Plant probiotics bacteria are live microbes that promote soil health and plant growth and build the stress-tolerant capacity to the plants. They benefit the plants by increasing nutrient absorption and release of stress-related phytohormones. These plant probiotic bacteria serve a better purpose to the plant when compared to chemical fertilizers. Use of chemical fertilizers such as arsenic and cadmium can lead to soil acidification and even release of harmful gases such as methane which further pollutes the environment.Corynebacterium spp., Bacillus spp., Lactobacillus spp., and Cytobacillus spp. The results were also examined using various bioinformatics tools for accuracy in their phylogenetic pattern.Different bacterial species were isolated from the agricultural fields of Tattiannaram, Telangana, and identified as the efficient rhizosphere bacteria with the essential qualities of plant growth promotion by evaluating the nitrogen-fixing ability on a selective media and various other methods. Upon the molecular characterization of the isolates, they were identified as The recognized species of plant probiotics have established roles in promoting plant growth and strengthening plant immunity. This research introduces an innovative methodology for evaluating and investigating recently identified bacterial isolates, focusing on their distinctive plant probiotic attributes. Through harnessing the potential of advantageous microorganisms and comprehending their interaction with plants and soil, our objective is to formulate inventive approaches to elevate crop productivity, enhance soil richness, and foster environmentally sustainable and robust agricultural methodologies. These characteristics exhibit promising potential for future incorporation into plant systems, fortifying growth and development, and underscoring their distinctive significance within the realm of agriculture.The online version contains supplementary material available at 10.1186/s43141-023-00615-5. Soil, the organic geological skin of the earth, is essential for life and habitat to an incredible microbial community. Microbes have mostly been regarded as stewards of the biosphere . Soil baBacilli and Pseudomonas being the most common genera reported [Plants interact with a range of microbes in both aerial and subsoil tissues, establishing associations that can be harmful (pathogenic), neutral, or advantageous to the host plant. Several studies have shown evidence of the ability of microbes to boost plant development in various cultivated species. One such criterion is the development of plant growth-promoting bacteria. Plant growth-promoting bacteria (PGPB) are one of the most generalized findings of plant probiotic microbes (PPM) in soil, having reported –17. Planreported . Upon coreported . Severalreported .Fusarium oxysporum is treated by Bacillus subtilisssp [Probiotics are living microbial species utilized to improve the health and vitality of the host. Microorganisms employed for probiotic purposes can be found in nature, but they must be correctly identified, isolated, and studied for virulence . Plant ptilisssp .. Plant tilisssp , 27.Haas and Keel coined the term plant probiotic bacteria (PPB) to describe a group of microorganisms that benefit plants and meet three necessary attributes that when combined result in improved plant protection: (i) niche colonization effectiveness and competitiveness, (ii) ability to induce systemic resistance (ISR) in their hosts, and (iii) existence of specific antagonistic traits on pathogens .Agriculture and forestry are constantly being impacted by increasing population, soil degradation, environmental degradation, and global warming . This haIn long-term agricultural production, nitrogen fertilizers are frequently used to boost early crop output. Long-term administration of nitrogen fertilizers, on the other hand, can alter the plant soil microbe system by altering the composition of vegetation and soil microbial populations –36. NitrThe entire methodology followed during the work is represented in Fig. Soil samples were gathered from the Tattiannaram fields in Telangana, India Fig. . TattianThe rhizosphere is considered an important plant nutrient source where most of the microbes make up this as a resident club. The first step in developing a producer strain is the isolation of concerned microorganisms from their natural habitats. A set of highly selective procedures which allows the detection and isolation of microbes producing the desired metabolite constitute primary screening. However, this is possibly the most critical step since it eliminates the bulk of unwanted useless isolates, either nonproducers or producers of known compounds. As a part of the primary screening technique, the isolation of the bacteria was done using basic lab equipment and certain media. To analyze for the various microbes within the collected soils, the basic medium such as nutrient agar was used, believing that majority of microbes can be screened. Each soil sample was serially diluted and plated as per the standard protocols of microbial isolation techniques. After the growth observed on basic media, the isolates with different morphological features were subcultured and further tested for biochemical properties. The biochemical properties were analyzed by performing IMViC tests, catalase, coagulase, oxidase, starch hydrolysis, organic acid production tests and blood haemolysis test, etc. In detail, the IMViC test helps to narrow down the group of bacteria and provide information about their metabolic capabilities. Plant probiotic bacteria often produce organic acids through their metabolic processes. These organic acids can help in solubilizing minerals, making them more available to plants. The ability to produce organic acids like citric acid and malic acid can be a desirable trait in plant probiotic bacteria. The purpose of performing various biochemical tests was tabulated in Table 2PO4 0.1%, MgSO4.7H2O 0.05%, NaCl 0.05%, FeSO4 0.01%, Na2Mo 0.0005%, CaCO3 0.2%, agar 1.5%). First, the isolates were inoculated on solid medium. Later to estimate their growth levels, the isolates were re-examined on liquid medium. This growth was analyzed for 24 h and 48 h by colorimetric analysis at 600 nm. Bacteria that thrive on Jensen’s media might have the ability to fix nitrogen, making them potentially beneficial for plants. The organisms that have shown positive with Jensen’s media were further examined if they can also utilize and tolerate to different chemical exposures such as zinc, phosphorus, and iron using modified zinc solubilizing medium , modified phosphate-solubilizing medium 2 0.02%, MgSO4.7H2O 0.010%, FeSO4 0.00001%, pH 7), and modified iron-solubilizing media , respectively. The growth rate was analyzed by measuring the OD  which is set to 540 nm (nanometers) and 580 nm using a colorimeter. Plants often face challenges in accessing essential nutrients like zinc, iron, and phosphorus due to their limited solubility in the soil. Bacteria capable of solubilizing these minerals can enhance nutrient availability for plants. The organisms which have shown good growth rate and minimum standard deviation upon all the triplicate values were selected as the best isolates and moved for further examination. The selected isolates were inoculated to Ashby’s liquid medium  to confirm the nitrogen-fixing property [Later, the studies were carried out to screen out the nitrogen-fixing property using certain specific media such as Jensen’s medium ”), inputting the 16S rRNA sequence into the query box, adjusting parameters, and subsequently initiating the BLAST search . SubsequLater, the bootstrapping technique was employed as it is crucial for making well-informed evolutionary interpretations, as it addresses the inherent noise in genetic data and the complexities of evolutionary processes . Using tUpon screening the isolates by considering the basic traits, a total of 101 isolates were found initially. Colony morphological testing, gram staining, and biochemical tests  were performed. The organisms with similar traits were re-examined and eliminated. For the second stage of screening, 36 isolates were selected. The selected isolates were highlighted in Table Later, these 36 isolates were subjected to test their nitrogen-fixing capacity. Jensen’s media was used to detect the presence of nitrogen-fixing bacteria. This media was prepared in broth consistency and inoculated with all the selected 36 isolates. All the isolates were grown as triplicates, and the average of all the readings was tabulated as the results in Table The seven best isolates which proved their ability of nitrogen fixation was further moved to examination such as zinc solubilizers, phosphate solubilizers, and zinc solubilizers. The result was tabulated in Table The isolates that shown best within chemical analysis tests were further examined using Ashby’s nitrogen liquid medium in order to compare their main purpose to be a plant probiotic. Surprisingly, the isolate nos. KL-015, KL-076, and KL-089 have shown positive results indicating their potentiality for nitrogen fixation. Along with these isolates, remaining screened out isolates were subjected to the exposure to liquid medium of Ashby’s. The isolate which had shown growth in liquid medium  technique. Based on the gel amplicon picture, the isolates have shown the bands at app. 1000 kb Fig. . The posCTGCACTTCGGGATAAGCTTGGGAAACTGGGTCTAATACCGGATAGGAACCATCTTTAGTGTGATGGTTGGAAAGTTTTTTCGGTGTAGGATGAGCTCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGGCGACGGGTAGCCGGCCTGAGAGGGTGTACGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGGGGGATGAAGGCCTTCGGGTTGTAAACTCCTTTCGCTAGGGACGAAGCTTTTTGTGACGGTACCTAGATAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCTCGTAGGTGGTTTGTCGCGTCGTCTGTGAAATTCTGGGGCTTAACTCCGGGCGTGCAGGCGATACGGGCATAACTTGAGTGCTGTAGGGGTAACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTTACTGGGCAGTTACTGACGCTGAGGAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGGTGGGCGCTAGGTGTGAGGGTCTTTCTACGACTTTCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGG.AGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTATC.ACCCGCGGTGCATTAGCTAGTTGGTAGGGTAAAGGCCTACCAAGGCATTGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAAGATAGAGAGAGTAACTGATCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAAGATAAGTCTGATGTGAAAGCCCTCGGCTCAACCGAGGAACTGCATCGGAAACTGTCTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGCGCAATCCCAAGAGATTGGGAGTTCCCTTCGGGGACGCTAAGACAGG.GGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTCAGGGAAGAACAAGTACCGGAGTAACTGCCGGTACCTTGACGGTACCTGACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTCCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTCCTGACAACCCTAGAGATAGGGCGTTCCCCTTCGGGGGACAGGATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAA.The above sequences were used to run the BLAST separately, and later on, the top 10 sequences were used to construct the cladogram using Clustal Omega tool as shown in Fig. To select a bacteria as a potent plant probiotic, it must be beneficiary to humans also as the end result of this concept is growth of a healthy plant. As we know, the greatest ecological and agronomical advantages derived by plants from their contact with microbes are biological nitrogen fixation by bacteria. It is believed that the bacterial isolates which were finally identified within this work are the one which can convert the atmospheric nitrogen to ammonia using its nitrogenase enzyme, thus making it available for plant absorption. The presence of the nitrogenase enzyme decides the plant’s maturity and adds economic value too. The bacteria may also be capable to utilize various chemicals such as zinc which stimulates enzymes that are involved in the creation of some proteins. It aids in the production of chlorophyll and certain carbohydrates, the transformation of starches to sugars, and its availability in plant tissue aids in the plant’s resistance to low temperatures . Zinc is“Plant probiotics” is an emerging and intriguing concept within the realm of agriculture and plant science. This concept revolves around the idea of using beneficial microorganisms to enhance plant health and growth. This innovative approach delves into the analysis of plant immunity as well, aiming to better understand how these microorganisms can interact with plants’ natural defense mechanisms. Numerous studies have drawn attention to the significant role that plant probiotics can play. These studies have identified specific microorganism isolates that have demonstrated particularly promising results. These promising isolates are then subjected to a thorough screening process to assess their potential. This assessment involves employing techniques such as biopriming, which entails treating seeds with these beneficial microorganisms to facilitate their growth and establishment in the plant’s environment. Moreover, we are simultaneously working on optimizing the growth conditions and parameters for these beneficial microorganisms. This involves creating an environment that allows these microorganisms to thrive and interact harmoniously with the plant. The ultimate goal is to develop a technique that can reliably enhance plant health and performance. One aspect of research in this area focuses on investigating the specific compositions of microbial communities in the soil. Different combinations of microorganisms can have varying effects on soil fertility and subsequently impact the development of plants that grow in that soil. Through careful analysis, researchers seek to uncover the intricate relationships between these microorganisms and the overall health of both the soil and the plants. Early findings suggest that these microbial communities possess an adaptive capability, allowing them to adjust and thrive under different chemical conditions present in the soil. This adaptability opens up avenues to cultivate these microorganisms into potent plant probiotics that can potentially withstand a range of environmental challenges. In summary, the emerging field of plant probiotics holds immense promise for revolutionizing agriculture and plant cultivation.Additional file 1. BLASTN Results.


Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip. Summary: Myosin-X transports lamellipodin (RAPH1) to filopodia tips to regulate filopodia functions. Cell migration is essential during embryonic development, immune surveillance and wound healing. Misregulation of cell migration is implicated in multiple diseases, including inflammation and cancer. One hallmark of cell motility is a high degree of plasticity, allowing cells to adopt different morphologies and migration modes . A shareFilopodia are small and dynamic finger-like actin-rich protrusions (1–5 µm in length and 50–200 nm in width) and are often the first point of contact between a cell and its immediate surroundings. Filopodia contain cell surface receptors, such as integrins, cadherins and growth factor receptors, interacting with and interpreting various extracellular cues. Filopodia assembly is primarily driven by the linear polymerization of actin filaments with their barbed ends facing the plasma membrane . These fHere, we set out to identify novel MYO10 cargo molecules. Using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10-binding partner. Using structured illumination microscopy, we report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips; thus, RAPH1 is likely an MYO10 cargo. We map the RAPH1 MYO10-binding site to a previously uninvestigated RAPH1 sequence, and demonstrate that RAPH1 is a critical positive regulator of filopodia formation and stability in cells. Our results indicate that, in filopodia, RAPH1 is not required for integrin activation. Instead, RAPH1 regulates MYO10 filopodia formation and stability.FERM, or GFP–TLN1FERM , followed by mass spectrometry analysis , the main cargo-binding site in MYO10 . We perfanalysis A,B. TLN1analysis . We idenanalysis . Interesanalysis .FERM and that RAPH1 is biotinylated in cells expressing GFP–MYO10–BioID .RAPH1 is a member of the Mig-10/RIAM/lamellipodin (MRL) protein family, with MIG-10 being the of RAPH1 . RAPH1 wof RAPH1 , but itsof RAPH1 . In addiof RAPH1 ; Movie 1ΔF) in cells  within RAPH1. RAPH1 comprises several conserved domains, including a Ras-association (RA) and a pleckstrin homology (PH) domain. RAPH1 also contains known profilin-, VASP- and multiple putative SH3-binding sites A. FurtheΔF filopodia  showed that this construct could accumulate at the tip of MYO10-containing filopodia  MYO10 is required to target RAPH1 to filopodia tips, and (2) the MYO10–RAPH1 interaction contributes to formation of filopodia containing MYO10. We propose that MYO10 transports RAPH1 to filopodia tips, contributing to filopodia stability via yet unknown mechanisms, possibly involving RAPH1 interactions with other proteins such as VASP. However, our data do not fully exclude the possibility that RAPH1 simply diffuses to filopodia and that MYO10 only contributes to RAPH1 accumulation at filopodia tips without direct transport. Testing this would require performing two-color single-molecule imaging of MYO10 and RAPH1 to see whether these proteins move toward filopodia tips together. However, we find that RAPH1 is not very abundant in filopodia when the MYO10 FERM domain is missing, suggesting that RAPH1 is likely to be actively transported by MYO10.RAPH1 is presumably in a complex with MYO10, VASP and actin at filopodia tips. In this scenario, MYO10 could tether RAPH1 to filopodia tips using its motor domain, providing resistance against the retrograde actin flow in filopodia . Once tein vivo.Interestingly, both MYO10 and RAPH1 have been implicated separately as positive regulators of cancer cell migration and invasion in similar contexts . In addi−1 final concentration), and sorted for green fluorescence using a fluorescence-assisted cell sorter (FACS). All cell lines tested negative for mycoplasma. Cells were not authenticated.U2-OS osteosarcoma cells and MDA-MB-231 cells were grown in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FCS) . U2-OS cells were purchased from DSMZ . MDA-MB-231 cells were provided by ATCC. The U2-OS MYO10-GFP lines were generated by transfecting U2-OS cells using Lipofectamine 3000 (Thermo Fisher Scientific), selected using geneticin , 1:1000], anti-His tag , and anti-tubulin . Rabbit polyclonal antibodies used in this study were anti-RAPH1 , anti-MYO10 , and anti-GFP . Biotinylated proteins were detected using Streptavidin conjugated with Alexa Fluor™ 555  or Alexa Fluor™ 647 , both provided by Thermo Fisher Scientific (S21381 and S21374). The bovine plasma fibronectin was provided by Merck (341631). DAPI  was provided by Thermo Fisher Scientific (D1306).U2-OS and MDA-MB-231 cells were transfected using Lipofectamine 3000 and the P3000TM Enhancer Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.47608 . The mScarlet-I-MYO10ΔF (MYO10ΔF-RFP) construct was previously described (145139). The GFP-VASP  plasmid was Addgene plasmid #54297 (deposited by Michael Davidson). The GFP-RIAM(1-666) construct was Addgene plasmid #80028  plasmid was Addgene plasmid #escribed  and is aThe GFP–MYO10–BioID construct was generated as follows. Flanking XbaI sites were introduced into BioID by PCR  and the resulting amplicon was then inserted into a unique XbaI site in the EGFPC1-hMyoX plasmid resulting in an EGFP-MYO10-(stop codon)-BioID fusion gene. The stop codon between MYO10 and BioID was then replaced with a codon encoding valine (GTA) using a quick-change mutagenesis kit from Agilent and following the manufacturer's instructions.ΔTBS [RAPH1 with amino acids (aa) 2–92 deleted] construct was created by inserting a custom gene block (IDT) in the EGFP-Lpd plasmid using the XhoI/HindIII sites. The sequence of the gene block is provided below:The GFP–RAPH15′-ATTAGACTCGAGCCGCGATGTGCTCTATAGAGCAGGAGCTCAGCAGCATTGGTTCAGGAAACAGTAAGCGTCAAATCACAGAAACGAAAGCTACTCAGAAATTGCCTGTTAGCCGACATACATTGAAACATGGCACCTTGAAAGGATTATCTTCTTCATCTAATAGGATAGCTAAACCTTCCCATGCCAGCTACTCCTTGGACGACGTCACTGCACAGTTAGAACAGGCCTCTTTGAGTATGGATGAGGCTGCTCAGCAATCTGTACTAGAAGATACTAAACCCTTAGTAACTAATCAGCACAGAAGAACCGCGTCAGCAGGCACAGTGAGTGATGCTGAAGTACACTCTATTAGTAATTCCTCCCATTCCAGCATCACTTCCGCAGCCTCCAGCATGGACTCTTTGGATATTGATAAAGTAACACGCCCTCAAGAGCTGGATTTGACACATCAAGGGCAGCCAATTACTGAGGAAGAACAGGCAGCAAAATTGAAAGCTGAGAAGATCAGAGTTGCCCTAGAGAAAATTAAAGAGGCACAAGTGAAAAAGCTGGTGATCAGAGTCCACATGTCTGATGACAGTTCTAAAACAATGATGGTGGATGAGAGGCAGACAGTAAGACAAGTACTGGATAACCTGATGGACAAATCCCACTGCGGTTATAGTTTAGACTGGTCACTGGTAGAAACCGTTTCTGAATTACAAATGGAGAGAATCTTTGAAGACCATGAAAACTTGGTTGAAAATCTTCTTAATTGGACAAGAGATAGCCAAAACAAGCTTATTAGA-3′.F2 construct (RAPH1 aa 535–868) was purchased from GenScript. The gene fragment was synthesized using gene synthesis and cloned into pGEX-4T-1 using the BamHI/XhoI sites.The RAPH1 fragments F1 (RAPH1 aa 1–535), F2 (RAPH1 aa 535–868), F3 (RAPH1 aa 868–1062), F4 (RAPH1 aa 1062–1250) and F5 (RAPH1 aa 536–587) constructs were purchased from GenScript. Briefly, the gene fragments were synthesized using gene synthesis and cloned into pcDNA3.1(+)-N-eGFP using the BamHI/XhoI sites. The GST–RAPH1Δ536-587 (RAPH1 aa 536-587 deleted) construct was created by inserting a custom gene block (IDT) in the EGFP-Lpd plasmid using the HindIII/KpnI sites. The sequence of the gene block is provided below:The GFP–RAPH15′-AAGCTTATATTTATGGAGCGTATAGAAAAATATGCACTTTTCAAAAACCCACAGAATTATCTTTTGGGGAAAAAGGAAACAGCTGAGATGGCAGATAGAAACAAAGAAGTCCTCTTGGAGGAATGTTTTTGTGGAAGTTCTGTAACTGTACCAGAAATTGAAGGAGTCCTTTGGTTGAAGGATGATGGCAAGAAGTCCTGGAAAAAGCGTTATTTTCTCTTGCGAGCATCTGGTATCTACTATGTTCCCAAAGGAAAAGCAAAGGTCTCTCGGGATCTGGTGTGCTTTCTCCAGCTGGATCATGTCAACGTTTATTATGGCCAGGACTATCGGAACAAATACAAAGCACCTACAGACTATTGTCTGGTGCTGAAGCATCCACAAATCCAGAAGAAATCTCAATATATCAAATACCTTTGTTGTGATGATGTGAGGACACTGCATCAGTGGGTCAATGGGATCCGCATTGCAAAGTATGGGAAGCAGCTCTATATGAACTACCAAGAAGCCTTGAAGAGGACAGAGTCAGCCTATGATTGGACTTCCTTATCCAGCTCCAGCATTAAATCGGAAGAGTCCAGCAAGGCCAGAATGGAGTCTATGAATCGGCCCTACACTTCACTTGTGCCCCCTTTATCCCCGCAACCTAAGATAGTCACCCCCTACACTGCTTCACAGCCTTCACCACCTCTACCTCCTCCGCCACCCCCACCTCCTCCTCCACCACCCCCTCCACCACCCCCTCCTCCCCCACTCCCCAGCCAGTCTGCACCTTCTGCAGGCTCAGCAGCCCCAATGTTCGTCAAGTACAGCACAATAACACGGCTACAGAATGCGTCTCAGCATTCAGGGGCCCTGTTTAAGCCGCCAACACCCCCAGTGATGCAGTCACAGTCAGTGAAGCCTCAGATCCTGGTACC-3′.The expression of RAPH1 was suppressed using 83 nM siRNA and Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions. siRNAs used were AllStars Negative siRNA control (cat. no. 1027418), RAPH1 siRNA #2  and RAPH1 siRNA #5  provided by Qiagen.Protein extracts were separated under denaturing conditions by SDS-PAGE and transferred to nitrocellulose membranes using a Mini Blot Module . Membranes were blocked for 30 min at room temperature using 1× StartingBlock buffer . After blocking, membranes were incubated overnight with the appropriate primary antibody (1:1000 in blocking buffer), washed three times in PBS, and probed for 1 h using a fluorophore-conjugated secondary antibody diluted 1:5000 in the blocking buffer. Membranes were washed three times using PBS over 30 min and scanned using an iBright FL1500 imaging system (Invitrogen).g for 5 min at 4°C. Clarified lysates were incubated with GFP-Trap magnetic or agarose beads for 1 h at 4°C. Complexes bound to the beads were isolated by centrifugation, washed three times with ice-cold lysis buffer, and eluted in Laemmli reducing sample buffer for 10 min at 95°C.Cells transiently expressing bait GFP-tagged proteins were lysed in a buffer containing 20 mM HEPES pH 7.4, 75 mM NaCl, 2 mM EDTA, 1% NP-40, as well as a cOmplete™ protease inhibitor tablet , and a phosphatase inhibitor mix (Roche cat. no. 04906837001). Lysates were then centrifuged at 15,000 Escherichia coli strain  was transformed with plasmids encoding the relevant His-tagged or GST-tagged proteins. Bacteria were grown at 37°C in LB medium supplemented with ampicillin . Protein expression was induced with IPTG  at 20°C. After 5 h, bacteria were harvested by centrifugation (20 min at 6000 g) and resuspended in resuspension buffer . Bacteria were then lysed by adding BugBuster  and a small spoonful of lysozyme . The suspension was mixed at 4°C for 30 min. Cell debris were then pelleted by ultracentrifugation  at 4°C for 1 h. His-tagged MYO10 FERM was purified using a Protino Ni-TED 2000 packed column  according to the manufacturer's instructions. The protein was eluted in multiple 1 ml fractions, supplemented with 1 mM AEBSF , and kept at 4°C until needed (for up to 1 week). For GST-tagged proteins, 600 µl of equilibrated glutathione–Sepharose 4B beads  was added to the supernatant and agitated for 1 h at 4°C. Beads were collected and washed four times with TBS supplemented with PMSF (1 mM). Protein-bound beads were stored at −80°C until needed.The BL-21(DE3) F2 Sepharose beads were incubated with 10 mM His-tagged MYO10FERM, and the mixture was rotated overnight at 4°C. Beads were then washed four times with TBS supplemented with 1 mM PMSF. Proteins bound to beads were then eluted in 2× Laemmli sample buffer at 80°C. Results were then analyzed by western blotting.GST and GST–RAPH1g, +4°C, 2 min). Biotinylated proteins were then incubated with streptavidin beads  for 1 h with rotation at +4°C. Beads were washed twice with 500 μl wash buffer 1 [10% (w/v) SDS], once with 500 μl wash buffer 2 , and once with 500 μl wash buffer 3 . Proteins were eluted in 40 μl of 2× reducing sample buffer  for 10 min at 90°C.U2-OS cells stably expressing GFP–MYO10 or GFP–MYO10–BioID were plated on fibronectin-coated plates in a medium containing 50 μM biotin for 24 h. After washing cells with cold PBS, cells were lysed, and debris were removed by centrifugation , gel lanes were sliced into five 2-mm bands. The slices were washed using a solution of 50% 100 mM ammonium bicarbonate and 50% acetonitrile until all blue color vanished. Gel slices were washed with 100% acetonitrile for 5–10 min and then rehydrated in a reducing buffer containing 20 mM dithiothreitol in 100 mM ammonium bicarbonate for 30 min at 56°C. Proteins in gel pieces were then alkylated by washing the slices with 100% acetonitrile for 5–10 min and rehydrated using an alkylating buffer of 55 mM iodoacetamide in 100 mM ammonium bicarbonate solution . Finally, gel pieces were washed with 100% acetonitrile, followed by washes with 100 μl 100 mM ammonium bicarbonate, after which slices were dehydrated using 100% acetonitrile and fully dried using a vacuum centrifuge. Trypsin  was used to digest the proteins (37°C overnight). After trypsinization, an equal amount of 100% acetonitrile was added, and gel pieces were further incubated at 37°C for 15 min, followed by peptide extraction using a buffer of 50% acetonitrile and 5% formic acid. The buffer with peptides was collected, and the sample was dried using a vacuum centrifuge. Dried peptides were stored at −20°C. Before liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis, dried peptides were dissolved in 0.1% formic acid. The LC-ESI-MS/MS analyses were performed on a nanoflow HPLC system  coupled to the Orbitrap Fusion Lumos mass spectrometer  equipped with a nano-ESI source. Peptides were first loaded on a trapping column and subsequently separated inline on a 15 cm C18 column . The mobile phase consisted of water with 0.1% formic acid (solvent A) and acetonitrile/water [80:20 (v/v)] with 0.1% formic acid (solvent B). Peptides were eluted with 40 min method: from 8% to 43% of solvent B in 30 min, from 43% to 100% solvent B in 2 min, followed by a wash for 8 min at 100% of solvent B. MS data was acquired automatically by using Thermo Xcalibur 4.4 software (Thermo Fisher Scientific). A data-dependent acquisition method consisted of an Orbitrap MS survey scan of mass range 350–1750 Raw data from the mass spectrometer were submitted to the Mascot search engine using Proteome Discoverer 1.5 (Thermo Fisher Scientific). The search was performed against the human database SwissProt_2021_02, assuming the digestion enzyme trypsin, a maximum of two missed cleavages, an initial mass tolerance of 10 ppm (parts per million) for precursor ions, and a fragment ion mass tolerance of 0.020 Dalton. Cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation was set as a variable modification.FERM and TLN1FERM datasets, two biological replicates were combined. Proteins enriched at least twofold in MYO10FERM over GFP and over TLN1FERM  and detected with more than ten spectral counts (across both repeats) were considered putative MYO10-binding proteins. The fold-change enrichment and the significance of the association used to generate the volcano Plots  and detected with over five spectral counts  were considered putative MYO10-binding proteins. To generate the MYO10no Plots A,B were The spinning-disk confocal microscope used was a Marianas spinning-disk imaging system with a Yokogawa CSU-W1 scanning unit on an inverted Zeiss Axio Observer Z1 microscope controlled by SlideBook 6 . Images were acquired using either an Orca Flash 4 sCMOS camera  or an Evolve 512 EMCCD camera . The objective used was a 100× oil  objective.The structured illumination microscope (SIM) used was DeltaVision OMX v4  fitted with a 60× Plan-Apochromat objective lens, 1.42 NA (immersion oil RI of 1.516) used in SIM illumination mode (five phases×three rotations). Emitted light was collected on a front-illuminated pco.edge sCMOS  controlled by SoftWorx.The confocal microscope used was a laser scanning confocal microscope LSM880 (Zeiss) equipped with an Airyscan detector (Carl Zeiss) and a 40× water (NA 1.2) or 63× oil (NA 1.4) objective. The microscope was controlled using Zen Black (2.3), and the Airyscan was used in standard super-resolution mode.For the filopodia formation assays, cells were plated on fibronectin-coated glass-bottom dishes (MatTek Corporation) for 2 h. Samples were fixed for 10 min using a solution of 4% PFA, then permeabilized using a solution of 0.25% (v/v) Triton X-100 for 3 min. Cells were then washed with PBS and quenched using a solution of 1 M glycine for 30 min. Samples were then washed three times in PBS and stored in PBS containing SiR-actin  at 4°C until imaging. Just before imaging, samples were washed three times in PBS. Images were acquired using a spinning-disk confocal microscope (100× objective). The number of filopodia per cell was manually scored using Fiji .2. Filopodia lifetimes were then measured by identifying and tracking all MYO10 spots using the Fiji plugin TrackMate . All live-cell imaging experiments were performed in normal growth medium, supplemented with 50 mM HEPES, at 37°C and in the presence of 5% COrackMate . In TracrackMate . The traU2-OS cells transiently expressing the constructs of interest were plated on high tolerance glass-bottom dishes  pre-coated first with poly-L-lysine  and then with bovine plasma fibronectin . After 2 h, samples were fixed and permeabilized simultaneously using a solution of 4% (w/v) PFA and 0.25% (v/v) Triton X-100 for 10 min. Cells were then washed with PBS, quenched using a solution of 1 M glycine for 30 min, and, when appropriate, incubated with the primary antibody for 1 h (1:100). After three washes, cells were incubated with a secondary antibody for 1 h (1:100). Samples were then washed three times and incubated with SiR-actin  at 4°C until imaging . Just before imaging, samples were washed three times in PBS and mounted in Vectashield (Vector Laboratories).To map the localization of each protein within filopodia, images were first processed in Fiji , and datThe preferential recruitment of protein to filopodia tips or shafts was assessed by calculating an enrichment ratio where the averaged intensity of the signal at the filopodia tip (bin 1–6) was divided by the averaged intensity at the filopodia shaft (bin 7–40).https://huygens.science.uva.nl/PlotsOfDifferences/) (https://github.com/guijacquemet/FiloMAP).Randomization tests were performed using the online tool PlotsOfDifferences (rences/)  preprintrences/) . Volcanorences/) . Superplrences/) . All numClick here for additional data file.10.1242/joces.260574_sup1Supplementary informationClick here for additional data file.


The treatment of chronic inflammation with systemically administered anti-inflammatory treatments is associated with moderate-to-severe side effects, and the efficacy of locally administered drugs is short-lived. Here we show that inflammation can be locally suppressed by a fusion protein of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO) and galectin-3 . Gal3 anchors IDO to tissue, limiting the diffusion of IDO-Gal3 away from the injection site. In rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis, the fusion protein remained in the inflamed tissues and joints for about 1 week after injection, and the amelioration of local inflammation, disease progression and inflammatory pain in the animals were concomitant with homoeostatic preservation of the tissues and with the absence of global immune suppression. IDO-Gal3 may serve as an immunomodulatory enzyme for the control of focal inflammation in other inflammatory conditions. An anti-inflammatory enzyme fused with a tissue-anchoring protein and injected into inflamed tissues ameliorates local inflammation without causing systemic immune suppression, as shown in multiple rodent models of inflammatory diseases. A major challenge in the treatment of chronic inflammatory diseases is the development of therapeutics to safely and specifically direct resolution of inflammation in a site-specific manner1. Anti-inflammatory drugs such as glucocorticoids are pleiotropic, non-specifically affecting numerous pathways1 and are consequently accompanied by issues of toxicity, resistance and a wide array of serious adverse effects such as infection and defective wound healing3. Additionally, systemic immune modulation leads to disease states such as hypertension, osteoporosis, obesity, cataracts and diabetes3. Biologic immunosuppressive drugs functioning through either cytokine blockade, cell depletion or cell surface receptor blockade provide improved specificity and can effectively modulate immune responses to halt disease progression in certain, but not all patients4. However, such treatments can also increase susceptibility to infections and disrupt tissue homoeostasis, leading to cancer, exacerbation of congestive heart failure and neurologic events, among other pathologies4. Critically, each of these therapeutics require life-long continual use and clinical options to resolve chronic inflammation and restore tissue homoeostasis remain to be developed5.Chronic inflammation, characterized by professional immune cell and resident tissue cell interactions, irreversibly damages tissues of the body and is an associated risk factor for a host of diseases such as cardiovascular disease, diabetes and cancer6. Catabolism of the essential amino acid tryptophan (Trp) by the cytosolic enzyme indoleamine 2,3-dioxygenase 1 (IDO) and the resultant production of kynurenine metabolites is a general regulator of inflammation in response to sterile and pathogenic inflammatory stimuli, acting on both innate and adaptive immune cells8. IDO catabolism of Trp is also a contributing factor to promoting fetal tolerance in pregnancy, warding off autoimmunity and avoiding immune elimination in some forms of cancer9. Trp insufficiency via IDO activates the metabolic stress sensor general control nonderepressible 2 (GCN2) to regulate immune cell cycle10, while kynurenine pathway metabolites activate anti-inflammatory programmes, such as kynurenine binding to the aryl hydrocarbon receptor (AHR)11. IDO expression in immune cells such as macrophages and dendritic cells has been demonstrated to suppress T cell activation and proliferation while activating and promoting phenotypic maintenance of regulatory T cells14. Additionally, the terminal product of the kynurenine pathway, nicotinamide adenine dinucleotide (NAD+), regulates innate immunity function in macrophages during aging and inflammation15. In total, actions of IDO serve as a key mechanism maintaining homoeostasis, suppressing autoimmunity and shutting down excess inflammation10. Informed by these data, we envisioned therapeutic delivery of exogenous IDO as a regulator of chronic inflammatory diseases.The capability to direct cellular metabolism to programme immune responses has recently emerged as a new avenue for therapeutic immunomodulation19, we recently demonstrated the utility of model enzymes fused to galectin-3 , a carbohydrate-binding protein, as a generalizable means to restrict enzyme diffusion via binding to tissue glycans20. Gal3 binds N-acetyllactosamine and other β-galactoside glycans, as well as glycosaminoglycans, which are highly abundant in mammalian tissues22, collectively representing a more universal target than a specific extracellular matrix protein. Thus, Gal3 fusion to enzymes represents a promising approach to retain local enzymatic function at an intended site of action. Building upon this success, we engineered the IDO-Gal3 fusion with the expectation of creating a tissue-anchored IDO administered as a localized anti-inflammatory therapeutic  to serve as positive feedback to further elevate expression9, endogenous Ido1 transcripts were quantified after treatment with IDO-Gal3 in both the LPS and psoriasis models  could also potentially contribute to the resolution of inflammation. In sum, s.c. administration of IDO-Gal3 ameliorated skin inflammation both prophylactically and therapeutically, using two different inflammatory insults acting through different specific receptors (CD14/TLR4 for LPS and TLR7 for imiquimod)26.Given the capacity for endogenous cellular IDO gene expression  and hard (bone) tissue destructionl29 Fig. , submandl29 Fig. . In vivol29 Fig. , whereasl29 Fig. . Retentil29 Fig. . The pril29 Fig. . Specifil29 Fig.  and thicl29 Fig. , were prl29 Fig.  and IL-1l29 Fig. , and chel29 Fig. . In contl29 Fig. , wherebyl29 Fig. , where tl29 Fig. . IDO-Gall29 Fig. . Further31 was utilized to test for the ability of IDO-Gal3 to reduce load-induced inflammatory joint damage. Intra-articular injection of IDO-Gal3 was locally retained, suppressed inflammation and spared joint tissue destruction , a form of osteoarthritis (OA), commonly arises after a ligament or meniscus tear, or following repeated overloading of a joint. A cyclic mechanical overloading PTOA mouse modelion Fig. , with meion Fig.  and day ion Fig.  and quanion Fig.  by ex viion Fig. . Weekly ion Fig.  and draiion Fig. , whereasion Fig. . Criticaion Fig.  and artiion Fig. . Additioion Fig.  and 20. We note that multiple injections of human IDO-Gal3 in mice led to the emergence of some anti-IDO-Gal3 antibodies 34. Intra-articular injection of IDO-Gal3 was locally retained, suppressed joint inflammation, reduced OA-associated pain and improved rat gait  vectors between NcoI and XhoI sites. IDO-Gal3 genetic and protein sequences are provided in E. coli (ThermoFisher), selected on Luria-Bertani (LB) ampicillin (50 μg ml−1) agar and incubated overnight at 37 °C. Isolated colonies from the plates were subcultured in 5 ml LB ampicillin broth overnight at 37 °C with orbital shaking. Successful transformants were confirmed by Sanger sequencing (Genewiz). Positive DNA sequences were then transformed into the kanamycin B resistant expression strain, Origami B (DE3) E. coli (Novagen) and selected on LB ampicillin with kanamycin B (15 μg ml−1) agar. Positive clones were picked, banked in LB with 10% w/v glycerol and used to preculture 5 ml of LB ampicillin and kanamycin. Overnight precultures were used to inoculate 1l 2× TY media  with ampicillin and kanamycin B at 37 °C and 225 r.p.m. in an orbital shaker until approximate exponential growth phase 600 nm = 0.6–0.8). IDO-Gal3 expression cultures were supplemented with 500 μM δ-Aminolevulinic acid (Sigma) at the time of inoculation. Recombinant protein expression was triggered using 0.5 mM isopropyl β-d-1-thiogalactopyranoside (ThermoFisher) and incubated for 18 h in an orbital shaker at 18 °C. Bacteria were washed and pelleted with PBS via centrifugation  with a superspeed centrifuge (ThermoFisher). Afterwards, the pellet was weighed, resuspended in 4 ml PBS per gram pellet with protease inhibitor (ThermoFisher) and disrupted by sonic dismembration . Following dismembration, lysates were treated with 20 units per gram pellet DNAse I (ThermoFisher) and 800 μg g−1 pellet lysozyme (ThermoFisher). The lysate was centrifuged  to remove the insoluble fraction and the supernatant was collected by decanting. Supernatant containing soluble recombinant protein was loaded into an α-lactose agarose (Sigma) affinity column pre-equilibrated with PBS. Columns were washed with 20 column volumes PBS and recombinant proteins were eluted with 100 mM β-d-lactose (Sigma) prepared in PBS. A final polishing step was performed by 200 kDa size exclusion chromatography on AKTA pure chromatography system (GE Life Sciences) to remove α-lactose and further isolate IDO-Gal3. Protein purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie staining. Endotoxin contaminants were removed by endotoxin removal solution (Sigma) following manufacturer instructions. Endotoxin content was analysed using Chromo-LAL kinetic chromogenic endotoxin quantification assay (Associates of Cape Cod) and determined to be below 0.1 EU ml−1 in all stocks.NanoLuc is the tradename of an engineered deep-sea shrimp luciferase variant developed by Promega CorporationE. coli was purchased from R&D Systems  as measured by its ability to oxidize l-tryptophan to N-formyl-kynurenine (NFK)). The specific activity of both proteins, IDO and IDO-Gal3, was measured before experiments to ensure maximal effect at the beginning of the assay, following the IDO manufacturer’s protocol. IDO-Gal3 was reacted in equimolar amounts to IDO in the standard protocol, and activities were compared using the unit pmol NFK min−1 pmol−1 IDO to more accurately compare activity.Recombinant human IDO expressed in α-lactose agarose affinity resin (Sigma-Aldrich). Proteins were eluted with a linear gradient of β-lactose (Sigma-Aldrich) in phosphate buffer.Affinity of IDO-Gal3 for lactose was determined using affinity chromatography in an AKTA Pure chromatography system (GE Life Sciences) equipped with consumer-packable glass column (GE Life Sciences) packed with CCATGGCGCACGCGATGGAAAACAGCTGGACCATCAGCAAAGAGTACCACATTGACGAGGAAGTTGGTTTCGCGCTGCCGAACCCGCAGGAAAACCTGCCGGACTTCTATAACGATTGGATGTTTATCGCGAAGCACCTGCCGGATCTGATTGAGAGCGGCCAGCTGCGTGAGCGTGTGGAAAAACTGAACATGCTGAGCATCGACCACCTGACCGATCACAAGAGCCAACGTCTGGCGCGTCTGGTTCTGGGTTGCATTACGATGGCGTACGTGTGGGGCAAAGGTCACGGCGACGTGCGTAAGGTTCTGCCGCGTAACATCGCGGTTCCGTACTGCCAACTGAGCAAGAAACTGGAACTGCCGCCGATTCTGGTGTATGCGGACTGCGTTCTGGCGAACTGGAAGAAGAAGGACCCGAACAAACCGCTGACCTATGAGAACATGGATGTGCTGTTCAGCTTTCGTGACGGTGATTGCAGCAAGGGCTTCTTTCTGGTGAGCCTGCTGGTTGAAATCGCGGCGGCGAGCGCGATCAAAGTGATTCCGACCGTTTTCAAGGCGATGCAGATGCAAGAGCGTGACACCCTGCTGAAAGCGCTGCTGGAAATCGCGAGCTGCCTGGAGAAGGCGCTGCAGGTGTTTCACCAAATTCACGATCACGTTAACCCGAAAGCGTTCTTTAGCGTGCTGCGTATCTACCTGAGCGGTTGGAAGGGCAACCCGCAGCTGAGCGACGGTCTGGTTTATGAGGGCTTCTGGGAAGATCCGAAAGAGTTTGCGGGTGGCAGCGCGGGTCAGAGCAGCGTGTTCCAATGCTTTGACGTTCTGCTGGGCATTCAGCAAACCGCGGGTGGCGGTCATGCGGCGCAGTTCCTGCAAGATATGCGTCGTTACATGCCGCCAGCGCACCGTAACTTCCTGTGCAGCCTGGAAAGCAACCCGAGCGTGCGTGAGTTTGTTCTGAGCAAAGGTGACGCGGGCCTGCGTGAAGCGTATGATGCGTGCGTGAAGGCGCTGGTTAGCCTGCGTAGCTACCACCTGCAGATCGTTACCAAATATATCCTGATTCCGGCGAGCCAGCAACCGAAAGAAAACAAGACCAGCGAGGACCCGAGCAAACTGGAGGCGAAGGGTACCGGCGGTACCGATCTGATGAACTTTCTGAAGACCGTGCGTAGCACCACCGAGAAGAGCCTGCTGAAAGAGGGTGGATCCGGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGAATTCGCGGACAACTTCAGCCTGCACGATGCGCTGAGCGGTAGCGGTAACCCGAACCCGCAGGGTTGGCCGGGTGCGTGGGGTAACCAACCGGCGGGTGCGGGTGGCTACCCGGGTGCGAGCTATCCGGGTGCGTATCCGGGTCAGGCTCCGCCGGGTGCGTACCCGGGCCAAGCTCCGCCGGGTGCTTATCCTGGTGCGCCGGGCGCGTACCCGGGTGCGCCGGCGCCGGGCGTGTACCCGGGTCCGCCGAGCGGTCCGGGCGCGTATCCGAGCAGCGGCCAGCCGAGCGCGCCGGGTGCGTATCCGGCGACCGGCCCGTATGGTGCGCCGGCGGGTCCGCTGATTGTTCCGTATAACCTGCCGCTGCCGGGTGGCGTGGTTCCGCGTATGCTGATCACCATTCTGGGCACCGTGAAGCCGAACGCGAACCGTATCGCGCTGGACTTCCAACGTGGTAACGATGTTGCGTTCCACTTTAACCCGCGTTTTAACGAGAACAACCGTCGTGTGATTGTTTGCAACACCAAACTGGACAACAACTGGGGCCGTGAGGAACGTCAGAGCGTGTTCCCGTTTGAGAGCGGCAAGCCGTTCAAAATTCAAGTGCTGGTTGAACCGGACCACTTTAAGGTGGCGGTTAACGATGCGCACCTGCTGCAGTACAACCACCGTGTTAAGAAACTGAACGAAATCAGCAAACTGGGCATCAGCGGTGACATTGATCTGACCAGCGCGAGCTATAACATGATTCTCGAG, with restriction sites underlined.The sequence used was GSGGGSGGSGGSGGEFADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSAPGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYNMILEHHHHHHMAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWMFIAKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLVLGCITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPILVYADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVSLLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKALQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGFWEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQDMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDACVKALVSLRSYHLQIVTKYILIPASQQPKENKTSEDPSKLEAKGTGGTDLMNFLKTVRSTTEKSLLKEG−1 lipopolysaccharide (LPS) in 40 μl, after 24 h or 120 h post IDO-Gal3 treatment, to assess local and distal modulation of inflammation. At 2 h after LPS administration, animals were euthanized and the injection site collected.B6 mice were administered 2.1 μg IDO-Gal3 in 40 μl PBS subcutaneously  at the region of the hock and challenged with 2 ng gβ, IFN-γ and IL-6) using Applied Biosystems QuantStudio 12K Flex Real-Time PCR System. Results are presented as the ratio of gene expression to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) expression determined by the relative quantification method. Treatment groups were normalized to PBS only group.Soft tissue was separated from bone and stored in RNAlater RNA stabilization reagent (Qiagen) in preparation for qPCR. Soft tissues were homogenized and RNA purified using RNeasy Protect mini kit (Qiagen). Complementary DNA was synthesized from RNA using the High-Capacity cDNA Reverse Transcriptase kit (ThermoFisher) for use in qPCR in accordance with manufacturer instructions. qPCR analysis was run with primers specific for pro-inflammatory cytokines  overnight. After fixation, tissues were washed in deionized water and decalcified by storing in 10% ethylenediaminetetraacetic acid (EDTA) at 4 °C for 3 weeks. Samples were assessed every 2–3 d for stiffness and EDTA solution replenished. Tissues were submitted in 70% ethanol to the University of Florida Molecular Pathology Core for processing, paraffin embedding, sectioning, mounting and staining with haematoxylin and eosin (H&E). Tissues were imaged using a Zeiss Axiovert 200M with a ×20 objective lens through the multidimensional acquisition module. Eleven images were taken per tissue and scored by two blinded independent individuals on the basis of cellular infiltration  and epidermis hypertrophy .n = 3). At 30 min after injection, the mice were euthanized, the hock and tibia tissue regions excised and flash frozen using liquid nitrogen. These samples were then submitted to the Southeast Center for Integrated Metabolomics at the University of Florida for mass spectrometric analysis of kynurenine levels.IDO-Gal3 (2.2 μg) in 40 μl PBS or 40 μl PBS alone was injected into the hock site of B6 mice  was collected in microtainer serum separator tubes and centrifuged at 1,500 r.p.m. for 5 min at room temperature. The serum was collected and stored at −20 °C. Cytokine analysis was performed using a Luminex 200 system running xPONENT 3.1 software (Luminex) following manufacturer instructions.37. Briefly, mice were anaesthetized and their backs were shaved, followed by application of depilatory cream to remove any remaining fur. Each day for 14 d total, 5% IMQ cream  was applied to the backs of the mice. On the 3rd day of IMQ application, mice were subcutaneously injected with five 10 μg doses of IDO-Gal3 in sterile saline distributed evenly throughout the back, a molar equivalent of NL-Gal3 delivered in the same volume, or a sterile saline control (n = 12 per group). Disease severity was measured each day using a modified version of the Psoriasis Area and Severity Index (PASI) where area of effect is not taken into account. Erythema (redness), scaling and thickening were scored independently and assigned a score on a scale of 0 to 4: 0, none, 1: slight, 2: moderate, 3: marked, 4: very marked. The cumulative score was reported as a measure of the severity of inflammation .Pre-clinical modelling of psoriasis was carried out in 8–12-week-old female C57BL/6j mice (Jackson Laboratory) in accordance with the Institutional Animal Care and Use Committee (IACUC) at the University of Florida. The model used was a modified version of a previously reported modelIn vivo imaging of fluorescently tagged IDO-Gal3 was carried out in 8–12-week-old female C57BL/6j mice (Jackson Laboratory) in accordance with the IACUC at the University of Florida. Before injection, IDO-Gal3 was incubated with IRDye 680RD NHS Ester  following manufacturer instructions. Mice received a 40 μl  subcutaneous injection of fluorescently labelled IDO-Gal3 to the hock. Immediately following injection and every subsequent 24 h following injection, mice were imaged using an IVIS Spectrum in vivo imaging system (PerkinElmer). Fluorescent images were captured using the AF680 emission filter, subject size 1.5 cm, 0.2 s exposure time, field of view B (6.6 cm), medium binning (factor of 8) resolution and a 1F/Stop aperture. Relative fluorescent intensities were represented by a pseudo colour scale ranging from red (least intense) to yellow (most intense).−1 cm−2 sr−1). The specific amount of protein in tissue was determined by comparison with a standard curve of NanoLuc-Gal3 activity.NanoLuc-Gal3 (164 pmol) in 40 μl PBS was injected subcutaneously into the hock of B6 mice. At the prescribed time points, animals were euthanized in accordance with approved protocols. Organs and tissues of interest were collected, weighed, processed, incubated with furimazine and bioluminescence quantified by a luminometer. Bioluminescence images were acquired using an IVIS Spectrum in vivo imaging system. Living Image software v4.3.1 (PerkinElmer) was used to acquire the data immediately after furimazine administration. Exposure time for the bioluminescence imaging was 1 s. Regions of interest (ROIs) were quantified as the average radiance (photons sListeria monocytogenes InIAM (strain 10403s). Tissue bacteria burdens in the liver and spleen were used as a metric for infection where the liver is local for gut infection and the spleen represents systemic spread of infection. On the day before infection, 10-week-old naïve C57BL/6 mice (Taconic Biosciences) received IDO-Gal3 injection subcutaneously (hock), while control mice received sterile saline injection. The following day, mice were orally infected with 2 × 109 colony-forming units (c.f.u.) of Listeria monocytogenes per mouse. To prepare for infection, bacteria were cultured overnight in BHI broth at 37 °C, shaking at 220 r.p.m. Before infection, a subculture of 2 ml of the bacteria and 18 ml BHI broth was cultured under the same conditions until reaching an OD600 = 0.8. Bacteria were pelleted, resuspended in 500 μl of sterile PBS, and 50 μl of bacteria was pipetted onto a small square of white bread and fed to each mouse individually. Once the entire piece of bread was eaten, mice were returned to their cage. At day 7 post infection, mice were euthanized, followed by collection of the spleen and liver. Tissue was homogenized, suspended in 1% saponin for 1 h, then plated at serial dilutions from undiluted to 1:1,000. BHI Agar plates were treated with streptomycin to limit non-specific bacterial growth, as this strain of L. monocytogenes is streptomycin resistant. C.f.u.s were counted at 24 h post-plating.Immune suppression from IDO-Gal3 was evaluated in response to oral infection with 9P. gingivalis strain 381 and 2.5 × 109Aggregatibacter actinomycetemocomitans strain 29522 (ATCC) resuspended in 2% low-viscosity carboxy-methyl-cellulose (Sigma-Aldrich). Oral lavage was repeated every week for 5 weeks. Each week, 1 d before the first day of infection (prophylactic) or 1 d after the last day of infection (therapeutic), 10 μl of IDO-Gal3 was injected into the submandibular space using a 30-gauge insulin syringe (Becton Dickenson). Each week of infection, on the first day of infection, before infection, microbial sampling of the oral environment was performed with calcium alginate swabs (ThermoFisher). One week following the last infection, the mandibles were collected to evaluate soft tissue soluble mediator expression and bone morphometric analysis.All mice were lavaged with 25 μl 0.12% chlorhexidine gluconate (3 M) for 3 d. On days 4, 5, 6 and 7, mice received a 25 μl oral lavage with 2.5 × 10β, IL-10 and MCP1 according to manufacturer protocols. Data were acquired on a Luminex 200 system running xPONENT 3.1 software (Luminex) and analysed using a 5-paramater logistic spline-curve fitting method using Milliplex Analyst v5.1 software (Vigene Tech). Data are presented as pg ml−1 normalized to total protein .Mandibles with both soft tissue and bone were subjected to bead beating at two 2 min intervals with 2 min of cooling in between using 1.0-mm-diameter zirconia silica beads (BioSpec) in cell extraction buffer (ThermoFisher). The buffer was prepared with a protease inhibitor cocktail  and PMSF protease inhibitor (Abcam) to allow for dissociation and lysis of all soft tissue while leaving the hard tissues intact. MILLIPLEX Multiplex assays (EMD Millipore) were used to probe resulting lysates for IL-6, IL-13 ROI was set with standardized dimensions of 1.5  × 4.0  × 0.9 mm . Anatomical landmarks were used for the standardized positioning of the ROI: frontal plane, the roof of the furcation area between mesial and distal roots of the upper first molar; sagittal plane, anterior limit was the distal aspect of the mesial root of the first molar. The thickness of the ROI on the transversal plane was set to 50 slices (900 μm) and counted towards the palatal /medial direction beginning from the image that included the centre of the upper first molar in its transversal width. A standardized threshold was set to distinguish between non-mineralized and mineralized tissues where total volume and total thickness of the ROI were calculated. Mean trabecular bone volume (BV) and thickness (BT) analysis assessed the percentage of mineralized tissue  within the total volume/thickness  of the ROI and is presented as the BV/TV ratio (mean trabecular bone volume) or BT/TT ratio (mean trabecular thickness). Vertical bone loss is the distance from the cementoenamel junction (CEJ) to the alveolar bone and was calculated at 12 sites over three molars and averaged to calculate the average vertical bone loss in mm.Mandibles were fixed in 4% buffered formalin for 24 h, stored in 70% alcohol and scanned at 18 μm resolution using a micro-CT system (Skyscan). Three-dimensional images were reconstructed and the resulting images re-oriented spatially using anatomical landmarks with the NRecon and DataViewer software (Skyscan). A standardized 5.4 mmP. gingivalis 16S, A. actinomycetemocomitans 16S and total 16S using real-time PCR. The percentage of A. actinomycetemocomitans 16S and P. gingivalis 16S within the total 16S compartment was calculated using the following formula: Ct value of total 16S/Ct value of A. actinomycetemocomitans or P. gingivalis 16S. 16s rRNA For: AGA GTT TGA TCC TGG CTC AG; Rev: ACG GCT ACC TTG TTA CGA CTT; Pg For: CTT GAC TTC AGT GGC GGC AG; Rev: AGG GAA GAC GGT TTT CAC CA; Aa For: GTT TAG CCC TGG CCG AAG; Rev: TGA CGG GCG GTG TGT ACA AGG.Genomic DNA was isolated from microbial sampling of the oral environment using a DNeasy kit (Qiagen) according to manufacturer instructions. The gDNA was then probed for 30 that applies cyclic mechanical loading to the knees of aged (6 months) mice, causing mechanical damage and consequent inflammation and cartilage degradation31. The mice were anaesthetized and placed in a fixture with the knee in flexion; loading (9 N) was applied axially for 500 cycles and loading sessions were done on the mice 5 times per week during the experiment. IDO or IDO-Gal3 was prepared at 143 μM, and 20 μl of each treatment was injected intra-articularly at the start of each week of the study, with each knee receiving 4 total treatments. At the end of the 4-week study, mice were euthanized for analysis of gene expression and histopathology.IDO-Gal3 activity was assessed in a post-traumatic osteoarthritis mouse modelPharmacokinetics of IDO and IDO-Gal3 retention after local injection at the disease site was assessed by intravital imaging over the course of 7 d. Both proteins were labelled with Li-Cor IRDye 680RD NHS ester (Li-Cor Biosciences) to visualize and measure protein knee retention. Mice were subjected to mechanical loading for 2 weeks before each treatment was administered via intra-articular injection. Intravital imaging was performed  immediately following injection and every subsequent 24 h. The fluorescence signal measured at the joint over time was normalized to the initial measurement for each knee and an exponential decay was individually fit for each specimen. The AUC/bioavailability for each joint was calculated from the best-fit line of exponential decay. After 7 d, the mice were euthanized and an ex vivo image was taken of each joint with the surrounding skin removed to increase measurement sensitivity.TaqMan qPCR in the knee joint and the popliteal lymph node that drains the knee joint. Following euthanization, knees and popliteal lymph nodes were excised. Combined joint tissue from the synovial wall and articular surface  and the popliteal lymph nodes were homogenized with bead pulverization in Qiazol. RNA was extracted and purified using the RNeasy Plus mini kit from Qiagen and quantified using NanoQuant plate from Tecan in a microplate reader . The RNA was converted to cDNA using the iScript Synthesis kit from Bio-Rad (Hercules). Gene expression was calculated by the ΔΔCt method, normalizing to GAPDH and beta-actin (ACTB). TaqMan reagents were purchased from ThermoFisher and used according to provided protocols, using appropriate primers .Gene expression was evaluated by 38. OARSI scoring was based on medial and lateral tibial plateaus 39, and a generic score was concurrently assigned on the basis of H&E features and the safranin O staining of the tibial plateaus according to the DJD methodology .Tissue samples were fixed in 10% formalin and decalcified in 20% EDTA for 7 d. A standard 8 h cycle of graded alcohols, xylenes and paraffin wax was used to process tissues before embedding and sectioning at 5 μm thickness. Sections were mounted on positively charged glass slides and stained with H&E using the Gemini autostainer (ThermoFisher). Safranin O staining was performed using the StatLab staining kit. Sections were imaged using Leica SCN400 slide scanner. Each joint was evaluated by at least two mid-frontal sections for both H&E and safranin O stains. A board-certified veterinary pathologist conducted histopathologic interpretations under blinded conditionsSixteen male Lewis rats  were acquired from Charles River Laboratories. Rats were acclimated to the University of Florida housing facilities for 1 week. After acclimation, rats underwent baseline gait and von Frey behavioural testing. Following baseline behavioural testing, all rats received medial collateral ligament plus medial meniscus transection (MCLT + MMT) surgery to their right hind limb. Gait data were collected on weeks 3, 5 and 7 post-surgery using a Phantom Miro Lab320 (Phantom Camera Control 3.0), while von Frey testing was conducted weekly after surgery. At 8 weeks post-surgery, rats received a unilateral saline or IDO-Gal3 injection. Rats underwent von Frey testing the day following injection and gait testing 2 d after injection. Both gait and von Frey data were collected weekly until euthanasia.−1) (Patterson Veterinary) intra-operatively and every 12 h for 48 h. Rats were grouped into a saline injection cohort (n = 7) or IDO-Gal3 injection cohort (n = 8). At 8 weeks after OA induction, rats received 30 µl unilateral injections of either sterile saline or IDO-Gal3 (33 pg µl−1) in the operated knee using sterile allergy syringes  (Becton Dickinson). First, rats were anaesthetized using 2.5% isoflurane (Patterson Veterinary) and the operated knee was aseptically prepped as described above. Then the needle was inserted through the patellar ligament following the patellar groove into the joint space. The knee was flexed and the injection site was cleaned with sterile gauze and 70% ethanol.MCLT + MMT surgery rats were anaesthetized in a 2.5% isoflurane (Patterson Veterinary) sleep box. Rats were then aseptically prepped with betadine surgical scrub (Purdue Products) and 70% ethanol in triplicate, and transferred to a sterile field with anaesthesia maintained via mask inhalation of 2.5% isoflurane. During MCLT + MMT surgery, a 1–2 cm midline skin incision was made along the medial aspects of the rat knee and the skin was retracted to reveal the medial collateral ligament. The medial collateral ligament was transected and the knee was placed in the valgus orientation to stretch the medial compartment and expose the medial meniscus. The medial meniscus was cut radially, and absorbable 5–0 vicryl braided sutures (Ethicon) were used for muscle closure and 4–0 ethilon nylon monofilament sutures (Ethicon) were used for skin closure. Rats recovered post-operatively in a warming box until weight bearing on all limbs. For pain management, rats received a subcutaneous injection of buprenorphine  was applied to the plantar region of each hind foot. First, the 4.0 g von Frey filament was applied. A less-stiff filament was applied if a paw withdrawal occurred, and a stiffer filament was applied if a paw withdrawal did not occur. Using these data, the force at which rats were equally likely to withdraw or tolerate was calculated via Chaplan’s approximation1.Tactile sensitivity was assessed by measuring the 50% paw withdrawal threshold determined using the Chaplan up-down method for von Frey filaments−1 anti-CTXII, 8.9 ng µl−1 anti-IL-6 or 357 ng µl−1 anti-MCP1 and then moved to static incubation at 4 °C overnight. Particles were then washed three times in PBS containing 2% BSA and 2 mM EDTA (capture buffer), with final antibody amounts per particle measured to be 0.33 ng Ab µg−1 particle (anti-CTXII), 0.33 ng Ab µg−1 particle (anti-IL-6) and 13.0 ng Ab µg−1 particle (anti-MCP1).Commercially available streptavidin-functionalized particles  were coated with biotinylated antibodies for CTXII , IL-6 and MCP1 . Here, particles were washed 3 times in PBS, incubated for 2 h on a tube revolver at room temperature in antibody mixes that contained either 8.9 ng µl40. Briefly, 300 µg of antibody-conjugated magnetic particles  were suspended in 10 µl of saline, then injected in the operated and contralateral knee. After 2 h incubation in the knee, particles were collected via 5 repeated 50 μl PBS washes of the knee, with collected fluid combined and particles in the fluid isolated via a magnetic separator. Collected particles were washed twice with capture buffer, then incubated for 15 min in the 100 mM Glycine-Tris buffer (pH 3.1) containing 2% BSA and 2 mM EDTA (release buffer). Following biomarker release, magnetic particles were isolated by magnetic separation and the pH of the supernatant was adjusted to 8.3 for enzyme-linked immunosorbent assays (ELISA). CTXII was then measured in the supernatant using the Cartilaps CTXII ELISA kit  according to manufacturer instructions, and CCL2 was quantified using a rat CCL2 ELISA kit (KRC1012 Life Technologies) according to manufacturer instructions and previously described modifications41. IL-6 was quantified using ELISA developed in the laboratory using biotin anti-rat IL-6 antibody (517703) and purified (coating) anti-rat IL-6 antibody . Coating antibody was diluted in 100 mM NaHCO3 and 34 mM Na2CO3 (pH 9.5), placed in coated microwells , incubated for 30 min on a plate shaker then overnight at 4 °C, washed 5 times with PBS with 0.5% Tween-20 (wash buffer), blocked for 1 h with PBS containing 2% BSA and 10% heat-treated bovine serum, and finally washed 5 times with wash buffer. Samples and standards were pre-incubated with anti-IL-6 detection antibody (30 min at room temperature and then overnight at 4 °C), then added to ELISA plate and incubated for 3 h. The plate was then washed 5 times, and 100 μl of avidin-HRP  was added to the plate and incubated for 30 min. The plate was again washed 5 times, with 100 μl of tetramethylbenzidine substrate added for 15 min, followed by 100 μl of stop solution (diluted sulfuric acid). Absorbance was read at 450 and 650 nm. Particles were quantified in 60 μl of capture buffer, with particle suspensions read for absorbance at 450 nm using Synergy 2 Multi-Mode microplate reader, as previously described40.Following euthanasia via exsanguination, magnetic capture was performed in both the operated and contralateral knee, as previously describedFollowing magnetic capture, operated and contralateral knees were dissected and placed in 10% neutral buffered formalin (ThermoFisher) for 48 h at room temperature. Following fixation, knees were decalcified using Cal-Ex (ThermoFisher) for 5 d at room temperature, dehydrated through an ethanol ladder and embedded in paraffin wax via vacuum infiltration. Then, 10 µm frontal sections were acquired, with at least one section taken at every 100 µm through the loading region of the medial meniscus. Slides were stained with toluidine blue.n = 8) or bilateral injections of NL-Gal3  (n = 8) in both operated and contralateral knees. (Surgeries and injections were conducted the same way as described above). Knees were flexed, then injected with 50 µl furimazine . Immediately after injection, knees were flexed again and luminescence was measured with IVIS using a 1 s (and 60 s) exposure time in field of view D. Furimazine injections and IVIS imaging were repeated at 1, 2, 4, 8, 12, 16, 20, 24 and 28 d post NL or NL-Gal3 injection. For analysis, a ROI was drawn around the largest luminescent signal and copied to create an identical sized ROI for all knees.Sixteen male Lewis rats  were acquired from Charles River Laboratories. Rats were acclimated to the University of Florida housing facilities for 1 week. Then rats underwent MCLT + MMT surgery. After 8 weeks, rats received a 50 µl injection of either Nano-Glo Luciferase (NL) or Nano-Glo Luciferase Galectin-3 . Rats were IVIS imaged immediately following injection, then at 1, 2, 4, 8, 12, 16, 20, 24 and 28 d post-injection. After imaging on day 28, rats were euthanized, and both operated and contralateral knees were dissected for joint tissue distribution analysis via IVIS. In vivo joint retention was measured in the operated and contralateral knees of 16 male Lewis rats. At 8 weeks post MCLT + MMT surgery, knees were aseptically prepped and rats received bilateral injections of NL   at room temperate for roughly 1 min. After incubation, tissues were removed from the 24-well plates and luminescence was measured with IVIS using auto-exposure in field of view C. For assessment, individual ROIs were drawn around the patellar tissue, tibial tissue, femoral tissue and meniscus. The ROI for each tissue was copied to create an identical sized ROI for the respective tissue.U-tests on psoriasis data. Study-specific analyses are reported in figure captions.Statistical analyses were performed using GraphPad Prism 8 and 9, with the following exceptions. Surgically induced osteoarthritis data were analysed using R Analytics 4.0.4 with RStudio 2022, and MATLAB 2020b was used for Mann-Whitney Further information on research design is available in the Supplementary InformationSupplementary methods and figures.Reporting Summary


Caenorhabditis elegans. We show that orthologs of GPA2 and GPB5, together with thyrotropin-releasing hormone (TRH) related neuropeptides, constitute a neuroendocrine pathway that promotes growth in C. elegans. GPA2/GPB5 signaling is required for normal body size and acts through activation of the glycoprotein hormone receptor ortholog FSHR-1. C. elegans GPA2 and GPB5 increase cAMP signaling by FSHR-1 in vitro. Both subunits are expressed in enteric neurons and promote growth by signaling to their receptor in glial cells and the intestine. Impaired GPA2/GPB5 signaling causes bloating of the intestinal lumen. In addition, mutants lacking thyrostimulin-like signaling show an increased defecation cycle period. Our study suggests that the thyrostimulin GPA2/GPB5 pathway is an ancient enteric neuroendocrine system that regulates intestinal function in ecdysozoans, and may ancestrally have been involved in the control of organismal growth.In vertebrates, thyrostimulin is a highly conserved glycoprotein hormone that, besides thyroid stimulating hormone (TSH), is a potent ligand of the TSH receptor. Thyrostimulin is considered the most ancestral glycoprotein hormone and orthologs of its subunits, GPA2 and GPB5, are widely conserved across vertebrate and invertebrate animals. Unlike TSH, however, the functions of the thyrostimulin neuroendocrine system remain largely unexplored. Here, we identify a functional thyrostimulin-like signaling system in  Glycoprotein hormones are key neuroendocrine factors that control diverse physiological processes, such as development, reproduction, energy homeostasis and growth , 2. In vDrosophila melanogaster and the mosquito Aedes aegypti  and thyrotropin-releasing hormone (TRH), have been identified in C. elegans and were found to have conserved physiological roles, such as in the control of reproduction and growth , respectively. GPLA-1 and GPLB-1 resemble vertebrate GPA2 and GPB5 and are widely conserved in nematodes  and gplb-1 (ibt4) knockout mutants display a significantly shorter body length compared to wild-type animals  mutant carries a substitution of the glycine residue between the second and third cysteine (G63E), which is highly conserved and important for proper cysteine knot formation (gpla-1 (ok2127) mutant contains a large deletion, removing the start codon and the complete first exon of the gene  cells. We co-expressed FSHR-1 with a cAMP response element (CRE)-luciferase reporter in HEK cells and quantified its activation by assessing bioluminescence levels in the presence of luciferin substrate  and the enteric muscles of the hindgut, which are involved in defecation  or hmc and the enteric muscles (gplb-1).To gain further insight into the role of thyrostimulin-like signaling in growth regulation, we investigated the expression patterns of  defects 2E, show defects , 49, 50, elegans . These i elegans –55. We afecation –57. Takefshr-1 under its endogenous promoter recapitulated the reported expression of the receptor in the intestine and in multiple neurons in the head  and exp-1(ox276) mutants are also significantly smaller than wild-type animals  have a carboxytail extension, referred to as the “seatbelt”, that wraps around the alpha subunit to stabilize heterodimer configuration , 79, 80.erodimer , 28, 82.erodimer , 38, 82.solution . Howeversolution . Based osolution . Severalsolution , in whicC. elegans FSHR-1, is abundantly expressed in the alimentary canal  superfamily that is required for intestinal calcium and pH oscillations  plates seeded with Escherichia coli OP50 bacteria. All experiments were performed using 1-day adult hermaphrodites, unless mentioned otherwise. Wild-type (N2-Bristol), IBE7 flr-2 (ut5), IBE24 flr-2 (ok2127), IBE1 fshr-1 (ok778) and EG1285 lin-15B&lin-15A (n765) oxIs12 [unc-47p::GFP + lin-15(+)] strains were obtained from the Caenorhabditis Genetics Center . CB156 unc-25 (e156) and EG276 exp-1 (ox276) strains were a kind gift from the lab of W.R. Schafer . Deletion alleles for gpla-1 (ibt1) and gplb-1 (ibt4) were obtained by CRISPR/Cas9 genome editing, as described below. A full list of strains used in this study can be found in All fshr-1 (a isoform), gpla-1, and gplb-1 (a isoform) were cloned in front of a SL2 trans-splicing site and fluorescent reporter gene.All fluorescent reporter and rescue constructs were made using the MultiSite Gateway Three-Fragment cloning system (Invitrogen). Genomic DNA or cDNA sequences of fshr-1 (3110 bp), gpla-1 (4058 bp), and gplb-1 (3589 bp) were cloned from genomic DNA of wild-type C. elegans. Tissue-specific rescue transgenes were generated using promoter regions of rab-3 (1208 bp) (rgef-1 (3660 bp)  (mir-228 (2217 bp)  , 66, rge3660 bp) , ges-1  –63, and 2217 bp) . All confshr-1 in HEK cells was obtained by directionally cloning the fshr-1 cDNA into the pcDNA3.1/V5-His-TOPO vector (Invitrogen). The cDNA sequence of the fshr-1a gene isoform was amplified by PCR using cDNA from mix-staged wild-type C. elegans as template. The forward primer included a ‘CACC’ sequence at the 5’ end that introduced a partial Kozak sequence for increased translation efficiency in mammalian cells.The plasmid for heterologous expression of gpla-1(ibt1) and gplb-1(ibt4) knockout alleles were generated by CRISPR/Cas9-mediated deletion of the gpla-1 or gplb-1 open reading frames. For each gene, two crRNA sequences were designed by looking for PAM sites (NGG) that were in the vicinity of the double stranded break site. After analysis of putative off-target sites (http://crispor.tefor.net/), we selected the highest scoring sequences based on their predicted on-target activities. Repair templates were designed to be in-frame and were codon-optimized for C. elegans (https://www.genscript.com/tools/codon-frequency-table). To simplify selection of the successfully edited worms, we used a Co-CRISPR technique in which the dpy-10 gene was also knocked out by CRISPR editing (The  editing . We used editing  for the  editing Table S2gpla-1 (ibt3 [His::GPLA-1]) knockin allele was made by CRISPR/Cas9 mediated insertion of a His-tag at the N-terminus of the gpla-1 open reading frame, using a similar strategy as described above and the crRNA and repair template listed in The Below, the sequences of the gene knockouts and knockins are shown with flanks on the (-) strand. Exons are in grey, with start and stop codons in red, the remaining gene sequences after CRISPR gene editing are underlined, and with the inserted gene sequence in blue.gpla-1(ibt1) deletion:ATGGGCTCCAAAGCACGAGCACGACGACGTTTAAGTTGTTTTTTAAGCGTTTTTGTTGTGACATGCTTATTACAGTACTGCACAGCAGGTGTTACTAAGAATAATAGTTGCAAAAAAGTTGgtacgtcacgaaatctactaaacttcgatcagtgtcctttaaatttttttttcagGAGTGGAGGAACTTATAGATGAAGAAGGCTGTGATTTGATGATAATTCGAATCAATCGATGCAGTGGGCATTGCTTCTCATTTACATTTCCTAATCCCTTAACGAAAAAATATTCAGTGCATGCGAAGTGCTGCCGGATGGTTGAATGGGAAATGgttagtattttttaactacagaaatgacttctgaaaatttataacaaagttattacggaagccaaaattctgggaatgtgttttgcgcaacatgtaaaaaaaatctcgtagcgaaagctacagtaattctctaaataactactgtagtgcttgcgtcgaattacggacttgatttgcgatatatccttcgtttctctgtattactttctcatttttgttttttttttaaacattctatcgaaaaattgatgattaattcatttcgaaagccgagcccgtaaatcgtcacaagcgctacagtagtcatgtaaagaattactgtatcgctacgagatgttttaatattgttttccaagaatcaatttttatttttcagCTTGAAACAGAATTAAAATGTTCCAAAGGAAACCGAAATCTTCGAATACCATCTGCAACACAATGTGAATGTTTTGATTGTCTTGTTCGATAGttgcagtttccatctctttcatttctcattcttgaatctcttgagttttacctgcataatagatttaatttattttctctcctccagtgccacattcatccacatttccataaaatctcgttcccttaattatttctataataatctcgttgatacgggcatctaaaaatgcaaaaaatcaaagcgaaagaaggatgactgacataaattgtctcaattctttaaattctatttttaacttttcagcctataccttctcaaatggplb-1(ibt4) deletion:tgttgacgttaagctcttttgaaaaaaATGCTTATATTTCCCGTGATCACTATACTTCATATATTTTTGgtaactttttgaaatatcttctaaaatgaatttaatatgtaacaatattctaacaaaatttttaatattgatcagatagtttttccaaatttaagtattaaccaggcgcgaaattttccgaattttaggccaaaaatacggtgcccggtctcgacacgaatttttttattaggtaaaaatgggtgtgtgcctttaaagagtactgtaactttaaactttcgttgctgcggaacttttgtcgacttttcatagctattagataaaaataaaaaaatattcaatattttcaacaaatctttagaaaaactatgtaaaatcgataaaaattctgcaacaaaaatttgaagttacagtactctttaaaggcacacactcgattgtatttaacaaaaaaagtcgtgtcgagaccggttaccgtaatttttgcgcaaatcggaataatttcgcgcttgggtaataagcatcacatctccaactaatttaaaagcaaaagtgtgatttttaaattcagATTTCGGTTGAATCTGGAAAAGAATGCGAGTTTGCAATGCGATTGGTCCCAGGATTCAATCCACTTCGTCAAGTTGATGCAAATGGAAAAGAATGCCGAGGAAACGTGGAATTGCCATTTTGCAAGGGTTACTGTAAGACTAGCGAGgtgaatttccttttttttccgattcaaaaataatccaattaaatttcagAGTGGCACCCATGGCTTTCCACCACGAGTTCAAAATAGTAAAGTGTGCACATTGGTCACCACTTCAACTCGAAAAGTAGTTCTTGATGATTGTGATGATGGAGCCGATGAGAGTGTCAAGTTTGTAATGGTTCCACATGGAACTGATTGTGAATGTTCTGCAGTTCCACTTGAACAACATCATTCATAAattatcatacattcattcaaaaattcatcgaataaataaaagttttgtggpla-1(ibt3 [His::GPLA-1]) insertion:ttctttaaattctatttttaacttttcagcctataccttctcaaatgATGCATCACCATCACCATCACGGCTCCAAAGCACGAGCACGACGACGTTTAAGTTGTTTTTTAAGCGTTTTTGTTGTGACATGCTTATTACAGTACTGCACAGCAGGTGTTACTAAGAATAATAGTTGCAAAAAAGTTGgtacgtcacgaaatctactaaacttcgatcagtgektcctttaaatttttttttcagGAGTGGAGGAACTTATAGATGAAGAAGGCTGTGATTTGATGATAATTCGAATCAATCGATGCAGTGGGCATTGCTTCTCATTTACATTTCCTAATCCCTTAACGAAAAAATATTCAGTGCATGCGAAGTGCTGCCGGATGGTTGAATGGGAAATGgttagtattttttaactacagaaatgacttctgaaaatttataacaaagttattacggaagccaaaattctgggaatgtgttttgcgcaacatgtaaaaaaaatctcgtagcgaaagctacagtaattctctaaataactactgtagtgcttgcgtcgaattacggacttgatttgcgatatatccttcgtttctctgtattactttctcatttttgttttttttttaaacattctatcgaaaaattgatgattaattcatttcgaagccgagcccgtaaatcgtcacaagcgctacagtagtcatgtaaagaattactgtatcgctacgagatgttttaatattgttttccaagaatcaatttttatttttcagCTTGAAACAGAATTAAAATGTTCCAAAGGAAACCGAAATCTTCGAATACCATCTGCAACACAATGTGAATGTTTTGATTGTCTTGTTCGATAGttgcagtttccatctctttcatttctcattcttgaatctcttgagttttacctgcataatagatttaatttattttctctcctccagtgccacattcatccacatttccataaaatctcgttcccttaattatttctataataatctcgttgatacgggcatctaaaaatgcaaaaaatcaaagcgaaagaaggatgactgacataaattgtctcaamyo-2p::mCherry, unc-122p::dsRed, or unc-122p::GFP) and 1-kb DNA ladder (Thermo Scientific) as carrier DNA.Germline transformations were carried out by injecting constructs into the syncytial gonad of young adult worms together with a co-injection marker  and resulting Z-stack projections were created and analyzed with ImarisViewer (v9.7.2) software. Adult worms were immobilized by mounting in 50 mM Sodium Azide solution in M9 buffer on a 2% agarose pad and covered with a glass cover slip. Expression patterns were confirmed in at least two independent transgenic strains. DVB and RME expression was confirmed by crossing with marker strain EG1285, which marks GABAergic neurons . Other chttps://github.com/jwatteyne/WormSizer). Synchronized L1 larvae were placed onto freshly seeded NGM plates one day post-synchronization and kept at 20°C until assaying. After 65 hours on food, 20 to 30 well-fed day one adults were transferred from the plates and anesthetized in 20 µL of 10 mM tetramisole hydrochloride solution (Sigma-Aldrich) in Milli-Q water on a 2% agarose pad. Images were captured with a ZEISS Axio Observer.Z1 at 5x magnification. Body size was measured from images of anesthetized day 1 adult worms on at least two independent days. After skeletonization, delineating the worm’s outline and midline, the worm’s total length and middle width were calculated using the calibration factor (pixels/µm) of the pictures. For the volume estimation, the midline of the worm was separated in 30 segments of the same size. The volume for each segment was determined by using the formula for the frustum of a cone, taking the natural shape of a worm into consideration. The summation of all these volumes was described as the worm’s total volume. To assess growth throughout adulthood, body size measurements were executed for 5 subsequent days (day 1 to day 5 adulthood) for at least two independent time periods.Body size parameters  of day one adults were measured using the custom WormSizer MATLAB script , luminal width was determined by measuring the average width (in µm) of the intestine at two different points in the posterior lumen. Body size parameters and luminal widths were plotted, and significance levels were calculated with GraphPad Prism 8 software.C. elegans N-terminal 6xHis-tagged GPLA-1 and non-tagged GPLB-1. Recombinant proteins were purified from cell lysate by affinity chromatography in PBS buffer  and analyzed by Tricine-SDS-PAGE on a 8 – 20% gel in Tris-Glycine running buffer and by subsequent Western blot using anti-His monoclonal antibodies. A similar approach was used to synthesize individual GPLA-1 and GPLB-1 proteins. N-terminal 6xHis-tagged GPLA-1 was expressed in HEK293 cells and purified by affinity chromatography. Non-tagged GPLB-1 was expressed in HEK cells and purified using ion exchange chromatography. Both recombinant GPLA-1 and GPLB-1 protein samples were analyzed by Tricine-SDS-PAGE.Recombinant proteins were synthesized and purified by OriGene™ Technologies . To generate recombinant GPLA-1/GPLB-1, HEK293 cells were transfected with expression vectors encoding 2, in Dulbecco’s Modified Eagle’s Medium (DMEM)/Nutrient F-12 Ham (Gibco) supplemented with 10% heat inactivated Fetal Bovine Serum  and 1% Penicillin/Streptomycin (Gibco). Co-transfection with the cAMP indicator CRE(6x)-luciferase and pcDNA3.1/fshr-1a was done in a 1:1 ratio when cells reached 60-70% confluency. Transfection medium contained Opti-MEM , CRE(6x)-luciferase and receptor plasmid DNA, Plus Reagent (Invitrogen) and Lipofectamine LTX (Invitrogen). One day post-transfection, fresh culture medium was added to the transfected cells. Two days post transfection, each well of a Bio-One CELLSTAR™ 96-well, Flat Bottom Microplate (Greiner) was loaded with a dilution series of recombinant protein compounds in 200 µM 3-isobutyl-1-methylxanthine (IBMX), which inhibits cAMP hydrolysis. Compound buffer (PBS with 10% glycerol) without hormone was added to the IBMX medium as a ligand-free negative control. Cells were detached, counted, pelleted and resuspended to a concentration of 10E-06 cells/mL in IBMX medium. Each well of the compound plate was supplemented with 50 µL of the cell suspension (50 000 cells/well) and the plate was incubated at 37°C for 3.5 h. Cells were then loaded with 100 µL SteadyLitePlus substrate (PerkinElmer) and incubated on a shaking plate for 15 minutes under dark conditions at RT. Finally, luminescence was measured twice for 5s (at 0s and 5s) per well at 469 nm on a Mithras LB 940 luminometer (Berthold Technologies). Measurements were performed in triplicate in at least 4 independent experiments. Luminescence values were plotted and significance levels were calculated with GraphPad Prism 8 software.The cAMP bioluminescence assay quantifies ligand-induced bioluminescent responses by measuring changes in intracellular cAMP levels after receptor activation. HEK293T cells were cultured in monolayer at 37°C in a humidified atmosphere with 5% COhttps://github.com/NathanDeFruyt/DefecationAnalysisBeetsLab). The length of one DMP cycle was defined by the time elapsed between two subsequent contractions of the posterior body wall muscles (pBocs). The average cycle length was calculated over 10 defecation cycles for each worm. aBoc frequency was defined as the ratio of aBoc over pBoc. Defecation parameters were plotted and significance levels were calculated with GraphPad Prism 8 software.The defecation motor program (DMP) was analyzed as previously described . AnimalsC. elegans GPLA-1 and GPLB-1 sequences were used as queries to identify sequences with highest similarity in selected model systems representative for their phylum by the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) database. The multiple sequence alignment program Clustal Omega  was used to align sequences, and conserved residues were identified using BoxShade .The H. Sapiens GPA2 and GPB5 and The original contributions presented in the study are included in the article/SK, MI, JW, LS and IB designed the research. SK, MI, SD and EV performed the experiments. SK, MI and IB analyzed data. IB and LS supervised the project and were responsible for funding acquisition. SK and IB wrote the paper. All authors contributed to the article and approved the submitted version.