The pericentric satellite III (SatIII or Sat3) and II tandem repeats recently appeared to be transcribed under stress conditions, and the transcripts were shown to play an essential role in the universal stress response. In this paper, we review the role of human-specific SatIII copy number variation (CNV) in normal stress response, aging and pathology, with a focus on 1q12 loci. We postulate a close link between transcription of SatII/III repeats and their CNV. The accrued body of data suggests a hypothetical universal mechanism, which provides for SatIII copy gain during the stress response, alongside with another, more hypothetical reverse mechanism that might reduce the mean SatIII copy number, likely via the selection of cells with excessively large 1q12 loci. Both mechanisms, working alternatively like swings of the pendulum, may ensure the balance of SatIII copy numbers and optimum stress resistance. This model is verified on the most recent data on SatIII CNV in pathology and therapy, aging, senescence and response to genotoxic stress in vitro. All eukaryotic genomes are highly repetitive. The estimated total fraction of repetitive sequences in human genome varies from approximately one-half  to over The scientific society converges that the terms “junk DNA”  or “selfHistorically, DNA fractions containing repeats were isolated by high-speed density gradient centrifugation of bulk DNA. Due to their different AT/GC content compared to the bulk DNA, repetitive DNA formed additional bands, giving rise to the term “satellite DNA” (satDNA) ,17.Human satellite DNA  is an esThe experimental facts listed below are obtained on different satellite DNA classes located in different genome regions. It remains an open question to what extent the data obtained for different satellite DNA classes  and locations  are relevant to the other classes and locations. When generalizing the empirical information and constructing a model, we postulated that the underpinning facts are universal.There are several approaches to the classification of repetitive sequences in human genomes. They can be based on function , size 2 or a com“Constitutive” heterochromatin in the pericentromeric areas has turned out to be just a historical term. The transcription of satellite DNA has been reported in different biological contexts ,33,34, iThe best characterized anti-stress mechanism, which is triggered by a variety of stressors, involves the transcriptional upregulation of a set of genes coding for molecular chaperones . AlthougIn vertebrates, heat shock genes are under the control of a family of transcription factors, the so-called heat shock factors, HSF1 to HSF4 ,47, eachOne of the most striking effects of stress is the rapid and reversible redistribution of HSF1 into a few nuclear structures termed nuclear stress granules which form primarily on the 9q12 locus and chromosome Y  and secoThe nuclear stress bodies (or nSBs) were discovered in the late 1980s and very soon after were associated with cellular response to stress factors ,54. TheyHominoidea superfamily. Interestingly, structures comparable to nSBs were found in Drosophila and called omega speckles [The nuclear stress bodies assemble on blocks of satellite III DNA , inducinspeckles . SimilarHowever, the adaptive stress response includes not only the activation of a series of protective genes, but also the temporal silencing of a wide variety of other genes. This process requires non-coding RNAs transcribed from satellite III repeats. The intriguing underpinned mechanism, though still partially elusive, is generally outlined at present, and HSF1 is a central player here, as well.The mechanisms for local nuclear stress bodies having an effect on the global landscape of nuclear genome expression remain hypothetical in general. The global stress response, in addition to HSF inducible gene transcriptional stimulation, which is beyond this review, is composed of global transcriptional repression, modulating splicing activities, and HSF-independent transcriptional de-repression of genes located in the vicinity of nuclear stress bodies.The activation of SatIII arrays participates in global stress-induced, genome-wide down-regulation of genome expression through transient sequestration of transcription factors ,61.Nuclear stress bodies may be viewed as transcription factories comprising a natural amplification of RNA pol II promoters. Although the number of transcription units is not yet defined, both the extent of the SatIII arrays and the size of HSF1 foci suggest that thousands of transcriptional units may be simultaneously activated . Thus, tIt was directly demonstrated that the recruitment of CREBBP to nSBs is SatIII-dependent, and that the loss of SatIII transcripts relieves the heat shock-induced transcriptional repression of at least a few target genes. Conversely, forced expression of SatIII transcripts resulted in the formation of nSBs and transcriptional repression even without a heat shock .Thus, SatIII transcripts were shown to serve as a “sink”, as originally proposed , to recrThe gene expression in heat-shocked human cells is also affected by transient targeting of specific splicing factors to SatIII RNAs, such as hSF2 or SRSF1 (serine/arginine rich splicing factor 1), other SRSFs and KHDRBS1  . The splSplicing profiles are controlled by the relative abundance of antagonistic hnRNP and SR proteins. It is plausible that SatIII RNAs, by sequestering specific RNA binding proteins into nSBs, may shift splicing decisions, for instance, toward the synthesis of molecules involved in the cell defense to stress .Alternative splicing affects more than 90% of cellular transcripts. The SRSFs recruited to nSBs are rapidly de-phosphorylated upon thermal stress exposure. The phosphorylation states of SRSFs are known to affect splicing patterns. During stress recovery, CDC-like kinase 1 (CLK1) is recruited to nSBs and accelerates the re-phosphorylation of SRSF9, thereby promoting target intron retention. A transcriptomic analysis revealed that the depletion of HSatIII lncRNAs, resulting in the elimination of nSBs, promoted splicing of 533 retained introns during thermal stress recovery. Thus, SatIII-dependent nSBs serve as a conditional platform for phosphorylation of SRSFs by CLK1 to promote the rapid adaptation of gene expression through intron retention (IR) following thermal stress exposure . Among tcis-acting effects or by creating nuclear domains in which genes escaping heat-induced transcriptional repression would relocate. Transcriptional de-repression of genes adjacent to nSBs through position effects presumably starts from the loss of epigenetic repressive marks at pericentromeric loci following heat shock, which could abolish the transcriptional repression exerted by pericentromeric heterochromatin on the activity of the promoters of gene present in the cis- or possibly the trans- position through chromatin opening and recruitment of transcription factors [Activation of SatIII facilitates transcription of nearby genes through  factors .Several examples have been reported in the literature in which gene inactivation is indeed associated with repositioning of repressed genes in the vicinity of these large blocks of heterochromatin. Based on these observations, a model has been proposed in which the repositioning of repressive genes in the vicinity of heterochromatin would be necessary for the maintenance of their repressed status through a position effect mechanism . Due to their highly repetitive nature, tandem repeats are known as a principal source of genome instability . Human sOpen chromatin conformation necessary for transcription strongly assists in changing the copy numbers of satellite DNA due to unequal exchanges. Although the stress-induced satellite III DNA transcription with the formation of nSBs occurs primarily on chromosomes 9  and Y 5, chromosGenerally, senescence  and, espThe human satellite II (HSatII) is the most differentially expressed satellite subfamily in epithelial cancers . It consIt still remains obscure to what extent these conclusions of non-canonical repeat transcription accompanied by reverse transcription-based copy gain could be extrapolated for SatIII and other types of moderate tandem repeats in different cancer and non-cancer conditions, including prolonged stress, senescence and pathology. A limited and undigested array of facts obtained in human and animal studies is available.P < 0.001) was found between the 1q copy gain with a 1q12 breakpoint and hypomethylated state of SatII sequences [Somatic copy number alterations were analyzed using comparative genomic hybridization (CGH) in 75 cases of hepatocellular carcinoma in China. The most common alterations involved gains of 1q and 8q and a loss of 16q (50%), losses of 4q and 17p and a gain of 5p (40%) and losses of 8p and 13q (30%) . These fequences . ObviousSamples from 33 gastric cancer patients were collected in Saudi Arabia and analyzed using CGH compared to 15 normal gastric samples from the same population. The study revealed frequently copy number gains within several chromosomal regions including 1q12 among the most frequent . Other fMyodes glareolus exposed to environmental radionuclides within the Chernobyl Exclusion Zone compared to animal subjects from adjacent non-contaminated areas. Notably, 18S rDNA and Msat-160 copy numbers were positively correlated in the genomes of bank voles from uncontaminated areas, but not in the genomes of animals inhabiting contaminated areas [Indirect evidence in favor of stress-based pericentric satellite repeat copy gain was acquired in animal studies on rodents. Although satellite III are primate-specific tandem repeats, there are similar pericentric repeats of MSat-160 in arvicoline rodents. Expansion of rDNA and MSat-160 pericentromere satellite repeats was revealed in the genomes of bank voles ed areas .Cellular senescence was first described by Hayflick and Moorhead in the 1960s  as the iCellular senescence results from a variety of stresses . SeemingAn accelerated aging model also corroborates the existence of SatIII transcription in aging/senescence. In fibroblasts derived from patients with Hutchinson–Gilford progeria syndrome, a complete loss of the heterochromatic marks was followed by the expression of chromosome 9 specific SatIII sequences .A progressive expansion of satellite III (1q12) copies was observed in replicative senescence and during natural aging  for det below.Summarizing, we propose a universal mechanism to launch a process of augmentation of SatIII (1q12) and other pericentric tandem repeat copy numbers in chronic stress (Chernobyl zone), replicative senescence, normal and accelerated aging and various cancers. We hypothesize that in all these conditions, the satellite repeat copy gain is associated with stress-induced transcription of non-coding RNAs from the satellite DNA, with subsequent reverse transcription and cDNA reintegration. What can be the possible consequences of SatII/III copy gain for the cell? As we can imagine, an increment of the satellite units obviously results in elevated transcription yielding more SatIII RNAs and thus strengthening the stress response. Hence, the cell line presumably becomes more resistant to the forthcoming stresses.p-value = 0.034 [For tumor cells, higher stress resistance means declined response to anti-cancer therapy and worse prognosis. Survival data of 46 samples of colorectal cancers were analyzed to compare tumors with SatII gain vs. no gain. Kaplan–Meier tests showed a significant reduction in overall survival in the SatII gain cases, with a median overall survival of 1096 vs. 1881 days; log-rank  = 0.034 . The patThe animal model described above also corroborates the hypothetically increased viability of organisms carrying increased numbers of satellite copies in their genomes. In the conditions of heightened radiation background, bank vole genomes with increased Msat-160 copy numbers have received a selective advantage in the successive generations .The location of satellite III stretches in chromosome Y described above  may refln = 840) of patients with schizophrenia, a disease in which elevated reactive oxygen species levels and declined antioxidant statuses in the brain and peripheral tissues was reported [P << 10−17) [However, experimental facts began to appear that the increasing abundance of pericentric satellites in the cell pool is a process lasting to a certain limit rather than ad infinitum, and possibly, reversible. Thus, in a large sample  .There is nothing surprising about that. The stress reaction drastically changes the transcriptional landscape on a whole-genome scale. Expression of heat shock proteins is boosted, whereas most other genes undergo global silencing and aberrant splicing. Substantially, all the housekeeping and specialized processes are aborted or changed because of depletion of the transcription machinery parts. It helps to save resources, protect the chromosomes against damage and reduce the vulnerability of cellular machinery until the stress impact has finished. However, this response is transient by its very nature. This is just the lesser of two evils, and the longer it lasts, the more deleterious it becomes.A low level of SatIII RNAs is detectable even in unstressed cell with an approximately 10,000-fold induction from the baseline after thermal shock or another stress treatment such as free radicals, heavy metals, ultraviolet or hyper-osmotic stress . In caseIn a series of experiments when HeLa cells were transiently transfected with antisense oligos for the knockdown of SatIII transcript, exposed to a heat shock (42 °C for 1 h), and cell viability was measured after heat shock release, the blockage of SatIII transcription led to a significant reduction (~70%) in cell survival as compared to cells that were transiently transfected with the control oligos and exposed to a heat shock. However, the SatIII antisense oligos did not affect the survival of cells that were not exposed to the heat shock, suggesting that the SatIII transcripts are required for survival only under thermal stress condition. The authors next wanted to estimate whether forced expression of SatIII transcripts would alter the viability of cells that are not exposed to a heat shock. Transient expression of a SatIII mammalian expression construct (pcDNA-SatIII) resulted in a paradoxically lower survival rate as compared to cells transfected with an empty vector or the bacterial expression vector SatIII repeats. Intriguingly, cells that expressed the cloned SatIII repeats exhibited significantly lower survival rates when exposed to heat shock, suggesting a toxic effect of Sat3 transcripts when ectopically expressed . These eDirect findings that evidence vulnerability of excessively long SatIII arrays were obtained in cytogenetic studies focused on human 1q12 blocks and their nuclear repositioning in health and pathology.It has long been known that within the interphase nucleus, human chromosomes are spatially organized as chromosome territories (CT) or domains ,107,108.The CT position is not fixed rigidly, but changes in response to environmental impacts, such as ionizing radiation (IR), reactive oxygen species and other deleterious impacts, demonstrating regular spatial shifts ,127. DurThe 1q12 site harbors numerous satellite II/III arrays. SatIII homology with human genome database was analyzed and the repeat was found to be located on eight human chromosomes: 1, 2, 7, 9, 10, 16, 22 and Y. Fragments of the Y chromosome with low homology are rare. The remaining chromosomes do not contain fragments homologous to the SatIII probe. More than half of the 1000 homologous SatIII sequences found in the database are localized within the first human chromosome 1q12 region. The first chromosome fragments found in the database contain dozens of tandem repeats homologous to the SatIII. Fragments of the remaining chromosomes according to the database contain fewer number of the SatIII units . A fragment of the pericentromeric satellites III was studied using DNA probe PUC1.77  specificThe adaptive response induced by low-dose ionizing radiation involves an alteration of the chromatin spatial configuration that is necessary to re-shape the expression profile of the genome in response to stress. The 1q12 heterochromatin loci movement from the periphery to the center of the nucleus is a marker of the chromatin configuration change. It was hypothesized that a large 1q12 domain could affect chromatin movement, thereby hindering the translocation and inhibiting the adaptive response (AR) .The change in the SatIII (1q12) position in the nucleus under the stresses and the absence of this transposition in some cells were found in a number of studies ,137,138.r1 and r2) value, the distance and angle between the signals (d and α), the signal area and the radius and the nucleus area. The radius vector r is normalized to the value of the nucleus radius and changes from 0 (nucleus center) to 1 (nucleus surface). Most signals are located in the area corresponding to r-values > 0.75; that is, in lymphocytes from healthy donors, 1q12 loci detected by the SatIII DNA probe are located near the nuclear envelope.The 1q12 regions are detected using Fluorescence In Situ Hybridization (FISH) as two fluorescent signals. In lymphocytes, the position of the FISH signal in the projection plane (circle) depends on how the nucleus is located on the slide during the sample preparation. Image processing includes determining the gravity center of the signal, the signal radius vector (r > 0.75) deep into the nucleus and converge with each other. A similar 1q12 loci movement was also observed when a proliferative stimulus PHA had been applied [Low-dose IR or hydrogen peroxide  significantly changes the 1q12 loci position in the nucleus. In response to stress, 1q12 loci move from the perimembrane region  exposed lymphocytes during longer cultivation (7 days) after irradiation. During the cultivation, some cells died and had signs of apoptosis and necrosis. The mean SatIII content was almost twice reduced as compared to the start of cultivation. At the same time, the population appeared to contain predominantly cells with small 1q12 size. Thus, in response to oxidative stress, the pool of cells underwent selection by SatIII content. Cells with low repeat abundance had an advantage. Since this repeat is distributed throughout the 1q12 site, it may be assumed that primarily cells with large 1q12 loci occupying a large nucleus volume died due to the stress. The same effect was then shown for MSC .Five human skin fibroblast (HSF) lines demonstrated the SatIII increase in the genomes of older cells during replicative senescence, suggesting the copy gain of the satellite repeats. The fewer copies the HSF genome had harbored before the experiments started, the more numbers of passages this line underwent before division was arrested. The copy gain appeared to be inversely proportional to the baseline SatIII content. These findings also corroborate the assumptions of SatIII expansion and toxicity of large SatIII clusters .Forces and molecular mechanisms that shape the radial configuration of the 1q12 loci under the stress action remain elusive, with prospective comprehension of the role of dynamic interactions of chromatin with the nuclear lamina-associated protein complexes ,141,142,We presented above a series of facts suggesting that there may exist a widespread or even universal mechanism of stretching the mammalian pericentric satellite tandem repeat clusters in the conditions of frequent or permanent cellular stresses. The mechanism was directly demonstrated in cancer cells, and indirect evidence was obtained in human aging and animal population studies. Extrapolating, the satellite cluster shall sooner or later become excessively long.As scrutinized by means of an example of human 1q12 loci, too long satellite arrays make the cell stress-intolerant, dying after further stress impacts. It results in positive selection for cells with short satellite arrays, which can elongate during the next stress impacts, and so the cycle repeats.Thus, we propose here a model of alternating growth of satellite DNA arrays and sweep them out by negative cell selection or via another mechanism yet to be elucidated. By analogy with swings of the pendulum, we call it the “pendulum model” .Further studies are required to address the natural question regarding the origin of the minor fraction of stress-resistant cells with short satellite clusters existing or appearing among the cells with long clusters lacking stress resistance and perished by stressors. The existence of such fraction is proven by the high variance of the satellite DNA content observed in samples with an elevated average abundance of satellite DNA. The first speculative explanation is that, by an unknown cause, a subset of cell avoids the satellite expansion, serving as a reserve stock for future. Alternatively, a mechanism of excision, unequal exchange or another avenue to remove the excessive satellite repeats acts to produce, at low frequency, cells with shortened satellite DNA clusters in every cell division.Below is an attempt to verify the pendulum model by applying it to some published experimental data obtained in various conditions.In a sample of 557 subjects aged 2 to 91 years, a trend of increasing mean SatIII content with age can be clearly seen . A diffeGrowing SatIII content with age is coherent with the idea of satellite repeat expansion during habitual stresses throughout the lifetime. At the same time, conspicuous is the fact that the variance is also increasing with age, as SD gradually grows from 2.7 in Children to 8.5 in the Senile group. Moreover, the minimum value in the Adult 1–3 and Senile groups is even less than that in Children, suggesting that the low-copy cells are produced from high-copy parental cells rather than retained through a series of cell divisions in a minor fraction of subjects who have avoided strong stresses during the lifetime .Five human skin fibroblast (HSF) lines were exposed to potassium Cr(VI) salt, which is known to induce oxidative stress with severity depending on the Cr(VI) concentration ,150,151.In subjects who had a chronic occupational exposure to low-dose ionizing radiation, the picture was similar to aging. While in the control cohort, SatIII content varied from 9 up to 33 pg/ng , in the gamma neutron-irradiated cohort, SatIII varied from 10 to 40 pg/ng (24 ± 6 pg/ng), and in the tritium-exposed cohort, it ranged from 1 to 176 pg/ng (29 ± 26 pg/ng). Again, as in the case of aging, growth of the mean value with stress was followed by elevated variance and wider range. Moreover, the minimum value in the tritium exposed cohort was less than the least value in controls, suggesting that the “reverse swing of the pendulum”—clearance of large 1q12 loci had occurred in some individuals .P > 0.2), while patients without hypoxia in medical history, i.e., with genetic background of the pathogenesis, surprisingly contained far fewer copies than healthy controls (P << 10−17), as noted above. SatIII copy numbers in schizophrenia cases negatively correlated with the PNSS  score that shows disease severity. It is not clear whether low abundance of satellite DNA was a direct cause of more severe signs or merely an indicator of stronger stress the patient had experienced.Oxidative stress was found to account for at least part of cases of schizophrenia . The SatIII content also did not depend on the type of neuroleptics used (P > 0.1) [Interesting results were obtained studying the therapy effect on the SatIII content in leukocytes. Antipsychotic therapy is known to induce oxidative burst . PatientP > 0.1) . Once moWe accumulated and reviewed an amount of data large enough to hypothesize that the transcription of pericentric 1q12 loci is involved in the standard stress response in human cells. We believe that a process of copy gain is a universal component of this response. However, the stress-induced SatII/III repeat expansion occurs to a certain limit. After the limit is reached, another mechanism is activated to reduce the number of SatIII copies. The mechanisms underlying the augmentation and removing excessive satellite repeats remain obscure. Nothing is known to us how the cell detects the limit to switch on the copy number reduction machinery. Perhaps, comparison with microsatellite expansion in unique gene promotors might help. However, to what extent the pericentric satellite stress-induced expansion is similar to oligonucleotide expansion resulting in some hereditary diseases  remains to be studied. The effects of stress-induced transcription from satellite DNA in 1q12 locus are an almost undiscovered field waiting for future studies.


Immune cell infiltration into tumors, intratumoral cellular organization, and the cell-specific expression patterns of chemokines and chemokine receptors greatly influence the efficacy of immunotherapeutic treatment strategies. In our recent review article, we shined a light on the deciding role of the chemokine network between immune mediated tumor regression or immune evasion of the tumor. Current T cell centric immunotherapeutic strategies primarily rely on increasing cellular activation and decreasing cellular inhibition, with the overall goal of enhancing effector cell function. These strategies neglect to account for the presence of the T cells within the tumor, hardly boosting immune cell infiltration. Chemokines and chemokine receptors are the regulators of recruitment, migration, and intratumoral compartmentalization. Yet, utilizing the chemokine network to recruit immune cells that will drive tumor regression is not a straightforward path, as tumor cells often hijack these pathways in the effort of immune evasion. Many novel therapeutic strategies involving chemokine targeting are under trial for many diverse tumor types. As a field, we can learn from both the successes and failures of these trials in order to push forward the next generation of immunotherapeutic strategies that include augmented T cell trafficking. The efficacy of checkpoint inhibitors in the treatment of cancer is linked to the degree of T cell infiltration in the tumor, specifically, the intratumoral localization of effector T cells relative to tumor cells, dendritic cells, and immune suppressive cell populations . ChemokiThese considerations collectively contribute to the subtyping of the immune presence in tumors as either immune desert, excluded, or inflamed. Analysis of tumor histology can determine which of these three subtypes a tumor can be categorized as, allowing for a more customized immunotherapeutic approach that target the specific mechanisms that prevent the antitumor response. Immune desert tumors have little-to-no immune infiltration. Immune deserts indicate a lack of pre-existing anti-tumor immunity, and patients with these tumors would benefit from the induction of tumor-specific T cells . The excNeutralizing chemokines that will enhance immunosuppression, andIncorporating chemokine receptors into T cell adoptive transfers to direct T cell trafficking post transfer.Despite the nuanced role of chemokines, intervention into this network is highly promising in combination with checkpoint inhibitors. In recent years, multitudes of clinical trials have included chemokine interventions. These trials can be generalized into two categories:NCT02826486) found that objective response rate, overall survival, and disease control rate were all improved in patients that received the combined drug regimen [Recruitment of myeloid-derived suppressor cells (MDSCs) into the tumor is a major mechanism of immune evasion by inhibiting T cell function by expressing arginase and inducible nitric oxide synthase and secreting immunosuppressive cytokines, including transforming growth factor-β (TGF-β) and IL-10 . The preIn the sera of colorectal cancer patients there is a noted increase in CCL15 and a striking intratumoral presence of MDSCs expressing its receptor CCR1 . InteresNCT03161431) will treat participants with melanoma for 21 days with SX-682, a CXCR1/2 inhibitor, as a monotherapy - then with pembrolizumab, an FDA approved immunotherapy for melanoma. All participants will be monitored for 3 months. After the monotherapy stage of the trial is complete, the next participants will receive SX-682 and pembrolizumab concurrently. These combination therapy participants will be evaluated for 2 years. SX-682 is an orally available allosteric inhibitor of CXCR1 and CXCR2 that has shown promise in promoting tumor regression pre-clinically. Specifically, SX-682 administration decreased MDSCs infiltration, increased T cell infiltration, enhanced T cell activation and cytotoxicity, promoted responsiveness to PD-1 blockade, limited tumor growth, and enhanced overall survival in mouse models of squamous cell carcinomas4. SX-682 holds much promise for success because of these diverse processes impacted by these pathways.The CXCR1 and CXCR2 axes, including the ligands CXCL1, CXCL2, CXCL5, and CXCL8, also contribute to migration and recruitment of MDSCs –16. TogeNCT02946671 and NCT02476123). The published report from one of these trials notes a decrease in intratumoralTregs and an increase in intratumoral CD8+ T cells [The recruitment of regulatory T cells (Tregs) into tumors also contributes to inhibition of effector T cell function. High levels of expression of CCR4 on Tregs lends rationale to the use of the anti-CCR4 antibody, mogamulizumab, in the treatment of solid tumors. Two Phase I trials combined mogamulizumab with the anti-PD-1 antibody nivolumab in the treatment of various solid tumors ( T cells . Though In the context of adoptive T cell transfer, chemokines can influence the recruitment of the T cells to the tumor and the intratumoral localization following infiltration. In prior discussions, much attention has been given to enhancing T cell recruitment. No less important, though, is the role of chemokines in properly localizing transferred T cells within the tumor and in supporting a microenvironment in which T cells can function. It has been previously reported that CD8+ T cells that are adoptively transferred into tumor bearing mice, rely on CXCR3 for tumor entry . AdditioThe CXCR1 and CXCR2 inhibitor SX-682 has already proven to promote tumor clearance following adoptive cell transfer of tumor antigen specific T cells in mouse models of squamous cell carcinomas . In thesThe findings highlighted here teach an important lesson - in the conception of the next generation of immunotherapeutic strategies we cannot label chemokine axes as “helpful” or “harmful” in a binary manner. In moving forward, we are faced with the great challenge of appreciating chemokine axes for orchestrating a complex interplay of cell types. In the example of CXCR1 and CXCR2, it is evident that the chemokine ligands of these receptors are notably high in a number of tumors. This is initially advantageous to the tumor as MDSCs are recruited to support an immune suppressive environment. One strategy to combat this is to inhibit the function of these receptors on MDSCs to prevent recruitment. Another strategy relies on taking back these chemokine pathways by overexpressing these receptors in CAR T cells armed to fight tumor growth. The strategies may not need to be mutually exclusive, in fact the most promise for success appears to be in a combined approach. The neutralization of chemokine pathways in tumor and immune suppressor cells, the overexpression of chemokine receptors on T cells engineered for adoptive transfer, and the administration of immune checkpoint inhibitors should be considered complementary tactics.


Mstn leads to hypermuscularity, the deletion of Gdf11 results in abnormal skeletal patterning and organ development. The perinatal lethality of Gdf11-null mice, which contrasts with the long-term viability of Mstn-null mice, has led most research to focus on utilizing recombinant GDF11 proteins to investigate the postnatal functions of GDF11. However, the reported outcomes of the exogenous application of recombinant GDF11 proteins are controversial partly because of the different sources and qualities of recombinant GDF11 used and because recombinant GDF11 and MSTN proteins are nearly indistinguishable due to their similar structural and biochemical properties. Here, we analyze the similarities and differences between GDF11 and MSTN from an evolutionary point of view and summarize the current understanding of the biological processing, signaling, and physiological functions of GDF11 and MSTN. Finally, we discuss the potential use of recombinant GDF11 as a therapeutic option for a wide range of medical conditions and the possible adverse effects of GDF11 inhibition mediated by MSTN inhibitors.Growth differentiation factor 11 (GDF11) and myostatin (MSTN) are closely related TGFβ family members that are often believed to serve similar functions due to their high homology. However, genetic studies in animals provide clear evidence that they perform distinct roles. While the loss of  A growth factor protein called GDF11 has potential for rejuvenation of heart, brain, and muscle tissues, but first its relationship with a twin growth factor (MSTN) must be unraveled. Although GDF11 and MSTN are almost identical in amino acid sequence, MSTN controls growth of skeletal muscles, and GDF11 regulates bone and organ patterning. To untangle what differentiates these proteins, Yun-Sil Lee and Joonho Suh at Seoul National University, South Korea, reviewed their evolution and function. They report that GDF11 and MSTN are the result of duplication of an original gene and have evolved to perform different roles. They cover some of the beneficial effects reported with GDF11 supplementation, such as tissue rejuvenation and increased lifespan, but caution that negative effects have been reported. Further investigation may illuminate growth and development processes, and therapeutic potential. Among the TGFβ family members, there are two highly homologous proteins, GDF11 and myostatin (MSTN), which share 89% sequence identity in their mature form but exhibit distinct endogenous functions. While Gdf11 is expressed broadly in numerous tissues, Mstn is expressed primarily in skeletal muscle4. The functional divergence of GDF11 and MSTN is indicated by the fact that their mutation in animals leads to the development of largely dissimilar features. For instance, while the genetic deficiency of MSTN leads to a hypermuscular phenotype in various species8, homozygous deletion of Gdf11 generates defects in axial skeletal patterning and organ development in mice9. However, unlike the relatively consistent reports of the function of MSTN in suppressing skeletal muscle growth, the reports of GDF11 function, particularly those examining the postnatal role of GDF11, remain highly controversial. One of the main reasons for this controversy lies in the fact that Gdf11-null mice, unlike Mstn-null mice, show perinatal lethality9, leading most studies to utilize recombinant proteins that cannot fully recapitulate the complex endogenous functions of GDF11. Importantly, in contrast to studies that utilized recombinant GDF11 or MSTN proteins, those that applied genetic knockdown, knockout, or conditional knockout techniques revealed relatively unvarying results despite their being fewer in number, and most have reported the positive roles of GDF11 and the negative roles of MSTN in the regulation of the development of various tissues. In this review, we first present the similarities and differences between GDF11 and MSTN from an evolutionary point of view and summarize the insights obtained to date regarding the biological processing, signaling mechanisms, and physiological functions of GDF11 and MSTN during development, adulthood, and aging. We also discuss the potential of recombinant GDF11 protein as a therapeutic option for various clinical conditions and the possible adverse effects of GDF11 inhibition mediated by MSTN inhibitors.Cytokines of the transforming growth factor β (TGFβ) family, including activins, growth differentiation factors (GDFs), bone morphogenetic proteins (BMPs), and TGFβs, have been extensively implicated in the regulation of developmental patterning, cellular proliferation and differentiation, and the maintenance of tissue homeostasisGDF11 and MSTN10. For instance, in Caenorhabditis elegans, daf-7 was shown to encode a homolog of GDF11 and MSTN, while in fruit flies (Drosophila melanogaster), myoglianin (Myo) was found to exhibit the highest sequence homology to GDF11 and MSTN13. An important question that arose from these identifications was whether the divergence of GDF11 and MSTN occurred at the time of the emergence of vertebrates. To provide an explanation, a phylogenetic study was conducted in various invertebrate and vertebrate species, and importantly, the amphioxus (Branchiostoma belcheri)14, which is an invertebrate known to be the closest relative of the vertebrates, was included in the analysis -like metalloproteinases, which dissociate the propeptides from the mature C-terminal dimers, thus freeing the ligands for receptor binding  of the proteins  and subsequently recruit type 1 receptors, activin receptor-like kinase 4 (ALK4) or ALK5 to form a heteromeric receptor complex to elicit downstream signaling via phosphorylation of SMAD2 and/or SMAD3 , follistatin-like 3 (FSTL3/FLRG), growth and differentiation factor-associated serum protein 1 (GASP1), GASP2, latent TGF-β binding protein 3 (LTBP3), and decorinMstn-knockout pups32, implying that GDF11 and MSTN may function as endocrine signaling molecules. However, our previous findings in mosaic mice in which Mstn was deleted exclusively in posteriorly located muscles highlighted the important paracrine function of MSTN in addition to its endocrine action in regulating muscle mass33. Furthermore, whether circulating GDF11 levels have physiological relevance was formerly questioned based on a result showing that the molar concentration of circulating GDF11 was approximately 500 times less than that of MSTN34. Because GDF11 and MSTN circulate mostly in inactive, latent forms35, their local activation patterns and localization of antagonists may largely contribute to distinct physiological effects of GDF11 and MSTN. Therefore, due to the complex modes of action of GDF11 and MSTN, considerable caution is required for the interpretation of the results of tissue-specific deletion of GDF11 or MSTN in conditional knockout mice.Both GDF11 and MSTN circulate in the blood, and maternal deficiency of MSTN was shown to stimulate additional muscle growth in Mstn/Gdf11 positively regulates the development of diverse tissues and functions similarly to vertebrate GDF11 rather than MSTN  methods have provided evidence that ancestral TN Table . Ancestrmyo, which is a homolog of GDF11 and MSTN, is strongly expressed in muscle and glial cells and has been shown to promote neuronal development and remodeling41, prevent age-related muscular dysfunction16, and extend the lifespan in Drosophila42. Specifically, RNAi-mediated knockdown of myo in glia13 or muscle16 in Drosophila resulted in neuronal remodeling defects or exacerbated age-related climbing defects accompanied by premature death, respectively. Furthermore, a recent study suggested that Myo extends lifespan in flies by exerting protective functions in muscle homeostasis through regulating 26S proteasome function42. However, whether Myo regulates muscle mass in flies requires further investigation due to the existence of conflicting reports. As an illustration, while Demontis et al.16 showed no changes in muscle mass, body weight, and feeding behavior upon either muscle-specific Myo suppression or overexpression, Augustin et al.43 demonstrated that muscle-specific silencing of Myo increased larval weight and body-wall muscle size. More recently, Upadhyay et al.44 re-examined the same mutant flies and suggested that Myo deficiency did not result in larger muscles and that Myo is functionally distinct from vertebrate MSTN in terms of regulating muscle size. They also reported that Myo promotes imaginal disc growth in Drosophila44. Meanwhile, depletion of Myo through RNAi injection in third-instar cricket nymphs prevented the normal molting cycle and metamorphosis and led to reductions in nymph body size and weight, although the extended developmental period of the RNAi-treated nymphs eventually led them to exhibit a larger final insect size45. Injection of RNAi targeting myo into either fifth- or sixth-instar nymphs resulted in developmental arrest and death, highlighting the crucial role of Myo in promoting proper insect development45. Similar functions of Myo were also reported for cockroaches46.The insect gene Mstn/Gdf11 gene is expressed in diverse tissues, including muscle, hepatopancreas, eyestalk, heart, gill, and stomach, with the highest expression detected in the heart17. Endogenous expression of Mstn/Gdf11 in shrimp muscle has been shown to peak immediately after molting, a period when significant growth occurs without restriction by a hard exoskeleton17. Interestingly, downregulation of the shrimp Mstn/Gdf11 gene by tail-muscle injection of sequence-specific dsRNA led to a significantly impaired growth rate 17, an effect opposite to that observed after suppression of MSTN in vertebrates. Likewise, a separate study on shrimp revealed that silencing of the Mstn/Gdf11 gene by tail-muscle injection of dsRNA significantly diminished growth and the survival rate15, indicating that ancestral MSTN/GDF11 in invertebrates is a positive regulator of growth and development, unlike vertebrate MSTN. Moreover, Zhuo et al.47 identified long (428 amino acids) and short (420 amino acids) forms of banana shrimp MSTN and FmMSTN, and the long form was positively correlated with a larger size in shrimp. Injection of dsRNA targeting FmMstn into these shrimp impaired their normal molting cycle but also resulted in the enlargement of the pleopod muscles47, which contradicts earlier findings. Further analysis and quantitation of the muscle fiber size in different muscle types are required to fully elucidate the effects of shrimp MSTN/GDF11 on controlling muscle development.In shrimp, the ancestral GDF11 and MSTN exhibit distinct expression patterns and functions. In mice, Mstn is initially expressed in the myotome compartment of somites at E9.5 and continues to be expressed in skeletal muscles to repress hyperplasia or increase the number of muscle fibers during development4. On the other hand, Gdf11 is predominantly expressed in the mouse tail bud at E9.5 and specifies the positional identity of the skeleton along the anterior-posterior axis9. Correspondingly, Gdf11-null mice exhibit anterior homeotic transformations of the vertebrae by displaying an increase in the number of thoracic and lumbar vertebrae and vertebrosternal ribs48. It should be noted that Gdf11 and Mstn double-mutants  exhibited more extensive homeotic transformations of the axial skeleton than Gdf11-null mice, indicating that GDF11 and MSTN have some redundant functions related to the control of skeletal patterning49. GDF11 has also been shown to mediate proper craniofacial development, as Gdf11-null mice display high (60%) penetrance of cleft palate50. In further support of this role of GDF11, a recent study identified a family with orofacial clefting and vertebral/rib hypersegmentation harboring a dominant-negative missense mutation in GDF11, in which an arginine is substituted for a glutamine at the furin protease cleavage site (R298Q)51. Additional analysis confirmed that mutant GDF11 (R298Q) is not processed into the active form, indicating that this mutation is the underlying cause of the phenotypes observed in this family51. An earlier study reported that GDF11 also promotes tooth development and that electroporation-mediated transfer of the Gdf11 gene to the amputated pulp of canine teeth enhances reparative dentin formation52. Furthermore, our group recently demonstrated that GDF11, in contrast to MSTN, facilitates osteogenesis during embryonic development and showed that compared to that in newborn wild-type mice, bone mass is diminished in newborn Gdf11-null mice and enhanced in newborn Mstn-null mice26.During the embryonic development of vertebrates, Gdf11-null embryos demonstrated that GDF11 plays a crucial role in the development of various organs. Specifically, analysis of Gdf11-null mice revealed that the majority of these mice experience complete renal agenesis and failure of ureteric bud outgrowth from the Wolffian duct53. These mice were further shown to exhibit malformations of the stomach characterized by a two-fold reduction in the thickness of the gastric wall and a decreased number of gastric rugae , a smaller spleen, and an abnormally shaped pancreas54, implying that GDF11 is essential for proper morphogenesis of the foregut-derived organs. Gdf11 deficiency also resulted in the greater expansion of islet progenitor cells as well as the impairment of β-cell differentiation in the pancreas54. In the olfactory epithelium and retina, GDF11 was shown to inhibit neurogenesis by either repressing progenitor cell proliferation or altering progenitor cell fate56. This conclusion was supported by the significantly increased number of olfactory epithelium neurons and retinal ganglion cells in mice lacking GDF11 and the contrasting patterns observed in mice deficient in FST, an antagonist of GDF11. However, a delay in neuronal differentiation and gliogenesis was later reported in the spinal cord of Gdf11-null mice, suggesting that GDF11 promotes the temporal progression of neurogenesis in the developing spinal cord57. In addition, using chicken embryos, Gamer et al.58 demonstrated that implantation of beads soaked in human recombinant GDF11 protein into early wing buds led to a dramatic truncation of the limbs due to suppression of both myogenesis and chondrogenesis. In contrast, a recent analysis of Gdf11-null embryos at E15.5 and Gdf11-null sternal chondrocytes revealed that chondrocyte maturation was impaired under Gdf11-deficient conditions26. Moreover, skeletal muscle-specific deletion of Gdf11 using conditional knockout techniques resulted in no differences in muscle mass and fiber type, indicating that the functions of GDF11 and MSTN in the skeletal muscles are most likely divergent49. Additional investigation of the skeletal muscles of Gdf11-null embryos will further clarify the role of GDF11 in myogenesis during development.Multiple studies that utilized Mstn deletion were shown to have a substantial increase in skeletal muscle mass, with individual muscle groups growing to approximately twice the normal size4. A significant increase in muscle mass was also observed in humans, cattle, sheep, and dogs with naturally occurring mutations in the MSTN gene8. Further analysis of Mstn-null mice revealed that MSTN inhibits both skeletal muscle fiber hyperplasia during early development and hypertrophy in adults. The direct role of MSTN in postnatal suppression of muscle fiber hypertrophy was demonstrated by the severe loss of muscle mass induced by systemic overexpression of MSTN in adult mice35 and the increase in muscle mass in adult mice treated with a monoclonal anti-MSTN antibody59. Paradoxically, circulating MSTN levels were shown to decrease with age in humans, implying that this decline is likely a secondary effect of age-related muscle loss60.The primary function of MSTN became evident when mice homozygous for 62, Rodgers et al.63 more recently argued that recombinant MSTN proteins stimulate C2C12 proliferation, emphasizing that the source of the recombinant MSTN protein can impact the outcome of an experiment. Furthermore, primary myoblasts isolated from both Mstn-null mouse embryos and adult mice were shown to exhibit a significantly increased proliferation rate64, and skeletal muscle regeneration after toxin-induced injury was significantly enhanced in Mstn-null mice66, indicating that endogenous MSTN suppresses satellite cell proliferation, differentiation, and muscle regeneration.Previous studies that applied recombinant MSTN proteins have presented mixed results regarding the role of MSTN in satellite cells. For instance, while several early studies showed that treatment with recombinant MSTN proteins inhibited C2C12 myoblast proliferationMstn-null mice, which survive to adulthood, Gdf11-null mice die shortly after birth, causing difficulties in identifying the role of GDF11 in adult tissue homeostasis. To overcome this limitation, Sinha et al.67 injected recombinant GDF11 proteins into aged mice and demonstrated that GDF11, in contrast to MSTN, acts as a rejuvenating factor in skeletal muscle. The aged mice treated with recombinant GDF11 proteins displayed striking improvements in muscle regeneration, exercise endurance, grip strength, myofibrillar and mitochondrial morphology, neuromuscular junctions, and the genomic integrity of muscle stem cells67. Furthermore, a more recent investigation in annual fish revealed that the application of GDF11 recombinant proteins boosted antioxidant enzyme activity in muscle, thus prolonging the lifespan68. Gdf11 expression levels were also shown to increase in slow-twitch muscles of aged mice after 6 weeks of treadmill running69. However, multiple studies have failed to reproduce these results, showing that GDF11 is deleterious towards muscle repair. For example, Egerman et al.70 argued that treatment with recombinant GDF11 proteins significantly impaired muscle regeneration and satellite cell expansion in mice through a downstream signaling pathway identical to that utilized by MSTN. Likewise, Hinken et al.71 showed that the recombinant GDF11 protein repressed satellite cell expansion, while Hammers et al.72 demonstrated that recombinant GDF11 and MSTN proteins decreased the myotube diameter through the canonical SMAD2/3 pathway. Zhou et al.73 also observed that injection of recombinant GDF11 protein into older rats significantly hindered muscle regeneration and function and induced tissue fibrosis. In addition, several other recent studies have shown that exogenous GDF11 treatment inhibits muscle growth75 and reduces strength76, while overexpression of the GDF11 propeptide, which antagonizes both mature GDF11 and MSTN, exerts beneficial effects77.In contrast to Gdf11 expression levels were shown to peak in skeletal muscles in mice between 4 and 8 weeks of age, which is when the most dramatic postnatal muscle development occurs20. This expression pattern is similar to that observed in shrimp, in which the expression of Mstn/Gdf11, which was shown to promote growth unlike vertebrate MSTN, peaks immediately after molting17. Moreover, Gdf11 expression levels were revealed to increase even further in Mstn-null mice during periods of rapid muscle growth20. To date, only one study has applied conditional knockout techniques in mice to investigate the postnatal functions of GDF11 in regulating skeletal muscle mass49. The study demonstrated that skeletal muscle-specific targeting of Gdf11 had no significant effect on muscle mass, fiber number, or fiber type, demonstrating that GDF11 and MSTN exhibit distinct functions in controlling muscle size49. Additional examinations focusing on genetic approaches will further advance the understanding of the role of GDF11 in muscle biology.Although most reports strongly suggest that exogenous GDF11 supplementation exerts an inhibitory effect on skeletal muscle growth and regeneration, further studies that employ genetic loss-of-function approaches are needed to fully elucidate the endogenous function of GDF11 in skeletal muscles. Interestingly, Mstn-null mice revealed that the absence of MSTN had no effect on heart weight but significantly decreased the end systolic diameter and increased fractional shortening78. Lim et al.79 consecutively demonstrated that Mstn-null mice subjected to ligation of the left anterior descending artery to induce myocardial infarction (MI) exhibited accelerated recovery of the ejection fraction, reduced cardiac fibrosis, and lower mortality, indicating that MSTN negatively affects cardiac function. Likewise, senescent MSTN-deficient mice were shown to display improved fractional shortening, smaller left ventricular diastolic and systolic diameters, and decreased cardiac fibrosis80, although an earlier study reported that MSTN has no significant effect on cardiac hypertrophy or fibrosis81. Additionally, transgenic mice overexpressing MSTN in cardiomyocytes exhibited interstitial fibrosis and impaired cardiac function82. Surprisingly, tamoxifen-induced, cardiomyocyte-specific deletion of Mstn in adult mice was reported to provoke severe cardiac hypertrophy and heart failure83. The authors of the study assumed that the much greater severity of the cardiac phenotype observed in the conditional knockout mice than in the straight knockout mice was due to the distinct modes of compensation83. To clarify the function of MSTN in hearts and whether the cardiac phenotypes of MSTN deficiency are influenced by the enhancement of skeletal muscle mass, additional examinations are needed.Despite its establishment as a potent inhibitor of skeletal muscle growth, MSTN has also been implicated in the regulation of cardiac tissue growth and function. A recent study that analyzed hearts of adult 84 performed heterochronic parabiosis experiments in mice and identified GDF11 as a rejuvenating factor that circulates in plasma. The group utilized both proteomics (SOMAmer) and western blot analysis to determine that the circulating levels of GDF11 decline with age, reporting that restoration of youthful levels through daily intraperitoneal injections of recombinant GDF11 proteins reverses age-related cardiac hypertrophy84. Specifically, GDF11 rather than MSTN stimulated the dose-dependent inhibition of cardiac myocyte hypertrophy in vitro84. In further support of these results, Poggioli et al.85 showed that circulating GDF11/MSTN levels diminish with age in multiple mammalian species, and administration of recombinant GDF11 proteins dose-dependently decreased cardiac mass in both young and old mice after only 9 days. Likewise, Du et al.86 demonstrated that Gdf11 expression levels decline in aged hearts and that either targeted myocardial delivery of the Gdf11 gene or recombinant GDF11 protein enhanced cardiac function and effectively reduced infarct size after ischemic injury in aged mice, providing support for the anti-aging function of GDF11. The association of plasma GDF11/MSTN levels with cardiovascular outcomes and overall deaths in humans was also reported using SOMAmer technology, which revealed that in patients with stable ischemic heart disease, increased GDF11/MSTN levels were associated with decreased rates of cardiovascular events, left ventricular hypertrophy, and overall death87. Mechanistically, GDF11 was shown to increase intracellular calcium levels and activate SMAD2/3 to prevent cardiomyocyte hypertrophy88.In 2013, Loffredo et al.84, Smith et al.89 showed that treatment of old mice with recombinant GDF11 proteins had no effect on cardiac mass, structure, or function. Moreover, recombinant GDF11 protein caused pathological hypertrophic signaling in neonatal rat ventricular myocytes, contradicting the classification of GDF11 as an anti-aging factor89. The same group subsequently published a dose-range study  performed in young mice that underwent transverse aortic constriction (TAC) surgery and reported that although treatment with recombinant GDF11 proteins reduced pathological cardiac hypertrophy and fibrosis and improved cardiac function, the highest dose (5.0 mg/kg) led to severe cachexia and premature death, and they also issued a warning against the use of recombinant GDF11 proteins as a therapy90. Recombinant GDF11 protein treatment was also recently shown to increase the levels of reactive oxygen species in isoproterenol-treated H9C2 cells (rat heart-derived cardiomyoblast cell line)91 and impair cardiac function in old mice75. In addition, Egerman et al.70 pointed out that the SOMAmer and antibody used in the initial study by Loffredo et al.84 were nonspecific, claiming that circulating GDF11 levels actually increase with age and are a pro-aging factor. However, Poggioli et al.85 later proposed that the levels of GDF11 detected by Egerman et al.70 were in fact the levels of immunoglobulin light chain, generating further controversy regarding the circulating levels of GDF11. Applying a novel immunoplexed liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay, Schafer et al.60 more accurately measured the circulating levels of GDF11 and reported that GDF11 levels remain constant in healthy adults throughout the lifespan. The study revealed that in older adults with severe aortic stenosis, higher GDF11 levels were associated with comorbidity and frailty60. Adding further controversy, a novel detection method using a parallel reaction monitoring (PRM) LC-MS/MS assay combined with immunoprecipitation recently showed that circulating levels of both GDF11 and MSTN significantly decline with age in female mice92.However, other groups have failed to observe the rejuvenating effects of GDF11 in cardiac tissues. After following the protocol used in a previous reportMyh6-Cre transgene, Garbern et al.93 generated a conditional knockout mouse model in which Gdf11 was targeted exclusively to cardiomyocytes and demonstrated that the mice exhibited progressive left ventricular dilation and a decrease in left ventricular systolic function at the age of 6 months. However, the authors also noted the adverse effects of the Cre recombinase itself and the possible compensatory expression of Gdf11 in noncardiomyocytes, which prevented the clear interpretation of the mechanism underlying the above results93. Apparently, further genetic analysis with avoidance of Cre toxicity is required to delineate the endogenous role of GDF11 in cardiac tissues.In contrast to the large number of studies that investigated the effects of recombinant GDF11 proteins, only a single recent study has addressed the function of GDF11 based on cardiac-specific genetic deletion in mice. Using a Mstn is broadly expressed throughout the adult rat central nervous system, including most neurons, axons, oligodendrocytes, astrocytes, and ependymal cells, suggesting that MSTN may play a crucial role in the brain94. Regarding the role of MSTN in the nervous system, examination of adult Mstn-null mice revealed that these mice exhibit increases in the number and size of axons and a delay in their age-related reduction95. Furthermore, MSTN-deficient mice were shown to display enhanced myelin thickness in motor axons and an increase in the number of sensory axons96. These mice were also shown to present a smaller brain size than wild-type mice at the age of 4 months, but the mechanism of brain size regulation by MSTN remains unclear97. Meanwhile, conflicting reports exist regarding the effects of recombinant MSTN proteins on neuronal cells. For instance, while Kerrison et al.98 showed that recombinant MSTN proteins dose-dependently enhanced the survival of retinal ganglion cells and neurite outgrowth, others demonstrated that recombinant MSTN proteins decreased the formation of neuronal colonies56 or suggested that MSTN inhibits neurogenesis in the olfactory system99.Despite the scarcity of information on the function of MSTN in the brain, a recent study showed that Gdf11-deficient mice has led to multiple studies that investigated the effects of recombinant GDF11 proteins on adult neurogenesis, demonstrating that GDF11 is a pro-neurogenic and pro-angiogenic factor. Shortly after Loffredo et al.84 reported GDF11 as a rejuvenating agent that protects the aged heart, the same group proposed that GDF11 exerts anti-aging effects on the brain, which was supported by the improvement of the cerebral vasculature and the enhancement of neurogenesis after the treatment of old mice with recombinant GDF11 proteins100. A separate experiment also showed that systemic delivery of recombinant GDF11 proteins enhanced hippocampal neurogenesis and vasculature in old mice by acting on brain endothelial cells, and only GDF11 but not MSTN promoted VEGF secretion in brain endothelial cells101. Likewise, a single injection of recombinant GDF11 protein was shown to improve short-term visual memory in middle-aged mice through upregulation of SOX2 expression102. Furthermore, treatment with recombinant GDF11 proteins was shown to promote neurogenesis and angiogenesis in mouse and rat models of stroke104 and in a mouse model of Alzheimer’s disease, revealing GDF11 as a potential therapeutic option for neurodegenerative disorders105. In contrast, in vitro data on the effects of recombinant GDF11 protein exposure to neural stem cell lines demonstrated that GDF11 suppresses cell proliferation and migration, suggesting that GDF11 should be a target for pharmacological blockade107. While the majority of studies presented the beneficial effects of recombinant GDF11 treatment on the mature nervous system, additional analysis utilizing genetic knockdown or conditional knockout of Gdf11 will further advance the understanding of the role and action mechanism of endogenous GDF11 in the adult brain.The perinatal lethality observed in Mstn-null mice were shown to exhibit enhanced bone mineral density in various parts of the skeleton108. In humans, genetic polymorphisms in MSTN were demonstrated to be associated with peak bone mineral density109. The effects of MSTN on bone may be both direct and indirect through the influence of skeletal muscle. While the indirect positive effect of enhanced skeletal muscle mass on bone strength was evidenced in Mstn-null mice110, the direct inhibition of osteoblast differentiation and stimulation of osteoclast formation by MSTN were also reported114. Surprisingly, despite the relatively low expression of Mstn in primary mouse osteoblasts and osteoclast precursors under physiological conditions, siRNA-mediated knockdown or genetic knockout of Mstn noticeably altered the differentiation rate of these cells113, highlighting the significant role of MSTN in the direct regulation of bone cells.The deficiency of MSTN has been described to result in not only an enlargement in skeletal muscle mass but also an increase in bone mass. In this regard, 115 demonstrated that circulating GDF11 levels were significantly diminished in both aged humans and patients with osteoporosis, and Gdf11 expression levels were substantially downregulated in the bone marrow of aged mice and mice with osteoporosis. Additionally, the group showed that treatment with recombinant GDF11 proteins significantly promoted osteoblast differentiation and inhibited adipogenesis of bone marrow mesenchymal stem cells115, emphasizing the pro-osteogenic role of GDF11, which is in contrast with the function of MSTN. However, Lu et al.116 subsequently published results showing the opposite results, indicating that recombinant GDF11 proteins inhibited osteoblast differentiation of bone marrow mesenchymal stem cells through a downstream signaling pathway identical to that of MSTN and that injection of recombinant GDF11 proteins suppressed bone formation in mice. In the same year, Liu et al.117 also reported similar findings and demonstrated that recombinant GDF11 treatment led to bone loss in both young and aged mice through impairment of osteoblast differentiation and increased osteoclast formation. Later, the same group further showed that recombinant GDF11 negatively affects bone healing in mice119. Moreover, in postmenopausal women, increased levels of circulating GDF11 were associated with decreased bone mineral density, demonstrating the inhibitory effect of GDF11 on bone formation120.As opposed to the consistent reports on the inhibitory function of MSTN on osteogenesis, the reports of the effects of GDF11 on adult bone homeostasis are controversial. In 2015, Zhang et al.26. Our findings revealed that both time-specific ubiquitous deletion and limb mesenchyme-specific deletion of Gdf11 resulted in diminished bone mass in young adult mice, suggesting that GDF11 endogenously promotes bone development, in contrast to MSTN26. Furthermore, both Gdf11-null osteoblasts and wild-type osteoblasts subjected to siRNA-mediated Gdf11 knockdown exhibited impaired differentiation and mineralization, while the opposite effects were observed in wild-type osteoblasts transfected with full-length GDF11 cDNA26. Whether there is a difference in the cellular and physiological outcomes of treatment with the full-length cDNA and mature forms of the GDF11 protein requires further investigation. A previous study demonstrated that skeleton-specific transgenic overexpression of the GDF11 propeptide, which is capable of inhibiting both mature forms of GDF11 and MSTN, enhanced bone formation in mice during embryogenesis and postnatal development121. However, these mice were shown to exhibit a posterior homeotic transformation in the cervical vertebra, resulting in transformation of the seventh cervical vertebra into a thoracic vertebra, which is the exact opposite of the anterior homeotic transformations observed in Gdf11-null mice48. Furthermore, the authors did not provide information on the relative expression of endogenous Gdf11 and the transgene during developmental stages, limiting the potential for the clear interpretation of the results122. Additional examination focused on genetic studies in mice will lead to additional insights into the endogenous mechanism of action of GDF11 in postnatal bone remodeling.In an attempt to avoid the controversy surrounding the effects of recombinant GDF11 proteins, our group has recently applied conditional knockout strategies in mice to examine the endogenous function of GDF11 in osteogenesis85 pointed out the existence of batch-to-batch variations in the concentrations of recombinant GDF11 proteins, which was also confirmed by the manufacturer, and suggested that differences in protein sources, protein refolding efficiencies, and protein concentrations all possibly contributed to the disparity in the outcomes. Likewise, Rodgers and Eldridge34 highlighted the possible significant influence of the source and quality of recombinant proteins on experimental results and indicated that both Sinha et al.67 and Egerman et al.70, who reported opposing results, utilized bacterially generated recombinant proteins that may be less effective or even produce different effects depending on the folding status. In support of this claim, Rodgers et al.63 demonstrated that recombinant MSTN proteins produced in bacteria and eukaryotes behave differently in the regulation of C2C12 myoblast proliferation. Most importantly, even though differences in the type 1 receptor-binding residues and signaling potency between mature GDF11 and MSTN have been reported25, it is difficult to rule out the fact that recombinant GDF11 and MSTN proteins cannot be effectively distinguished due to their high sequence similarity, revealing the possibility that the responses mediated by recombinant GDF11 protein treatment actually reflect the endogenous functions of MSTN. Apparently, due to the large discrepancy in the reported effects of recombinant GDF11 protein treatment, further establishment of experimental settings that generate more reliable outcomes as well as different strategies for GDF11 supplementation will be needed for further consideration of GDF11 as a therapeutic option.After the initial reports of GDF11 as a rejuvenating agent for the heart, skeletal muscle, and brain, numerous groups have evaluated the effects of recombinant GDF11 protein administration on various tissues Table . However123, unexpectedly promoted rapid increases in hematocrit, hemoglobin, red blood cells, and late-stage erythropoiesis, which were suggested to be caused by suppression of endogenous GDF11124, although recent studies using conditional knockout techniques refuted this mechanism126. In addition, our group has recently demonstrated that transgenic overexpression of FST, an endogenous inhibitor of MSTN, GDF11, and activins, substantially enhanced muscle mass but induced spontaneous tibial fractures due to a reduction in bone mineral density, implying that inhibition of GDF11 may have adverse effects on bone26. Therefore, possible side effects triggered by both exogenous administration and endogenous inhibition of GDF11 and the means to resolve them should be evaluated with caution in order to enhance the potential for GDF11 to be applied in clinical settings.Ample studies that noted the detrimental effects of recombinant GDF11 protein injection have also indicated GDF11 as a potential target for pharmacological blockade. However, the relatively little information is available on the endogenous functions of GDF11 in regulating adult physiology, which indicates the need for a cautious approach in the development of GDF11 inhibitors. In fact, sotatercept (ACE-011), an ACVR2A fusion protein originally designed by Acceleron Pharma to increase bone mineral densityGdf11-null mice, in contrast to the long-term viability of MSTN-deficient mice, led to complications in characterizing the role of GDF11 in adult tissues and caused many groups to utilize recombinant GDF11 proteins to identify its postnatal function. However, difficulties in biochemically distinguishing between the recombinant GDF11 and MSTN proteins as well as variations in the quality of recombinant proteins aroused much controversy regarding the effects of GDF11 treatment. Indeed, while numerous studies have presented the beneficial physiological outcomes after supplementation with recombinant GDF11 proteins, providing a rationale for its therapeutic application, an equally large number of studies have also underscored its harmfulness, demonstrating GDF11 as a potential therapeutic target for inhibition. Therefore, future studies should focus on implementing genetic knockdown or conditional knockout techniques, which may be more promising approaches to differentiate the endogenous functions of GDF11 and MSTN and their regulatory mechanisms. Furthermore, reliable research strategies to improve the consistency of test results are needed to support the progression of GDF11 therapy or GDF11 inhibitors to clinical trials.The remarkable sequence similarity between GDF11 and MSTN led to the assumption that the two molecules are functionally redundant. However, multiple genetic studies in mice provide clear evidence that they play distinct roles under a range of physiological conditions. Notably, the perinatal lethality observed in Table S1


Stroke remains the number one cause of morbidity in the United States. Within weeks to months after an ischemic event, there is a resolution of inflammation and evidence of neurogenesis; however, years following a stroke, there is evidence of chronic inflammation in the central nervous system, possibly by the persistence of an autoimmune response to brain antigens as a result of ischemia. The mechanisms underlying the involvement of macrophage and microglial activation after stroke are widely acknowledged as having a role in ischemic stroke pathology; thus, modulating inflammation and neurological recovery is a hopeful strategy for treating the long-term outcomes after ischemic injury. Current treatments fail to provide neuroprotective or neurorestorative benefits after stroke; therefore, to ameliorate brain injury-induced deficits, therapies must alter both the initial response to injury and the subsequent inflammatory process. This review will address differences in macrophage and microglia nomenclature and summarize recent work in elucidating the mechanisms of macrophage and microglial participation in antigen presentation, neuroprotection, angiogenesis, neurogenesis, synaptic remodeling, and immune modulating strategies for treating the long-term outcomes after ischemic injury. In the United States, stroke is the number one cause of morbidity and the third highest cause of death. It is estimated that over 800,000 people have a stroke each year, equating more than one stroke-per-minute. Of these 800,000 strokes, 87% are ischemic in nature, resulting from a clot or mass blocking a blood vessel, and 13% are hemorrhagic caused by a weakened blood vessel that ruptures . The ensDespite decades of research, tissue plasminogen activator (tPA) remains the only drug approved by the U.S. Food and Drug Administration for treating stroke. Effective only if administered 3–4.5 h after the onset of stroke, the vast majority of patients are not able to receive tPA [Current treatments fail to provide neuroprotective or neurorestorative benefits; therefore, to ameliorate brain injury-induced deficits, therapies must target both the initial response to injury and the subsequent inflammatory process . Recent Microglia are the resident macrophages in the central nervous system (CNS) and are a part of the mononuclear phagocyte system. Similar to other tissue-specific macrophages of embryonic origin, microglia originate from the yolk sack and are established approximately at the same time as neurons during the early prenatal period, where they participate in the developmental maintenance of neurons throughout life . HoweverMacrophages that reside in the CNS-border, which includes the choroid plexus, perivascular spaces, and subdural meninges, express distinct transcriptional signatures from microglia and infiltrating macrophages ,16. Throhigh populations are identified as macrophages and CD11b+CD45low populations as microglia. However, it is important to note that microglia can upregulate CD45 after ischemic stroke and become indistinguishable from CD11b+CD45high cells in the border regions of the CNS or the periphery [Although current advances in technology have defined microglia and macrophages as having transcriptionally distinct profiles, identification of microglia and macrophages in the brain through the use of various cellular markers remains unclear and contentious in the literature . Many greriphery ,22. In this review, we will distinctly denote microglia and macrophage nomenclature based on the methods by which each research study used to make its conclusions, with additional comments on the authors’ chosen nomenclature and any potential discrepancies with the study’s interpretation resulting from that choice.After the onset of brain ischemia, microglia undergo morphological and functional changes in the penumbra, while macrophages also infiltrate the brain parenchyma and migrate toward the infarct area. Studies have shown that activated microglia can have both a beneficial and detrimental effect during all stages of ischemic stroke; however, the timing and kinetics at which specific anti- and proinflammatory events occur may influence the nature of the outcome . ActivatWhile the detrimental effects of microglia have been emphasized in many studies, Szalay et al. showed that a selective elimination of microglia using a CSF1R antagonist PLX3397 prior to a middle cerebral artery occlusion (MCAO) led to a 60% increase in infarct size. The increase in infarct volume was reversed with microglial repopulation . SimilarThere has been evidence of infiltration of peripheral monocytes to the brain in response to DAMPs produced after ischemic stroke, with the presence of neutrophils and macrophages in the site of CNS injury ,46,47,48hi (CCR2+) proinflammatory monocyte migration to injury sites. The increase in CCL2 (MCP-1) expression in the ischemic hemisphere leads to an increased infarct volume [lo monocytes that do not express CCR2, but express the CX3CR1 receptor for CXC3CL1 fractalkine, develop into M2 macrophages after recruitment to normal tissues [This switch in macrophage phenotype has been explored in ischemic models. For example, monocyte chemoattractant protein-1 (MCP-1) possesses cytokine-like properties and plays a significant role in macrophage and Ly-6Ct volume ,52, whilt volume ,54. In c tissues . This unIt is important to note that the M0/M1/M2 trichotomy is an oversimplification of activation states as the status of macrophages may include overlapping functional phenotype as well as different ones. For example, the M2 phenotype includes subpopulations such as M2a, M2b, M2c, and Mox, each with distinct physiological functions . Not allhi (CCR2+) macrophages express the M1 phenotype in the ischemic hemisphere, but convert to an alternate phenotype at the injury site 48 h after stroke. These proinflammatory cells downregulate Ly6C and CCR2 expression, enhancing the resolution of inflammation by releasing vascular endothelial growth factor and TGF-β [Depending on the ischemic milieu, macrophages can transform to different functional phenotypes in the hemisphere of infarction . In respnd TGF-β ,60. Zhannd TGF-β . Similarnd TGF-β . Microglia are known to influence angiogenesis, whereas the elimination of microglia or deficiency in macrophage colony-stimulating factory causes a reduction in retinal vasculature ,63. DuriIt is often reported that ischemic stroke can lead to synaptic dysfunction; however, it is known that microglia and macrophages participate in synaptic remodeling and refining neural circuitry . MicroglMacrophage and microglial activation have been linked to changes in neurogenesis following ischemic stroke in mammals ,73. It hMicroglia and macrophages may participate in regulating neurogenesis by supporting axonal regrowth and regeneration to allow for functional recovery after stroke. Their production of local trophic gradients helps to stimulate axonal sprouting toward the infarct area . MicroglThe increase in neurogenesis after stroke may only be transient, and similar to what is seen in traumatic brain injury models, appears to be disrupted by a secondary inflammatory response . While tAlthough neurons in the stroke lesion core cannot be rescued, improvement in stroke outcome can be achieved through reduction in secondary brain injury due to inflammation. Based on our developing knowledge of microglia and macrophage involvement in the pathophysiological processes of stroke, many novel therapeutic approaches have emerged to promote remodeling of the injured brain in stroke models by altering the activation phenotypes of these cells. Modulating microglial and macrophage activation and polarization through use of various pharmacological and small molecules has been a popular area of study . MinocycThe nature of the ischemic environment is a key determinant in driving microglia and macrophage function and their activation phenotype. For example, microglia and macrophages are highly susceptible to energy deficits, leading to activation and recruitment; thus, metabolic status of the lesion environment is a major factor in determining the nature of microglial response ,100. A nMicroglia and macrophage activation could also be modulated toward the M2 phenotype through post-stroke treatment with docosahexaenoic acid (DHA) administered immediately after reperfusion and daily for three days thereafter. Treatment with DHA also significantly inhibits infiltration of neutrophils, T, and B lymphocytes . A polarAngiogenesis can also be stimulated by modulating microglial polarization toward M2 through AMPK signaling after stroke. The treatment of human umbilical vein endothelial cells (HUVECs) in vitro with conditioned media collected from BV-2 microglial cells in culture incubated in berberine, an isoquinoline alkaloid extract from traditional Chinese medicine, facilitated angiogenesis of HUVEC cells . MoreoveAdditionally, the use of partial major histocompatibility complex (MHC) class II constructs as a novel treatment for ischemic stroke presents hopeful outcomes . These cIt is also worth mentioning that there is accruing evidence suggesting oxidative stress activated mechanisms playing a role in the impairment of function after stroke. It is known that M1 microglia produce free radicals and oxidants, leading to long-term deficits in stroke patients ,113,114.Stem cell therapies for stroke have the potential to provide neurorestorative benefits. The type of stem cell and route of administration determines the efficacy of stem cell-based therapies in mediating a therapeutic effect . The theNeural stem cell (NSC) transplants have been shown to improve behavioral outcomes and angiogenesis in rat models of ischemic stroke, leading to studies interrogating human NSCs (hNSC) ,117. GraBone marrow stem cells (BMSC) have been shown to migrate to the site of ischemia and differentiate into neural cells . More reUmbilical cord blood stem cells (UCBSC) offer many advantages over other types of stem cells. The immune properties of UCBSCs allow for increased tolerance for human leukocyte antigen mismatches and decreased incidence of graft-versus-host disease as well as ease of procurement and availability ,127. NonA significant subset of stroke patients experience progressive cognitive decline. Levine et al. conducted a prospective study of cognitive function in 23,572 patients aged 46 years or older who were followed for an average of six years post stroke . These iint are a subset of microglial cells that can also upregulate antigen presentation and activation markers [After an ischemic event, antigen-presenting cells (APCs) accumulate in the brain parenchyma and express MHC class I and II cell surface molecules ,134,135. markers ,139,140. markers , demonst markers ,142,143.The volume of infarction and severity of stroke is correlated with the concentration of neural antigens found in the serum of patients after stroke ,145. AutRegulation of microglial function as a means to attenuate neurological dysfunction seen in chronic stroke has become a promising strategy in pre-clinical research . Cell trPositron emission tomography imaging studies in patients with chronic stroke and cognitive decline revealed persistent microglia activation . PreclinTransplantation of fetal microglia has been demonstrated to improve ischemia-induced functional changes and apoptotic events after stroke. Ischemia was induced in rats by MCAO and human microglial cells (HMO6) from fetal telencephalon tissue were transplanted into a treated group . AnimalsFetal microglial transplantation may improve stroke outcomes by modulating inflammation and facilitating angiogenesis. Expression of IL-1β is high in HMO6 and has been shown to increase VEGF mRNA expression in HMO6 cell lines when co-cultured with a mesenchymal stem cell line B10 . White mMicroglia derived from iPSCs are an important consideration for translational neuroimmunology research and hold great potential for therapy. iPSC-microglia are able to recapitulate the inflammatory-modulating properties of brain-resident microglia by resembling the in vivo phenotypical profiles of microglia within stroke lesions and responding to IL-13 stimulation . While pExogenous microglia also have the potential to protect against neuronal damage after stroke by exhibiting an affinity for ischemic brain lesions. Injection of exogenous microglia was show to promote CA1 cell survival by migrating to the CA1 cell layer and increasing the expression of BDNF and GDNF in the ischemic hippocampus . Cell thIschemic injury is characterized by the time dependent activation of microglia and infiltration of macrophages into the penumbral tissue. Lack of effective treatment options is partially attributed to the dynamic pathophysiology of ischemic stroke; however, future research aimed at elucidating mechanisms underlying the involvement of these cell types in a time-dependent manner is a promising strategy for treating the long-term outcomes after ischemic injury.The studies reviewed here suggest the importance of microglia and macrophages in both neuroprotection and neurogenesis after stroke and the potential use of these cell types for therapy, especially for treating long-term cognitive deficits. Further discrimination of this relationship may include careful distinction between different populations of macrophage and microglia due to their temporal differences after the onset of stroke. Currently in the literature, there is a lack of consistent agreement in nomenclature used to distinguish differences between microglia and macrophage populations. This aspect will be important to accurately describe the role of these cells over time, shortly before and following the induction of stroke.While alternative activation and modulation of the macrophage and microglial response may stimulate neurogenesis, angiogenesis, and synaptic remodeling in the injured brain, it is worth considering other immune cells, factors, and molecules that are influenced by inflammatory microglia and macrophages. Investigation of functional recovery and improvement of long-term cognitive outcomes may be dependent on the relationship between macrophage and microglia with MHC class II molecules, T cells, and the presence of neuroantigens.Understanding the timing and kinetics at which specific anti- and proinflammatory events occur will affect stroke outcome, and delineating the role of macrophage and microglia and their signaling pathways over time could provide a host of candidates for therapeutic interventions after an ischemic event. Transplantation studies demonstrate the potential of targeting signaling pathways and using microglia in gene therapy as a vehicle for the transfer of therapeutic genes as a promising therapy for treating chronic stroke.The role of microglia and macrophages in stroke-induced neurogenesis is multifaceted and complex: production of trophic factors is essential for the migration of newborn neurons; however, activation of microglia and macrophages can mediate inflammation that is detrimental to neurogenesis. Their interaction with other immune cells such as lymphocytes contributes to the complexity of the microenvironment generated by DAMPs and neuroantigens, and consequently alters the macrophage and microglial response to injury . Since t


Pneumonia remains the leading infectious cause of child mortality worldwide. Despite highly effective interventions to prevent and treat pediatric pneumonia, globally, it is estimated that only 60% of children under 5 years of age with symptoms of pneumonia are taken to a healthcare provider . HealthcThe ITIP project was conducted at Kamuzu Central Hospital and Bwaila District Hospital in Lilongwe, Malawi between June 2016 and April 2019 and included two prospective, double-blind, randomized controlled clinical trials evaluating the duration of amoxicillin dispersible tablet (DT) treatment for non-severe community-acquired pneumonia among children 2–59 months of age. Children in the ITIP1 fast-breathing pneumonia trial were randomized to either placebo DT (intervention) or amoxicillin DT (control) administered twice daily for 3 days, and children in the ITIP2 chest-indrawing pneumonia trial were randomized to either 3 (intervention) or 5 (control) days of twice-daily amoxicillin DT, with those in the 3-day group receiving placebo for the last 2 days of study participation. Adherence was defined as the proportion of study drug doses taken by the enrolled child of those that were expected; children who were switched to a different course of antibiotics or otherwise experienced treatment failure were not expected to continue the original course of study drug, and thus doses after such events were not counted toward the adherence denominator. For example, if an ITIP1 study participant was switched to a different treatment course after dose 4 and ingested 3 out of 4 expected study drug doses, their adherence was 75%. Similarly, if an ITIP2 study participant was switched to a different treatment course after dose 6 and ingested 3 out of 6 expected study drug doses, their adherence was 50%.Upon enrollment and randomization, the blinded study staff prepared and administered the first dose of study drug and instructed the caregiver how to administer the remaining doses using customized job aids and patient instructions with pictograms . ChildreIn a secondary analysis of data from these two trials, we found the average percent of adherence to all treatment arms to be very high , with equivalence in clinical outcomes and acceptability of use [%, with eQualitative pediatric pneumonia research conducted in Malawi found that caregivers showed confusion around dosing and treatment durations, and found it more difficult to be adherent when multiple drugs required different dosing schedules or preparations, such as splitting pills, diluting medication in water, or preparing food to eat with the medication . This suWhile clinical trials evaluating antibiotic treatment may not be ideal to evaluate adherence reflective of real-world clinical and home settings given the increased follow-up and monitoring, they nevertheless may provide some useful information. Further studies evaluating adherence to DT antibiotic treatment for pediatric pneumonia in low-resource settings should be completed, integrating considerations and observations of healthcare administrators and providers, and caregivers. Additional work may be necessary to explore the barriers and facilitators to implementing solutions like easier-to-use DT formulations in these settings. Our finding of high adherence to DT antibiotics for pediatric pneumonia treatment in a clinical trial setting suggest that high adherence is possible, and addressing the gap between research and real-life implementation may be beneficial in successfully preventing deaths from pneumonia.


Nuclear factor, erythroid 2-like transcription factor 2 (Nrf2) and its cytoplasmic inhibitor, kelch-like ECH-associated protein 1 (Keap1), comprise a redox-responsive endogenous antioxidant defense module that orchestrates the expression of cytoprotective genes to maintain homeostasis , as wellOne the most impactful articles in this Special Issue is the review by Yagishita et al. on the current landscape of Nrf2 biomarkers in clinical trials . They anAmong the four compounds reviewed by Yagishita et al., sulforaphane is the most widely studied and the only one that has been associated with all six types of Nrf2 biomarkers . SulforaDespite the current challenges in the clinical application of Keap1/Nrf2 signaling modulators, interest in realizing the translational potential of this pathway remains very intense. This is evidenced by the results of a patent search from my group by Chartoumpekis et al., covering the period 2017–2020 and focusing on patents mentioning Nrf2 [The majority of studies in this Special Issue report on the protective effects of Keap1/Nrf2 signaling in specific tissues or organs, which is related to the concept of Nrf2 as “a multi-organ protector” . The mosIn an original research study, Chen et al. show that restraint stress, induced by limiting the ability of mice to move freely, alters expression of glucocorticoid bioavailability mediators, suppresses Nrf2 and promotes oxidative stress in the liver . SpecifiBesides the liver, the kidney is a major detoxification organ. Guerrero-Hue et al. review the protective roles of Nrf2 in renal disease . This isSolenocera crassicornis) have a protective effect against the renal toxicity of cyclophosphamide [In a preclinical research study in mice, Jiang et al. show that low-molecular weight peptides extracted from the head of a prawn  wild-type lung squamous cell carcinoma (LUSC) or lung adenocarcinoma (LUAD), whereas in LUAD, the effect of the epigenetic silencing of KEAP1 on its transcription was also observed in patients with epidermal growth factor receptor (EGFR)-mutated tumors [In the lung, the Keap1/Nrf2 pathway has a central role not only in maintaining homeostasis and protecting against various diseases  but alsod tumors . These rIn a research study in mice, Li et al. show that dimethyl fumarate alleviates dextran sulfate sodium-induced colitis . The pheReactive oxygen species are produced by mitochondria during activity in various muscle tissues, including the heart, where the role of Keap1/Nrf2 signaling in ischemia–reperfusion injury has been well established . In a stNeurons are particulary sensitive to oxidative stress, and they rely on their own antioxidant response as well as that of neighboring astrocytes to maintain homeostasis. In a review article, Kang summarizes recent studies on the relevance of Keap1/Nrf2 signaling for mitochondrial dynamics and/or mitophagy in various neurological diseases . Based oLastly, the roles of Keap1/Nrf2 signaling in endocrine tissues are also addressed in a set of studies in this Special Issue. In a study using mice, Cisterna et al. show that ex vivo treatment of explanted adipose tissue with ozone improves its preservation, which may be of relevance as an adjuvant treatment to improve graft survival in patients undergoing autologous fat transplantation . This phAnother endocrine tissue that can be transplanted is the pancreas, and specifically the pancreatic islet cells for the treatment of diabetes. The outcomes of islet cell transplantation (ICT) depend, in large part, on the number and viability of the transplanted islet cells, which, however, are generally in short supply. Therefore, methods to improve the viability of islet cells for transplantation are actively pursued. Jarrin Lopez et al. review the results of recent studies that employed dimethyl fumarate or the Nrf2-activating phytochemical epigallocatechin gallate for ICT, and they discuss Keap1/Nrf2 signaling as a potential target to ameliorate oxidative stress at every step of the standard ICT protocol .Finally, the thyroid gland has a special relationship with oxidative stress because it must not only defend itself against reactive oxygen species but it must also actively produce them as part of the thyroid hormone synthesis process. In a review article from my group, Thanas et al. discuss the latest studies on the roles of Keap1/Nrf2 signaling in the thyroid gland, which range from regulation of thyroid physiology to implication as a protective factor against diverse disease states including hypothyroidism, thyroid autoimmunity, thyroid enlargement (goiter), hyperthyroidism and thyroid carcinoma . In addiEven though preclinical research on Nrf2 is very active, and the first examples of clinical translation are already available, much remains to be learned about the basic mechanisms involved in the regulation and function of the Keap1/Nrf2 pathway. This is exemplified in the present Special Issue by two studies. The article by Ibrahim et al. employed RNA sequencing-based transcriptional profiling and quantitative proteomics to delineate the overlapping and differential genetic programs effected by Nrf2 and its closely related transcription factors Nrf1 and Nrf3 , recent In another study, Martin-Hurtado et al. review the very novel topic of an emerging partnership between Nrf2 and primary cilia . The priAntioxidants that are open for submissions with a direct or indirect focus on Nrf2, six of which have a clear translational/clinical focus . With such active research in the field, tangible further progress towards the elucidation of basic mechanisms, tissue/organ-specific functions and clinical translation can be credibly expected in the current decade.The collection of reviews and original research studies in this Special Issue illustrates the vibrant state of research into the various aspects of the Keap1/Nrf2 pathway, but also the persistent challenges in the translation of basic and preclinical research findings into new preventive or therapeutic strategies. At present, there are nine Special Issues in