{"paper_id":"74437eef-949e-487b-ab18-322037039eff","body_text":"Explor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 476\n© The Author(s)  2024.  This  is an Open  Access  article  licensed  under  a Creative  Commons  Attribution  4.0 International \nLicense (https://creativecommons.org/licenses/by/4.0/), which  permits  unrestricted  use,  sharing,  adaptation,  distribution \nand reproduction in any medium  or format,  for any purpose,  even commercially,  as long as you give appropriate  credit  to the \noriginal author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.\nExploration of Immunology\nOpen Access Review\nIL-6 signaling pathway differentiation for endometriosis and \ninflammatory diseases\nRyan D. Castle*\nIbis Optum Consulting, Ewa Beach, Hawaii 96706, USA\n*Correspondence: Ryan D. Castle, Ibis Optum Consulting, 5593 Cormorant Ave, Ewa Beach Hawaii 96706, USA. rcastle@\nibisoptum.com\nAcademic Editor: Giustino Varrassi, Paolo Procacci Foundation, Italy\nReceived: February 18, 2024  Accepted: May 30, 2024  Published: August 20, 2024\nCite this article:  Castle RD. IL-6 signaling  pathway  differentiation  for endometriosis  and inflammatory  diseases.  Explor \nImmunol. 2024;4:476–89. https://doi.org/10.37349/ei.2024.00153\nAbstract\nInterleukin-6  (IL-6)  plays a critical  role in the pathogenesis  of various  chronic  inflammatory  diseases, \ntracked across numerous fields of research. Despite this, a crucial aspect often overlooked in studies is the \ndifferentiation between IL-6 classic-signaling and trans-signaling, leading to potential confounding of their \nfindings;  less  than  half  of selected  IL-6  studies  included  differentiation.  This  review  delves  into  the \ndistinction  between IL-6 classic- and trans-signaling  and their role in chronic inflammatory  diseases and \nendometriosis.  The unique  pro-inflammatory  nature  of IL-6 trans-signaling,  contrasted  with the anti-\ninflammatory  character  of IL-6  classic-signaling,  presents  significant  implications  for  research \nmethodology,  particularly  in studies  investigating  anti-inflammatory  interventions  or interleukin \ninhibitors.  Diagnostic  failure  to account  for these  distinct  pathways  may inadvertently  misrepresent \nbeneficial  immune responses  or the efficacy  of interventions,  posing significant  challenges  in predicting \nhealth outcomes.  Interventions  that do not differentiate  these pathways  could face reduced  efficacy  or \nsafety. This review proposes adjustments to research methodologies and stresses the importance of careful \ninterpretation  of inflammatory  markers  in IL-6-related  research.  Examples  of differentiation  issues are \ndiscussed  across  the topics  of endometriosis  and multiple  inflammatory  diseases.  By addressing  this \nmethodological  issue,  researchers  could potentially  improve  patient  outcomes,  enhance  the efficacy  of \ninterventions, and contribute to public health advancements.\nKeywords\nInflammation, interleukin-6, endometriosis, cytokines, biomarkers, inflammatory diseases\nIntroduction\nInterleukin-6  (IL-6),  a pleiotropic  cytokine,  has drawn considerable  attention  for its central  role in the \npathogenesis  of various chronic inflammatory  diseases [1]. Its dual functionality  can be attributed  to the \nexistence of two distinct signaling mechanisms: IL-6 classic-signaling and IL-6 trans-signaling. IL-6 classic-\nsignaling has been recognized for its regulatory and protective roles, contributing to immune resilience [2]. \n\n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 477\nConversely,  IL-6  trans-signaling  has been  implicated  in instigating  and perpetuating  inflammatory \nresponses,  exacerbating  disease  progression.  Each of these pathways  carries  unique  physiological  and \npathological  implications,  however,  much  of the extant  literature  investigating  the efficacy  of anti-\ninflammatory  interventions  fails  to differentiate  between  them.  This  lack  of specificity  potentially \nconfounds  our understanding  of the therapeutic  effects  and hinders  the development  of precision \ninterventions [3].\nThe study of endometriosis, where conventional therapies are often dependent on early and accurate \ndiagnostics,  offers a valuable perspective toward the importance of the differential  effects of IL-6 classic- \nand trans-signaling [4]. From a clinical perspective, the reliance on IL-6 levels for diagnosing endometriosis \nis fraught with challenges.  The heterogeneity  of endometriosis  manifestations  means that IL-6 levels can \nvary widely among patients, influenced by disease stage, lesion location, and individual immune responses. \nUnreliable IL-6 measurements may lead to misdiagnosis or underdiagnosis, particularly in cases where IL-6 \nlevels  are  incongruent  with  clinical  symptoms.  This  discrepancy  complicates  the  development  of \nstandardized diagnostic criteria and can delay appropriate treatment interventions [5].\nThe  treatment  of endometriosis  often  involves  managing  symptoms  and  controlling  disease \nprogression  through  hormonal  therapies  and  surgical  interventions.  Given  IL-6’s  involvement  in \ninflammatory  processes,  therapies  targeting  IL-6 signaling  have been proposed  as potential  treatments. \nHowever,  without  accurate  and reliable  measurements  of IL-6 and an understanding  of its signaling \ncontext, it is challenging to predict therapeutic outcomes or tailor treatments to individual patient profiles \n[6].\nThis review seeks to underscore  the importance  of differentiating  between  IL-6 classic-  and trans-\nsignaling in the study of chronic inflammatory  diseases and in particular  endometriosis.  The subsequent \nsections will delve into the unique roles of these signaling pathways in inflammation  and health, explore \nhow their conflation could confound existing research, and propose potential directions for future studies \nin this  area.  Ultimately,  the aim is to enhance  our understanding  of IL-6  signaling  and facilitate  the \ndevelopment of more targeted and efficacious therapeutic interventions for chronic inflammatory diseases.\nIL-6 signaling: classic vs. trans\nIL-6,  a frequently  tracked  cytokine  in immunological  studies,  operates  through  two distinct  signaling \npathways: IL-6 classic-signaling and IL-6 trans-signaling [7]. The division of these pathways, first identified \nin 1994,  enables  IL-6  to assume  roles  ranging  from  modulating  inflammation  to bolstering  immune \ndefenses, a balance that is gaining recognition as vital for human health [8].\nIL-6 classic-signaling is limited to specific cells equipped with the membrane-bound IL-6 receptor (IL-\n6R), including hepatocytes and certain leukocytes. This signaling commences with IL-6 attaching to IL-6R, \nthen pairing with the universally expressed signal-transducing receptor protein, glycoprotein 130 (gp130). \nThis event triggers a succession of intracellular signals, culminating in the activation of diverse genes that \ndrive the cellular response to IL-6. The selective character of IL-6 classic-signaling  ensures its effects are \npredominantly  on cells possessing  the necessary  receptor  apparatus,  thereby  maintaining  a restricted \ninfluence.\nIn contrast, IL-6 trans-signaling involves the soluble IL-6R (sIL-6R). This receptor is unattached to the \ncell membrane and can circulate freely and bind to IL-6. The resulting sIL-6R/IL-6 complex can engage with \ngp130 on the surfaces  of practically  all cells, instigating  the same intracellular  signal cascade as classic-\nsignaling.  This  mechanism  broadens  IL-6’s  influence  on cells  lacking  the  membrane-bound  IL-6R, \ndrastically augmenting the potential scope of its action.\nThese two signaling  routes play contrasting  roles within the immune system and inflammation,  as \ndemonstrated in Figure 1. IL-6 classic-signaling  is tied to host defense functions, aiding the activation and \ndifferentiation of T cells and B cells-vital components of the immune response to infections that culminate \nin anti-inflammatory  action  [9]. Conversely,  IL-6 trans-signaling  has been linked  to pro-inflammatory \nresponses  and is considered  a major  player  in the pathogenesis  of numerous  chronic  inflammatory \ndiseases, such as rheumatoid arthritis (RA) and inflammatory bowel disease (IBD).\n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 478\nFigure 1. The different signaling pathways for IL-6\nNote. Reprint with permission from “IL-6-induced classic and trans-signalling” by Schaper F. [cited 2024 Feb 16]. Available from \nhttps://www.systembiologie.ovgu.de/en/-p-176.html\nDespite  these  crucial  distinctions  between  IL-6  classic-  and  trans-signaling,  many  research \ninvestigations  probing  anti-inflammatory  interventions  often  neglect  this  differentiation.  Failing  to \ndistinguish  between  these  signaling  mechanisms  when  assessing  IL-6  levels  could  lead  to data \nmisinterpretation,  and subsequently  an over- or under-estimation  of the efficacy  of anti-inflammatory \ninterventions. Such an oversight could unintentionally compromise the evaluation of potential side effects \nand the overall effectiveness of therapeutic strategies [10].\nFor example:\nIf an anti-inflammatory  IL-6 inhibitor  suppresses  both IL-6 classic-  and trans-signaling,  it may \ninadvertently  impede  beneficial  immune  responses,  leading  to unintended  consequences  like \nheightened susceptibility to infections.\n• \nConversely, interventions that selectively inhibit IL-6 trans-signaling,  could be successfully treating \ninflammatory symptoms but still present high IL-6 reports in undifferentiated biomarker tests [11].\n•\nNeglecting  these  differences  can potentially  obstruct  progress  in devising  effective  therapies  for \nchronic inflammatory  diseases,  and may also complicate  our understanding  of the relationship  between \ninflammation,  IL-6 signaling, and the treatment of chronic inflammatory  diseases. Hence, future research \ninitiatives  should  make deliberate  efforts  to distinguish  between  these  IL-6 signaling  pathways  when \ninvestigating anti-inflammatory interventions.\nLack of differentiation\nThe demarcation between IL-6 classic-signaling and IL-6 trans-signaling is essential to comprehending the \nmultifaceted  roles of IL-6 in health and disease.  However,  this distinction  is frequently  glossed  over in \nbiomedical research, especially in studies centered on chronic inflammatory disease and endometriosis.\n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 479\nA significant  contributor  to this oversight  is the technical  challenge  of differentiating  between these \ntwo signaling pathways in vivo. While in vitro studies can utilize techniques such as receptor blockade to \ndistinguish between IL-6 classic- and trans-signaling,  these methods are less practical in living organisms \ndue to the universal expression of gp130 and the potential unintended effects of blocking agents [12].\nAdditionally,  the most commonly employed assays for quantifying IL-6 levels in biological specimens \ndo not differentiate between IL-6 bound to soluble or membrane-bound  IL-6R, thus failing to encapsulate \nthe distinction between the two pathways.\nAnother element contributing to this oversight is the relatively recent elucidation of the disparate roles \nIL-6 classic-  and trans-signaling  play. While IL-6 has been a point of interest  in immunology  for several \ndecades,  the concept of trans-signaling  was introduced  only in the mid 1990s [13]. Given this relatively \nrecent evolution and the topic’s complexity, many researchers may not fully comprehend the significance of \ndifferentiating between these two signaling pathways when interpreting IL-6 measurements.\nThis failure to distinguish between IL-6 classic- and trans-signaling is especially relevant in behavioral \nmedicine, which frequently  employs anti-inflammatory  interventions  to enhance well-being  and prolong \nhealthspan.  These interventions,  encompassing  mindfulness  meditation,  yoga, and acupuncture,  among \nothers, have been shown to affect IL-6 levels. However, the specific impacts of these interventions on IL-6 \nclassic- vs. trans-signaling remain largely uncharted territory [14].\nA meta-analysis compared measurements of IL-6 in response to both inflammatory diseases and anti-\ninflammatory  interventions  [15]. This analysis  found that different  collections  of biomarkers  labeled as \nundifferentiated  “IL-6” were impacted with significant  effect sizes in response to both pro-inflammatory \nand anti-inflammatory  catalysts.  The study proposed that these metrics were in fact capturing  both IL-6 \ntrans and classic,  respectively,  and had thus been underreporting  the efficacy  of the anti-inflammatory \ntreatments.  When IL-6 trans and classic  were incorporated  into the analysis,  interventions  expected  to \nfocus on IL-6 trans ranked higher in efficacy, as seen in Figure 2.\nFigure 2. Biomarker measurements indicating an increase in pro-inflammatory  action (orange) via IL-6 trans from COVID-19, \nwhile anti-inflammatory actions (blue) were associated with melatonin, vitamin D3, and meditation. Conversely, meditation and \nvitamin D3 were associated with anti-inflammatory action (blue) via IL-6 classic\nNote. Adapted  with permission  from “Implications  for Systemic  Approaches  to COVID-19:  Effect  Sizes  of Remdesivir, \nTocilizumab,  Melatonin, Vitamin D3, and Meditation.”  by Castle RD, Williams MA, Bushell WC, Rindfleisch JA, Peterson CT, \nMarzolf J, et al. J Inflamm Res. 2021;14:4859–76 (https://doi.org/10.2147/JIR.S323356). CC BY-NC.\n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 480\nDifferentiating  between  IL-6  classic-  and trans-signaling  is a pivotal  aspect  of interpreting  IL-6 \nmeasurements  in research  but is frequently  overlooked,  particularly  in addressing  complex,  chronic \nconditions. The impacts of this lack of distinction can be found in the fields of endometriosis, neuroscience, \nand  many  chronic  inflammatory  diseases,  though  these  are  only  examples  of a far  broader  issue. \nAcknowledging and addressing this oversight is necessary to foster a more nuanced understanding of the \neffects of anti-inflammatory interventions, ultimately contributing to the formulation of more targeted and \neffective therapies.\nDiscussion\nUnpredictable IL-6 metrics in endometriosis research\nEndometriosis, a chronic inflammatory condition, affects approximately 10% of reproductive-aged women \nworldwide [16]. Characterized by the growth of endometrial-like  tissue outside the uterus, endometriosis \nresults  in pain  and  fertility  issues  among  the  afflicted.  The  molecular  and  cellular  mechanisms \nunderpinning the pathology of endometriosis have been the subject of intensive study, with IL-6 emerging \nas a pivotal cytokine in this context. Research has suggested that IL-6, especially through the trans-signaling \npathway,  contributes  to pain  in endometriosis  patients  by promoting  endometrial  lesions  [17]. \nFurthermore,  IL-6 can also facilitate  angiogenesis,  supporting  the survival  and proliferation  of ectopic \nlesions [18]. The nuanced roles of IL-6 classic- and trans-signaling  in endometriosis  bear implications  for \nthe diagnosis, treatment, and overall understanding of the condition.\nThe misinterpretation  of inflammatory  responses is a significant  concern stemming from this lack of \ndifferentiation.  The anti-inflammatory  nature of IL-6 classic-signaling,  particularly in tissue regeneration, \ncontrasts sharply with the pro-inflammatory tendencies of trans-signaling [8]. Consequently, an inaccurate \nassessment  of the inflammatory  milieu  in endometriosis  patients  could  arise,  potentially  misleading \nclinicians  in gauging  the severity  of the disease  or the efficacy  of therapeutic  interventions.  Given the \ncentrality  of IL-6 measurements  in endometriosis  research,  such misinterpretations  may also result in \ntherapeutic  strategies  that  inadvertently  suppress  the beneficial  effects  of classic-signaling,  thereby \npotentially  prolonging  patient  recovery  and undermining  tissue  repair  and regeneration  [19]. Without \nprecise  differentiation,  the  potential  for  IL-6’s  dual  nature  to serve  as a nuanced  biomarker  for \nendometriosis  regulation  and dysregulation  remains  largely  untapped,  reducing  treatment  efficacy \nassessments [20].\nThis oversight  is particularly  visible  in chronic  inflammatory  diseases  like endometriosis.  Figure 3 \ndemonstrates  the lack of studies  incorporating  differentiation  between  the signaling  pathways  of IL-6. \nSearches through PubMed were conducted on multiple search strings relating to endometriosis  and IL-6, \nwith an increasing focus on pathway differentiation. While endometriosis studies incorporating IL-6 were \ncommon,  only  a fifth  of those  studies  mentioned  signaling  pathways.  When  specific  pathways  were \narticulated, results became negligible. There were only eight results for a search of endometriosis and IL-6 \ntrans vs. classic, and only seven studies actually incorporated any differentiation between IL-6 trans and IL-\n6 classic.\nThis steep drop off reflects not only a significant  gap in the state of research, but a disproportionate \nlack of focus in ongoing research. Table 1 consists of a network analysis of the preceding and proceeding \ncitations of endometriosis studies relating to IL-6 examined the number of citations studies received and a \nsimilarity factor rating, based on how closely the studies list reflected the initial search parameter.\nThe number of citations  for papers related to endometriosis  and undifferentiated  IL-6 was 235% \nhigher than for endometriosis IL-6 trans.\n• \nThe similarity factor rating for undifferentiated IL-6 was 352% higher than for endometriosis and IL-\n6 trans.\n•\nThere is a clear and significant difference in number of citations for the top 40 articles related to either \ndifferentiated  or undifferentiated  IL-6 and endometriosis.  It is likely  this reflects  far less activity  and \n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 481\nFigure 3. Results of searches through PubMed (accessed 2024 Feb 17) for studies on endometriosis and IL-6, with and without \ndifferentiation\nongoing research into the differentiation  of IL-6 signaling  pathways within endometriosis  diagnosis  and \ncare. The low numbers  of similarly  clustered  studies  relating  to endometriosis  and differentiated  IL-6 \nsuggests problematic heterogeneity  in the different studies related to the topic. Diagrams using weighted \nclustering  can visually  depict the distance  between studies with or without differentiation.  Figure 4 and \nFigure 5 illustrates  how a network analysis  of IL-6 trans and endometriosis  produced  distant,  disparate \nclustering,  whereas the network analysis  of undifferentiated  IL-6 and endometriosis  is close and evenly \nclustered.\nTaken  together,  there  is a scarcity  of endometriosis  studies  specifically  incorporating  IL-6 \ndifferentiation,  a relatively low number of citations related to endometriosis  and any focus on IL-6 trans, \nand a low network analysis rating of IL-6 differentiated studies. These factors strongly suggest the study of \nendometriosis  diagnostics  and treatments  do not commonly incorporate  differentiation  of IL-6 signaling \npathways.  It is believed endometriosis  is indicative  of similar disparities  relating to many other chronic \ninflammatory diseases.\nThere are several potential consequences for this disparity. From a clinical perspective, the reliance on \nIL-6  levels  for  diagnosing  endometriosis  is common  and  highly  influential.  The  heterogeneity  of \nendometriosis manifestations means that IL-6 levels can vary widely among patients, influenced by disease \nstage, lesion location, and individual immune responses, requiring precise measurement.  Unreliable IL-6 \nmeasurements may lead to misdiagnosis or underdiagnosis, particularly in cases where undifferentiated IL-\n6 levels are incongruent  with clinical  symptoms  [21]. This discrepancy  complicates  the development  of \nstandardized  diagnostic  criteria and can delay appropriate  treatment  interventions.  Timely diagnosis  of \nendometriosis  is critical  for proper  care,  with serious  health  impacts  resulting  from misdiagnosis  or \nunderdiagnosis [22].\nMoreover, the treatment of endometriosis often involves managing symptoms and controlling disease \nprogression  through  hormonal  therapies  and surgical  interventions.  However,  without  accurate  and \nreliable  measurements  of IL-6 and an understanding  of its signaling  context,  it is challenging  to predict \ntherapeutic  outcomes  or tailor  treatments  to individual  patient  profiles.  For instance,  indiscriminate \nblockade of IL-6 could dampen its beneficial effects in tissue protection and repair, leading to unclear side \neffects and obscured therapeutic outcomes. For example, two different studies into the use of tocilizumab \nas an IL-6 inhibitor in rats produced comparable clinical results, but starkly different IL-6 measurements. \n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 482\nTable 1. The top 40 studies in a network analysis of searches for endometriosis  and IL-6 as well as endometriosis  and IL-6 \ntrans\nEndometriosis IL-6-trans Endometriosis IL-6\nCitations Refs Similarity Citations Refs Similarity\n7 0 100 42 31 100\n4 0 10.9 138 51 20.9\n16 57 9.4 52 25 20.7\n6 27 9.1 43 81 20.4\n15 73 7.7 131 64 19.4\n3 74 7.3 127 54 18.1\n11 61 6.8 85 49 18.1\n7 40 6.2 145 36 16.3\n13 37 6.2 49 54 16.2\n50 54 6 34 43 15.8\n16 45 6 38 38 14.7\n13 42 5.8 27 36 14.5\n7 54 5.6 60 24 14\n14 49 5.5 68 41 13.5\n13 148 5.5 177 66 13.4\n13 28 5.5 75 29 13.2\n8 69 5.4 37 67 12.8\n0 109 5.4 33 76 12.8\n43 162 5.3 134 70 12.6\n59 68 5.3 111 49 12.6\n15 48 5.2 32 89 12.4\n108 37 5.2 230 65 12.4\n9 43 5.2 41 28 12.2\n73 36 5.1 90 173 12.1\n52 44 5.1 228 24 11.9\n25 57 5.1 141 63 11.8\n27 128 5 137 57 11.8\n18 135 5 41 49 11.6\n239 125 5 47 49 11.6\n38 190 5 35 43 11.4\n50 46 4.9 53 54 11.3\n9 37 4.9 20 23 11\n6 46 4.9 282 271 11\n1 0 4.8 22 65 11\n1 45 4.8 63 28 11\n1 0 4.8 152 150 11\n1 87 4.8 57 21 10.9\n1 54 4.8 95 60 10.9\n13 227 4.8 64 114 10.8\n1 46 4.7 35 79 10.6\n1 21 4.7 81 160 10.6\n1,007 - 228.7 3,552 - 539.3\nMultiple search terms were used to identify synonyms and largest returns were utilized. Relative similarities were color coded \nfrom strong to weak on a respective blue-red spectrum. Bold numbers at the bottom indicate total sums of the fields above. -: \nblank cell\n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 483\nFigure 4. A visualization of the network analysis of search parameter “endometriosis IL-6-trans” as seen in Table 1, organized \nby citation. Clustering is by weighted similarity factor, studies that are more closely linked are more closely clustered. Created \nwith Connected Papers accessed 2024 Feb 18, which is connected to the Semantic Scholar Paper Corpus (licensed under \nODC-BY)\nOne study, which did not differentiate IL-6 trans and classic, reported no discernible IL-6 changes after the \nintervention  [23]. The other study, which included  signal differentiation  in its measurements,  reported \nsharp reductions in IL-6 trans [24]. If IL-6 measurements are not consistent, inflammation measurements \ncan be unreliable.\nThe challenges  extend  to monitoring  disease  progression  and treatment  efficacy.  IL-6  has been \ninvestigated as a biomarker for disease activity, however, variable IL-6 levels, influenced by both biological \nfluctuations  and measurement  inaccuracies,  can obscure  the correlation  between  cytokine  levels  and \ndisease state [25]. This uncertainty  complicates  the clinician’s  ability to make informed  decisions  about \ntreatment adjustments or to accurately predict disease prognosis.\nGiven  IL-6’s  involvement  in inflammatory  processes,  narrow  inhibitor  therapies  targeting  IL-6 \nsignaling  have been proposed  as potential  treatments  [26]. Research  into olamkicept  has focused  on \ntargeting  IL-6  trans-signaling  while  leaving  IL-6  classic-signaling  intact  [27]. The results  have  been \npromising  for  IBD  and  ulcerative  colitis,  reducing  clinical  inflammatory  symptoms  without  the \nimmunosuppressant side effects of total IL-6 inhibition. The success of precise targeting indicates the value \nof widespread differentiation.\nA practical  dimension  where  greater  differentiation  of IL-6 pathways  might  prove  valuable  is in \nsymptom management. Pain, a predominant symptom of endometriosis, has been linked to IL-6 mediated \nnerve  fiber  growth  in ectopic  lesions  [28]. By understanding  and targeting  the specific  IL-6 signaling \npathways responsible, pain management could see substantial advancements, enhancing patient quality of \nlife. Differentiated IL-6 inhibitors have been widely explored for the treatment of COVID-19, and a similar \nfocus may be warranted for endometriosis and other inflammatory diseases.\n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 484\nFigure 5. A visualization of the network analysis of the search parameter “endometriosis IL-6” as seen in Table 1, organized by \ncitation. Clustering is by weighted similarity factor, studies that are more closely linked are more closely clustered. Created with \nConnected Papers accessed 2024 Feb 18, which is connected to the Semantic Scholar Paper Corpus (licensed under ODC-BY)\nBy addressing  the oversight  in differentiation  several  benefits  could emerge that may significantly \nimprove  endometriosis  management.  Tailored  therapeutic  strategies  that specifically  target IL-6 trans-\nsignaling could mitigate the pro-inflammatory aspects of endometriosis more effectively without disrupting \nthe beneficial effects of classic-signaling [29]. Such specificity in treatment could herald improved outcomes \nand reduced  side effects.  Furthermore,  the diagnostic  precision  in endometriosis  can be significantly \nenhanced.  Accurately  differentiated  IL-6  signaling  can offer  clinicians  insights  into  disease  severity, \nprogression,  and earlier detection,  a critical component of endometriosis  care that can inform treatment \ndecisions and prognostic evaluations [30].\nImplications for chronic inflammatory illnesses\nAs seen in the focus on endometriosis, IL-6 plays a pivotal role in the immune response and has also been \nimplicated  in the pathogenesis  of a wide range of inflammatory  diseases,  including  rheumatoid  arthriti, \nsystemic lupus erythematosus (SLE), and IBD.\nIn the context of RA IL-6 trans is a key driver of inflammation  and joint destruction.  Some therapies \nthat inhibit IL-6, such as monoclonal  antibodies  against IL-6R, have demonstrated  significant  efficacy  in \nreducing disease activity and improving clinical outcomes. However, by inhibiting both signaling pathways, \nthese therapies may also interfere with the protective roles of IL-6, such as its involvement in host defense \nand tissue  regeneration.  A more selective  approach  that targets  only the trans-signaling  pathway  can \npreserve  the cytokine’s  homeostatic  functions  while  effectively  mitigating  inflammation  and disease \nprogression  [31]. Similarly,  in diseases  like IBD, where IL-6 levels  correlate  with disease  severity  and \ninflammation,  selectively targeting the trans-signaling  pathway could reduce intestinal inflammation  and \npromote mucosal healing, offering a novel approach to treatment that minimizes the risk of systemic side \neffects [32]. Differentiated targeting of IL-6 trans inhibition has shown significant promise as a useful anti-\ninflammatory intervention, with awareness of differentiation being the limiting factor in adoption [33].\nThe differentiation  of IL-6 signaling pathways also has profound implications  for the development of \ndiagnostic tools and the monitoring of disease activity in chronic inflammatory  conditions.  Inflammatory \nmarkers, including IL-6, play an essential role in diagnostics, and treatment decision-making. Elevated IL-6 \n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 485\nlevels have been associated  with disease activity  in several chronic inflammatory  diseases,  serving as a \nuseful biomarker for disease progression and response to therapy. However, the common lack of specificity \nin distinguishing  between the signaling pathways of IL-6 limits the utility of this cytokine as a biomarker. \nBy encouraging diagnostic assays that can differentiate between classic- and trans-signaling activities of IL-\n6, clinicians could gain valuable insights into the underlying mechanisms driving disease activity, allowing \nfor more personalized and precise treatment strategies. Such advancements in diagnostics could facilitate \nearly  intervention,  improve  disease  management,  and potentially  lead to better  patient  outcomes  by \nenabling the timely adjustment of therapies based on specific inflammatory profiles [34].\nThe precise  differentiation  of IL-6 signaling  pathways  holds significant  promise  for enhancing  the \ntreatment of chronic inflammatory diseases by providing a basis for the development of targeted therapies \nthat minimize  adverse  effects  while maximizing  therapeutic  efficacy  [35]. Moreover,  the differentiated \nmeasurement of IL-6 activity could improve the use of inflammatory markers in diagnostics and treatment \nmonitoring, leading to more informed clinical decision-making and improved patient care.\nLimitations\nThis review  presents  an examination  of the role of IL-6 signaling  in relation  to chronic  inflammatory \ndiseases and the complexities inherent in the differentiation between IL-6 classic-and trans-signaling. This \nhas significant  implications,  especially in the research of endometriosis,  but there are several limitations \nworth noting.\nWhile this review is comprehensive it is not exhaustive. Though it is among the most often measured, \nIL-6 is only one of many cytokines involved in immune response and inflammation. A focus solely on IL-6 \ndoes not provide  a full picture  of the dynamic  nature of immune reactions  and inflammation.  The very \nproblem  addressed  in this paper  also limits  the conclusions  that can be derived  from this paper.  The \ninvolvement of other cytokines and signaling pathways with IL-6 signaling is both complex and unclear.\nThe review also relies heavily on published literature, which may be subject to publication bias. Studies \nwith null or negative findings are less likely to be published, which may skew the body of literature toward \nstudies that report significant effects. This bias could potentially impact the interpretation and conclusions \nof this review [36].\nFinally, while this review highlights a methodological  issue in the measurement of IL-6 and proposes \npotential solutions, the practical implementation of these solutions may be challenging. The differentiation \nbetween IL-6 classic- and trans-signaling in research settings is not straightforward and requires technical \ndevelopment  [37]. Additionally,  the proposed methodologies  may not be feasible in all research settings \ndue to cost, technical complexity, or lack of resources.\nWhile this review provides a critical perspective  on the role of IL-6 in endometriosis  research, these \nlimitations should be taken into consideration when interpreting the findings and suggestions put forth.\nConclusions\nThe examination  of IL-6 signaling  pathways  and their distinct  roles in the pathology  of endometriosis \nhighlights a growing issue in the study of chronic inflammatory diseases. This differentiation is not merely a \nmolecular  distinction  but a pivotal  factor  that influences  the direction  of research,  the development  of \ndiagnostic criteria, and the formulation of therapeutic interventions for endometriosis and potentially other \nchronic inflammatory diseases. The current research landscape, as indicated by the analysis of publications \nand citation patterns, demonstrates a pronounced disparity in focus, with a very limited number of studies \naddressing the different roles of IL-6 signaling pathways. This gap underscores a significant oversight in the \nfield,  limiting  the understanding  of IL-6’s  dual roles  in inflammation  and tissue  regeneration  and its \nimplications for disease management.\nThese gaps in research reveal critical vulnerabilities in the diagnosis and treatment of endometriosis, a \ncondition marked by significant pain and fertility issues in women of reproductive age. The predominance \nof studies that fail to differentiate between IL-6 pathways may lead to a misinterpretation of the cytokine’s \n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 486\nrole in endometriosis,  potentially  delaying diagnosis or guiding therapeutic  strategies  that inadvertently  \nsuppress  beneficial  signaling  mechanisms.  This  oversight,  as highlighted  in the tocilizumab  studies,  \nhighlights  a critical need for a shift towards incorporating  IL-6 pathway differentiation  in endometriosis  \nresearch and inflammatory measurements.\nThe significance  of IL-6 differentiation  extends  beyond  the example  of endometriosis,  suggesting  \nbroader applications for the management of chronic inflammatory conditions. A more nuanced focus on IL-\n6 signaling such as that seen in olamkicept could inform biomarker interpretations for diseases like RA and \nIBD, where  IL-6 plays  a key role in pathogenesis.  The potential  for targeted  inhibition  of IL-6 trans-\nsignaling,  while  preserving  the protective  effects  of classic-signaling,  offers  a promising  avenue  for \ndeveloping treatments that are both effective and exhibit fewer side effects. Progress in this field is unlikely \nunless the significance of IL-6-trans-specific inhibitors is discussed more widely.\nAddressing the disparities in IL-6 research requires concerted efforts to prioritize studies that explore \nthe distinct functions of IL-6 signaling pathways. This entails not only increased discussion  and focus on \nresearch  in this area but also the adoption  of more advanced  diagnostic  tools capable  of distinguishing  \nbetween  IL-6 signaling  mechanisms.  Such tools  could  provide  clinicians  with critical  insights  into the \ninflammatory status of patients, facilitating more informed treatment decisions and potentially improving \npatient outcomes.\nThe improved  differentiation  of IL-6 signaling  pathways  presents  a critical  step in the research  and \ntreatment  of endometriosis  and other chronic inflammatory  diseases.  Bridging the current research gap \noffers  the promise  of more accurate  diagnostics,  comprehensive  therapeutic  strategies,  and a deeper  \nunderstanding of inflammatory pathologies. As the scientific community moves forward it is imperative to \nembrace  the complexity  of cytokine  signaling,  recognizing  its potential  to transform  the landscape  of \ndisease diagnosis and management. Through targeted research and clinical application, the nuanced roles \nof IL-6  trans  and  IL-6  classic  in health  and  disease  can  be better  understood,  paving  the  way  for  \nadvancements in patient care and treatment efficacy.\nAbbreviations\ngp130: glycoprotein 130\nIBD: inflammatory bowel disease\nIL-6: interleukin-6\nIL-6R: interleukin-6 receptor\nRA: rheumatoid arthritis\nsIL-6R: soluble interleukin-6 receptor\nDeclarations\nAcknowledgments\nDuring the preparation of this work, the author used ChatGPT 4 in order to assess readability and identify \ntypographical  errors. After using this tool/service,  the author reviewed and edited the content as needed \nand take full responsibility  for the content  of the publication.  The author  also thanks  to Pubmed  and \nConnected Papers.\nRDC:  Conceptualization,  Analysis,  Investigation,  Methodology,  Resources,  Validation,  Visualization,  \nAuthor contributions\nWriting—original draft, Writing—review & editing. The author read and approved the submitted version.\nConflicts of interest\nThe author declares that there are no conflicts of interest.\n\nExplor Immunol. 2024;4:476–89 | https://doi.org/10.37349/ei.2024.00153 Page 487\nEthical approval\nNot applicable.\nConsent to participate\nNot applicable.\nConsent to publication\nNot applicable.\nAvailability of data and materials\nNot applicable.\nFunding\nNot applicable.\nCopyright\n© The Author(s) 2024.\nReferences\nUciechowski  P, Dempke  WCM. Interleukin-6:  A Masterplayer  in the Cytokine  Network.  Oncology. \n2020;98:131–7. [DOI] [PubMed]\n1.     \nTanaka T, Narazaki M, Masuda K, Kishimoto T. Regulation of IL-6 in Immunity and Diseases. Adv Exp \nMed Biol. 2016;941:79–88. [DOI] [PubMed]\n2.     \n Nara H, Watanabe R. Anti-Inflammatory Effect of Muscle-Derived Interleukin-6 and Its Involvement in \nLipid Metabolism. Int J Mol Sci. 2021;22:9889. [DOI] [PubMed] [PMC]\n3.     \nSoliman AM, Fuldeore M, Snabes MC. 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