Hearing loss is a major risk factor for tinnitus, hyperacusis, and central auditory processing disorder. Although recent studies indicate that hearing loss causes neuroinflammation in the auditory pathway, the mechanisms underlying hearing loss–related pathologies are still poorly understood. We examined neuroinflammation in the auditory cortex following noise-induced hearing loss (NIHL) and its role in tinnitus in rodent models. Our results indicate that NIHL is associated with elevated expression of proinflammatory cytokines and microglial activation—two defining features of neuroinflammatory responses—in the primary auditory cortex (AI). Genetic knockout of tumor necrosis factor alpha (TNF-α) or pharmacologically blocking TNF-α expression prevented neuroinflammation and ameliorated the behavioral phenotype associated with tinnitus in mice with NIHL. Conversely, infusion of TNF-α into AI resulted in behavioral signs of tinnitus in both wild-type and TNF-α knockout mice with normal hearing. Pharmacological depletion of microglia also prevented tinnitus in mice with NIHL. At the synaptic level, the frequency of miniature excitatory synaptic currents (mEPSCs) increased and that of miniature inhibitory synaptic currents (mIPSCs) decreased in AI pyramidal neurons in animals with NIHL. This excitatory-to-inhibitory synaptic imbalance was completely prevented by pharmacological blockade of TNF-α expression. These results implicate neuroinflammation as a therapeutic target for treating tinnitus and other hearing loss–related disorders. Prolonged exposure to loud noises causes neuronal hyperexcitability and increases the risk of tinnitus. This study reveals that this type of tinnitus is mediated by noise-induced neuroinflammation; blockade of neuroinflammatory responses prevents noise-induced neuronal excitation/inhibition imbalance and tinnitus. Hearing loss is a widespread condition that affects approximately 500 million individuals . It is aIncreasing evidence indicates that NIHL and conductive hearing loss can lead to inflammatory responses, such as the activation of microglia and the release of proinflammatory cytokines –21, in tAlthough neuroinflammation is important in maintaining homeostasis of the central nervous system against external and internal insults, it can be detrimental if it becomes overactive or chronic . Persistn = 4 mice for each time point). Two hours of sound exposure resulted in long-lasting threshold shifts of up to 50 dB to the left ear, and weaker, temporary threshold increases in the right ear that recovered to less than 10 dB within 10 days. We then examined the expression of proinflammatory cytokines in AI of mice with NIHL 0.5, 1, 3, and 10 days post noise exposure (n = 4 mice for each time point). Real-time-PCR assays showed that the TNF-α mRNA level increased after noise exposure, with stronger increases in the left side  . The increase in TNF-α transcription peaked at 12 hours after the noise exposure (multiple comparison P < 0.001 for 12 hours versus control). Although the level declined over the ensuing days, it was still significantly higher than control at 1 day (P < 0.001) and 10 days (P < 0.001) after noise exposure. In contrast to the rapid increase of the TNF-α mRNA level, the expression of proinflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18) were significantly up-regulated bilaterally but only 10 days after noise exposure . There were no significant differences in the expression of these two proinflammatory cytokines between the left and right sides of AI .Tumor necrosis factor alpha (TNF-α) expression is elevated by loud noise exposure in the auditory periphery and cochlear nucleus –21, 39 aF4,30 = 26.76, P < 0.001). The increase was bilateral, and there were no differences between the two sides .Nod-like receptor protein 3 (NLRP3) is a main structural component of the inflammasome, a multiprotein complex responsible for the activation of inflammatory responses . MonauraF4,30 = 7.015, P < 0.001; bilateral difference, F1,30 = 11.133, P = 0.002; interaction, F4,30 = 3.562, P = 0.017; posthoc comparison to the control level, P < 0.05 for 1, 3, and 10 days). While the increase in the TNF-α protein level persisted in the right side, it decayed back to the baseline level in the left side by 10 days after the exposure .In contrast to the rapidly peaking TNF-α mRNA level, the TNF-α protein level was not significantly altered until 1 day after noise exposure, and the increase was more pronounced in AI of the right hemisphere than the left hemisphere . The noise-induced deramification was limited to the auditory cortex and was not observed in the neighboring visual cortex . Taken together, our results so far indicate that monaural exposure to loud noises results in more pronounced neuroinflammation in the contralateral AI, as indicated by increased expression of proinflammatory cytokines and activation of microglia.During neuroinflammatory responses, microglia are activated and change their morphology from the ramified shape to a nonramified, amoeboid shape , 44. We F1,59 = 6.551, P = 0.013; exposure effect, P < 0.001 for the wild-type group, P > 0.5 for the knockout group; right side, effect on genotype, F1,61 = 30.352, P < 0.001; exposure effect, P < 0.001 for the wild-type group, P > 0.05 for the knockout group). These results indicate that TNF-α knockout mice do not develop the type of neuroinflammatory response that we have observed in AI of wild-type mice after noise exposure.TNF-α is a central organizer of neuroinflammatory responses –51. Our F1,36 = 65.366, P < 0.001; pairwise comparison: P < 0.05 for all four frequencies tested). By contrast, noise exposure did not change gap detection performance in TNF-α knockout mice . A direct comparison of the gap detection performance after noise exposure indicates that it was significantly worse in wild-type mice than in TNF-α knockout mice . We also examined the prepulse inhibition (PPI) of the startle response as a measure of how well the mice could hear the tones used in the gap detection test and to determine if hearing loss contributed to the observed impairment in gap detection. While PPI performance was not changed by noise exposure in the TNF-α knockout mice , it was significantly improved in the wild-type mice after noise exposure . The lack of impairment in PPI performance in the control mice with monaural NIHL suggests that they could still detect the tones with the protected ear and that acoustic modulation of startle response is still intact in those mice. Furthermore, wild-type and TNF-α knockout mice showed similar ABR thresholds measured after the completion of the behavioral tests  and TNF-α knockout mice . Infusion of albumin did not have the same effect . Furthermore, the PPI performance was not significantly altered in wild-type or knockout mice by either recombinant TNF-α or albumin . These results suggest that the absence of noise-induced tinnitus in TNF-α knockout mice was due to the lack of TNF-α but not secondary developmental effects.To determine whether the absence of noise-induced tinnitus in TNF-α knockout mice was due to the lack of TNF-α or secondary developmental abnormalities, we infused recombinant TNF-α into the right AI of normal-hearing wild-type and TNF-α knockout mice. After 3 days of recovery, the infused animals underwent gap detection and PPI tests. Our results indicate that TNF-α infusion resulted in impaired gap detection at the frequency of 20 kHz in wild-type mice  and prevented an increase in TNF-α expression 12 hours after noise exposure . These results suggest that microglia are activated by noise exposure and contribute to the increase in TNF-α expression.Both microglia and neurons express TNF-α , 32, 54.F3,20 = 5.115, P = 0.009 amongst naïve, PLX [PLX3397], PLX/post noise, and post noise/PLX washout) without a treatment × frequency interaction . Pairwise comparisons indicated that gap detection was significantly improved by PLX3397 administration (P < 0.001). Noise exposure did not significantly alter gap detection performance in animals that had received PLX3397 (P = 0.33). Furthermore, after the washout of PLX3397 for 7 days, gap detection performance returned to the baseline level before PLX3397 administration (P = 0.005 comparing performances before and after washout and P = 0.24 comparing performances between naïve and after washout). The treatments had no significant effects on performance in the PPI test , suggesting that sound detection and startle performance were not altered by PLX3397 treatment or noise exposure. These results indicate that microglia contribute to noise-induced increase in TNF-α expression and tinnitus.To examine the effects of PLX3397 on noise-induced tinnitus, we performed noise exposure in a group of six mice that had been treated with PLX3397 for 21 days. Following the noise exposure, PLX3397 was given to the mice for another 10 days. Gap detection and PPI performances were examined at four stages: 1) before administration of PLX3397, 2) on the 21st day of the drug administration, 3) on the 10th day after noise exposure, and 4) 7 days after the withdrawal of PLX3397. Behavioral tests indicated that treatment with PLX3397 improved gap detection performance and prevented noise-induced tinnitus . A repeaF2,14 = 5.75, P < 0.05) and microglial morphological changes in AI 5 days after noise exposure . A similar dTT administration regime also completely prevented increases in the proinflammatory cytokines TNF-α, IL-1β, IL-18, and the inflammasome protein NLRP3 10 days after noise exposure . These results indicate that dTT administration after noise exposure prevents noise-induced increases in proinflammatory cytokines and microglial activation—two defining characteristics of neuroinflammation.To further investigate the role of neuroinflammation in tinnitus, we examined the effect of 3,6′-dithiothalidomide (dTT)—a TNF-α inhibitor that has been shown to reduce neuroinflammation—on noise-induced tinnitus. We treated mice with five daily injections of dTT starting within 1 hour after noise exposure was finished and found that dTT prevented the increase in TNF-α expression  or PPI . Furthermore, these dTT-treated and noise-exposed mice had elevated ABR thresholds that were not different from the thresholds of mice that received noise exposure without dTT treatment . Taken together, these results indicate that pharmacologically inhibiting neuroinflammation with dTT prevents noise-induced tinnitus.To examine the effects of dTT on noise-induced tinnitus, we treated mice with daily dTT administration for 5 days starting within 1 hour after the noise exposure. Gap detection and PPI performances were examined before and 10 days after the noise exposure. No significant differences were observed in either gap detection (n = 10) when compared to the sham-exposed control group (n = 16) , and this decrease was completely reversed by dTT treatment (n = 19) . The corresponding mIPSC amplitudes of the three groups were not significantly different . The frequency of mEPSCs increased in the noise-exposed and vehicle-treated group (n = 6) compared to the sham-exposed control group (n = 9) , and this too was prevented by dTT treatment (n = 5) . The three groups were not significantly different in their mEPSC amplitudes . These results confirm previous findings that NIHL results in increased excitatory and reduced inhibitory synaptic transmission, causing an excitatory-to-inhibitory synaptic imbalance, and suggest that this imbalance is largely mediated by noise-induced neuroinflammatory responses.TNF-α has been shown to change excitatory and inhibitory synaptic functions , 59–65. F1,193 = 1,796.414, P < 0.001; vehicle group, F1,136 = 690.958, P < 0.001). After noise exposure, the number of licks in sound trials decreased from 9.5 licks per trial before exposure to 9 licks per trial after exposure , whereas the licks in silent trials significantly increased for both dTT- and vehicle-treated groups , suggesting that the animals perceived phantom sounds . However, administration of dTT after noise exposure significantly reduced the number of licks in silence, suggesting that the drug alleviated tinnitus .To verify the role of neuroinflammation in noise-induced tinnitus, we used a different rodent model of tinnitus and our newly developed conditioning-based behavioral test . Awake rIn this study, we demonstrated a potential role of neuroinflammation in noise-induced tinnitus. We documented a series of neuroinflammatory events in the auditory cortex following noise exposure—a rapid bilateral increase of TNF-α mRNA levels, followed by a steady contralateral increase of TNF-α protein levels; a subsequent, predominantly contralateral change of microglia morphology; and an increased expression of additional proinflammatory cytokines IL-1β and IL-18. Microglial activation and proinflammatory cytokines—the two defining aspects of neuroinflammation—appear to depend on each other in noise-induced neuroinflammatory responses. Pharmacologically depleting microglia prevents noise-induced increases in TNF-α, whereas genetic deletion or pharmacological blockade of TNF-α prevents noise-induced changes in microglial morphology. This is consistent with existing literature showing that activated microglia release proinflammatory cytokines and that proinflammatory cytokines further activate microglia , 28, 44.Previous research reported that peripheral microglial activation and increased TNF-α expression occurred in the inner ear following noise trauma , 67–70. We exposed the left ear of the mice to a continuous 8-kHz tone at 112–114 dB SPL, centered at 8 kHz for 2 hours, with the right ear protected. Although the left ear was deafened rapidly within 20 minutes, the right ear continued to be exposed to the noise at a relatively loud level for an extended period during the noise exposure . Since aExposure to loud noise increases the expression of TNF-α and causes inflammatory responses in the cochlea , 67–70. Several types of brain cells can synthesize TNF-α, including microglia, neurons, and astrocytes , 54. We In the present study, noise-induced microglial deramification required an increase in TNF-α expression. Both genetic knockout of the TNF-α gene and pharmacological blockade of the increase of TNF-α expression prevented noise-induced microglial deramification. This is in contrast to blast-induced microglial deramification, which was not blocked by the same TNF-α blocker . It is pNIHL has been shown to up-regulate glutamatergic synapses and down-regulate gamma-aminobutyric acid (GABA) synapses in the auditory pathway , 6–10. THearing loss is a risk factor for tinnitus . We founAll procedures have been approved by University of California–Berkeley, University of Arizona, and Wayne State University IACUCs.tm1Gkl)  and Sprague-Dawley rats  were used in the experiments.C57BL/6J and TNF-α knockout mice  and xylazine  and maintained at 36.5°C with a homeothermic heating pad . Unilateral NIHL was induced by playing a continuous 112–114 dB SPL noise centered at 8 kHz through a custom-made piezoelectric earphone speaker to the left ear for 2 hours. The right ear was protected with sound attenuating clay. The sound level was measured with a Bruel and Kjaer 4135 condenser microphone .Under ketamine anesthesia, ABR tests were performed to assess hearing thresholds before and at various time points after noise exposure. ABR signals were acquired using the BioSigRP software on a TDT RX5 Sys3 recording rig. Tone pips  were delivered to a single ear through a cannulated speaker at a rate of 19 times per second. The speaker was calibrated to have <3% harmonic distortion and flat output in the entire frequency range . Five hundred recordings were averaged for each frequency intensity pair. ABRs were visually identified by an experienced experimenter who was “blind” to the animals’ group identities. The lowest sound level that evoked discernable ABRs was determined at each testing frequency and recorded as the ABR threshold for that frequency.PLX3397  was applied in a suspension containing 2% dimethyl sulfoxide (DMSO), 1.5% Tween80, and 1% carboxymethyl cellulose at a concentration of 5 mg/mL. The naïve mice were given daily 30-mg/kg PLX3397 injections (IP) for 21 days and were randomly assigned into PLX/NIHL or PLX/naïve groups. The control group was treated with injections containing 2% DMSO, 1.5% Tween80, and 1% carboxymethyl cellulose at 6 mL/kg. The TNF-α mRNA level in AI was detected 12 hours after noise exposure.dTT (ACME Bioscience) was prepared as a suspension in 1% carboxymethyl cellulose at a concentration of 50 mg/mL. The noise-exposed mice were randomly assigned into the NIHL/dTT group and were given daily 56-mg/kg dTT injections (IP). The other noise-exposed mice were assigned into the NIHL/vehicle group and treated with vehicle (1% carboxymethyl cellulose at 1.1 mL/kg) injection. The dTT and vehicle treatment started within 1 hour after noise or sham exposure and lasted for 5 days.Mice were anaesthetized with ketamine (100 mg/kg IP) and xylazine (10 mg/kg IP). An injection of recombinant TNF-α was placed stereotactically to the right auditory cortex through a burr hole that was made on the temporal ridge 1.75 mm anterior from the junction between the temporal ridge and the transverse suture. A glass micropipette filled with the recombinant mouse TNF-α  or mouse albumin fraction V (1 mg/in ACSF) solution was lowered down 500 μm from the pial surface, and 5 μL of solution was injected at 100 nL/min by pressure injection . The micropipette was then retracted 250 μm, and an additional 5 μL of solution was injected. To minimize leaking, the micropipette was left in place for 8 minutes before being withdrawn. Animals were allowed 3 days for recovery before undergoing gap detection and PPI tests.During the testing session, a mouse was caged in a plastic container with a mesh lid. The container was placed on a piezoelectric sensor in a sound attenuation chamber. Sounds were played through an open field speaker (FOSTEX FT17H) fixed above the container. The gap detection task measured the acoustic startle response elicited by a brief white noise pulse and its suppression by a preceding silent gap embedded in a continuous background sound. Each trial started with a carrier pure tone  played for a duration of 10–20 s. In uncued trials, the carrier tone was followed by a startle stimulus—a 50-ms white noise burst at 102 dB SPL. In cued trials, a 50-ms silent gap in the background sound was introduced starting 100 ms before the onset of the loud noise burst. In each testing session, the animal underwent a total of 500 trials (50% cued and 50% uncued). After each session, we calculated the startle response ratio, which is defined as the average startle amplitude in the silent gap-cued trials divided by the average amplitude in the uncued trials. A lower startle response ratio indicates better detection of the silent gap. A startle response ratio of 1 suggests that the animal failed to detect the silent gap.To assess an animal’s ability to hear a sound and perform an auditory task, animals underwent the PPI test before and after noise exposure. The test apparatus for the PPI task was identical to that of the gap detection. However, the test differed in that carrier tone was absent and a white noise burst was cued by a 50-ms pure tone pulse . In short, the PPI task tests an animal’s ability to detect a pure tone pulse in silence, while the gap detection task measures an animal’s ability to detect a silent gap in a continuous pure tone.Gap detection and PPI performances were stabilized over two daily sessions each before data were collected. We compared individual animals’ performance before and after noise exposure or drug administration. An increase of gap ratio accompanied by i) normal ABR threshold for the intact ear and ii) normal PPI behavior were taken to indicate tinnitus. Because both the gap detection task and the PPI task require normal hearing and hearing sensitivity was highly variable across animals above 32 kHz, only trials with carrier frequencies between 5 and 20 kHz were included in the final analysis.The behavioral test and experimental procedures were the same as previously reported . BrieflyMice were deeply anesthetized with isoflurane. The AI area tissue was collected and stored in 100 μL RIPA buffer  at −80°C. Tissues were homogenized in RIPA buffer by sonication. The total RNA was extracted by RNeasy Mini Kit . Immediately following extraction, the total RNA concentration and A260:A280 ratio of each sample were determined via NanoDrop 2000 (Thermo Scientific). The High Capacity cDNA Reverse Transcription kit (Thermo Fischer) was used to generate cDNA in a thermal cycler (ABI9700) for 2 hours at 37°C. Ten nanograms of cDNA were used in each reaction for real-time PCR using the CFX96 Real-Time PCR system . Threshold cycle (Ct) values of the target genes were normalized to the endogenous control gene . The primer sequences are listed as Mice were transcardially perfused under deep anesthesia with ice-cold PBS, followed by 4% paraformaldehyde. Brains were removed and fixed in the same fixative overnight at 4°C, equilibrated in 30% sucrose, and embedded in Tissue-Tek (Sankura Finetek). Frozen coronal sections (16 μm) were collected on gelatinized glass slides. After air drying, sections were washed in PBS and penetrated with 0.1% Triton-X at room temperature for 10 minutes. The samples were blocked with Dako Serum-free blocking buffer (Dako) and incubated with primary anti-IBA1 Polyclonal antibody  overnight at 4°C. The secondary antibody conjugated with Alexa Fluor 568 (Invitrogen) were incubated for 1 hour at room temperature to enable fluorescent detection. After rinsing with PBS, the sections were mounted with fluorescence mounting medium (Dako) and viewed under the Olympus BX40 microscope with a digital microscope camera . Immunofluorescent images were systematically and randomly sampled from the AI area with the experimenters “blind” to the sample groups. Sample images were smoothed with a median filter and thresholded to remove background pixels according to the control images. The filter parameters and the threshold values were fixed across all groups for each experimental condition. All the images for the same experiment were taken on the same day using the same parameters. Analysis of the IBA1 staining was performed with the software Image J. Based on area coverage, the microglial cell size and the cell body size were determined manually. The soma-to-whole cell size ratio of the IBA1-stained cells was used to measure microglial deramification in the AI area , 48.2/5% CO2) external solution . The brain was sectioned along the transverse plane into 300-μm slices with a vibrating microtome , and brain slices were stabilized in oxygenated external solution in a tissue chamber for 30 minutes at 32.5°C, followed by acclimation to room temperature (22.5°C) for at least 30 minutes before recordings. All recordings were performed at room temperature.Mice were deeply anesthetized with isoflurane and then decapitated. The brain was extracted and immediately placed in an oxygenated  at a rate of 2 mL/min. A fixed stage microscope  equipped with differential interference contrast optics and a 40x water-immersion objective was used to visualize individual neurons in the auditory cortex.The chamber was continuously perfused with artificial cerebrospinal fluid (ACSF) . In whole-cell configuration, the initial access resistance typically ranged from 15 to 23 MΩ and remained stable during the recording session.Series resistance was also continuously monitored with a brief voltage pulse. Recordings were accepted when a cell had a resting membrane potential of at least −68 mV and a series resistance of 15–25 MΩ (<20% change during the recording session). mEPSCs were recorded from pyramidal neurons in Layers 2/3 in oxygenated ACSF solution containing 1 μM tetrodotoxin (TTX), with the cell held at −80 mV. mIPSCs were recorded in oxygenated ACSF solution containing 1 μM TTX, 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 100 μM d-2-amino-5-phosphonovalerate (d-AP5), with the cell held at 0 mV. Signals were low-pass filtered at 2 kHz. All recordings were performed with Multiclamp 700B amplifier (Molecular Devices) and with pCLAMP10 software (Molecular Devices), and data were analyzed using MiniAnalysis software (Synaptosoft).Statistical analyses were conducted using MATLAB and SPSS (IBM). One-sample Kolmogorov–Smirnov tests suggest that our startle response ratio in gap detection and PPI tests did not significantly deviate from normal distributions . ANOVAs S1 DataThis file contains individual numerical values used to generate figures in this manuscript.(XLSX)Click here for additional data file.


Megachile rotundata) was interrupted at the red-eye stage and were transferred to 6°C with a 1-h pulse to 20°C and returned to 6°C. RNA was collected before, during, and after the temperature pulse and compared to pupae maintained at a static 6°C. The warm pulse is sufficient to cause expression of transcripts that repair cell membrane damage, modify membrane composition, produce antifreeze proteins, restore ion homeostasis, and respond to oxidative stress. This pattern of expression indicates that even brief exposure to warm temperatures has significant protective effects on insects exposed to stressful cold temperatures that persist beyond the warm pulse. Megachile rotundata’s sensitivity to temperature fluctuations indicates that short exposures to temperature changes affect development and physiology. Genes associated with developmental patterning are expressed after the warm pulse, suggesting that 1 h at 20°C was enough to resume development in the pupae. The greatest difference in gene expression occurred between pupae collected after the warm pulse and at constant low temperatures. Although both were collected at the same time and temperature, the transcriptional response to one FTR cycle included multiple transcripts previously identified under long-term FTR exposure associated with recovery from chill injury, indicating that the effects of FTR occur rapidly and are persistent.The response of ectotherms to temperature stress is complex, non-linear, and is influenced by life stage and previous thermal exposure. Mortality is higher under constant low temperatures than under a fluctuating thermal regime (FTR) that maintains the same low temperature but adds a brief, daily pulse of increased temperature. Long term exposure to FTR has been shown to increase transcription of genes involved in oxidative stress, immune function, and metabolic pathways, which may aid in recovery from chill injury and oxidative damage. Previous research suggests the transcriptional response that protects against sub-lethal damage occurs rapidly under exposure to fluctuating temperatures. However, existing studies have only examined gene expression after a week or over many months. Here we characterize gene expression during a single temperature cycle under FTR. Development of pupating alfalfa leafcutting bees ( Insects respond to temperature stress in a way that is non-linear and is influenced by life stage and previous thermal exposure . Cold toMegachile rotundata, the alfalfa leafcutting bee, is an extensively studied system for investigating indirect chill injury with a well-characterized, beneficial response to FTR  cold storage  were obtained from JWM Leafcutters, Inc. . Pre-pupae were stored at 6°C under darkness to maintain diapause until the start of the experiment. Prior to the experiment, pre-pupae were transferred to a 29°C incubator to initiate development in cell culture plates. Three replicate plates were used for each sampled time point. Pre-pupae were placed in two reporter plates to monitor development. After 50% of the bees in the reporter plates had developed to the red-eye stage , bees were transferred to FTR and CT treatments to interrupt development. The CT treated bees were stored at a CT of 6°C under darkness. The FTR treatment was exposed to 6°C with a warm pulse of 20°C which occurred between 11:00 and 12:00 with a 1 h ramp to 20°C, 1 h incubation at 20°C, and a 1 h ramp down to 6°C  extraction. mRNA was extracted using the Trizol protocol. Quality assessment and quantification were performed by Nanodrop and Qubit. RNA was shipped on dry ice to Georgia Genomics Facility for sequencing. Prior to sequencing quality was assessed using a Bioanalyzer. Paired-end libraries were generated from three replicates per time point. Illumina sequencing was performed on one high volume NextSeq 500 flowcell. Quality of the resulting sequence reads was assessed using FastQC (v0.11.7) . OverrepM. rotundata genome (accession: GCF_000220905.1) using Hisat2 (v2.1.0)  . Mapped (v2.1.0)  and asse(v2.1.0)  was used(v2.1.0)  and RStu(v2.1.0) . The R p(v2.1.0)  was used(v2.1.0) . Jensen–(v2.1.0) . Princip(v2.1.0)  on the F(v2.1.0) .M. rotundata genes were identified in the Apis mellifera genome  using standalone NCBI-BLAST+ (v2.8.1) (Orthologous (v2.8.1)  and a Py(v2.8.1) , 2009b u(v2.8.1)  and enri(v2.8.1) , OrthoDB(v2.8.1) , and Fly(v2.8.1) .M. rotundata identified by M. rotundata genome in the most recent genome release was applied by sequence alignment using NCBI-BLAST+ (v2.8.1)   to facil(v2.8.1)  and DB BM. rotundata genome (accession: GCA_000220905.1) leaving 11.0 million reads (28.3%) unmapped , Delta 11 acyl-CoA desaturase (LOC100881714), and desaturase/reductase enzymes  modify fatty acid chains to influence membrane fluidity. Intracellular lipid transport proteins include two long-chain fatty acid transporters , CRAL-TRIO domain lipid-binding transporters  is up-regulated between treatments at T2 versus CT. Other known cryoprotectants were not differentially expressed, including genes that synthesize trehalose, glycerol or other polyols, and sorbitol. Three membrane-bound ion transport channel proteins  including the HOX gene homothorax, dachsous which mediates imaginal disc development and cellular adhesion, and expanded which regulates Hippo signaling and cell proliferation during development . Analysis of enriched GO terms under FTR shows decreased expression of membrane-bound transport proteins during the warm pulse  versus up-regulated . This is a result of the disproportionate 71.74% down-regulation present in the original study. Common transcripts regulate transcription during development, are involved in metabolic processes, or are membrane-bound transport or signaling proteins , the North Dakota State University College of Science and Mathematics and the Department of Biological Sciences.The dataset supporting the conclusions of this article is included within the article . Sequence reads associated with the mRNA sequencing analysis are archived at NCBI (BioProject: PRJNA528472).ICB online.icz096_Supplementary_DataClick here for additional data file.


Kristi S. Anseth. Prof. Anseth is a Distinguished Professor and the Tony Tisone Endowed Chair in Chemical and Biological Engineering, Associate Professor of Surgery, and the Associate Director of the BioFrontiers Institute at the University of Colorado Boulder. She was the first engineer to be named a Howard Hughes Medical Institute (HHMI) Investigator and is one of a select few individuals elected to all three United States National Academies  as well as to the National Academy of Inventors for her major contributions to the fields of biomaterials, tissue engineering, and regenerative medicine.In this issue of Prof. Anseth earned her B.S. in Chemical Engineering with Highest Distinction from Purdue University where she performed undergraduate research in the laboratory of Prof. Nicholas Peppas. She completed her PhD in the Chemical Engineering department at the University of Colorado Boulder under the advisement of Prof. Christopher Bowman; her doctorate was completed in just 28 months and resulted in 10 first‐author publications. Upon graduation, she assumed postdoctoral research at the Massachusetts Institute of Technology with Prof. Robert Langer. In 1996, after just 1 year away from Boulder, she returned to her doctoral alma mater as faculty in the Department of Chemical Engineering at the University of Colorado.Repurposing traditional polymer chemistry approaches for applications in the biological sciences, a theme that permeates much of her research, Prof. Anseth's early efforts sought to synthesize and characterize biodegradable polymeric materials for controlled drug release. Pairing reaction kinetics and polymer network theory, she established powerful statistical models to predict bulk material degradation over time.Prof. Anseth has demonstrated that many of the same polymer chemistry methodologies used for controlled drug delivery can also be exploited to synthesize scaffolds for three‐dimensional 3D) cell culture. She was among the first to show that traditional photopolymerization reactions could be performed in the presence of live cells,D cell cuPopular Science's “Brilliant 10” and AIChE's “One Hundred Chemical Engineers of the Modern Era.”Recognizing that biological tissue composition varies tremendously over time, particularly during development and disease, Prof. Anseth has pioneered the creation of dynamic materials whose physical and chemical properties can be externally modulated on demand and in the presence of living cells.Prof. Anseth's innovative approach to engineering biomaterials continues to yield new understandings in fundamental biological processes as well as translational advances in a variety of applications. Taking advantage of photodegradable materials that can soften with the flick of a switch, she was the first to demonstrate the concept of “mechanical memory” whereby future cellular decisions are influenced heavily by past culture conditions.Bioengineering and Translational Medicine, Prof. Anseth served on the editorial boards for journals including PNAS, Biomacromolecules, and the Annual Review of Chemical and Biomolecular Engineering.Throughout all of her research success, Prof. Anseth makes a special point to give back to the community. She served as the President of the Materials Research Society, the Chair of the Frontiers of Engineering Program for the National Academy of Engineering, and was recently elected as a member of AIChE's Board of Directors. She has chaired and organized many conferences and symposia to promote graduate education and science alike and has helped identify future opportunities in biomaterials research on NSF steering committees. In addition to Above all, Prof. Anseth is a fantastic educator. She has mentored over 45 graduate students and 25 postdoctoral fellows in her laboratory, placing an even split of her trainees in academic and industry positions. Her uncanny ability to successfully educate both in the lab and the classroom has been recognized through the highest teaching awards given by her department, college, and university, as well as through a Camille Dreyfus Teacher‐Scholar Award. She remains a life‐long mentor to each of her mentees, always willing to take a call to celebrate the highs or help move past the lows. On behalf of her past, present, and future trainees, I express my sincerest gratitude for Kristi's scientific excellence and her unmatched selflessness to the biomaterials community.


Influenza virus continues to be a major health problem due to the continually changing immunodominant head regions of the major surface glycoprotein, hemagglutinin (HA). However, some emerging vaccine platforms designed by biotechnology efforts, such as recombinant influenza virus-like particles (VLPs) have been shown to elicit protective antibodies to antigenically different influenza viruses. Here, using biochemical analyses and cryo-electron microscopy methods coupled to image analysis, we report the composition and 3D structural organization of influenza VLPs of the 1918 pandemic influenza virus. HA molecules were uniformly distributed on the VLP surfaces and the conformation of HA was in a prefusion state. Moreover, HA could be bound by antibody targeting conserved epitopes in the stem region of HA. Taken together, our analysis suggests structural parameters that may be important for VLP biotechnology such as a multi-component organization with (i) an outer component consisting of prefusion HA spikes on the surfaces, (ii) a VLP membrane with HA distribution permitting stem epitope display, and (iii) internal structural components. The major influenza surface glycoprotein, hemagglutinin (HA), mediates viral entry by undergoing a prefusion to postfusion conformational change and some broadly neutralizing antibodies target conserved epitopes on stem regions displayed only on the prefusion state of HA6. Although the use of structure-guided design approaches is gaining more attention in efforts to evaluate epitope conformation and display with the goal to improve the immunogenicity and efficacy of nanoparticles for a number of viral systems9, efforts to invoke structure-guided design of membrane containing vaccine VLPs that are pleiomorphic in structure has not been studied to a great extent. This may be because many nanoparticle platforms are refractory to structural techniques, such as X-ray crystallography, due to possible sample flexibility and pleiomorphy that impede crystallization. Here, we refer to nanoparticles as particles without a membrane and virus-like particles (VLPs) as nanoparticles with a membrane. Cryo-electron microscopy (Cryo-EM) is a structural biology technique in which structures with membranes, flexibility and pleiomorphy can be studied10. Due to the pleiomorphy of influenza virus, it has been studied by cryo-EM methods13.Enveloped viruses, such as influenza, infect millions of people world-wide on an annual basis. During the 1918 pandemic the influenza virus killed 50 to 100 million people14.However, the 3D structural organization of influenza vaccine VLPs has not been addressed to a large extent nor has comparison between vaccine VLPs and virus particles. Thus, the structure guided-design and characterization of influenza VLPs has not been explored in great detail. Lack of such information on the 3D molecular structure of influenza VLPs hinders studies on the role of particle organization on facilitating and stabilizing HA epitope display for optimizing membrane-containing immunogens for influenza vaccines. Here, we focused on biochemical and cryo-electron microscopy analysis of recombinant H1 HA VLPs because this HA subtype represents viruses that have caused 1918 and 2009 influenza pandemics. Also, in previous studies, these VLPs produced very broadly protective responses including to challenge with lethal doses of influenza viruses expressing different HA subtypesWe report that VLPs were dominated by spherical morphologies that were similar in sizes to influenza virions. The VLPs could be generalized as being constructed of multiple structural components. HA spike components were projecting from the VLP surfaces and were embedded in the membrane component. HA molecules appeared to be in a prefusion conformation and immunoassays with antibodies indicated epitope display and integrity of conserved stem epitopes. Also, VLPs contained internal component material. In terms of overall future prospects, our findings suggest that the structure-guided evaluation and design of influenza HA VLPs could be used to optimize the design of VLPs to carry immune factors or other influenza antigens. Such designed VLPs could promote broadly protective immune responses to different strains and subtypes of seasonal and pandemic influenza viruses. In addition, influenza VLPs could be systems used to elucidate general design principles of VLP platforms for biotechnology vaccine efforts focused on viral glycoproteins.14. In order to determine the relative purity and protein composition of VLPs, SDS-PAGE analysis and mass spectrometry of protein bands were performed. Also, to study the organization of VLPs, they were imaged by cryo-electron microscopy. Proteomic analysis revealed that hemagglutinin (HA) was the most abundant protein in the sample  H1 and matrix (M1) as previously reportedple Fig. –c and HAple Fig. . A majorple Fig. . Based ople Fig. . Of noteple Fig. . Peptideple Fig. . The majple Fig. . HA appeBecause hemagglutinin, matrix (M1), and nucleoprotein (NP) proteins comprise the majority of the influenza structural proteins Fig. , we analBecause influenza virus particles can have both spherical and filamentous morphologies Figs , S4, theThe ellipticity of VLPs was assessed by the ratio of the major axis to the minor axis (i.e. aspect ratio), where a circle is an ellipse with an aspect of ratio of 1. By convention for an ellipse the major axis is the longest axis. The average aspect ratio ranged from 1.0 to 3.3 across all VLPs. However, the VLP population had a distribution that was highly skewed towards 1.0 Fig. . The larThe distribution of particle sizes and aspect ratios was visualized using heat-map analysis of the VLP lengths of the major axes versus the VLP aspect ratios Fig. . This waHA spikes were observed on the surfaces of VLPs Figs , S6. In To understand the 3D conformation of HA and how HA molecules were arranged in 3D over the surfaces of VLPs, cryo-electron tomography was used to obtain 3D structures of particles. Tomograms (3D volumes) of particles were examined by slicing through the volumes  and the organization and conformation of constituent hemagglutinin (HA) molecules, we studied VLPs made with the H1 HA from the 1918 influenza pandemic virus. VLPs were studied by biochemical analysis along with structural studies by cryo-electron microcopy and tomography coupled to image analysis. Our results indicated size variation among the VLPs but with most VLPs being ascribed to a spherical morphology. HA was the major component of the VLPs and hundreds of HA molecules were displayed on VLP surfaces with HA in an apparent prefusion state. Despite having an almost undetectable amount of the internal matrix (M1) protein, the VLPs did contain internal component densities. Inclusion of internal components (i.e. molecular cargo) was observed in all VLPs. We postulate based on mass spectrometry Table  that int17. In previous studies purified influenza virus particles were subjected to proteomic analyses using mass spectrometry to identify cellular proteins17. Analyses of viruses from mammalian and avian cells identified host proteins in categories such as cytoskeletal proteins, annexins, glycolic enzymes, and tetraspanins16. Other studies of virus-host protein interaction networks (i.e. interactome) for influenza virus identified cellular factors such as plakophilin 2  that could restrict influenza replication17. For our study, SDS-PAGE, immunoassays, cryo-electron microscopy and peptide-finger printing identified influenza H1 HA as the major component of 1918 influenza VLPs  composition maybe important because compositional studies of influenza virus particles from different cells identified cellular factors that may function in influenza virus assembly, entry, and replication14. Based on these results, we suggest that some cellular proteins of Sf9 cells could be involved in influenza VLP assembly, but HA remains the dominant component of the VLPs  were to HA, while the next highest matches of peptides were to cellular proteins of Sf9 cells: heat shock cognate 70 (n = 6), heat shock protein 70 A1 (n = 4), actin (n = 4) peptide matches. Only one peptide (n = 1) matched to the matrix protein M1 and only one peptide (n = 1) to an open reading frame from baculovirus Table . Matrix LPs Fig. . WhetherLPs Fig.  and onlyLPs Fig. . One not29. Furthermore, the dominant method for VLP production has been the co-expression of matrix M1 with one or all viral glycoproteins, HA, NA and M2. Influenza viruses can have spherical and filamentous morphologies12  have been implicated in virus assembly, budding, and morphologys12 Figs , S4. Int13. In this study, we present supplemental work of imaging influenza viruses to demonstrate the spherical and filamentous natures of influenza viruses under similar imaging conditions as VLPs  and nanoparticle production. On rare occasions, smaller VLPs were observed inside of larger ones by cryo-electron microcopy  induced long-lasting mucosal immunity against H3N2 viruses. This was termed as a mucosal adjuvant41. Thus, our observation of influenza VLPs with proposed internal cellular components would be on a continuum of future studies that maybe important in further engineering influenza VLPs with improved immunogenicity. This could be done by encapsulating conserved influenza epitopes or cellular proteins that can boost the immune responses by engineering in membrane-targeting signals such as GPI-anchors onto proteins. Such structure-guided design of novel influenza VLPs could lead to more efficacious influenza vaccines that protect against different strains and subtypes and aid universal influenza vaccines efforts47.The 3D structural observations on apparent plasticity of content packaging and arrangement of influenza vaccine VLPs as described in this work may have bearing on understanding some previously reported influenza VLP biotechnology engineering work that did not present detailed 3D cryo-EM analysis. For example, cellular proteins have previously been engineered to be assembled into influenza VLPs and other non-influenza viral epitopes have been fused to VLP glycoproteins48. In the case of influenza, some anti-HA antibodies can block fusion activity by binding to the prefusion state of HA and inhibiting the conformational changes needed to mediate fusion. Hence, there is great interest in displaying influenza HA and other viral glycoproteins in a prefusion state on vaccine immunogens such as membrane-containing VLPs49 and non-membrane containing nanoparticle complexes52. Thus, a number of vaccine efforts have focused on the development of more efficacious influenza vaccines that would elicit antibodies to the more conserved stem region of HA that is involved in mediating fusion55. One reported vaccine strategy to elicit broadly neutralizing antibodies to conserved HA epitopes in the prefusion state was to engineer HA proteins with the stem region only in that the HA head region was removed. Also, in another method chimeric HA proteins with antigenically different head regions but with conserved stem regions were used to boost stem antibodies57. Concerning the 1918 VLPs in this study, they were shown to be efficacious in animal models58, but the assessment of reactivity with stem antibodies and structural conformational state of HA within the VLPs were in not explored in detail. In this study, we showed by cryo-electron microscopy that HA on 1918 VLPs were consistent with a prefusion state  H1 VLPs were purified from a baculovirus expression system via the co-expression of HA from influenza virus A/South Carolina/1/1918 (H1N1) and matrix protein M1 of influenza virus A/New York/312/2001 (H1N1), respectively14. Total protein concentration was determined to be ~0.84 μg/μl based on bicinchoninic acid (BCA) protein assay (ThermoFisher). VLP purity and protein composition along with HA cleavage status were analyzed biochemically by methods such SDS-PAGE and subsequent peptide fingerprinting by mass spectrometry. Samples were analyzed under reducing conditions with the reducing agent dithiothreitol (DTT) at a final concentration of 100 mM and heated at 95 degrees Celsius for 10 minutes. Gels were stained over-night with coomassie blue stain  for protein band visualization and then subsequently subject to densitometry analysis. Gels were scanned and digitized into images with a gel documentation system (Enduro GDS). One-dimensional profiles of the gel lanes for VLP and molecular weight standards were calculated and areas under profile peaks were analyzed with the image processing software, Fiji60.Virus-like particles (VLPs) of the 1918 H1 VLP were prepared as previously reported61. The total numbers of PSMs from searches were 174 from the influenza database, 23 from the Sf9 cell database, and 1 from the baculovirus database. Within the influenza virus database search 99.4% of the PSM were to hemagglutinin and the highest match was 108 PSMs to an influenza hemagglutinin H1: Accession (Q9WFX3), Description (Hemagglutinin OS = Influenza A virus (strain A/Brevig Mission/1/1918 H1N1) GN = HA PE = 1 SV = 2 − [HEMA_I18A0]. Sequences were from Gene Bank: AF117241.1 for HA H1 of Influenza A virus (A/South Carolina/1/1918 (H1N1)); Q9WFX3.2 for HA H1 of influenza A virus (A/Brevig Mission/1/1918 (H1N1)). Sequence alignment between the H1 HA sequences of H1 of influenza A/South Carolina/1/1918 (H1N1) and H1 of A/Brevig Mission/1/1918 (H1N1) was done with the program EMBOSS-Lite with the sequences being 100% identical. Identified peptides from mass spectrometry was mapped onto the H1 HA (1918) ectodomain crystal structure from the Protein Data Bank (PDBID 1RD8)62 using Chimera software package63. Further details on the concepts of Peptide Spectrum Match (PSM) and coverage in mass spectrometry can be found in the user guide for the Proteome Discoverer software .For peptide finger printing the major band at ~75 KDa of the VLP sample was excised from the gel and subjected to protease digestion to create tryptic peptides that were analyzed by mass spectrometry (MS). Acquired MS/MS spectra were searched against and mapped on the expected 1918 H1 HA sequence. Spectra were searched against three constructed proteome databases: (i) influenza virus, (ii) Sf9 cells, and (iii) baculovirus using the program SEQUEST and Fixed Value Peptide Spectrum Match (PSM) validator algorithms in the Proteome Discoverer 1.4 software Reactivity of H1 HA of VLPs with antibodies was assessed by western blotting and ELISA. Purified rabbit polyclonal antibody raised against purified recombinant H1 HA of influenza A/California/07/2009  was used as primary antibody to probe for reactivity to H1 HA 1918 VLPs by Western blot analysis using standard procedures. Blots were probed with secondary goat anti-rabbit HRP and subsequently visualized using SuperSignal West Pico chemiluminescent substrate  to expose film. The H1 rabbit polyclonal is cross-reactive to different HA H1s, but specific for H1 subtype and thus recombinant H3 HA (A/Wisconsin/67/05 (H3N2) and recombinant H1 HA (A/PR/8 (H1N1) proteins were used as positive and negative HA subtype controls, respectively. Thus, HA H3 is an influenza HA from group 2 influenza virus (negative control) and H1 from APR8 was a cross-reactive H1 . All recombinant HA proteins were full-length uncleaved HA0 and purified from baculovirus expression systems (Protein Sciences Corporation).6. Antibody C179 was from Takara Bio USA. In addition, 1918 H1 HA VLPs were probed via ELISA with the pan H1 rabbit polyclonal that had shown reactivity in western blot. ELISA plates were coated with samples at a concentration of 2 μg/ml, before incubating with C179 antibody or rabbit pan H1 polyclonal antibody (1.5 ug/ml). Samples were 1918 H1 HA VLPs and H3 virus (A/Victoria/3/75 H3N2) and recombinant H3 HA (A/Wisconsin/67/05 (H3N2)) protein as negative controls because C179 does not react with H3 HAs. Subsequent detection was with a horseradish peroxidase conjugated to goat anti-mouse IgG and goat anti-rabbit IgG for detection. Binding was quantified by the addition of ABTS substrate  and optical density was measured at a wavelength of 405 nm. Samples were in triplicate.Reactivity of 1918 H1 HA of VLPs to a stem antibody that requires a conformation epitope was probed by ELISA using the monoclonal antibody C179, which has been shown to bind to the conserved stem region of HA H1 2009 pandemic virus64, except that VLPs were stained with 1.5% phosphotungtic acid. Images were collected on a Tecnai 12 electron microscopy with LaB6 illumination operating at 100 kV  at nominal magnifications of 52,000x and 110,000x. Images were recorded on a 4k × 4k OneView camera . For cryo-electron microcopy, 3.5 μl of unstained VLPs were applied to glow discharged 200 mesh R2/2 Quantifoil Cu grids  and plunge frozen using a Vitrobot Mark IV plunger . Samples were imaged under cryo conditions at 300 kV on a Titan Krios electron microscope . Images were collected using EPU software  on a 4k × 4k Gatan Ultrascan charge-coupled-device (CCD) camera  1.9 Å pixel size . Images were collected with a defocus range of −3 to −5 μm and the electron dose ranged from ~10 to 20 e−/Å2. Individual VLPs from 2D cryo images were further analyzed by 2D image analysis.Negative-staining electron microscopy of VLPs was similar to that reported previously for other samples2. Images within the tilt series were mutually aligned by using the gold particles as fiducial markers and the 3D volumes (tomograms) were reconstructed as implemented the IMOD software package65.VLP samples were mixed with 5 or 10 nm gold particles as fiducials and plunge-frozen as above. Tilt series were collected using a GIF-2002 energy filter  coupled with a 2k × 2k MultiScan Gatan Ultra CCD . For lower defocus data, tilt series were collected using a Falcon II direct-detector camera using a Volta phase plate (VPP) . Zero loss energy filtered data was collected with a 70 μm objective aperture at 3.3 Å pixel size  with a defocus range of −3.5 to −5.5 μm. Tilt series collected with VPP on Falcon II was acquired at 2.9 Å pixel size  with a −2.5 μm defocus. All single-axis tilt series data were collected over −/+ 60 degrees with 2° tilt increments using Tomography 4.0 software . Tilt series datasets were collected under low-dose conditions with a cumulative electron dose of ~100 electrons/Å60. The major axis and minor axis of the fit ellipsoid were recorded. Morphology was assessed by the VLP aspect ratio, which was the major (longer) axis divided by the minor (shorter) axis. A histogram of the aspect ratios of the VLP population and a heat map of the aspect ratios vs. major lengths were used to analyze the relation between VLP aspect ratios and major VLP lengths. Histogram and heat-map analyses were with the R Project for Statistical Computing software package. VLP sizes and morphologies from different regions of the heat-map were then visually compared via their 2D cryo-EM images.Size parameters such lengths and shape morphology were measured from 2D cryo-EM images of VLPs (N = 295). The dark membrane perimeters appearing in VLP images were modeled as ellipses. Ellipses with varying major and minor axes were semi-automatically fit to VLP membrane perimeters using the image processing software, Fiji67. For VLP analysis a circular average was computationally created and the minima and maxima of the pixel values were normalized to 0 and 1, respectively. Density values were plotted in a 1D profile curve for the membrane-spike region of the VLP. For spike (hemagglutinin) profile analysis, 75 spike regions were picked and aligned and averaged as implemented in EMAN268. Box sizes were 256 by 256 pixels resulting in particle images having multiple but visually separated HA molecules. The selected particle images were cycled through multiple rounds of alignment and averaging, thereby creating a 2D image average with apparent side-by-side delineated HA molecules. The 2D average was computational rotated so that the middle HA was oriented with its long-axis in a horizontal direction with a resulting 1D density profile of the selected middle HA spike and membrane region. Coordinates of the H1 HA ectodomain crystal structure (PDBID 1RD8)62 were overlaid on the HA 2D average from cryo-EM on the same scale in Chimera. Subtomogram averaging of HA surface spikes on VLPs were performed similar to as previously reported for ring structures using the software Relion69. Coordinates (xyz) for spike centers (684) were manually selected using IMOD and passed to Relion to extract 3D volumes (subtomograms) of HA spikes and to carryout subtomogram averaging. Post-processing as carried out in the Relion software package indicated a resolution of 26.5 angstroms. The 3D subtomogram average was compared to HA ectodomain coordinates  representing prefusion and postfusion HA structures from the protein data bank70.2D profile analyses were of VLPs and glycoprotein spikes regions. For VLP profiles, individual spherical VLPs with no neighboring obstructions were picked for profile assessment from 2D cryo-EM images. 1D line profile values were extracted from VLP and HA images using Bsoft65. Line-segments were saved as two points with x, y, z coordinates and distances calculated with an in-house python program script. The student t-test showed that there was not a statistically significant difference between HA distances measured by tangential or central sections of VLPs. Because VLPs tomograms revealed internal components (molecular cargo) inside the VLPs, the VLPs were subsequently categorized according to the density of molecular cargo present. Three categories were devised as being low (<25%), medium (>25% to <50%), and high (>50%) pertaining to the relative amount of higher density components relative to lower density space inside the same VLP. VLP segmentations of spike-membrane regions from internal components were carried out with segmentation features within the Chimera software package63. Supplemental semi-automated methods were developed and used to determine the relative amounts of internal densities in VLPs for supplemental analysis that is detailed in supplemental methods.3D volumes (i.e. subtomograms) of individual VLPs were extracted from the tomograms that contained multiples VLPs. A total of 353 VLP subtomograms were analyzed. Spacing between HA spikes were measured using line segments traced on both near tangential and central slices of VLP tomograms as analyzed in IMODDatasets are available from the authors upon request.Supplemental materialMovie S1Movie S2Movie S3


H17 pathway has enabled the development of targeted therapies used in the clinic today. Particularly, TNF inhibitors have become a benchmark for the treatment of numerous chronic inflammatory diseases such as psoriasis. Although being highly effective in psoriasis treatment, anti-TNFs can themselves induce psoriasis-like skin lesions, a side effect called paradoxical psoriasis. In this review, we provide a comprehensive look at the different cellular and molecular players involved in classical plaque psoriasis and contrast its pathogenesis to paradoxical psoriasis, which is clinically similar but immunologically distinct. Classical psoriasis is a T-cell mediated autoimmune disease driven by TNF, characterised by T-cells memory, and a relapsing disease course. In contrast, paradoxical psoriasis is caused by the absence of TNF and represents an ongoing type-I interferon-driven innate inflammation that fails to elicit T-cell autoimmunity and lacks memory T cell-mediated relapses.Chronic plaque psoriasis is a common debilitating skin disease. The identification of the pathogenic role of the TNF/IL-23/T Upon recognition of autoantigens, T-cells produce TH17-cytokines such as IL-17A, IL-17F, and IL-22, which drive the psoriatic phenotype by inducing keratinocyte hyperproliferation. In support of this, several single nucleotide polymorphisms cluster throughout this pathway including genes in the TNF/dendritic cell activation pathway  as well as in the T-cell activation , and TH17/TC17-differentiation pathways   , LFA-1 a4β1 , and the4β1 . More spand CCR4 , whereasand CCR6 . Among tand CCR6 , 28, andand CCR6  producedand CCR6 . Moreoveand CCR6 . Indeed,and CCR6 , 20. Act+ T-cell subsets across chronic inflammatory diseases antigens in psoriasis. As mentioned above, epidermal CD8+ T-cells in psoriasis are key effectors in psoriasis antigens by CD8+ T-cells is pathogenic in psoriasis.Psoriasis has been studied extensively from a genetics perspective, with HLA class I alleles known for more than 40 years to be heavily implicated . The HLAsoriasis , and thesoriasis , 30—thusStreptococcus pyogenes has been identified as an antigen target of primarily CD8+ T-cells , neutrophils, and fibroblasts. One of its major roles is to mount appropriate adaptive immune responses to tumors and pathogens. This is achieved through several mechanisms. Induction of DC-maturation leads to upregulation of CD40, CD80, CD83, and CD86 thereby potentiating T-cell receptor (TCR)-mediated responses and amplifying weak antigen affinity interactions . It alsoor sites . These por sites –52. In doduction , 54.H17 cells , anti-p19 (IL-23 specific), anti-IL-17A, and anti-IL-17 receptor antibodies. This pathway and its pathogenic mechanisms in classical plaque psoriasis are illustrated in Figure Detailed knowledge about the pathogenesis of chronic plaque psoriasis and the central role for the TNF/IL-23/T+ T-cells immunity in psoriasis. Indeed, depletion of pDCs or blocking type-I IFN signalling both inhibited psoriasis development confirming that its overexpression by pDCs reflects a critical early/acute event in the pathogenesis of psoriasis  activating dermal myeloid DC, (2) inducing their maturation by upregulating co-stimulatory molecules and HLA molecules, and (3) participating in T activatiAlmost two decades of clinical experience with anti-TNFs have provided considerable advances in our understanding of the biology of TNF. More than 2 million patients have been treated with anti-TNFs so far.de novo psoriasis. Though the side effect is a well-established phenomenon, its pathogenesis had remained elusive and only recently, the dysbalance of TNF and type-I IFN (yin-yang of TNF and IFNα) has been confirmed as a pathogenic mechanism underlying paradoxical psoriasis  or inhibiting TLR 7 and 9, thereby blocking pDC activation, could provide more suitable therapeutic options in patients that need continuation of their anti-TNF treatment. In this way, production of type-I IFNs by monocytes and stromal cells would remain intact and might allow sufficient immune responses toward infectious agents.Though newer treatments targeting IL-23 and IL-17 show better efficacy, today, anti-TNFs remain a gold-standard in psoriasis management. Yet important immunological side effects of TNF blockade, such as paradoxical psoriasis and lupus-like syndrome, may require premature discontinuation of an otherwise effective treatment option for patients. These side effects are caused by an unabated type-I IFN-driven immune response making it an intriguing target in these patients. While, anti-IFNs have not shown efficacy in chronic plaque psoriasis confirming the distinct inflammatory pathways in chronic and acute forms of classical psoriasis , they prDespite considerable advances in the understanding of paradoxical psoriasis and its pathogenesis, several questions are still unanswered. Downstream mechanisms that mediate the interferon-driven psoriatic phenotype of paradoxical psoriasis remain unknown as IFNα does not directly induce keratinocyte hyperproliferation. The identification of cytokines involved and their cellular source might provide additional novel targets for therapeutic intervention. In addition, biomarkers to predict side effects such as paradoxical psoriasis and lupus-like syndrome could help optimising the management of patients with chronic inflammatory diseases.AM and CC wrote and edited the manuscript and figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heart failure with preserved ejection fraction (HFpEF) is less well understood than heart failure with reduced ejection fraction (HFrEF), with greater diagnostic difficulty and management uncertainty.The primary aim is to develop an optimised programme that is informed by the needs and experiences of people with HFpEF and healthcare providers. This article presents the rationale and protocol for the Optimising Management of Patients with Heart Failure with Preserved Ejection Fraction in Primary Care (OPTIMISE-HFpEF) research programme.This is a multi-method programme of research conducted in the UK.OPTIMISE-HFpEF is a multi-site programme of research with three distinct work packages (WPs). WP1 is a systematic review of heart failure disease management programmes (HF-DMPs) tested in patients with HFpEF. WP2 has three components  that enable the characteristics, needs, and experiences of people with HFpEF, their carers, and healthcare providers to be understood. Qualitative enquiry (WP2a) with patients and providers will be conducted in three UK sites exploring patient and provider perspectives, with an additional qualitative component (WP2c) in one site to focus on transitions in care and carer perspectives. A longitudinal cohort study (WP2b), recruiting from four UK sites, will allow patients to be characterised and their illness trajectory observed across 1 year of follow-up. Finally, WP3 will synthesise the findings and conduct work to gain consensus on how best to identify and manage this patient group.Results from the four work packages will be synthesised to produce a summary of key learning points and possible solutions (optimised programme) which will be presented to a broad spectrum of stakeholders to gain consensus on a way forward.HFpEF is often described as the greatest unmet need in cardiology. The OPTIMISE-HFpEF programme aims to address this need in primary care, which is arguably the most appropriate setting for managing HFpEF. HFpEF is poorly recognised and managed in the community. As HFpEF is set to become the dominant form of heart failure (HF), it is vital that effective primary care-based programmes of identification and management are developed. The OPTIMISE-HFpEF programme of research aims to fill this void.1 Half of all HF cases may be attributable to HFpEF, and prevalence is rising at a rate of 1% annually.3 Although mortality for all-cause HF in the UK has modestly improved, no treatment has yet been shown to improve mortality and morbidity in HFpEF.5 Lack of evidence for pathophysiological mechanisms underpinning the disease, effective pharmacotherapies, and disease management programmes specifically targeting HFpEF hamper progress. It is perhaps unsurprising then that clinicians may, to coin Oktay and Shah, 'approach HFpEF with diagnostic and therapeutic nihilism'.6HFpEF is less well understood than HFrEF and is associated with greater diagnostic difficulty and management uncertainty.https://www.optimisehfpef.phpc.cam.ac.uk/), in line with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement.7 This project aims to explore the views of people with HFpEF and the multiple stakeholders involved in HFpEF care; phenotype a UK cohort; and undertake consensus methods to develop an optimised programme that would provide guidance to clinicians in diagnosing and managing HFpEF.This article presents the rationale and protocol for the OPTIMISE-HFpEF research programme . They do not address or troubleshoot many of the key issues debated in HFpEF research; for example, the lack of specificity of echocardiogram to accurately capture diastolic left ventricle dysfunction,25 the frequent absence of echocardiographic evidence of structural heart disease in invasively confirmed HFpEF,26 and the poor sensitivity of natriuretic peptides in HFpEF.28 All the latter are key components of HFpEF diagnostic algorithms in the UK. Moreover, they lack the necessary focus on exercise, which has pleiotropic effects on systemic endothelial function and therefore improves symptoms and quality of life.29General practice has a key role in all parts of the HF patient pathway, from initiating diagnosis to long-term management;30 In HFpEF, the need to establish this is ever more pressing given the increasing prevalence, the burden of multimorbidity, and the complex interplay of medical, psychological, behavioural, and social factors for this group.31 Optimally managing a vulnerable and complex population of older adults, in a pragmatic and patient-centred way, is arguably better addressed by generalists familiar with patient preferences and priorities and the application of comprehensive and multidimensional assessment.32Others have already argued an effective pathway to achieve cohesive primary and/or specialist co-management remains elusive and the current structure of care is suboptimal.HFpEF is poorly recognised and managed in the community. As HFpEF is set to become the dominant form of HF, it is vital that effective primary care-based programmes of identification and management are developed. The OPTIMISE-HFpEF programme of research aims to fill this void by: (1) finding out what patients and providers want; (2) understanding what people with HFpEF are like in the UK, through detailed phenotypic characterisation of a large cohort; and (3) exploring common problematic areas like transitions of care , hospitalisations, and the support of carers see . ResultsThe primary aim is to investigate HFpEF with the view to developing an optimised programme that is informed by the needs and experiences of people with HFpEF and healthcare providers. A secondary aim is to gauge stakeholder consensus on the optimised programme using online and face-to-face consensus methods.33 The resulting mixed-methods approach consisting of five WPs is cognisant of these key elements  has been adopted.ents see  and has Multiple sites were involved to enhance the diversity of the sample in each cohort see .>50%); or (iii) have an echocardiogram reporting structural heart disease or diastolic dysfunction without moderate to severe systolic dysfunction. Although the European Society of Cardiology guideline is used to diagnose HFpEF9 in WP2b, this pragmatic definition was employed given the literature detailing the challenges of identifying patients with HF from primary care records.36Participants in WP2a and WP2b must meet the following inclusion criteria: adult patients with diagnosed or suspected HFpEF, and the ability to communicate in English. Diagnosed or suspected HFpEF was defined as anyone diagnosed with non-valvular HF that (i) were not diagnosed with left ventricular systolic dysfunction or have a documented ejection fraction (EF) <50%; or (ii) have a reported ‘normal’ or preserved EF . In brief, the NIHR CRN offers a range of support to help researchers plan, set up, and deliver research. In this case, assistance was sought to gain access to general practices that were provided with an electronic medical record screening algorithm to enable identification of potential participants. A GP within the practice screened against the inclusion and exclusion criteria before inviting eligible patients. Patients interested in taking part were asked to return an expression of interest form to the research team. The research team contacted those interested to discuss the study in detail  before written informed consent was sought see . PurposiA patient advisory group is active within the study, and continues to provide input.https://www.crd.york.ac.uk/prospero/). Eighteen studies met the inclusion criteria, representing 1866 patients with HFpEF. All studies were assessed against a ‘disease management programme taxonomy’ and scored for complexity, intensity, and bias. The review concluded HF-DMPs may improve mortality, hospitalisation rates, self-care, and quality of life in patients with HFpEF; however, further research specifically tailored to HFpEF is required.WP1 was a systematic review of controlled studies of HF-DMPs that included patients with HFpEF. Four bibliographic databases were searched, yielding 6089 titles that were screened against an agreed protocol .In WP2a and WP2c, sociodemographic data, interview notes and/or reflections, and interview recordings will form the main data sources. In WP2c, the caring relationship and arrangement will be documented and the Carer Support Needs Assessment Tool (CSNAT) completed.20 Refined characterisation of a representative, real-world cohort will help with effective targeting of the components in the final programme of care.WP2b will collect sociodemographic, behavioural, clinical, and quality-of-life data at baseline, 6, and 12 months . The aimThe results of findings from all the WPs will be integrated to produce a position article that will form the basis of the questions presented in the modified Delphi online elicitation approach. This methodology produces ratings and rankings of statements as well as qualitative online discussion data from expert panel members.https://www.optimisehfpef.phpc.cam.ac.uk/) and in line with the principles outlined in the International Conference on Harmonisation Good Clinical Practice guideline.38 WP2a and WP2c will use a semi-structured interview template that will guide the interview process while also allowing flexibility for exploration of specific issues. WP2b has a comprehensive Manual of Operations and Procedures to standardise practice in relation to outcome measure collection.All study procedures will be conducted in accordance with the respective protocols  and Microsoft Excel (version 14.0.7237.5000) software. A thematic framework (coding scheme) will be developed, before indexing, charting, describing, and interpreting the data.41 The analysis will be both inductive (data-driven) and deductive (theory-driven), with burden of treatment and minimally disruptive medicine models employed as sensitising frameworks.43 WP2c will follow a similar analysis pathway; deductive analysis will be driven by burden of treatment theory  and the CSNAT (carers).The main data source in WP2a and WP2c will be qualitative semi-structured interview recordings and verbatim transcripts. These will be analysed using the framework methodWhere possible, descriptive statistics  will be presented for all WPs. In WP2b, pre-specified baseline comparisons will determine differences by sex, body mass, and presence of frailty. In addition to robust clinical information, the cohort will be described according to patient-reported measures on symptoms, self-management, HF specific quality of life, and physical activity. Reported physical activity will be validated against information from activity monitors regarding both level of activity and time spent sedentary. A similar analysis will be conducted with data from the 6- and 12-month follow-up to determine changes in variables from baseline. Data on outcomes  will be collected over the 12-month period.45Analysis in WP3 will be managed via an online platform, which enables quantitative, categorical, and text data analysis from all rounds of the elicitation and is conducted in four stages: preliminary data analysis, analysis of agreement, data modelling, and analysis of changes in responses.Dissemination of the study findings will include at least one peer-reviewed article from each WP, and presentations at appropriate primary care and specialist conferences.This multi-method programme aims to explore the views of the multiple parties involved in HFpEF care; to characterise a UK cohort of HFpEF patients; and to explore hospitalisation, transitions of care, and carer burden with a view to informing the development of an optimised programme of care. The optimised programme would be developed using consensus methods via an online platform that would refine programme components and preliminarily assess feasibility. The final programme would then undergo further testing in a cohort of patients with confirmed HFpEF.The strengths of this programme are the patient, carer, and health professional focus and the particular emphasis on pragmatic, primary care-based solutions. Heart failure is usually identified and managed long term in the primary care setting, however there is an absence of much needed guidance and support available for GPs. The main limitation of this project relates to testing the findings of the consensus work (WP3); this would be subject to future funding.29 However, much of this research is in the highly specialist or tertiary setting, and there is a dearth of literature on the role of generalists in HFpEF identification, diagnosis, and management. Smeets and colleagues have proposed a multi-component optimisation intervention in the primary care setting (OSCAR-HF pilot study); this, however is in HF generally as opposed to HFpEF specifically.46There is a growing body of research in HFpEF owing to its status as the 'greatest unmet need in cardiology’.HFpEF is an underrecognised condition that poses many challenges for clinicians. This programme seeks to understand these problems from the perspective of patients, their carers, and the healthcare professionals who look after them, with a view to establishing an optimised programme of management that is cognisant of these issues.


Specifically, loss of LV contractions is associated with progressive synovitis and erosions. Furthermore, draining lymph node expansion is a biomarker of arthritic progression, and both macrophages and lymphatic endothelial cells express inducible nitric oxide synthase (iNOS), which disrupts LV contraction and transport of immune cells to the draining lymph nodes. Therefore, to directly assess these relationships, we tested the hypothesis that TNF-Tg mice with global genetic ablation of iNOS , were examined with (1) ultrasound to determine popliteal lymph node (PLN) volume and (2) near-infrared imaging (NIR) to assess popliteal LV contraction frequency, and differences between genotypes were assessed at 3, 4, 5, and 6 months of age. Knees and PLN were harvested at 4 months in females and 6 months in males, to assess synovitis, bone erosions, and cellular accumulation in PLN sinuses via histology.iNOS−/−× TNF-Tg and TNF-Tg compared to their WT and iNOS−/− counterparts at 2 and 3 months, respectively. Subsequently, TNF-Tg PLNs continue to increase in volume, while iNOS−/−× TNF-Tg did not increase in volume from the initial timepoints. WT and iNOS−/− PLN volume was unchanged throughout the experiment. LV contraction frequency was increased at 4 months in females and 5 months in males, in the iNOS−/−× TNF-Tg mice compared to the TNF-Tg. Synovitis and erosions were moderately reduced in iNOS−/−× TNF-Tg versus TNF-Tg knees in females, while no differences in knee pathology were observed in males.Initially, an increase in PLN volume was observed for both female and male iNOSGenetic iNOS ablation maintains draining lymph node volume and LV function during TNF-induced inflammatory arthritis and is associated with moderately decreased joint inflammation and damage. Rheumatoid arthritis (RA) is a chronic, debilitating, inflammatory disease of diarthrodial joints that affects 1.0% of the adult population worldwide [2+ flux which shortens myosin filaments and nitric oxide (NO) release which lengthens the filaments regulates lymph fluid movement via a series of contractions. Pathologic disruptions of these contractile events may contribute to buildup of fluid during chronic inflammation.During the course of inflammatory arthritis, excess fluid, cells, and macromolecules produced in inflamed joints are cleared from the interstitium by the synovial lymphatic system . Importa+/CD21hi B cells filled sinuses, and a loss of vessel contraction and stagnant CD11b+ cells within the vessel [Recent studies in the tumor necrosis factor-transgenic (TNF-Tg) mouse model of RA demonstrated a critical role of the lymphatic system in joint homeostasis . Detailee vessel , 9. The e vessel , 7, 9. He vessel , 10. ImpAnother finding of particular relevance to the potential contribution of the lymphangion in RA is that lymphatic vessels (LVs) undergo significant pathologic changes throughout the course of arthritis, which ultimately leads to the loss of lymphatic function. It is well known that monocytoid cells migrate to and proliferate in the synovium during inflammatory arthritis , 13. Int−/− mice were obtained from Jackson Laboratories (strain: B6.129P2-Nos2tm1Lau/J) and were crossed with TNF-Tg mice to obtain iNOS−/−× TNF-Tg+/−. All studies were performed with littermate wild-type or iNOS−/− controls. Due to the observed temporal differences in male and female TNF-Tg pathology [The present work was conducted with prior approval of the University of Rochester Medical Center University Committee for Animal Resources. The 3647 tumor necrosis factor-transgenic mouse lines were originally obtained from Dr. George Kollias , 20, areathology , male anB-mode and power Doppler ultrasound imaging was performed with the VisualSonics Vevo3100 using the MX700 probe  on the left and right PLN. LN volumes and normalized power Doppler (PD) signal were collected as previously described , 21. BriNear-infrared imaging was performed as previously described , 21, 22.At the terminal endpoints , popliteal lymph nodes and knees were fixed in neutral-buffered formalin and processed for paraffin-embedded histology. Sagittal sections of the knees and ankles were obtained, and one section from the medial compartment was stained with hematoxylin and eosin (H and E), and for tartrate-resistant acid phosphatase (TRAP), as we have previously described . B220-PE−/− presence) when appropriate. When significant three-way interaction effects were observed, Tukey’s post hoc tests were performed to investigate specific differences. When significant interaction effects were not observed, custom contrast t tests were performed between TNF-Tg mice with and without a functional iNOS gene at each timepoint with an adjusted alpha level of 0.016 to account for the three comparisons. A one-way ANOVA to assess differences in PLN histology was performed. A Wilcoxon sign rank test was used to test knee histology parameters.All statistical analyses were performed in JMP Pro 13 . All continuous variables were analyzed for normality via a Shapiro-Wilk test. For outcomes that were not normally distributed, a rank transformation was performed before further analysis. Mixed models for the longitudinal variables weight, PLN volume, NPDV, and contraction frequency with a repeated structure were used to assess the interactions between different combinations of factors . Importantly, there were no differences between male and female iNOS−/− or iNOS−/−× TNF-Tg mice at peak LN disease .Given the previously described sexual dimorphism in the LNs of the TNF-Tg mouse line , we inve−/− on this phenotype, we performed NIR lymphatic imaging in both male and female mice (Fig. −/−× TNF-Tg mice compared to TNF-Tg mice (Fig. −/−× TNF-Tg mice maintained their contraction frequency. This relationship disappeared at 6 months, in which iNOS−/−× TNF-Tg mice displayed a loss of contraction frequency compared to iNOS−/− mice.To understand LV function throughout the course of inflammatory-erosive arthritis, and the effect of iNOSice Fig. . NIR imaice Fig. a. In mal−/−× TNF-Tg female mice, and significantly, more cells were present in their sinuses compared to WT mice (Fig. −/−× TNF-Tg mice (Fig. −/−× TNF-Tg PLNs (Fig. −/−× TNF-Tg PLN sinuses, we conclude that iNOS unlikely regulates the migration of B cell into the sinuses.In previous work, we described the significant influx of B cells in draining LN and their migration from the follicular area into the LN sinuses , 9, 26. ice Fig.  A–E. Intice Fig. E. The loice Fig. F–J. To aLNs Fig. A, B. How−/−× TNF-Tg mice. Histomorphometry of the pannus tissue revealed a trend of decreased synovitis with significantly fewer immune cell clusters in iNOS−/−× TNF-Tg compared to TNF-Tg female knees (Fig. To assess the effects of iNOS ablation on TNF-induced inflammatory-erosive arthritis, we performed histomorphometry on the knees from 4-month-old female and 6-month-old male TNF-Tg and iNOSees Fig. a–f. Howeees Fig. g–l. OsteThe mechanistic impact of iNOS in rheumatoid arthritis is very challenging to elucidate due to the pleiotropic and contrasting functions of NO in the maintenance of homeostasis and in other settings, the promotion of inflammation. While initial reports of iNOS inhibition in animal models of acute arthritis were promising , 28, cliPreviously, our lab identified distinct phases of PLN volume in TNF-Tg mice that were associated with discrete stages of knee arthritis , 11. In This prevention of LN expansion, however, may not necessarily be beneficial for arthritic disease outcomes in TNF-Tg mice. Our histologic results indicate moderate attenuation of the arthritis phenotype in female mice and no alteration in the phenotype in male mice. The failure to achieve a robust reduction in arthritis may be explained by multiple factors. First, the inflammatory arthritic phenotype in TNF-Tg mice is largely non-antigen driven . Therefo−/−× TNF-Tg LN, suggesting that at these timepoints, B cell translocation may not play a role in LN expansion. Further, when we qualitatively analyzed B220-stained LNs, there were no remarkable differences between TNF-Tg and iNOS−/−× TNF-Tg LNs.B cell accumulation in the follicles and sinuses was also shown to be important in expanding and collapsing TNF-Tg LNs , 8. To i−/− LN volume at baseline when analyzed independent of TNF-Tg. However, there was no difference in TNF-Tg and iNOS−/−× TNF-Tg LN volume at the earliest timepoints suggesting that in the setting of inflammation, there is an iNOS independent phase of expansion.Our study design limited the breadth of our conclusions because we utilized a pan genetic ablation of iNOS and a cell-targeted approach may have elucidated specific cellular effects of iNOS on TNF-induced arthritis. This is particularly important in understanding the role of iNOS on the vascular system as NO is a potent vasodilator, and iNOS was found at increased levels in RA patients and has been implicated in vascular endothelial dysfunction . FurtherIn conclusion, we identified iNOS independent and dependent phases of LN expansion in an inflammatory-erosive arthritis model. This data suggests iNOS may play a role in LN responses throughout the course of arthritic progression.


Understanding macrophage responses to biomaterials is crucial to the success of implanted medical devices, tissue engineering scaffolds, and drug delivery vehicles. Cellular responses to materials may depend synergistically on multiple surface chemistries, due to the polyvalent nature of cell–ligand interactions. Previous work in our lab found that different surface functionalities of chemically modified alginate could sway macrophage phenotype toward either the pro-inflammatory or pro-angiogenic phenotype. Using these findings, this research aims to understand the relationship between combined material surface chemistries and macrophage phenotype. Tumor necrosis factor-α (TNF-α) secretion, nitrite production, and arginase activity were measured and used to determine the ability of the materials to alter macrophage phenotype. Cooperative relationships between pairwise modifications of alginate were determined by calculating synergy values for the aforementioned molecules. Several materials appeared to improve M1 to M2 macrophage reprogramming capabilities, giving valuable insight into the complexity of surface chemistries needed for optimal incorporation and survival of implanted biomaterials. Macrophages are functionally diverse cells with a multitude of roles in immunity , diseaseClassically activated, M1, macrophages are said to be pro-inflammatory and cytotoxic ,13. As cWith the application of wound healing and successful implant incorporation, the significance of macrophage plasticity is made evident by examining the timeline of macrophage presence during the FBR . MacrophMany techniques for macrophage reprogramming have been developed that focus on either the chemical or environmental stimuli to which macrophages are exposed ,15,32,33Given the complexity of biological systems and the immune system in particular, the idea that the most effective material surface might be made up of several chemistries is reasonable . HoweverThe focus of this work was to examine the synergistic effects of a few promising material surface chemistries described above. In order to more accurately describe the phenotype of macrophages in vitro, a newer nomenclature will be used throughout this paper that is based on the molecule used to activate the cells, for example M(LPS) and M(IL-4) . By modiExperiments were performed with a minimum of three replicates. Results were compared to controls of unmodified alginate as well as singly modified samples in previous work . All matMW) alginate  were chosen based on the results of previous research in this lab . A . A 9]. AAlginate is an appealing biomaterial for numerous wound healing and tissue engineering applications. It is a naturally occurring, relatively inexpensive polymer, it has low toxicity and good biocompatibility, and is easily gelled under mild conditions with divalent cations . The strIn this study, we aimed to find combined modifications that would improve the biocompatibility of alginate by reprogramming macrophages towards an M2-like phenotype. Based on the equation used to calculate synergy, values greater than one for urea:nitrite and less than one for both TNF-α and nitrite production would be desired. These values are most important for M(LPS) macrophages as this is the phenotype we are aiming to reprogram. M(LPS) macrophages tend to mediate the inflammatory stages of the foreign body response to biomaterials, which we are attempting to minimize. TNF-α production synergy values tended to be less than or equal to one for all phenotypes studied. This may be due, in part, to the fact the TNF-α, must diffuse through the alginate coating in order to be measured. As Several different observations were noticed when examining synergistic values of TNF-α production in concert with urea:nitrite. With respect to the M(0) macrophage synergy values, sulfonic acid/epoxide and nitro/epoxide resulted in antagonistic synergy values for TNF-α production. Perhaps unsurprisingly, the pairwise modifications with epoxide resulted in increases in TNF-α production synergy values for M(IL-4) cells. Epoxide was chosen for this study because it resulted in low permeability and high TNF-α production. Sulfonic acid combinations are also able to increase TNF-α production synergy values for M(IL-4) cells and increased urea:nitrite values. Sulfonic acid/epoxide was also able to improve urea: nitrite values, indicating the combinations of these modifications shifted M(0) cells towards an M2 phenotype. Ester combinations resulted in improved urea:nitrite values and did not have an observable synergistic effect on TNF-α for M(0) cells. Future work in examining the synergy of these materials in a dose dependent context may elucidate the relationship of these modifications on shifting macrophage phenotypes.By including the TNF-α diffusion study, this work also examined the changes in diffusive properties of alginate due to combined surface modifications. Ideally, combined modifications would result in a lower mass transport of TNF-α than in the case of single modifications. This would be indicated by synergy values less than one. Modifications sulfonic acid/ketal, nitro/ketal, and oxime/nitro all had values below one. Ketal lowering diffusion was expected since it was previously observed to have low permeability for TNF-α . InteresPair-wise modifications of alginate resulted in synergistic and antagonistic responses for M(IL-4), M(LPS) and M(0) cells for both nitrite and TNF-α secretion. There was no observed advantage or disadvantage of these modifications in terms of arginase activity. In combining the data measuring the shifts in macrophage phenotype with that examining TNF-α diffusion through the modified alginates, improved materials for artificial organs can be obtained. Interestingly, certain modifications appear to influence the synergistic index for TNF-α diffusion, such as ketal, which lowered the index for both pair-wise combinations in which it was included. This observation held for material mediated shifts in macrophage phenotype, e.g., ester for TNF-α secretion from M(0) cells. These results further demonstrate the importance of chemical moieties on biomaterials in achieving appropriate cellular responses. Although this study is not conclusive, it provides valuable insight into the impact of multiple surface modifications on macrophage phenotype. This library is also not large enough to offer much in the way of justified reasoning for the synergistic effects of the combined modifications. However, it provided evidence that maximum macrophage reprogramming may require a multitude of stimuli, and further supported the findings that simply altering the surface chemistry of a biomaterial may have a significant impact on its biocompatibility.