IDO1 scavenges reactive oxygen species in myeloid-derived suppressor cells to prevent graft-versus-host disease.

Tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) also has an immunological function to suppress T cell activation in inflammatory circumstances, including graft-versus-host disease (GVHD), a fatal complication after allogeneic bone marrow transplantation (allo-BMT). Although the mononuclear cell expression of IDO1 has been associated with improved outcomes in GVHD, the underlying mechanisms remain unclear. Herein, we used IDO-deficient (Ido1-/-) BMT to understand why myeloid IDO limits the severity of GVHD. Hosts with Ido1-/- BM exhibited increased lethality, with enhanced proinflammatory and reduced regulatory T cell responses compared with wild type (WT) allo-BMT controls. Despite the comparable expression of the myeloid-derived suppressor cell (MDSC) mediators, arginase-1, inducible nitric oxide synthase, and interleukin 10, Ido1-/- Gr-1+CD11b+ cells from allo-BMT or in vitro BM culture showed compromised immune-suppressive functions and were skewed toward the Ly6ClowLy6Ghi subset, compared with the WT counterparts. Importantly, Ido1-/-Gr-1+CD11b+ cells exhibited elevated levels of reactive oxygen species (ROS) and neutrophil numbers. These characteristics were rescued by human IDO1 with intact heme-binding and catalytic activities and were recapitulated by the treatment of WT cells with the IDO1 inhibitor L1-methyl tryptophan. ROS scavenging by N-acetylcysteine reverted the Ido1-/-Gr-1+CD11b+ composition and function to an MDSC state, as well as improved the survival of GVHD hosts with Ido1-/- BM. In summary, myeloid-derived IDO1 enhances GVHD survival by regulating ROS levels and limiting the ability of Gr-1+CD11b+ MDSCs to differentiate into proinflammatory neutrophils. Our findings provide a mechanistic insight into the immune-regulatory roles of the metabolic enzyme IDO1.

Gut microorganisms and their metabolites modulate the severity of acute colitis in a tryptophan metabolism-dependent manner.

PURPOSE: Growing evidence shows that nutrient metabolism affects inflammatory bowel diseases (IBD) development. Previously, we showed that deficiency of indoleamine 2,3-dioxygenase 1 (Ido1), a tryptophan-catabolizing enzyme, reduced the severity of dextran sulfate sodium (DSS)-induced colitis in mice. However, the roles played by intestinal microbiota in generating the differences in disease progression between Ido1+/+ and Ido1-/- mice are unknown. Therefore, we aimed to investigate the interactions between the intestinal microbiome and host IDO1 in governing intestinal inflammatory responses. METHODS: Microbial 16s rRNA sequencing was conducted in Ido1+/+ and Ido1-/- mice after DSS treatment. Bacteria-derived tryptophan metabolites were measured in urine. Transcriptome analysis revealed the effects of the metabolite and IDO1 expression in HCT116 cells. Colitis severity of Ido1+/+ was compared to Ido1-/- mice following fecal microbiota transplantation (FMT). RESULTS: Microbiome analysis through 16S-rRNA gene sequencing showed that IDO1 deficiency increased intestinal bacteria that use tryptophan preferentially to produce indolic compounds. Urinary excretion of 3-indoxyl sulfate, a metabolized form of gut bacteria-derived indole, was significantly higher in Ido1-/- than in Ido1+/+ mice. Transcriptome analysis showed that tight junction transcripts were significantly increased by indole treatment in HCT116 cells; however, the effects were diminished by IDO1 overexpression. Using FMT experiments, we demonstrated that bacteria from Ido1-/- mice could directly attenuate the severity of DSS-induced colitis. CONCLUSIONS: Our results provide evidence that a genetic defect in utilizing tryptophan affects intestinal microbiota profiles, altering microbial metabolites, and colitis development. This suggests that the host and intestinal microbiota communicate through shared nutrient metabolic networks.

Inherently variable responses to glucocorticoid stress among endogenous retroviruses isolated from 23 mouse strains.

Active participation of endogenous retroviruses (ERVs) in disease processes has been exemplified by the finding that the HERV (human ERV)-W envelope protein is involved in the pathogenesis of multiple sclerosis, an autoimmune disease. We also demonstrated that injury-elicited stressors alter the expression of murine ERVs (MuERVs), both murine leukemia virus-type and mouse mammary tumor virus (MMTV)-type (MMTV-MuERV). In this study, to evaluate MMTV-MuERVs' responses to stress (e.g., injury, infection)-elicited systemic glucocorticoid (GC) levels, we examined the GC-stress response of 64 MMTV-MuERV promoters isolated from the genomes of 23 mouse strains. All 64 promoters responded to treatment with a synthetic GC, dexamethasone (DEX), at a wide range from a 0.6- to 85.7-fold increase in reporter activity compared to no treatment. An analysis of the 10 lowest and 10 highest DEX responders revealed specific promoter elements exclusively present in either the three lowest or the two highest responders. Each promoter had a unique profile of transcription regulatory elements and the glucocorticoid response element (GRE) was identified in all promoters with the number of GREs ranging from 2 to 7. The three lowest DEX responders were the only promoters with two GREs. The findings from this study suggest that certain MMTV-MuERVs are more responsive to stress-elicited systemic GC elevation compared to the others. The mouse strain-specific genomic MMTV-MuERV profiles and individual MMTV-MuERVs' differential responses to GC-stress might explain, at least in part, the variable inflammatory responses to injury and/or infection, often observed among different mouse strains. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Interaction of CD99 with its paralog CD99L2 positively regulates CD99L2 trafficking to cell surfaces.

Mouse CD99 and its paralog CD99-like 2 (CD99L2) are surface proteins implicated in cellular adhesion and migration. Although their distributions overlap in a wide variety of cells, their physical/functional relationship is currently unknown. In this study, we show the interaction between the two molecules and its consequence for membrane trafficking of mouse (m)CD99L2. The interaction was analyzed by bimolecular fluorescence complementation, immunoprecipitation, and fluorescence resonance energy transfer assays. When coexpressed, mCD99 formed heterodimers with mCD99L2, as well as homodimers, and the heterodimers were localized more efficiently at the plasma membrane than were the homodimers. Their interaction was cytoplasmic domain-dependent and enhanced mCD99L2 trafficking to the plasma membrane regardless of whether it was transiently overexpressed or endogenously expressed. Surface levels of endogenous mCD99L2 were markedly low on thymocytes, splenic leukocytes, and CTL lines derived from CD99-deficient mice. Importantly, the surface levels of mCD99L2 on mCD99-deficient cells recovered significantly when wild-type mCD99 was exogenously introduced, but they remained low when a cytoplasmic domain mutant of mCD99 was introduced. Our results demonstrate a novel role for mCD99 in membrane trafficking of mCD99L2, providing useful insights into controlling transendothelial migration of leukocytes.

Divergent and dynamic activity of endogenous retroviruses in burn patients and their inflammatory potential.

Genes constitute ~3% of the human genome, whereas human endogenous retroviruses (HERVs) represent ~8%. We examined post-burn HERV expression in patients' blood cells, and the inflammatory potentials of the burn-associated HERVs were evaluated. Buffy coat cells, collected at various time points from 11 patients, were screened for the expression of eight HERV families, and we identified their divergent expression profiles depending on patient, HERV, and time point. The population of expressed HERV sequences was patient-specific, suggesting HERVs' inherent genomic polymorphisms and/or differential expression potentials depending on characteristics of patients and courses of injury response. Some HERVs were shared among the patients, while the others were divergent. Interestingly, one burn-associated HERV gag gene from a patient's genome induced IL-6, IL-1ß, Ptgs-2, and iNOS. These findings demonstrate that injury stressors initiate divergent HERV responses depending on patient, HERV, and disease course and implicate HERVs as genetic elements contributing to polymorphic injury pathophysiology.

REViewer: a tool for linear visualization of repetitive elements within a sequence query.

A species-specific population of arrangements of repetitive elements (REs), called RE arrays, exists in the human and mouse genomes. We developed an RE analytical tool, named REViewer, for visualizing RE occurrences within RE arrays and other genomic regions as an interactive line map. REViewer utilizes an RE reference library which is established with two RE types: 1) REMiner-generated undefined REs and 2) RepeatMasker-derived defined REs. RE occurrences within queries are visualized as a line map using these two RE types. The REViewer's controller provides analytical options, such as zoom, customization of axis unit, and RE type selection. The functionality of REViewer was evaluated using the human chromosome Y sequence. The REViewer is determined to be an efficient tool that facilitates visualization of up to 6000 REs in RE arrays and other genomic regions. The maximum query size is linked to the RE mining tools (e.g., REMiner, RepeatMasker), not to REViewer.

ERE database: a database of genomic maps and biological properties of endogenous retroviral elements in the C57BL/6J mouse genome.

Endogenous retroviral elements (EREs), a family of transposable elements, constitute a substantial fraction of mammalian genomes. It is expected that profiles of the ERE sequences and their genomic locations are unique for each individual. Comprehensive characterization of the EREs' genomic locations and their biological properties is essential for understanding their roles in the pathophysiology of the host. In this study, we identified and mapped putative EREs (a total of 111 endogenous retroviruses [ERVs] and 488 solo long terminal repeats [sLTRs]) within the C57BL/6J mouse genome. The biological properties of individual ERE isolates (both ERVs and sLTRs) were then characterized in the following aspects: transcription potential, tropism trait, coding potential, recombination event, integration age, and primer binding site for replication. In addition, a suite of database management system programs was developed to organize and update the data acquired from current and future studies and to make the data accessible via internet.

REMiner-II: a tool for rapid identification and configuration of repetitive element arrays from large mammalian chromosomes as a single query.

Genes occupy ~3% of the human and mouse genomes whereas repetitive elements (REs), whose biologic functions are largely uncharacterized, constitute greater than 50%. A heterogeneous population of RE arrays (arrangement structures) is formed by combinations of various REs in mammalian genomes. In this study, REMiner-II was refined from the original REMiner for a more efficient identification and configuration of RE arrays from large queries (e.g., human chromosomes) using an unbiased self-alignment protocol. Chromosome-wide RE array profiles for the entire sets of human and mouse chromosomes were obtained using REMiner-II on a personal computer. REMiner-II provides 10 adjustable parameters and three data output modes to accommodate different experimental settings and/or goals. Examination of the human and mouse chromosome data using the REMiner-II viewer revealed species-specific libraries of complexly organized RE arrays. In conclusion, REMiner-II is an efficient tool for chromosome-wide identification and characterization of RE arrays from mammalian genomes.

Age-dependent and tissue-specific structural changes in the C57BL/6J mouse genome.

We tested the hypothesis that structural changes in the genome parallel age- and organ-specific phenotypes in conjunction with the differential transposition activities of retroelements. The genomes of the liver from C57BL/6J mice were larger than other organs, coinciding with an increase in genomic copies of certain retroelements. In addition, there were differential increments in the genome size of the liver with increasing age, which peaked at 5 weeks. The findings that the genome structure of an individual is variable depending on age and organ type in association with the transposition of retroelements may have broad implications in understanding biologic phenomena.

REMiner: a tool for unbiased mining and analysis of repetitive elements and their arrangement structures of large chromosomes.

Repetitive elements (REs) constitute a substantial portion of the genomes of human and other species; however, the RE profiles (type, density, and arrangement) within the individual genomes have not been fully characterized. In this study, we developed an RE analysis tool, called REMiner, for a chromosome-wide investigation into the occurrence of individual REs and arrangement of clusters of REs, and REMiner's functional features were examined using the human chromosome Y. The algorithm implemented by REMiner focused on unbiased mining of REs in large chromosomes and data interface within a viewer. The data from the chromosome demonstrated that REMiner is an efficient tool in regard to its capacity for a large query size and the availability of a high-resolution viewer, featuring instant retrieval of alignment data and control of magnification and identity ratio. The chromosome-wide survey identified a diverse population of ordered RE arrangements, which may participate in the genome biology.

ROP-ESAT6-CFP10 as Tuberculosis-Specific Antigen in Interferon Gamma Release Assay.

OBJECTIVE: The ROP-ESAT6-CFP10 antigen (Changzhou Niujin Shisong Biotech [CBI], China) was recently developed using recombinant overlapping peptide (ROP) technology. We used ROP-ESAT6-CFP10 as a tuberculosis (TB)-specific antigen and compared it with existing interferon-gamma release assays (IGRAs). METHODS: Healthy volunteers and patients who were diagnosed with TB within a one-year period were enrolled. Samples were tested with QuantiFERON-TB Gold (QFT; QIAGEN Sciences Inc., USA), T-SPOT.TB (Oxford Immunotec, UK), and ELISpot using ROP-ESAT6-CFP10 as a TB-specific antigen (ROP-TB). For ROP-TB, two concentrations (1 µg and 5 µg) of ROP-ESAT6-CFP10 were used as TB-specific antigens. Agreement between assays was evaluated. RESULTS: A total of 35 TB patients and 20 healthy volunteers were evaluated. Agreement between T-SPOT.TB and ROP-TB 1 µg, QFT and ROP-TB 1 µg, and ROP-TB 1 µg and ROP-TB 5 µg/mL were 79.1% (kappa=0.483), 76.7% (kappa=0.557), and 95.3% (kappa=0.894), respectively. The median number of spots between the T-SPOT.TB and ROP-TB assays in the TB patients had no significant difference. CONCLUSIONS: ELISpot using newly developed ROP-ESAT6-CFP10 showed good agreement with T-SPOT.TB and QFT. Since ROP technology can lower the manufacturing cost, ROP-ESAT6-CFP10 might work as a good source of TB-specific antigen for IGRAs.

Prevalent de novo somatic mutations in superantigen genes of mouse mammary tumor viruses in the genome of C57BL/6J mice and its potential implication in the immune system.

BACKGROUND: Superantigens (SAgs) of mouse mammary tumor viruses (MMTVs) play a crucial role in T cell selection in the thymus in a T cell receptor (TCR) Vß-specific manner and SAgs presented by B cells activate T cells in the periphery. The peripheral T cell repertoire is dynamically shaped by the steady induction of T cell tolerance against self antigens throughout the lifespan. We hypothesize that de novo somatic mutation of endogenous MMTV SAgs contributes to the modulation of the peripheral T cell repertoire. RESULTS: SAg coding sequences were cloned from the genomic DNAs and/or cDNAs of various tissues of female C57BL/6J mice. A total of 68 unique SAg sequences (54 translated sequences) were identified from the genomic DNAs of liver, lungs, and bone marrow, which are presumed to harbor only three endogenous MMTV loci (Mtv-8, Mtv-9, and Mtv-17). Similarly, 69 unique SAg sequences (58 translated sequences) were cloned from the cDNAs of 18 different tissues. Examination of putative TCR Vß specificity suggested that some of the SAg isoforms identified in this study have Vß specificities different from the reference SAgs of Mtv-8, Mtv-9, or Mtv-17. CONCLUSION: The pool of diverse SAg isoforms, generated by de novo somatic mutation, may play a role in the shaping of the peripheral T cell repertoire including the autoimmune T cell population.

Lipopolysaccharide stress induces cell-type specific production of murine leukemia virus type-endogenous retroviral virions in primary lymphoid cells.

Some murine-endogenous retroviruses, making up ∼10 % of the mouse genome, are induced during the course of experimental sepsis in which lipopolysaccharide (LPS), a pathogenic component of gram-negative bacteria, often plays a critical role. In this study, we investigated whether LPS stress induces the production of murine leukemia virus type-endogenous retrovirus (MuLV-ERV) virions in primary lymphoid cells. LPS treatment of cells (single-cell suspensions and sorted B- and T-cells) isolated from seven lymphoid organs of C57BL/6J mice resulted in a differential increase in the production of MuLV-ERV virions in most cells examined. Interestingly, among the 34 unique MuLV-ERV U3 sequences cloned from the viral genomic RNAs, the nuclear respiratory factor 1 (transcription factor) element was present only in the 20 U3 sequences that were derived from the LPS-induced MuLV-ERV U3 bands. Using the U3 sequences as a probe, 55 putative MuLV-ERV loci were mapped onto the C57BL/6J mouse genome and 15 of them retained full coding potential. Furthermore, one full-length recombinant MuLV-ERV originating from a locus on chromosome 13 was determined to be responsive to LPS stress. The findings from this study suggest that LPS stress differentially activates MuLV-ERV virion production in lymphoid organs in a cell type- and MuLV-ERV-specific manner. Further investigation is needed to define the role of MuLV-ERVs in the LPS signalling pathway(s) in general, as well as in the pathogenesis of sepsis.

Identification of a unique library of complex, but ordered, arrays of repetitive elements in the human genome and implication of their potential involvement in pathobiology.

Approximately 2% of the human genome is reported to be occupied by genes. Various forms of repetitive elements (REs), both characterized and uncharacterized, are presumed to make up the vast majority of the rest of the genomes of human and other species. In conjunction with a comprehensive annotation of genes, information regarding components of genome biology, such as gene polymorphisms, non-coding RNAs, and certain REs, is found in human genome databases. However, the genome-wide profile of unique RE arrangements formed by different groups of REs has not been fully characterized yet. In this study, the entire human genome was subjected to an unbiased RE survey to establish a whole-genome profile of REs and their arrangements. Due to the limitation in query size within the bl2seq alignment program (National Center for Biotechnology Information [NCBI]) utilized for the RE survey, the entire NCBI reference human genome was fragmented into 6206 units of 0.5M nucleotides. A number of RE arrangements with varying complexities and patterns were identified throughout the genome. Each chromosome had unique profiles of RE arrangements and density, and high levels of RE density were measured near the centromere regions. Subsequently, 175 complex RE arrangements, which were selected throughout the genome, were subjected to a comparison analysis using five different human genome sequences. Interestingly, three of the five human genome databases shared the exactly same arrangement patterns and sequences for all 175 RE arrangement regions (a total of 12,765,625 nucleotides). The findings from this study demonstrate that a substantial fraction of REs in the human genome are clustered into various forms of ordered structures. Further investigations are needed to examine whether some of these ordered RE arrangements contribute to the human pathobiology as a functional genome unit.

Tropism, cytotoxicity, and inflammatory properties of two envelope genes of murine leukemia virus type-endogenous retroviruses of C57BL/6J mice.

Envelope (env) proteins of certain endogenous retroviruses (ERVs) participate in various pathophysiological processes. In this study, we characterized pathophysiologic properties of two murine leukemia virus-type ERV (MuLV-ERV) env genes cloned from the ovary of C57BL/6J mice. The two env genes (named ENV(OV1) and ENV(OV2)), with 1,926 bp coding region, originated from two MuLV-ERV loci on chromosomes 8 and 18, respectively. ENV(OV1) and ENV(OV2) were ~75 kDa and predominantly expressed on the cell membrane. They were capable of producing pseudotype murine leukemia virus virions. Tropism trait and infectivity of ENV(OV2) were similar to the polytropic env; however, ENV(OV1) had very low level of infectivity. Overexpression of ENV(OV2), but not ENV(OV1), exerted cytotoxic effects and induced expression of COX-2, IL-1ß, IL-6, and iNOS. These findings suggest that the ENV(OV1) and ENV(OV2) are capable of serving as an env protein for virion assembly, and they exert differential cytotoxicity and modulation of inflammatory mediators.

Hydrogen-rich water reduces inflammatory responses and prevents apoptosis of peripheral blood cells in healthy adults: a randomized, double-blind, controlled trial.

The evidence for the beneficial effects of drinking hydrogen-water (HW) is rare. We aimed to investigate the effects of HW consumption on oxidative stress and immune functions in healthy adults using systemic approaches of biochemical, cellular, and molecular nutrition. In a randomized, double-blind, placebo-controlled study, healthy adults (20-59 y) consumed either 1.5 L/d of HW (n = 20) or plain water (PW, n = 18) for 4 weeks. The changes from baseline to the 4th week in serum biological antioxidant potential (BAP), derivatives of reactive oxygen, and 8-Oxo-2'-deoxyguanosine did not differ between groups; however, in those aged ≥ 30 y, BAP increased greater in the HW group than the PW group. Apoptosis of peripheral blood mononuclear cells (PBMCs) was significantly less in the HW group. Flow cytometry analysis of CD4+, CD8+, CD20+, CD14+ and CD11b+ cells showed that the frequency of CD14+ cells decreased in the HW group. RNA-sequencing analysis of PBMCs demonstrated that the transcriptomes of the HW group were clearly distinguished from those of the PW group. Most notably, transcriptional networks of inflammatory responses and NF-κB signaling were significantly down-regulated in the HW group. These finding suggest HW increases antioxidant capacity thereby reducing inflammatory responses in healthy adults.

Quantitative Electrode Design Modeling of an Electroadhesive Lifting Device Based on the Localized Charge Distribution and Interfacial Polarization of Different Objects.

Electroadhesive devices can lift materials of different shapes and various types using the electrostatic force developed at the interface between the device and the object. More specifically, the electrical potential generated by the device induces opposite charges on the object to give electrostatic Maxwell force. Although this technology has a great deal of potential, the key design factors based on the fundamental principles of interfacial polarization have yet to be clearly identified. In this study, we identify that the lifting force is quantitatively related to the total length of the boundary edges of the electrodes, where the induced charges are selectively concentrated. We subsequently propose a model equation that can predict the electrostatic lifting forces for different object materials as a function of the applied voltage, impedance, and electrode-boundary length. The model is based on the fact that the amount of induced charges should be concentrated where the equipotential field distance is minimal. We report that the impedance magnitude is correlated with the electroadhesive lifting forces by analyzing the impedance characteristics of objects made of different materials (e.g., paper, glass, or metal), as attached in situ to the electroadhesive device.

Endogenous retroviruses in systemic response to stress signals.

Infection of germline cells with retroviruses initiates permanent proviral colonization of the germline genome. The germline-integrated proviruses, called endogenous retroviruses (ERVs), are inherited to offspring in a Mendelian order and belong to the transposable element family. Endogenous retroviruses and other long terminal repeat retroelements constitute ~8% and ~10% of the human and mouse genomes, respectively. It is likely that each individual has a distinct genomic ERV profile. Recent studies have revealed that a substantial fraction of ERVs retains the coding potentials necessary for virion assembly and replication. There are several layers of potential mechanisms controlling ERV expression: intracellular transcription environment (e.g., transcription factor pool, splicing machinery, hormones), epigenetic status of the genome (e.g., proviral methylation, histone acetylation), profile of transcription regulatory elements on each ERV's promoter, and a range of stress signals (e.g., injury, infection, environment). Endogenous retroviruses may exert pathophysiologic effects by infection followed by random reintegration into the genome, by their gene products (e.g., envelope, superantigen), and by altering the expression of neighboring genes. Several studies have provided evidence that ERVs are associated with a range of pathogenic processes involving multiple sclerosis, systemic lupus erythematosus, breast cancer, and the response to burn injury. For instance, the proinflammatory properties of the human ERV-W envelope protein play a central role in demyelination of oligodendrocytes. As reviewed in this article, recent advances in ERV biology and mammalian genomics suggest that ERVs may have a profound influence on various pathogenic processes including the response to injury and infection. Understanding the roles of ERVs in the pathogenesis of injury and infection will broaden insights into the underlying mechanisms of systemic immune disorder and organ failure in these patients.

Cosegregation of CD14 locus and polymorphic alleles of glucocorticoid receptor and protocadherins into CD14 knockout mouse genome.

Targeted mutagenesis technology has been used to investigate the biological characteristics of genes. We incidentally observed a discrepancy in the size of the glucocorticoid receptor (GCR) between CD14 knockout (KO) mice and backcross controls. The CD14 KO mice were generated using 129S4/SvJae-derived J1 embryonic stem cells, C57BL/ 6J donor blastocysts, and C57BL/6J backcross strain. In this study, the extent of genomic heterogeneity of the CD14 KO mice, potentially affecting their phenotype, was characterized in comparison to C57BL/6J controls. There were polymorphic alleles of the GCR gene in CD14 KO and C57BL/6J mice: a tandem repeat of 8 CAGs and 17 CAGs in the transactivation domain, respectively. The subsequent finding of eight CAGs in all 129 substrains examined and colocalization of CD14 and GCR genes on the same contig on chromosome 18 suggested that the GCR allele in the J1 embryonic stem cell genome cosegregated with the targeted CD14 locus. Interestingly, all three clusters of the protocadherin family, central genes in determining neuronal networks, were mapped between the CD14 and GCR loci. Further analyses revealed numerous nonsynonymous coding single-nucleotide polymorphisms within the protocadherin family between CD14 KO and C57BL/6J mice. In addition, heterogeneous profiles of endogenous retroviruses, which constitute approximately 10% of the genome, were observed between them. These findings suggest that cosegregation of genes flanking the targeted locus leads to a substantial level of genetic heterogeneity in CD14 KO mice compared with their backcross controls. Phenotypic changes observed in some KO mice may not be as definitive as expected.

Skewed Dendritic Cell Differentiation of MyD88-Deficient Donor Bone Marrow Cells, Instead of Massive Expansion as Myeloid-Derived Suppressor Cells, Aggravates GVHD.

Graft-versus-host disease (GVHD), a life-threatening complication after bone marrow transplantation (BMT), is induced by activation of alloreactive donor T cells. Our previous study demonstrated that transplantation of myeloid differentiation factor 88 (MyD88)-deficient knockout (KO) bone marrow (BM) resulted in aggravation of GVHD. Here, to understand the cellular mechanism, we performed longitudinal in vivo imaging and flow cytometric analyses followed by transcriptome and functional examination of donor MyD88-KO BM progenies in GVHD hosts, using a major histocompatibility complex-matched but minor histocompatibility antigen-mismatched C57BL/6→BALB.B model. In GVHD hosts with MyD88-KO BMT, donor BM-derived CD11b+Gr-1+ cells were found to undergo cell death, a fate significantly different from the explosive expansion shown by the wild type (WT) counterparts, and also from the moderate expansion of the WT or MyD88-KO BM-derived cells in non-GVHD hosts. It was also revealed that MyD88-KO CD11b+Gr-1+ cells preferred differentiation into CD11c+ dendritic cells (DCs) to expansion as myeloid-derived suppressor cells in GVHD hosts or in high inflammatory in vitro conditions. These CD11c+ DCs comprised the majority of MyD88-KO CD11b+Gr-1+ apoptotic cells in GVHD hosts. Their ability to cross-present alloantigens of host origin contributed to the enhancement of T cell alloreactivity, causing GVHD aggravation and eventually death through the killing function of activated T cells. These results provide insights into the roles of MyD88 in myelopoiesis of donor BM and the protective effects in GVHD hosts, helpful information for development of a strategy to control GVHD.