L-012. We observed a more intense luminescence signal in the hind paws of Pstpip2 −/− mice compared with that of WT mice ( Fig. 1L ), suggesting that ROS overproduction may be partially involved in the genesis of inflammation and osteolysis. Taking these results together, we demonstrated that the 5-bp deletion in Pstpip2 induces a CRMO-like phenotypes, characterized by progressive swelling of the hind paws accompanied by severe bone damage. The above results suggest that the occurrence and aggravation of this CRMO-like disease are strongly correlated with the increase in the frequency of myeloid cells, the level of inflammatory cytokines and the production of ROS. Deletion of Morrbid significantly inhibits the CRMO-like disease progression caused by the 5-bp deletion in Pstpip2 Morrbid is a long non-coding RNA (lncRNA) that is specifically highly expressed in mature, short-lived myeloid cells and is conserved between humans and mice. We and others have shown that Morrbid -deficient mice exhibit a drastic reduction in mature myeloid cells, such as eosinophils, neutrophils, and monocytes, in blood and tissues. By regulating its downstream pro-apoptotic gene Bcl2l11 in cis , Morrbid regulates apoptosis and controls the lifespan of these cells [ 19 ] . We therefore asked about the expression levels of both Morrbid and Pstpip2 in murine myeloid cells. Using scRNA-seq, we observed that both Morrbid and Pstpip2 were specifically highly expressed in myeloid cells such as Mono, GMP, and NE ( Fig. 2A ). Download figure Open in new tab Figure 2 Loss of Morrbid significantly inhibits the CMO disease progression. ( A ) Expression of Pstpip2 and Morrbid in the scRNA-seq dataset of BM cells from hind paws in WT mice. Mono, monocytes; Neu, neutrophils; GMP, granulocyte-macrophage-progenitors. ( B ) Schematic for the rationale and hypothesis in this study. Red dashed boxes indicate the relevant major myeloid cell types, and red dashed arrows indicate increased or decreased cell number and/or proportion in mutants compared to the WT mice. ( C ) Schematic for the construction of the compound mutant Morrbid −/− Pstpip2 −/− mice. ( D ) Disease severity was scored (scale from 0 to 10) by visual inspection of the hind paws of Pstpip2 −/− and Morrbid −/− Pstpip2 −/− mice. n=8 per group. ( E ) Representative images of the hind paws of 20-week-old Pstpip2 −/− and Morrbid −/− Pstpip2 −/− mice. ( F ) Representative images and quantification (size in diameter) of the popliteal lymph nodes of 20-week-old Pstpip2 −/− and Morrbid −/− Pstpip2 −/− mice. n=3 for Pstpip2 −/− , n=4 for Morrbid −/− Pstpip2 −/− . ( G ) Representative H&E images of hind paw sections of Pstpip2 −/− and Morrbid −/− Pstpip2 −/− mice. scale bar, 200 μm. ( H ) Frequencies of Neu, Mono, and Lymph in the peripheral blood of Pstpip2 −/− and Morrbid −/− Pstpip2 −/− mice. n=8 per group. ( I ) Representative IHC images of hind paw sections of Pstpip2 −/− and Morrbid −/− Pstpip2 −/− mice. Stain in brown: neutrophil marker Ly6G; scale bar, 200 μm. ( J ) IL-1β and MIP-1α (CCL3) concentration in hind paw lysates detected by ELISA. n=5 per group. ( K ) Representative micro-CT of hind paw bones from 20-week-old Pstpip2 −/− and Morrbid −/− Pstpip2 −/− mice. Gray images show total bone tissue while pseud-ocolor images provides difference between high-density (in yellow) and low-density (in blue) area. ( L ) Representative histologic sections of hind paws from Pstpip2 −/− and Morrbid −/− Pstpip2 −/− mice stained by TRAP (a marker for osteoclasts; arrows), the dashed line marks BM cavity region. scale bar, 100 μm. ( M ) Measurement and quantification of superoxide in Pstpip2 −/− and Morrbid −/− Pstpip2 −/− mice. The mice were injected (i.p.) with L-012 chemiluminescent probe before imaging. Two-tailed Student’s t -test, mean±SEM;* p <0.05,** p < 0.01,*** p <0.001,**** p <0.0001. In view of the massive expansion of myeloid cells in the peripheral blood and hind paws sites of the Pstpip2 −/− mice, we hypothesize that Morrbid deletion retards progression of CRMO-like syptoms ( Fig. 2B ). We investigated whether ablation of Morrbid in the Pstpip2 −/− mice would be sufficient to observe the rescue, and thus, DKO mice were generated by crossing Morrbid +/− mice with Pstpip2 +/− mice. Pstpip2 −/− mice were used as controls for further study of the function of Morrbid in osteomyelitis ( Fig. 2C ). We found that Morrbid knockout significantly attenuated and delayed the development of hind paw swelling caused by Pstpip2 depletion using our scoring system ( Fig. 2D ). Furthermore, the effect was durable rather than temporary, as evidenced by our observations in the hind paws of two groups of 20-week-old mice ( Fig. 2E ). We dissected popLN of both groups of mice, measured diameters and performed statistics, and found that Morrbid −/− Pstpip2 −/− mice had apparently smaller popliteal lymph nodes compared with Pstpip2 −/− mice, suggesting that inflammation in the surrounding tissues was controlled ( Fig. 2F ). These phenotypes indicated that deletion of Morrbid effectively rescued the development of inflammatory response in CMO mice. We next addressed the effects of Morrbid on immune cells in CMO. H&E staining of the mice hind paws showed that Morrbid −/− Pstpip2 −/− mice had significantly reduced infiltration of immune cells and inflammatory granulomas ( Fig. 2G ). The hemogram analysis for peripheral blood from the two groups of mice showed that the Morrbid −/− Pstpip2 −/− mice had a significantly reduced frequency of neutrophils and monocytes and increased frequency of lymphocytes as compared with the Pstpip2 −/− mice ( Fig. 2H ). IHC staining of the hind paws revealed a significant reduction in the number of neutrophils in Morrbid −/− Pstpip2 −/− mice ( Fig. 2I ). The excessive activation of immune cells releases a mass of cytokines and enhances the self-feedforward of inflammation [ 34 ] . The hind paw homogenates of the two groups of mice were obtained for enzyme linked immunosorbent assay (ELISA) with the aim of measurement of the protein levels of IL-1β and MIP-1α (CCL3) in hind paws. The results showed that the two cytokines from Morrbid −/− Pstpip2 −/− mice were all significantly lower than that from Pstpip2 −/− mice ( Fig. 2J ). These data suggest that effective reduction of inflammation in the hind paws of CRMO-like mice could be achieved by Morrbid deletion, and the reduction of myeloid cells and cytokines in the lesions are important for mitigating the CRMO-like symptoms. In addition to this, we also quantified bone damage in the DKO mice. By micro-CT, we confirmed that knockout of Morrbid in Pstpip2 −/− mice resulted in an evident reduction in low-density areas and significantly decreased the degree of osteolysis ( Fig. 2K ). Bone tissue homeostasis is synergistically maintained as supported by osteoblastic bone formation and osteoclastic bone resorption [ 35 ] . Tartrate-resistant acid phosphatase (TRAP), as a specific marker of osteoclasts, plays a critical role in the synthesis and degradation of bone tissue [ 36 – 39 ] . By staining TRAP in hind paw sections from both groups of mice, we found that Morrbid −/− Pstpip2 −/− mice had a decreased number of osteoclasts and an increased complete bone marrow cavity in the hind paw accordingly ( Fig. 2L ). These results suggest that the rescue of bone damage in Pstpip2 −/− by Morrbid deletion is likely due to inhibition of OC formation. We also evaluated the effect of Morrbid deletion on ROS production. We observed a significant reduction in ROS luminescence signal in Morrbid −/− Pstpip2 −/− mice compared with that in Pstpip2 −/− mice after intraperitoneal injection of L-012 ( Fig. 2M ). In conclusion, deletion of Morrbid significantly reduced the frequencies of myeloid cells and the levels of inflammatory cytokines in the hind paws of CMO mice, which effectively inhibited the symptoms and progression of osteomyelitis. sc-RNA seq reveals the pathogenesis of Pstpip2 -deficient CRMO and that loss of Morrbid relieves autoinflammatory symptoms in Pstpip2 −/− mice scRNA-seq analysis was performed on hind paws of four groups of mice (WT, Morrbid −/− , Pstpip2 −/− and DKO). Muscle cells were removed and only cells from the bone marrow were included for the library construction and sequencing procedure (see methods for details). After standard Seurat annotation pipeline and filtering mesenchymal cells, around 8,000 to 10,000 hematopoietic cells (CD45 + ) per group were included for further annotation analysis after quality control. Annotation of cells by canonical cell marker genes grouped all cells into 11 cell populations, including GMP, common lymphoid precursors (CLP), hematopoietic stem and progenitor cells (HSPC), and Plasmablasts, monocytes, neutrophils, osteoclasts, mast cells, plasmacytoid dendritic cells (pDC), and lymphoid cells (T and B cells) ( Fig. 3A, B , Supplemental Figure 1A ). Download figure Open in new tab Download figure Open in new tab Download figure Open in new tab Download figure Open in new tab Supplementary Figure 1. Supplementary Figure Additional computational analysis of scRNA-seq datasets from the four genotype mice. ( A ) Expression profiles of marker genes used for annotating the BM cells in the scRNA-seq datasets. ( B and C ) Volcano plots showing DEGs in each cell-type compartment from Pstpip2 −/− mice compared with that from WT and DKO mice. ( D ) KEGG enrichment analysis of GMP2 and Cxcl2_NE among different groups. ( E ) GO enrichment analysis of osteoclast/monocytes (OMC) among different groups. ( F ) Scatter plots showing DEGs in the indicated comparison. ( G ) Circle plot (left) and heatmap (right) of the relative number and strength of interactions in DKO compared with Pstpip2 −/− mice; the up-regulated intercellular interactions are shown in red; the down-regulated intercellular interactions are represented in blue. ( H and I ) Heatmap for overall signal patterns of WT compared with Pstpip2 −/− mice; and for Pstpip2 −/− mice compared with DKO mice. ( J ) Expression (left) and quantification (right) of representative transcription factors in the four genotype mice. Transcriptional factors Nfkb2, Rela, Bcl11a and Runx1 are included. ( K ) RNA velocity in all BM cells from the four groups of mice. ( L ) Developmental trajectories of GMP, NE and OC in four groups of mice. Developmental initiation points are marked by blue boxes. Download figure Open in new tab Figure 3 scRNA-seq analysis of the BM cells from the hind paws of four genotype mice. ( A ) Uniform manifold approximation and projection (UMAP) visualization of the BM cells (n=35,790); each dot represents a cell, and a color indicates a cell-type as indicated. (B ) Separate UMAP plots of BM cells from the four genotype mice. ( C ) Stacked chart of the proportion of each cell-type in the four genotype mice. ( D ) Heatmap for the stemness scores of BM cells from the four groups of mice by CytoTRACE. Higher predicted values (dark red) suggest state with higher stemness (less differentiated and more progenitor-like). Note that monocytes, neutrophils and GMP from Pstpip2 −/− appear more immature compared to the myeloid cells form other three genotypes. ( E and F ) The total and various types of myeloid cells inflammatory gene signature scores and ROS metabolic gene signature scores of the BM cells from four groups of mice. Myeloid-lineage cells including GMP, monocytes, neutrophils, osteoclasts were included for scoring the total score. The individual scores were also computed as indicated. ( G ) Utilizing the scRNA-seq datasets we also measured the activity of osteoclasts by computing differentiation score, proliferation score, ubiquitination score, endoplasmic reticulum stress score and apoptosis score of osteoclasts from the four groups of mice. Datasets from each genotype group had all of these cell populations, but the overall proportion of myeloid cells was higher in Pstpip2 −/− mice than in WT and in Morrbid −/− Pstpip2 −/− mice ( Figure 3C ). We asked the effect of Pstpip2 mutation and Morrbid deletion on the development states of different cell types. We visualized the cellular stemness of individual cells by using cellular trajectory reconstruction analysis (CytoTRACE) using gene counts and expression. Results show that the OC, GMP and Neu prediction score in Pstpip2 −/− mice are higher than that in WT and DKO mice, suggesting their stemness level are enhanced ( Figure 3D ). These cells from Pstpip2 −/− were at a more primitive level, suggesting the time intervals before the occurrence of apoptosis were likely larger, and their survival time may be extended. However, loss of Morrbid predisposed these myeloid cells to undergo apoptosis earlier, as supported by the lower stemness scores of these cells in DKO mice ( Figure 3D ). Consistent with results in Fig. 1l and Fig. 2m , Pstpip2 −/− mice has higher inflammatory and ROS metabolic scores on total cells and all myeloid cell types than WT and DKO mice ( Figure 3, E-F ). Compared with WT and DKO, Pstpip2 −/− has higher differentiation score, proliferation score, ubiquitination score, endoplasmic reticulum (ER) stress score and apoptosis score ( Figure 3G ). We performed analyses of DEGs in cell clusters between different groups. The results showed that matrix metalloproteinase genes ( Mmp3 and Mmp13 ) and serum amyloid gene ( Saa3 ) were upregulated in multiple cell populations from Pstpip2 −/− mice compared to that from WT mice ( Supplemental Figure 1B ). And high expression of these genes was positively correlated with inflammatory disease progression [ 40 – 45 ] . In contrast, the expression of these genes was significantly reduced in DKO mice ( Supplemental Figure 1C ). However, lncRNA Malat1 and the proteoglycan 4 gene ( Prg4 ) were up-regulated in DKO mice ( Supplemental Figure 1C ). Multiple studies have shown the role of lncRNA Malat1 in bone and cartilage diseases and osteogenic differentiation [ 46 – 48 ] . Proteoglycan encoded by Prg4 was reported to function as a cartilage surface lubricant and involve in repairing articular cartilage damage in mouse models of inducible full-thickness injury [ 45 , 49 ] . These results suggest that the expression of these genes is upregulated in Pstpip2 −/− mice, and loss of Morrbid inhibits the expression level of these genes and alleviates the symptoms. We then focused on three cell types from the scRNA-seq datasets, GMP, OC, and NE. These cells were specifically extracted and clustered again to distinguish their subsets. Based on the expression of typical marker genes, we annotated a total of four cell subsets in GMP, four cell subsets in OC, and five cell subsets in NE ( Figure 4A ①, 4B ①, 4C ①). Gene Ontology (GO) enrichment analysis was used for all cell subsets to understand the function of each cell type. GMP seemed to be related to the catabolism of collagen. OC was highly associated with the response to IL-1 and reactive oxygen species metabolism process. The chemotaxis-related functions of NE were significantly enriched ( Figure 4A ②, 4B ②, 4C ②). These biological functions suggest a key role for these three cell types in CRMO-like disease progression. Download figure Open in new tab Figure 4 Characteristics of inflammatory cells and osteoclasts in Pstpip2 −/− and Morrbid −/− Pstpip2 −/− mice. ( A-C ) Myeloid-linage cells including GMP, OC and NE were extracted for further analysis. Four genotype mice were included: WT, Pstpip2 −/− , Morrbid −/− and DKO. GMP, OC and NE cells from Figure 3A were extracted for further analysis as shown. ①UMAP plots of GMP, OC and NE respectively;②GO enrichment analysis of different cell types as indicated; ③Stacked charts of the various cell subsets proportion in GMP, OC and NE respectively;④Scatter plots showing DEGs in the indicated comparison. Each point represents a gene;⑤Heatmap of TF-RAS in GMP, OC and NE respectively. ( D ) Circle plots showing cell-cell communications between cell subtypes in the BM scRNA-seq datasets from WT, Pstpip2 −/− , and DKO mice. ( E ) Overall signal intensities based on Outgoing (x-axis) and Incoming (y-axis) strengths. Of note, a much higher basal level of signals was determined in Pstpip2 −/− (∼10-fold higher). According to the comparison of the proportion of each cell subtype between WT and Pstpip2 −/− mice, we paid attention on the cell subtypes with large differences between these two groups, namely GMP2, OMC and Cxcl2_NE ( Figure 4A ③, 4B ③, 4C ③), and carried out further analysis. The results of differential gene analysis between different groups of these cell subsets showed that inflammation and peroxidation related genes, such as Saa3 , Grina , Mmp13 and Prdx1 , were significantly upregulated in Pstpip2 −/− . Oppositely, the expression of genes involved in bone protection and remodeling, such as Suco and Prg4 , were significantly higher in WT and DKO ( Figure 4A ④, 4B ④, 4C ④). Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of GMP2 and Cxcl2_NE and GO analysis of OMC showed that the pathways and functions of cell metabolism, rheumatoid arthritis and osteoclast differentiation were more significant in Pstpip2 −/− compared with WT and DKO ( Supplemental Figure 1, D-E ). These results further supported a mechanism by which loss of Pstpip2 induces CMO and loss of Morrbid inhibits CRMO-like progression. The Pstpip2 −/− had Il1b_OC and Acp5_OC, which were not present in the WT, and the proportion and number of these two OC subsets in DKO were obviously lower than those in Pstpip2 −/− . We therefore inferred that these two subsets of OC may be the major osteoclast population that play an osteolytic role in CMO ( Figure 4A ③, 4B ③, 4C ③). The differences between the two subtypes of OC were analyzed in the comparison between Pstpip2 −/− and DKO. However, Acp5_OC in the DKO could not be enriched due to the few numbers of cells. Then we compared the differences in Il1b_OC. DEG analysis showed that inflammation-related genes, such as Mmp13 , Grina and S100a8 , were highly expressed in Pstpip2 −/− , and osteoprotective genes, Prg4 , were significantly upregulated in DKO ( Supplemental Figure 1F ). GO enrichment analysis showed that OC in this subtype was associated with immune disorders and the emergence of some chronic inflammatory diseases ( Supplemental Figure 1F ). All the above results suggest that the proportion and function changes of myeloid cells play prominent roles in the progression of CRMO-like symptoms in Pstpip2 −/− , and DKO mice reverses these effects. We next discuss the states of cell-to-cell interactions between different groups in these cell types using CellChat. The number of interactions was greater in the Pstpip2 −/− than in the other two groups ( Figure 4D ). Comparison of the number and strength of intercellular interactions between Pstpip2 −/− and DKO mice (DKO vs. Pstpip2 −/− ) showed that most intercellular interactions were enhanced in Pstpip2 −/− (in blue). Only a few of interactions were enhanced in DKO (in red) ( Supplemental Figure 1G ). These results suggest that cell activity is higher in CRMO-like diseased tissues. In the pairwise comparison of the overall signaling patterns, the signaling pathways with differential intensities were mainly affected in Acp5_OC, including MIF, VISFATIN, SPP1, CSF3, TGFβ and GDF signaling pathways. These signaling pathways were significantly enhanced in Acp5_OC of Pstpip2 −/− ( Supplemental Figure 1H ). Comparing the differences in GMP signaling pathways among the three groups, we found that TGFβ, SEMA3, KIT, APRIL and BAFF signaling pathways were more intense in DKO ( Supplemental Figure 1I ). In addition, the intensities of the incoming and outgoing interactions as a whole in Pstpip2 −/− were higher than that in WT and DKO. The interaction strength of NE of the various cell types in the DKO was similar to that in WT, and the interaction strength of OC of the various cell types in the DKO was lower than that in Pstpip2 −/− ( Figure 4E ). These results show that intercellular communication is enhanced in CMO, especially in some signaling pathways related to myeloid cell activation and inflammation occurrence. By visualizing the transcriptional factor regulon activity score (TF-RAS) of different cell types in each group, we explored the transcription factor activities of different cell types in three groups. We found that some transcription factors that are usually highly expressed in inflammatory diseases, Nfkb2, Rela, Jund, Atf4 and Esrra, had higher scores among the three cell types of Pstpip2 −/− ; Ctcf, Elf1, Fli1, Bcl11a, and Runx1 had higher TF-RAS in the WT and DKO ( Figure 4A ⑤, 4B ⑤, 4C ⑤), which are partially associated with bone protection in some diseases. Similar results were observed for the distribution and expression levels of these transcription factors ( Supplemental Figure 1J ). These results suggest that the pathogenesis of CRMO-like autoinflammation may be associated with the changes of some transcription factors and the activation of some pathways (e.g., NF-κB). Taken together, the inter-cellular mechanism of CRMO caused by Pstpip2 mutation is complicated, involving various aspects such as the level of gene expression, transcription factors and developmental stage regulation. Loss of Morrbid regulates myeloid cells from multiple points, and ultimately inhibits inflammation progression and bone damage in CMO. Experimental validation in the four genotype mice Based on the results of the above, we have an unbiased and comprehensive understanding of pathology of CRMO-like diseases and the involvement of Morrbid in the disease. To verify our results concluded above, we further performed experiments including the flow cytometry and qRT-PCR. We extracted whole bone marrow cells from the four genotype mice (WT, Morrbid −/− , Pstpip2 −/− and DKO) and measured the frequencies of different types of myeloid cells and osteoclast precursors (OCP) in bone marrow by flow cytometry. Compared with WT and Morrbid −/− Pstpip2 −/− mice, the frequencies of myeloid cells (Gr1 + CD11b + ), monocytes (c-Kit + /c-Kit − CD11b + Ly6C + ), and OCP (c-Kit + CD11b lo Csf1r + ) were all significantly higher or tended to be higher in the bone marrow of Pstpip2 −/− mice ( Figure 5A-D ). A similar trend was observed in neutrophils (c-Kit + /c-Kit − CD11b + Ly6G + ) ( Figure 5B-C ). These results are consistent with the previous results showed in scRNA-seq dataset that elevated myeloid cell frequencies may contribute to CRMO-like diseases and that loss of Morrbid inhibits CRMO progression by repressing such elevations. Download figure Open in new tab Figure 5 Experimental analysis using flow cytometry and qRT-PCR for validating findings from scRNA-seq analysis. ( A-D ) Representative flow cytometry profiles in BM samples from the four groups of mice (left) and quantification of the results (right). ( A ) myeloid cells (Gr1 + CD11b + ); ( B ) pre-mature monocytes (c-Kit + CD11b + Ly6C + ) and neutrophils (c-Kit + CD11b + Ly6G + ); ( C ) mature monocytes (c-Kit − CD11b + Ly6C + ) and neutrophils (c-Kit − CD11b + Ly6G + ); ( D ) osteoclast precursors (c-Kit + CD11b low Csf1r + ). n≥4 animals for each group. ( E ) The expression levels of indicated genes in hind paw lysates, determined by qRT-PCR. n≥3 animals for each group. Two-tailed Student’s t test, mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns, not statistically significant. We extracted total RNA from the hind paws of mice with different genotypes and measured expression levels of some genes by qRT-PCR. The level of Il1b expression in the hind paw of mice was similar to our previous data for IL-1β ( Figure 5E ). There was a study shown that NLRP3 inflammasome is involved in the pathogenesis of CRMO-like disease in Pstpip2 cmo/cmo , not only by converting pro-IL-1β to active IL-1β, but also by ROS production and cellular stress [ 14 ] . In our data analysis of cellular transcription factors, we also found that transcription factor Nfkb2, which is related to the NLRP3 pathway, was significantly active in the tissues of Pstpip2 −/− mice hind paws. The expression level of Nlrp3 in our four groups of mice was measured and confirmed that its important role in CRMO ( Figure 5E ). We also examined the expression levels of other inflammation-related genes (e.g., Mmp13 , Acp5 , Csf1r , Prdx1 , Prg4 ) and gene encoding representative transcription factors (e.g., Nfkb2 , Rela , Bcl6 ) identified by scRNA-seq. These results were similar to those obtained with single-cell transcriptome analysis, which indicate that loss of Morrbid alleviated the inflammatory symptoms and bone tissue damage in CRMO. This alleviation is associated with down-regulation of some genes within the NF-κB signaling pathway that accelerates inflammatory progression. Discussion Chronic non-bacterial osteomyelitis is an inflammation of bone tissue without bacterial infection. When it is active and occurs in multiple foci, it is referred to as CRMO [ 50 ] . Patients with CRMO usually present with recurrent fever and bone pain, and swelling and deformity may occur in the affected area [ 8 ] . Here we described a new Pstpip2 mutant mouse strain with 5-bp loss in the protein-coding region, interrogated the pathogenesis of CRMO-like phenotypes by scRNA-seq analysis, and demonstrated the efficacy of targeting Morrbid for mitigating CRMO. In summary, our findings provided in-depth insights into the relevance and function of lncRNA Morrbid in the context of AID and CRMO, and demonstrated Morrbid is a target for inhibiting osteomyelitis. PSTPIP2, an adaptor protein that located in myeloid cell membranes, regulates membrane curvature and effects the morphology of cell membrane, stabilizing the cytoskeleton and regulating the biological functions of myeloid cells, including endocytosis, podosome formation, cell division and movement. PSTPIP2 negatively regulates the activation of phagocytes and inhibits the occurrence of inflammation. In osteoclasts, PSTPIP2 promotes the decomposition of osteoclasts and regulates the dynamic homeostasis of osteoblasts and osteoclasts [ 15 ] . In previous studies, the cmo mutation in Pstpip2 inactivates the PSTPIP2 protein, resulting in uncontrolled bone-destroy functions of OC. Mice with Pstpip2 mutation develop CRMO lesions and show a massive increase in myeloid cells at the lesion location. The overproduction of osteoclasts causes bone damage at the lesion site and severely destroys the basic structure of bone. Massive infiltration of neutrophils and monocytes/macrophages leads to excess release of proinflammatory cytokines. In addition, the abundant NADPH oxidase in phagocytes produces a large amount of ROS, which further promotes the progression of inflammation and osteolysis. Biased myelogenesis is also indicated the perpetual cycle for the progression of CRMO in the Pstpip2 cmo/cmo . However, the efficacy of depleting inflammatory cells in CRMO has not been tested until this study. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used as early treatment for CRMO [ 11 , 51 ] . When inflammation continues to worsen, off-label empirical therapies, including bisphosphonates and TNF-α antagonists, are used [ 11 , 51 ] . However, there are significant differences in the response to these drugs among different patients [ 51 ] . There is currently no standardized treatment regimen and no FDA-approved drug for the treatment of CRMO [ 8 , 11 ] . Therefore, it is imperative to develop effective CRMO inhibition methods. Morrbid as a lncRNA controls myeloid cell lifespan. Myeloid cells deficient in Morrbid exhibited increased apoptosis. Our experiments confirmed that deletion of Morrbid in Pstpip2 −/− mice significantly reduced the number of myeloid cells at the lesion site, lowered inflammatory cytokines and ROS levels, and alleviated bone damage. The reduced expression of Morrbid suggests control over the pathogenic mechanisms of CRMO, ultimately suppressing inflammation and bone damage symptoms associated with CRMO disease. The high conservation of Morrbid between humans and mice suggests the possibility of Morrbid as a target to inhibit the development of the disease. How to effectively and specifically target Morrbid will be a challenge to be overcome. In addition, the main cell type driving the initiation of CRMO in Pstpip2 −/− mice (or Pstpip2 cmo/cmo ) remains controversial. The prominent role of myeloid cells in the progression of CRMO has been observed in previous studies and this study. Previous studies held the view that neutrophil or monocyte lineage drives the initiation of CRMO [ 11 , 14 , 17 , 52 ] . Our study together with single-cell RNA sequencing data confirmed an increased frequency of multiple types of myeloid cells, especially neutrophils and osteoclasts, in the lesions and peripheral blood of mice with CRMO. By scRNA-seq of the four genotype mice, Pstpip2 −/− mice showed increased inflammatory levels and ROS production, significantly upregulating the expression of genes that promote inflammation. Furthermore, Pstpip2 −/− mice enhanced intercellular communication and increased the activity of transcription factors related to NK-κB pathway. The deficiency of Morrbid resulted in a comprehensive inhibitory effect in these aspects. However, further studies are required to determine which type of myeloid cell plays a dominant role in the CRMO initiation, and the primary cell type exerts inhibitory effects when deletion of Morrbid . Based on the experimental results and single-cell RNA-seq data analyses, we tend to suggest that neutrophils play a key role in the process of the inflammation aggravation. The excess release of inflammatory cytokines further promotes the activation of monocytes into osteoclasts, leading to osteolysis. PSTPIP2 binds to its interaction partners and plays a key role in membrane ligation processes in eukaryotic cells [ 15 ] . It is widely involved in cell endocytosis, division and morphological regulation [ 15 , 16 , 53 ] . It is mainly expressed in cells of the monocyte or macrophage lineage, and is highly relevant to the migration of macrophages and the decomposition of osteoclasts [ 15 ] . Morrbid not only has the ability to regulate myeloid cell apoptosis [ 19 ] , but also has an expression profile highly similar to Pstpip2 in mice. Importantly, the study of Morrbid in osteoclast formation and function is still lacking now. Our study fills a gap in this area. However, the mechanism by which Morrbid functions and its effects on PSTPIP2 interaction partners need to be further explored. Methods Animals We used wild-type (WT), Morrbid −/− , Pstpip2 −/− , and Morrbid −/− Pstpip2 −/− mice in this study. All mice were on C57BL/6 background. Morrbid −/− and Pstpip2 −/− mice were generated by and purchased from Cyagen Biosciences (Suzhou, China). Morrbid −/− mice were generated by knockout of six exons of Morrbid . Pstpip2 −/− mice were generated by a 5-bp deletion in Pstpip2 exon 2. Morrbid −/− Pstpip2 −/− mice were generated by crossing Morrbid −/− mice with Pstpip2 −/− mice. All animal experimentations were performed in accordance with protocols approved by the Animal Care and Use Committee of Tianjin Medical University. Histopathology The hind paws were fixed in 4% paraformaldehyde (#p1110, Solarbio) for 24 h and decalcified in EDTA Decalcified Solution (#BL616B, biosharp) for 2 to 3 weeks, followed by paraffin embedding and histological cutting. The slice thickness was 4 μm. Hematoxylin and eosin (H&E) staining was used to detect the infiltration of immune cells and inflammatory granulomas. For immunohistochemistry (IHC) staining, sections were stained with Ly6G (E6Z1T) Rabbit mAb (#87048, Cell Signaling Technology) and counterstained with hematoxylin. H&E staining and IHC staining of slides were stained using the BOND automatic system (Leica) and scanned in Aperio (Leica). The image postprocessing and analysis were done in Aperio ImageScope software (Leica). For tartrate-resistant acid phosphatase (TRAP) staining, paraffin embedding and tissue sectioning were performed as previously described. Slides were stained according to the TRAP staining kit (#G1050, Servicebio) operating standard, scanned in 3DHISTECH scanner (DANJIER, China), and images were processed in 3DHISTECH SlideViewer software (DANJIER, China). Hemogram detection of peripheral blood Heparinized capillary tubes (#2501, KIMBLE) were accessed to the fundi of mice eyes and rotated to cut the venous plexus. After sufficient blood were drawn, it is transferred to centrifuge tubes with anticoagulant (#110403015, BKMAM). The number and frequency of blood cells were detected by automatic blood cell analyzer (URIT, China). Micro-computed tomography Hind paws were scanned by in vivo micro-computed tomography Skyscan 1276 (Bruker). Scanning parameters were voltage, 50 kV; current, 200 μA; filter, 0.25 mm aluminum; pixel size, 9 mm; exposure time, 650 to 800 ms; rotation step, 0.4° for 180° total; object to source distance, 81.719 mm; and camera to source distance, 158.176 mm; with time of scanning, 7 min. Reconstruction of virtual slices was performed in NRecon software 1.7.3 (Bruker) with setup for smoothing = 3, ring artifact correction = 6, and beam hardening correction = 30%. Intensities of interest for reconstruction were in the range of 0.0030 to 0.1500. For reorientation of virtual slices to the same orientation, the DataViewer 1.5.4 software (Bruker) was used. CTvox 3.3.0 (Bruker) was used for Micro-CT image processing. ROS detection To assess ROS production in vivo, mice were intraperitoneal (i.p.) injected with luminescence reporter L-012 (#120-04891, FUJIFILM Wako) in final concentration 25 mg/kg dissolved in PBS with concentration of 20 mmol/L. Luminescence signal was acquired by IVIS Spectrum (PerkinElmer), with the exposure parameters set to automatic. The quantification of photon counts was performed in Living Image Software (PerkinElmer). ELISA Hind paw tissue was cut into small pieces and homogenized with tissue homogenizer (60 HZ, 60 s) in 1 ml RIPA Lysis buffer (#E122-01, Genstar) containing 1 mM PMSF (#B111-01, Genstar). After two rounds of centrifugation (each 12,000 rpm, 10 min, 4°C) the lysates were snap-frozen in liquid nitrogen and stored in −80°C. Frozen tissue homogenates described above were thawed, total protein concentration was measured by Nanodrop (Thermo). Enzyme linked immunosorbent assay (ELISA) was performed according to manufacturer’s instructions using Mouse IL-1β ELISA Kit (#EK201B, MULTI SCIENCES) and Mouse CCL3/MIP-1a ELISA Kit (#EK261, MULTI SCIENCES) to detect the concentrations of IL-1β and CCL3/MIP-1α in the tissue homogenates. Flow cytometry The bone marrow cells were flushed out with FACS buffer. Single-cell suspensions were treated with red blood cell lysis buffer, stained, and analyzed using BD LSRFortessa (BD Biosciences). For myeloid cell analysis, staining with antibodies against CD11b (#101224, BioLegend) and Gr-1 (#108408, BioLegend). For monocyte and neutrophil analysis, cells were incubated with biotinylated antibodies against c-Kit (#105812, BioLegend), CD11b, Ly-6C (#128024, BioLegend), Ly-6G (#127608, BioLegend). For osteoclast precursor (OCP) analysis, staining with antibodies against c-Kit, CD11b and Csf1r (#135511, BioLegend). Real-time quantitative PCR Hind paws were cut into small pieces and homogenized in 1 mL TRIzol using tissue homogenizer (60 HZ, 60 s). Total RNA was isolated from the hind paws with TRIGene (#P118-05, Genstar) according to the manufacturer’s instructions. In brief, 200 μL of chloroform was added to the 1 mL of TRIGene, and the samples were incubated at room temperate for 5 min following vortexing. After centrifugation, the aqueous phase was transferred to a new DNase/RNase-free tube (BIOFIL), and equal volumes of isopropanol were added. After incubation at room temperature for 10 min, the RNA was pelleted by centrifugation, and then the RNA was washed two times in 75% ethanol before resuspension in DEPC water. 1 μg of RNA was reverse-transcribed to cDNA using StarScript III RT MasterMix (#A233, Genstar) according to the manufacturer’s instructions. Real-time quantitative PCR was performed using a 2×RealStar Power SYBR qPCR Mix (#A314, Genstar) and samples were run on a LightCycler ® 480 Instrument II (Roche). For each sample, transcript levels of tested genes were normalized to Actb. Bulk RNA sequencing (RNA-seq) Analysis For RNA extraction, total RNA was extracted from the tissue using TRIzol ® Reagent according the manufacturer’s instructions. Then RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA sample (OD260/280=1.8∼2.2, OD260/230≥2.0, RIN≥6.5, 28S:18S≥1.0) was used to construct sequencing library. For library preparation and sequencing, RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions (Illumina, San Diego, CA). The RNA-seq transcriptome library was prepared following Illumina® Stranded mRNA Prep, Ligation from Illumina (San Diego, CA) using 1μg of total RNA. Shortly, messenger RNA was isolated according to polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer firstly. Secondly double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol. Libraries were size selected for cDNA target fragments of 300 bp on 2% low range ultra agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by Qubit 4.0, paired-end RNA-seq sequencing library was sequenced with the NovaSeq 6000 sequencer (2×150 bp read length). For quality control and read mapping, the raw paired end reads were trimmed and quality controlled by fastp with default parameters. Then clean reads were separately aligned to reference genome with orientation mode using HISAT2 software. The mapped reads of each sample were assembled by StringTie in a reference-based approach. For differential expression analysis and functional enrichment, to identify DEGs between two different samples, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. RSEM was used to quantify gene abundances. Essentially, differential expression analysis was performed using the DESeq2 or DEGseq. DEGs with |log2FC|≥1 and FDR≤0.05 (DESeq2) or FDR≤0.001 (DEGseq) were considered to be significantly different expressed genes. In addition, functional-enrichment analysis including GO and KEGG were performed to identify which DEGs were significantly enriched in GO terms and metabolic pathways at Bonferroni-corrected P-value≤0.05 compared with the whole-transcriptome background. GO functional enrichment and KEGG pathway analysis were carried out by Goatools and KOBAS, respectively. scRNA-seq and analysis of the scRNA-seq datasets Cell preparation After harvested, tissues were washed in ice-cold RPMI1640 and dissociated using Collagenase Ⅱ (Sigma 9001-12-1, V900892-100MG) and DNaseⅠ (Sigma 9003-98-9, DN25-1G). Cell count and viability was estimated using fluorescence Cell Analyzer (Countstar ® Rigel S2) with AO/PI reagent after removal erythrocytes (Solarbio R1010). Finally, fresh cells were washed twice in the RPMI1640 and then resuspended at 1×10 6 cells per ml in 1×PBS and 0.04% bovine serum albumin. scRNA-seq library construction and sequencing Single-cell RNA-Seq libraries were prepared using SeekOne ® Digital Droplet Single Cell 3’ library preparation kit (SeekGene Catalog No. K00202). Briefly, appropriate number of cells were mixed with reverse transcription reagent and then added to the sample well in SeekOne ® chip. Subsequently Barcoded Hydrogel Beads (BHBs) and partitioning oil were dispensed into corresponding wells separately in chip. After emulsion droplet generation reverse transcription were performed at 42 [for 90 minutes and inactivated at 80[for 15 minutes. Next, cDNA was purified from broken droplet and amplified in PCR reaction. The amplified cDNA product was then cleaned, fragmented, end repaired, A-tailed and ligated to sequencing adaptor. Finally, the indexed PCR were performed to amplified the DNA representing 3’ polyA part of expressing genes which also contained Cell Barcode and Unique Molecular Index. The indexed sequencing libraries were cleanup with SPRI beads, analyzed by Qubit (Thermo Fisher Q33226) and Agilent 4200 TapeStation (Agilent G2991BA). The libraries were then sequenced on illumina NovaSeq 6000 with PE150 read length. Identifying the marker genes of single cell The FindAllMarkers function was used to identify marker genes of single cells [ 54 ] . Aggregation-mediated cell clusters were identified based on DEGs exhibiting log fold changes (logFCs) and aggrephagy-related genes. Analysis of transcription factors SCENIC was employed as a computational tool to simultaneously reconstruct gene regulatory networks and identify stable cell states from single-cell RNA-seq data [ 55 ] . The inference of the gene regulatory network was based on coexpression analysis and DNA motif analysis, followed by the examination of network activity in individual cells to determine their cellular status [ 55 , 56 ] . We analyzed transcription factors (TFs) using the pySCENIC package [ 57 ] . Cell–cell communication analysis The signaling inputs and outputs among the different cell types and aggrephagy-mediated cell clusters were assessed using the CellChat package [ 58 ] . The netVisual_circle function was employed to evaluate the strength of cell[cell communication networks within specific subsets of cells [ 58 ] . Pseudotime analysis The core concept of CytoTRACE is that cells with a high level of differentiation express fewer genes, while cells with a low level of differentiation express more genes [ 59 ] . We used the CytoTRACE package to predict the differentiation status of cell subsets. Statistical analysis The p values were calculated in GraphPad Prism 9. Comparisions between 2 groups were determined by unpaired t test (two-tailed) for data. Comparison of multiple groups were determined by two-way ANOVA with Tukey-Kramer or Bonferroni multiple comparisons test. Most of experiments in this study were repeated 2 or 3 times independently and representative data were shown. Symbol meanings are as follows: ns, not statistically significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. “n” in figure legends represent number of experiment repeats, points in column scatter plots represent biological replicates. Conclusion Although PSTPIP2 has not been implicated with a direct role in human inflammatory diseases, Pstpip2 cmo/cmo is widely used for testing inflammation regulation and a classic model for CRMO. In the study, we described a new Pstpip2 frameshift mutations mice strain and confirmed Pstpip2 loss-of-function causes a CRMO disease in mice. More importantly, we demonstrated that loss of Morrbid through various layers of mechanisms inhibited the occurrence of the disease ( Figure 6 ). Our results not only demonstrated the link between Morrbid and AIDs and the function of Morrbid in a new special type of myeloid cells, osteoclasts, but also conducted single-cell transcriptome sequencing technologies to reveal the CRMO pathogenesis induced by loss of Pstpip2 and the intervention by the loss of Morrbid . In conclusion, our study describes that CRMO occurrence relies on multiple layers of inflammatory myeloid cells at the single-cell level and provides a theoretical and preclinical basis for Morrbid as a therapeutic target for autoinflammatory diseases. Download figure Open in new tab Figure 6 Mouse models with genetic mutation in Pstpip2 are one of the most important and fantastic models for studying autoinflammatory diseases (AIDs) including osteomyelitis. We generated a 5-bp deletion in Pstpip2 for testing Morrbid involvement in the AIDs. Our genetic analysis and pathological analysis including scRNA-seq profiling demonstrate that Morrbid is an effective target for anti-inflammation and loss of Morrbid mitigate osteomyelitis induced by loss of Pstpip2 . Data availability Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. The raw sequencing data from this study have been deposited in the Genome Sequence Archive in BIG Data Center ( https://bigd.big.ac.cn/ ), Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under the accession number: PRJCA026323. Authors’ contributions and materials ZC and QH conceived and designed the study, drafted and wrote the manuscript. ZC and JD wrote the computational scripts for sequencing analysis and visualized the results. QH performed most of the experiments and analysis. Funding This work was supported by grants from Tianjin Municipal Education Commission Scientific Research Program Projects (#2022KJ194 to DG). Declarations section Competing interests ZC is a scientific advisor to Beijing SeekGene BioSciences Co. Ltd. Other authors declare no potential conflict of interest. View this table: View inline View popup Download powerpoint Supplementary Table Primer sequences used for quantitative RT-PCR are listed below: Acknowledgments We thank members of Cai laboratory and colleagues of Tianjin Medical University for their technical and administration support, as well as for their helpful suggestions improve the manuscript. Footnotes ↵ # Co-first-authors References [1]. ↵ Krainer , J. , Siebenhandl , S. , Weinhäusel , A. , Systemic autoinflammatory diseases , J Autoimmun 109 ( 2020 ) 102421 . OpenUrl CrossRef PubMed [2]. ↵ Szekanecz , Z. , McInnes , I. 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Share Depicting pathogenesis of osteomyelitis by single cell RNA-sequencing and an involvement of Morrbid in the autoinflammatory disease Qingran Huo , Jiayu Ding , Hongxi Zhou , Hang He , Lorie Chen Cai , Jingjing Liu , Ge Dong , Zhigang Cai bioRxiv 2025.01.23.634426; doi: https://doi.org/10.1101/2025.01.23.634426 Share This Article: Copy Citation Tools Depicting pathogenesis of osteomyelitis by single cell RNA-sequencing and an involvement of Morrbid in the autoinflammatory disease Qingran Huo , Jiayu Ding , Hongxi Zhou , Hang He , Lorie Chen Cai , Jingjing Liu , Ge Dong , Zhigang Cai bioRxiv 2025.01.23.634426; doi: https://doi.org/10.1101/2025.01.23.634426 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Immunology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)