We identified 322 (1.21%) tet(X) positive samples from 12,829 human microbiome samples distributed in four continents  using retrospective data from worldwide. These tet(X) genes were dominated by tet(X2)-like orthologs but we also identified 12 samples carrying novel tet(X) genes, designed tet(X45), tet(X46), and tet(X47), were resistant to tigecycline. The metagenomic analysis indicated these tet(X) genes distributed in anaerobes dominated by Bacteroidaceae (78.89%) of human-gut origin. Two mobile elements ISBf11 and IS4351 were most likely to promote the transmission of these tet(X2)-like orthologs between Bacteroidaceae and Riemerella anatipestifer. tet(X2)-like orthologs was also developed during transmission by mutation to high-level tigecycline resistant genes tet(X45), tet(X46), and tet(X47). Further tracing these tet(X) in single bacterial isolate from public repository indicated tet(X) genes were present as early as 1960s in R. anatipestifer that was the primary tet(X) carrier at early stage (before 2000). The tet(X2) and non-tet(X2) orthologs were primarily distributed in humans and food animals respectively, and non-tet(X2) were dominated by tet(X3) and tet(X4). Genomic comparison indicated these tet(X) genes were likely to be generated during tet(X) transmission between Flavobacteriaceae and E. coli/Acinetobacter spp., and ISCR2 played a key role in the transmission. These results suggest R. anatipestifer was the potential ancestral source of tet(X). In addition, Bacteroidaceae of human-gut origin was an important hidden reservoir and mutational incubator for the mobile tet(X) genes that enabled spread to facultative anaerobes and aerobes.The emergence of IMPORTANCE The emergence of the tigecycline resistance gene tet(X) has posed a severe threat to public health. However, reports of its origin and distribution in human remain rare. Here, we explore the origin and distribution of tet(X) from large-scale metagenomic data of human-gut origin and public repository. This study revealed the emergency of tet(X) gene in 1960s, which has refreshed a previous standpoint that the earliest presence of tet(X) was in 1980s. The metagenomic analysis from data mining covered the unculturable bacteria, which has overcome the traditional bacteria isolating and purificating technologies, and the analysis indicated that the Bacteroidaceae of human-gut origin was an important hidden reservoir for tet(X) that enabled spread to facultative anaerobes and aerobes. The continuous monitoring of mobile tigecycline resistance determinants from both culturable and unculturable microorganisms is imperative for understanding and tackling the dissemination of tet(X) genes in both the health care and agricultural sectors. The first generation of tetracycline antibiotics consisted of tetracycline, chlortetracycline, and oxytetracycline and were put into clinical practice in 1952 . Howevertet(X) displaying tigecycline insusceptibility was discovered on an R plasmid from a B. fragilis isolate of human origin. This was the earliest occurrence of an antibiotic resistance gene (ARG) that directly inactivated tetracyclines (tet(X) gene was only functional under aerobic growth conditions because it is a flavin dependent monooxygenase that requires FAD, NADPH, Mg2+, and O2 to inactive almost all of the tetracycline class (tet(X1) and tet(X2) were confirmed on a transposon from Bacteroides thetaiotaomicron of human origin and shared 61.7 and 99.5% amino acid identity with tet(X), respectively. To date, tet(X)/tet(X2) genes have already spread to 16 countries/regions covering five continents  (−1) (A third-generation tetracycline (tigecycline) was approved in the United States in 2005 and its use in the European Union and China was authorized in 2006 and 2010, respectively , 5. Tigecyclines . The tetne class . In 2001tet(X3) and tet(X4) genes in 2019 posed a severe threat to public health (tet(X) orthologs have been identified and include tet(X5)–tet(X14) (–tet(X3), tet(X4), tet(X6), and tet(X14), first detected in Acinetobacter baumanii, Escherichia coli, Myroides phaeus, and Empedobacter stercoris, respectively. The tet(3.2) and tet(X5) gene were identified from an Empedobacter brevis isolate of shrimp origin and an Acinetobacter baumannii isolate of human origin, respectively. All the tet(X7)–tet(X13) orthologs were identified directly from gut-derived metagenomic libraries, but their host bacteria were unknown. Epidemiological studies (tet(X) orthologs were dominated by tet(X3) and tet(X4) that were primarily detected from Acinetobacter spp. and E. coli, respectively. Furthermore, tet(X3)/tet(X4) samples from humans, animals, and meat for consumption revealed a prevalence of 0.3–66.7% and the highest level of 66.7% was detected from pig cecum samples from abattoirs (tet(X3)/tet(X4) in animal isolates  (tet(X)-carrying clinical isolates  –14. Thes –14. The studies , 16 indi the tetX–tet(X13) the tetX–tet(X13)lude tetX–tet(X14)tet(X) genes have been primarily identified using traditional cultural methods and this imposed limitations on their identification including the loss of uncultured bacteria, low-throughput and long processing times. Although the tet(X7)–tet(X13) orthologs were found directly from gut-associated samples using metagenomic sequencing, the bacterial hosts, relative abundance and propagative characteristics were absent (mcr-1 (tet(X3) (25.4%) in poultry samples (tet(X) in public databases (tet(X) genes from these species-level genome bins and tracing their distribution in assembly isolates from public repository may provide a new perspective for source tracking of the global spread of tet(X).The e absent . Nonethet  had emerged as early as 1960 and the Riemerella anatipestifer was its potential ancestral source. In addition, Bacteroidaceae of human gut origin were a hidden reservoir and mutational incubator for mobile tet(X) genes that enabled spread to facultative anaerobes and aerobes.In the current study, we utilized these data mining techniques and discovered that tet(X) orthologs reported in a previous study (tet(X)-like orthologs that shared 98.71–100% identity with tet(X2) (Acc. No. AJ311171) . The remaining 3.73% (12/322) shared < 98.20% identical with the known tet(X). Since there was not a criterion for assignment of tet(X) orthologs in previous studies (tet(X2)-like orthologs as tet(X2.2) to tet(X2.16). These orthologs represented a large proportion of samples  from 19 countries, and they likely represent a relatively comprehensive assessment of tet(X2)-like orthologs. We therefore tentatively used the lowest cutoff of 98.20% between tet(X2)-like orthologs [tet(X2.15) vs tet(X2.16)] for assignment of novel tet(X) orthologs. In addition, when tet(X)s were annotated as one group of tet(X), such as tet(X2) , they should be grouped into one clade using the neighbor joining typing method (tet(X)s have been assigned (tet(X) orthologs and their subtypes shared less than 98.20% amino acid identity with their closest neighbors in the phylogeny designed tet(X45), tet(X45.2), tet(X45.3), tet(X46), tet(X46.2), and tet(X47) . Most of these tet(X) orthologs found from metagenomic analysis were not present in the NCBI database with the exceptions of tet(X2) (Acc. No. AJ311171), tet(X2.4) (Acc. No. JQ990987) and tet(X46.2) (Acc. No. KU547718.1) (Table S3).A total of 202,265 metagenome-assembled genomes (MAGs) that were reconstructed from 12,829 metagenomic samples of human-microbiome origin in previous studies 2022. We dous study  , tet(X46), and tet(X47) groups found from metagenomic analysis of human-gut origin (E. coli JM109 were resistant to tigecycline (MICs 8 to 16 ml/liter) and exhibited high MIC to the fourth-generation antibiotics omadacycline (MICs 16 to 32 ml/liter) and eravacycline (MICs 2 to 4 ml/liter) . In addition, all the non-tet(X2) exhibited resistance to tetracycline (MICs 128 to 256 ml/liter), doxycycline (MICs 32 to 64 ml/liter), and minocycline (MICs 16 to 32 ml/liter) (Table S4). All the tet(X2) like orthologs were susceptible to tigecycline (MICs 0.25 to 1 ml/liter) but resistant to tetracycline (MICs 8 to 256 ml/liter). Among the 23 tet(X) variants, 22 of them were resistant to doxycycline (MICs 8 to 64 ml/liter) and minocycline (MICs 2 to 32 ml/liter), excluding tet(X2.10) that intermediate to doxycycline (MIC 1 ml/liter) and minocycline (MIC 0.5 ml/liter) (Table S4). All of these tet(X2) like orthologs exhibited MIC of omadacycline from 0.5 to 16 ml/liter, and eravacycline from 0.0125 to 4 ml/liter (Table S4). Three tet(X2) like orthologs tet(X2), tet(X2.4) and tet(X2.15) showed relatively higher MICs to omadacycline (8 to 16 ml/liter) and eravacycline (1 to 4 ml/liter).All the t origin  in the Etet(X)s carrying MAGs were detected in 19 countries including Europe (n = 12), Asia (n = 4) and America (n = 3) but were absent in Oceanica and Africa (tet(X) in European countries (0–9.40%) was more complex than Asian (0–3.17%) and American countries (0–2.44%) although the positive rates for tet(X) in these three continents were not significantly (P > 0.05) different . The prevalence of tet(X) was highest in Spain (9.40%), followed by Germany (5.14%), France (3.82%), Denmark (3.34%) and China (3.17%) and the remaining countries were < 3% (The 322 tet(X)-carrying MAGs carried a range of 82–108,870 contigs (Table S8) and based on these contigs in each MAG the bacterial taxonomy assignment indicated that all the MAGs could be assigned to five phyla and was dominated by Bacteroidetes  followed by Firmicutes , Proteobacteria , Candidatus Melainabacteria , and Fusobacteria   (tet(X) carrying Bacteroidaceae MAGs could be further divided into 14 Bacteroides species that were dominated by Bacteroides vulgatus , Bacteroides uniformis , Bacteroides dorei , Bacteroides ovatus , Bacteroides fragilis  and Bacteroides caccae  (Table S7).The 322 , 2/322) . Of whictet(X2)-like positive MAGs were distributed across these 19 tet(X) positive countries. tet(X2.4) , tet(X2) , tet(X2.5) , and tet(X2.2)  totaled 96.11% (297/309) and prevailed over other tet(X2)-like orthologs  . Only 81.08% (240/296) of these four predominant tet(X) carrying MAGs could be assigned at genus level and most of them were also identified in Bacteroides , followed by Prevotella  and Alistipes  (Table S8). In addition, the non-tet(X2) orthologs included 75% (9/12) that were distributed in China and United States. Of which, tet(X45.2) was the most prevalent ortholog (tet(X2) carrying MAGs were distributed in Europe and the single tet(X46.2) ortholog was carried by an Enterococcaceae from Italy. Almost all tet(X)-carrying MAGs were collected from human stools excluding only one tet(X2.4)-carrying MAG identified as an Enterococcus spp. from an oral cavity sample (Table S8).The ortholog . The remtet(X) carrying MAGs included 196 from the 4,390 MAGs and their average abundance in human microbiomes has been calculated in previous study (Table S8) (tet(X)-positive MAGs was 5.97 ± 3.89 and significantly higher than that of the total 4,390 MAGs (1.76 ± 3.74) (The 322 able S8) . The ave ± 3.74) .tet(X)s in culturable isolates, we examined 774,435 whole genome sequences of bacterial isolates present in GenBank and only 0.12%  carried an ORF with > 70% amino acid identity with the known tet(X) genes including the novel ones found in the current study . The 70% sequence identity cutoff was chosen to ensure that no other gene family would incorrectly annotate as new tet(X) members (tet(X) genes were found in 17 bacterial families that were dominated by aerobes including Moraxellaceae , Enterobacteriaceae , and Weeksellaceae . The Bacteroidaceae  were also an important anaerobic carrier for tet(X2) like orthologs (tet(X) orthologs were most prevalent and included tet(X2)-like , tet(X3)  and tet(X4)  (Table S9). Interestingly, different bacterial families from variant hosts were preference for carrying specific tet(X) ortholog  and most Weeksellaceae  isolates carried tet(X2)-like orthologs and were primarily from human samples . In addition, all tet(X3) were detected from Acinetobacter spp. and almost all tet(X4)  were carried by E. coli and these were primarily from food animals  including pigs, chickens, ducks, cattle, and geese.To further trace the distribution of these  members . The Phy members . These tortholog . Almost tet(X2)-like orthologs were detected prior to 8.41 ± 6.17 years ago and earlier than tet(X3) (4.00 ± 1.12) and tet(X4) (2.38 ± 1.34). To be noted, the collection dates of three R. anatipestifer isolates from U.K. duck samples were prior to 1980. One isolate (BioSample: SAMN09912225) was collected in 1966 and was positive for tet(X12) and a tet(X2)-like gene that only differed in a single amino acid from tet(X2) was designed tet(X2.17). The two other isolates were collected in 1976. One (BioSample: SAMN09912224) carried a tet(X2.17) gene and another (BioSample: SAMN09912221) carried a tet(X) gene shared 99.45% similarity with tet(X47), designed tet(X47.2).The tet(X) orthologs were produced by Bayesian phylogenetic inference (tet(X) during 1966–2018. The most recent common ancestor (MRCA) of all tet(X) from this study was approximately from 1944 to1964, and the tracer analysis indicated that the presence of tet(X) most likely occurred in 1956 A.D. (95% highest posterior distributor). Two main lineages that originated from tet(X2.17) and tet(X12), respectively, were observed in this phylogeny and both tet(X) orthologs were collected from the Riemerella anatipestifer of duck origin from the United Kingdom in 1966 A.D.Date for lineage divergence of the earliest occurred nference . The anatet(X)-carrying contigs ranging from 1,190 to 931,600 bp were retrieved from the metagenome and bacterial-isolate data. These contigs were grouped into 455 clusters that carried a range of 1–48 contigs . In each cluster, longer contig shared more than 97% coverage and more than 97% similarity with shorter contig. The high coverage and similarity of these contigs indicated that these tet(X) could spread among each cluster (Table S10). This indicated tet(X) orthologs could spread among a great diversity of hosts including human, animal and environment (tet(X) hosts. tet(X2)-like tet(X3), tet(X4), tet(X7), and tet(X47) have been found in humans as well as tet(X2)-like, tet(X3), tet(X4), and tet(X6) in pigs. However, only tet(X2)-like and tet(X3) orthologs could transfer between these two hosts (tet(X2)-like orthologs could hitch a great diversity of vehicles to spread between humans and pigs and these included Bacteroides spp., R. anatipestifer and Chryseobacterium spp. The remaining tet(X) genes were spread only via special species between different hosts. For instance, the tet(X3) gene could only be transited by Acinetobacter spp. and spread between pigs and other hosts including pigeons, cattle, geese, ducks and humans (tet(X4) in the genomic array rdmC-tet(X4)-△ISCR2 could spread among wild birds, humans, pigs, chickens and the environment  gene and these included ermF, aadK, tet(Q), and blaOXA-347 that conferred resistance to erythrocin, streptomycin, tetracycline, and ampicillin, respectively (erm(F) gene was the most frequent to flank the tet(X) gene (n = 152), and their genomic environment were clustered into two types according to their relative position: ermF located upstream  and downstream  of tet(X) gene -tet(X2)/tet(X2.2)-aadK (n = 24) (Bacteroides origin (aadK (930 bp) was replaced by another aminoglycoside ARG aadS (903 bp) in CTnDOT and these two ARGs shared a 96.77% identity at the nucleotide level. Interestingly, the tet(X) orthologs and their genomic contexts were more diverse when ermF was located immediately downstream of tet(X). These tet(X) genes included 12 orthologs that were dominated by tet(X2.4) and tet(X2.5) (tet(X) genes were able to spread among anaerobes, aerobes and facultative anaerobes .Genomic annotation and comparisons indicated that other ARGs frequently flanked the ectively  and b. O(X) gene  and b. T(n = 24) . This sts origin , but theet(X2.5) , Text S1tet(X) emerged as early as 1960s in R. anatipestifer of duck origin, which was earlier than that had been previously reported in 1980s (tet(X)s orthologs likely occurred 9900 years ago (7887 BC) (tet(X) genes but speculated it occurred only 65 years ago (1956 A.D.) that was 4 years following the introduction of tetracycline in clinics. This was consistent with the streptomycin, erythrocin and florfenicol, in which their resistance also emerged over the ensuing years following their introduction in clinic (tet(X) orthologs from public repositories on a large scale  and the earliest emergence of 28 tet(X) variants was selected to perform MRCA estimations (tet(X) variants was an important factor for the chronogram phylogeny construction using the Bayesian evolutionary analysis, which should be more reasonable than previous study (tet(X) emergency between current and previous studies (tet(X) variants that spanned a longer time, and the coverages of some tet(X) variants carrying contigs were unknown (Table S3), which may affect the identification accuracy. Thus, these results could only provide an alternative model for the emergence of tet(X).One of the most significant findings in current study was that 7887 BC) . Howeverimations . The timus study . Meanwhi studies . HoweverFlavobacteriaceae have been recognized as a potential ancestral source of the tigecycline resistance gene tet(X) (tet(X) orthologs [tet(X2.17) and tet(X12)] harbored by R. anatipestifer that is also a bacterial species member belonging to Flavobacteriaceae family. Meanwhile, before 2000 only a total of seven isolates were confirmed as tet(X) gene carrier and five of them were also identified as R. anatipestifer . Among the 896 tet(X)-carrying isolates from public repository, 71 tet(X)-carrying were Flavobacteriaceae including R. anatipestifer that accounted for a large number  versus all other Flavobacteriaceae members (R. anatipestifer harbored a great diversity of tet(X) variants and only 23.94% (17/71) carried the known tet(X)s including tet(X12), tet(X14), tet(X2.17) and tet(X47.2) (tet(X) orthologs that shared 94.9–99.7% similarity with their most closely related tet(X) ortholog (Table S9). A recent study indicated the poultry pathogen R. anatipestifer appears to be a reservoir for tet(X) tigecycline resistance .The e tet(X) . We foun members . Additiot(X47.2) . The remsistance . These itet(X) and their flaking genomic contexts was observed when the ermF gene was present downstream of tet(X) (tet(X) genomic contexts from MAGs and culturable isolates yielded 11 genomic backbones that were associated with the formation of non-tet(X2) orthologs found in the current study (tet(X2) orthologs were likely to be generated from tet(X2)-like orthologs during their transmission between anaerobes or between anaerobes and facultative anaerobes (Bf11 and IS4351 played important roles in their transmission between anaerobes that was dominated by Bacteroides spp. where a mobile cyclic structure was speculated based on genomic Types I - IV (tet(X45), tet(X46), and tet(X47) groups were likely to be generated during the transmission of these mobile structures. In addition, genomic comparisons indicated that these tet(X) genes were also able to spread between Flavobacteriaceae and E. coli as well as between Flavobacteriaceae and Acinetobacter sp.  and the new tet(X) orthologs were likely to be produced during their transmission. The high similarity of the nucleotide sequences flanking tet(X3) and tet(X4) (Flavobacteriaceae and ISCR2 played a key role in this process (A great diversity of nt study . The nontet(X) prevalence in the human microbiome using a large-scale survey of 12,829 samples (–tet(X2)-like and two new non-tet(X2) orthologs have been identified directly in the human stool samples. Since there was not standard for assignment of the newfound tet(X) orthologs, certain conflict for tet(X) numbering have been published in previous papers (tet(X) orthologs in current study. Thus, we temporarily set up a criterion for the assignment of tet(X) orthologs. This maybe not comprehensive as the assignment for mobile colistin resistance (mcr) genes (pd-help@ncbi.nlm.nih.gov) to confirm and allocate the allele numbers for new mcr gene. The assignment of new mcr alleles would not be confused in this platform and an allele numbers assignment platform for tet(X)s should be established urgently.The human microbiome plays an important role in public health. Here, we first determine the  samples –22. A tos papers , 27, andr) genes  which hatet(X) at 1.21%  that was higher than for E. coli and K. pneumoniae from hospital isolates  (tet(X). Our results were similar to an epidemiological study that detected the blaNDM and mcr-1 genes directly from samples that was higher than for the E. coli isolates -like genes carrier in human microbiomes were dominated by the Bacteroidaceae in contrast to previous epidemiological studies where tet(X3) and tet(X4) were primarily carried by A. baumannii and E. coli, respectively -carrying MAGs (5.97 ± 3.89) annotated as Bacteroidaceae prevailed over species-level genome bins (1.76 ± 3.74) in the human microbiome. This was likely the reason for the absence of tet(X3) and tet(X4) in our human microbiome analyses. Although tet(X) genes are inactive in anaerobes, the high abundance of tet(X2)-carrying MAGs and a variety of non-tet(X2)-like orthologs found in the current study indicated that the Bacteroidaceae were an important reservoir and mutational incubator for the mobile tet(X) orthologs in the human microbiome (Bacteroidaceae could generate new non-tet(X2) orthologs with tigecycline inactivation functions, and the comparison of the tet(X) genomic environment suggested that these non-tet(X2) enabled transfer to facultative anaerobes and aerobes  genes, and the earliest emergence of high-level tigecycline resistance genes tet(X3) and tet(X4) were likely to be 1975 and 1971, respectively, both of which were earlier than the clinical introduction of tigecycline in 2005. This was further evidence that the use of even older antibiotic tetracycline may contribute to the resistance to newer antibiotics (tet(X) distributions from culturable isolates indicated that tet(X2)-like and non-tet(X2) orthologs were prevalent in anaerobes and aerobes respectively (tet(X) is active only in an aerobic environment, the non-tet(X2)-like orthologs with tigecycline inactivate function tended to be captured by aerobes under tetracycline selective pressure (tet(X2) orthologs primarily distributed in aerobes but the high prevalence of tet(X2)-like orthologs in Bacteroidaceae from human microbiome need to be further explored.We have demonstrated  and tetX were likectively . Since tpressure . This watet(X)-carrying MAGs were estimated using bioinformatic methods that may not be as precise as cultural methods, but such an approach has proved feasible (tet(X)-carrying contexts from MAGs in the current study were likely to be chromosome-borne. This differed with tet(X3)/tet(X4) that are present in a variety of plasmids and ISCR2 was an essential element for their mobilization (Bacteroidaceae of these tet(X)s orthologs was primarily mediated by the CTnDOT-like conjugative transposon and ermF-related IS elements including ISBf11 and IS4351. CTnDOT has been reported to harbor ermF, tet(X1) and tet(X2) in Bacteroides  in the Type III genomic contexts have also been previously identified (CR2 element belonging to the IS91 family has been described in the first report of tet(X3) and tet(X4), both downstream and upstream of tet(X3). This inserted sequence (IS) element could form a mobile amplicon and this was demonstrated using inverse PCR experiments (Acinetobacter towneri isolate flanking region of tet(X6) (tet(X6) and indicated that this IS element can play an important role in transmission of non-tet(X2) orthologs with tigecycline inactivation functions in the Flavobacteriaceae, E. coli and Acinetobacter.Taxonomic assignments for feasible , 32. Anofeasible . Therefolization . Transmiteroides  and conjteroides . Therefod Vibrio . The eryisolates . The IS entified  and reveeriments  and in a tet(X6) . We alsotet(X) gene. The tet(X) gene emerged as early as 1960 and the R. anatipestifer was an ancestral source of tet(X). The tet(X3)-carrying Acinetobacter spp. and tet(X4)-carrying E. coli were prevalent in food animals and these two tet(X)s were likely formed during the transmission of tet(X)s between Flavobacteriaceae and E. coli/Acinetobacter, and ISCR2 played a key role in the transmission. The tet(X2)-like orthologs enriched in the anaerobes that was dominated by Bacteroidaceae of human-gut origin and could transfer between these anaerobes. The mobile elements CTnDOT, ISBf11, and IS4351 played important roles in the transmission. The low-level tigecycline resistance tet(X2)-like gene could mutate to high-level tigecycline resistant determinants that could spread to facultative anaerobes and aerobes. Bacteroidaceae of human-gut origin was an important reservoir and mutational incubator for tet(X) that could spread to facultative anaerobes and aerobes.In conclusion, we concluded an analysis on integrated human gut meta genome and global bacterial isolates to trace the origin and distribution of de novo-assembled and binned into metagenome-assembled genomes (MAGs) for the exploration of new bacterial species in previous studies (––tet(X) orthologs from these 202,265 MAGs using BLAST using an 80% identity and 70% hit length cutoff. The prevalence of tet(X) in 31 countries were plotted using R version 3.5.3. Phylogenetic analysis for amino acid sequences of all tet(X) gene products was constructed using neighbor joining with the default parameters in Mega X Version 10.0.5  genes. EcoRI and a SalI sites were then added 5′ and 3′ respectively . The synthesized tet(X) genes were cloned into plasmid vector pBAD24 and transformed into competent E. coli JM109 as described in our previous study (E. coli JM109+pBAD24-tet(X4) and E. coli JM109+pBAD24, were used as positive and negative controls, respectively, as previously described . The United States FDA criteria was employed to interpret tigecycline breakpoints for E. coli and MIC ≥4 mg L−1 was considered non-susceptible while eravacycline and omadacycline were uninterpreted with no breakpoint. E. coli ATCC 25922 was used as the quality control strain.Tigecycline resistance for these gene products was assessed by synthesis of full-length nucleotide sequences of all detected us study . The traescribed . The MICtet(X)-carrying MAGs from three previous studies (–tet(X)-carrying MAG was taxonomically profiled using Kraken2 -carrying MAGs were performed using an automatic PhyloPhlAn (3.0) pipeline s in culturable bacteria isolates, a total of 774,435 bacteria assembled whole genome sequences were downloaded from the NCBI database as of 7 November 2020. tet(X)-like open reading frames (ORFs) were determined using BLATX against all the tet(X)s variants mentioned in current study with a minimum similarity of 70 and 100% coverage. The collection date, origin, countries, and the bacterial host of the tet(X)-positive isolates were retrieved according to their Biosample Number. The phylogenetic structure for the tet(X)-carrying isolates were also performed using PhyloPhlAn (3.0) pipeline mentioned above.To further trace the spread of all tet(X)s, the earliest emergency of tet(X) variants (with collection date) with a 388 amino acid (aa) length were applied to generate a chronogram using Bayesian evolutionary analysis version 1.10 -carrying contig were extracted from the MAGs of metagenomic analysis and isolates from public repository. CD-HIT was employed to group tet(X)-carrying full length contigs using a cutoff with a minimum similarity of 97% over 97% of the query coverage (tet(X)-carrying contigs were annotated using Prokka (The coverage . These tg Prokka  and in cg Prokka  and ISfig Prokka  database


The H7N9 avian influenza virus (AIV) that emerged in China have caused five waves of human infection. Further human cases have been successfully prevented since September 2017 through the use of an H7N9 vaccine in poultry. However, the H7N9 AIV has not been eradicated from poultry in China, and its evolution remains largely unexplored. In this study, we isolated 19 H7N9 AIVs during surveillance and diagnosis from February 2018 to December 2019, and genetic analysis showed that these viruses have formed two different genotypes. Animal studies indicated that the H7N9 viruses are highly lethal to chicken, cause mild infection in ducks, but have distinct pathotypes in mice. The viruses bound to avian-type receptors with high affinity, but gradually lost their ability to bind to human-type receptors. Importantly, we found that H7N9 AIVs isolated in 2019 were antigenically different from the H7N9 vaccine strain that was used for H7N9 influenza control in poultry, and that replication of these viruses cannot, therefore, be completely prevented in vaccinated chickens. We further revealed that two amino acid mutations at positions 135 and 160 in the HA protein added two glycosylation sites and facilitated the escape of the H7N9 viruses from the vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control. Human infection with H7N9 virus has been successfully prevented since the application of an H7N9 vaccine in poultry in September 2017 in China; however, the H7N9 virus has not been eradicated from poultry. In this study, we evaluated the genetic and biologic properties of H7N9 viruses detected in poultry in China from February 2018 to December 2019. We found that the H7N9 viruses gradually lost their binding to human-type receptors and were antigenically different from the H7N9 vaccine strain that was used in China for H7N9 influenza control in poultry. We further uncovered the genetic changes that facilitate the escape of the H7N9 viruses from vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control. H7N9 influenza viruses emerged in China in early 2013 and caused five waves of human infections from 2013–2017, with a total of 1568 cases, of which 615 were fatal . After fTo control H7N9 influenza virus, an H5/H7 bivalent inactivated vaccine was initiated in China in September 2017. The H5 seed virus (Re-8) contains the HA and NA genes from the clade 2.3.4.4 virus A/chicken/Guizhou/4/2013 (H5N1) and its six internal genes from the high-growth A/Puerto Rico/8/1934 (H1N1) (PR8) virus and has been used in China since 2015 , and theAlthough the prevalence of H7N9 virus in poultry has declined considerably and human infection has been essentially eliminated since the application of the H7N9 poultry vaccine, the H7N9 influenza virus has not been eradicated from poultry; it has caused a few disease outbreaks in chickens and zoo birds in northern China in 2018 and 2019 . When vaIn total, 19 H7N9 AIVs were isolated: 13 H7N9 AIVs were isolated from 71,018 samples that were collected in China from February 2018 to December 2019 during our routine surveillance, and six viruses were isolated from the samples that were submitted to our laboratory for disease diagnosis . The virTo investigate their genetic relationship, the whole genomes of the 19 H7N9 AIVs were fully sequenced . All 19 AIVs had a similar amino acid motif of -PKRKRTAR/G- at their HA cleavage site, which meets the criterion for highly pathogenic AIVs ; the virThe HA gene of the 19 H7N9 viruses share 97%–100%; NA gene 97.6%–100%, and matrix (M) gene 97.1%–100% identity at the nucleotide level; they cluster in phylogenetic trees with the H7N9 HPAIVs that were previously detected in 2017 Figs  and S4A.We previously reported that the H7N9 HPAIVs isolated in China between February 2017 and January 2018 formed nine genotypes  and the α-2, 6-sialylglycopolymer [Neu5Acα2-6Galb1-4GlcNAcb1-pAP (para-aminophenyl)-alpha-polyglutamic acid (α-PGA)] , 37. Chiwww.antigenic-cartography.org/).Antigenic analysis of H7N9 viruses was performed by using HI tests with antisera generated in chickens against different inactivated viruses, including H7-Re2, CK/LN/SD014/2018, and CK/IM/SD010/2019. The HI assay was performed as previously described . AntigenThe commercial trivalent H5/H7 inactivated oil-emulsified vaccine based on the H5 Re-11, Re-12, and H7-Re2 viruses was developed as previously reported by Zeng et al. .5EID50 of H7N9 viruses. Pharyngeal and cloacal swabs were collected from all surviving birds on Days 3 and 5 post-challenge (p.c.) and titrated in eggs. The birds were observed for signs of disease and death for two weeks.Groups of 10 three-week-old female SPF chickens were inoculated intramuscularly with 0.3 ml of the vaccine or with an equal volume of PBS as a control. Three weeks post-vaccination, antisera were collected from the birds and the HI titers were measured by using the homologous vaccine strain and challenge viruses. The chickens were then challenged i.n. with 10The 3D structure of the HA protein was obtained by using SWISS-MODEL; the image of the HA protein was drawn by using Pymol software as described previously .The PR8-IM19 reassortant virus, containing the HA and NA genes from an H7N9 virus (CK/IM/SD010/2019 (IM19)) and the six internal genes from the PR8 virus, was rescued by reverse genetics as previously reported . Using tViruses were concentrated by ultracentrifugation and purified by sucrose gradient centrifugation as described previously ,41. The S1 FigThe total number of H7N9 human cases as of 31 January 2021. The dashed lines indicate October 1st of each year. The red arrow indicates when H5/H7 vaccine administration to poultry was initiated in China.(TIF)Click here for additional data file.S2 Fig(A) and PB1 (B) were rooted to A/chicken/Rostock/45/1934 (H7N1). The viruses sequenced in this study are shown in red and blue in the phylogenetic trees.The phylogenetic trees of PB2 (TIF)Click here for additional data file.S3 Fig(A) and NP (B) were rooted to A/chicken/Rostock/45/1934 (H7N1). The viruses sequenced in this study are shown in red and blue in the phylogenetic trees.The phylogenetic trees of PA (TIF)Click here for additional data file.S4 Fig(A) and NS (B) were rooted to A/chicken/Rostock/45/1934 (H7N1). The viruses sequenced in this study are shown in red and blue in the phylogenetic trees.The phylogenetic trees of M (TIF)Click here for additional data file.S5 FigThe dots show the potential N-glycosylation sites at the corresponding positions in the HA protein of H7N9 viruses. Abbreviations: CK, chicken; DK, duck; PCK, peacock; AH, Anhui; FJ, Fujian; GX, Guangxi; HeB, Hebei; IM, Inner Mongolia; LN, Liaoning; NX, Ningxia; SaX, Shaanxi; SX, Shanxi.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Acinetobacter sp., Pseudomonas sp., and Empedobacter sp. To date, mechanistic details underlying the improvement of catalytic activities of new TetX enzymes are not available.Tigecycline is a tetracycline derivative that constitutes one of the last-resort antibiotics used clinically to treat infections caused by both multiple drug-resistant (MDR) Gram-negative and Gram-positive bacteria. Resistance to this drug is often caused by chromosome-encoding mechanisms including over-expression of efflux pumps and ribosome protection. However, a number of variants of the flavin adenine dinucleotide (FAD)-dependent monooxygenase TetX, such as Tet(X4), emerged in recent years as conferring resistance to tigecycline in strains of Enterobacteriaceae, E. coli strains bearing the tet(X4) gene. Comparison between the structures of Tet(X4) and Tet(X4)-tigecycline complex and those of Tet(X2) showed that they shared an identical FAD-binding site and that the FAD and tigecycline adopted similar conformation in the catalytic pocket. Although the amino acid changes in Tet(X4) are not pivotal residues for FAD binding and substrate recognition, such substitutions caused the refolding of several alpha helixes and beta sheets in the secondary structure of the substrate-binding domain of Tet(X4), resulting in the formation of a larger number of loops in the structure. These changes in turn render the substrate-binding domain of Tet(X4) more flexible and efficient in capturing substrate molecules, thereby improving catalytic efficiency.In this study, we found that Tet(X4) exhibited higher affinity and catalytic efficiency toward tigecycline when compared to Tet(X2), resulting in the expression of phenotypic tigecycline resistance in Our works provide a better understanding of the molecular recognition of tigecycline by the TetX enzymes; these findings can help guide the rational design of the next-generation tetracycline antibiotics that can resist inactivation of the TetX variants.The online version contains supplementary material available at 10.1186/s12915-021-01199-7. The abusive usage of antibiotics in the past few decades resulted in widespread drug resistance, a clinical and public health problem that poses a significant threat to human health. As a last-resort antibiotic, tigecycline was approved for clinical use by FDA in 2005 and is still effective against multiple-resistant (MDR) pathogens , 2. HoweIn this study, we characterized the mechanism underlying the degradation of tigecycline by Tet(X4), resolved the crystal structure of the Tet(X4) and tigecycline-Tet(X4) complex, and revealed the substrate basis of Tet(X4)-mediated catalysis. Compared to the structure of Tet(X2), the secondary structure of the substrate-binding domain of Tet(X4) deconstructed a large number of α-helix and β-sheet which resulted in the loss of various internal contact points in the structure, rendering the substrate-binding domain more flexible in allowing access of the tigecycline molecule to flavin adenine dinucleotide (FAD) for oxidation.E. coli strain BW25113 carrying the plasmid pBAD-18-tet(X4) was found to exhibit an 8–64-fold increase in MIC of various tetracycline antibiotics when compared with the host strain  and higher substrate-binding affinity (KM) by Tet(X4) when compared to Tet(X2). The increased activity of Tet(X4) toward tigecycline is similar to that of the previously reported tigecycline resistance-conferring enzyme Tet(X7) . TheBacteroides thetaiotaomicron and Tet(X7)  from Pseudomonas aeruginosa. Only one monomer was observed in the crystallographic asymmetric unit in all structures. The monomer includes 369 amino acids of Tet(X4) , namely, Asn12 to Gln383. The final R factor and R free values of the refined structures varied from 16.63 to 18.53% and 19.97 to 23.88%, respectively.We solved the X-ray crystal structure of Tet(X4) at a resolution of 1.78 Å, with the key dataset and refinement statistics being shown in Table S27, Glu46, Arg47, Gly57, Gly58, Arg117, Leu139, Asp311, Pro318, and Val324) are conserved in Tet(X2) and Tet(X4). Moreover, multiple sequence alignment (MSA) revealed that these residues are also conserved among all Tet(X) variants  and Tet(X4)-tigecycline structures, FAD is bound non-covalently to an IN-conformation such as those of Tet(X) and TetX7) Figs.  and 3 [7 Figs.  aAccommodation of tigecycline in the binding pocket of Tet(X4) was investigated by X-ray diffraction studies of tigecycline-soaked Tet(X4) crystals. Analysis of the Tet(X4)-tigecycline complex structure revealed specific interactions between the tigecycline hydroxylation product (T1C) and the isoalloxazine of FAD in the large active site cavity Fig. A. Electr192  establishes a hydrogen-bonding bridge connecting the hydroxyl group of Ser238 to the carbonyl O atom of Gln192; this type of interaction was also observed in the TetX-minocycline complex structure [5 and O4 atoms  also forms a hydrogen bond (3.2 Å) with the hydrophilic region (C21) of T1C  with the hydroxyl sites at C11, C21, C10, and C91 of T1C. Since T1C does not have any specific group at C5, C6, C8, and C9, only the functional group of C7 makes van der Waals contacts with the side chains of Met215, Asn371, and Met375 (within 4.0 Å). Furthermore, the hydrophobic segment of T1C (C41–C7) forms several interactions with the side chains of hydrophobic residues Phe224, Pro318, and Phe319. These observations indicate that recognition of tigecycline by the active site of Tet(X4) mainly involves targeting of the conserved hydrophilic substituents in the A ring and C10, C11, and C12 of T1C. The Tet(X4)-tigecycline complex structure is highly identical to the Tet(X2)-tigecycline complex structure, with RMSD at 0.4 Å. T1C adopts similar conformations that interact with Gln192, Arg213, Phe224, Pro318, Gly321, and Met375, which is observable in both the Tet(X4)-tigecycline and Tet(X2)-tigecycline complex structures  was found to have increased from 22.6 to 52.9 Å2 in the Tet(X4)-tigecycline complex structure, indicating that the loop exhibits higher mobility for ligand binding and release. This observation also shows that the loop (H314-G323) plays an important role in substrate recognition by Tet(X4).The RMSD of the Cβ positions between the tigecycline complex structure and TetX4) is 0.1 Å, suggesting that insertion of the tigecycline molecule did not cause dramatic conformational changes in the Tet(X4) monooxygenase structure  and structure of Tet(X2) revealed the difference between the substrate-binding domain of the two enzymes Fig. . First, 10-helix) and β-sheet declined to 30.8% and 40.4%, respectively (Table The changes in the secondary structure of TetX4) have been supported by CD scan and FTIR analysis (Table  have bee282S or V329M into Tet(X2) by site-directed mutagenesis. Residues 282 connected α9 and β16 -producing strain. Consistently, the tigecycline-catalyzing efficiency of L282S and V329M also increased 1.5–3.5 folds when compared to Tet(X2). These findings indicate that amino acid substitutions could cause changes in the secondary structures of Tet(X4) and improve the catalytic activity of the enzyme.Compared to Tet(X2), the variant residues in TetX4) are not in vital sites for FAD binding and substrate recognition, but such amino acid changes might drastically affect the secondary structure of Tet(X4). To confirm our hypothesis, we introduced Lβ16 Fig. ; when th are not n Figs. S, Table 33% Fig. S, Table 33% Fig. S, the amitet(X) genes, which encode tetracycline inactivation enzymes such as TetX4, were found to be responsible for conferring resistance to the latest-generation tetracycline antibiotics among MDR Gram-negative pathogens [tet(X) variants, such as tet(X5-14), have emerged and disseminated extensively [Since their discovery in the 1940s, tetracyclines have become the key antimicrobial agents in agricultural, veterinary, and clinical applications , 21. As athogens , 10. Furensively , 26. HowE. coli strains bearing the tet(X4) gene. Interestingly, Tet(X4) shared a highly identical FAD-binding site with that of Tet(X2), with tigecycline being accommodated in a similar conformation in the catalytic cavity. Compared to Tet(X2), the amino acid changes in Tet(X4) are not the pivotal residues for FAD-binding and substrate recognition. However, these amino acid substitutions, such as L282S and V329M, might cause the refolding of several alpha helixes and beta sheets in the secondary structure of the substrate-binding domain of Tet(X4), allowing a larger number of loops to form in the structure. These changes render the substrate-binding domain of Tet(X4) more flexible and efficient in capturing substrates and thereby improving catalysis. Loop regions as the most flexible parts of protein structures often play an important role in protein functions by interacting with the solvent and substrates [In this work, we showed that Tet(X4) exhibited a high affinity toward tigecycline and catalyzed tigecycline more efficiently than Tet(X2). These functional properties of Tet(X4) are responsible for causing the phenotype of tigecycline resistance in bstrates . The finStructure-guided approaches have been proven to be effective in assisting the discovery of antibiotic analogs and inhibitors , 30. The282S and V329M, in Tet(X4) which might cause the refolding of several alpha helixes and beta sheets in the secondary structure of the substrate-binding domain, allowing a larger number of loops to form in the structure. These changes render the substrate-binding domain of Tet(X4) more flexible and efficient in capturing substrates and thereby improving catalysis. These findings facilitate the design of next-generation tetracycline which can resist degradation by Tet(X4).In conclusion, we investigated the structural basis of the high catalytic activity of Tet(X4) on tigecycline, which is one of the last line antibiotics used to treat multidrug-resistant bacterial infections. We found that Tet(X4) exhibited a high affinity toward tigecycline and catalyzed tigecycline more efficiently than Tet(X2). These functional properties were due to carriage of specific amino acid substitutions, such as Ltet(X4) gene was amplified from genomic DNA of ST767 E. coli strain [EcoR I /Sal I restriction sites was sub-cloned into the plasmid vector pBAD18-kan (kanamycin resistance), which contained the arabinose pBAD promoter. Another tet(X4) gene PCR product with the BamH I/Xho I restriction sites was ligated to a modified pET-M vector which contained 3C protease cleave site beyond the His6 tag. Subsequently, the recombinant plasmid pBAD-18-tet(X4) was transformed into competent cells of E. coli BW25113, followed by antibiotic susceptibility tests. The recombinant plasmid pET-M-tet(X4) was transformed into competent E. coli BL21 (DE3) cells for protein expression and purification. We also constructed two tet(X2)-bearing vectors as control. Mutations were introduced into the tet(X2) gene using the QuickChange Site-Directed Mutagenesis Kit (Stratagene).The full-length i strain  by two pE. coli strain BW25113 harboring the tet(X4)-bearing pBAD18-kan vector was performed. Minimum inhibitory concentrations (MICs) were determined according to Clinical and Laboratory Standards Institute (CLSI) procedures using Mueller–Hinton broth microdilution method [E. coli strain ATCC 25922 was used as quality control.Antibiotic susceptibility test of n method . E. colig for 5 min and resuspended in lysis buffer  and then broken by high-pressure homogenization. The soluble fractions were passed through a Ni-nitrilotriacetic acid (NTA) column, rinsed with 25 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 30 mM imidazole, and finally eluted with 25 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 300 mM imidazole. The eluted proteins were concentrated using the Amicon Ultra-15  centrifugal filter device. The purified enzyme was incubated with 3C protease at 4 °C overnight to remove the His6 tag. The target proteins were further purified by gel filtration chromatography  in a buffer of 20 mM Tris (pH 7.5), 150 mM NaCl, and 2 mM DTT (Dithiothreitol). The desired fractions were collected and concentrated. The purity of the protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)  containing 100 μg/mL ampicillin was inoculated into a 5-mL overnight culture, followed by incubation with shaking at 37 °C until an optical density of 0.6 at 600 nm (OD600) was reached. The expression of enzymes was induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16°C for 16 h. The cells were harvested by centrifugation at 11,300×2, and 0.5 mM nicotinamide adenine dinucleotide phosphate (NADPH). UV-visible spectroscopy measurements were performed in triplicate at 400-nm wavelength light, using a UV-1900 UV-Vis spectrophotometer (Shimadzu) for measurement for 3 min at room temperature. Initial reaction velocities were determined by linear regression using the UVProbe 2.70 Software and fitted to the Michaelis–Menten equation by GraphPad Prism 8.Each 500-μL reaction was prepared with 100 mM TAPS buffer at pH 8.5 with 0–50 μM substrate, 5 mM MgClTet(X4) protein was concentrated to 22 mg/mL and crystallized by sitting drop vapor diffusion at 16 °C in 0.2 M ammonium acetate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, and 30% (w/v) polyethylene glycol 4000. Tigecycline was soaked into the crystals by incubating the Tet(X4) crystals in a reservoir buffer for 30 min. The crystals were cryoprotected by 25% glycerol in reservoir buffer for 10 s and flash-cooled in liquid nitrogen. Diffraction data were collected at 100 K on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility . The dif−1 (wavenumber). Typically, eight scans were collected and averaged for a single spectrum with a resolution of 4 cm1. The background was corrected before scanning the samples. The FITR spectra of Tet(X2), Tet(X4), L282S, and V329M were collected in a buffer of 20 mM Tris (pH 7.5) and 150 mM NaCl; the absorbance of the buffer was subtracted from the spectra of Tet(X2), Tet(X4), L282S, and V329M. The concentrations of Tet(X2), Tet(X4), L282S, and V329M in the assay were 20–30 mg/mL.Infrared spectra were recorded on a PerkinElmer Spectrum 100 instrument using an attenuated total reflection (ATR) sampling accessory as described in previous studies , 41. Bri−1 corrected with baseline. The CD spectra of Tet(X2), Tet(X4), L282S, and V329M protein solutions were acquired, and changes in CD spectral results were analyzed. The secondary structures were determined by BeStSel [CD spectra were measured by a Jasco J-1500 spectropolarimeter at 25 °C. CD measurements in a spectral range of 260 to 200 nm were performed, with an interval of 1 nm and scanning speed of 50 nm min BeStSel , 43.Additional file 1: Figure S1. Size-exclusion chromatogram of Tet(X4), peak 2 is corresponded to Tet(X4); the inset shows reduced SDS–PAGE analysis of the purified proteins.Additional file 2: Figure S2. (A) Chemical structure of tigecycline. Rings B-D is responsible for β-diketone chromophore that was circled by the orange rectangle. (B) In vitro absorbance scan at wavelength between 300nm and 500 nm taken at 60 seconds intervals, covering the Tet(X4) protein, NADPH, MgCl2, and tigecycline. The rainbow shape illustrates the spectral change over time. Time-dependent decrease in absorbance from 370 nm to 420 nm indicates enzymatic disruption of the characteristic tigecycline β-diketone chromophore and consumption of NADPH.Additional file 3: Figure S3. Mass spectrometry analysis of enzymatic reactions with tigecycline as substrate. (A), Reaction without enzyme at 0 minutes; (B), Reaction without enzyme at 30 minutes; (C), Reaction of Tet(X4) with tigecycline as substrate at 0 minutes; (D), Reaction of Tet(X4) with tigecycline as substrate at 30 min.Additional file 4: Table S1. Crystallographic data and refinement statistics.Additional file 5: Figure S4. (A) Representative mean spectra of Tet(X2) protein solution shown in the range 1600–1700 cm-1 after baseline correction and vectorial normalization; (B) Curve-fitting analysis of amide I band of Tet(X2) protein solution; (C) Representative mean spectra of Tet(X4) protein solution shown in the range 1600–1700 cm-1 after baseline correction and vectorial normalization; (D) Curve-fitting analysis of amide I band of Tet(X4) ; (E) Representative mean spectra of the L282S mutant protein solution shown in the range 1600–1700 cm-1 after baseline correction and vectorial normalization; (F) Curve-fitting analysis of amide I band of the L282S mutant protein; (G) Representative mean spectra of the V329M mutant protein solution shown in the range 1600–1700 cm-1 after baseline correction and vectorial normalization; (H) Curve-fitting analysis of amide I band of the V329M mutant protein.Additional file 6: Figure S5. (A) Circular dichroic spectral profiles of Tet(X2) protein solution measured at wavelength between 200 nm and 260 nm; the raw CD spectrum is shown; (B) Circular dichroic spectral profiles of Tet(X4) protein solution measured at wavelength between 200 nm and 260 nm; (C) Circular dichroic spectral profiles of the L282S mutant protein solution measured at wavelength between 200 nm and 260 nm; (D) Circular dichroic spectral profiles of the V329M mutant protein solution measured at wavelength between 200 nm and 260 nm.Additional file 7: Table S2. Primers were used in this study.Additional file 8. Validation reports of the structure of Tet(X4) and Tet(X4)-tigecycline complex.


Rationale: In obesity the fine-tuned balance of macrophage phenotypes is disturbed towards a dominance of pro-inflammatory macrophages resulting in exacerbation and persistence of inflammation and impaired tissue repair. However, the underlying mechanisms are still poorly understood.Methods: Impact of obesity on macrophage differentiation was studied in high fat diet induced obese and db/db mice during skin inflammation and wound repair, respectively. Mechanisms of S100A9-mediated effects on macrophage differentiation was studied on in vitro generated macrophages by genomic and proteomic approaches. The role of S100A9 on macrophage differentiation was investigated by pharmacological inhibition of S100A9 during skin inflammation and wound repair in obese and db/db mice.Results: We demonstrate an overexpression of S100A9 in conditions of obesity-associated disturbed macrophage differentiation in the skin. We show that saturated free fatty acids (SFA), which are increased in obesity, together with S100A9 induce TLR4 and inflammasome-dependent IL-1β release in macrophages which in turn amplifies S100A9 expression initiating a vicious cycle of sustained S100A9 overexpression in skin inflammation in obesity. We reveal a yet unrecognized impact of obesity-associated S100A9 overexpression on macrophage differentiation. S100A9 binding to TLR4 and activation of NFkB attenuates development of M2-like macrophages and induces pro-inflammatory functions in these cells. Consequently, inhibition of S100A9 restores disturbed M2-like macrophage differentiation in mouse models of obesity-associated skin inflammation and wound repair. Similarly, breaking the vicious cycle of S100A9 overexpression by dietary reduction of SFA restored M2-like macrophage activation. Improvement of skin inflammation and wound repair upon reduction of S100A9 by pharmacological inhibition or by reduction of SFA uncovers the pathogenic role of S100A9 overexpression in obesity.Conclusion: This study identifies S100A9 as a previously unrecognized vital component in obesity-associated disturbed macrophage differentiation and subsequent impaired regulation of inflammation and wound repair. The findings open new opportunities for therapeutic implications for inflammatory diseases and wound repair in obesity. Inflammation is a reaction of the host to infection or tissue damage with the physiological aim to restore tissue homeostasis Dysbalances of M1/M2 differentiation have been observed in obesity. Numerous studies show that numbers of M1-like macrophages in adipose tissue increase in obesity inducing a pro-inflammatory microenvironment that contributes to chronic systemic low-grade inflammation Obesity is associated with hyperglycemia, dyslipidemia, hyperinsulinemia, elevation of free fatty acids (FFA), strong accumulation of adipose tissue, and increased levels of pro-inflammatory adipokines, cytokines and chemokines 2+binding protein of the S100 family 38). In our previous study, we observed an increase of S100A9 during skin inflammation in obesity S100A9, also known as MRP14, is a CaIn the present study we explore the mechanisms of obesity-associated overexpression of S100A9. We reveal a yet unrecognized impact of S100A9 on M2-like macrophage differentiation contributing to disturbed macrophage polarization and subsequent impaired resolution of skin inflammation and tissue repair in obesity.Since in obesity proper macrophage differentiation is disturbed, we asked for the mechanisms of obesity-associated dysregulation of macrophage differentiation and its consequences for inflammation and tissue repair in the skin. Interestingly, we observed a pathological S100A9 overexpression in conditions of disturbed macrophage differentiation. First, we induced an acute skin inflammation in high fat diet (HFD)-induced obese mice or lean control mice, to mimic the situation of an obesity-associated increased inflammatory response in the skin. Mice were fed a HFD for 15 weeks resulting in increased body weight, impaired glucose metabolism and elevated serum levels of SFA A. Upon adb/db mice, the latter representing an established model of obesity-associated impaired wound healing with disturbed macrophage differentiation db/db mice, S100A8 and S100A9 expression is rapidly induced upon injury  and in obese mice, respectively  might be an additional yet unrecognized source of S100A9 during inflammation or tissue repair  and fibroblasts (detected by CD90) express only very low level of S100A9 protein. Gating on CD45+/Ly6G-/F4/80+ cells indicate that macrophages also express S100A9 protein only on a low level .ex vivo with obesity-associated mediators as described for keratinocytes and S100A9 gene expression in the dWAT was analyzed . Indeedrol mice B. We alse Figure C. In pare Figure D in the Finally, to prove the importance of S100A9 and elevated SFA as driver of the IL-1β-mediated upregulation of S100A9 we blocked their activity during IMQ-driven skin inflammation in short-HFD. First, we analyzed short-HFD mice after blocking S100A9 function by paquinimod at day 4 when inflammation had established and inflamed skin of short-HFD mice is characterized by elevated levels of IL-1β and S100A9. Indeed, release of IL-1β and gene and protein expression of S100A9 in inflamed skin of short-HFD-mice treated with paquinimod is reduced to the level of chow control mice Figure E-G. ThisSince our data indicate that SFA are one important driver of S100A9 overexpression in obesity we investigated whether reduction of SFA restores M2 differentiation. Consistent with the lack of S100A9 overexpression Figure J-K, shorin vitro and in vivo data demonstrate that sustained overexpression of S100A9 in skin inflammation in obesity is mediated by SFA that initiate and drive a positive feedback loop between skin cells and the dermal macrophage compartment via the induction of an inflammasome-dependent IL-1β release  In the present study, we show that obesity-associated overexpression of S100A9 in addition to its known chemotactic and pro-inflammatory actions impairs macrophage differentiation that contributes to amplification of skin inflammation and delayed resolution as well as impaired skin repair in obesity.db/db mice at times when M2 activation and inflammatory resolution had already occurred in the respective control mice suggesting an unknown role of S100A9 in the control of macrophage activation. In vitro experiments with murine peritoneal macrophages demonstrate that addition of exogenous recombinant S100A9 disturbed IL-4-mediated differentiation into M2-like macrophages. These cells showed a gene expression profile with a mixture of typical M1 and M2 genes but important genes of M2-macrophage differentiation including relmα and CD301 S100A9 belongs to the family of danger-associated molecular patterns induced upon infection, injury or inflammation to initiate the first rapid inflammatory response in vivo in situations of tumor burden or inflammatory challenge in vivo in multiple forms. This plasticity might contribute to their diverse and sometimes opposite functions It has been described that S100A8 and S100A9 exist as homodimers but preferentially form the S100A8/A9 heterodimer  in the presence of Zn2+ and Ca2+ TLR4 and RAGE have been described as receptors for S100A9 Proteome analysis indicates that S100A9 interfere with a multitude of pathways during M2-like macrophage differentiation underlining the impact of S100A9 on macrophage differentiation. Thus, S100A9 induces several pro-inflammatory pathways while Stat6, one of the master regulators of M2-like macrophage differentiation in vitro findings of impaired M2-differentiation by S100A9, we observed disturbed M2-like macrophage differentiation in skin inflammation and wound healing when S100A9 expression is pathologically increased. A crucial role for S100A9 in the control of macrophage function was also described by Dessing et al. They observed an increased activity of M2-like macrophages associated with fibrosis in a renal ischemia/reperfusion injury model in S100A9-knockout (KO) mice db/db mice prove the novel role of S100A9 in the control of macrophage polarization. S100A9 was blocked by paquinimod which inhibits the binding of S100A9 to its receptors, TLR4 and RAGE db/db mice which are characterized by a disturbed M1-M2 balance and impaired wound healing, resembling some features of non-healing wounds in diabetic patients db/db mice. In parallel, wound repair was improved in these mice. Again, we administered paquinimod two days after wounding to avoid affecting the known chemotactic activity of S100A9. Hence, we assume that less severe skin inflammation and improved wound healing in paquinimod-treated mice are attributed to the restored activation of M2-macrophages due to blocking of S100A9 activity in the later phase of inflammation. We acknowledge that we do not provide direct evidence of disturbed M2-macrophage differentiation as ultimate cause for increased skin inflammation and delayed wound repair in obese mice. However, restored activation of M2 macrophages after blocking S100A9 indicates an association. In this context, several studies have demonstrated the decisive role of M2 macrophages in regulating inflammation and tissue repair by improving inflammatory processes and wound healing through targeted support of M2 macrophage activation Consistent to our db/db mice suggesting that obesity per se does not affect S100A9 expression. S100A9 secretion is induced during infection, injury and inflammation. Neutrophils and monocytes have been described as cellular source (34). However, in the present study we focus on the effect of S100A9 on M2-like macrophage differentiation which takes place at a time when neutrophils and monocytes had been mostly removed suggesting that S100A9 derived from these cells might play only a minor role in the control of M2-like macrophage differentiation. Consistent with studies showing high expression of S100A9 in keratinocytes in psoriatic skin lesions and in wounds In the present study we found almost no expression of S100A9 in skin of lean, obese and in vitro data suggest an important role of SFA in the control of S100A9 expression we used short-HFD mice where mice were fed a HFD enriched in SFA for only 6 weeks. The short-HFD mice had a normal body weight and glucose metabolism but showed elevated serum levels of SFA. They therefore represent an excellent model to investigate the role of SFA independent of other HFD-induced metabolic changes such as hyperglycemia or insulin resistance. Indeed, we detected increased IL1-β amounts in inflamed skin of short-HFD-fed mice and increased activity of caspase-1. Various studies report the capability of SFA to activate NLRP3 inflammasome and release of IL-1β in cells in response to a stimulation with the TLR4 agonist lipopolysaccharide in vivo. Abrogation or downregulation of IL-1β release after reduction of SFA in short-HFD-fed mice demonstrate the requirement of elevated SFA level for IL-1β promotion in this context. IL-1β is an important regulatory cytokine in the onset of inflammation processes and activates immune and tissue cells ex vivo skin cultures. Consistently, sustained overexpression of S100A9 in skin inflammation in short HFD-mice and obese mice coincides with an increase of IL-1β in myeloid cells isolated from the lesional skin. Consequently, inhibition of S100A9 or reduction of SFA as one driver of S100A9 overexpression in obesity breaks this viscous cycle of S100A9 overexpression.Typical obesity-associated mediators such as high SFA, glucose or insulin do not directly change S100A9 expression in keratinocytes and the dWAT. However, TNFα and IL-1β stimulated S100A9 expression and the combination of both resulted in a synergistic effect. Various pathways and transcription factors including NFkB, MAPK, p53, HIF1α, Hbx, and Stat3 have been described to regulated S100A9 expression In summary, in obesity overexpression of S100A9 impairs appropriate macrophage activation and polarization preventing resolution of skin inflammation that finally contributes to a sustained and amplified skin inflammation and impaired tissue repair in obesity Figure .Four weeks old male C57BL/6 mice were fed with HFD ) or chow diet (4.5% crude fat) for 6 or 15 weeks Skin inflammation was induced by topical application of 125 mg IMQ  on the shaved back of C57BL/6 mice including the dorsal side of the ears. Severity score representing the sum of erythema and scaling of the back skin  was determined by two independent researchers. Ear thickening was determined using a caliper. Treatment with the Q compound paquinimod  was started 10 h after IMQ application. It was administered via drinking water to achieve a concentration of 20 mg/kg/d.db/db mice (13 weeks old) under anesthesia. Treatment with paquinimod  at a concentration of 20 mg/kg/d was started two days after wounding. Wounds were harvested at indicated time points post wounding.Wound healing study: Wounding was performed with full-thickness 6 mm punch biopsies on the back of C57BL/6 mice or Myeloid cells were isolated after enzymatic digestion of skin by 0.15 mg/ml liberase  and 0.12 mg/ml DNase  as described Epidermal cells were isolated upon segregation of the epidermis from dermis by overnight incubation with 0.25% trypsin .Dermal WAT was mechanically removed from the dermis. For isolation of SVF and dWAT dWAT was digested by 0.15 mg/ml liberase (Roche) for 1 h at 37°C. Tissue homogenate was filtered through a 70 µm filter and centrifuged at 4000 rpm for 10 min. The floating cell layer containing adipocytes was isolated and washed once. The cell pellet represents the SVF and was washed once with PBS.Total RNA from homogenized skin biopsies and cells was isolated using the Relia Prep RNA Tissue Miniprep System  according to the manufacturer′s protocol. cDNA was generated from a total of 500 ng RNA with LunaScript RT Supermix  as described in the manufacturer′s protocol. Quantitative real-time PCR was performed with LunaUniversal qPCR Mastermix (NEB) according to the manufacturer′s instructions see .Before microarray analysis RNA integrity and concentration were examined on an Agilent Fragment Analyzer  using the HS RNA Kit (Agilent) according to the manufacturer's instructions. Microarray analysis was conducted at the Core Unit DNA Technologies . cRNA was prepared from 100 ng of total RNA hybridized to Gene Chip Clariom S arrays  according to the manufacturer's instructions. The arrays were scanned with a third generation AffymetrixGeneChipScanner 3000. For data analysis, Affymetrix Gene Chip data were extracted from fluorescence intensities and were scaled in order to normalize data for inter-array comparison using Transcriptome Analysis Console (TAC) 4.0.2 software according to manufacturer's instruction (Thermo Fisher Scientific).6) from two different mice were isolated and cultured at 8 x 105 cells/ml in RPMI 1640 medium (Thermo Fisher Scientific) containing 10% FCS (ThermoFisher Scientific), 1% penicillin/streptomycin (Biochrom AG), and 10 ng/ml M-CSF (Miltenyi) for 24 h. M2-like macrophage differentiation was induced by addition of 20 ng/ml IL-4 (Miltenyi) in presence or absence of 1.5 µg/ml recombinant murine S100A9 homodimer . Cells were lysed with scioExtract buffer after 30 min according to the manufacture's protocol  and stored in liquid nitrogen. Protein labelling and analysis was performed by Sciomics GmbH. Upon labelling with scioDye 2 samples were analyzed on a scioDiscover antibody microarrays (Sciomics) targeting 1,352 different proteins with 1,821 antibodies. Afterwards microarrays were incubated with a pan-phospho-antibody. Slide scanning was conducted using a Powerscanner . Spot segmentation was performed with GenePix Pro 6.0 . Acquired raw data were analyzed using the linear models for microarray data (LIMMA) package of R-Bioconductor after uploading the median signal intensities. For normalization, a Cyclic Loess normalization was applied. Differences in protein abundance or phosphorylation level between sample groups are presented as log-fold changes (logFC) calculated for the basis 2. Pathway analysis was performed with KEGG database.Mouse peritoneal cells (1×10Skin was homogenized in ProcartaPlex® Cell Lysis Buffer (Thermo Fisher Scientific) containing 1 mM PMSF (Miltenyi) using 5 mm Stainless Steel Beads  and TissueLyser LT (Qiagen). Lysates were centrifuged for 10 min at 16,000 x g, 4°C. Proteins were separated by 8-16% SDS PAGE  and blotted onto nitrocellulose membrane. Anti-S100A9 antibody , anti-rpl26 (Sigma), anti-GAPDH , anti-p-Stat6, anti-p-Erk, anti-p-NFkB, anti-p-p38  were used. After overnight incubation and washing steps anti-rabbit IRDye680 labelled antibody (Invitrogen) was added followed by washing and detection at LI-COR Odyssey Fc . Signals of investigated proteins were normalized to reference proteins Rpl26 or GAPDH.Murine IL-1ß, TNFα, IL-6 were detected by ELISA according to the manufacturer´s protocol (Thermo Fisher Scientific) and detected using Synergy HT .Cells were incubated with antibody mixture including anti-F4/80-PE (BioRad), anti-Ly6G-FITC, anti-CD11b-PECy7, anti-CD45-PerCPCy5.5 and anti-Ly6C-APC or, anti-CD301-APC or anti-CD206 APC  for 15 min and subsequently, 0.4 µl zombie dye (Biolegend) was added for 5 min. For detection of S100A9 protein intracellular staining was performed using the True-Nuclear™ Transcription Factor Buffer Set. Cells were first stained with antibody mixture including anti-CD45-PerCPCy5.5, anti-CD11b-PECy7, anti-Ly6G-APC, anti-F4/80-PE or anti-CD45-PerCPCy5.5, CD31-PE, CD90-PE-Cy7 for 15 min, washed and fixed for 45 min before intracellular staining with goat anti-S100A9 antibody (R&D) for 30 min, washing and addition of secondary antibody  for further 30 min.TMII or BD FACSLyric™ and data analysed with BD FACSDivaTM Software or BD FACSuite™ Application . Using forward and sideward scatter single cells were gated. Living singlets were chosen from zombie-negative cells as described Flow cytometry was performed with BD FACSCantoTissue sections were stained by Masson trichrome staining. Immunofluorescence staining was performed with a S100A9 antibody (R&D) and anti-goat-Alexa546 (Invitrogen). Images were captured using KEYENCE BZ-9000 fluorescence microscope .Mouse myeloid cells were generated from peritoneal lavage cells of C57BL/6 mice. Cells were cultured at 8 x 105 cells/ml in RPMI 1640 medium (Thermo Fisher Scientific) containing 10% FCS (ThermoScientific), 1% penicillin/streptomycin (Biochrom GmbH), and 10 ng/ml M-CSF (Miltenyi) for 24 h. If indicated peritoneal-derived myeloid cells were stimulated as follows: 1.5 µg/ml recombinant murine S100A9 homodimer (R&D), 10 ng/ml TNFα (Miltenyi), or 10 ng/ml IL-1β (Miltenyi). In addition, cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) containing 5 mM glucose (Invitrogen), 10% FCS (ThermoScientific), 1% penicillin/streptomycin (Biochrom GmbH), and 5 ng/ml M-CSF (Miltenyi) for 24 h and then stimulated with 25 mM glucose. In co-stimulation experiments S100A9 homodimer was added 3 hours after addition of 25 mM glucose. For stimulation with FFA cells were incubated with 500 µM PA, SA or OA (Sigma-Aldrich) complexed to bovine serum albumin (BSA) as previously described Differentiation of M1- and M2-like macrophages was induced in peritoneal-derived cells by addition of 100 ng/ml LPS (Sigma) / 20 ng/ml INFγ (Milteny) and 20 ng/ml IL-4 (Miltenyi) for 24 h, respectively. If indicated, S100A9 homodimer (1.5 µg/ml), S100A8 homodimer , or S100A8/A9 heterodimer  was added during M2-like differentiation. NFkB, MAPK, PI3K, TLR4 and RAGE signaling was inhibited by addition of 4 µM BMS-345541 , 10 nM Cobitinimib , 5 µM Apitolisib (Selleckchem), 1 µM CLI-095 (Invivogen) and 10 µM RAGE antagonistic peptide (R&D), respectively.Human peripheral blood monocytes were isolated by magnetic bead isolation of CD14+ cells from blood of healthy volunteers in compliance with institutional ethical use protocols (number of the vote 428/16-ek). CD14+ monocytes were seeded in RPMI  supplemented with 1% penicillin/streptomycin, 1% glutamine (Biochrom AG) and 10% heat inactivated FCS (Biochrom AG) at 0.5 × 106 cells per well in the presence of 50 ng/ml M-CSF (Miltenyi).Keratinocytes: A spontaneously immortalized mouse keratinocyte cell line was kindly provided by M. Magin , in 5% CO2 at 37°C Mouse skin was cultured in supernatants from peritoneal cells after stimulation with FFA, S100A9 homodimer or co-stimulation with FFA and S100A9 homodimer and all controls as described above. For neutralization of IL-1β and TNFα activity 200 ng/ml IL-1 receptor antagonist (Invitrogen) or 1 µg/ml aTNFα (R&D) were added.ex vivo with 10 ng/ml IL-1β in the presence of NFkB inhibitor  or MAPK inhibitor . Skin samples were analyzed after 24 h of culture. Epidermis was separate by incubation with 0.25% trypsin (Sigma) for 1 h at 37°C. Dermal WAT was mechanically removed.Mouse skin was stimulated Serum aliquots (60 µl) were mixed with 1 ml methanol and centrifuged at 13,000 × g for 2 min to precipitate proteins. Lipids were extracted from the supernatant by the addition of 1 ml chloroform and water according to Bligh and Dyer Statistical analysis for two-group comparisons regarding normally distributed metrical data was performed using two-tailed Student's t test. Normality was tested by D'Agostino & Pearson Normality test or Shapiro-Wilk-Test (n≤4). Where normality was absent, Mann-Whitney test was used. For statistical comparison of more than two groups, ANOVA Test was used. As post-hoc test in case of significance of the ANOVA, Dunnet's T3 multiple comparison test was used. Calculations were done using GraphPad Prism version 7.02. P-values of 0.05 or smaller were consider statistically significant. The different degrees of significance were indicated as followed: * P < 0.05; ** P < 0.01; *** P < 0.001All animal experiments were performed according to institutional and state guidelines and were approved by the Committee on Animal Welfare of Saxony .Supplementary figures and table.Click here for additional data file.


Acute exacerbation (AE) of interstitial pneumonia (IP) is the most fatal complication after lung resection for lung cancer. To improve the prognosis of lung cancer with IP, the risk factors of AE of IP after lung resection should be assessed. S100 calcium-binding protein A4 (S100A4) is a member of the S100 family of proteins and is a known marker of tissue fibrosis. We examined the usefulness of S100A4 in predicting AE of IP after lung resection for lung cancer.This study included 162 patients with IP findings on preoperative high-resolution computed tomography scan who underwent curative-intent lung resection for primary lung cancer between April 2007 and March 2019. Serum samples were collected preoperatively. Resected lung tissue from 76 patients exhibited usual IP (UIP) pattern in resected lung were performed immunohistochemistry (IHC). Relationship between S100A4 and the incidence of AE of IP and short-term mortality was analyzed.P < 0.001), with a sensitivity of 93.8% and a specificity of 75.3% at the cutoff value of 17.13 ng/mL. Multivariable analysis revealed that a high serum S100A4 level (> 17.13 ng/mL) was a significant risk factor for AE of IP . A 1-year overall survival (OS) was significantly shorter in patients with high serum levels of S100A4 (75.3%) than in those with low serum levels . IHC staining revealed that fibroblasts, lymphocytes, and macrophages expressed S100A4 in the UIP area, and the stroma and fibrosis in the primary tumor expressed S100A4, whereas tumor cells did not.The receiver operating characteristic area under the curve for serum S100A4 to predict postoperative AE of IP was 0.871 (95% confidence interval [CI], 0.799–0.943; Serum S100A4 had a high predictive value for postoperative AE of IP and short-term mortality after lung resection.The online version contains supplementary material available at 10.1186/s12890-021-01554-y. Interstitial pneumonia (IP), mostly idiopathic pulmonary fibrosis (IPF), is associated with an increasing risk of lung cancer , 2. Appr−/− mice [S100 calcium-binding protein A4 (S100A4) is a member of the S100 family of proteins and is a known marker of tissue fibrosis . Reporte−/− mice . Based oTNM Classification for Lung Cancer [This study was approved by the Ethics Committee of Hiroshima University Hospital . Patient consent was obtained by using informed consent documents with an opt-out process. The study was carried out in accordance to institutional guidelines which is established based on the Declaration of Helsinki. Patients in whom IP had been diagnosed on preoperative high-resolution computed tomography (HRCT) and had undergone lung resection for primary lung cancer between April 2010 and March 2019 were included in this study. Twenty patients for whom preoperative serum samples were unavailable were excluded from this study. Among included patients, patients with UIP pattern in resected specimen was underwent immunohistochemistry (IHC). Flowchart of patient selection was shown in Additional file g Cancer .Fig. 1SeA 16-row multidetector CT was used to obtain chest images. We used the following parameters for high-resolution images of the lungs: 120 kVp, 200 mA, 1–2 mm section thickness, 512 × 512 pixel resolution, 0.5–1.0 s scanning time, a high-spatial reconstruction algorithm with a 20 cm field of view, and mediastinal  and lung  window settings. ILD was defined radiologically according to the American Thoracic Society (ATS), European Respiratory Society (ERS), Japanese Respiratory Society (JRS), and Latin American Thoracic Association classifications (ALAT) and the patterns were classified as usual IP (UIP), possible UIP pattern, and inconsistent with UIP .Hybrid video-assisted thoracic surgery was conducted as an approach method . PostopeTNM Classification of Lung Cancer [The pathological stage of lung cancer is based on the eighth edition of the g Cancer . Becauseg Cancer : (1) eviTo conduct the IHC study, we used 3 μm-thick tissue sections from representative formalin-fixed paraffin-embedded (FFPE) blocks of IP and the primary tumor and an immunohistochemical autostainer  with the ultraView Universal DAB Detection Kit (Roche-Ventana Diagnostics). Rabbit anti-human S100A4 monoclonal antibody , diluted 250 times with an antibody diluent (Roche-Ventana Diagnostics) were used as the primary antibody. Representative FFPE blocks of IP area and primary tumor were selected by pathologists (K.K and Y.T) who did not know the patients’ outcomes. The expression of S100A4 in IP area and primary tumor was evaluated.t-test. To evaluate the independent risk factors for AE of IP, a logistic regression model was used. Sex , presence of collagen disease, percent predicted vital capacity , percent diffusing capacity for carbon monoxide  [P value of < 0.05 was considered statistically significant for all analyses.Data were presented as median and interquartile range (IQR) for continuous variables and n (%) for categorical variables. Categorical variables were compared using a chi-square test. Continuous variables were analyzed using the unpaired ; ≤ 40%) , serum lP < 0.001). There were no differences in the values of S100A4 between histological subtype of NSCLC . The incidence of AE of IP more than grade IIIa was also higher among patients with high S100A4 levels (16.0%) than among those with low S100A4 levels . The incidence of any grade of respiratory complications was also higher among patients with high S100A4 levels (42.0%) than among those with low S100A4 levels . Among the patients with high S100A4 levels, the 30-day mortality rate was 8.0% (P = 0.002) and 90-day mortality rate was 16.0% (P < 0.001); both rates were 0% among the patients with low S100A4 levels  with high S100A4 levels but in only 2 (1.8%) with low S100A4 levels , male (P = 0.010), low %VC (≤ 80%) , high KL-6 (≥ 1000) , and operative time  were significant predictive factor of AE of IP. In the multivariable analysis, high S100A4 level , male (P = 0.033), low %VC (≤ 80%) , and operative time  were significant predictive factor of AE of IP  than in those with lower S100A4 levels   that serExtracellular S100A4 was also shown to activate lung fibroblasts , and theSeveral researchers have examined whether S100A4 could be a therapeutic target. Niclosamide, an approved antihelminthic drug for the treatment of tapeworm infections, inhibited metastasis formation in a mouse model of colon cancer by blocking S100A4 expression and its effects on the WNT/CTNNB1 signaling pathway . In a moThis study had several limitations. First, at present, S100A4 cannot be easily measured in daily clinical practice. Second, this study was retrospective and conducted at a single institute. The number of included patients was small—although, being from a single institution, the cohort was relatively large—and in multivariable analysis, the well-known risk factors of AE of IP, such as DLCO or UIP pattern on preoperative CT, were not significant predictive factors. Therefore, confirmation by a large trial, such as Japan Clinical Oncology Group 1708 , is neceAmong patients with high serum levels of S100A4, AE of IP occurred more frequently and the short-term mortality rate after surgery was higher than those with low serum levels of S100A4. Although a study with a larger sample size is necessary to confirm our findings, the serum level of S100A4 appears to be a significant predictor of AE of IP after lung resection for lung cancer.Additional file 1: Fig. S1. Flowchart of patient selection. A total of 182 patients underwent curative-intent resection for primary lung cancer. Serum samples were obtained before resection in 162 patients, and the S100A4 level was measured. UIP was diagnosed in surgically resected specimens from 76 patients, and these specimens were subjected to immunohistochemistry for S100A4.Additional file 2: Fig. S2. Representative computed tomographic images of IP. Representative images of UIP pattern (A), possible UIP pattern (B), and pattern inconsistent with UIP (C) according to the ATS, ERS, JRS, and ALTA classifications.Additional file 3: Fig. S3. Representative image of UIP pattern. UIP pattern in resected specimens was diagnosed based on the following features, according to the guidelines of IP from the ATS, ERS, JRS, and ALTA: (1) evidence of marked fibrosis/architectural distortion and honeycombing in a predominantly subpleural/paraseptal distribution; (2) presence of patchy involvement of lung parenchyma by fibrosis; (3) presence of fibroblast foci (*); and (4) absence of features suggesting an alternative diagnosis.Additional file 4: Fig. S4. ROC curve of KL-6 level to predict AE of IP. ROC curve analysis of the level of KL-6 in predicting postoperative AE of IP .Additional file 5: Table S1. Computed tomography findings of interstitial pneumonia.Additional file 6: Table S2. Characteristics of patients who underwent immunohistochemistry.


The phosphatidylinositol 3-kinase (PI3K) is frequently hyperactivated in cancer and plays important roles in both malignant and immune cells. The effect of PI3Kα inhibitors on the tumor microenvironment (TME) remains largely unknown. Here, we investigated the modulation of the TME by a clinical PI3Kα-specific inhibitor CYH33.The activity of CYH33 against a panel of murine tumors in the immune-competent context or athymic mice was detected. Single-cell RNA sequencing and multi-parameter flow cytometry were performed to determine the immune profiling of TME. The effect of CYH33 on immune cells was conducted with primary murine cells.+T and CD4+T cells, while attenuating M2-like macrophages and regulatory CD4+T cells. Increase in memory T cells was confirmed by the induction of long-term immune memory on CYH33 treatment. Mechanistically, CYH33 relieved the suppressed expansion of CD8+T cells via preferential polarization of the macrophages to the M1 phenotype. CYH33 promoted fatty acid (FA) metabolism in the TME, while FA enhanced the activity of CD8+T cells in vitro. The combination of CYH33 with the FA synthase (FASN) inhibitor C75 synergistically inhibited tumor growth with enhanced host immunity.CYH33 exhibited more potent antitumor activity in immune-competent context. CYH33 enhanced the infiltration and activation of CD8CYH33 induces immune activation and synergizes with FASN inhibitor to further promote the antitumor immunity, which gains novel insights into how PI3K inhibitors exert their activity by modulating TME and provides a rationale for the concurrent targeting of PI3K and FASN in breast cancer treatment. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin pathway is one of the most frequently dysregulated signaling pathways in human cancer.+CD25highFoxP3+ regulatory T cells (Tregs) contribute to the inhibition of cytotoxic CD8+T cells and tumor progression.+T cells.Tumor progression depends not only on the intrinsic hallmarks of cancer cells but also on the formation of a milieu that escapes immune surveillance. Regulatory immune cells including myeloid-derived suppressor cells (MDSCs), M2-like tumor-associated macrophages (TAMs), and CD41 phase arrest.CYH33 is a novel PI3Kα-selective inhibitor discovered by our group with a distinctive structure, which exhibits superior anticancer activity both in vitro and in vivo compared with alpelisib.+T cells mediated by M2-like macrophages by reprogramming macrophages. CYH33 treatment also upregulated fatty acid (FA) metabolism, which was accompanied with increased free FA (FFA) level and the activity of CD8+T cells. The combination of CYH33 with the FA synthase (FASN) inhibitor C75 further enhanced the immunogenic effect of CYH33 and led to synergistic antitumor efficacy.In this study, we screened the activity of CYH33 in a panel of murine tumor cells and allografts inoculated in immune-competent mice. Deep immune profiling of tumor tissue by single-cell RNA-seq (scRNA-seq) and flow cytometry demonstrated that CYH33 enhanced the infiltration and activation of T cells as well as modulated the myeloid cells. CYH33 may release the suppression of CD8CYH33 was provided by Shanghai HaiHe Biopharma Co., Ltd. Alpelisib was purchased from Dalian Meilun Biotechnology . C75 was purchased from MedChemExpress . All compounds were dissolved in dimethyl sulfoxide  at the concentration of 10 mM and stored at −20°C. For in vivo experiments, CYH33 was dissolved in normal saline containing 0.5% Tween 80  and 1% CMC-Na .4T1, EMT6, LLC1, B16F10, KLN-205, A20, and CT26 cells were obtained from the American Type Culture Collection . Colon 26 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank . PY8119 cells were generously provided by Dr. Suling Liu . U14 and MC38 cells were obtained from Cobioer Biosciences . All cells were maintained in the medium suggested by the suppliers.control cells-ODtreated cells)/(ODcontrol cells-ODDay0 cells)×100%. GI50 values were obtained using four parameter concentration-response curve fitting with SoftMaxPro .Cell proliferation was evaluated by sulforhodamine B (SRB) assay as described previouslyCell lysates were prepared and subjected to western blotting with primary antibodies against AKT (No. 4691), phosphor-AKT , phosphor-AKT  , and β-Actin . Images were captured with the ImageQuant LAS 4000 system .3. Mice were administered orally with vehicle, CYH33, and alpelisib or intraperitoneally injected with C75. Body weight and tumor volume were measured and recorded twice per week. The tumor volume (V) was calculated using the formula: V=0.5 × length (mm) ×width (mm)2. Relative tumor volume (RTV) was calculated as the ratio of tumor volume at the given time point versus tumor volume at the initial treatment. The treatment to control ratio (T/C) was calculated as the following formula: T/C (%) = (TRTV/CRTV)×100%, where TRTV and CRTV represented the RTV of treatment and vehicle control group, respectively. The synergy of the combination of CYH33 and C75 in vivo was determined using the Bliss independence model, namely fractional product method, as described previously.24 26Female Balb/c, Balb/c nude, C57BL/6, and DBA/2 mice aged 6–8 weeks old were obtained from Shanghai Institute of Materia Medica . Tumor cells were subcutaneously inoculated into the right side of flank. Animals were randomized to receive vehicle or tested compounds when tumor volume reaches about 50 mmTumor-bearing mice were sacrificed after drug treatment for 7 days. Single-cell suspension was prepared as previously described with modifications.Single-cell suspension prepared from tumor tissue were pre-incubated with anti-CD16/32 monoclonal antibody  and then stained with antibodies against membrane markers for 45 min at 4℃. Dead cells were marked using Fixable Viability Stain 510 . For intracellular staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set  or the Intracellular Fixation & Permeabilization Buffer Set  following the manufacture’s instruction. Antibodies were added and incubated for 1 hour at 4℃. Intracellular cytokine staining was performed 4–6 hours after ex vivo stimulation with leucocyte activation cocktail in the presence of GolgiStop  at 37℃. All the fluorescently labeled antibodies used for staining are listed in 10.1136/jitc-2021-003093.supp1Supplementary data10.1136/jitc-2021-003093.supp2Supplementary data+ tumor-infiltrating leucocytes were enriched from the cell suspension using CD45 (TIL) MicroBeads  according to the manufacture’s instruction. The single-cell suspension of tumor cells or isolated CD45+ cells with high cell viability was loaded and single cells were captured by the BD Rhapsody Single-cell Analysis System. Single-cell cDNA was prepared using BD Rhapsody cDNA Kit (No. 633773). The constructed cDNA libraries were sequenced with the Illumina NovaSeq PE150 platform and each unique molecular identifier was captured at least six times. For analysis, the sequencing data were processed using Rhapsody pipeline (V.1.8). The expression matrix of each sample was used for further analysis using the R package Seurat (V.3.1.4)Tumor samples were processed using the mouse tumor dissociation kit . CD45The depletion of T cells and macrophages were conducted as previously described.+T, CD4+T, and CD4+CD25−T cells were enriched from cell suspension using EasySep Mouse CD8+T cell Isolation Kit , Mouse CD4+T cell Isolation Kit , or EasySep Mouse CD4+CD25+ Tregs Isolation Kit II , respectively. Tregs were induced from CD4+CD25-T cells for 5 days as described.31Fresh murine spleen tissue was cut into small species and subjected to digestion using mouse Spleen Dissociation Kit  according to the manufacture’s protocol. CD8+T cells were cultured for 72 hours in 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL of interleukin 2 (IL-2) . CD4+T cells and Tregs were stimulated and cultured for 72 hours as described.31 32For proliferation assay, T cells were labeled with 5 µM carboxyfluorescein succinimidyl amino ester  for 15 min in serum-free medium. CD8+T cells, palmitic acid /bovine serum albumin  complex was prepared as reported.+T cells cultured in glucose-free medium  were incubated with 25 µM oleic acid and 12.5 µM palmitic acid or vehicle control for 72 hours.To detect the effect of FFA on the CD8+T cells, CFSE-labeled CD8+T cells were cultured alone or cocultured with the M2-polarized bone-marrow-derived macrophages (BMDMs) (at a 2:1 ratio) in 1640 medium supplemented with β-mercaptoethanol (0.05 mM), anti-CD3/CD28 dynabeads , and colony-stimulating factor-1 (CSF-1)  for 72 hours. The proliferation of T cells was measured by flow cytometry.To detect the effect of TAM on the proliferation of CD8Bone marrow-derived monocytes were collected from 5 to 6 weeks old Balb/c mice and cultured in 1640 medium supplemented with 10% FBS and 20 ng/mL of CSF-1 for 6–7 days to obtain M0 BMDMs. Induction of M1 or M2 polarization was performed as previously described.Total RNA was extracted using the RNeasy Mini Kit  according to the manufacture’s protocol. cDNA was synthesized with total RNA using a reverse transcription kit . Quantitative PCR (qPCR) was performed with the cDNA and specific primers using SYBR Green Master Mix . The primers used were listed in http://software.broadinstitute.org/gsea/index.jsp.RNA sequencing was performed by WuXi AppTec using the Illumina-HiSeq. Differentially expressed genes were identified with p value less than 0.01. Pathway enrichment analysis was carried out using GSEA, Tumor tissues were fixed in 4% paraformaldehyde. Paraffin embedding, H&E staining and immunohistochemistry against CD45, CD4, CD8, CD11b, F4/80, CD206, cleaved-caspase 3, and Ki67 were conducted by Shanghai ZuoCheng Bio Company . Slides were observed under a Leica DM6 B microscope equipped with sCMOS camera .Total lipids were extracted from tumor tissue as described.All statistical analyses were conducted using GraphPad Prism V.7  or R (V.3.5.1). Differences between two groups were calculated by unpaired two-tailed Student’s t-test. Differences among multiple groups were calculated by one-way or two-way analysis of variance. Differences were considered statistically significant when p value was less than 0.05. Data are presented as mean±SD unless otherwise noted. For each parameter of all data presented, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.50 over 10 µM. In comparison, CYH33 exhibited moderate antiproliferative activity against PY8119 and EMT6 cells with a GI50 of 2.29 µM or 3.16 µM, respectively. Consistent with previous studies,We previously reported that CYH33 overcame TAM-mediated resistance to radiation therapy, suggesting that CYH33 may execute its activity by reshaping the TME.To evaluate the efficacy of CYH33 in vivo, three lines of cells were inoculated to immune-competent or nude mice. CYH33 at 20 mg/kg markedly suppressed the growth of 4T1, PY8119 or EMT6 tumors in immune-competent mice with T/C values of 25.5%, 18.0% or 42.5%, respectively . Alpelis50 failed to be significantly correlated with that in vivo represented as the T/C value. This observation was consistent with that in 4T1 cells, indicating that CYH33 may execute its activity beyond direct action on tumor cells.To determine the efficacy of CYH33 in tumors originating from other tissue types in immune-competent mice, a panel of murine tumor cells was screened in vitro and in vivo. As shown in − tumor-like cells, with an expression profile similar to that of Krt18+ cells but with low Krt18 expression. To determine the cluster distribution among different treatment groups, UMAP plot of each sample was shown , which is the indicative marker of T-cell exhaustion, was observed in CD8+T cells  injection of anti-CD8 or anti-CD4 antibodies, respectively  and M2-polarized (IC50=1.658 µM) BMDMs in a dose-dependent manner, whereas little effect on the proliferation of M1-polarized BMDMs was observed. We next detected the effect of CYH33 on the polarization of BMDMs toward M1 or M2, respectively  among class I PI3K isoforms, it strongly inhibited PI3Kδ kinase activity (IC50=78.7 nM) and PI3Kδ-medicated signaling at pharmacological concentrations,+T cells, which might be due to preferential polarization toward M1 instead of M2 in the presence of CYH33. These findings are in line with our previous report that CYH33 decreased the proportion of CD206-positive cells.et alPI3K inhibitors regulate immune cells and influence the interaction between tumor and immune cells.of Tregs . Selecti+T cells in nutrient-deficient TME. Supplementation of FFA results in augmented expansion of CD8+T cells.+T cells in the tumor tissue. It is noteworthy that C75 treatment enhanced the FFA content while inhibited the FA synthesis. The level of FFA is determined by the balance between the anabolism and catabolism of cellular lipid. C75 treatment has been found to activate lipolysis and enhance the efflux of FFA.The PI3K/AKT pathway plays important roles in a variety of metabolic processes by direct phosphorylation of metabolic enzymes or regulation of various transcription factors,In conclusion, CYH33 elicited antitumor immune response against multiple tumors that originated from different tissue types in an immune-competent context via modulating the landscape of immune cells and reprograming the FA metabolism. Combination with C75 further promoted tumor immunogenicity, which demonstrated that the regulation of metabolism by PI3K inhibition could reshape the microenvironment. Our findings gain novel insights into how PI3K inhibitors exert their activity by modulating the tumor-immune interaction, and provide a rationale for the concurrent targeting of PI3K and FASN inhibitor in breast cancer treatment.