Serious games are now widely used in many contexts, including psychological research and clinical use. One area of growing interest is that of cognitive assessment, which seeks to measure different cognitive functions such as memory, attention, and perception. Measuring these functions at both the population and individual levels can inform research and indicate health issues. Attention is an important function to assess, as an accurate measure of attention can help diagnose many common disorders, such as attention-deficit/hyperactivity disorder and dementia. However, using games to assess attention poses unique problems, as games inherently manipulate attention through elements such as sound effects, graphics, and rewards, and research on adding game elements to assessments  has shown mixed results. The process for developing cognitive tasks is robust, with high psychometric standards that must be met before these tasks are used for assessment. Although games offer more diverse approaches for assessment, there is no standard for how they should be developed or evaluated.To better understand the field and provide guidance to interdisciplinary researchers, we aim to answer the question: How are digital games used for the cognitive assessment of attention made and measured?We searched several databases for papers that described a digital game used to assess attention that could be deployed remotely without specialized hardware. We used Rayyan, a systematic review software, to screen the records before conducting a systematic review.The initial database search returned 49,365 papers. Our screening process resulted in a total of 74 papers that used a digital game to measure cognitive functions related to attention. Across the studies in our review, we found three approaches to making assessment games: gamifying cognitive tasks, creating custom games based on theories of cognition, and exploring potential assessment properties of commercial games. With regard to measuring the assessment properties of these games , we found three approaches: comparison to a traditional cognitive task, comparison to a clinical diagnosis, and comparison to knowledge of cognition; however, most studies in our review did not evaluate the game’s properties .Our review provides an overview of how games used for the assessment of attention are developed and evaluated. We further identified three barriers to advancing the field: reliance on assumptions, lack of evaluation, and lack of integration and standardization. We then recommend the best practices to address these barriers. Our review can act as a resource to help guide the field toward more standardized approaches and rigorous evaluation required for the widespread adoption of assessment games. Whether in the lab or in the world, detecting stimuli and responding to them, both consciously and unconsciously, involves many cognitive functions. One of these important cognitive functions is stimuli” .internal mechanisms of attention can be divided into multiple types. Common areas of attention include selective attention (how people attend to relevant information and ignore irrelevant information), divided attention (when people attend to multiple things at once), and sustained attention (the ability to focus on something for a continuous amount of time) [These of time) ,3.There are also models of attentional control that describe the difference between involuntary and voluntary attention. Attentional control is related to inhibition, shifting, and updating. Inhibition involves preventing irrelevant stimuli from impairing performance, shifting refers to the allocation of attention to whatever is most relevant at the time, and updating is how people encode new information into working memory .Measuring and understanding attention and attentional control are important, as attention is a major cognitive function that influences human development and mental health. Furthermore, as attention is related to a variety of cognitive deficits . Research on attention has often relied on specific cognitive tasks, such as the Eriksen flanker task [Measuring attention, and other aspects of cognition such as memory and perception, is often done using ker task  and the ker task , which hker task , and attker task .Cognitive assessment tasks have specific standards that must be met before they are widely used, especially in clinical settings. They are expected to have certain psychometric properties, such as validity (how well they measure what they claim to measure), reliability (how consistent the test is), sensitivity (how well they identify true positives), and specificity (how well they identify true negatives).Although cognitive assessment tasks are standardized and highly used, they do have some limitations. They can be expensive, as many require trained experts to administer the tasks . They arSea Hero Quest [quality when developing assessment games. In fact, studies have shown that some game elements are associated with lowered enjoyment compared with traditional tasks [Games have the potential to improve the quality and quantity of collected data by increasing participants’ engagement in the moment (better data) and by engaging many more people over longer periods (more data) . For exaro Quest , researcro Quest  noted thal tasks ,17.Game elements can also hinder the assessment properties of a task. Cognition is complicated, and traditional tasks are heavily studied before researchers can be confident that they measure what they claim to measure in a consistent way . Even a small change in a task must be studied to understand its effects .gamelike stimuli such as cartoon characters has resulted in decreased performance compared with standard tasks, possibly because it is more difficult to differentiate between complicated graphical stimuli than simple colored shapes [The use of games to measure cognitive processes related to attention poses unique issues. Through their use of graphics, stimuli, and visual feedback, games inherently manipulate the player’s attention, which has been shown to be problematic when using games for assessment. For example, Wiley et al  found thd shapes .Attention can also interact with games based on individual differences. A study by Delisle and Braun  found thtraining and intervention [assessment. Although games for training and assessment are often grouped together, recent research suggests that gamification may not be the best approach for assessment [There has been considerable research interest in the use of gamification  on cognrvention , with lesessment . AlthougOur eligibility criteria required each included paper to be published before March 1, 2021, a peer-reviewed journal article or conference proceeding, primary research , and written in English.digital game used for the assessment of attention-related processes that could be used remotely. For this criterion, we used the following definitions:In addition, each paper needed to include a game, we chose to follow the original researchers’ intentions. If the authors of a paper referred to an assessment as a game, gamified, or some other variation, we included the paper.Digital game: As there are many ways to define a Attention: We included papers related to attention and attentional control. Assessment: We were interested in studies that sought to measure attention, for purposes of either detection or diagnosis, research, or monitoring cognitive changes. Studies focused on treatment, training, or interventions were excluded, as were studies on educational and work assessments .Remote: As the goal of digitizing assessment is often to increase its scale, accessibility, and reach , we incl(gamif* OR game OR games) AND (cognit* OR neuropsych* OR assessment OR memory OR executive function OR attention* OR impulse control OR processing speed OR inhibition OR anxiety OR depression)) AND DOCTYPE (ar).We searched the titles and abstracts of papers across several databases, chosen for their relevance to games user research and psychology: ACM Digital Library, IEEE Xplore, PubMed, PsycINFO, Scopus, and Web of Science. Our keywords were used in a search string adapted to the requirements of each database but generally followed the same format. In addition, where possible, we added the requirement that the returned records be articles published in English. We set no lower time limit but did require that the included papers be published before March 1, 2021. To facilitate other potential systematic reviews, we cast a wide net with our search terms and included other cognitive processes. For example, for Scopus, the search strings were as follows: TITLE-ABS  AND (attention*)).As a final quality check, we also screened the first 100 results from Google Scholar using the search string: , the population examined , and the sample size of the study.We also listed a number of details about each assessment, including its intended purpose, a general description of the assessment, and if it was focused on a specific disorder . We were also interested in how each assessment was measured, particularly how it was evaluated, if it was compared with any traditional cognitive tasks or a clinical diagnosis, and any results from the evaluation.We listed details about gameplay, giving a general description of each game and listing any game mechanics used . We also noted how the game was developed , the expertise of individuals involved in its development , any evaluation of the game and the results , and the authors’ motivation for using a game.The first 2 authors collected the data from each paper in a spreadsheet, with each author responsible for half of the papers. The first author then reviewed each paper and the spreadsheet to ensure uniform data collection. These data then informed the qualitative synthesis presented in our results. The wide range of methods used in the included papers precluded any meta-analysis or meaningful quantitative analysis. At most, we provided the summary statistics. We intended for this review to provide an overview of how research is conducted in the field and focus on the methodology of each paper.Our initial search was conducted in December 2019, with an updated search conducted in March 2021 to include any new publications. The two searches resulted in a set of 91,968 records. We used Mendeley Reference Management software, which automatically deleted 38,179 duplicate records. We then manually deleted remaining 4424 duplicates, resulting in a final set of 49,365 records to review.the Olympic Games or game as in animal game.The first author’s initial screening excluded 46,969 records. These exclusions were made quickly based on brief searches through titles and abstracts. For example, many records referenced The first 2 authors then reviewed the remaining 2396 papers in more detail and excluded a further 2326 papers. At this stage, common exclusions included papers that addressed cognitive processes other than attention , papers A total of 78 papers were selected for analysis; however, we were unable to obtain the full text of 4 papers from any digital library, interlibrary loan, or attempting contact with the authors, leaving a final set of 74 papers for the review .The full details of the papers included in this review can be found in The 74 papers in our review were published in journals from a variety of fields . Of these 74 papers, 33 (45%) papers were from psychology and medical publications, 26 (35%) papers were from interdisciplinary publications, 13 (18%) papers were from computer science publications, and 2 (3%) were from education publications .The earliest study in our review was from 2000. Most papers were published in the late 2010s, with 14 papers published in 2018 and 13 in 2019 . Two papThe selected papers included 40,154 participants, with sample sizes ranging from 5 to 16,233 participants.The majority of studies (35) focused on children, with 12 papers focusing on older adults and 25 on a general adult population. In addition, 2 papers had participants from across the human life span, with both children and adults of all ages.Although the majority of papers (34) looked at a general population, 3 main disorders were also studied: 16 papers on dementia and general cognitive impairment, 11 on ADHD, and 7 on dyslexia. Furthermore, 6 papers examined other disorders .The papers studied many different aspects of attention and attentional control: inhibition (25 papers), sustained attention (23 papers), visual attention (19 papers), selective attention (14 papers), switching (9 papers), updating (7 papers), divided attention (6 papers), orienting (2 papers), and attentional bias (1 paper). Furthermore, 23 papers measured multiple types of attention. In addition, 5 papers did not specify what type of attention was measured nor could it be inferred from the information in the papers.The papers in our review listed several reasons for using a game to assess cognition: to address the limitations of traditional tests (32 papers), to increase participant motivation (22 papers), to engage children (18 papers), and because of previous research (7 papers). In addition, 7 papers listed multiple reasons, whereas 7 papers did not list a motivation for using a game.The most common reason for using a game was to improve motivation and engagement . For example, Thirkettle et al  created whack-a-mole game for delirium screening in emergency departments, noting that it would be particularly useful to have an automated cognitive test given the busy and demanding nature of emergency rooms.In total, 32 papers used games to address the limitations of traditional assessments, as they can be costly in terms of time and resources, require special expertise, lack ecological validity, and cannot be widely deployed. For example, Brown et al  collecteOur review found three different approaches to developing games for assessment purposes: gamifying cognitive tasks (33 papers), creating custom games based on theories of cognition (37 papers), and exploring potential assessment properties of commercial games (4 papers). One study used both a gamified task and a commercial game.magic power points. This example uses points, graphics, and themes to gamify the task, but any one game element or combination of elements can be used for gamification. For example, Lumsden et al [Gamifying a traditional cognitive task is a common approach for making a game for assessment. This approach involves adding points, graphics, and other game elements to a traditional task. Typically, these tasks are digitized , and then the game elements are layered over the top of the basic task. As an example of gamification from our review, Johann and Karbach  created en et al  created en et al  used man21 Tally, described as “blackjack played in two dimensions,” based on theories of divided attention.Another popular approach is to create a custom game based on theories of cognition or other previous research. For example, several papers in our review created games to detect dyslexia based on theories of visual-spatial attention ,96,100. taskified game. Other custom games, such as the EVO game by Anguera et al [EVO was designed specifically to resemble a commercial action video game, with a focus on “high-level art, music, feedback and storylines.”The custom games in our review are diverse, ranging in appearance and complexity. Some are fairly similar to gamified tasks in their simplicity and approach. For example, although Chesham et al  did not ra et al , are morMusVis, a game to detect dyslexia using language-independent methods. They based their design choices on the visual and auditory processing abilities of individuals with dyslexia. On the other hand, some papers in our review did not offer a clear rationale for how they designed their custom game and why they expected it to work.These custom games are also diverse in their development. Some games are very literature- and hypothesis-driven, whereas others offer less rationale for their design. For example, Rauschenberger et al  created Four papers used preexisting commercial games. In these cases, the games were not created by the researchers; rather, the researchers explored the potential assessment properties of a game. For example, Intarasirisawat et al  used TetAs another example, Houghton et al  used CraAs digital games have not been traditionally used for cognitive assessments, they need to be evaluated for their relation to cognition; for example, researchers may want to know whether people with and without ADHD display different mouse behaviors  or if scOur review found three approaches to evaluating the assessment aspect of games: comparison to a traditional task (31 papers), comparison to a clinical diagnosis (14 papers), and comparison to knowledge of cognition (11 papers). An additional 4 papers compared game results with both a traditional task and a clinical diagnosis, and 1 study used both clinical diagnoses and comparisons to normative data. Of these papers, we further identified 4 that used machine learning to evaluate assessment. Thirteen papers did not evaluate the assessment aspect of the game.EXPANSE, which gamified the dot probe task, go/no-go task, Stroop task, trail making task, and Wisconsin Card Sorting Test. Participants were asked to complete both the standard tasks and game-based versions.The most common approach to evaluating the assessment aspect of a game is to compare the results of a game with the results of an established cognitive task. For example, if the scores from a game are designed to measure response inhibition, researchers may want to compare those scores with those from a go/no-go task. If the patterns of responses are similar, it is likely that the game measures response inhibition in a similar way to a go/no-go task. This process is what Chicchi Giglioli et al  followedAlthough this process offers the most direct comparison between tasks and their gamified components, of the 33 papers that gamified a traditional task, only 14 evaluated the game by comparing it with the task.whack-a-mole game designed to measure response inhibition. Although this game was not based on a specific task, it calculated correlations between performance on the game and performance on a standard Stroop task, which also measures response inhibition. Similarly, Baniqued et al [Custom and commercial games may also be compared with traditional tasks. For example, Tong et al  created ed et al  had partAnother approach to evaluation is to compare the results of a game with a clinical diagnosis or questionnaire. For example, if a game is designed to measure selective attention, researchers may want to look at how scores on the game differ between children with and without ADHD. As children with and without ADHD typically display different patterns of selective attention, the game can be assessed to determine if it discriminates between children with and without ADHD. If game performance differs between the 2 groups, it may be measuring selective attention. Alternatively, it may be picking up on some other feature of cognition that differs between children with and without ADHD; therefore, this approach needs to be used carefully.why it works than if it works. Most of the games in our review used this evaluation approach for situations in which diagnosis was the goal. For example, Peijnenborgh et al [Timo’s Adventure, had clinical validity by showing that children with and without ADHD had significant performance differences.If the intent of the game is to diagnose ADHD, it matters less gh et al  demonstrAs another example, Fukui et al  recruitecanonical results. Thirkettle et al [The results of a game can also be compared with the theories of cognition. For example, if a game is designed to examine attentional blink phenomena, we can refer to the literature on patterns of attentional blink to determine if the results from the game make sense. These comparisons can occur in different ways, for example, comparisons with normative data or a specific theory. Brown et al  used a sle et al  comparedle et al  comparedAs an additional finding, we found 4 papers that used machine learning in their evaluation. These studies compared the results of a game with the results of a cognitive task or clinical diagnosis and then used machine learning to build a classification model for the game results. For example, Jung et al  used macWhen using digital games for assessment, it is important to evaluate the games themselves. As discussed earlier, many papers in our review discussed using games as assessment tools with the motivation to increase participant engagement with testing. Thus, researchers may want to know if a game is more enjoyable than a traditional task ,90 or ifIn our review, only 25 studies evaluated some aspects of gameplay. The majority of studies (n=41) did not evaluate game features. Furthermore, 8 papers did not formally report an evaluation of the game but suggested that some evaluation was done  and game properties . The papers in our review that evaluated the assessment properties used three approaches: comparison to a traditional cognitive task, comparison to a clinical diagnosis, and comparison to knowledge of cognition; however, most studies did not evaluate the game properties.From our review, we identified three barriers to the progress in using games for cognitive assessment. We propose recommendations to address these barriers and offer ideas for further research.There are three barriers to making substantial progress in using games for cognitive assessment. The first barrier we identified is that the literature currently perpetuates assumptions about how users interact with assessment games. Second, there is a lack of evaluation of these games. Third, there is no clear standard for integration across the field.Although the papers in our review did not explicitly state that assumptions about games informed their choices, our results revealed some patterns. In total, 13 papers did not evaluate the assessment aspect of the game. Of these papers, the vast majority were about gamified tasks (9 papers). In addition, of the 33 papers that gamified a traditional task, only 14 evaluated the game by comparing it with the task. This lack of evaluation for gamified tasks may be due to the assumption that adding simple elements such as points and graphics will not interfere with performance on the basic task.In addition, 40 papers chose to use a game for assessment because of a potential increase in participant engagement and enjoyment; however, this choice was rarely followed up with an evaluation of how enjoyable participants found the game. In fact, of these papers, only 18 evaluated game play. This assumption that a game of any type or quality will be engaging and will yield better results than a traditional task is pervasive across the literature, despite evidence to the contrary. For example, Wiley et al  analyzedMore than half  of the papers in our review did not evaluate any aspect of game play, and 17% (13/74) of papers did not evaluate any assessment properties. This lack of evaluation is problematic for the advancement of cognitive assessment games. For the games in our review to be seriously considered as assessment tools in the way that standard cognitive tasks are perceived, they need to be evaluated and validated in the same way.Only 21 papers from our review evaluated both assessment and game properties. As Levy et al  noted, “The final barrier to progress is integration across the field. Currently, there is little guidance on how assessment games should be made, evaluated, and used. Although every project will be different , for the field to advance, there needs to be integration at the level of the game structure. At the structural level, researchers should develop a clear understanding of how different game mechanics in assessment games  interact with user performance and experience. Some research is being conducted in this regard; for example, multiple studies have shown a classic speed-accuracy trade-off when points are included in an assessment game ,39. Thisbest practices from the papers in our review. These include using clearly defined goals to guide the development of a game, ensuring robust evaluation, and working with interdisciplinary teams.To help advance the field, we identified the Describing a clearly stated motivation for using a game over a traditional assessment can justify the use of games in this context. For example, motivation may be to increase engagement with children or reduce dropout rates for long-term monitoring. The game can then be evaluated to determine if it meets the motivation . This evaluation is important because the field is new enough that the results are often not generalizable.in-the-wild. As an example from our review, McKanna et al [Clearly articulating a clear long-term goal can set guideposts for the development and evaluation of games for assessment. For example, if the goal is to use a game as a neuropsychology tool, researchers can focus their efforts on making a standardized game that meets robust standards for validity and reliability and has a large normative data set . If the na et al  identifiEvaluating the assessment capacity of assessment games is necessary to establish their validity. Given the new variables that any game element introduces to an assessment, moving forward, a focus on robust evaluation needs to be prioritized for any cognitive assessment game. We can look at cognitive psychology for best practices when evaluating tasks. For example, when developing a new parametric go/no-go task, Langenecker et al  measuredMeasuring the player experience within assessment games is necessary to justify the use of games over traditional assessment tasks. In our review, we identified three approaches to evaluating assessment games: comparison to a task, comparison to a clinical diagnosis, and comparison to theories of cognition. Comparisons to a task and to theories of cognition can help determine construct validity. A comparison with a clinical diagnosis may measure the sensitivity. However, there are many other issues that need to be addressed. For example, does the game work for people from different cultures or with different educational backgrounds? Are there practice effects that may interfere with repeated use? Some of the studies in our review addressed questions such as these; for example, Rello et al  assessedIt may be useful to develop best practices around how and what should be evaluated when developing an assessment game. This list should include evaluating the assessment aspects in ways similar to cognitive psychology methods, but it also needs to include evaluating the game aspects. Knowledge on how to evaluate games can come from games user research.Smart Aging by Bottiroli et al [Integrating knowledge across the disciplines of psychology, clinical sciences, game design, or user experience will help ensure robust results. Regardless of the goals of the assessment game, the best result will often come from an interdisciplinary team. Levy et al  noted thli et al , a game There are some limitations to our systematic review. We did not search every database for papers; for example, we ruled out using Springer Link because we could not search by title and abstract. We selected our databases based on the relevance to the field and focused on using a mix of computer science– and psychology-related databases. Similarly, we attempted several combinations of search terms. Some yielded too many results for a feasible review. We aimed to search as comprehensively as we could realistically manage. We struck an appropriate balance with 49,365 records to review after duplicates were removed.We also used cross-referencing and Google Scholar to check for additional papers that met our criteria; however, it is still possible that we missed some papers.Our review also only addressed papers that used games to assess cognitive processes related to attention to keep this review to a manageable scope. Many other studies have assessed memory and other cognitive functions using games. We intend to cover memory in another review, and future work should cover games that are used to assess more complicated cognitive functions, such as decision-making.game in the title or abstract of the paper. We may have missed papers in our review those that used what authors defined as a task but still implemented game elements, such as points. In addition, we may have included papers that used what authors defined as a game but could be considered a task.Another limitation of our selection criteria is our reliance on authors to define their work as a game or task. Our search criteria depended on the inclusion of the word Finally, our review only covers published work and thus, carries the risk of publication bias. As our review focused on the methodologies used by studies and not the outcomes, this issue is not a significant concern.We conducted a systematic review to answer the following question: How are digital games used for the cognitive assessment of attention made and measured? We searched a wide range of databases to identify an initial set of 49,365 papers, which we then narrowed to a set of 74 papers that we reviewed in detail. From these studies, we identified three unique approaches for developing a game for assessment. We also identified that, across the field, the focus tends to be on development rather than evaluation. Assumptions about how the application of games to cognitive tasks should improve assessment are widespread but perhaps not widely demonstrated. Our review can act as a resource to help guide the field toward more standardized approaches and rigorous evaluation required for the widespread adoption of assessment games.


The chronic neurological aspects of traumatic brain injury, post‐stroke syndromes, long COVID‐19, persistent Lyme disease, and influenza encephalopathy having close pathophysiological parallels that warrant being investigated in an integrated manner. A mechanism, common to all, for this persistence of the range of symptoms common to these conditions is described. While TNF maintains cerebral homeostasis, its excessive production through either pathogen‐associated molecular patterns or damage‐associated molecular patterns activity associates with the persistence of the symptoms common across both infectious and non‐infectious conditions. The case is made that this shared chronicity arises from a positive feedback loop causing the persistence of the activation of microglia by the TNF that these cells generate. Lowering this excess TNF is the logical way to reducing this persistent, TNF‐maintained, microglial activation. While too large to negotiate the blood‐brain barrier effectively, the specific anti‐TNF biological, etanercept, shows promise when administered by the perispinal route, which allows it to bypass this obstruction. This article is about how the neurological aspects of long COVID , can be best understood, and treated. For this purpose, the literature on TNF and brain function, both physiological and disease‐associated, and how this is being applied to various long‐term neurodegenerative conditions, is discussed.  AMPAα‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acidBBBblood‐brain barrierDAMPdamage‐associated molecular patternsHMGB1high‐mobility group box 1PAMPpathogen‐associated molecular patternsPOCDpost‐operative cognitive deficitPTSDpost‐traumatic stress disorderrTNFrecombinant TNFTBItraumatic brain injuryTLRsToll‐like receptors1Having been a party to the literature on the cytokine concept of disease pathogenesis since its inceptionDespite the advent of awareness of pathogen‐associated molecular patterns (PAMPs) and damage (or danger) associated molecular patterns (DAMPs), and the activities of the cytokines thus induced through Toll‐like, and functionally similar pattern recognition receptors in increased concentrations (discussed below), these earlier mindsets can also dominate research into both these non‐infectious and infectious conditions, preventing them being discussed together, using a common knowledge base. As the title infers, this commentary is intended to foster this cytokine approach becoming widespread enough to allow a broader understanding of shared features of what are, traditionally, seen as profoundly different illnesses, with very different causes. In particular, discussion focusses on how a number of chronic neurological changes can be common to certain conditions, be they infectious or non‐infectious.2It seems appropriate to expand here on the above PAMP and DAMP terminology, since it is central to understanding the pathophysiology of both infectious and non‐infectious diseases, including their influence on neurological function. Further detail of this concept, attributable to JanewayClinical similarities within acute infectious diseases can also be rationalized within this framework. At the Dana‐Faber Institute in Boston, when rTNF was being tested for killing tumors in patients,3To illustrate the DAMP principle more broadly, the wide spectrum of involvement of high‐mobility group box 1 (HMGB1) warrants brief discussion. This DNA‐binding protein is evolutionarily conserved  or, as discussed below, non‐infectious influences such as hypoxia or trauma, can alter brain function in the same way.Clearly, all the above functions are vulnerable to TNF being outside its physiological range when it is over‐induced by either PAMPs or DAMPs that can enter, or be generated in, the brain. The capacity of microglia, via the cytokines these cells generate, to influence neuronal function, are regularly reviewed.Once TNF, IL‐1β, IL‐6, IL‐4 and other host‐origin cytokines were identified, interacting pathways proposed, and laboratory reagents available, an awareness of their roles in states such as infectious and inflammatory disease, sickness behavior, ageing, and neurodegeneration dominated their literatures. Consequently, the infectious disease world often demonstrates little awareness that, at lower concentrations, these cytokines have many essential roles in brain physiology. Homeostatic normal synaptic plasticity and scaling as well as associated glutamate control, neurogenesis, neuromodulation, synaptic function, memory,6The next challenge is to understand why such neurophysiological change can be rendered chronic, whether in post‐stroke syndromes, Lyme disease, COVID‐19, and influenza encephalopathy survivors, whereas systemic cytokine levels and associated acute symptoms do not persist. In 2007 it was reported that TNF generation in the mouse, after intraperitoneal injection of bacterial LPS, persists in the CSF for very much longer (at least 10 months) than in the serum (6 h).These intriguing sets of observations and proposals are consistent with the reportin vivo in two of these three quoted studies, it seems pressing for the techniques in the previous paragraph to be applied to influenza, persistent Lyme disease and long COVID brains. This concept is also consistent with the endotoxin tolerance being observed systemically but not in the brain. Importantly, it implies that perispinally administered etanercept (discussed later) can deactivate these microglia by inactivating this excess TNF, hence removing the positive feedback loop that causes its production to persist. In this way these PAMP and DAMP‐induced cytokines, and not the pathogen, ischemia or trauma directly, are argued here to be the essential cause of most of the commonly seen neurological dysfunction in the persistent stage of all these illnesses. Logically, therefore, this principle applies to the persistent cerebral phases of COVID‐19, influenza or Lyme disease as well as to encephalopathies seen post‐stroke or in TBI survivors.A precedent for the existence of long‐term microglial activation, with a DAMP rather than a PAMP the original cause, has been reported in ischemic stroke,77.1Having already covered similar ground with long COVID,7.2Since these are non‐infectious conditions, their pathophysiology is exclusively DAMP‐driven, mostly by hypoxia and the products of tissue damage. Non‐infectious inflammatory conditions such as post‐stroke syndromes8A cluster of chronic symptoms form the syndrome observed when cerebral TNF becomes inappropriately excessive for the normal function of a particular cerebral site. Below, we discuss fatigue, neurogenic pain, delirium, aggressiveness, and suicide tendency as examples. The ingredients in a particular mix can be expected to depend on the site and pattern of excessive induction of TNF, independent of whether the driving force is a PAMP or a DAMP Figure . Even wi8.1Two of the clinical states in which loss of capacity to appreciate taste or smell are commonly reported, post‐stroke and long COVID, are discussed throughout this text. One of this pair being infectious, and the other not, nicely fits the argument made throughout that the pathogenesis of the syndromes discussed here is independent of whether their inducer was a PAMP or a DAMP. Moreover, the literature explains loss of taste, taste distortion8.2Fatigue is an undisputed and regularly documented component of long COVID, persistent Lyme disease and viral infections as diverse as influenza and a range of arbovirus infections. As with delirium and aggression, the presence of fatigue fits the pattern of the later stages of these infectious conditions sharing symptoms with non‐infectious neurodegenerative states such as post‐TBIDbp, Tef, Hlf, and the period genes Per1, Per2 and Per3, all of which are involved in controlling circadian rhythm. They reasoned that this suppression, through influencing sleep cycles, provides a causal link between TNF and the fatigue of disease. In 2012, we summarized the implications of these changes in clock gene function for mitochondrial dysfunction, and thus reduced systemic and cerebral energy availability.Considering that the original evidence that TNF would induce fatigue was inadvertent, it is very impressive indeed. In 1988 open trials comprising 115 treatments in 50 patients, recombinant TNF (rTNF) was infused over 24 h in the hope of causing tumor regression. Quite unexpectedly, this induced fatigue so severe that hospital discharge was considerably delayed.8.3Neurogenic pain, a term encompassing both neuropathic (nerve damage) and central (CNS‐origin) pain, is commonly seen in both long COVID‐19 diseaseAs observed in other vector‐spread viral diseases, such as Chikungunya and Dengue, persistent Lyme disease can include arthralgia. Typically this does not respond readily to antibiotics, implying it is not directly caused by the pathogen.8.4Delirium is variously described as an acute and profound disturbance of thinking, memory, orientation, perception and emotion. Transient delirium is common in intensive care units, and has been perceptively described as an extreme manifestation of the sickness behavior caused by TNF and the other cytokines it induces.8.5Behavioral changes, such as aggressiveness, are also recorded in persistent Lyme disease.The relevant literature on suicidal tendencies, plausibly termed self‐aggression in this context, also warrants summarizing here. Risk of suicide is reportedly increased in many of the conditions being discussed here, both infectious  or PAMPs .As discussed earlier, changes in cerebral function, often subtle, but sometimes disabling, can become persistent. Inactivating chronically activated microglia by removing the excess TNF that maintains this stateAs referenced earlier in the present text, post‐stroke syndromes, as well as other non‐infectious neurodegenerative diseases, typically exhibit the same fatigue, neurogenic pain, delirium and aggressiveness as are recorded in persistent Lyme and long COVID‐19. This is consistent with the chronic cerebral TNF generation that maintains microglia in a chronically activated state, rather than the initial pathogen, being a plausible therapeutic target in persistent Lyme disease. Gabapentin, generally regarded as an anticonvulsant, is a synthetic analogue of gamma aminobutyric acid. Hampered by dose‐limiting side effects, it has nevertheless been used, with some success, against chronic neurogenic pain, including in Lyme disease.10Glutamate is a key physiological excitatory neurotransmitter in virtually all activities of the nervous system, yet in excess it is extremely harmful. As we expanded upon 5 years agoVarious alphaviruses cause the same chronic neurological sequelae as seen in COVID‐19 and influenza. The glutamine analogue 6‐diazo‐5‐oxo‐L‐norleucine (DON), albeit too toxic to be a practical drug, is a useful experimental agent. As does TNF,11With the realization that TNF is as central to normal signaling and pathophysiology in the central nervous system, as we have reviewed,in vivo brain distribution of etanercept, a molecule with a size comparable to albumin, after perispinal injection in a rat model.The perispinal route consists of a shallow injection of the dose into the cerebrospinal venous system.Therefore treatment with perispinally administered etanercept, plus five minutes tilting head‐down , can be predicted, from literature discussed earlier, to de‐activate the chronically TNF‐generating microglia, kept activated by chronically increased cerebral TNF.The initial observation that perispinal treatment with a specific anti‐TNF biological typically causes such a rapid return to normal neurological function was as striking as it was unexpected across the field. Certainly, large players can find the phenomenon more convenient to dismiss than to attempt to understand. My invited 2017 editorial to comment on a Clinical Advisory issued by the American Academy of Neurology (AAN)in vitro cerebral context, exposure of a human neuroblastoma cell line to TNF was shown to promote oxygen radical‐mediated caspase‐1 activation and thence IL‐1β secretion.Attention is drawn to the curious phenomenon of the same encephalopathies being often defined only in TNF or IL‐1β terms. Whatever their origin, infectious and non‐infectious encephalopathies are, beyond the nature of the initial inducer, very similar in pathogenesis. Yet certain encephalopathy publications148,150 and IL‐8.Not surprisingly, therefore, anti‐TNF inhibits IL‐1 generation.In conclusion, it is obvious from the literature on the pathogenesis and therapy of all of the conditions discussed here, systemic and cerebral, infectious and non‐infectious, that they are yet to be considered in an integrated manner. Largely, silos still reign. Aspects of the depth of this need for productive cross‐fertilization are cognitively expressed in a recent commentary with a more clinical perspective.The author declares no conflict of interest or financial involvement in the research discussed here.


Pro-inflammatory TNF is a highly gene-selective inhibitor of the gene expression program of tissue repair and wound healing macrophages. Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn’s Disease. Previously, we and others found that TNF blocks the emergence and function of alternative-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balance of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages, we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis, and signaling pathway deconvolution. We found that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way, dependent on JNK signaling via the type 1 TNF receptor on specific populations of alternative-activated macrophages. We further determined that JNK signaling has a profound and broad effect on activated macrophage gene expression. Our findings suggest that TNF’s anti-M2 effects evolved to specifically modulate components of tissue and reparative M2 macrophages and TNF is therefore a context-specific modulator of M2 macrophages rather than a pan-M2 inhibitor. A temporal and spatial balance between pro- and anti-inflammatory immune responses ultimately defines the final outcome of infection, wounding, and tissue repair. In general, the passage from an inflammatory response associated with microbial clearance, cellular damage, and cell death that evolves towards tissue repair and homeostasis is termed resolving inflammation . By contTNF is a pro-inflammatory cytokine targeted by different biologic drugs including Infliximab, a monoclonal antibody directed against both soluble and transmembrane bound forms of TNF, and Etanercept, an engineered soluble TNF receptor . The ratRecently, an additional and unexpected connection between TNF blockade and resolving inflammation was exposed using molecular genetic approaches. In mice lacking the type 1 tumor necrosis factor receptor  or TNF (encoded by Tnf), diverse inflammatory responses were associated with a striking enhancement of macrophages with M2 characteristics , 2021. MAn implication of the discovery of the anti-M2 effect of TNF is that in clinical settings where suppression of the pro-inflammatory effects of TNF is the intended mechanism-of-action, M2 macrophages will be enhanced in number or function , 2012. TSignaling from TNFR1 involves three main pathways: NF-κB signaling, MAPK signaling and the caspase-8 pathway, which intersects with the receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and RIPK1 pathways to regulate necroptosis and apoptosis . ActivatRetnla (encoding RELMα), as a paradigmatic gene induced by IL4+IL13 and negatively regulated by TNF , a chard by TNF . When pr by 24 h . When TNIL4+IL13 , indicatRetnla mRNA, TNF blocked RELMα protein expression  expression of IL4+IL13+TNF stimulated BMDMs compared with IL4+IL13–treated ones . Type 2 mRNAs were also increased with IL4+IL13 but decreased from baseline with the supplementation of TNF . Type 3 mRNAs were not increased by IL4+IL13 but diminished by TNF at baseline  and therefore are not conventional M2 genes, as they are not induced by IL4+IL13. We verified these regulatory modalities by qRT-PCR , immune regulation , and phagocytosis ; this pattern is consistent with the original observations about TNF restricting M2 macrophages, as TNF targets key mRNAs needed for the execution of tissue repair and wound healing  had a decreased chromatin accessibility with IL4+IL13+TNF stimulation  to estimate the rate of transcription of selected TNF-regulated genes by measuring the nascent unspliced mRNA in comparison with the conventional spliced transcript. The critical control for PT RT–PCR is the omission of reverse transcriptase in parallel matched samples to quantify contaminating genomic DNA . We adder Fn1 PT , indicatr Fn1 PT . M2 genemulation . These fTnfr1a−/− BMDMs with IL4+IL13, TNF or IL4+IL13+TNF and measured oxygen consumption rate  production in comparison to LPS treatment  with IL4+IL13+TNF compared with IL4+IL13 treated BMDMs. In general, the phosphoproteome changes were primarily linked to TNF stimulation and resulted in an up-regulation of phosphopeptides with IL4+IL13+TNF . Ser25 ppression  ruling opression . Two of pression . In earlpression . JNK1/2 pression . Based opression .Phosphoproteomic results related to Fig 6A.Table S1 Retnla expression  double knockout is lethal . Furtherogenesis . We ther enzymes . When we enzymes . To bypa enzymes .We next tested if pharmacological JNK inhibition after BMDM development was completed would block the anti-M2 activity of TNF and “mirror” the phenotype of the TNFR1 knockout. Such a hypothesis would be supported in the cases where JNK was an obligatory factor in the TNFR signaling pathway but not required for IL4R signaling. We compared the effects of JNK-IN-8 versus a noncovalent ATP competitive JNK inhibitor (SP600125) on JunB and c-Jun phosphorylation after BMDM stimulation with IL4+IL13 or IL4+IL13 with concurrent TNF stimulation. The idea behind this was to use SP600125 to transiently inhibit JNK and evade the profound effects of JNK-IN-8 on CSF1R signaling. However, SP600125 had minimal effects on JunB and c-Jun phosphorylation compared with JNK-IN-8, which by contrast was a complete inhibitor of the downstream targets of JNK .Mertk or Type 3 genes such as Arhgap19) (Retnla)  . Second,(Retnla) . These f(Retnla) . Thus, e(Retnla) , JNK sig(Retnla) . Third, (Retnla) . JNK sigRetnla were completely suppressed by TNF, whereas other targets were partly inhibited (Mgl2) or blocked in a time-dependent way (Arg1). TNF is therefore a context-specific modulator of M2 macrophages rather than a pan-M2 inhibitor.Our experiments were initiated with the idea of establishing the mechanistic basis of the anti-M2 effects of TNF; in part, we reasoned that as anti-TNF biologics have a vast spectrum of activities in RA and IBD patients, we should be able to elucidate a signaling framework that connects successful anti-TNF therapy with the number and function of M2 macrophages. In previous experimental systems, we and others showed that many hallmark M2 genes were negatively regulated by TNF , 2021. HRetnla expression. However, in pathology where too much TNF is driving the pathology, anti-TNF has beneficial effects; most likely through induction of M2, as demonstrated in this article. Thus, we can place our findings about the specificity of the anti-TNF M2 pathway in clinical context; the outcomes of anti-TNF biologic therapy in RA and IBD in terms of enhancement of the number and function of M2 macrophages are likely variable and patient specific. An additional finding in this regard concerns the fact that cytokines such as IL1β and IL36 can achieve similar effects to TNF. Although we used this information to understand the signaling pathway that controlled M2 expression, a broader interpretation is that inflammatory lesions replete in combinations of IL1β, IL36, and TNF may have additive anti-M2 effects.So far, accurate measurements of inflammatory gene expression in resolving information have been made in the skin wound healing model . This moAt the cellular level, our scRNAseq data highlighted that IL4+IL13 stimulated cultures of BMDMs are heterogenous and that there is no clear pattern of M2 gene expression within the cluster distribution, nevertheless we observed the IL4+IL13 stimulated group mainly in the cluster 1, 2, and 6. Our experiments agree with recent work that shows that a seemingly homogenous bone marrow culture expanded in CSF1 or GMCSF, which was unstimulated, has an underlying heterogeneity in gene expression and cytokine responsiveness . We obseInterrogation of the JNK and Jun family of bZIP proteins in macrophages has intrinsic challenges. Mice lacking JNK1 and JNK2, c-Jun, and JunB are all embryonic lethal and JNK inhibition during macrophage development is similarly lethal. Conditional deletion of genes encoding JunB and JNK1/2 has revealed different effects on macrophage polarization pathways . HoweverStat6−/− , and Tnfr1a−/− mice were mated and maintained under specific pathogen-free conditions at the animal facility of the Max Planck Institute of Biochemistry. Both Stat6−/− and Tnfr1a−/− mice were on a C57BL/6J background. Use of mice for breeding and organ isolation was approved by the Government of Upper Bavaria. For the in vivo wound healing model 10–12 wk old female wild-type BALB/c mice were used and were approved by the North Rhine–Westphalian State Agency for Nature, Environment, and Consumer Protection (LANUV) and the University of Cologne (reference number 84-02.04.2017.A019). Mice, which were used for the isolation of peritoneal macrophages, were maintained in specific pathogen-free conditions under protocols approved by the animal care committees of the Regierungspräsidium Freiburg and the Institutional Animal Care and Use Committee of Johns Hopkins University, in compliance with all relevant ethical regulations.For BMDM culture, male C57BL/6J (Wt), 2.BMDMs were grown in DMEM  composed of 10% FBS , penicillin/streptomycin and CSF1. The BMDMs were cultured for 7 d on 150 mm TC-treated culture dishes  in the presence of endotoxin-free baculovirus-expressed recombinant human CSF1 generated in-house. Every other day 3 ml of new CSF1-containing medium was added to the culture. On day 7, BMDMs were harvested with a cell scraper . After counting, the cells were seeded in the presence of 100 ng/ml CSF1 and stimulated the next day. All cell culture procedures were carried out at 37°C with 5% COEscherichia coli O111:B4 , 10 μg/ml Etanercept , 20 μM JNK-IN-8 , and 10 μM SP600126 . TNF was always added at the same point as IL4+IL13 , 10 ng/ml IL13 , 10 ng/ml TNF , 10 ng/ml IL1β , 33.3 ng/ml IL36α , 33.3 ng/ml IL36β , 33.3 ng/ml IL36γ , 5 ng/ml LPS from 3 except .BMDMs were lysed in TRIzol  for subsequent RNA isolation according to the manufacturer’s instructions. cDNA was synthesized using SuperScript IV reverse transcriptase , random hexamers  and oligo(dT) primers , and analyzed by qRT-PCR. Retnla primers were purchased from QIAGEN. All values were normalized to GAPDH . Primer sequences can be found in GSE169348 and GSE185380.RNA samples were subjected to mRNA-seq and quantified using Qubit 2.0 according to the manufacturer’s instructions. RNA was polyA enriched. Libraries were prepared and sequenced on an Illumina NextSeq 500 platform. Raw mapped reads were processed in R to determine differentially expressed genes and generate normalized read counts. RNAseq data are available in BMDMs were suspended in FACS buffer (PBS + 1% FBS) and incubated with Fc block . Cell surface marker were stained with anti-F4/80  and for live/dead staining with LDFA . Afterwards, cells were washed, permeabilizated with Foxp3/Transcription Factor Staining Buffer Set , blocked with 2% normal mouse serum (Invitrogen) and stained for intracellular Arg1  and Retnla  expression. Cells were washed twice and then resuspended for analysis on a BD LSR Fortessa FACS flow cytometer. Gating strategy is shown in Healthy wild-type mice were injected i.p. with PBS or 12 mg/kg bodyweight Etanercept three times every other day for a total of 6 d. On day 6, the mice were euthanized and the peritoneum was flushed with PBS. SPMs were analyzed by flow cytometry using the gating strategy shown in 10–12-wk-old female wild-type BALB/c mice were anesthetized by i.p. injection of Ketanest/Rompun . Littermates of the same sex were randomly assigned to experimental groups. The back skin was shaved, and full-thickness punch biopsies were created on the back using a standard biopsy puncher (Stiefel). Wounds were excised at indicated time points after injury and processed to generate single-cell suspensions for flow cytometry following an established protocol .Mice were treated i.p. with 12 mg/kg bodyweight Etanercept  or vehicle (NaCl) in a total volume of 200 μl every third day starting 3 d prior wounding and were continued every 3 d until the mice were euthanized.Single-cell suspensions of wound tissue were prepared by a combination of enzymatic digestion  and mechanical disruption . Excised wound tissue was sectioned with a scalpel, placed in DMEM medium with 30 μg/ml Liberase TM Research Grade , and incubated at 37°C for 90 min (shaking). Digested wound tissue was mechanically disrupted for 5 min using the Medimachine System. Cells were passed through 70 and 40 μm cell strainer, and washed with PBS/1% BSA/2 mM EDTA. Fc receptors were blocked with mouse SeroBlock FcR  and cells were stained with fluorescein isothiocyanate-conjugated anti-CD45 , allophycocyanin conjugated anti-CD11b , and phycoerythrin-conjugated anti-F4/80  in PBS/1% BSA/2 mM EDTA. Dead cells were excluded using 7 amino-actinomycin D . Cells were sorted using a FACSAria III cell sorting system (BD) equipped with FACSDiva software (BD). Sorting strategy is shown in 5 flow cytometry-sorted CD45+CD11b+F4/80+ wound macrophages were lysed in RLT lysis buffer  and total RNA was isolated using the RNeasy Micro Kit (QIAGEN) according to the manufacturer’s instructions. To obtain one data point at 2, 4, and 7 d post injury (dpi), four wounds from one mouse were pooled; at 14 dpi eight wounds from two mice were pooled. Reverse transcription of isolated RNA was performed using the High Capacity cDNA RT Kit . Amplification reactions (triplicates) were set up using the PowerSYBR Green PCR Master Mix  and quantitative real-time PCR (qRT-PCR) was validated with the QuantStudio 5 real-time PCR system (Thermo Fisher Scientific). The comparative method of relative quantification (2−∆∆Ct) was used to calculate the expression level of the target gene normalized to Rps29. Primer sequences can be found in For quantitative real-time PCR analysis, 2 × 10Wild-type BMDMs were stimulated with 10 ng/ml IL4, IL13, and TNF. Cells were scraped and stained for live/dead staining with LDFA. The scRNAseq workflow was based on the previously described Smart-seq2 protocol  with thehttp://www.R-project.org) using Seurat v. 3  with Ensembl 98 splice sites as support . Uniquelrat v. 3  with defrat v. 3  and interat v. 3  as a dimrat v. 3 , selectiPT qRT-PCR was done like previously described . Wild-tyLysates were prepared from BMDMs on ice in RIPA buffer  containing protease and phosphatase inhibitors . Proteins were separated on Tris–HCl gradient gels  and transferred to nitrocellulose . Membranes were blocked in 5% BSA  and probed with primary antibodies overnight at 4°C. Anti-pStat6 (56554), Stat6 (5397), pJNK (9251), JNK (9252), pERK (9101), ERK (9102), p-p38 (9211), p38 (9212), Nos2 (2977), pJunB (8053), JunB (3753), p-cJun (9261), and cJun (9165) antibodies were obtained from Cell Signaling Technology. Irg1 antibody (ab222411) was purchased from Abcam. As loading control anti-Grb2 antibody (610112) was used from BD. Membranes were washed and probed with secondary antibodies at a 1:10,000 dilution and developed using chemiluminescence reagents .6 BMDMs were plated into each well of Seahorse cell culture plates and preincubated at 37°C for a minimum of 45 min in the absence of CO2 in Seahorse XF DMEM medium  supplemented with glutamine, glucose and pyruvate. After analysis with the Seahorse Cell Mito Stress Test Kit , normalization was made by protein quantification.Real-time oxgen consumption rate and extracellular acidification rates measurements were made with a Seahorse XF HS Mini Analyzer (Agilent Technologies). 0.04 × 10Nitric oxide was measured with Griess reagent according to 4 BMDMs treated for 24 h were washed in PBS and then lysed in 10 mM Tris–HCl, pH 7.4,10 mM NaCl, 3 mM MgCl2, and 0.1% Igepal CA-630 . Nuclei were spun down and then resuspend in 25 μl TD (2× reaction buffer), 2.5 μl TDE1 (Nextera Tn5 Transposase) and 22.5 μl nuclease-free water, and incubated for 30 min at 37°C. DNA was purified with the QIAGEN MinElute PCR Purification Kit . PCR amplification was performed with the NEBNext High-Fidelity 2× PCR Master Mix  using custom Nextera PCR Primers containing barcodes. Adaptors were removed with AMPure XP beads according to manufacturer’s protocol. Libraries were quantified with the Qubit and submitted for sequencing with a HISeq 3000 (Illumina) by the staff at the Deep-sequencing Facility at the Max-Planck-Institute for Immunobiology and Epigenetics. Sequenced samples were trimmed with Trimmomatic (https://cran.r-project.org/web/packages/wordcloud2/vignettes/wordcloud.html), with word size proportional to enrichment −log10P-value. Coverage tracks were visualized with Spark (Preprint). Comparison of accessibility of specific transcription factor sites across treatments was performed with deepStats .Libraries were prepared using the Nextera DNA library Prep Kit (Illumina) adapting a published protocol . Brieflymmomatic  and mappmmomatic . Coveragmmomatic . Open chmmomatic , whereasmmomatic  with at mmomatic . Bed filth Spark Preprint2PO4, final conc. 1 mM) were added to the rest of the digested lysate. Lysates were shaken, then spun down for 3 min at 2,000g, and supernatants were incubated with TiO2 beads for 5 min at 40°C . Beads were washed five times with isopropanol and 5% TFA, and phosphopeptides were eluted off the beads with 40% acetonitrile (ACN) and 15% of ammonium hydroxide (25% NH4OH) on C8 stage tips. After 20 min of SpeedVac at 45°C, phosphopeptides were desalted on poly(styrenedivinylbenzene)-reversed phase sulfonate (SDB-RPS) stage tips and resolubilized in 5 µl 2% ACN and 0.3% TFA and injected in the mass spectrometer (MS).To enrich for phosphorylated peptides, we applied the Easy Phos protocol developed in the Mann laboratory , 2018. I6 ions was first performed, followed by 10 data-dependent MS/MS scans with higher energy collisional dissociation . Dynamic exclusion of 40 s and the Apex trigger from 4 to 7 s was enabled.Samples were loaded onto 50-cm columns packed in-house with C18 1.9 μM ReproSil particles (Dr. Maisch GmbH), with an EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to the MS . A homemade column oven maintained the column temperature at 60°C. Phosphopeptides were eluted with a 140 min gradient starting at 5% buffer B  followed by a stepwise increase to 20% in 85 min, 40% in 35 min, 65% in 10 min and 80% in 2 × 5 min at a flow rate of 300 nl/min. Samples were measured in data-dependent acquisition with a (TopN) MS method in which one full scan  at a target of 3 × 10MS raw files were processed by the MaxQuant version 1.5.38  and frag2-transformed. Samples that did not meet the measurement quality of the overall experiment were excluded. Quantified proteins were filtered for at least 100% of valid values among three or four biological replicates in at least one condition. Missing values were imputed and significantly up- or down-regulated proteins were determined by multiple-sample test . The MS-based proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository and are available via ProteomeXchange with identifier PXD028667  . Summed XD028667 .Wild-type BMDMs were stimulated with IL4+IL13+TNF for 15 min  or with The significance of differences in the experimental data were determined using GraphPadPrism software. All data involving statistics are presented as mean ± SEM. The number of replicates and the statistical test used are described in the figure legends.GSE169348, GSE185380, and PXD028667 were used.Data from


Follicular diameter, E2 concentration, follicle survival, antral-like cavity formation, and ovulation were measured to assess the effects of dexamethasone. In our work, TNF-α inhibited in vitro follicular growth and E2 secretion in a dose-dependent manner. Based on the results of the present research, we concluded that the addition of dexamethasone partially counteracts the repressive effect of TNF-α on follicle growth and E2 secretion during in vitro culture of the preantral follicles of mice. Thus, the findings in this paper suggest that dexamethasone may act as a therapy by counteracting the effects of TNF-α in PCOS patients. These results provide a new foundation for exploring the treatment of PCOS patients with dexamethasone.As a proinflammatory cytokine, tumor necrosis factor-α (TNF-α) is central to the female reproductive tract and affects various phases of follicular development and uterine cycles. High levels of TNF-α play a vital role in the pathogenesis of polycystic ovary syndrome (PCOS) in patients. Clinicians know that dexamethasone can inhibit the induction of androgen by suppressing the adrenal glands which improves the status of the endocrine system in PCOS patients. We hypothesize that dexamethasone has much more functionality and can exert a therapeutic effect by antagonizing TNF-α. We added TNF-α to the follicular culture medium to simulate the high TNF-α levels observed in the endocrine environment of PCOS patients. Dexamethasone was added to the medium to determine if it could counteract the inhibitory effect of TNF-α on follicular growth and 17β-estradiol (E As an inflammation-associated cytokine, tumor necrosis factor-α (TNF-α) is connected to various biological processes, including inflammation, cell apoptosis, and necrosis . It alsoPolycystic ovary syndrome (PCOS) is a complex endocrinopathy in women of reproductive age with a prevalence of approximately 6–8% women worldwide . AdditioClinicians know that treatment with dexamethasone is beneficial to PCOS patients. The combination of dexamethasone and clomiphene citrate has been observed to increase ovulation and pregnancy rates in PCOS patients that are resistant to clomiphene citrate , 14. TheThis study was approved by the Experimental Animal Ethics Committee of our organization (SUMC2014-014). Treatment of the mice complied with the Guide for the Care and Use of Laboratory Animals by the National Institute of Health  and the rules of the National Animal Protection of China. All female Kunming mice used in the present study were in the preadolescence stage (PND12-14). They were purchased from the Animal Center of Shantou University Medical College.HEPES MEM , α-minimal essential medium , and insulin-transferrin-selenium mix  were obtained from Gibco, Invitrogen. Ten percent fetal bovine serum (FBS) was obtained from Thermo Fisher Scientific . Human pituitary follicle-stimulating hormone (FSH) was kindly donated by the Reproductive Center at the First Affiliated Hospital of Shantou University Medical College. TNF-α  and epidermal growth factor  were purchased from PeproTech. Human chorionic gonadotropin  was obtained from BioVision. Dexamethasone  and mineral oil  were obtained from Sigma. Human tubal fluid (HTF) was obtained from Sage Science.We isolated mouse preantral follicles from ovaries as described previously –19. PrepThe preantral follicles were washed twice with a follicular separation solution {HEPES-MEM+10% fetal bovine serum (FBS)}, which was followed by one wash with a culture medium, and then they were grown as droplet cultures. The follicles selected for inclusion in the present study had the following characteristics: (1) the diameter of the follicles ranged from 120 to 150 μm, (2) viable oocytes were clearly in the center of the follicle, and (3) the basement membranes were intact and had attached theca cells . All steAll preantral follicles were grown in vitro in a culture medium containing α-MEM, 10% FBS, and 10 μL/mL ITS   in an incubator set to 37°C with 5% COOn day 12, 5 IU/mL of HCG and 5 ng/mL of EGF were added to the culture medium to induce ovulation and meiotic resumption  and those without FSH served as the negative control (FSH−) , 10 ng/mL (T10), 15 ng/mL (T15), and 20 ng/mL (T20), respectively , 1 μg/mL (D1), and 10 μg/mL (D10). The final concentration of absolute ethyl alcohol in the culture fluid did not exceed 0.1%.On day 1, dexamethasone was added to samples already containing 100 mIU/mL of FSH and 15 ng/mL TNF-α, to determine its impact on the effects of 2 secretion  follicle survival, (4) antral-like cavity formation, and (5) ovulation. Furthermore, the first polar body (PB) excretion ratio was used as an indicator of oocyte nuclear maturation. It was calculated by dividing the number of MII oocytes by the number of ovulated follicles.To assess the influences of FSH, TNF-α, and dexamethasone on follicle growth, the following variables were observed and measured: (1) follicle diameter, (2) the concentration of EThe morphological changes were evaluated in accordance with previous studies , 27, and2 level was conducted. Estradiol EIA kits from Cayman  were used according to the manufacturer’s instructions, to measure the E2 concentrations in the recovered culture fluid. The culture fluid of non-viable follicles was excluded from the analyses.The culture fluid was refreshed every 2 days. The collected medium was pooled and frozen at −80°C until an assay of its EP < 0.05 was considered statistically significant.All tests were repeated a minimum of three times. One-way ANOVA was used to evaluate the differences between means using a post hoc test. Differences in percentages were compared using a chi-square test using the software SPSS19.0. Data were expressed as the mean ± SD. A 2. On day 12, the surviving follicle fraction between the FSH− and FSH+ groups were 9.3% and 92.8%, respectively. The percentage of follicles forming the antral-like cavities in the FSH− group was almost 0%, whereas it reached 63.8% in the FSH+ group. The ovulation fraction was 9.1% on the 13th day after HCG stimulation in the FSH− group but attained 72.4% in the FSH+ group. With the extension of the cumulus, each COC had mucification . However, when 1 μg/mL of dexamethasone was added to the cultures, the survival rate of the follicles increased from 25.5 to 49.4% and the concentration of E2 in the culture medium increased from 815.6 ± 54 to 1258 ± 89.8 pg/mL (P < 0.05) on day 12. There was no observed change in either follicle diameter or antral-like cavity formation with the addition of dexamethasone. These data and results indicate that dexamethasone counteracts the inhibitory effects of TNF-α. The total number of follicles that ovulated in the T15, T15+D0.1, T15+D1, and T15+D10 groups was 22.3%, 25.3%, 43.2%, and 23.1%, respectively, whereas the percentages of the surviving follicles that ovulated was 87.5%, 85.3%, 87.4%, and 87.5%, respectively (Table Dexamethasone partially counteracted the effects of TNF-α in a dose-dependent manner Fig. . TNF-α i2 and explains the indirect effect of dexamethasone on E2 secretion. In addition, no germinal vesicle was observed in the oocytes of each experimental group, and there were no statistically significant differences in the PB rates among the groups. There was no evidence in the present study that dexamethasone and TNF-α had an effect on nuclear maturation. The addition of 1 μg/mL dexamethasone alone had no effect on follicle growth and E2 secretion (Fig. .Granulosa cells of the surviving follicles in the TNF-α group grew only sparsely. At a concentration of 1 μg/ml, the dexamethasone promoted the proliferation of the granulosa cells, which are responsible for the secretion of Eion Fig. ; Table 32 secretion.The present study successfully established an in vitro preantral follicle culture system. This is because the follicles traversed the preantral, antral, and preovulatory phases by day 12 of the experiment, which is consistent with the in vivo growth patterns of follicles. In addition, we proved that the addition of FSH is beneficial and essential to the in vitro development of follicles. As described in Fig. 2 secretion, which is consistent with previous studies [2 production in a dose-dependent manner. The minimum effective concentration at which TNF-α inhibits follicle growth and hormone secretion is 5 ng/mL as previously reported [2. These results are consistent with those of previous studies [Data collected from these experiments illustrate that the administration of TNF-α inhibits follicle growth and E studies , 29, 30.reported , but our studies .Hara et al. argued that TNF-α primarily affects antral and not preantral follicles . However15 group. The addition of dexamethasone could only partially reverse the TNF-α inhibition of follicular growth and E2 secretion Although the diameter of the follicles did not increase, dexamethasone did promote the proliferation of granulosa cells and increased the secretion of E2 in the follicles treated with TNF-α. These results suggest that in a high TNF-α environment, dexamethasone can improve the levels of steroids, thereby increasing the survival of follicles. However, treatment with 1 μg/mL dexamethasone alone did not change the development of the follicles or affect steroid hormone production. The addition of dexamethasone preserves the follicular microstructure of the ovary [2 production. These aspects require detailed research.In addition, our results show that dexamethasone partially counteracts the inhibitory effect of TNF-α on follicle growth. From the results, the addition of dexamethasone increased the survival of TNF-α-treated follicles, especially in the TIt is believed that hyperandrogenism in PCOS is, to some extent, due to excess production of adrenalin by the adrenal glands and that this complex of metabolites in PCOS results in therapeutic challenges . It is w2. The addition of dexamethasone partially counteracted these inhibitory effects of TNF-α. The results of the present study suggest that dexamethasone may exert a therapeutic effect because it is antagonistic to TNF-α. Based on these results, there is a new foundation from which to conduct research on the application of dexamethasone in PCOS patients with high TNF-α levels.In our in vitro follicle culture system, TNF-α inhibited the growth of preantral follicles and inhibited the secretion of E


Background: Exploring the potential biological relationships between heart failure with preserved ejection fraction (HFpEF) and concomitant diseases has been the focus of many studies for the establishment of personalized therapies. Hypertension (HTN) is the most common concomitant disease in HFpEF patients, but the functional connections between HFpEF and HTN are still not fully understood and effective treatment strategies are still lacking.Methods: In this study, tandem mass tag (TMT) quantitative proteomics was used to identify disease-related proteins and construct disease-related networks. Furthermore, functional enrichment analysis of overlapping network modules was used to determine the functional similarities between HFpEF and HTN. Molecular docking and module analyses were combined to identify therapeutic targets for HFpEF and HTN.Results: Seven common differentially expressed proteins (co-DEPs) and eight overlapping modules were identified in HFpEF and HTN. The common biological processes between HFpEF and HTN were mainly related to energy metabolism. Myocardial contraction, energy metabolism, apoptosis, oxidative stress, immune response, and cardiac hypertrophy were all closely associated with HFpEF and HTN. Epinephrine, sulfadimethoxine, chloroform, and prednisolone acetate were best matched with the co-DEPs by molecular docking analyses.Conclusion: Myocardial contraction, energy metabolism, apoptosis, oxidative stress, immune response, and cardiac hypertrophy were the main functional connections between HFpEF and HTN. Epinephrine, sulfadimethoxine, chloroform, and prednisolone acetate could potentially be effective for the treatment of HTN and HFpEF. Heart failure with preserved ejection fraction (HFpEF), which is a complex syndrome characterized by a normal left ventricular ejection fraction and abnormal diastolic function, accounts for more than 50% of heart failure (HF) patients . CurrentPatients with HFpEF that are diagnosed with hypertension (HTN) and coronary heart disease are regarded as having vascular-related HFpEF . StudiesDisease network construction provides a solution to explore the relationships between diseases , where mThe Dahl salt-sensitive (DS) rat model has been implemented for the study of HFpEF . Toward n = 6), the HFpEF group , and the control group . All experiments were reviewed by the Animal Ethics Committee of the Shandong University of Traditional Chinese Medicine (Ethics No. SDUTCM2018071501).Specific pathogen-free 6-week-old male DS rats  were obtained from the Charles River Animal Laboratory . Rats were housed in groups of six rats per cage under controlled conditions  and with free access to water and food. After a week of adaptation, the rats were randomly divided into the following three groups: the HTN group , respectively, and the rats in the control group were randomly sacrificed after 7 or 11 weeks of the control diet. Pentobarbital  was used for anesthesia in rats, and the left ventricle (LV) was collected from each rat and stored at −80°C.n = 3, HFpEF group n = 3, control group (euthanized at 7 weeks) n = 3, control group (euthanized at 11 weeks) n = 3] were collected for TMT quantitative proteomics. Previous studies reported that data with three samples in each group could reliably be statistically analyzed  was used for washing three times. In addition, an appropriate amount of eluent  was added, and the filtrate was collected and lyophilized. One hundred microliters of 0.1 M TEAB buffer was used for reconstitution, and 41 μL of TMT labeling reagent was dissolved in acetonitrile. The mixture was mixed at room temperature for 2 h, and 8% ammonia was added to stop the reaction. An equal volume of the labeled sample was used for mixing and freeze-drying after desalting.The Bradford protein quantification kit  was used to determine the protein concentration. DB protein dissolving solution  was added to the protein sample to a volume of 100 μL, trypsin and 100 mM buffer were added, and mixing and digestion were performed at 37°C for 4 h. Then, pancreatin and CaClMobile phase A solution  and mobile phase B solution  were prepared. The freeze-dried powder was dissolved in solution A and centrifuged at 12,000 rpm for 10 min at room temperature. An L-3000 HPLC system and a water VEHC 18  were used for this study, and the column temperature was set to 45°C. Details of the elution gradient are shown in 6, and the maximum injection time of the C-trap was 20 ms. The top 40 precursor ions were selected for the full scan, and the higher-energy collision dissociation (HCD) method was used for the fragment, which contributed to the secondary mass spectrometry detection. The isolation window of MS2 spectrum is 2 m/z. HCD spectrum ranged from 120 to m/z (precursor ion) × z (charge number) + 100 m/z. The resolution was 45,000 (200 m/z), the maximum capacity of the C-trap was 5 × 104, the maximum injection time of the C-trap was 86 ms, the threshold intensity was 1.2 × 105, and the dynamic exclusion range was 20 s.Mobile phase A solution  and phase B solution  were prepared. One microgram of the supernatant from each fraction was used for the test. The UHPLC system was upgraded with the EASY-nLC 1200 system. We prepared both the pre-column  and the analytical column . The elution conditions for liquid chromatography are shown in 1. The search parameters included trypsin as the enzyme used to generate peptides with a maximum of two missed cleavages permitted. A precursor mass tolerance of 10 ppm was specified along with a 0.05 Da tolerance for MS2 fragments. Except for the TMT labels, carbamidomethyl (C) was set as a fixed modification. The variable modifications were oxidation (M) and acetyl (protein N-term). A peptide and protein false discovery rate of 1% was enforced using a reverse database search strategy. The quantitative values of proteins obtained from two pairs of samples were examined using the t-test, and the p-values were calculated. Fold change >1.1, fold change <0.91, and P-value < 0.05, were considered to filter differentially expressed proteins (DEPs). Proteomic data is provided as MS/MS raw files were processed using the MASCOT engine  embedded into Proteome Discoverer software, and searched against the UniProt database, including Uniprot_RattusNorvegicus_36080_20180123 sequences2 was used to predict protein interactions and functional associations. The PPI networks of HFpEF- and HTN-DEPs were obtained under controlled parameters (interaction score >0.4). PPI networks were analyzed using Cytoscape (version 3.6.1)3.The STRING database (version 10.5)4. The P-value was set at <0.05 for GO and KEGG pathway enrichment, as is standard in the field  pathway enrichment analyses (version 6.8)he field .5. Furthermore, the modules were divided by Molecular Complex Detection 6. The modules were obtained under controlled parameters . The parameters for Cytoscape were set as default, as recommended by previous studies  studies .7, which integrates the relationships between gene products, diseases, chemicals, and environments. Furthermore, related MM symptoms of the co-DEPs were observed in SymMap8 and in previous publications.The association between the co-DEPs and cardiovascular diseases was analyzed using the Comparative Toxicogenomics Database (CTD)9, through the target search. Molecular docking analysis is a common strategy for drug discovery. The structures of the co-DEPs were downloaded from the Protein Data Bank (PDB)10, and the PDB IDs of the proteins are shown in 11, and the CIDs are shown in Small-molecule compounds related to the co-DEPs were observed from the DrugBank12 and previous publications , regulation of protein dephosphorylation , response to lead ion , negative regulation of catalytic activity , and regulation of phosphorylation . Blood microparticles , extracellular exosome , extracellular space , mitochondrial inner membrane , and extracellular matrix  related cell compositions were significantly enriched. Furthermore, the main enriched molecular functions were protein homodimerization activity , protein serine/threonine phosphatase inhibitor activity , protein binding , chaperone binding , and endopeptidase inhibitor activity . The enriched KEGG pathways were prion diseases  and the complement and coagulation cascades  , cardiac muscle contraction , muscle contraction , fatty acid beta-oxidation , and cardiac myofibril assembly . The main components were extracellular exosome , mitochondrion , striated muscle thin filament , mitochondrial inner membrane , and a band . The molecular functions of HTN-PEGs were mainly enriched in protein homodimerization activity , actin filament binding , tropomyosin binding , oxidoreductase activity , and actin monomer binding . The enriched KEGG pathways were adrenergic signaling in cardiomyocytes , carbon metabolism , cardiac muscle contraction , starch and sucrose metabolism , and mineral absorption   . With re3637201) .In total, from the PPI network of HFpEF-DEPs  and thatP-Value 5.59E-08), acute-phase response , regulation of the heart contraction force , muscle contraction , and response to lead ion . Extracellular exosome , mitochondrion , blood microparticle , extracellular space , and a band  related cell compositions were significantly enriched. Furthermore, the main enriched molecular functions were protein homodimerization activity , calcium ion binding , identical protein binding , actin filament binding , and tropomyosin binding . The enriched KEGG pathways included adrenergic signaling in cardiomyocytes , prion diseases , cardiac muscle contraction , complement and coagulation cascades , and adrenergic signaling in cardiomyocytes  (The overlapping modules between the HFpEF and HTN networks are shown in 0014194) . Finally0014194) .Twelve small-molecule compounds were obtained from the DrugBank, including prednisolone acetate, bismuth subsalicylate, phenoxymethylpenicillin, polyethylene glycol, prednisolone, chloroform, salicylic acid, epinephrine, triptorelin, benzylpenicillin, propofol, and sulfadimethoxine. The binding affinity between the co-DEPs and the small-molecule compounds are shown in Heart failure with preserved ejection fraction is a complex syndrome that includes many types of clinical phenotypes. Huge pathophysiological differences exist among patients with different clinical HFpEF phenotypes, and no treatment strategy is suitable for all patients with HFpEF . ExploriIn this study, TMT-labeled quantitative proteomics was used to identify HFpEF- and HTN-related proteins. The functional links between HFpEF and HTN were analyzed at the network, module, and protein levels. Furthermore, molecular docking was used to determine precision medicine targets for HFpEF and HTN treatment. Seven co-DEPs were found among the HFpEF- and HTN-DEPs identified, including Hp, Tf, COQ9, Acat1, Timm44, Abcb6, and Prnp. Notably, Hp levels are closely related to hypertension and heart failure . MoreoveFever and anemia were found to be important co-DEP-related MM symptoms, which is consistent with previous findings . The comHeart failure with preserved ejection fraction and HTN shared eight overlapping modules, and the main biological functions enriched in these modules were myocardial contraction, energy metabolism, apoptosis, oxidative stress, immune response, and cardiac hypertrophy. We also found that post-translational modification and regulation of actin filaments could play an important role in HFpEF and HTN. Previous research has shown that phosphorylation of cardiac myosin-binding protein-C influences the progress of cross-bridge detachment and that deficient phosphorylation leads to diastolic dysfunction . FurtherThe study also suggested that chloroform, epinephrine, sulfadimethoxine, and prednisolone acetate could be effective drugs for treating HTN and HFpEF. In addition, epinephrine, sulfadimethoxine, and prednisolone acetate have been widely used in many clinical diseases, but chloroform was not approved drug for human use. It suggested that chloroform maybe an candidate drugs for HTN-HFpEF. Previous studies have shown that gut microbiota dysfunction is closely related to the development of HTN and HFpEF , and somAccording to the enrichment results of the overlapping modules, myocardial contraction was the most important biological function shared between HFpEF and HTN. HTN influences the structure and function of the heart, suppresses myocardial contractions, and increases the prevalence of HFpEF . PreviouHere, we found that energy metabolism is closely related to HFpEF and HTN. These findings are consistent with earlier observations . Acta1, Furthermore, apoptosis was found to be an important biological function in HTN and HFpEF. Activation of apoptosis can lead to cardiac dysfunction , and inhIn our study, oxidative stress was important in both HFpEF and HTN. Myocardial fibrosis, the major factor leading to myocardial remodeling, was found to be a common pathological mechanism among HFpEF and HTN. Previous studies using an animal model of HFpEF and HTN have confirmed that the regulation of oxidative stress contributes to the inhibition of myocardial fibrosis . SimilarImmune responses are activated in both HFpEF and HTN . AlthougThe results showed that cardiac hypertrophy, which is associated with diastolic function, was significantly associated with HFpEF and HTN . AngioteSeven co-DEPs were observed between the HFpEF-DEPs and HTN-DEPs, including Hp, Tf, COQ9, Acat1, Timm44, Abcb6, and Prnp. These co-DEPs were closely related to the main functional similarities of HFpEF and HTN, including myocardial contraction, energy metabolism, apoptosis, oxidative stress, immune response, and cardiac hypertrophy. These co-DEPs may serve as biomarkers and drug targets for HFpEF and HTN. Furthermore, epinephrine, sulfadimethoxine, chloroform, and prednisolone acetate may serve as precision medicines for the treatment of HTN and HFpEF. Our study provides several targets for of the development of personalized therapies and precision medicines to treat HFpEF and other comorbidities.There are some limitations to this study. Proteins with low expression levels or those showing insignificant changes could have been ignored in the analyses. Furthermore, these results need to be validated through fundamental research and clinical trials. Further animal experiments will help to explore the function of these drugs in HTN and HFpEF, and small-scale clinical trials will contribute to identifying whether these drugs have similar effects in patients with HTN and those with HFpEF.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by the Animal Ethics Committee of Shandong University of Traditional Chinese Medicine.GZ, JC, and CW conceived the study, acquired the data, and wrote the manuscript. PJ designed the experiments and interpreted the data. YW and YZ performed the experiments and statistical analysis. YJ and XL designed the study and revised the manuscript. All authors read and approved the final manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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