DNA methylation signatures reveal that distinct combinations of transcription factors specify human immune cell epigenetic identity.

Epigenetic reprogramming underlies specification of immune cell lineages, but patterns that uniquely define immune cell types and the mechanisms by which they are established remain unclear. Here, we identified lineage-specific DNA methylation signatures of six immune cell types from human peripheral blood and determined their relationship to other epigenetic and transcriptomic patterns. Sites of lineage-specific hypomethylation were associated with distinct combinations of transcription factors in each cell type. By contrast, sites of lineage-specific hypermethylation were restricted mostly to adaptive immune cells. PU.1 binding sites were associated with lineage-specific hypo- and hypermethylation in different cell types, suggesting that it regulates DNA methylation in a context-dependent manner. These observations indicate that innate and adaptive immune lineages are specified by distinct epigenetic mechanisms via combinatorial and context-dependent use of key transcription factors. The cell-specific epigenomics and transcriptional patterns identified serve as a foundation for future studies on immune dysregulation in diseases and aging.

Surfaceome analysis of extracellular vesicles from senescent cells uncovers uptake repressor DPP4.

Senescent cells are beneficial for repairing acute tissue damage, but they are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) and proliferating cells (P-EVs) and found that P-EVs were readily taken up by proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not. We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We found that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to selectively prevent uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP4-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.

Flow Cytometry of Synaptoneurosomes (FCS) Reveals Increased Ribosomal S6 and Calcineurin Proteins in Activated Medial Prefrontal Cortex to Nucleus Accumbens Synapses.

Learning and behavior activate cue-specific patterns of sparsely distributed cells and synapses called ensembles that undergo memory-encoding engram alterations. While Fos is often used to label selectively activated cell bodies and identify neuronal ensembles, there is no comparable endogenous marker to label activated synapses and identify synaptic ensembles. For the purpose of identifying candidate synaptic activity markers, we optimized a flow cytometry of synaptoneurosome (FCS) procedure for assessing protein alterations in activated synapses from male and female rats. After injecting yellow fluorescent protein (YFP)-expressing adeno-associated virus into medial prefrontal cortex (mPFC) to label terminals in nucleus accumbens (NAc) of rats, we injected 20 mg/kg cocaine in a novel context (cocaine+novelty) to activate synapses, and prepared NAc synaptoneurosomes 0-60 min following injections. For FCS, we used commercially available antibodies to label presynaptic and postsynaptic markers synaptophysin and PSD-95 as well as candidate markers of synaptic activity [activity-regulated cytoskeleton protein (Arc), CaMKII and phospho-CaMKII, ribosomal protein S6 (S6) and phospho-S6, and calcineurin and phospho-calcineurin] in YFP-labeled synaptoneurosomes. Cocaine+novelty increased the percentage of S6-positive synaptoneurosomes at 5-60 min and calcineurin-positive synaptoneurosomes at 5-10 min. Electron microscopy verified that S6 and calcineurin levels in synaptoneurosomes were increased 10 min after cocaine+novelty. Pretreatment with the anesthetic chloral hydrate blocked cocaine+novelty-induced S6 and calcineurin increases in synaptoneurosomes, and novel context exposure alone (without cocaine) increased S6, both of which indicate that these increases were due to neural activity per se. Overall, FCS can be used to study protein alterations in activated synapses coming from specifically labeled mPFC projections to NAc.SIGNIFICANCE STATEMENT Memories are formed during learning and are stored in the brain by long-lasting molecular and cellular alterations called engrams formed within specific patterns of cue-activated neurons called neuronal ensembles. While Fos has been used to identify activated ensemble neurons and the engrams within them, we have not had a similar marker for activated synapses that can be used to identify synaptic engrams. Here we developed a procedure for high-throughput in-line analysis of flow cytometry of synaptoneurosome (FCS) and found that ribosomal S6 protein and calcineurin were increased in activated mPFC-NAc synapses. FCS can be used to study protein alterations in activated synapses within specifically labeled circuits.

OA susceptibility in mice is partially mediated by the gut microbiome, is transferrable via microbiome transplantation and is associated with immunophenotype changes.

OBJECTIVES: The Murphy Roths Large (MRL)/MpJ 'superhealer' mouse strain is protected from post-traumatic osteoarthritis (OA), although no studies have evaluated the microbiome in the context of this protection. This study characterised microbiome differences between MRL and wild-type mice, evaluated microbiome transplantation and OA and investigated microbiome-associated immunophenotypes. METHODS: Cecal material from mixed sex C57BL6/J (B6) or female MRL/MpJ (MRL) was transplanted into B6 and MRL mice, then OA was induced by disruption of the medial meniscus surgery (DMM). In other experiments, transplantation was performed after DMM and transplantation was performed into germ-free mice. Transplanted mice were bred through F2. OARSI, synovitis and osteophyte scores were determined blindly 8 weeks after DMM. 16S microbiome sequencing was performed and metagenomic function was imputed. Immunophenotypes were determined using mass cytometry. RESULTS: MRL-into-B6 transplant prior to DMM showed reduced OA histopathology (OARSI score 70% lower transplant vs B6 control), synovitis (60% reduction) and osteophyte scores (30% reduction) 8 weeks after DMM. When performed 48 hours after DMM, MRL-into-B6 transplant improved OA outcomes but not when performed 1-2 weeks after DMM. Protection was seen in F1 (60% reduction) and F2 progeny (30% reduction). Several cecal microbiome clades were correlated with either better (eg, Lactobacillus, R=-0.32, p=0.02) or worse (eg, Rikenellaceae, R=0.43, p=0.001) OA outcomes. Baseline immunophenotypes associated with MRL-into-B6 transplants and MRL included reduced double-negative T cells and increased CD25+CD4+ T cells. CONCLUSION: The gut microbiome is responsible in part for OA protection in MRL mice and is transferrable by microbiome transplantation. Transplantation induces resting systemic immunophenotyping changes that correlate with OA protection.

Blending Indigenous and western science: Quantifying cultural burning impacts in Karuk Aboriginal Territory.

The combined effects of Indigenous fire stewardship and lightning ignitions shaped historical fire regimes, landscape patterns, and available resources in many ecosystems globally. The resulting fire regimes created complex fire-vegetation dynamics that were further influenced by biophysical setting, disturbance history, and climate. While there is increasing recognition of Indigenous fire stewardship among western scientists and managers, the extent and purpose of cultural burning is generally absent from the landscape-fire modeling literature and our understanding of ecosystem processes and development. In collaboration with the Karuk Tribe Department of Natural Resources, we developed a transdisciplinary Monte Carlo simulation model of cultural ignition location, frequency, and timing to simulate spatially explicit cultural ignitions across a 264,399-ha landscape within Karuk Aboriginal Territory in northern California. Estimates of cultural ignition parameters were developed with Tribal members and knowledge holders using existing interviews, historical maps, ethnographies, recent ecological studies, contemporary maps, and generational knowledge. Spatial and temporal attributes of cultural burning were explicitly tied to the ecology of specific cultural resources, fuel receptivity, seasonal movement patterns, and spiritual practices. Prior to colonization, cultural burning practices were extensive across the study landscape with an estimated 6972 annual ignitions, averaging approximately 6.5 ignitions per Indigenous fire steward per year. The ignition characteristics we document align closely with data on historical fire regimes and vegetation but differ substantially from the location and timing of contemporary ignitions. This work demonstrates the importance of cultural burning for developing and maintaining the ecosystems present at the time of colonization and underscores the need to work collaboratively with Indigenous communities to restore ecocultural processes in these systems.

Viruses in sanctuary chimpanzees across Africa.

Infectious disease is a major concern for both wild and captive primate populations. Primate sanctuaries in Africa provide critical protection to thousands of wild-born, orphan primates confiscated from the bushmeat and pet trades. However, uncertainty about the infectious agents these individuals potentially harbor has important implications for their individual care and long-term conservation strategies. We used metagenomic next-generation sequencing to identify viruses in blood samples from chimpanzees (Pan troglodytes) in three sanctuaries in West, Central, and East Africa. Our goal was to evaluate whether viruses of human origin or other "atypical" or unknown viruses might infect these chimpanzees. We identified viruses from eight families: Anelloviridae, Flaviviridae, Genomoviridae, Hepadnaviridae, Parvoviridae, Picobirnaviridae, Picornaviridae, and Rhabdoviridae. The majority (15/26) of viruses identified were members of the family Anelloviridae and represent the genera Alphatorquevirus (torque teno viruses) and Betatorquevirus (torque teno mini viruses), which are common in chimpanzees and apathogenic. Of the remaining 11 viruses, 9 were typical constituents of the chimpanzee virome that have been identified in previous studies and are also thought to be apathogenic. One virus, a novel tibrovirus (Rhabdoviridae: Tibrovirus) is related to Bas-Congo virus, which was originally thought to be a human pathogen but is currently thought to be apathogenic, incidental, and vector-borne. The only virus associated with disease was rhinovirus C (Picornaviridae: Enterovirus) infecting one chimpanzee subsequent to an outbreak of respiratory illness at that sanctuary. Our results suggest that the blood-borne virome of African sanctuary chimpanzees does not differ appreciably from that of their wild counterparts, and that persistent infection with exogenous viruses may be less common than often assumed.

Female reproduction and viral infection in a long-lived mammal.

For energetically limited organisms, life-history theory predicts trade-offs between reproductive effort and somatic maintenance. This is especially true of female mammals, for whom reproduction presents multifarious energetic and physiological demands. Here, we examine longitudinal changes in the gut virome (viral community) with respect to reproductive status in wild mature female chimpanzees Pan troglodytes schweinfurthii from two communities, Kanyawara and Ngogo, in Kibale National Park, Uganda. We used metagenomic methods to characterize viromes of individual chimpanzees while they were cycling, pregnant and lactating. Females from Kanyawara, whose territory abuts the park's boundary, had higher viral richness and loads (relative quantity of viral sequences) than females from Ngogo, whose territory is more energetically rich and located farther from large human settlements. Viral richness (total number of distinct viruses per sample) was higher when females were lactating than when cycling or pregnant. In pregnant females, viral richness increased with estimated day of gestation. Richness did not vary with age, in contrast to prior research showing increased viral abundance in older males from these same communities. Our results provide evidence of short-term physiological trade-offs between reproduction and infection, which are often hypothesized to constrain health in long-lived species.

The Microbiome in Osteoarthritis: a Narrative Review of Recent Human and Animal Model Literature.

PURPOSE OF THE REVIEW: The microbiome has recently emerged as a powerful contributor to health and illness in chronic, systemic disorders. Furthermore, new microbiome niches beyond traditional gut locations are frequently being described. Over the past 5 years, numerous pivotal studies have demonstrated associations between changes in various microbiome niches and the development of osteoarthritis (OA). Herein, we review the most impactful recent literature, including microbiome associations with disease and the potential therapeutic value of microbiome manipulation. RECENT FINDINGS: The gut microbiome of human OA patients is enriched in specific bacterial clades, most notably Streptococcus, which correlates with OA pain, Firmicutes, and others. Most studies have focused on knee OA, although one publication demonstrated positive associations with 3 gut microbiome clades in hand OA. OA can be easily distinguished from RA by evaluating differences in oral microbiome composition. Most studies have also demonstrated a reduction in richness of the gut microbiome (alpha diversity) associated with OA. Several studies have identified bacterial signatures within human knee and hip cartilage, synovial fluid, and synovial tissue and have described changes in these patterns occurring with the development of OA. In animal models of OA, high-fat diet-induced obesity has been the most well-studied OA risk factor associated with changes in the microbiome, with numerous bacterial clades changed within the gut microbiome and associated with OA. Also in animal models, various oral supplementations, including dietary fiber, probiotics including Lactobacillus species, and cecal microbiome transplantation have all shown improvements in OA histopathology or cartilage healing. Microbiome changes are strongly associated with the OA disease process and with individual OA risk factors related to both the gut microbiome and the microbial DNA patterns in the joint. Microbiome-directed interventions have the potential to prevent or reduce the progression of OA. Future studies should investigate the mechanistic underpinnings of these microbiome associations and further define the therapeutic potential of microbiome augmentation.

Cytomegalovirus infection reduced CD70 expression, signaling and expansion of viral specific memory CD8+ T cells in healthy human adults.

BACKGROUND: Cytomegalovirus (CMV) infection leads to effector memory CD8+ T cell expansion and is associated with immune dysfunction in older adults. However, the molecular alterations of CMV-specific CD8+ T cells in CMV infected healthy young and middle-aged adults has not been fully characterized. RESULTS: We compared CD8+ T cells specific for a CMV epitope (pp65495-503, NLV) and an influenza A virus (IAV) epitope (M158-66, GIL) from the same young and middle-aged healthy adults with serum positive for anti-CMV IgG. Compared to the IAV-specific CD8+ T cells, CMV-specific CD8+ T cells contained more differentiated effector memory (TEM and TEMRA) cells. Isolated CMV-specific central memory (TCM) but not naïve (TN) cells had a significant reduced activation-induced expansion in vitro compared to their IAV-specific counterparts. Furthermore, we found that CD70 expression was reduced in CMV-specific CD28+CD8+ TCM and that CD70+ TCM had better expansion in vitro than did CD70- TCM. Mechanistically, we showed that CD70 directly enhanced MAPK phosphorylation and CMV-specific CD8+ TCM cells had a reduced MAPK signaling upon activation. Lastly, we showed that age did not exacerbate reduced CD70 expression in CMV- specific CD8+ TCM cells. CONCLUSION: Our findings showed that CMV infection causes mild expansion of CMV-NLV-specific CD8+ T cells, reduced CD70 expression and signaling, and proliferation of CMV-NLV-specific CD8+ TCM cells in young and middle-aged healthy adults and revealed an age-independent and CMV infection-specific impact on CD8+ memory T cells.

FANCJ compensates for RAP80 deficiency and suppresses genomic instability induced by interstrand cross-links.

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ's catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.

Phased diploid genome assembly with single-molecule real-time sequencing.

While genome assembly projects have been successful in many haploid and inbred species, the assembly of noninbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short- or long-read approaches. The phased diploid assembly enabled the study of haplotype structure and heterozygosities between homologous chromosomes, including the identification of widespread heterozygous structural variation within coding sequences.

Task-shifting to improve the reach of mental health interventions for trauma patients: findings from a pilot study of trauma nurse training in patient-centered activity scheduling for PTSD and depression.

Despite high rates of posttraumatic stress disorder (PTSD) and depression among traumatically injured patients, engagement in session-based psychotherapy early after trauma is limited due to various service utilization and readiness barriers. Task-shifting brief mental health interventions to routine trauma center providers is an understudied but potentially critical part of the continuum of care. This pilot study assessed the feasibility of training trauma nurses to engage patients in patient-centered activity scheduling based on a Behavioral Activation paradigm, which is designed to counteract dysfunctional avoidance/withdrawal behavior common among patients after injury. Nurses (N = 4) and patients (N = 40) were recruited from two level II trauma centers. A portion of a one day in-person workshop included didactics, demonstrations, and experiential activities to teach brief intervention delivery. Nurses completed pre- and posttraining standardized patient role-plays prior to and two months after training, which were coded for adherence to the intervention. Nurses also completed exit interviews to assess their perspectives on the training and addressing patient mental health concerns. Findings support the feasibility of training trauma nurses in a brief mental health intervention. Task-shifting brief interventions holds promise for reaching more of the population in need of posttrauma mental health care.

Acid Ceramidase Deficiency in Mice Results in a Broad Range of Central Nervous System Abnormalities.

Farber disease is a rare autosomal recessive disorder caused by acid ceramidase deficiency that usually presents as early-onset progressive visceral and neurologic disease. To understand the neurologic abnormality, we investigated behavioral, biochemical, and cellular abnormalities in the central nervous system of Asah1P361R/P361R mice, which serve as a model of Farber disease. Behaviorally, the mutant mice had reduced voluntary locomotion and exploration, increased thigmotaxis, abnormal spectra of basic behavioral activities, impaired muscle grip strength, and defects in motor coordination. A few mutant mice developed hydrocephalus. Mass spectrometry revealed elevations of ceramides, hydroxy-ceramides, dihydroceramides, sphingosine, dihexosylceramides, and monosialodihexosylganglioside in the brain. The highest accumulation was in hydroxy-ceramides. Storage compound distribution was analyzed by mass spectrometry imaging and morphologic analyses and revealed involvement of a wide range of central nervous system cell types (eg, neurons, endothelial cells, and choroid plexus cells), most notably microglia and/or macrophages. Coalescing and mostly perivascular granuloma-like accumulations of storage-laden CD68+ microglia and/or macrophages were seen as early as 3 weeks of age and located preferentially in white matter, periventricular zones, and meninges. Neurodegeneration was also evident in specific cerebral areas in late disease. Overall, our central nervous system studies in Asah1P361R/P361R mice substantially extend the understanding of human Farber disease and suggest that this model can be used to advance therapeutic approaches for this currently untreatable disorder.

Severe fire weather and intensive forest management increase fire severity in a multi-ownership landscape.

Many studies have examined how fuels, topography, climate, and fire weather influence fire severity. Less is known about how different forest management practices influence fire severity in multi-owner landscapes, despite costly and controversial suppression of wildfires that do not acknowledge ownership boundaries. In 2013, the Douglas Complex burned over 19,000 ha of Oregon & California Railroad (O&C) lands in Southwestern Oregon, USA. O&C lands are composed of a checkerboard of private industrial and federal forestland (Bureau of Land Management, BLM) with contrasting management objectives, providing a unique experimental landscape to understand how different management practices influence wildfire severity. Leveraging Landsat based estimates of fire severity (Relative differenced Normalized Burn Ratio, RdNBR) and geospatial data on fire progression, weather, topography, pre-fire forest conditions, and land ownership, we asked (1) what is the relative importance of different variables driving fire severity, and (2) is intensive plantation forestry associated with higher fire severity? Using Random Forest ensemble machine learning, we found daily fire weather was the most important predictor of fire severity, followed by stand age and ownership, followed by topographic features. Estimates of pre-fire forest biomass were not an important predictor of fire severity. Adjusting for all other predictor variables in a general least squares model incorporating spatial autocorrelation, mean predicted RdNBR was higher on private industrial forests (RdNBR 521.85 ± 18.67 [mean ± SE]) vs. BLM forests (398.87 ± 18.23) with a much greater proportion of older forests. Our findings suggest intensive plantation forestry characterized by young forests and spatially homogenized fuels, rather than pre-fire biomass, were significant drivers of wildfire severity. This has implications for perceptions of wildfire risk, shared fire management responsibilities, and developing fire resilience for multiple objectives in multi-owner landscapes.

Large-mode-area fibers operating near single-mode regime.

Lower NA in large-mode-area fibers enables better single-mode operation and larger core diameters. Fiber NA has traditionally been limited to 0.06, mostly due to the control tolerance in the fabrication process. It has been recognized recently that transverse mode instability is a major limit to average power scaling in fiber lasers. One effective method to mitigate this limit is to operate nearer to the single-mode regime. Lower fiber NA is critical in this since it allows relatively larger core diameters which is the key to mitigate the limits imposed by nonlinear effects. We have developed a fabrication process of ytterbium-doped silica glass which is capable of highly accurate refractive index control and sufficient uniformity for LMA fibers. This process is also capable of large-volume production. It is based on a significant amount of post-processing once the fiber preforms are made. We have demonstrated 30/400 and 40/400 LMA fibers with a NA of ~0.028 operating very close to the single-mode regime. The second-order mode cuts off at ~1.2µm and ~1.55µm respectively. We have also studied issues related to bend losses due to the low NA and further optimization of LMA fibers.

Polarizing ytterbium-doped all-solid photonic bandgap fiber with ~1150µm(2) effective mode area.

We demonstrate an Yb-doped polarizing all-solid photonic bandgap fiber for single-polarization and single-mode operation with an effective mode area of ~1150µm(2), a record for all-solid photonic bandgap fibers. The differential polarization mode loss is measured to be >5dB/m over the entire transmission band with a 160nm bandwidth and >15dB/m on the short wavelength edge of the band. A 2.6m long fiber was tested in a laser configuration producing a linearly polarized laser output with a PER value of 21dB without any polarizer, the highest for any fiber lasers based on polarizing fibers.

Quantitative mode quality characterization of fibers with extremely large mode areas by matched white-light interferometry.

Quantitative mode characterization of fibers with cores much beyond 50µm is difficult with existing techniques due to the combined effects of smaller intermodal group delays and dispersions. We demonstrate, for the first time, a new method using a matched white-light interferometry (MWI) to cancel fiber dispersion and achieve finer temporal resolution, demonstrating ~20fs temporal resolution in intermodal delays, i.e. 6µm path-length resolution. A 1m-long straight resonantly-enhanced leakage-channel fiber with 100µm core was characterized, showing ~55fs/m relative group delay and a ~29dB mode discrimination between the fundamental and second-order modes.

Ytterbium-doped large-mode-area all-solid photonic bandgap fiber lasers.

Single-mode operation in a large-mode-area fiber laser is highly desired for power scaling. We have, for the first time, demonstrated a 50µm-core-diameter Yb-doped all-solid photonic bandgap fiber laser with a mode area over 4 times that of the previous demonstration. 75W output power has been generated with a diffraction-limited beam and an efficiency of 70% relative to the launched pump power. We have also experimentally confirmed that a robust single-mode regime exists near the high frequency edge of the bandgap. These fibers only guide light within the bandgap over a narrow spectral range, which is essential for lasing far from the gain peak and suppression of stimulated Raman scattering. This work demonstrates the strong potential for mode area scaling of in single-mode all-solid photonic bandgap fibers.

Sex-Linked Discrepancies in C57BL6/J Mouse Osteoarthritis are Associated With the Gut Microbiome and are Transferrable by Microbiome Transplantation.

OBJECTIVE: Females have reduced osteoarthritis (OA) in surgical models. The objective of the current study was to evaluate a sex-linked gut microbiome in the pathogenesis of OA. METHODS: We induced OA via destabilization of the medial meniscus surgery in adult male and female C57BL6/J mice with and without opposite-sex microbiome transplantation. Eight weeks later, animals were euthanized, and OA severity, synovitis, and osteophyte scores were determined. Serum lipopolysaccharide was measured chromogenically, and serum cytokines were quantified via multiplex immunoassay. Cecal microbiome profiles were generated using 16S deep sequencing. RESULTS: Males had worse OA histology (3.5x, P = 6 × 10-7 ), synovitis (2.4x, P = 5 × 10-4 ), and osteophyte scores (3.7x, P = 3 × 10-4 ) than females. Male-into-female transplantation worsened all outcomes (histology 1.8x, P = 0.02; synovitis 2.0x, P = 3 × 10-5 ; osteophyte 2.1x, P = 0.01) compared to females, whereas female-into-male transplantation improved all outcomes except for synovitis (histology 0.53x, P = 2 × 10-4 ; osteophyte 0.28x, P = 5 × 10-4 ) compared to males. In the gut microbiome analysis, 44 clades were different in at least one group comparison; 5 clades were correlated with the Osteoarthritis Research Society International score (Lactobacillus R = -0.40, Aldercreutzia R = -0.40, rc4_4 R = -0.55, Sutterella R = -0.37, and Clostridiales R = 0.36). In the cytokine analysis, 10 analytes were different in at least one group comparison; 3 were different in two groups (female and female-into-male transplants vs male comparisons, all reduced in female and female-into-male transplants), including interleukin-12 (0.66x, P = 0.02; 0.66x, P = 0.02, respectively), eotaxin (0.74x, P = 5 × 10-6 ; 0.57x, P = 0.03), and tumor necrosis factor ⍺ (0.49x, P = 0.03; 0.52x, P = 0.009). CONCLUSION: Sex-linked differences in the mouse gut microbiome are associated with OA outcomes, are reversible by opposite-sex microbiome transplantation, and are associated with serum cytokine changes.