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Single-Cell RNA-Sequencing Identifies Infrapatellar Fat Pad Macrophage Polarization in Acute Synovitis/Fat Pad Fibrosis and Cell Therapy.

ABSTRACT: The pathogenesis and progression of knee inflammatory pathologies is modulated partly by residing macrophages in the infrapatellar fat pad (IFP), thus, macrophage polarization towards pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes is important in joint disease pathologies. Alteration of M1/M2 balance contributes to the initiation and progression of joint inflammation and can be potentially altered with mesenchymal stem cell (MSC) therapy. In an acute synovial/IFP inflammation rat model a single intra-articular injection of IFP-MSC was performed, having as controls (1) diseased rats not receiving IFP-MSC and (2) non-diseased rats. After 4 days, cell specific transcriptional profiling via single-cell RNA-sequencing was performed on isolated IFP tissue from each group. Eight transcriptomically distinct cell populations were identified within the IFP across all three treatment groups with a noted difference in the proportion of myeloid cells across the groups. Largely myeloid cells consisted of macrophages (>90%); one M1 sub-cluster highly expressing pro-inflammatory markers and two M2 sub-clusters with one of them expressing higher levels of canonical M2 markers. Notably, the diseased samples (11.9%) had the lowest proportion of cells expressing M2 markers relative to healthy (14.8%) and MSC treated (19.4%) samples. These results suggest a phenotypic polarization of IFP macrophages towards the pro-inflammatory M1 phenotype in an acute model of inflammation, which are alleviated by IFP-MSC therapy inducing a switch towards an alternate M2 status. Understanding the IFP cellular heterogeneity and associated transcriptional programs may offer insights into novel therapeutic strategies for disabling joint disease pathologies.

SUBMITTER: Kouroupis D

PROVIDER: S-EPMC8615266 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTo investigate the in vitro and in vivo anti-inflammatory/anti-fibrotic capacity of IFP-MSC manufactured as 3D spheroids. Our hypothesis is that IFP-MSC do not require prior cell priming to acquire a robust immunomodulatory phenotype in vitro in order to efficiently reverse synovitis and IFP fibrosis, and secondarily delay articular cartilage damage in vivo.MethodsHuman IFP-MSC immunophenotype, tripotentiality, and transcriptional profiles were assessed in 3D settings. Multiplex secretomes were assessed in IFP-MSC spheroids [Crude (non-immunoselected), CD146+ or CD146- immunoselected cells] and compared with 2D cultures with and without prior inflammatory/fibrotic cell priming. Functionally, IFP-MSC spheroids were assessed for their immunopotency on human PBMC proliferation and their effect on stimulated synoviocytes with inflammation and fibrotic cues. The anti-inflammatory and anti-fibrotic spheroid properties were further evaluated in vivo in a rat model of acute synovitis/fat pad fibrosis.ResultsSpheroids enhanced IFP-MSC phenotypic, transcriptional, and secretory immunomodulatory profiles compared to 2D cultures. Further, CD146+ IFP-MSC spheroids showed enhanced secretory and transcriptional profiles; however, these attributes were not reflected in a superior capacity to suppress activated PBMC. This suggests that 3D culturing settings are sufficient to induce an enhanced immunomodulatory phenotype in both Crude and CD146-immunoselected IFP-MSC. Crude IFP-MSC spheroids modulated the molecular response of synoviocytes previously exposed to inflammatory cues. Therapeutically, IFP-MSC spheroids retained substance P degradation potential in vivo, while effectively inducing resolution of inflammation/fibrosis of the synovium and fat pad. Furthermore, their presence resulted in arrest of articular cartilage degradation in a rat model of progressive synovitis and fat pad fibrosis.Conclusions3D spheroids confer IFP-MSC a reproducible and enhanced immunomodulatory effect in vitro and in vivo, circumventing the requirement of non-compliant cell priming or selection before administration and thereby streamlining cell products manufacturing protocols.

Project description:BackgroundThe infrapatellar fat pad (IPFP) and synovium are reported to act as a functional unit, with emerging roles in the pathophysiology of knee osteoarthritis (KOA). Circular RNAs (circRNAs) are involved in the pathogenesis of KOA, especially in cartilage homeostasis regulation. Nevertheless, the regulatory mechanisms of circRNAs in the KOA IPFP/synovium unit remain to be elucidated. Therefore, the current study aimed to investigate alterations in the expression of circRNAs and predict their functions in the KOA IPFP/synovium unit using bioinformatics analysis.MethodsIn brief, 6 synovium and IPFP specimens were collected, in which 3 from patients with KOA and 3 from controls. Then, circRNA sequencing was conducted on 2 KOA synovium and IPFP specimens as well as 1 control to investigate the expression profiles of circRNAs. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome signaling pathway analyses were employed to predict the functions of the differentially expressed circRNAs. Based on the miRNA sponge theory, we constructed a circRNA-miRNA network to predict the molecular regulatory mechanism of these circRNAs. Venn analysis was performed to confirm the circRNAs and miRNAs expressed in both synovium and IPFP. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was also used to validate the expression levels of circRNAs that were co-expressed in both synovium and IPFP.ResultsA total of 65 and 72 circRNAs were differentially expressed in KOA synovium and IPFP, respectively (fold change ≥2, P<0.05). After obtaining the parental genes of differentially expressed circRNAs, the top 10 enrichment GO entries, KEGG pathways, and Reactome pathways were annotated. Furthermore, hsa_circ_0005265 was found to be down-regulated in both synovium and IPFP, which was validated by qRT-PCR. The circRNA-miRNA network was created to annotate the probable regulatory mechanisms of the differentially expressed circRNAs, which consequently confirmed 2 target miRNAs (hsa-miR-6769b-5p and hsa-miR-1249-5p) associated with hsa_circ_0005265 in both synovium and IPFP.ConclusionsOur outcomes bring us closer to understanding the potential mechanism of the IPFP/synovium unit in the progression of KOA and finding new molecular targets for KOA therapy.

Project description:BackgroundInterpretation of patellar ligament (PL) ultrasonography may be difficult, as hypoechoic or heterogenous echogenicity are common findings. Verifying suspected disease of equine PLs by histopathology is also problematic as descriptions of normal PL vascularity and histology are scarce. The current study describes the PL and infrapatellar fat pad (IFP) vascular pattern from computed tomography scans of barium perfused normal equine specimens (n = 8; age 10 days to 18 years), as well as routine histology to serve as a reference for future investigations into PL pathology and IFP disease.ResultsThe PLs received a bipolar blood supply. Vascular architecture consisted of numerous distinct longitudinal vessels with several horizontal connections, which branched into extensive latticeworks of smaller vessels throughout the ligaments. Several vascular connections between the PLs and the IFP were identified. One distinct longitudinal vessel was seen entering each of the IFP lobes at the distocranial aspect, branching extensively into lobar vascular networks which anastomosed by several horizontal branches at the mid portion of the IFP where the two lobes merge. Histologically, there were large variations in PL interfascicular endotenon thickness, vascularity and fatty infiltration; these parameters increased with age for the intermediate and medial PL. Areas of metaplastic tenocytes / chondroid metaplasia were identified in all investigated adult medial PLs; in 2/7 in the intermediate PL and in 4/7 in the lateral PL. The adult IFP consisted of white unilocular adipose tissue, organized in lobules separated by thin connective tissue septa increasing in thickness towards the periphery and the distocentral aspect.ConclusionsThe equine PLs and IFP are highly vascularized structures with ample vascular connections suggestive of crosstalk. This, together with the large variation in PL endotenon thickness, vascularity and fatty infiltration, should be taken into consideration when assessing potential PL histopathology as these changes increase with age and are found in horses without clinical signs of stifle disease. Metaplastic tenocytes / chondroid metaplasia should be considered a normal finding throughout the medial PL and is not age dependent. The role of the equine IFP in stifle disease has yet to be elucidated.

Project description:Background Infrapatellar fat pad (IPFP) quality has been implicated as a marker for predicting knee osteoarthritis (KOA); however, no valid quantification for subtle IPFP abnormalities has been established. Purpose To investigate whether MRI-based three-dimensional texture analysis of IPFP abnormalities could help predict incident radiographic KOA. Materials and Methods In this prospective nested case-control study, 690 participants whose knees were at risk for KOA were included from the Pivotal Osteoarthritis Initiative MRI Analyses incident osteoarthritis cohort. All knees had a Kellgren-Lawrence grade of 1 or less at baseline. During the 4-year follow-up, case participants were matched 1:1 to control participants, with incident radiographic KOA as the outcome. MRI scans were segmented at the incident time point of KOA (hereafter, P0), 1 year before P0 (hereafter, P-1), and baseline. MRI-based three-dimensional texture analysis was performed to extract IPFP texture features. Least absolute shrinkage and selection operator and multivariable logistic regressions were applied in the development cohort and evaluated in the test cohort. The area under the receiver operating characteristic curve (AUC) was used to evaluate the discriminative value of the clinical score, IPFP texture score, and MRI Osteoarthritis Knee Score. Results Participants were allocated to development (n = 500, 340 women; mean age, 60 years) and test (n = 190, 120 women; mean age, 61 years) cohorts. In both cohorts, IPFP texture scores (AUC ≥0.75 for all) showed greater discrimination than clinical scores (AUC ≤0.69 for all) at baseline, P-1, and P0, with significant differences in pairwise comparisons (P ≤ .002 for all). Greater predictive and concurrent validities of IPFP texture scores (AUC ≥0.75 for all) compared with MRI Osteoarthritis Knee Scores (AUC ≤0.66 for all) were also demonstrated (P < .001 for all). Conclusion MRI-based three-dimensional texture of the infrapatellar fat pad was associated with future development of knee osteoarthritis. ClinicalTrials.gov registration no.: NCT00080171 © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Fischer in this issue.

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Single-Cell RNA-Sequencing Identifies Infrapatellar Fat Pad Macrophage Polarization in Acute Synovitis/Fat Pad Fibrosis and Cell Therapy.

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