Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied. Glossina spp.) are the vectors that transmit unicellular protozoa of genus Trypanosoma that cause human African trypanosomiasis (HAT), also known as sleeping sickness and animal African trypanosomiasis . They are obligate blood-feeders that serve as a ‘transporters’ and provide an environment where the parasite can differentiate, multiply, and become infective to mammals. Some studies have revealed that the ingestion of trypanosomes during a blood meal does not always result in mature infections3. Preference for tsetse fly mammalian hosts  can differ greatly depending on the species of Glossina, wildlife, and geographical locations6. Tsetse flies may originally have been reptile feeders lining in forests, later becoming adapted to feeding on mammals. Earlier reports indicate that warthogs facilitated tsetse flies to migrate from the ancient forest habitat (possible origin) to the Savannah habitat7. The morsitans subgenus is found in woodland Savannah and bushy areas where there is plenty of game animals. A review of the natural hosts of 17 tsetse species and subspecies from different regions of Africa collated from published and unpublished work from 1953 to 1991, comprising altogether 47, 697 blood meals revealed that Glossina morsitans mainly prefer warthog as a blood meal source and bovids were found to be important especially Kudu, buffalo, bushbuck, and elands8. The morsitans subgenus can change behavior and live off domestic animals, especially cattle, in the absence of game animals. This behavior has been reported in Sudan ; and in Nigeria, where these tsetse flies used to live along a busy cattle trekking route passing through a densely populated and heavily cultivated area, well outside its usual range9. It has also been observed that G. morsitans do not readily change its habitats to feed on man and his domestic animals and is one of the species that disappear when the human population in an area grows10. On the other hand, the palpalis subgenus, usually residing in riverine vegetation, are closely associated ecologically with reptiles but opportunistically feed on mammals in their habitats including humans10. Given the difference in blood composition between various mammalian hosts, blood meals ingested from different host species could affect the structure of gut microbiota in tsetse flies, which could explain some of the previously observed geographical variations11. Blood source and composition may, therefore, be essential factors that modulate vector competence, through complex interactions between nutrients, immunity and bacterial communities. An earlier study found that delipidation of serum has a similar effect as complete removal of dietary serum from pig and cattle blood and significantly reduced the maturation rate of T. congolense infections in G. morsitans12. Another study found that blood from different sources resulted in differences in infection rates in G. m. centralis and G. m. morsitans indicating that the presence of species-specific factors in the blood affects trypanosome survival in tsetse13.Tsetse flies  and cytochrome c oxidase I (COI), are widely used for blood meal identification of mosquitoes, black flies, tsetse flies and fleas17. Gel analysis can give a broad classification of species by the size of the amplicons but is not as specific as sequencing the amplicon. Recently with the introduction of high-throughput sequencing, multiple amplicons from multiple environmental samples can be analyzed in a single sequencing run, making identification of vertebrate species in the environment19. This metabarcoding approach is an efficient and sensitive tool for biodiversity monitoring and can be expanded to the identification of blood meal sources since it is less arduous and has a high-cost performance.Additionally, this gene has an evolutionary rate that is almost similar to that of mitochondrial DNA itself20. One important factor contributing to this refractory phenotype is the endogenous bacterial microbiome, which plays a role in the structural integrity of the peritrophic matrix  during larval development21. The colonization of the gut by microbial communities may or may not increase tsetse fly resistance against trypanosome invasion. Previous work suggests that Sodalis and Wigglesworthia  can modulate trypanosome development22. Wigglesworthia must be present in the larval stage during the maturity of tsetse flies for their immune system to properly develop and function23. The artificial elimination of Wigglesworthia results in flies being sterile and a compromised immune system leading to increased susceptibility to trypanosome infection24. A recent study found that disruption of Wigglesworthia folate production resulted in reduced sensitivity to trypanosome infection25.Tsetse flies harbor a natural ability to clear trypanosome infections (refractory phenotype), resulting in only few flies (<5%) that successfully transmit trypanosomesSodalis is associated with increased susceptibility to trypanosome infection in tsetse flies and that some Sodalis genotypes are related to the ability of trypanosomes to establish infection in the tsetse fly midgut and that the density of these bacteria also influences susceptibility27. The presence of another tsetse fly symbiont, Wolbachia, is associated with cytoplasmic incompatibility (CI). A recent study demonstrated that mating Wolbachia infected male G. m. morsitans with uninfected female leads to arrested embryonic development where deformed embryos aborted without hatching into larva28. The presence of some bacteria can affect the appearance of others in the midgut of tsetse flies. In G. morsitans group, Wolbachia and Spiroplasma co-infections do not occur in nature, and Spiroplasma might be affected by the presence of Wolbachia29.In contrast, the presence of Kosaconia cowanni Zambiae inhibits trypanosome in the midgut of G.m. morsitans30. Tsetse flies may ingest bacteria within the environment, particularly from the skin surface of hosts during blood meals32. The diversity and composition of these bacteria significantly vary with different regions, tsetse populations, and species. A recent study found that flies residing in geographically separated niches had different Spiroplasma infection prevalence33. Other bacteria found in tsetse flies are environmentally acquired, such as members of the phyla Actinobacteria Bacteroidetes, Firmicutes, and Proteobacteria are found consistently in different tsetse species captured in geographically distinct localities36. In most cases, however, these microbes account for less than 1% of tsetse’s entire enteric microbiota34, and their effect on the biology of tsetse flies is still unclear.Moreover, the presence of some other bacterial species can hamper trypanosome infection in the midgut of tsetse flies. A recent study observed that In a previous study, we have analyzed the trypanosome species in tsetse flies caught in Zambia and Zimbabwe using the DNA metabarcoding method. In this study, we applied a similar approach for the simultaneous identification of mammalian blood meal sources and bacterial microbiome in the same group of samples. The method makes use of multiple PCR of three target regions, (1) 16 S rRNA genes for bacterial analysis and (2) mitochondrial 12 S rRNA genes for mammalian host analysis coupled to high-throughput amplicon sequencing. Such an approach could be applied to study the different epidemiological variables with reduced cost and without compromising on the sensitivity and specificity of species identification. Such an approach could also help to provide more data and information essential for the formulation of new or better strategies for controlling the transmission of African trypanosomes.Glossina morsitans centralis. As a pilot study, we had analyzed 32 tsetse flies collected using Epsilon traps in April 2014 from Hurungwe Game reserve (near Mana pools) in Zimbabwe in order to evaluate the next-generation sequencing approach for blood meal host identification. This group of flies were analyzed as a pilot study. For this group of samples, information on tsetse fly species and sex was not available. All flies had been preserved in individual tubes containing silica gel beads before being crushed. Isolation of DNA was performed using the DNA Isolation Kit for mammalian blood (Roche USA) as per the manufacturer’s protocol for extraction of DNA from Buffy coat. However, the red blood cell lysis step was bypassed to allow lysis of all cells in the solution at once, including trypanosomes. The DNA samples were stored at −80 °C until analysis.We analyzed 85 tsetse flies collected from Zambia, along the Kafue national park border in June 2017 , 0.16 µL of 5 U/μL KAPA Taq polymerase , 0.4 µL of DMSO, and 1 µL of template DNA. The temperature and cycling profile included incubation at 95 °C for 10 min followed by 37 cycles as follows: 95 °C for 30 sec, annealing for 1 min at 60 °C for 12 S mitochondrial RNA PCR  and 55 °C for bacterial 16 S RNA PCR , extension at 72 °C for 2 min, and a final extension at 72 °C for 10 min. After the first PCR, Agencourt AMPure XP beads  were used to purify the amplicon away from free primers, primer dimer species, and PCR reagents. PCR, amplicons were viewed on 2% agarose gels. A negative template control was included in each set of PCR reactions.Initial PCR reactions were carried out with adapter-ligated primers, where respective multiplexing adapter sequences were added to the 5′ end of each respective primer set , 4 µL of 5X buffer, and 2 µL of template (amplicon from first PCR). The temperature and cycling profile included incubation at 95 °C for 3 min, followed by 11 cycles as follows: 95 °C for 30 sec, 61 °C for 1 min, 72 °C for 2 min, final extension at 72 °C for 10 min. To enable the sequencing of all amplicons in this study in one run, we used unique sets of dual index primers for each sample for each PCR analysis . A PCR clean-up was done using Agencourt AMPure XP beads and products viewed on 2% agarose gels.The second PCR was done in 10 µL reactions containing 1 µL of 10 µM Illumina dual-index primer mix (i5 and i7 primers), 1.2 µL of 25 mM MgCl38. Equal volumes of each sample amplicons from the two PCR analyses were pooled into one library. The library pool was purified using the Wizard SV Gel and PCR Clean-Up System  by cutting out bands of interest to separate them from primer dimers and post PCR reagents. Quantification of each of the libraries was done using a Qubit dsDNA HS assay kit and a Qubit fluorometer . The concentration of the library was then adjusted to a final concentration of 4 nM using nuclease-free water and applied to the MiSeq platform . Sequencing was performed using a MiSeq Reagent Kit v3 for 300 base pairs, paired-end  and a PhiX DNA spike-in control added to improve the data quality of low diversity samples, such as single PCR amplicons. For trypanosome species detection assay, positive template controls comprising an artificial mixture of T. congolense and T. brucei rhodesiense DNA from cultured parasites were also included in the sequencing library. The Raw data of this study is available in the Sequence read archive (SRA) database under submission numbers SUB6078288 and SUB6078557.The processing of the amplicon library for sequencing was prepared as outlined in a previous study38. For mammalian species analysis, pre-processing of reads was done using the amplicon toolkit  pipeline39. The pre-processing involved trimming of primers, removal of sequences less than 200 bp, and merging pair-end reads. Reads were merged using the VSEARCH option for merging a long overlap region between the paired reads (~270 bp), allowing for merging of staggered read pairs. When reads did not merge, the forward read was rescued for downstream processing since the full amplicon insert could be covered by one read-only. For bacterial microbiome analysis, read merging was done using the AMPtk pipeline’s USEARCH merging option with the customized parameters: fastq_pctid set to 80, , minhsp set to 8, and fastq_maxdiffs set 10 to limit the number of mismatches in the alignment to 10, and fastq_pctid set to 300. For bacterial microbiome analysis, only merged reads were considered for downstream processing.Analysis for the identification of trypanosome species in all the samples had been conducted in an earlier study using a new approach utilizing internal transcribed spacer 1 (ITS1) PCR and next-generation amplicon sequencingAll merged reads were processed using DADA2 v1.12 in the R environment, including filtering low quality reads, removal of reads shorter than 100 bases pairs, read error correction, clustering and chimera removal resulting in Amplicon sequence variant (ASV) table representing all unique sequence clusters and their frequency per sample. About 42% of the samples from Kafue and 50% of the samples from Hurungwe had over half of their reads passing pre-processing analysis. For bacterial microbiome analysis, 77% of the samples had 90% of their reads passing pre-processing analysis. . The significance of taxonomic composition and abundance of bacterial species between homogeneous groups was then tested using PERMANOVA. The differentially abundant taxa among the different groups of flies were estimated using DESeq2 (v.1.14.1)41 by performing the Wald significance test with a parametric fit type and multiple-inference correction by B-H method. Abundance plots for all bacterial genera (DESeq2 transformed counts) were plotted in the R environment. The final ASV tables and taxonomic files for mammalian host species analysis and 16 S bacterial analysis are provided  and Shannon diversity values (estimator of species richness and species evenness in a community with more weight on species richness) were computed with the significance between groups tested using the non-parametric Kruskal–Wallis rank test. The variation in the taxonomic composition and abundance of bacterial species in different groups of flies  was estimated by beta diversity analysis using principal coordinates analysis (PCoA) with the estimation done using Bray-Curtis dissimilarity distance matrix to estimate the compositional dissimilarity between two different groups or sites, based on counts. The homogeneity of group variances was first done using the G. m. centralis) tested positive for human, cattle, dog, bush pig, African buffalo, warthog, greater kudu, rat, and bat  caught in Hurungwe, Zimbabwe had the mouse and human sequences detected  pointed to a limited bacterial diversity of species between the different groups of flies as indicated by the Shannon diversity index  the high abundance of Wolbachia 16 S rRNA gene copies in infected female flies compared to males  , that of Wolbachia was 95.3% (CI 88.5–98.2%), and that of Wigglesworthia was 100% (CI 95.6–100%).Analysis of the bacterial microbiome in wild-caught dex Fig.  and alsodex Fig. . The bacdex Fig. . Howeverdex Fig. . This diles Fig. . The bacBacillus, Pseudomonas, Corynebacterium, Paracoccus, and Acinetobacter, among others. , it was difficult to analyze the diversity of other bacteria families, which had very low read counts.Other bacteria detected included 42. Our results on the wildlife animals identified in this study, i.e., warthog, bush pig, African buffalo, African hunting dog, and greater kudu is in agreement with previous studies including observations made in Luangwa Valley, Zambia that reported similar mammalian hosts in G. morsitans44. This indicates that wild animals (mainly found in woodland Savannah and bushy areas) constitute a significant source of blood meal for tsetse flies found in Kafue, Zambia, and Hurungwe, Zimbabwe. However, the samples in this study were sampled at only one time-point in the year for each site (June 2017 for the Kafue samples and April 2014 for the Hurungwe samples). Since the presence of different mammalian hosts  is variable during different seasons of the year, this may influence the number and type of mammalian blood meal host species detected in tsetse flies and their trypanosome infection rates. Due to the nature of opportunistic feeding habits, the blood meal host species can differ even in the same locality where different animals are distributed unevenly either due to natural causes or some human activities, e.g., livestock grazing and wildlife restriction.Our results give insight into the broad range of mammalian hosts from which tsetse flies obtain their blood meal. The flies caught in this study were collected in areas that interface human settlements and areas set aside for wild animals. Specifically, in the Kafue area, samples were collected on a road traveled by residents. This could explain the high numbers of flies with human blood meals, further supporting the submission that tsetse flies can change feeding preferences in the absence of wild animals or the presence of an alternative hostTrypanosoma brucei rhodesiense is still present in the area45. A study in Zambia’s eastern region reported infection in dogs with T. congolense, T. b. brucei and T. b. rhodesiense suggesting that dogs are reservoirs of trypanosomes in Zambia46. More study is needed to determine the variation of mammalian hosts and specific trypanosome species prevalence to identify distinct reservoirs important for different regions. The detection of rat DNA is essential because rats are not thought to be relevant blood meal sources for the tsetse fly. The finding that rat and bat are blood meal sources of G. morsitans reinforces the observation that tsetse flies are opportunistic feeders. Although this is the first report of detection of DNA from fruit bat in wild-caught tsetse flies, an experimental study in 1941 had reported that G. morsitans and G. tachinoides readily fed on captured bats (Nycteris hispida)47. The presence of human infective trypanosomes in bats has not been reported, but this could be possible since an earlier study reported that experimental infection of insect-eating bats (Tadarida condylura) with T. b. rhodesiense and T. b. brucei produced a much more chronic infection than in mice48. Our data provide evidence that bats are exposed to the risk of infection by tsetse flies in the field and more studies are needed to investigate the existence of natural trypanosome infections in bats. Our finding that rats are blood meal hosts for tsetse flies was consistent with other studies that have reported T. brucei infection in rats in west Africa50, pointing to a possibility that wild rats may be unlooked as reservoirs of trypanosomes.Recently, a human case of trypanosomiasis was reported in the Kafue area (an old trypanosomiasis focus), indicating that Wigglesworthia, Wolbachia and Sodalis) were the most abundant comprising 99.7% of the total reads obtained while all other bacteria identified comprised only 0.3% of the total reads. This explains the results obtained from alpha and beta diversity analysis that show limited bacteria species diversity (both in abundance and richness). In this study, we report a high prevalence of Sodalis (57.7%) in G. m. centralis from Kafue, Zambia compared to the results of a previous study conducted in Luambe National Park, Zambia, that found a lower Sodalis prevalence of 17.5% in G. m. morsitans, 1.4% prevalence in G. pallidipes, but a higher prevalence in G. brevipalpis (93.7%)51. Additionally, the prevalence of Wolbachia (95.3%) was higher than a previous study that found 68.1% in G. tachinoides and 58.5% in G. m. submorsitans52. As earlier stated, due to sampling at only one specific time-point of the year, we cannot rule out the effect of seasonal changes on the prevalence of bacterial symbionts. We observed that the Sodalis and Wolbachia 16 S gene copies significantly different in infected male flies and infected female flies. These abundance values cannot be interpretable as actual bacterial cell numbers, due to the nature of amplicon data and the presence of multiple copies of 16 S rRNA genes in bacteria. However, information on which representative sequences have significantly different copy-number counts between groups can provide useful information and guide the generation of hypotheses. Therefore, there is need for more studies on the infection rates of Sodalis and Wolbachia in infected G. morsitans. The prevalence of Wigglesworthia glossinidius was 100% as expected since it is an obligate symbiont necessary for the immunity and reproduction of tsetse flies. Our study had some limitations in such as the low number of trypanosome infected flies as well as low and uneven numbers in groups of flies with mixed, single, or no blood meal detected. Nonetheless, the results of this study are in agreement with the observation that symbiotic bacteria account for up to 99% of tsetse fly’s entire enteric microbiota34. The environmentally-acquired bacteria in this study accounted for only 0.3% of the total read counts. The taxonomic identities of some of these bacteria were in agreement with previous studies that also reported Bacillus, Pseudomonas, Corynebacterium, and Acinetobacter in wild-caught tsetse flies54. We also detected some that are commonly found in soil and water, indicating surface contaminating bacteria (from the environment). These bacteria, however, accounted for less than 0.1% of the total read count. Owing to the low read numbers obtained for non-symbiotic bacteria, and limited samples, we could not correlate any genus with blood meal source, sex, or status of trypanosome infection. Our results seem to indicate that blood meal sources from different hosts have little effect on the general gut microbiome composition of tsetse flies. However, the imposing abundance of the three symbiotic bacteria hampers the accurate detection and analysis of other bacteria; thus, the difficulty in identifying any significant associations. Studies on other vectors indicate changes in bacterial flora with different blood meals, as has been demonstrated recently in mosquitos (Aedes aegypti)55. Future studies should focus on more sensitive detection and analysis of non-symbiotic bacteria in tsetse flies to shed light on the relationship between bacterial microbiome, trypanosome infection, blood meal source, and other factors and their importance in aspects such as vector competence, immunity, reproduction, etc.Since tsetse flies are obligate blood-feeders, they are less likely to obtain bacteria from the diet since blood is largely considered to be an entirely sterile environment. However, tsetse flies still acquire bacteria from external sources. The results obtained from this study support this observation were the three known symbionts of tsetse flies (Supplementary information.Supplementary information2.


Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica in Lucilia illustris Meigen, 1826 , and the quantitative dynamics of the two bacterial species along the insect life-stages and transfer to beef and pork host tissues using qPCR gyrase b specific primers. The content of both bacterial species increased along the insect life stages. W. chitiniclastica was detected in all developmental stages independent of the feeding substrate. I. indica was measurable with 102 gene copies ng−1 DNA threshold starting from the third instar larvae when feeding on beef, and from the egg stage with a 102× higher representation when using the pork substrate. The transfer of bacterial species to both tissues occurred after 3 colonization days except for I. indica that was visible in beef liver only during day 5. Considering the utilization of pork tissues as human analogues, these quantitative microbial dynamics data provides first insect-specific bacterial candidates as potential colonization biomarkers in forensic investigations.For the last decades, forensic microbiology became an emerging complementary tool in criminalistics. Although the insect-microbe interactions regarding pathogen transmission were extensively studied, only scarce information is available on bacterial transfer from necrophagous insects to host tissues. Our data provides the first report on the occurrence of  Recently, studies involving the investigation of necrophagous insect’s microbiome emerged9, trying to elucidate the interaction of bacteria associated with different insect species. To date, the majority of the studies that have focused on investigating the insect microbiome mostly addressed problems like antimicrobial resistance and pathogen transmission15.The application of entomology in the field of forensic sciences related to the postmortem interval (PMI) estimation is well established since decades17, trying to add important data that can be applied in the field of forensic sciences. None, to our knowledge, targeted the specific amplification and quantification of Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica transfer from Lucilia illustris Meigen, 1826  immature stages to the colonized tissues. These two species are gram-negative and aerobic bacteria, belonging to Proteobacteria phylum, Xanthomonadaceae family, being first isolated and described from necrophagous insect species. W. chitiniclastica was first identified and described from the flesh fly Wohlfahrtia magnifica  (Diptera: Sarcophagidae)18, while I. indica was also first isolated from adult flesh flies19. These bacterial taxa were also reported from hospital cases involving sepsis20, bacteraemia21, myiasis22, their identification and investigation having medical importance in certain clinical cases.Very few studies have focused on the bacterial characterization and microbiome metagenomic assessment from different necrophagous insect species23 or during autopsies, the human bodies are either clean of insect eggs, larvae and/or adults, or those go unnoticed. Information on the transfer of insect-specific bacteria could be very useful in proving a previous presence of the necrophagous insect species on the decomposed body, even from the earliest moment of colonization. This can be accomplished by quantitatively analyzing the bacterial content, which, as it appears from the investigation carried out in this article, changes according to the insect developmental stage and/or tissue diet. By relating the bacterial quantity variation to the different insect developmental stages, and by correlating these variations with the colonization time, bacterial species insect-associated could be used in the near future as PMI biomarkers.Sometimes, in unforeseen circumstancesL. illustris immature life stages  and in the quantitative analysis of the bacterial transfer to the feeding substrate in function of the different larval stages. This article provides first insights into the bacterial transfer quantification having potential implications in the field of forensic sciences. The importance of the present study also resides in the fact that these bacterial taxa were identified from different necrophagous insect species, sampled from different geographical locations26, so that the data obtained during this survey can be of use to the international scientific community, due to their role as possible PMI biomarkers in the absence of insects. Nevertheless, before any conclusion is drawn, numerous studies must be carried out, using even other insect-specific bacteria, and even other insect species that colonize decomposed remains.In this respect, the main objective of the present research consisted in the quantitative investigation of two insect-specific bacterial taxa throughout Lucilia illustris Meigen, 1826  was selected for the survey considering its synanthropic environmental occurrence and high abundance during summer months, being also an earliest primary carrion colonizer. L. illustris adults were sampled three different times (approx. 30 specimens/sampling time) from the urban area of Bucharest (Romania) (N44°26′49.349′ E26°2′45.352″) during summer (July 2017).L. illustris eggs were placed in small sterilized rearing jars of 21 × 12 cm in diameter  and the immature stages were reared under the same constant laboratory conditions, in the presence of 375 g of liver, being sampled daily. Non-colonized rearing jars containing the same amount of pork or beef liver were used as control and kept under the same experimental aseptic conditions . After oviposition, L. illustris immature developmental stages  and fresh and aged liver samples. Approximately 0.3 g of eggs and one individual larvae of each stage were sampled for DNA extraction. Liver tissue samples were collected prior to insect colonization and simultaneously with the daily insect sampling.Insect and liver tissue were collected daily in triplicate along 2-weeks period, considering all Liver tissue and insect samples were preserved in 200 µl Tris-EDTA pH 8 (TE) buffer at −20 °C for DNA extraction, and in 75% ethanol for taxonomic identification.27 under a stereomicroscope Stemi2000 , and confirmed by COI barcoding.Insect adults and immature stages were identified using the taxonomic identification keys provided by LehrerWohlfahrtiimonas chitiniclastica and Ignatzschineria indica in insects and liver tissues was carried out by qPCR specific bacterial gyrase subunit B gene (gyrB) fragments. The primers  modified protocol29. DNA concentration and purity were measured with a BiodropDuo UV/VIS Spectrophotometer . For insects, the specimens were washed in 70% ethanol and sterile 10 mM phosphate-buffered saline (PBS) solution to remove all exterior contaminants, and homogenized for 12 min in the presence of 2 mm ZR Bashing Beads  using a SpeedMill PLUS Cell Homogenizer  prior to total DNA extraction.Quantification of rs Table  were desI. indica or 50 °C for W. chitiniclastica, and 30 sec at 72 °C, were carried out using a Mastercycler ep gradient S thermocycler PCR . The melting curve analysis was used as amplification control, being included at the end of each program. Quantification was performed by interpolation in a standard regression curve of cycle threshold (Ct) values generated from samples of known concentration of DNA template. Standard curves  Master Mix (ThermoFisher Scientific), 10 µM of each forward and reverse primer, 100 ng DNA template and nuclease free water in a total volume of 10 µl. The amplification reactions comprising an initial incubation step of 10 min at 95 °C followed by 40 cycles of 15 sec at 95 °C, 30 sec at 49 °C for W. chitiniclastica ranged between 7.5 × 10−4 ng/μL–7.5 × 10−9 ng/ μL, and 3.7 × 10−2–10−8 ng/μL for I. indica (R2 > 0.99). No-template controls were included in all PCR runs. Samples were run in triplicates, with a Ct threshold value over 34 not considered.The DNA decimal dilutions series for 30:The gene copy number per reaction was calculated aswhere the length represents the base-pair length of the PCR product, and the plot was performed between the logarithmic scale (base 10) of copy gene number and the experimental time.gyrB) fragments. The corresponding amplicon length covered 140 bp for W. chitiniclastica and 262 bp for I. indica. Both PCR amplification , specific primer pairs for each bacterial species  collected from the pork liver feeding substrate, with a maximum bacterial content reached starting with the first instar larvae, about 103x higher than in the egg samples. At the same time, W. chitiniclastica exhibited comparable amounts between the three larval stages .ges Fig. , startin2). Further, the maximum abundances for W. chitiniclastica were recorded for the second and third larval stages (104.2–106.4).For the beef liver substrate, a lower bacterial transfer was recorded for the first and the second instar larvae, with comparable abundances for the egg and first instar larvae samples , and maximum content recorded for the third instar larvae (106.1), showing a 102.72 × increase as compared to the egg samples, and also an upward trend throughout larval development.In the case of ica Fig.  the bactI. indica identified from the insect immature stages reared on beef liver was visible only in the third instar larvae (103.2), exhibiting 10−1.89× lower quantities compared to the third larval stages fed with pork liver diet. During the earlier insect life stages, I. indica was not observed given the very low abundances, overall being present in 50% lower abundance than in the insect specimens reared on the pork liver.On the other hand, W. chitiniclastica and I. indica transfer occurred after three experimental days, with a similar dynamics trend during days 3 and 4 (105.1–105.3), 102.47x higher for I. indica than W. chitiniclastica in day 5. The higher bacterial content registered from the pork liver belonged to I. indica in day 5 (106.4).In the case of the pork liver substrate Fig. , both W.W. chitiniclastica was present in the beef liver , reaching comparable levels as in the pork liver.ver Fig.  startingI. indica was accounted for the beef feeding substrate during the first 4 experimental days, in accord with the first developmental stages of L. illustris specimens . I. indica was observed only during day 5 (102.6), presenting 10−1.95 × lower content than W. chitiniclastica.No identification of Although the abundance profile varied between pork and beef feeding substrates, the highest bacterial content for the two taxa investigated was reached for the insect specimens reared on pork liver. Moreover, the initial content of both bacterial taxa could not be detected in the first experimental days in the liver samples, nor from the liver used as reference.32. For the most part, the decomposition begins immediately after the negative signs of life are installed, manifested by the biochemical changes and the proliferation of bacteria from the gastrointestinal tract. All of these physical and biochemical changes attract a diversity of necrophagous insect species, which come to colonize the respective cadaver in a certain time sequence. When the temperatures are higher, a decomposed body attracts faster Diptera and/or Coleoptera species that reproduce and feed on the remains33. Starting with the initial moment of carcass colonization by different necrophagous insect species , a microbiome transfer from the insect species to the colonized remains also takes place34.The decomposition dynamics of human and animal carcasses is dependent on the cause of death, ante mortem circumstances, geographical location, meteorological parameters as well as specific conditions encountered at the decomposition environmental site39. However, more is to be known regarding the transfer of insect-specific bacterial species throughout the developmental cycle, as well as their transfer to the colonized tissues/substrates. To date, those studies that focused on characterizing the bacterial diversity from different insect species used both classical microbiological cultivation40 or/and modern molecular high throughput 16S rRNA gene sequencing43, as investigation techniques.For the insect species the periods of activity, colonization patterns and feeding preferences have been well researched24 and calliphorids7 were used to investigate the insect microbiome content, focusing primarily on the pathogen transmission17 and antimicrobial resistance44. Moreover, studies concerning the bacterial communities involved their relationships and role played during the developmental stages of different insect species40.Given their medical, veterinary and forensic importance, muscidsMusca domestica Linnaeus, 1758 (Diptera: Muscidae) was often used as experimental model for these types of studies given this species frequently association with human living environments, easily transmitting different pathogens. As such, the research focused mainly on characterizing the bacterial diversity and on the identification of bacterial pathogens40 from this muscid species.In this regard, Wohlfahrtia magnifica  (Diptera: Sarcophagidae) were used to investigate the bacterial content. Tóth and collaborators17 studied the bacteria associated with W. magnifica given the myiasis-causing potential of this fly species in domestic animal, especially in Eurasia. During their study it was emphasized that Ignatzschineria spp. is most probably associated with the insect larvae foregut. At the same time, both Ignatzschineria and Wohlfahrtiimonas were noticed to have a strong chitinase activity, that may play an important role during the fly developmental period17. Even though Tóth and collaborators17 did not performed a quantitative investigation, both bacterial species were identified from W. magnifica larval stages. In our experiment not only that the bacterial species were identified, but their quantitative presence was analyzed, demonstrating that these taxa are insect specific and that their in-depth study can lead to a better understanding of their involvement as insect colonization biomarkers.However, other insect species, such as Hermetia illucens  (Diptera: Stratiomyidae) life stages6. The main purpose of Zheng and collaborators6 study was to analyze the transmission of pathogens throughout H. illucens developmental cycle. Nevertheless, the bacterial diversity characterization revealed Ignatzschineria spp. presence in the analyzed specimens. This identification proves once again this bacterial association with fly species belonging to different Diptera families.Furthermore, the bacterial diversity was also investigated from the black soldier fly 45 researched the larvae mass microbiome belonging to different Calliphoridae species ) and revealed Xanthomonadaceae as an abundant family, though no taxonomic level up to genera was presented. Nevertheless, their study showed that Xanthomonadaceae increased throughout the decomposition process. These results are similar to our data on the investigated and quantified bacterial taxa that exhibited a similar increasing time pattern both in the insect immature stages and colonized liver.Weatherbee and collaborators42 focused on studying the bacterial diversity associated with the third instar larvae of the stable fly Stomoxys calcitrans  (Diptera: Muscidae). The results showed Ignatzschineria among the most abundant bacterial genera in the larvae specimens. At the same time, Ignatzschineria was more abundant in the fly larvae specimens than in the living substrates, similar to our findings, were I. indica prevailed in L. illustris immature stages. Other synanthropic species, such as the oriental latrine fly, Chrysomya megacephala  was used for the microbiome characterization during the developmental cycle43. During the respective study43, both Ignatzschineria and Wohlfahrtiimonas were identified from the insect samples, though, unlike other experiments, these genera were not encountered among the prevailing gut bacteria. Nevertheless, once again it is demonstrated that these bacteria are present in different Diptera species, which may recommend them as universal biomarkers for the insect colonization time.A more recent study46, using Proteus mirabilis in M. domestica and the green fly Lucilia sericata   the presence of Ignatzschineria and Wohlfahrtiimonas was revealed. They used qPCR assays to quantitatively monitor P. mirabilis and to evaluate for how long different type strains persist in the fly’s digestive tract, but also used 454 pyrosequencing assays to characterize the entire microbiome. Similar to our study, the authors used qPCR as a method of investigation, though targeting a different bacterial taxon. However, the microbiome characterization by 454 pyrosequencing revealed Wohlfahrtiimonas in L. sericata specimens. Furthermore, Ignatzschineria genus was identified as prevalent bacteria in green bottleflies46 sampled during summer months, consistent to our findings of Ignatzschineria indica from L. illustris.During another study that investigated the persistence of antibiotic resistant bacteria performed by Wei and collaboratorsW. chitiniclastica was identified as a common bacterial taxon from both culture isolates and clone library samples during Gupta and collaborators study24, Ignatzschineria spp. being also identified during the survey. Both Ignatzschineria and Wohlfahrtiimonas were identified during another Gupta and collaborators25 investigation on bacterial content from adult sarcophagids intestinal gut. Moreover, both taxa were among the prevalent cultured bacteria identified from both flesh flies’ adults and larvae samples, although Ignatzschineria was common in both culture and clone sequences.7 investigated the bacterial content from Lucilia sericata  and Lucilia cuprina  species . During their study the horizontal transmission of bacteria was more noticeable than the trans-generational inheritance. Although during their experiment the taxonomic identification was performed up to the genera level, Ignatzschineria was encountered among the first five dominant genera identified from the two insect species, while no records of Wohlfahrtiimonas were mentioned. Moreover, as the authors stated, data on bacteria associated with Lucilia species is limited, and though several years had passed since their study, scarce in-depth investigations were directed towards the research of bacteria associated with green bottleflies. In this regard, the current study presents innovative and preliminary data on W. chitiniclastica and I. indica that may help to expand the research in the forensic field, regarding the necrophagous insect-associated bacterial investigations.Even though most of the studies performed to date concerned mainly bacteria that are muscids-associated, green flies were also considered for research. In this respect, Singh and collaboratorsIgnatzschineria and Wohlfahrtiimonas with different insect species. The presence and quantification of both bacterial genera in necrophagous insect species can provide data on body colonization even in the absence of adults and/or immature stages. This could happen, as previously mentioned, during the crime scene investigation or autopsy sampling where, regardless of the reason, the insect evidence is overlooked, especially at the beginning of insect colonization (i.e. egg clusters). However, this information could be used in the absence and/or presence of entomological evidence, in order to bring additional information about the colonization time, throughout the bacterial quantitative time related presence.All these previous studies revealed the presence and frequent association of Ignatzschineria and Wohlfahrtiimonas taxa were identified from insects and liver tissue samples, with comparable values for the pork liver substrate, suggesting that they can be used as potential bacterial markers for insect colonization, supporting the base hypothesis of this experiment.In the current survey, both Although further investigations targeting these bacterial species are required to confirm their role as colonization biomarkers using various feeding substrates, larvae tissues and conditions, this report highlights for the first time the applicative potential in forensic sciences of these two bacterial species.Supplementary Figure S1.


Low birthweight (LBW) is a significant public health issue, and maternal smoking is the most prevalent preventable cause of LBW. But there is limited evidence on association of LBW among children and cigarette smoke exposure in mothers in China. In this cross-sectional study, we try to explore if the LBW in children is positively associated with mothers’ prenatal cigarette smoke exposure.We selected 8, 586 mothers and their singleton children in 2018 in Songjiang district, Shanghai. Birthweight of children and gestational weeks of mother was identified by birth records in the hospital, we classified mothers’ prenatal cigarette smoke status into the first-hand smoke (FHS) exposure and the second-hand smoke (SHS) exposure. We use SAS 9.1.3 software to calculate the prevalence of children’s LBW and the prevalence of mothers’ prenatal cigarette smoke exposure including FHS and SHS. Chi-square test and logistic regression were used to analyze the difference.In 8, 586 women, The prenatal FHS and SHS exposure prevalence was 0.9 and 20.8%, respectively. The mean birthweight of children was 3315.5 g with a standard deviation of 497.2 g, the mean birthweight was 167.7 g and 66.1 g lower in children born to mothers with prenatally FHS and SHS exposure compared with those children whose mother were not exposed, respectively. The children’s LBW prevalence was 4.7% in this study. By comparing with children whose mother were not exposed, the LBW prevalence was higher among children whose mother were prenatally exposed to FHS , and SHS .Children’s LBW is positively associated with mothers’ prenatal tobacco smoke exposure both for FHS and SHS. So implementing tobacco control measures is crucial to lower smoking prevalence among women, and decrease smoking prevalence of their family members as well as work fellows. The birthweight less than 2500 g is defined as Low birthweight (LBW), LBW continues to be a significant global public health issue . It is eCigarette smoking in childbearing aged women hand its associated adverse outcomes have been a common concern since the 1960s , 4, 10. China is the largest tobacco producer and consumer in the world, the Global Adult Survey conducted in 2010 indicates that there are 300 million adult smokers in China, and 1 million deaths each year are due to tobacco consumption –20. The In this paper, we conducted a cross-sectional study in Songjiang district, a rural area of Shanghai, China. We aim to learn the prevalence of prenatal cigarette smoke exposure among mothers, both for FHS and SHS, and the prevalence of low birthweight in children, and to explore if prenatal cigarette smoke exposure among mother is positively related with the low birthweight among children.We conducted this study during March and September in 2018 in Songjiang district of Shanghai. PASS software was applied for sample size calculation, we assumed that the prevalence of low birthweight was 5% in Songjiang district, considering an inspection level (α) of 0.05 and a permissible error (δ) of 0.005 , at least 7299 children should be sampled and recruited. Take population size of kindergarten into consideration, we finally selected 20 kindergartens in this study. We applied a cluster sampling method to select study population. First, we randomly selected 20 kindergartens out of all 115 kindergartens in Songjiang District. Second, we recruited all singleton children and their mothers from the 20 selected kindergartens. Overall, a total number of 8, 626 children as singleton and their mothers were recruited in this study. Finally, 8, 586 pairs of mothers and children completed the questionnaire interview and were included in the final analysis. Songjiang Maternal and Child Health-care Hospital Institution Review Board authorized the ethic approval of this study (IRB#20171203), and ahead of the questionnaire interview, research coordinators orally communicated with each mother and their children, and then signed the informed consent papers.‘Have you ever smoked at least one cigarette every day for over six months during your pregnancy?’, ‘Does anyone smoke around you in workplace or at home for at least 10 minutes each day during pregnancy?’,‘which way is your child being delivered, vaginal or cesarean?’, ‘Is your child delivered before 37 gestational week?’, ‘what is your body weight and height before pregnancy?’, ‘what is the birthweight of your child? ’, ‘have you been diagnosed as gestational diabetes mellitus (GDM) during the pregnancy? ’ etc.). Part three covered 7 questions which were extracted from the birth records of the mothers and singletons to verify the self-reported information . Part four included information for follow-up contact of mothers and their children.In this study, questionnaire for data collection covered 4 parts . We recorded mothers’ education as completed schooling years and classified it into 3 categories including primary school or illiterate (0–6 year), school of junior high (7–9 years), school of senior high (10–12 years), and college and above (> 12 years). Family yearly income (CNY) was categorized into 4 categories .In this study, prenatal first-hand smoke exposure (FHS) was defined as a mother who smoked at least one cigarette each day for over 6 months before and during pregnancy , 19, andWe performed the data analysis by SAS software (version 9.1.3). In this study, data was described as frequency and (or) prevalence for qualitative variables and means as well as standard deviations (SD) or median as well as interquartile range (IQR) for quantitative variables. Chi-square test was applied to examine the difference of prevalence of prenatal FHS and SHS exposure among mothers with different demographic characteristics, and student’s t test or Wilcoxon Rank Sum test were applied to examine children’s birthweight difference between mothers with and without prenatal cigarette smoke exposure, and we also employed Chi-square test to examine the prevalence difference of low birthweight (LBW) as well as preterm birth in children between mothers with and without prenatal tobacco smoke exposure. We applied univariable and multivariable logistic regression to calculate the odds ratios (OR) and 95% confidence interval (95% CI) of the LBW prevalence in children between whose mothers with prenatal cigarette smoke exposure and whose mothers without, and to explore the association of prenatal tobacco smoke exposure among mothers and LBW prevalence in children. Directed acyclic graphs (DAGs) method, a graphical tool which provide a way to visually represent and better understand the key concepts of exposure, outcome, confounding and bias, was applied to identify potential confounders in this study, and the confirmed confounders were then adjusted in multivariable logistic regression analysis.In this study, mothers’ age ranged from 19 to 48 with an average age of (32.2 ± 3.9) years old, the majority of mothers (71.1%) had an education over college, approximately 50% of mothers had family income over 300, 000 CNY by year, and 52.9% of mothers were local residents. Ahead of pregnancy, 61.5% of mothers had a normal body weight, 17.4% of them were overweight or obesity. 6.2% of mothers were diagnosed as gestational diabetes mellitus (GDM) in pregnancy.The prevalence of prenatal FHS exposure among 8, 586 mothers was 0.9%. The prenatal FHS exposure prevalence among mothers of local residents 1.2%) was significantly higher than mother of no-local residents (0.6%), and the prevalence of prenatal FHS exposure was higher in mother with GDM (1.9%) than mothers without (0.9%). Mothers had higher prevalence of prenatal FHS exposure with age over 36 years (1.3%) or under 25 years (2.5%). And the prevalence of prenatal FHS exposure among mothers with education of college and above was lower than mothers with education of illiterate, primary school, school of junior high and senior high. Table .2% was sIn this study, 1, 785 out of 8, 586 women exposed to SHS prenatally with a prevalence of 20.8%. The prenatal SHS exposure prevalence in mothers aged < 30 years (25.4%) was higher than those aged over 30 years (18.2%). Mothers with a BMI < 18.5 or > 27 had higher prevalence of prenatal second-hand smoke exposure. Mothers with education of junior high or senior high schools were highly exposed to second-hand smoke prenatally than those with an education of illiterate, primary, as well as college and above. Mothers with local residency (24.1%) had higher prenatal second-hand smoke exposure prevalence than mothers without local residency status (17.1%), and the prenatal second-hand smoke exposure prevalence in mothers with GDM (27.0%) was higher than mothers without GDM (20.4%). Table In this study, 8, 586 singleton children included 4, 595 boys (53.5%). The average birthweight of children born to mothers with prenatal FHS exposure was lower than children born to mothers without prenatal FHS exposure, the difference was statistically significant . Likewise, a lower average birthweight was identified among children whose mothers exposed to SHS prenatally compared with whose mothers without prenatal SHS exposure . Meanwhile, both in boys and girls, the average birthweight of children whose mothers were prenatally exposed to tobacco smoke was significantly lower than children whose mothers were not prenatally exposed tobacco smoke. Table The LBW prevalence in children was 4.7%  in this study. Children born to mothers with prenatal FHS exposure had significantly higher prevalence of LBW than children whose mothers were not prenatally exposed (12.4% vs 4.6%). Similarly, the prevalence of LBW was significant higher in children whose mothers with prenatal SHS exposure than children born to mothers without prenatal SHS exposure (8.4% vs 3.7%). Table Table In this study, we identified that mothers’ prenatal tobacco smoke exposure could increase the LBW occurrence in children. Children born to mothers with prenatal FHS exposure had significantly higher prevalence of LBW than children born to mothers without prenatal FHS exposure , even after the adjustment of potential confounders . Likewise, Children born to mothers with prenatal SHS exposure also had higher prevalence of LBW than children born to mothers without prenatally exposed to SHS , even with the adjustment of potential confounders . Table 9, 5.53].It is well documented that tobacco smoke can lead to lung and other cancers, heart diseases, stroke and many other diseases , and tobBirthweight is an easily obtained measure of fetal development, and low birthweight interferes with newborn survival and negatively affects both childhood and adult life . In thisPrevious studies demonstrate that low birthweight is associated with social and cultural issues, preterm birth, tobacco use, malnutrition in gestation, sexual transmitted diseases and other maternal infections, as well as maternal lifestyle choices –4. In thIn this study, we noticed an elevated prevalence of low birthweight among children whose mothers were prenatally exposed to FHS or SHS. The LBW prevalence was 2.91 times higher among children whose mothers were with prenatal FHS exposure, and 2.35 times higher among children born to mothers with prenatal SHS exposure. According to previous studies, the association between mothers’ prenatal tobacco smoke exposure and LBW in children was confounded by preterm birth, GDM of mother, social-economic status, as well as pre-pregnancy overweight –38. In oThis study is the first attempt in China to estimate the association of LBW in children and prenatal FHS exposure in mothers. The large sample size is a key strength of this study, we sampled 8, 586 singleton children and their mothers in this study which accounting for approximately 20% of those preschool children, and almost 80% of total newborns in 1 year in Songjiang district of Shanghai, so study results could be generalized to Songjiang district or other rural areas of Shanghai, China. In this study, the tobacco smoke exposure information, as well as the gestational information including the gestational week and birthweight of children, pre-pregnancy BMI of mother, and the GDM was verified by their birth records in the hospital, which reduced the recall bias in some degree. Meanwhile, potential confounding factors were identified by directed acyclic graphs and subsequently adjusted in logistic regression model is another strength of this study, which ensures a relatively unbiased association between children’s LBW prevalence and mothers’ prenatal tobacco smoke exposure.This study has some limitations. First, the study design of a cross-sectional study may induce some recall bias and only allow the calculation of prevalence. Second, the detailed information of prenatal first-hand smoke and second-hand smoke exposure among mothers such as daily tobacco consumption, SHS exposure hours were not available, made it impossible to estimate the dose-effect association between prenatal cigarette smoke exposure in mothers and low birthweight in children. Third, the self-reported information of prenatal tobacco smoke exposure among mothers might be under reported and lead to a potential risk of under-estimating the odds ratios between mothers’ prenatal tobacco smoke exposure and LBW in children. So the incorporation of some improvements should be considered in the follow-up studies.Children’s LBW is positively associated with mothers’ prenatal tobacco smoke exposure to both FHS and SHS. Therefore, implementing tobacco control measures is crucial to lower smoking prevalence among women, and decrease smoking prevalence of their family members as well as work fellows in Shanghai, China.Additional file 1. Questionnaire for prenatal tobacco smoke exposure in mother and low birthweight in their children.


The immune system plays critical roles in promoting tissue repair during recovery from neurotrauma but is also responsible for unchecked inflammation that causes neuronal cell death, systemic stress, and lethal immunodepression. Understanding the immune response to neurotrauma is an urgent priority, yet current models of traumatic brain injury (TBI) inadequately recapitulate the human immune response. Here, we report the first description of a humanized model of TBI and show that TBI places significant stress on the bone marrow. Hematopoietic cells of the marrow are regionally decimated, with evidence pointing to exacerbation of underlying graft-versus-host disease (GVHD) linked to presence of human T cells in the marrow. Despite complexities of the humanized mouse, marrow aplasia caused by TBI could be alleviated by cell therapy with human bone marrow mesenchymal stromal cells (MSCs). We conclude that MSCs could be used to ameliorate syndromes triggered by hypercytokinemia in settings of secondary inflammatory stimulus that upset marrow homeostasis such as TBI. More broadly, this study highlights the importance of understanding how underlying immune disorders including immunodepression, autoimmunity, and GVHD might be intensified by injury. Sequelae of TBI and soft tissue injuries resulting from polytrauma include changes in cerebral metabolism, excitotoxicity, induction of emergency hematopoiesis, infiltration of fluid and inflammatory cells into the brain, systemic cytokine storm and localized cytokine release, and activation of microglia, the resident macrophage of the central nervous system8. The immune system plays critical roles in promoting tissue repair and clearance of dying neurons during recovery from neurotrauma but is also responsible for the deleterious inflammation that kills healthy neurons. Systemic immunodepression and vulnerability to infection often follow the acute phase of TBI wherein hypercytokinemia and inflammation resolve, placing patients at increased risk of pneumonia and sepsis9. Thus, understanding the contribution of the immune system to recovery is critical to development of efficacious therapies for neurotrauma11.Traumatic brain injury (TBI) is a major contributor to long-term disability and mortality in children and adults, and, despite intensive efforts, there are no effective treatments for TBI13. CD4+ and CD8+ T cells are recruited to the human brain after TBI and can be detected 3–26 days after injury15. Despite this increased accessibility to the central nervous system, CD4+ type 2 helper T (Th2) cells may not need to infiltrate the brain to modulate glial response, as they act via secretion of cytokines that circulate systemically18. Importantly, T cells capable of producing IFN-γ, like CD4+ type 1 helper T (Th1) cells, have been linked to increased microglial activation, including promotion of elevated microglial motility and phagocytic activity21. It was recently shown that programmed cell death-1 (PD-1) is upregulated by neurotrauma22. PD-1 targeting activates the epithelium of the choroid plexus to permit trafficking of monocytes into the brain, and this process depends upon IFN-gamma production by Th1 cells23. Not surprisingly, cytokines are altered in the brain parenchyma and peripheral blood in response to TBI, stroke, and neurodegerative disease25. Overexpression of cytokines in murine models has been shown to be neuroprotective in part through alternative activation of microglia, reducing neurodegeration in diseases such as Alzheimer’s, but the clinical consequences of cytokine perturbation in TBI are still incompletely understood30.Healthy brains normally have low numbers of surveilling peripheral immune cells within the parenchyma; nonetheless, peripheral immune cells do infiltrate the CNS, accumulate near areas of pathology, and can modify microglial polarization31. Consistent with these data, a meta-analysis of 28 other studies indicate that MSCs exert a positive effect on locomotor recovery32. While MSCs and MSC potency-enhancing factors improve outcomes in animal models associated with inflammation such as TBI, full restoration of cognitive function in animal models of TBI remains elusive. Our recent reports suggest that mechanical cues may contribute uniquely to the intracellular signaling that initiates MSC repression of inflammatory phenotypes34. Exposure of human MSCs to fluid shear stress typical of frictional forces present on the luminal lining of arteries (15 dyne/cm2) stimulates a broad range of anti-inflammatory signaling  and enhances MSC potency in suppression of cytotoxic TNF-α production by activated immune cells from the spleen35. In particular, we find evidence for prostaglandin E2, a metabolic product of COX2 enzyme activity, as a key mediator of shear-amplified efficacy and improved therapeutic potency36.Bone marrow mesenchymal stromal cells (MSCs) possess unique potential as neurotrauma therapeutics in part because their innate function is to regulate the hematopoietic system during homeostasis and in response to injury and infection. In addition to supporting hematopoietic stem cell (HSC) self renewal in the bone marrow, MSCs are thought to actively home to damaged tissue, attract immune cells by release of chemokines, and repress T, B, NK, macrophage, and dendritic cell inflammatory phenotypes via direct contact and paracrine signaling. We and others have shown that learning and memory are improved by administration of MSCs after TBI. Specifically, our studies show that MSCs promote hippocampal neurogenesis and improved neurocognitive function in mice after TBIWe utilize a humanized mouse model of TBI to examine chief components of the human immune system likely to contribute to outcome following trauma. Unexpectedly, we find that graft-versus-host interactions in the bone marrow and variation in human chimerism between animals complicates interpretation of immune response to neurotrauma. Despite these limitations, the model suggests that TBI exacerbates alloreactivity and rejection of host marrow and/or host niche components, leading to marrow destruction. The effect was more pronounced in injured mice that did not receive MSC therapy, suggesting that physiologic stress associated with injury could exacerbate pathology but that MSCs conferred some protection from TBI-induced immune activation in the marrow. Herein, our data demonstrate a role for T cells in bone marrow fitness following neurotrauma and suggest that, with judicious use, the humanized mouse could enable identification of human immune subsets important for neural protection and repair, as well as those that contribute to systemic disease and increased susceptibility to infections that cause patient morbidity after TBI.scid IL2Rɣnull (NSG) mice  within 48 h of birth were exposed to sublethal irradiation (100 cGy). Three hours after myeloablative conditioning, mice were anesthetized on ice and were infused via facial vein with a total of 2.5 × 105 primary human umbilical cord blood CD34+ cells . Briefly, commercially enriched CD34+ cells were thawed from cryopreservation and resuspended in 15 µl of sterile saline per neonate for intravenous transplantation using a Hamilton glass syringe, as reported in our prior study37. After cell infusion, pups were gently warmed and returned to the mother. All transplantation experiments were approved by and conducted in compliance with guidelines from the the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Health Science Center.Newborn NOD-38, as reported previously33. Thawed MSCs were plated at 1 × 105 cells/ml, and medium was changed every 3 days. At 80% confluence, cells were passaged by treatment with TrypLE Express  into IBIDI channels (μ-Slide I 0.4) at a density of 2–6 × 104 cells/cm2 for mouse TBI experiments.Bone marrow stromal cells were derived from whole bone marrow from independent human donors . Mononuclear cells from whole bone marrow were enriched in the buffy layer of Ficoll-Paque. Cells were resuspended for immediate expansion in complete culture medium consisting of MEM-α , 20% fetal bovine serum , 2 mM L-glutamine , 100 units/ml penicillin , and 100 μg/ml streptomycin . Nonadherent cells were removed after 2 days. Adherent colonies were expanded further and frozen as Passage 1. MSCs were profiled for expression of surface markers consistent with minimal guidelines established by the International Society for Stem Cell Therapy2 laminar WSS on the culture surface, by peristaltic pump  for 3 hr35. Flow rate required to achieve this force assumed steady laminar flow through a three-dimensional rectangular pipe 39. We determined WSS at the bottom center of the channel as Human MSCs were allowed to attach for 18 h on gas-permeable polymer coverslips within microfluidic channel slides . We applied unidirectional flow rates of 11.4 ml/min, corresponding to 15 dyne/cm2. MSCs (1 × 107 cells/kg) suspended in culture medium were injected via tail vein within 2 h of WSS exposure. Between 4 and 8 experiments conducted across different days were used to generate data for analysis of immune cells in the brain and hematopoietic tissues. Between 6 and 15 mice were prepared per treatment group. Each mouse appears as a single data point in graphical displays. All animal experiments were approved by and conducted in compliance with guidelines from the IACUC at the University of Texas Health Science Center.Experimental traumatic brain injuries were delivered on exposed brain in 5–8 month old male and female NSG mice by a Leica Impactor 1 CCI device. Injury was produced by administration of a single impact on the right parietal association cortex with a 3 mm impactor tip. Depth of deformation was 1.0 mm, and velocity was 5 m/s with 200 ms dwell time. Control (sham) mice were treated with cranial incision and craniotomy alongside animals undergoing CCI. MSC therapy included delivery of low passage (P2–5) bone marrow MSCs that were cultured under static conditions or preconditioned for 3 h with WSS of 15 dyne/cm40. Briefly, blood was collected from the retroorbital plexus at 6-week intervals after human umbilical cord CD34+ cell transplantation. White blood cells were enriched by 1% dextran sulfate-PBS-EDTA separation and treatment with RBC lysing buffer . Long bones of both legs were isolated and cleaned of all muscle 7 days after surgical procedures. One pair of femur and tibia were placed in 4% paraformaldehyde for histopathology and the other pair were crushed in PBS with a mortar and pestle, incubated with RBC lysing buffer, followed by filtration through a 70 μm cell strainer in preparation for flow cytometry. Lymph nodes were digested with a mixture of collagenase and DNAse and filtered through a 70 μm cell strainer. Spleens were macerated in 2% FBS-PBS for filtration in a 70 μm cell strainer, and leukocytes were enriched by removal of erythrocytes with RBC lysing buffer. The brain was collected 7 days after injury, and microglia were isolated using a Neural Dissociation kit . The resulting single cell suspension was washed and cleaned of myelin using 30% Percoll in HBSS, followed by a CD11b/c enrichment using a MACS kit . The myeloid-enriched cells were then prepared for flow cytometric analysis using the protocol described below.Peripheral blood and bone marrow isolation was performed as previously describedSubsets of human and murine cells were identified by immunostaining for flow cytometric analysis immediately following isolation of tissues. Cell preparations for analysis of T regulatory cells were fixed and permeabilized, whereas, all other panels were unfixed samples. Antibodies for detection of chimerism, T regulatory cells, murine myeloid derived suppressor cells (MDSCs), human MDSCs, and microglia are outlined in Table All data were analyzed with SigmaPlot 12.5 for statistical significance and are reported as individual points for each independent biological sample where feasible, with mean ± SEM. All data were evaluated for normality and variance to determine appropriateness of parametric versus non-parametric statistical tests. One-way ANOVA and either the Holm-Sidak method or Tukey test for multiple comparisons were used to evaluate differences between cell numbers, frequencies of immune cell subsets, and spleen size. Linear regression was used to evaluate the correlation between immune cell frequencies and spleen size. Pearson’s chi-square was used to assess whether bone marrow loss was independent of chimerism. The t-test or Mann–Whitney Rank Sum test was used to evaluate the relationship between marrow aplasia and chimerism. Significance levels of p < 0.05, < 0.01, or < 0.001 are denoted in graphs by a single, double, or triple asterisk, respectively.41. T cell development depends upon epithelially derived interleukin 7 (IL-7) and can be supported by coadministration of IL-7 or, alternatively, engraftment into the neonate43. Thus, NSG were transplanted as neonates with CD34+-enriched human cord blood HSCs and monitored for chimerism into adulthood  or vehicle control (media only) 24 h after surgery. MSCs were either cultured under standard (static) conditions or preconditioned by fluid WSS for 3 h prior to recovery for intravenous infusion. Animals receiving one of these four treatments  were euthanized at a sub-acute (7-day) post-procedure time point. One vehicle control mouse died at 6 days after injury and was found to have a partially calcified fracture to the left tibia. Similarly, a static cultured MSC mouse died 4 days after injury. Non-humanized C57BL/6 mice underwent CCI or sham surgery for evaluation of a subset of outcomes. Chimerism and lineage composition of the peripheral blood of humanized mice was evaluated   toward decreased hCD4+ and Treg cells in the bone marrow and lymph nodes of injured vehicle control animals  in conjunction with more profound inflammatory response in the injured mice. Analysis of other tolerogenic immune cells, including myeloid derived suppressor cells from human and mouse, did not reveal significant changes in frequency ; thus, the bone marrow is a major reservoir for T cells69. Inflammatory syndromes characterized by uncontrolled activation and proliferation of T lymphocytes and macrophages often present early in life following viral infection or other immune activated conditions such as autoimmune disease and malignant lymphoma70. One such disease, hemophagocytic lymphohistiocytosis or macrophage activation syndrome, is caused by hypercytokinemia. Hemophagocytic lymphohistiocytosis has recently been modeled in humanized NSG mice and has shown that transplant regimes which enhance T cell development also lead to an abundance of hemosiderin-containing hemophagocytic histiocytes in the bone marrow, which is accompanied by lethal disease71. Consistent with this report, humanized NSG mice in our study also exhibited extensive histiocytosis in the marrow, and we found that mice with higher levels of human T cell contribution suffer a higher incidence of bone marrow loss. The identity of the cytokines driving marrow pathology is unclear, but putative candidates include those triggered by TBI such as the pro-inflammatory cytokines TNFα, IL-1β, IL-6, and IL-18 (inducer of IFN-γ), and some anti-inflammatory cytokines including IL-1072. Yet, some apparently pro-inflammatory Th1 cytokines can also have anti-inflammatory functions. For example, the Th1 secreted cytokine, IL-10, can modulate CD4+ T cell functions by down-regulation of IL-273 but can also create a tolerogenic environment to allo-antigens74. Similarly, TNF-α priming can enhance Treg suppressive function to attenuate GVHD75. A future priority will be to elucidate how these cytokines contribute to recovery after TBI. Collectively, our data point to the bone marrow as an organ susceptible to inflammation and raises the possibility that elevated activation of T cells and associated inflammatory cytokine production could trigger autoreactivity of murine histiocytes toward niche cells of the marrow, further impairing the bone’s ability to support hematopoiesis.T cells are chief contributors to manifestation and severity of GVHD and act by secreting cytokines that lead to disease onset, especially in the context of cytokine storm+ T cells, including Treg cells, were modestly decreased in the bone marrow and lymph nodes of injured vehicle control animals. The implications of this trend are somewhat difficult to predict in the absence of careful analysis of T cell phenotype and activity. Chronic and acute GVHD are characterized by activation and proliferation of conventional CD4+ T cells. Both natural and induced Treg cells restrain CD4+ conventional T cell proliferation and attenuate GVHD by multiple mechanisms, including IL-2, IL-10 and TGF-β76. Indeed, one of the most effective therapies for GVHD is to increase Treg frequency, such as by IL-2 therapy77. Thus, the decrease in Treg frequency observed in the injured vehicle control mice in our study suggests that TBI would produce greater inflammation in part by reducing Treg in the bone marrow and lymph nodes. However, a cautious approach at characterization of the Treg cells in the NSG mouse would necessarily include intracellular staining for IL-10 and in vitro measurement of Treg suppressor activity in T effector cell proliferation assays. Not surprisingly, MSCs have been shown to promote Treg expansion79 and substantial evidence supports their application to treat GVHD80. Of special relevance to the WSS-treated MSCs in our study is that MSC-derived TGF-β and prostaglandin E2 are key regulators of Treg cell induction82. Clinical consequences of cytokine perturbation in TBI are incompletely understood; however, based upon these data, it is feasible that settings of secondary inflammatory stimulus that upset marrow homeostasis like TBI could contribute to T-cell mediated hypercytokinemia resulting in loss of self-tolerance or immunodepression.CD484. Likely through paracrine signaling, MSCs could modulate inflammatory T cell responses but could also suppress death of hematopoietic cells in the bone marrow. Consistent with the protective effects of MSCs on hematopoiesis, exogenous administration of secreted factors including platelet derived growth factor and TPO confer radioprotective effects on the bone marrow via reduction of apoptosis in multipotent hematopoietic stem and progenitor cells as well as mature blood lineages such as megakaryocytes85. Perhaps not coincidentally, TPO has also been shown to inhibit neuronal cell death86, further supporting existing evidence in the literature that paracrine signaling from MSCs produces positive pleiotropic effects on multiple organ systems.Several mechanisms could have contributed to improved bone marrow condition after MSC therapy. MSCs secrete a spectrum of soluble molecules that alter the local milieu to contribute to angiogenesis, cytoprotection, tissue repair, cell growth, and inflammatory suppression+ helper cells, which are found in greater numbers in the injured brain15. In humans, the frequencies of lymphoid and myeloid lineages vary across individuals, but typically the T lymphocyte population accounts for 10–25% of the leukocytes in the peripheral blood. We demonstrate a mean engraftment of approximately 50% T cells in the human graft for mice that met minimum requirements for chimerism. If only 50% of the white blood cells in the blood were human, that would translate to 25% human T cells in the humanized mouse blood. This T cell frequency falls within the range of a normal human peripheral blood sample. We suggest that the abundance of human T cells makes this a useful model for studying T cell contributions to recovery from neurotrauma. However, we acknowledge that the humanized model should be optimized and that certain limitations exist for study of certain immune cell subsets, such as myeloid lineages. Indeed, due to the rarity of human monocytes, modeling of the human innate immune response is likely not feasible. We were unable to resolve significant differences in neuroinflammation following injury, but ample evidence supports that immune response can play a role in recovery from traumatic CNS injury. In an independent report by Carpenter and colleagues, a similar humanization model showed that spinal cord injury resulted in different neuroinflammatory profiles before and after stable engraftment of the human immune system87. Neuroinflammation could be influenced by T cell activity, as it was shown in a non-humanized model that administration of T cells reactive to myelin protein antigens results in improved recovery in spinal cord and optic nerve injuries90. Stimulated T cells directly produce endogenous neurotrophins including BDNF91 and promote elevated neurotrophin production by neurons, outgrowth of neuronal axons, and modulation of microglia phenotype via secretion of IL-4, a prototypical Th2 cytokine94. Some of the protective function of T cells also derives from their effect on astrocytes. Study of two major cytokines produced by Th1 and Th2 cells, interferon-gamma and IL-4, respectively, suggests that a balanced Th1 and Th2 cytokine response is necessary to protect two chief functions of astrocytes, glutamate clearance and thiol secretion95. Together, numerous reports support a role for cytokines produced by Th1 and Th2 cells in neuroprotection and repair in CNS injury. Thus, we suggest that the robust engraftment of T cells in the humanized mouse presents a unique opportunity to identify the principal T cell subtypes that respond to TBI, elucidate how cells of the CNS engage with T cells following injury, and understand the role of cytokines in neural protection and repair.Some limitations of the current mouse model could be viewed as unique opportunities to better understand the human immune system. For example, T cells dominated the graft, and many of these were CD496. Destruction of bone marrow by irradiation or chemotherapy could be done prior to experimental TBI; however, mice are incredibly vulnerable to opportunistic infection, inflammatory response due to tissue damage, respiratory distress, dehydration, bleeding, and death in the weeks following myeloablation97. A more precise and informative strategy could include transplantation of T cell depleted hematopoietic progenitors or post-transplant depletion of immune cell subsets via antibody-based blocking, immunosuppressive agents, pretreatment with liposomal clodronate, and/or genetic targeting of the human immune cells99.Direct effects of marrow destruction on neurological outcomes of TBI are still unclear. Previous attempts to address the impact of the hematopoietic system on TBI outcomes have included approaches such as splenectomy. Splenectomy is a straightforward surgery that is tolerated well by rodents and has been shown to improve outcomes after TBIOverall, the humanized mouse is a complex model system that is made more challenging to evaluate by variation in human chimerism and interactions between mouse and human cells. Nevertheless, with careful experimental design and cautious expectations, we believe that this tool could present unique opportunities for modeling human immune response to therapeutic interventions for neurological disease and injuries. We examined several immune cell subsets thought to be modulated in response to TBI and MSC therapy and have identified a compelling role for T cells in control of bone marrow fitness after neurotrauma. Our study supports premise for use of the humanized mouse model of TBI to identify components of the human immune system that drive positive response to cellular therapies, drug candidates, and methods for protection against nosocomial infection. More broadly, this research also argues for new appraisal of how autoimmunity and GVHD might be intensified by trauma.Supplementary file1 (PDF 20193 kb)


Osteoarthritis (OA) is the most common joint disorder that has had an increasing prevalence due to the aging of the population. Recent studies have concluded that OA progression is related to oxidative stress and reactive oxygen species (ROS). ROS are produced at low levels in articular chondrocytes, mainly by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and ROS production and oxidative stress have been found to be elevated in patients with OA. The cartilage of OA-affected rat exhibits a significant induction of p47phox, a cytosolic subunit of the NADPH oxidase, similarly to human osteoarthritis cartilage. Therefore, this study tested whether siRNA p47phox that is introduced with poly  (PLGA) nanoparticles (p47phox si_NPs) can alleviate chondrocyte cell death by reducing ROS production. Here, we confirm that p47phox si_NPs significantly attenuated oxidative stress and decreased cartilage damage in mono-iodoacetate (MIA)-induced OA. In conclusion, these data suggest that p47phox si_NPs may be of therapeutic value in the treatment of osteoarthritis. Osteoarthritis (OA) is the most prevalent form of joint disease, a hallmark of which is cartilage loss . HoweverThe NOX family of NADPH oxidases is a major source of ROS production under various pathological conditions. Currently, this family is known to include seven members that utilize various combinations of subunits to form active enzyme complexes: NOX1, NOX2, NOX3, NOX4, NOX5, dual oxidase 1 (DUOX1), and DUOX2 ,11,12. OUpon exposure to cytokines, synovial NOX is known to produce superoxide anions that activate multiple signaling pathways and lead to the expression of inflammatory genes in rheumatoid arthritis synovial fibroblasts ,15,16. MLocal treatment via intra-articular injection is an appropriate strategy due to the fact that OA only affects the joints. When small molecular drugs have been introduced into intra-articular space, they have been easily and quickly removed by blood vessels and lymphatics . TherefoThus, the present study investigated the roles of p47phox in ROS production and cartilage damage in an OA model by using NPs.Male Sprague–Dawley rats weighing 120–140 g at the time of OA induction were used. Under brief isoflurane anesthesia, the animals were intra-articularly injected in the left knee with 2 mg of mono-iodoacetate  that was dissolved in 20 µL saline; controls received saline only (day 0). MIA was delivered through the left patellar tendon by using a 30-G needle.6) were seeded into 10 mm diameter dishes and cultured for 10 days in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). The medium was changed every 2 days [We used a scalpel to excise cartilage from the femoral condyles and posterior patellar surfaces of OA patients that were treated at Chungnam National University Hospital (CNUH) . The cartilage was cut into 2 mm thick pieces, and the digested suspension was passed through a 40 µM pore-size cell strainer to isolate individual chondrocytes. Cells were counted by using a cell counter. Cells (5 × 10y 2 days .v/v) paraformaldehyde for 2 days, decalcified in a Calci-Clear solution  for 2 days, sectioned in the coronal plane (4 µm thickness), embedded in paraffin wax, and used to prepare slides after staining with hematoxylin. Then, the slides were sequentially dehydrated in 70%, 80%, 90%, and 100% ethanol. Finally, sections were cleared in xylene. A light microscope and a digital camera were used to capture and evaluate the histopathological features of the articular cartilage.Cartilage from humans with OA was frozen, sectioned to a 4-μm thickness, and fixed in 4% paraformaldehyde (PFA). MIA-injected knees were fixed in 4%  for 1 h to prevent nonspecific binding, the sections were incubated with primary antibodies  and diluted in a blocking buffer. Immunostaining was performed by using the avidin–biotin peroxidase complex (ABC) method, as reported previously ,34. The Superoxide anion levels in the spinal cord were determined by using dihydroethidium , as described previously . CartilaAn MTT assay was performed in order to test the cell viability caused by siRNA-encapsulated PLGA nanoparticles as per the manufacturer’s instructions. Detailed procedures can also be found in previous reports .w/v; Thermo Fisher Scientific, Waltham, MA, USA) was directly added into the primary emulsion and further emulsified by sonication for 1 min to form a W1/O/W2 double emulsion. The resulting product was then diluted with 6 mL of 1% PVA1500 (w/v) and stirred magnetically for 3 h at room temperature to evaporate the DCM in a fume hood. Finally, the PLGA NPs were collected by centrifugation at 15,000× g for 15 min at 4 °C, washed twice with deionized water, and freeze-dried with 10 vials.p47phox siRNA-encapsulated PLGA NPs were prepared from a PLGA nanoparticle synthesis service from the Nanoglia company  with minor modifications, as reported previously ,35. A coTwo milligrams of lyophilized particles were dispersed in 1 mL of deionized water to determine the size distribution, zeta potential and polydispersity index (PDI) with the Zetasizer Nano ZS , and the diameter and shape were determined with scanning electron microscopy . The each 20 µM of siRNA-encapsulated PLGA nanoparticles, were collected and incubated in a Eppendorf tube with 250 µL of PBS, incubated at 37 °C for 48 h. At the designated time, 200 µL of the released medium was taken and replaced by the same amount of a fresh buffer. The amount of the p47phox siRNA was measured in the released buffer by using NanoDrop (Thermo Fisher Scientific). The accumulated release percentage of the p47phox siRNA and the entrapment efficiency were evaluated according to a previous report .p-values of less than 0.05 were considered statistically significant. All statistical analyses were performed by using GraphPad Prism 6 .The data are expressed as mean ± standard error of mean (SEM). The statistical significance between multiple groups was compared by a one-way analysis of variance (ANOVA) followed by an appropriate multiple comparison test. Chondrocyte clustering is a histological sign of late-stage OA ,37; thisThe progression of OA is significantly related to oxidative stress and ROS . The majThe most common experimental OA model features intra-articular injections of MIA, which is a metabolic inhibitor, into the knee joints of rats . This prBecause the cartilage damage that was caused by MIA was associated with ROS production, the expression levels of p47phox were measured. Animals injected with 2 mg of MIA exhibited an increased expression of p47phox by day 3 C,D. NextThe present study also assessed whether the inhibition of p47phox would affect cartilage damage in the rat model of MIA-induced OA. The efficient delivery of p47phox siRNA into the joints by using a gene delivery system requires the consideration of several factors. Thus, the present study employed poly (PLGA) copolymers; these are biodegradable in and biocompatible with humans, and some products from PLGA are widely used in many Food and Drug Administration (FDA)-approved drugs. There are currently 15 FDA-approved PLA/PLGA-based drug products that are available on the US market .Our research group previously demonstrated that p38 MAPK siRNA that is encapsulated in PLGA NPs attenuates spinal nerve ligation-induced neuropathic pain . MoreoveNext, the p47phox siRNA release profiles from p47phox si_NPs were analyzed by using a cumulative percentage approach. The results showed that p47phox si_NPs gradually released siRNA; this cumulative release peaked at 48 h after injection. After an initial burst of release (53.2%) at 24 h, the release of siRNA was consequently observed in a sustained manner at different times. The percentage of encapsulation efficiency was calculated as the amount of siRNA released from the lyophilized PLGA NPs/the amount of siRNA initially taken to prepare the NPs × 100. Encapsulation efficiency was 36.4 ± 0.17% G. We setTo address the question whether siRNA-encapsulated nanoparticles could work in cartilage, the determination of the effective dose was firstly performed. Different doses of siRNA p47phox NPs—0.2, 0.4, and 0.8 µM—were applied to the MIA-induced OA . The mecNext, the present study investigated whether p47phox si_NPs could attenuate the loss of proteoglycan and calcification of articular cartilage on day 3 after MIA injection. Compared to scrambled siRNA-loaded NPs, treatment with p47phox si_NPs reduced the thickness of the subchondral bone plate and attenuated the loss of cartilage lacunae B. To obsThe signaling pathways by which ROS contribute to the pathophysiology of OA are complex and require further investigation. In general, antioxidant therapies are inefficient treatments for the relief of OA symptoms, whereas antioxidant drugs have shown promising in vitro results; thus, further human studies are required. In the present study, MIA-induced OA chondrocytes produced ROS, a result that is consistent with findings from MIA-treated human chondrocytes. Excessive ROS production causes apoptotic cell death in OA chondrocytes; once induced, ROS are synthesized at a constant rate for a substantial period of time. The present results demonstrated a marked increase in the release of ROS in MIA-induced OA chondrocytes. Excessive levels of intracellular ROS are due to oxidative stress and enhanced levels of oxidative markers, which are the primary causes of cell damage and death. Thus, the inhibition of ROS-mediated injuries can reduce oxidative stress, protect chondrocytes, and treat OA. The present results demonstrated that the suppression of MIA-mediated injuries attenuated oxidative stress and protected chondrocytes. Therefore, the present findings contribute to the expanding understanding of proteins that are involved in the regulation of p47phox activation, and they also provide further insights regarding possible mechanisms that are involved in the regulation of cellular fates following OA.The permeability transition pore is generally regarded as the major ROS target inside mitochondria . The oxiThe activation and regulation of the NADPH oxidase are controlled by the phosphorylation of its cytosolic component (p47phox) on serine subunits that are located between Ser303 and Ser379 . NotablyOA is a systemic inflammatory disorder that most commonly targets the joints. The synovial fluid in patients with OA contains large numbers of neutrophils and macrophages, which suggests that these factors may contribute to tissue injury. NADPH oxidase activity and the phosphorylation of p47phox are markedly increased in neutrophils from patients with OA. This upregulation could be due to the actions of pro-inflammatory cytokines, such as TNF-α, which is found in high concentrations in the synovial fluids of these patients .Our study presented, for the first time, that p47phox siRNA-encapsulated PLGA NPs could have therapeutic effects on OA patients, leading to reduced chondrocytic cell death and cartilage damages. Additionally, the administration of NPs to the knees of patients with OA may influence their in vivo efficacy. However, these considerations are beyond the scope of this study and will be investigated in future work from our research group.In the present study, the successful encapsulation of siRNA into PLGA NPs resulted in near-monodispersed and spherical particles with a low polydispersity index, and these particles maintained their effects for an extended duration. Furthermore, the present study demonstrated that the p47phox siRNA-encapsulated PLGA NPs acted synergistically to reduce ROS production in an animal model of OA; the formulated PLGA NPs provided a sustained release of siRNA. Additionally, the capability of the manufactured NPs to permit sustained release suggests the potential for a delivery system that could reduce dosing frequency to a weekly regimen. Therefore, p47phox siRNA PLGA NPs may represent a promising novel therapeutic avenue for the treatment of OA.