Sb supergene in the fire ant Solenopsis invicta determines the form of colony social organization, with colonies whose inhabitants bear the element containing multiple reproductive queens and colonies lacking it containing only a single queen. Several features of this supergene — including suppressed recombination, presence of deleterious mutations, association with a large centromere, and “green-beard” behavior — suggest that it may be a selfish genetic element that engages in transmission ratio distortion (TRD), defined as significant departures in progeny allele frequencies from Mendelian inheritance ratios. We tested this possibility by surveying segregation ratios in embryo progenies of 101 queens of the “polygyne” social form (3512 embryos) using three supergene-linked markers and twelve markers outside the supergene.The Significant departures from Mendelian ratios were observed at the supergene loci in 3–5 times more progenies than expected in the absence of TRD and than found, on average, among non-supergene loci. Also, supergene loci displayed the greatest mean deviations from Mendelian ratios among all study loci, although these typically were modest. A surprising feature of the observed inter-progeny variation in TRD was that significant deviations involved not only excesses of supergene alleles but also similarly frequent excesses of the alternate alleles on the homologous chromosome. As expected given the common occurrence of such “drive reversal” in this system, alleles associated with the supergene gain no consistent transmission advantage over their alternate alleles at the population level. Finally, we observed low levels of recombination and incomplete gametic disequilibrium across the supergene, including between adjacent markers within a single inversion.Sb supergene is a selfish genetic element capable of biasing its own transmission during reproduction, yet counterselection for suppressor loci evidently has produced an evolutionary stalemate in TRD between the variant homologous haplotypes on the “social chromosome”. Evidence implicates prezygotic segregation distortion as responsible for the TRD we document, with “true” meiotic drive the most likely mechanism. Low levels of recombination and incomplete gametic disequilibrium across the supergene suggest that selection does not preserve a single uniform supergene haplotype responsible for inducing polygyny.Our data confirm the prediction that the The online version of this article (10.1186/s12863-018-0685-9) contains supplementary material, which is available to authorized users. Selfish genetic elements are heritable entities that spread in populations despite having no or even negative fitness consequences for the host organism –4. AmongTransmission ratio distortion increasingly is viewed as an important evolutionary force driving such diverse phenomena as the origin of meiotic asymmetries in oogenesis , the dynAn additional explanation for the apparent low incidence of segregation distortion and other forms of TRD is suggested by the fact that relatively few studies have surveyed wild populations specifically for evidence of subtle drive, including detailing the nature and extent of variation among progenies , 31–33. Solenopsis invicta is a highly eusocial insect that has emerged as an important model for social evolution and is of interest with regard to the potential for TRD, including segregation distortion. This species, which is an invasive pest in the southern USA and elsewhere  segregation) to 1.0 (complete segregation distortion) [k can be less than 0.5, in the latter case depending solely on the identity of the focal allele. Therefore, for some segregation analyses in which we wished simply to compare the average magnitude of distortion among all the study loci , we set k as the proportion of gametes bearing the allele in a segregating progeny that was present at a frequency equal to or greater than 0.5 (unpolarized k). In the majority of segregation analyses, however, k refers to alleles marking the supergene in the Sb-linked loci or to specific alleles at the non-supergene loci (polarized k).Segregation ratios for gametes bearing drive elements typically are represented by the parameter tortion) , 17. Howk = 0.5; e.g., [k values expected by chance (variation from 1:1 segregation due solely to sampling error) were taken as the limits for statistical significance of the observed values in one- or two-tailed tests.We tested for significant departures from Mendelian proportions (TRD) at each segregating locus within each progeny by means of one-tailed exact binomial tests . ThSb supergene in our embryo progenies was evaluated further by considering the three supergene-linked loci  simultaneously. The expected frequency of departures from Mendelian ratios at Sb occurring by chance in the absence of TRD, given our sample sizes, was estimated in a first multilocus simulation analysis that accounted for the observed correlations in segregation ratios between these markers (see below). A second, far more conservative, multilocus simulation analysis that disregarded the correlations between marker segregation ratios also was conducted. See Additional file The frequency and significance of TRD involving the k values between supergene and non-supergene loci considered as separate classes. In a first set of analyses, we tested whether proportions of significant deviations from 1:1 ratios  differed between the two classes by conducting a permutation test in which differences in these proportions between paired loci, members of which belonged to the same or to different classes, were compared to differences between paired loci for which class identity of each member was assigned randomly (1000 permutations); assignments were constrained so that the numbers of within- and between-class pairs in the empirical data were preserved in each permutation replicate. We complemented these permutation analyses by conducting Mann-Whitney tests that compared the observed differences in numbers of segregating progenies with k ≥ 0.65 between paired markers of the same or alternate classes  differed between the two classes. A bootstrap test for differences in mean k values was conducted by constructing 5000 bootstrap samples of k values for each class of loci using the online program STATKEY [k values was subtracted from the mean for the supergene values and the 95th percentile of the 5000 differences was taken as the one-tailed confidence limit for the null hypothesis that k values for the supergene loci did not exceed those for the other loci. Further information on these analyses is given in Additional file We compared both the proportions of departures from 1:1 segregation ratios and the distributions of  STATKEY ; for eacGp-9 alleles, the results of this resampling procedure yielded unbiased overall population allele frequency estimates for this gene.Next, a resampling procedure was undertaken to estimate the experiment-wide frequencies of supergene-associated alleles within segregating progenies; this was done to evaluate the overall population-level effect of any allele-specific transmission advantages . Second, inviability proportions for the pool of 24 progenies shown to have significant supergene TRD by simultaneous consideration of all three supergene loci were compared to the proportions for the remaining 77 progenies using the non-parametric Mann-Whitney Test.The relationship between degree of deviation from Mendelian ratios at supergene-linked markers and extent of failure of eggs to embryonate in a progeny was determined in order to examine the possibility that female gamete killing is the mechanism for segregation distortion e.g., , 89, 90), 9089, 9 the USA . Two sepHexp = 0.286–0.819 , with per-locus ulations , and Hexred_ant to 101 for six of the microsatellite markers and Gp-9. Means of > 30 embryos per progeny were successfully genotyped at each microsatellite locus, with a mean of 34.5 embryos scored for Gp-9 , a result expected given that most polygyne queens mate with monogyne males , whereas a single queen (1.0%) mated with a single diploid male that was heterozygous at Gp-9. Among the 6.9% of mother queens that mated multiply, most mated with two males (6.0%) and the remaining one (1.0%) evidently with three males. Two of these polyandrous queens mated exclusively with Gp-9B-bearing males, while the remaining five mated with males of each Gp-9 haplotype.Based on comparisons of mother queen and offspring embryo genotypes, several different types of progenies were recognized ; a ; a Gp-9Br) between single queens and their mates was 0.041 . This result accords with the minimal nuclear genetic differentiation reported between geographically adjacent polygyne and monogyne populations in the USA [r = 0.069), as reported previously for invasive polygyne S. invicta [The estimated mean pairwise coefficient of relatedness . In contrast, genotype frequencies at all three supergene loci, C294, Gp-9, and i_126, departed significantly from HWE , with moderate to large negative estimates of FIS indicative of excess heterozygosity . Excess heterozygosity is expected at supergene markers because homozygosity for either supergene haplotype effectively is lethal in queens of the polygyne form [Genotype frequencies for the 113 study queens matched the frequencies expected under Hardy-Weinberg equilibrium (HWE) at all twelve loci not linked to the supergene  between pairs of marker loci are depicted in Fig. c for only five marker pairs do not overlap with 0.5, the value for freely recombining loci. The three lowest estimates involve all of the pairwise comparisons of the supergene markers , with the significance of disequilibrium withstanding correction for multiple comparisons for all three pairs  , with two of the supergene-linked marker pairs showing the lowest probabilities of equilibrium . Only these two pairs retained statistical significance after correction for multiple comparisons (both p < 0.025). The minor discrepancies between the results for eggs and males presumably can be attributed to most males having originated from monogyne rather than polygyne mothers. An important point regarding the five i_126 and seven C294 alleles we identified is that haplotypes with the i_126230 and, especially, the C29492 alleles almost always also contained the b allele of Gp-9 in both eggs and males .c) and gametic disequilibrium (D*) [c values significantly < 0.5, gametic disequilibrium was significantly negatively correlated with recombination frequency in both eggs and males . For the pairs of freely recombining markers (c = 0.5), no association between recombination rate and disequilibrium was observed , as expected given that recombination erodes disequilibrium to yield reduced estimates that vary stochastically   are showy .C294 and Gp-9), is re-enforced by genotypic data from the 113 polygyne mother queens. All of these queens were Bb heterozygotes at Gp-9, yet seven (6.2%) were apparent homozygotes for the 92 allele of C294. Although we cannot rule out the existence of rare null alleles or back mutation at locus C294, the observed pattern is consistent with the evidence that supergene recombinants are produced infrequently between the SB and Sb chromosomes and further suggests that individuals with such haplotypes can survive to become reproductives in the wild. Detectable but typically low levels of recombination between inverted homologous regions, occurring by means of double crossovers or gene conversion, have been widely documented in diverse taxa [The important finding based on inferred egg haplotypes that some recombination occurs and that disequilibrium is not absolute across the supergene, even for markers located within the same inversion  among the study loci  segregation ratios, based on binomial tests conducted on each locus segregating in the 101 study progenies, are reported in Table :1 segregoci Fig. . Binomiai_129 . Based on the bootstrap confidence limits, the proportions of progenies with significant deviations from 1:1 ratios at this and all other non-supergene loci did not differ from the proportions expected by chance in the absence of TRD ; not surprisingly, this pair also displayed the fourth lowest  recombination frequency (c), just after the three supergene-linked locus pairs  fell well above the entire range of differences for the 1000 permuted pairs (maximum = 0.093), while the mean difference for paired loci in which both were members of a single class (0.048) fell below the range for the permuted pairs (minimum = 0.063). Mann-Whitney tests on the differences in frequencies of segregating progenies with k ≥ 0.65 (indicative of important departures from Mendelian ratios) between paired markers of the same or alternative classes support the preceding inference of relatively more instances of TRD for supergene loci—the median difference for paired loci of the alternative classes (0.123) was significantly higher than that for paired loci of the same supergene status (0.042) . Evidence that the supergene loci also displayed elevated k values overall compared to the other loci comes from our finding that the mean bootstrap k for the former class exceeds that for the latter by 0.015, with the lower one-tailed confidence limit (0.008) greater than zero, the value expected under the null hypothesis that supergene k values generally do not exceed k values for the other markers. Indeed, only three of the 5000 bootstrap replicate mean differences (0.06%) were less than or equal to zero.Comparison of the supergene and non-supergene loci as distinct classes confirmed that the former displayed both higher proportions of meaningful departures from 1:1 segregation ratios and overall elevated k) at the three supergene loci with those expected by chance, given our specific sample sizes, are shown in Fig. k values compared to those expected. Specifically, values of k for ten progenies segregating at C294 fell within the lower and upper 2.5% tails for the simulated progenies and thus are judged to deviate significantly from Mendelian ratios , thirteen progenies segregating at Gp-9 fell within the tails (5.05 expected), and 20 progenies segregating at i_126 fell within the tails (4.25 expected). The results of this simulation thus suggest that significant deviations from Mendelian ratios occur at each supergene marker in three to five times more progenies than expected in the absence of TRD. For the non-supergene locus i_129, three segregating progenies fell within the 5% tail, which does not differ significantly from the fewer than two expected by chance .Results of a simulation analysis for comparing the distributions of observed segregation proportions (Sb supergene (and with a hypothesized drive locus) , eleven progenies displayed excesses and six deficiencies for the Gp-9b supergene allele (2.5 of each expected by chance), and nine progenies displayed excesses and eight deficiencies for the i_126 supergene alleles (2.2 of each expected by chance). Thus, drive reversal at the homologous region of the SB chromosome appears to be as potent as the predicted drive associated with the Sb supergene.An unexpected feature of the Mendelian deviations for the supergene loci evident from the above is that not all departures feature a surplus of the allele associated with the us) Fig. . Indeed,Sb supergene drive system.Considering all three supergene loci simultaneously, 24 of the 101 progenies (23.8%) exhibited significant distortion at one or more loci . The proportions of progenies expected to exhibit such departures in the absence of drive, obtained via multilocus simulations explicitly incorporating our sample sizes, are shown along with the observed proportion in Fig. C294, Gp-9, and i_126, respectively . Thus, the effects of Sb drive and reversal-of-drive on supergene frequency appear to be relatively balanced in our study population.A resampling procedure undertaken to estimate the overall (population) frequencies of supergene alleles within segregating progenies showed that alleles associated with the supergene did not gain a consistent transmission advantage over their alternate alleles. Specifically, these frequencies were very close to 0.5 at all three supergene loci: 0.497, 0.501, and 0.502 for k values for individual alleles at each of the twelve non-supergene loci revealed no evidence of consistent, directional segregation distortion at any of these loci, with the middle quartiles of k values for all adequately sampled alleles overlapping 0.5 . Indeed, median k values for Sb-linked alleles for all nestmate queens (not just those with significant TRD) did not differ significantly according to queen colony of origin . Thus, there is no evident colony-level effect on the tendency of queens to produce eggs deviating from Mendelian ratios of Sb.Queens producing progenies that exhibited significant drive  at the three supergene markers originated from ten of the twelve polygyne source colonies. The frequency of such queens varied from 11.1% (1/9) to 50.0% (2/4) per colony in the ten with any, and variation in this frequency across all twelve colonies was not significant (χp = 0.352). Further, queens producing non-Mendelian ratios were no more closely related to their mates than were other queens . With respect to the supergene status of their mates, monandrous queens producing non-Mendelian supergene ratios mated with Sb or SB males at frequencies similar to queens producing Mendelian ratios , a result not affected by polarity of k in the progenies , not significantly different from the proportion of such queens producing Mendelian ratios  for pairs of nestmate queens and congruence in their progeny’s degree of deviation from 1:1 supergene segregation ratios (∆k) were examined to learn if relatedness is predictive of similarities in such deviations. No association was detected  required to explain observed k values in our TRD study differs strikingly from the pattern actually measured in the supplementary test units  was recovered intact from the 40 test units after 48 h, with only one unit maintaining fewer than 88% and most units (23) maintaining all of the embryos provided to them . Moreover, comparison of non-embryonation proportions for the 24 progenies implicated as having supergene-associated TRD with the proportions for the remaining 77 progenies revealed no significant difference between the groups . Given that female gamete mortality may be expected to lead to a positive association between level of distortion and frequency of non-embryonation, these analyses offer no support to the hypothesis that this mechanism underlies significant supergene TRD in polygyne S. invicta.Finally, association of the level of segregation distortion at the supergene loci with the extent of egg embryonation failure in a progeny was examined to investigate if female gamete dysfunction may underlie such distortion e.g., , 89, 90), 9089, 9 . Based Social b (Sb) supergene on chromosome 16  in the fire ant S. invicta plays a major role in determining the form of social organization, most importantly, whether colonies contain a single or multiple reproductive queens. Remarkably, other features of the supergene suggest that it is a selfish genetic element and raise the possibility that it may act as a transmission ratio distorter [Sb supergene contains multiple inversions and is pericentric, so that recombination consequently is greatly reduced; the centromere of chromosome 16 is exceptionally large, possibly reflecting a role in chromosomal competition for inclusion in the egg during meiosis; the supergene contains deleterious mutations and abundant transposable elements; and the supergene mediates two known selfish features—“green-beard” behavior and a bias in the development of diploid larvae toward the queen rather than worker caste [Sb supergene transmission at the expense of its homolog, thus aiding its persistence in populations despite its negative fitness effects on individual fire ant sexuals.The istorter , 57: theer caste , 65, 67.k values) averaged higher for alleles at all three supergene loci than for alleles at any of the non-supergene loci. These results demonstrate the existence of supergene-associated TRD at modest yet significant levels, and further reveal substantial inter-progeny variation in the segregation ratios. This latter finding is important because such variation rarely has been studied in wild populations [Our survey of segregation ratios in embryo progenies of 101 polygyne queens using three supergene-linked markers and twelve additional markers located across the genome revealed that significant departures from Mendelian ratios occurred at the supergene in 3–5 times more progenies than expected in the absence of TRD. By comparison, the non-supergene loci displayed lower proportions of progenies with significant departures, with the mean proportion close to that expected in the absence of TRD with Type I error. Additionally, the magnitude of departures from 1:1 ratios Sb supergene , but often instead favored the alternate, non-supergene-linked allele(s) on the homologous SB chromosome. Such drive reversal [SB/Sb) queens evidently affects the expression of supergene-associated drive. As might be expected given the relatively common occurrence of drive reversal in our system, supergene haplotypes evidently gain no consistent transmission advantage over their alternate haplotypes at the population level. From this we conclude that Sb-linked drive and SB-linked reversal-of-drive appear to be at a balanced equilibrium in invasive polygyne S. invicta , 30Sb suGp-9 and microsatellite marker C294 are located near one another within the largest of three inversions that collectively have been assumed to demarcate the supergene boundaries. Microsatellite i_126 is located outside any known inversions on the opposite arm of the chromosome near the centromere, more than a third of the length of the chromosome from the two other markers. The patterns of recombination and gametic disequilibrium we observed, with the adjacent marker pair displaying very low recombination rates and high disequilibrium and the other pairs displaying slightly higher recombination and lower disequilibrium, are fully congruent with this structural information. The presence of several adjacent or overlapping inversions, the proximity of one of them to the centromere , an reduced , 60 likeomosome  are heterozygotes for the 92 allele of C294, and an excess number are heterozygotes for the 230 allele of i_126 (60.2% observed vs. 42.1% expected under Hardy-Weinberg equilibrium).The ination  in order to minimize the possibility that differential mortality during embryogenesis, rather than events surrounding oogenesis, were responsible for any distortion. Nonetheless, there existed a short window during which endogenous or worker-mediated embryo mortality, or other factors, could have altered transmission ratios. One explanation for such TRD, differential fertilization success/zygote viability due to egg-sperm compatibility issues associated with an evolutionary arms race involving male gametes’ access to eggs , in direct contradiction to predictions of this hypothesis .Clarification of the specific mechanism of supergene-associated TRD in osition 4-60 h in s . Specifically, the equal numbers of significant deviations favoring or disfavoring supergene-bearing eggs in progenies fathered by SB males (eleven of each), as well as the similarity in distributions of k for progenies fathered by either SB or Sb males, speaks against any role for diminished supergene homozygote (or heterozygote) embryo viability in explaining the TRD we observed  in about one-quarter of these test progenies were selective worker cannnibalism the primary cause of the TRD observed in our main experiment.Intervention by the workers tending embryos in our isolation units, via cannibalism or neglect, could conceivably have played a role in TRD, behavior that might be viewed as a form of competition between embryos with and without the supergene for maternal  care and resources . Such TS. invicta occurring before fertilization and thus constituting segregation distortion, and the possibility exists that it may in fact be “true” meiotic drive. True meiotic drive takes advantage of the arena for competition provided by intrinsic asymmetries in meiotic cell fate during oogenesis [S. invicta (averaging 34% of chromosome length) suggest a history of competition between homologs for preferential access to the meiotic spindle assembly and for segregation into an oocyte . Indeed, because of their roles as sites of attachment to the microtubule lattice, variant centromeres and heterochromatic neocentromeric elements are viewed as definitive genomic substrates for mediating non-Mendelian meiotic segregation [S. invicta) [Several considerations are consistent with TRD in polygyne ogenesis ; thus, iogenesis . In contogenesis . Aside f drive”; , 109), aregation , 111. Noregation , 113. Alinvicta)  suggestsS. invicta queens. This is because parallel transmission biases were detected at pairs of supergene markers separated by as much as 10 Mb, whereas biased gene conversion is expected to act locally [Finally, we note that biased gene conversion during meiosis is an unlikely explanation for the observed TRD in polygyne A hallmark of true meiotic drive and other TRD systems is the presence of intragenomic conflict related to the transmission advantage of drive elements coupled with the negative organismal fitness effects associated with possession of these elements. This conflict reflects the fact that drive homologs experience a direct transmission disadvantage, whereas unlinked genes elsewhere in the genome suffer adverse consequences by being transmitted to suboptimally adapted individuals while not benefitting from a transmission advantage , 24. As S. invicta queens may be linked to the presence of variants segregating at such modifier loci. One relatively simple scenario for the genetic mechanism posits multiple unlinked suppressor loci that segregate allelic variants with additive effects to counter Sb drive  into the SB haplotype background, which could preserve allelic congruence across our markers, also are unlikely to explain drive reversal, based on the general rarity of this form of recombination between large inverted and non-inverted segments [Sb drive complex in a haplotype (SB) lacking inversions  and than found at non-supergene loci. Unexpectedly, significant embryo TRD involved not only excesses of alleles associated with the Sb supergene (and with a hypothesized drive element contained within it), but also recurrent excesses of the alternate alleles on the homologous SB chromosome. The common occurrence of such drive reversal in this system is consistent with the evolution of a complex genomic architecture featuring multiple suppressors of supergene drive, such that the Sb haplotype evidently gains no consistent transmission advantage over its homolog at the population level. Evidence points to “true” meiotic drive as the most likely mechanism for the TRD patterns we observed. These findings are important because they reveal the presence and distribution of TRD, a factor mediating diverse evolutionary phenomena, in a wild population in which it was predicted, and they suggest its proximate causes.The SB ancestry in the native range would confirm historical recombination and demonstrate that selection does not preserve a single uniform supergene haplotype for regulating a derived form of fire ant social organization.We further observed low levels of recombination and incomplete gametic disequilibrium across the supergene, even between adjacent markers within the same inversion. Discovery of recombinant supergenes bearing traces of Additional file 1:Table S1. Polygyne Solenopsis invicta nest information, mother queen characteristics, and numbers of embryos genotyped in progenies. (PDF 200 kb)Additional file 2:Text S1. Methods―additional information. (PDF 155 kb)Additional file 3:Figure S1. Protocol for collecting progeny embryos for Sb supergene transmission ratio distortion (TRD) study in Solenopsis invicta. Families (progenies) of diploid embryos were obtained from individual mother queens initially isolated in plaster-bottomed petri dishes for three to four weeks with several thousand adult workers (colony fragments) (i). Queens confirmed to be mated (producing worker brood) at the end of this period were then isolated with 2–3 workers in plastic plaster-bottomed specimen cups (isolation cups) (ii). Each such queen was removed from the cup after 12 h then frozen in a − 80 °C freezer (iii). Eggs laid by the queen were maintained in the cup with the workers for an additional 48 h (by which time they were embryos within the egg coat), then transferred into a gelatin capsule and placed immediately in a − 80 °C freezer pending DNA extraction (iv). Thirty-six haphazardly selected embryos per progeny were sampled for genotyping at Gp-9 and 14 microsatellite loci (v). (TIF 1353 kb)Additional file 4:Table S2. Characteristics of marker loci genotyped in polygyne queens and their progeny embryos . (PDF 10 kb)Additional file 5:Figure S2. The social chromosome  of polygyne Solenopsis invicta. Depiction is based on reference genome build Si_gnH_C3 of a haploid SB male from the USA [Sb chromosome (two of which overlap) are shown by blue bars, and the positions of three supergene-linked marker loci  and the centromere are indicated . The region of reduced recombination on Sb chromosome 16, estimated by mapping to the new reference build 2796 RADseq SNPs from 92 haploid sons of a heterozygous SB/Sb queen [ the USA . LocatioSb queen , is indiAdditional file 6:Text S2. Results―additional information. (PDF 144 kb)Additional file 7:Table S3. Proportions of haplotypes with a supergene-associated allele that also bear such an allele at another supergene locus. (PDF 97.8 kb)Additional file 8:Figure S3. Associations between recombination frequency (c) and gametic disequilibrium (D*) in progeny embryos (eggs) and males. The five locus pairs with values of c significantly less than 0.5 are indicated by the larger circles identified in the legend (the top three listed pairs are supergene loci). Locus pairs involving red_ant are excluded because of small sample sizes. (TIF 363 kb)Additional file 9:Figure S4. Associations of binomial probabilities of Mendelian segregation ratios between supergene loci. Colored lines represent the least squares regression lines fitted to the three sets of values. Samples sizes, Spearman ρ and associated probability values (after Bonferroni correction for multiple tests), and R2 (coefficient of determination) values are shown in the inset. (TIF 669 kb)Additional file 10:Figure S5. Distributions of segregation proportions (k) in embryo progenies for 56 alleles of twelve non-supergene-linked microsatellite loci. Boxes represent interquartile ranges, whereas whiskers indicate the range limits. Within boxes, black horizontal bars represent the medians and red dots the means. Five alleles at four of the loci that segregated in only a single progeny are not included. The three alleles with boxes not overlapping 0.5 occurred in very few segregating progenies (numbers shown in blue). As expected for alleles that segregate in Mendelian ratios, estimates of the mean deviations from 1:1 ratios (│0.5 - k│) tend to decrease with larger numbers of segregating progenies studied . (TIF 1481 kb)Additional file 11:Figure S6. Segregation proportions (k) in polygyne queen embryo progenies fathered by males lacking or bearing the Sb supergene. Values that differ significantly from 1:1 segregation ratios at one or more of the three supergene loci  are indicated by the yellow dots. The distributions of progeny k values, which represent weighted means across the supergene loci for each progeny, do not differ significantly with respect to the supergene-related haplotype of the fathers . (TIF 157 kb)Additional file 12:Figure S7. Genetic relatedness between pairs of nestmate queens (r) in relation to their patterns of supergene TRD. Queen pairs are classified into three types: pairs in which one or both queens did not display significant TRD, pairs in which both queens displayed TRD of the same polarity , and pairs in which the queens displayed TRD of opposite polarity  . (A) Distributions of r values for queens of the different types; sizes of the symbols indicate the relative numbers of identical r values . Similar results showing that queens with significant TRD do not display unusually high or low relatedness to nestmate queens with TRD were observed as well for the seven individual source colonies from which these queens originated. (B) Box and whisker plots of the data. The boxes represent the interquartile ranges while the whiskers represent the 90th percentiles. Medians for each type are zero; means are indicated by the red dotted lines. Pairs of queens in which both members displayed TRD were neither significantly more nor less closely related to one another than were pairs of nestmate queens not displaying TRD (see main text). (TIF 631 kb)Additional file 13:Text S3. Genetic mechanism of drive reversal―additional information. (PDF 135 kb)Additional file 14:Figure S8. Numbers of eggs/embryos successfully maintained by 2–3 polygyne workers over a 48 h period in supplementary tests. Dark red portions of the bars indicate the differences between initial and final numbers of intact eggs/embryos present in each test unit, corresponding to MTRD values if the losses are due to worker cannibalism . The smaller plot contains boxplots comparing distributions of presumptive MTRD values for our 101 TRD study progenies and 40 supplementary test units. MTRD for the former was estimated from the unpolarized k values ; these are the Mean Absolute Deviation from the Mean (red), Mean Absolute Deviation from the Median (gold), and Median Absolute Deviation from the Median (green). The boxplots and dispersion statistics show that MTRD values in our supplementary test units are too low and unvarying to support the hypothesis that worker intervention during embryogenesis rather than segregation distortion (meiotic drive) primarily caused significant supergene TRD in our main experiment. (TIF 1564 kb)Additional file 15:Figure S9. Hypothetical scenario for additive genetic mechanism of Sb supergene drive reversal in polygyne Solenopsis invicta. The presence of seven suppressor loci segregating wild-type alleles (ineffective in suppressing Sb drive) along with suppressor alleles is depicted as an example of such a multilocus system, with several different multilocus haplotypes arising from recombination and segregation in the population illustrated. The inset depicts the distribution of multilocus haplotypes with varying proportions of suppressor alleles expected in an equilibrium population of 5000 haplotypes with the seven suppressor loci in gametic equilibrium, equal allele frequencies, and no selection. Haplotypes with no or very few suppressor alleles allow Sb drive to prevail, those with high or maximal numbers of suppressor alleles overwhelm Sb drive to cause drive reversal, and those with intermediate proportions of suppressor alleles (the most common circumstance―inset) neutralize Sb drive to favor Mendelian segregation ratios. The suppressor loci are shown in the region of SB chromosome 16 homologous to the supergene  Double crossover between the wild-type Sb and SB social chromosomes in the segment between markers i_126 and Gp-9 transferred the complete drive complex (locus) from Sb to SB without altering the congruence of alleles at all three supergene markers observed in progenies with both drive and reversal-of-drive. Two novel recombination products were generated, a SB chromosome containing the drive locus and a Sb chromosome without it, and these presumably must be paired in a queen to yield drive reversal in her progeny (pairing of wild-type SB with wild-type Sb yields supergene (Sb) drive; pairing leading to any of six different drive-locus+ or drive-locus− homozygotes is expected to yield Mendelian proportions, although at least some of these homozygotes are expected to be lethal genotypes in queens; and pairing leading to either of two drive-locus+/drive locus– heterozygotes on otherwise SB or Sb haplotypes in homozygous condition is expected to yield drive that is undetectable). (B) Sequence differentiation between SB (N = 60) and Sb (N = 20) social chromosomes from native (South American) S. invicta, as measured by differences in the pooled SB nucleotide diversity (π) and the median values of absolute sequence divergence (dxy) between individual chromosomes of each type along 5 kb non-overlapping windows. Values significantly greater than zero , consistent with recombination of Sb segments into SB haplotypes, are rare and involve small segments. Asterisks indicate three such instances of likely recombination of ≈5-10 kb-size elements. Positions of the three inversions contained within the supergene are shown by the blue bars. (C) Linkage disequilibrium  in native S. invicta along the SB social chromosome (N = 60). Outside of the centromere, LD is weak, as expected given the lack of sizeable inversions and predicted resultant free recombination. Data for Panels B and C were obtained from whole-genome sequence assemblies . Centro


The stellate ganglia are the predominant source of sympathetic innervation to the heart. Remodeling of sympathetic nerves projecting to the heart has been observed in several cardiovascular diseases, and sympathetic dysfunction contributes to cardiac pathology. Wistar Kyoto rats are a common model for the study of cardiovascular diseases, but we lack a profile of the baseline transcriptomic and neurochemical characteristics of their cardiac sympathetic neurons. Most studies of cardiovascular disease have used male animals only, but in the future both male and female animals will be used for these types of studies; therefore, we sought to characterize the transcriptome of male and female stellate ganglia and to correlate that with catecholamine and acetylcholine content in the heart. We have generated a dataset of baseline RNA expression in male and female Wistar Kyoto rat stellate ganglia using RNA-seq, and have measured neurotransmitter levels in heart and stellate ganglia using HPLC and mass spectrometry. We identified numerous gene expression differences between male and female stellates, including genes encoding important developmental factors, receptors and neuropeptides. Female hearts had significantly higher neurotransmitter content than male hearts; however, no significant differences were detected in expression of the genes encoding neurotransmitter synthetic enzymes. Similarly, no statistically significant differences were identified between the sexes in cardiac tyrosine hydroxylase levels. As researchers increasingly recognize the importance of studying both male and female animals in disease models, it becomes increasingly clear that a sound understanding of the baseline physiology needs to be established in both sexes, in line with ongoing efforts of the NIH . StellatRats, like mice, are a prominent model organism used in studies of cardiovascular diseases. Most studies, however, use only male rats to investigate the mechanisms that underlie pathology. This is true for studies of a broad range of diseases including heart failure, arrhythmias, and hypertension that utilize a variety of different rat strains –7. RecenOur study aims to provide baseline transcriptome and neurochemistry data on male and female adult Wistar Kyoto (WKY) rats, which are a common strain used in cardiovascular studies. RNA-seq analysis of stellate ganglia identified numerous sex-based differences in gene expression and several of these were confirmed by real-time PCR. Sex differences were also identified in cardiac neurotransmitter levels, with higher levels of both norepinephrine (NE) and acetylcholine (ACh) identified in female atria and elevated NE found throughout the female ventricle. Comparison with our previous mouse data suggests limited overlap between the two species in genes that exhibit sex-based differences in expression.ad libitum access to food and water. Male rats were consistently heavier than female rats . Rats were sedated with isoflurane, and the heart and sympathetic ganglia were rapidly dissected. Left and right atria and ventricles were separated, as were the left and right stellate and superior cervical ganglia (SCG). The left ventricles were further separated into apex, mid, and base sections. Tissues collected for RNA were placed in RNAlater (Qiagen) and stored at -20°C. Tissues from a second set of animals collected for neurochemical analyses were snap frozen and stored at -80°C.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the National Academies Press (Ed 8). All procedures were approved by the Oregon Health & Science University Institutional Animal Care and Use Committee (protocol number IP00001366). Male and female Wistar Kyoto (WKY) rats (age matched 16–18 weeks old) were obtained from Jackson Laboratories, and maintained on a 12 h: 12 h light/dark cycle with RNA was extracted from ganglia using the RNAqueous Micro kit (Ambion) according to the manufacturer’s instructions as previously described. The DNAhttp://multiqc.info/). The files were imported into ONPRC’s DISCVR-Seq[The quality of the raw sequencing files were evaluated using FastQC combinedISCVR-Seq, LabKey[ISCVR-Seq Server-bISCVR-Seq was usedISCVR-Seq was usedISCVR-Seq. Two-pasISCVR-Seq was usedISCVR-Seq was utilGene-level raw counts were filtered to remove genes with extremely low counts in many samples following the published guidelines. SpecifiGene-level differential expression analysis was performed in open source software R. Gene-leGapdh endogenous control, Rn99999916_s1; Th, Rn00562500_m1; Ret, Rn01463098_m1; Hpca, Rn04222958_g1; Penk, Rn00567566_m1; Nr4a2, Rn00570936_m1; Htr2b, Rn00691836_m1; Htr3b, Rn00573408_m1; Egr1, Rn00561138_m1.Select expression differences were confirmed using additional RNA from same right stellate ganglia that were sequenced. Reverse transcription was performed using the iScript reverse transcription supermix (BioRad) according to the manufacturer’s instructions. The following TaqMan Gene Expression Assays (Thermo-Fisher) were used for real-time PCR analysis: Frozen tissue was used for neurotransmitter analysis as described previously. All tisGraphing and statistical analyses were performed using Microsoft Excel and GraphPad Prism v6. Binary comparisons between males and females for individual genes (qPCR) were performed using Student’s t-tests. Comparison of two data sets with unequal variances used an unpaired t-test with Welch’s correction. Neurotransmitter levels across different parts of the same hearts were compared using two-way ANOVA with repeated measures, with p values adjusted for multiple comparisons.Fig 1). In addition, a higher concentration of NE was observed across all regions of the heart in females compared to males. In right atria, a higher concentration of ACh was also observed in females compared to males, proportional to the increase in NE. Surprisingly, the NE metabolite DHPG was not detectable in any of the rat heart tissue samples, although the DHPG standards were readily detected.To characterize baseline neurochemical characteristics of adult WKY rat hearts, norepinephrine (NE) and acetylcholine (ACh) were measured in atria, the right ventricle, and the base, mid-wall and apex of the left ventricle. A base to apex gradient in NE concentration is a well-established feature of cardiac physiology  and our Table 1). The dopamine metabolite DOPAC was significantly higher in left female stellate ganglia compared to males, and female left stellates also had a significantly lower NE to dopamine ratio than male left stellates. Male and female right stellate ganglia exhibited similar levels of all detected catecholamines and their metabolites, in contrast to the sex differences identified in cardiac catecholamine content.Catecholamines and their metabolites were also quantified in left and right stellate ganglia. Both NE and dopamine were detected in rat stellates, with similar levels in male and female ganglia . Genes associated with sympathetic neurotransmission were among the most highly expressed genes, consistent with our previous studies in mice[S1 Table). No significant differences were detected in the expression of catecholaminergic genes such as Th (tyrosine hydroxylase), Dbh (dopamine beta hydroxylase), Slc18a2 (vesicular monoamine transporter 2), or Slc6a2 (norepinephrine transporter), or other genes associated with neurotransmission (S2 Table). Differential expression analysis identified 22 genes more highly expressed in males, and 20 genes more highly expressed in females with FDR-adjusted p < 0.05, but the scale of fold difference between the sexes was small for some of the genes, particularly the ones on autosomes (Table 2). Differentially expressed genes were dispersed across many gene families, with no significant pathway enrichment observed after Panther v13[To identify transcriptomic differences between male and female stellate ganglia, and determine if those contributed to the differences in cardiac neurotransmitter content, RNA-seq was performed using RNA from adult right stellate ganglia. Separation of the sexes was observed during multidimensional scaling (MDS) analysis to determine how the expression profiles of each sample compared to all others Fig . Genes as in mice, 9. Siminther v13 and DAVInther v13..We previously carried out RNA-seq comparing male and female mouse stellate ganglia, and theTable 2) were selected for confirmation by qPCR, including Penk , Nr4a2 , Hpca , Egr1 , and Htr2b and Htr3b (Serotonin receptors). Th mRNA was assayed by qPCR as a gene that was not different between the sexes in mouse or rat, and Ret mRNA was quantified as a gene that exhibited differential expression in mouse stellates but similar expression in male and female rat stellates. PCR confirmed significantly higher expression of Penk, Hpca and Htr3b in the stellate ganglia of male rats, and significantly higher expression of Egr1 and Htr2b in the stellate ganglia of female rats . Th and Ret mRNA were similar in both sexes confirming RNA-seq results.The non-X/Y genes that were different between the sexes in WKY rats included genes encoding developmental growth factors and serotonin receptors. A subset of genes that were in the top 50 most differentially expressed , from the same rats . The expression pattern of decreased Penk and Htr3b in female rats compared to males was conserved in stellate and the SCG, but there was no significant difference between the sexes in the expression of Hpca, Egr1, or Htr2b in the SCG.To determine if the sex differences in gene expression observed in .Given the increased concentration of NE content in female hearts, and the lack of any differences in noradrenergic gene expression, we quantified TH protein in the left ventricle by western blot, assessing TH in the base and the apex  Figs. THIn the present study we generated a reference transcriptome of stellate ganglia in male and female adult WKY rats with associated tissue neurochemistry. Statistically significant differences were detected between the sexes both in terms of gene expression and cardiac neurotransmitter levels. The most abundant genes within all ganglia related to neurotransmission, and these genes were present at identical levels in male and female ganglia. Genes that were differentially expressed between the sexes in rat stellate ganglia were distinct from those that were differentially expressed in male and female mouse stellates. Most of the genes that were differentially expressed in stellates were expressed at similar levels in male and female SCG, suggesting sex differences that were specific to the stellate. Neurotransmitter levels were similar in male and female stellate ganglia, but NE levels were significantly higher across all regions of female hearts compared to male hearts. No physiological parameters were quantified in this study, which focused on transcriptomics and neurochemistry.There is a gradient of NE content and sympathetic nerve density from the base to the apex of the heart that is conserved across species , 31–33. in vitro or in vivo enhances expression of TH and other synthetic enzymes, which can lead to higher catecholamine content and blood pressure in male animals [). Although expression of the VMAT1 gene trended toward an increase in female stellate, it’s unlikely that this contributed to the difference in cardiac NE since its expression was near the limit of detection (3.5 CPM) whereas VMAT2, the major neuronal vesicular monoamine transporter, was expressed at similarly high levels (1600 CPM) in male and female ganglia.It is unclear what factors contributed to the higher NE content in female hearts, especially since NE content was identical in male and female stellate ganglia, which contain most of the sympathetic neurons projecting to the heart. The mal animals , 43. In Since NE content differed in the heart but not ganglia, we quantified TH protein in the left ventricle to determine if local changes in TH content could account for the differences in NE levels. TH levels were variable between animals of both sexes and no significant differences were observed between the sexes. We attempted to quantify TH activity using a colorimetric assay developed by Vermeer and colleagues , but it Penk, which encodes the precursor protein giving rise to enkaphalins, was significantly lower in both the stellate and SCG in female rats. The enkephalins are expressed by a subset of sympathetic neurons [Egr1 mRNA was higher in stellate ganglia of female rats. Egr1 is a transcription factor that stimulates expression of the NE biosynthetic enzymes TH and DBH[Egr1 expression. Although we did not measure estrogen levels, it is unlikely that hormonal signaling can account for the stellate-specific differences in Egr1, Hpca, and Htr2b mRNA, which are expressed at similar levels in male and female SCG. Target-induced signaling is more likely to explain selective differential expression, and expression of the serotonergic receptors Htr2b and Htr3b may be related to sex differences in innervation associated with nursing[The genes that exhibited the greatest differential expression between sexes were those associated with the sex chromosomes, as expected. Other genes that were significantly different between the sexes were diverse, yet potentially important. The proenkephalin gene  neurons , 49 and  neurons . In contH and DBH, 52. EgrH and DBH, 54, raih nursing. It is u, providing confirmation of the core stellate ganglion transcriptome. Although we identified sex-specific differences in the expression of many genes, those associated with noradrenergic transmission were expressed at similar levels in male and female ganglia. The identification of differences in tissue neurotransmitter content highlights the need to include both sexes in experiments, and to carefully characterize target tissues as well as the ganglia that innervated them.Our male-female comparison data in WKY rat ganglia complement other investigations of stellate transcriptomes. A recent study by Bardsley and colleagues compared the stellate ganglion transcriptome of male Wistar and SHR rats , 9. ThatS1 FigActb, Tubb3 and Hprt1) for reference. Genes specific to sympathetic neurons are among the most highly expressed in the stellate.Right stellate ganglion expression levels of genes in both male and female rats using normalized Log10CPM values (Counts Per Million); Mean ± SEM (n = 12). Select genes important in sympathetic function are presented in addition to 3 commonly used housekeeping genes ((PDF)Click here for additional data file.S2 FigAverage expression across all samples ±SEM (n = 12) expressed as Log10 Counts per Million (CPM).(PDF)Click here for additional data file.S3 FigAverage expression across all samples ±SEM (n = 12) expressed as Log10 Counts per Million (CPM).(PDF)Click here for additional data file.S4 FigAverage expression across all samples ±SEM (n = 12) expressed as Log10 Counts per Million (CPM).(PDF)Click here for additional data file.S5 FigAverage expression across all samples ±SEM (n = 12) expressed as Log10 Counts per Million (CPM).(PDF)Click here for additional data file.S6 FigAverage expression across all samples ±SEM (n = 12) expressed as Log10 Counts per Million (CPM).(PDF)Click here for additional data file.S7 FigAverage expression across all samples ±SEM (n = 12) expressed as Log10 Counts per Million (CPM).(PDF)Click here for additional data file.S8 FigAverage expression across all samples ±SEM (n = 12) expressed as Log10 Counts per Million (CPM).(PDF)Click here for additional data file.S9 FigEqual amounts of protein from 6M and 6F hearts were separated on 4–12% gels and blotted for TH and then GAPDH.(PDF)Click here for additional data file.S10 FigEqual amounts of protein from 6M and 6F hearts were separated on 4–12% gels and blotted for TH and then GAPDH.(PDF)Click here for additional data file.S1 TableThe 100 most highly expressed genes in both sexes listed side by side for comparison.(XLSX)Click here for additional data file.S2 Tablelog2FC is the fold change of gene expression in log2 scale (fold difference) male vs. female rats. For example, log2FC of 1 corresponds to fold difference of 2 (up-regulated) vs. female and log2FC of -1 corresponds to fold difference of 0.5 (down-regulated) vs. female. P-value is the empirical Bayes moderated p-value for that particular contrast. FDR is the false discovery rate adjusted p-value.Contains gene identities, raw count expression data, normalized CPM expression data, and statistics for comparing male and female rats. (XLSX)Click here for additional data file.S3 TableThe data from Sheet 1 that were compiled to generate (XLSX)Click here for additional data file.


Betaine is a key metabolite of the methionine cycle and known for attenuating alcoholic steatosis in the liver. Recent studies have focused on the protection effect of betaine in mitochondrial regulation through the enhanced oxidative phosphorylation system. However, the mechanisms of its beneficial effects have not been clearly identified yet. Mitochondrial dynamics is important for the maintenance of functional mitochondria and cell homeostasis. A defective mitochondrial dynamics and oxidative phosphorylation system have been closely linked to several pathologies, raising the possibility that novel drugs targeting mitochondrial dynamics may present a therapeutic potential to restore the cellular homeostasis. In this study, we investigated betaine’s effect on mitochondrial morphology and physiology and demonstrated that betaine enhances mitochondrial function by increasing mitochondrial fusion and improves cell survival. Furthermore, it rescued the unbalance of the mitochondrial dynamics from mitochondrial oxidative phosphorylation dysfunction induced by oligomycin and rotenone. The elongation properties by betaine were accompanied by lowering DRP1 and increasing MFN2 expression. These data suggest that betaine could play an important role in remodeling mitochondrial dynamics to enhance mitochondrial function and cell viability.  Mitochondria are essential organelles for providing the majority of cellular energy ATP through oxidative phosphorylation (OXPHOS), maintaining homeostasis, oxygen metabolism, cell survival and apoptosis  which facilitates fission to activate apoptosis and mitofusins (MFNs) which mediates fusion to have an anti-apoptotic effect  is an essential nutrient that has been studied for cytoprotective properties such as an osmolyte in cell volume maintenance and correction of a defective cellular methylation against environmental stress  and other chemicals were purchased from Sigma-Aldrich unless otherwise specified.TM transfection reagent. After 24 hours of transfection, cells were pretreated with or without mitochondrial inhibitors for 2 hours and then co-incubated with betaine for additional 24 hours.Human HCC (Huh7) cells were transfected with the dsRed2-MLS vectors using the TurbofectDanio Rerio) husbandry and experimental protocols complied with institutional guidelines were approved by local ethics boards  (Westerfield mito:EGFP) zebrafish  zebrafish embryos were anesthetized in 0.02% tricaine, and mounted with 3% methylcellulose. Z-series of images  were collected with an LSM 700 confocal laser microscope (Carl Zeiss) and presented as a stacked image. The mitochondrial length was analyzed as average lengths from 10 mitochondria of individual cells using ImageJ .For mitochondria imaging of Huh7 cells, dsRed2-MLS transfected Huh7 cells were seeded onto 0.1% poly-D-lysine-coated coverslips and cultured for 24 hours. For the live imaging of mitochondria in zebrafish embryos, embryos at 1 dpf (day post fertilization) were pretreated with 2 μM oligomycin for 15 minutes and then co-incubated with betaine for 6 hours at 28.5°C. Tg for 30 min. Images were taken using an LSM710 confocal microscope. The signals were examined using identical settings to obtain the same sensitivity.Cell viability was assessed with MTT. Absorbance was measured at 550 nm with a SpectraMax® M5 microplate reader.Whole-cell lysates (30 μg) were loaded on 10–15% SDS-PAGE and incubated with primary antibodies against β-actin, DRP1, GFP and MFN2 (Santa Cruz), and then incubated with the HRP-conjugated secondary antibody (Thermo Scientific). Signals were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) using LI-COR (LI-COR).n = 3). The statistical significance of the experimental differences was determined with two-way analysis of variance. P values less than .05 were considered statistically significant, and significance is indicated on the graphs with asterisks.The data are presented as the mean and standard deviation of the results from three independent experiments (3)3N+CH2COO− and known as a methylamine  dysfunction. We used mitochondrial inhibitors for disruption of mitochondrial respiratory chain function through inhibition of electron transport at Complex I (rotenone) or blockage of ATP synthesis through inhibition of ATP synthase (oligomycin)  zebrafish was used to observe the change of mitochondrial dynamics for studying mitochondrial physiology and pathogenesis in vivo  zebrafish for representing the mitochondrial dynamics. 1 dpf zebrafish embryos were treated with 0, 5, 25 mM betaine for 6 hours. Live cell imaging of the embryos revealed the normal mitochondria morphology and the average length of mitochondria was ∼ 1 μm (While we showed that betaine has the ability to extend mitochondrial networks s ∼ 1 μm . Betainein vivo model for testing pharmaceutical mitochondria-targeted drugs (Pinho et al. The zebrafish model system has been characterized to the effect of mitochondrial inhibitors identifying morphological and functional phenotype on zebrafish development and provides a potential Mitochondrial dynamics reveal the overall health condition of mitochondria. Healthy mitochondria constantly fuse and divide to keep a dynamic network under physiological conditions (Nunnari and Suomalainen The dysregulation of mitochondrial fusion and fission is associated with mitochondrial dysfunction and found in several human diseases, such as neurodegenerative diseases like CMT3A, ataxia and Huntington’s disease (Reddy et al. Our results demonstrated for the first time that betaine itself can promote the mitochondrial fusion and restore fragmented into elongated and tubular mitochondria over mitochondrial OXPHOS dysfunction induced by oligomycin and rotenone. This enhancing fusion activity of betaine helped to improve mitochondrial function and increase the chance of the survival . IntriguIt was suggested that betaine enhances AKT pathway and decreases AMPK activation (Apicella et al. In conclusion, our results indicate that betaine can regulate mitochondrial dynamics through fission and fusion events and betaine-mediated mitochondrial changes positively reverse from mitochondrial OXPHOS dysfunction to normal physiological status. Therefore, discovery for new compounds like betaine having enhancing ability of mitochondrial fusion, DRP1 inhibitors, or Mfn2 agonists are of great interest for the therapeutic potential for the treatment of mitochondrial dysfunction-related diseases.


Immunopathologic examination of the lungs of mouse models of experimental influenza virus infection provides new insights into the immune response in this disease. First, there is rapidly developing perivascular and peribronchial infiltration of the lung with T-cells. This is followed by invasion of T-cells into the bronchiolar epithelium, and separation of epithelial cells from each other and from the basement membrane leading to defoliation of the bronchial epithelium. The intraepithelial reaction may involve either CD8 or CD4 T-cytotoxic cells and is analogous to a viral exanthema of the skin, such as measles and smallpox, which occur when the immune response against these infections is activated and the infected cells are attacked by T-cytotoxic cells. Then there is formation of B-cell follicles adjacent to bronchi, i.e., induced bronchial associated lymphoid tissue (iBALT). iBALT reacts like the cortex of a lymph node and is a site for a local immune response not only to the original viral infection, but also related viral infections (heterologous immunity). Proliferation of Type II pneumocytes and/or terminal bronchial epithelial cells may extend into the adjacent lung leading to large zones filled with tumor-like epithelial cells. The effective killing of influenza virus infected epithelial cells by T-cytotoxic cells and induction of iBALT suggests that adding the induction of these components might greatly increase the efficacy of influenza vaccination. Multicolor flow cytometry has revolutionized analysis of the components of protective immune responses. However, flow cytometry alone fails to capture important aspects of the interactions between immune cells and the tissues they respond in, and the process of immunopathology and/or repair taking place. Although often used simply to provide a basis of scoring the degree of inflammation associated with responses against pathogens, histological examination can be a powerful tool to reveal novel insight into mechanisms underlying health and disease that cannot be appreciated through even sophisticated flow cytometry approaches alone. In this review, we will briefly discuss how studies utilizing five mouse models of influenza permit dissection of the different components of the immune response in experimentally induced influenza infection  summari.Mouse models of influenza are widely used in influenza immunology research. One strength of this translational model is that the pathology of viral pneumonia is similar to humans (as will be discussed). Additional benefits of a wealth of available research tools, transgenic strains, as well as gene deficient animals far outweigh the well-recognized and acknowledged caveats of the model ,3. The m−/−CXCR3−/− mice, that develop improved CD8 T cell memory against influenza, reveals not only increased T-cell mediated cytotoxicity against infected cells, but also increased BALT formation and epithelial proliferation [+ FoxP3+ regulatory cells (Treg) during influenza infection by treating WT mice with anti-CD25 antibody (clone PC62) to deplete this subset prior to infection. This work clearly demonstrates increased inflammation, epithelial cell toxicity, greater induced bronchial associated lymphoid tissue (iBALT) formation and markedly increased proliferation of bronchial epithelial cells and type II pneumocytes in the absence of Tregs [In the first two models, memory CD4 T cells specific for influenza were passively transferred to either wild-type (WT) or to Severe Combined Immunodeficient (SCID) mice that lack adaptive immune cells. The adoptive hosts were challenged with virus to investigate the mechanisms by which memory CD4 T cells participate in clearing infection. These studies reveal a role for cytotoxic CD4 T-cells in elimination of virus infected bronchial epithelium and type II pneumocytes . In the feration . FinallyCD8 T cells are considered the major subset of antigen-specific immune cells with cytotoxic potential, but recent studies have clearly identified that major histocompatibility complex (MHC) class II restricted CD4 T cells may also be cytotoxic ,10,11. M50 of the A/PR8 that is lethal to mice not receiving memory HNT cells, but against which the transfer of HNT cells protects. After considering the ‘take’ of the donor cells, this transfer reconstitutes mice with a quantity of cells that is consistent with the total number of virus-specific memory CD4 T cells that can be detected in an influenza-primed mouse [Unprimed WT BALB/c mice received the same number of either naive or memory transgenic CD4 T cells that express a T cell receptor for an epitope of the A/PR8 influenza virus (referred to as HNT transgenics) by intravenous injection. The same day the recipients were challenged with a dose of 10,000 EIDed mouse .50) that is nevertheless lethal to all SCID hosts not receiving memory cells. Memory CD4 T cells promote robust reduction of viral titer in SCID mice during the first week of otherwise lethal doses of influenza, but without contributions from other components of the adaptive immune system, such as virus-specific antibody or CD8 T cells, viral titers are not fully cleared [To refine the above analysis and focus on the relationship between protective memory CD4 T cells and histological changes observed during protective responses against influenza, we took advantage of a transfer model using SCID hosts. As SCID mice are more susceptible to infection than are WT mice, we used a lower dose of A/PR8 virus than used in the experiments discussed above 2500 EID00 EID50 Histological analysis past the second week of infection also revealed unexpected findings that help to explain the cause of virus-associated death in SCID hosts, and that might suggest a novel role for memory CD4 T cells in regulating lung epithelial proliferation. By week 3, bronchial epithelial cells extend into the adjacent pulmonary parenchyma, most likely derived from focal proliferation of epithelial cells along terminal air sacs or alveolar walls with squamous metaplasia. By week 4 I, the deTo determine the effect of reaction of memory CD4 T-cells with soluble antigen in the lung in the absence of infection, WT C57BL/6 mice that were given memory CD4 T cells specific for ovalbumin protein (generated from OT-II transgenic mice) were challenged with ovalbumin intranasally in the absence of any kind of adjuvant signal . We reasAlthough the eliciting antigen  diffuses from the bronchus into the lung, the reactive T-cells migrate from the vessels so that the antigen-T-cell reaction takes place in the perivascular space resulting in the perivascular mononuclear cell reaction observed. This may be compared to a delayed hypersensitivity skin test, and the inflammation peaks at 24 to 48 h after antigen challenge. This ‘delayed hypersensitivity reaction’ in the lungs mediated by memory CD4 T cells responding to antigen does not extend to the bronchi. In contrast, WT and SCID mice receiving virus specific memory CD4 T cells prior to infection with influenza (as in the model discussed above) show marked alveolar peribronchial and bronchial inflammation, presumably in this case due to reaction of memory cells with peptide expressed on the surface of influenza-infected cells. Thus, memory CD4 T cells may mediate T-cell cytotoxicity in the lung when directed to virus infected cells or delayed-type hypersensitivity when directed to soluble protein antigens. It is still unclear if distinct subsets within the memory population mediate each of these responses, nor is it entirely clear what kinds of inflammatory signals are needed to initiate these outcomes.50 of A/PR8, which causes only mild disease, or with 5000 EID50, a dose resulting in the death of 50% of the WT mice. Interestingly, this dose, though lethal was not high enough to produce acute lethal necrotic bronchitis observed classically. In contrast to the WT mice, only 20% of the IL-10 deficient mice died demonstrating a detrimental role for IL-10 production in this model [IL-10 is a pleotropic cytokine also known as cytokine synthesis inhibitory factor. In the context of infection, it is generally produced by effector CD4 and CD8 T cells, and it acts via STAT3 signaling to downregulate expression of Th1 cytokines and CD4 T-cell activation , but canis model . No diffHistological analysis at day 8 revealed that invasion of the bronchial epithelium by lymphocytes F and des−/−CXCR3−/− mice on a C57BL/6 background were primed with a sublethal dose (500 EID50) of the X31 strain of influenza (H3N2). X31 is murine influenza strain that drives milder disease and is commonly used to prime mice in order to test aspects of the immunity raised following challenge with the more pathogenic viral strain A/PR8. After 90 days, the primed mice were rechallenged with a supralethal (50 LD50) dose of A/PR8 (H1N1) to assess heterosubtypic immunity, which is largely mediated by virus-specific memory CD4 and CD8 T cells [−/−CXCR3−/− were able to clear the secondary challenge by 8 days post-infection [The studies were conducted to determine if CD8 T cell responses to discrete chemokine signals (mediated by the receptors CCR5 and CXCR3) within the lung altered their positioning and subsequent memory fate. Indeed, improved CD8 memory was seen in mice deficient for both CCR5 and CXCR3 , that ha T cells . Interes−/−/CXCR3−/− mice [−/−/CXCR3−/− mice. There was also marked mononuclear cell infiltrate around the bronchi, and extensive proliferation of epithelial cells from the terminal bronchi into the adjacent alveoli. Again, these epithelial cells stain for both TTF and protein C [The rechallenged mice were sacrificed at days 0, 3, 7, and 14 days and their lungs examined. This allowed for a unique investigation of how major chemokines associated with classic Th1/Tc1 responses shape protective responses against influenza, which are of interest to vaccine design. Inflammation and epithelial proliferation occurred in both WT and chemokine receptor-deficient mice group but were quantitatively much greater in the CCR5−/−/CXCR3−/− mice are linked to improved memory CD8 T cell generation. These results also indicate that robust iBALT formation could be a critical aspect of improved vaccine strategies.One major difference in the lungs of mice undergoing primary versus secondary infection with influenza is the presence of iBALT in the latter. The prominent iBALT in primed mice stains heavily for B-cells whereas staining for T cells is scant. The presence of larger unlabeled cells suggests the presence of F4/80 negative dendritic cells or other antigen presenting cells within the iBALT. These studies suggest that modulating chemokine-based trafficking within the lung is a viable strategy to improve influenza-primed memory CD8 T cell generation, with perhaps broader than expected consequences during recall challenge. Further studies are required to determine whether the distinguishing aspects of the response in CCR550) and challenged after 35 days with a supralethal dose of A/PR8 . The contribution of Tregs to controlling the immune response during secondary influenza infection was examined in untreated mice as well as in mice treated with anti-CD25 antibody (clone PC61) prior to virus re-challenge. A single dose of 100 μg of this antibody clone given intraperitoneally preferentially depletes Tregs that express very high levels of the IL-2 receptor alpha chain (CD25) under steady state conditions [Finally, a protocol was established to investigate the extent to which FoxP3+ regulatory CD4 T cells impact the outcome of primary and heterosubtypic influenza infection. Again, in addition to employing other means to assess the immune response, histological changes were examined to gain a more complete picture for how Tregs modulate the host response. Wildtype C57BL/6 mice were primed with a relatively high sublethal dose of X31 virus  and viral exanthema including smallpox and measles . In contConventional influenza vaccines protect against infection by inducing circulating antibody that blocks the interaction of the virus with potential target cells. Every year a new seasonal vaccine is made to protect against the strains that are predicted to circulate. There are two major problems with this approach: First, the vaccine epitopes are highly mutable surface proteins. This means that there are often mis-matches between the vaccine and the prevalent circulating strains due to poor prediction. Second, although blocking antibody may prevent infection, it is likely that a vaccine that induces influenza directed CD4 and CD8 T cell responses will produce longer-lasting and more effective immunity, not only to the immunizing strain, but also to other more virulent strains than the presently used vaccines ,51. We wIndeed, many antigens expressed by vaccinia virus vectors have generated strong antigen-specific T-cell and antibody responses. Immunization with vaccinia vectors is most effective when the antigen is presented in association with MHC class I on the surface of epidermal cells. When vaccinia infects epithelial cells the vaccinia antigens are presented by the endogenous pathway and T-cell cytotoxicity rather than delayed-type hypersensitivity is induced . Indeed,In fact, evidence from both human and animal studies indicates that such Vaccinia-based vaccination against influenza can induce robust, protective immune responses. A fusion construct of nucleoprotein and matrix was found to be safe and immunogenic in chickens using MVA . Further®, administered intranasally has been approved by the Centers for Disease Control and Prevention for the 2018–2019 influenza season after a 2-year hiatus for evaluation after changing the type A H1N1 component. As shown above intranasal challenge not only induces cytotoxic T-cells, but also formation of iBALT that serves as a local site for a secondary antibody response in the lung. As stated above vaccinia readily infects lung epithelium of mice and careful vaccinia strain selection is required to reduce fatal infections. A potential benefit of intranasal vaccination using a low virulence recombinant vaccinia virus incorporating major influenza antigens is that this strategy may circumvent pre-existing neutralizing influenza specific antibody and provide a means to efficaciously induce universal influenza immunity. Of course, we stress again that a major determinant of the successful application of any intranasal vaccination approach is that it must be accomplished with negligible inflammatory or proliferative effects in the lung, such as those described in this review.Another striking aspect of our observations is the development of local immunity that occurs following intranasal infection. It makes sense that an optimized vaccine against a given pathogen should target the induction of protective immune cells at the site of infection. In the case of influenza, the site of infection is the lung, but the field’s understanding of immunity at mucosal sites is still incomplete. Nevertheless, a live attenuated influenza vaccine, FLUMISTIn summary, our studies find that through histological analysis of lungs from influenza-infected mice, basic mechanisms that are associated with viral control can be observed that are not always evident in flow cytometry, ELISPOT-based studies, or other common approaches to dissect and characterize responding immune cells. While the most effective universal influenza vaccine should induce both antibody and T-cells, as synergy among immune mechanisms will provide the best protective response , our obs


Human muscle-derived stem cells (hMDSCs) have been shown to regenerate bone efficiently when they were transduced with Lenti-viral bone morphogenetic protein 2 (LBMP2). However, whether the age of hMDSCs and the animal host affect the bone regeneration capacity of hMDSCs and mechanism are unknown which prompted the current study.We isolated three gender-matched young and old populations of skeletal muscle stem cells, and tested the influence of cells’ age on in vitro osteogenic differentiation using pellet culture before and after Lenti-BMP2/green fluorescent protein (GFP) transduction. We further investigated effects of the age of hMDSCs and animal host on hMDSC-mediated bone regeneration in a critical-size calvarial bone defect model in vivo. Micro-computer tomography (CT), histology, and immunohistochemistry were used to evaluate osteogenic differentiation and mineralization in vitro and bone regeneration in vivo. Western blot, quantitative polymerase chain reaction (PCR), and oxidative stress assay were performed to detect the effects of age of hMDSCs on cell survival and osteogenic-related genes. Serum insulin-like growth factor 1 (IGF1) and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured with an enzyme-linked immunosorbent assay (ELISA).RANKL and lower level of IGF-1.We found LBMP2/GFP transduction significantly enhanced osteogenic differentiation of hMDSCs in vitro, regardless of donor age. We also found old were as efficient as young LBMP2/GFP-transduced hMDSCs for regenerating functional bone in young and old mice. These findings correlated with lower phosphorylated p38MAPK expression and similar expression levels of cell survival genes and osteogenic-related genes in old hMDSCs relative to young hMDSCs. Old cells exhibited equivalent resistance to oxidative stress. However, both young and old donor cells regenerated less bone in old than young hosts. Impaired bone regeneration in older hosts was associated with high bone remodeling due to higher serum levels of hMDSC-mediated bone regeneration was not impaired by donor age when hMDSCs were transduced with LBMP2/GFP, but the age of the host adversely affected hMDSC-mediated bone regeneration. Regardless of donor and host age, hMDSCs formed functional bone, suggesting a promising cell resource for bone regeneration.The online version of this article (10.1186/s13287-018-1066-z) contains supplementary material, which is available to authorized users. Aging has a significant impact on human health and can result in a multitude of problems. Reduced stem cell number or dysfunction is associated with age-related degenerative diseases. Since stem cells have great potential for the treatment of age-related degenerative diseases, such as osteoporosis, fracture non-union, and large bone defects, they are likely candidates for use in elderly individuals to restore loss of tissue homeostasis due to degenerative disease. Therefore, it is important to investigate the effects of donor and host cell aging on stem cell-mediated tissue regeneration.The effects of age of stem cells on their self-renewal and differentiation capacities have been studied for both non-human animal and human cells; however, discrepancies exist between different studies. Both bone marrow mesenchymal stem cells (BMMSCs) and adipose-derived stem cells (ADSCs) isolated from aged rats have been shown to exhibit increased cell senescence and a trend of increasing p38 and p53 levels with age compared to neonatal and young rats . HoweverIn fact, there are very few studies which have investigated the effects of host age on bone tissue regeneration. It has been shown that reduced bone graft efficiency in aged animals is related to loss of WNT3A protein, but supplemental liposome-reconstituted WNT3A protein restored the bone formation potential . The ageHuman MDSCs (hMDSCs) have been shown to efficiently repair critical-size bone defects when transduced with lentiviral-bone morphogenetic protein 2 (LBMP2) . Also, hThe use of human tissues was approved by the Institutional Review Board (IRB) of the University of Pittsburgh, and all animal experiments and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh. Similarly, research performed at the University of Texas Health Science Center at Houston  was approved by the UTHealth Institutional Biosafety Committee.The University of Pittsburgh and UTHealth were accredited by the Association For Assessment and Accreditation of Laboratory Animal Care (AAALAC). The use of animals for in vivo study followed the guidelines of the “Basel Declaration” and “ethical guidelines” of the International Council for Laboratory Animal Science (ICLAS).Six populations of hMDSCs were isolated via a modified preplate technique, as previously described , from skA lentiviral vector encoding the human BMP2 gene, under the control of the human cytomegalovirus (CMV) promoter and with a GFP tag separated by an internal ribosome entry site (IRES) from the target gene, was constructed in collaboration with Dr. Bing Wang’s laboratory. The GFP tag facilitates monitoring of transduction efficiency and the use of fluorescence-activated cell sorting (FACS) to select transduced cells. The LBMP2/GFP viral vector was packaged using 293T cells .Human MDSCs were transduced with LBMP2/GFP in the presence of polybrene (8 μg/ml) at passage 8–10 for 16 h. Twenty-four hours after transduction, transduction efficiency was observed using fluorescence microscopy and found to be about 50–60%. Cells were passaged twice after transduction and then subjected to cell sorting using FACS to isolate cells having the GFP tag. After sorting, the cells were expanded in proliferation media. Supernatants were collected from different passages of each population, and BMP2 secretion levels were measured using a BMP2 quantikine ELISA kit .http://www.ihcworld.com/_protocols/special_stains/von_kossa.htm) to verify pellet mineralization. Osteocalcin immunohistochemistry using a mouse anti-human osteocalcin primary antibody  was also performed, as previously reported  , and mouse anti-beta actin  as primary antibodies. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse  and goat anti-rabbit  secondary antibodies were used to detect mouse and rabbit primary antibodies, respectively. Bio-Rad Clarity™ and Clarity Max™ Western ECL-blotting substrates were used to reveal target protein bands. The Bio-Rad Chemidoc Touch system was used to capture digital images. Band densities were quantified using Image Lab software from Bio-Rad and then normalized to that of actin.t test was used to analyze and compare quantitative data between young and old donors and young and old hosts. For data with high standard deviations, we used the Wilcoxon rank sum non-parametric test. A value of P < 0.05 was considered statistically significant.One-way analysis of variance (ANOVA) or the Student In order to test whether the age of donor hMDSCs affects their osteogenic potential and bone regenerative capacity, we isolated three gender-matched pairs of young and old hMDSCs. We transduced each population of the three young and old hMDSC pairs with LBMP2/green fluorescent protein (LBMP2/GFP) under the same conditions using a multiplicity of infection (MOI) of 8. We measured levels of BMP2 produced by the LBMP2/GFP-transduced cells after sorting via FACS for GFP and subsequent cell culture. The BMP2 secretion levels ranged between 1 and 6 ng/million cells/24 h for young and old cells  based on the donor’s gender and cell proliferation rate in vitro for comparisons. When we transplanted LBMP2/GFP-transduced hMDSCs in young mouse hosts following critical-size calvarial bone defect injury, we found that bone regenerative capacity using old LBMP2/GFP-transduced hMDSCs was as good as that observed using young LBMP2/GFP-transduced hMDSCs  cells were observed when three of the six populations of donor LBMP2/GFP-transduced hMDSCs  were transplanted into young animal hosts compared to old animal hosts  in young versus old hosts. We also compared GFP+ young versus old donor cell numbers in both the young and old hosts. In young hosts, significantly more GFP+ old compared to young donor cells were quantified for two of the three donor pairs (pair 1 and 3), while significantly more GFP+ young compared to old donor cells were quantified for the third pair , while no significant differences were found for pair 1 or 3 . However, no consistent differences were observed for contributions of young versus old donor cells, suggesting the donor cells’ contributions to the new bone were not affected by the age of the donor  using anti-GFP antibody to trace donor cells to determine their contribution to bone regeneration with respect to age of the animal host. For young versus old hosts, significantly more GFP-positive  staining to reveal bone remodeling. TRAP-positive (violet red) cells were found in every mouse host, indicating newly formed bone underwent remodeling Fig. a. QuantiP = 0.097).To investigate whether systemic factors affect hMDSC-mediated bone regeneration in young and old animal hosts, we performed ELISAs to measure serum levels of three different factors related to bone formation and remodeling, including insulin-like growth factor I (IGF1), sclerostin, and receptor activator of nuclear factor kappa B ligand (RANKL). Serum IGF1 levels were significantly lower in old animal hosts when compared to young hosts Fig. c. No sig2O2) in culture for 24 h. We found no significant differences between young and old hMDSCs in the three gender-matched pairs of donor hMDSCs  of hydrogen peroxide (HWe observed significantly lower levels of phosphorylated p38 mitogen-activated protein kinase (pp38MAPK) in old hMDSCs compared to young hMDSCs Fig. a, b. No In this study, we investigated the roles of donor cell age and host age on hMDSC-mediated osteogenesis and bone regeneration in vivo. Our results indicate that both young and old hMDSCs underwent osteogenesis in vitro as evidenced by microCT, Von Kossa staining, and osteocalcin staining. In two gender-matched pairs, old hMDSCs exhibited less osteogenic differentiation than young hMDSCs after LBMP2/GFP transduction; the third pair showed no differences. Conversely, in vivo, we found that old donor cells were able to regenerate new bone in the critical-size defect model in both young and old animal hosts as efficiently as young hMDSCs. However, bone regenerative capacity was reduced in old animal hosts compared to young animal hosts when using either young or old hMDSC populations. Although bone formation was lower in old animal hosts, all the cells formed functional bone in both young and old animal hosts. This finding is very meaningful clinically as stem cells are most likely to be used in older individuals. Our results support the feasibility of using hMDSCs in bone regeneration applications regardless of the age of the donor stem cells. It is generally accepted that aging causes a reduction in the number of tissue stem cells and cell senescence, thereby resulting in loss of tissue homeostasis. However, very few studies have investigated the effects of donor age on stem cell-mediated bone regeneration –6. Thus In this study, we compared three pairs of gender-matched young and old cells with respect to their in vitro osteogenic capacities, and found that young and old hMDSCs exhibited no differences in osteogenic potentials for two of the three pairs (pair 2 was the exception). However, the young hMDSCs from pair 2 were growing very rapidly when compared to the other cells. The higher mineralized pellet size observed with that cell population may be the result of more rapid proliferation and an increased number of cells during initial osteogenic differentiation, thus resulting in bigger mineralized pellets. LBMP2/GFP transduction significantly enhanced the osteogenic potential of both young and old hMDSCs, although the BMP2 secretion levels by the transduced cells varied somewhat. We did observe that old cells exhibited less osteogenic differentiation than young cells in two pairs; however, this was not observed in the third pair. These observations indicate that hMDSC function is not consistently impaired by age. Indeed, our results are similar to those of a previous study in rabbits, which concluded that MDSC function did not decline with age . It has Despite the differences in the in vitro osteogenic differentiation of hMDSCs, in vivo induction of new bone regeneration was not impaired after transplantation of old LBMP2/GFP-transduced hMDSCs into critical-size defects compared to transplantation of gender-matched young LBMP2/GFP-transduced hMDSC in both young and old animal hosts. This finding could not be explained by a difference in BMP2 secretion levels, as the mean BMP2 secretion level was lower in old LBMP2/GFP-transduced hMDSCs than in young hMDSCs. Rather, these results imply that the bone regenerative capacity of hMDSCs was not negatively affected by donor cell age after LBMP2/GFP transduction.Additionally, in vitro oxidative stress testing showed that old cells exhibit oxidative resistance abilities similar to those of young hMDSCs. This finding was also supported by our Western blot analysis, which showed that old cells express similar levels of p16INK4a (cell cycle inhibitor) and pAKT  when compared with young cells. The protein p16INK4a is a generally accepted molecular marker for senescence and aging , 19. It + osteoclasts in the new bone area in old animal hosts may be explained by the relatively higher serum RANKL levels in old animal hosts, which contribute to higher bone remodeling activity. It has been shown that high levels of serum RANKL are associated with lower bone mass in children [Importantly, we found that the age of the animal host did affect hMDSC-mediated bone regeneration. We found both young and old donor LBMP2/GFP-transduced hMDSCs regenerated less bone in old animal hosts when compared to young animal hosts. Additionally, our findings showing more TRAPchildren .Furthermore, we also found that serum IGF1 levels were significantly lower in old animal hosts than young animal hosts. IGF1 is a well-known bone growth factor. IGF1 in bone matrix maintains bone mass via activation of the mechanistic target of rapamycin (mTOR) in mesenchymal cells , 26. TheFew studies have investigated the role of animal host age in bone regeneration. It has been reported that BMMSCs and cortical bone BMP2 levels decrease during aging ; howeverWe also found that old hMDSCs expressed similar levels of RUNX2, OSX, SOX9, IGF1, and IGF2 compared to young hMDSCs. After LBMP2/GFP transduction, the expression levels of these genes were also similar and no significant differences were found between young and old hMDSCs, which may explain why the bone regenerative capacity of old hMDSCs in vivo was not compromised. Notably, COX2 expression was lower in old hMDSCs than in young hMDSCs for both untransduced and LBMP2/GFP-transduced hMDSCs. This finding is consistent with a previous study which showed that a decrease in COX2 in aged mice is associated with delayed fracture healing in the mice . Our preLastly, our results also demonstrate that, although bone regeneration mediated by hMDSCs was impaired in old animal hosts, significant amounts of new bone formation were still seen in the defect area in old animal hosts . The newly formed bone was found to be functional, as shown by both Herovici’s staining and H&E staining. Therefore, the relative degree of impaired bone regeneration observed in old animal hosts should not hinder the application of hMDSCs for bone regeneration, and perhaps, additional time is needed to repair bone in old animals when compared to young hosts.Taken together, our study reveals that old donor hMDSCs are as efficient as young donor hMDSCs for regenerating bone in young and old mice. The maintenance of the bone regenerative capacity of old LBMP2/GFP-transduced hMDSCs may be associated with lower levels of ppMAPK38, high levels of GPX 1(after LBMP2/GFP transduction), the ability to express similar levels of osteogenesis-related genes, and equivalent capacity to resist oxidative stress compared to young cells. The relative decrease in new bone formation in old hosts may be attributed to faster bone remodeling due to circulating factors in the serum of young hosts. LBMP2/GFP transduction may overcome the age-related decline in bone regeneration mediated by hMDSCs, as new functional bone was formed in the defect area regardless of donor cell and host age. Whether BMP2 or other BMPs regulate cell cycle activity in aging hMDSCs and, therefore, rescue aging-associated defects in their bone regenerative capacity warrants further investigation. Therefore, hMDSCs are very promising as a cell source for promoting bone regeneration, regardless of donor and host age.Additional file 1:Figure S1. Young and old hMDSCs exhibit similar oxidative stress resistance. Young and old hMDSCs were cultivated in media containing 500 and 650 μM H2O2 for 24 h in the presence of PI (which labels dead cells) and imaged using a live imaging system. The number of surviving cells and cell survival rate was quantified. (A and B) Comparison of cell survival of the young 1 and old 1 donor cells at 500 μM and 650 μM H2O2 respectively. (C and D) Comparison of cell survival of the young 2 and old 2 donor cells at 500 and 650 μM H2O2 respectively. (E and F) Comparison of cell survival of the young 3 and old 3 donor cells at 500 μM and 650 μM H2O2 respectively. No differences were observed at either concentration for young and old donors. Notably, all hMDSCs were highly resistant to oxidative stress using H2O2, with survival rates at 650 μM being > 50% for most of the cell populations tested. Table S1. primers information. (DOCX 296 kb)